Patent Publication Number: US-2023136909-A1

Title: Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 3-hydroxyisobutyrate or methacrylic acid related thereto

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation application of U.S. patent application Ser. No. 15/979,313, filed May 14, 2018, which is a continuation of U.S. patent application Ser. No. 15/133,150, filed Apr. 19, 2017, now U.S. Pat. No. 10,000,758, which is a continuation application of U.S. patent application Ser. No. 14/071,339, filed Nov. 4, 2013, now U.S. Pat. No. 9,346,902, which claims the benefit of U.S. Ser. No. 61/766,660 filed Feb. 19, 2013, and U.S. Ser. No. 61/722,657 filed Nov. 5, 2012, the content of each of which is incorporated herein by reference in its entirety. 
    
    
     1. SUMMARY 
     Provided herein are methods generally relating to metabolic and biosynthetic processes and microbial organisms capable of producing organic compounds. Specifically, provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 3-hydroxyisobutyrate and/or methacrylic acid (MAA). Also provided herein are non-naturally occurring microbial organisms and methods thereof to produce optimal yields of 3-hydroxyisobutyrate and/or MAA. 
     In a first aspect, provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In certain embodiments, the methanol metabolic pathway comprises one or more enzymes selected from the group consisting of a methanol methyltransferase; a methylenetetrahydrofolate reductase; a methylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolate cyclohydrolase; a formyltetrahydrofolate deformylase; a formyltetrahydrofolate synthetase; a formate hydrogen lyase; a hydrogenase; a formate dehydrogenase; a methanol dehydrogenase; a formaldehyde activating enzyme; a formaldehyde dehydrogenase; a S-(hydroxymethyl)glutathione synthase; a glutathione-dependent formaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Such organisms advantageously allow for the production of reducing equivalents, which can then be used by the organism for the production of 3-hydroxyisobutyrate or MAA using any one of the 3-hydroxyisobutyrate or MAA pathways provided herein. 
     In a second aspect, provided herein is a non-naturally occurring microbial organism having (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a 3-hydroxyisobutyrate pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate pathway enzyme expressed in a sufficient amount to produce 3-hydroxyisobutyrate. In certain embodiments, the 3-hydroxyisobutyrate pathway enzyme is selected from the group consisting of succinyl-CoA transferase, ligase, or synthetase; methylmalonyl-CoA mutase; methylmalonyl-CoA epimerase; methylmalonyl-CoA reductase (aldehyde forming); methylmalonate semialdehyde reductase; and methylmalonyl-CoA reductase (alcohol forming). 
     In a third aspect, provided herein is a non-naturally occurring microbial organism having (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a MAA pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a MAA pathway enzyme expressed in a sufficient amount to produce MAA. In certain embodiments, the MAA pathway enzyme is selected from the group consisting of succinyl-CoA transferase, ligase, or synthetase; methylmalonyl-CoA mutase; methylmalonyl-CoA epimerase; methylmalonyl-CoA reductase (aldehyde forming); methylmalonate semialdehyde reductase; 3-hydroxyisobutyrate dehydratase; and methylmalonyl-CoA reductase (alcohol forming). 
     In other embodiments, the organism having a methanol metabolic pathway, either alone or in combination with a 3-hydroxyisobutyrate or MAA pathway, as provided herein, further comprises a formaldehyde assimilation pathway that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In some of embodiments, the formaldehyde assimilation pathway comprises a hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, dihydroxyacetone synthase or dihydroxyacetone kinase. In certain embodiments, provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol dehydrogenase expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a formaldehyde assimilation pathway. In certain embodiments, the organism further comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis. In certain embodiments, the formaldehyde assimilation pathway enzyme is selected from the group consisting of a hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, dihydroxyacetone synthase and dihydroxyacetone kinase. 
     In some embodiments, the organism further comprises one or more gene disruptions, occurring in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 , and/or amino acids by said microbial organism, wherein said one or more gene disruptions confer increased production of 3-hydroxyisobutyrate or MAA in said microbial organism. In some embodiments, one or more endogenous enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2  and/or amino acids by the microbial organism, has attenuated enzyme activity or expression levels. In certain embodiments, the organism comprises from one to twenty-five gene disruptions. In other embodiments, the organism comprises from one to twenty gene disruptions. In some embodiments, the organism comprises from one to fifteen gene disruptions. In other embodiments, the organism comprises from one to ten gene disruptions. In some embodiments, the organism comprises from one to five gene disruptions. In certain embodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 gene disruptions or more. 
     In another aspect, provided herein are methods for producing 3-hydroxyisobutyrate, comprising culturing any one of the non-naturally occurring microbial organisms comprising a methanol metabolic pathway and a 3-hydroxyisobutyrate pathway provided herein under conditions and for a sufficient period of time to produce 3-hydroxyisobutyrate. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. 
     In yet another aspect, provided herein are methods for producing MAA, comprising culturing any one of the non-naturally occurring microbial organisms comprising a methanol metabolic pathway and a MAA pathway provided herein under conditions and for a sufficient period of time to produce MAA. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. 
    
    
     
       2. BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    shows exemplary metabolic pathways enabling the extraction of reducing equivalents from methanol. The enzymatic transformations shown are carried out by the following enzymes: 1A) a methanol methyltransferase, 1B) a methylenetetrahydrofolate reductase, 1C) a methylenetetrahydrofolate dehydrogenase, 1D) a methenyltetrahydrofolate cyclohydrolase, 1E) a formyltetrahydrofolate deformylase, 1F) a formyltetrahydrofolate synthetase, 1G) a formate hydrogen lyase, 1H) a hydrogenase, 1I) a formate dehydrogenase, 1J) a methanol dehydrogenase, 1K) a formaldehyde activating enzyme, 1L) a formaldehyde dehydrogenase, 1M) a S-(hydroxymethyl)glutathione synthase, 1N) a glutathione-dependent formaldehyde dehydrogenase, and 1O) a S-formylglutathione hydrolase. In certain embodiments, steps K and/or M are spontaneous. 
         FIG.  2    shows exemplary 3-hydroxyisobutyrate and MAA pathways, which can be used to increase 3-hydroxyisobutyrate or MAA yields from carbohydrates when reducing equivalents produced by a methanol metabolic pathway provided herein are available. The enzymatic transformations shown are carried out by the following enzymes: 2A) succinyl-CoA transferase, ligase, or synthetase; 2B) methylmalonyl-CoA mutase; 2C) methylmalonyl-CoA epimerase; 2D) methylmalonyl-CoA reductase (aldehyde forming); 2E) methylmalonate semialdehyde reductase; 2F) 3-hydroxyisobutyrate dehydratase; and 2G) methylmalonyl-CoA reductase (alcohol forming). 3-hydroxyisobutyrate production can be carried out by 2A, 2B, 2C, 2D and 2E; 2A, 2B, 2D and 2E; or 2A, 2B and 2G. MAA production can be carried out by 2A, 2B, 2C, 2D, 2E and 2F; 2A, 2B, 2D, 2E and 2F; or 2A, 2B, 2G and 2F. 
         FIG.  3    shows an exemplary formaldehyde assimilation pathway. The enzymatic transformations are carried out by the following enzymes: 3A) a hexulose-6-phosphate synthase, and 3B) a 6-phospho-3-hexuloisomerase. 
         FIG.  4    shows an exemplary formaldehyde assimilation pathway. The enzymatic transformations are carried out by the following enzymes: 4A) a dihydroxyacetone synthase, and 4B) a dihydroxyacetone kinase. 
     
    
    
     3. DETAILED DESCRIPTION OF THE INVENTION 
     3.1 Definitions 
     As used herein, the term “non-naturally occurring” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism&#39;s genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a 3-hydroxyisobutyrate or MAA biosynthetic pathway. 
     As used herein, “3-hydroxyisobutyric acid” (IUPAC name 3-hydroxy-2-methylpropanoic acid), is the acid form of 3-hydroxyisobutyrate, and it is understood that 3-hydroxyisobutyrate and 3-hydroxyisobutyric acid can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH. The chemical structure of 3-hydroxyisobutyric acid. acid is shown below: 
     
       
         
         
             
             
         
       
     
     As used herein, “methacrylic acid” (MAA) having the chemical formula CH 2 ═C(CH 3 )CO 2  (IUPAC name 2-methyl-2-propenoic acid), is the acid form of methacrylate, and it is understood that methacrylic acid and methacrylate can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH. The chemical structure of MAA is shown below: 
     
       
         
         
             
             
         
       
     
     A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein. 
     As used herein, the term “isolated” when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring. 
     As used herein, the terms “microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. 
     As used herein, the term “CoA” or “coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation. 
     As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen. 
     As used herein, the term “gene disruption,” or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention. The phenotypic effect of a gene disruption can be a null mutation, which can arise from many types of mutations including inactivating point mutations, entire gene deletions, and deletions of chromosomal segments or entire chromosomes. Specific antisense nucleic acid compounds and enzyme inhibitors, such as antibiotics, can also produce null mutant phenotype, therefore being equivalent to gene disruption. 
     As used herein, the term “growth-coupled” when used in reference to the production of a biochemical product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism. The term “growth-coupled” when used in reference to the consumption of a biochemical is intended to mean that the referenced biochemical is consumed during the growth phase of a microorganism. 
     As used herein, the term “attenuate,” or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein. Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given pathway to function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host microbial organism to survive, reproduce or grow. Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but does not necessarily mimic complete disruption of the enzyme or protein. 
     “Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid. 
     It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism. 
     The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. 
     Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as  E. coli  and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the  E. coli  metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements. 
     An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor. 
     Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5′-3′ exonuclease and  Drosophila  DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa. 
     In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others. 
     A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene. 
     Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having 3-hydroxyisobutyrate or MAA biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. 
     Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences. 
     Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters: Match: 1; mismatch: −2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences. 
     3.2 Microbial Organisms that Utilize Reducing Equivalents Produced by the Metabolism of Methanol 
     Provided herein are methanol metabolic pathways engineered to improve the availability of reducing equivalents, which can be used for the production of product molecules. Exemplary product molecules include, without limitation, 3-hydroxyisobutyrate and MAA, although given the teachings and guidance provided herein, it will be recognized by one skilled in the art that any product molecule that utilizes reducing equivalents in its production can exhibit enhanced production through the biosynthetic pathways provided herein. 
     Methanol is a relatively inexpensive organic feedstock that can be derived from synthesis gas components, CO and H 2 , via catalysis. Methanol can be used as a source of reducing equivalents to increase the molar yield of product molecules from carbohydrates. 
     The present invention relates generally to biosynthetic processes, and more specifically to organisms having methacrylic acid biosynthetic capabilities. 
     Methyl methacrylate (MMA) is an organic compound with the formula CH 2 ═C(CH 3 )CO 2 CH 3 . This colorless liquid is the methyl ester of methacrylic acid (MMA) and is the monomer for the production of the transparent plastic polymethyl methacrylate (PMMA). 
     The principal application of methyl methacrylate is the production of polymethyl methacrylate acrylic plastics. Also, methyl methacrylate is used for the production of the co-polymer methyl methacrylate-butadiene-styrene (MBS), used as a modifier for PVC. Methyl methacrylate polymers and co-polymers are used for waterborne coatings, such as latex paint. Uses are also found in adhesive formulations. Contemporary applications include the use in plates that keep light spread evenly across liquid crystal display (LCD) computer and TV screens. Methyl methacrylate is also used to prepare corrosion casts of anatomical organs, such as coronary arteries of the heart. 
     Methacrylic acid, or 2-methyl-2-propenoic acid, is a low molecular weight carboxylic acid that occurs naturally in small amounts in the oil of Roman chamomile. It is a corrosive liquid with an acrid unpleasant odor. It is soluble in warm water and miscible with most organic solvents. Methacrylic acid polymerizes readily upon heating or treatment with a catalytic amount of strong acid, such as HCl. The resulting polymer is a ceramic-looking plastic. Methacrylic acid is used industrially in the preparation of its esters, known collectively as methacrylates, such as methyl methacrylate. The methacrylates have numerous uses, most notably in the manufacture of polymers. 
     Most commercial producers apply an acetone cyanohydrin (ACH) route to produce methacrylic acid (MAA), with acetone and hydrogen cyanide as raw materials. The intermediate cyanohydrin is converted with sulfuric acid to a sulfate ester of the methacrylamide, hydrolysis of which gives ammonium bisulfate and MAA. Some producers start with an isobutylene or, equivalently, tert-butanol, which is oxidized to methacrolein, and again oxidized to methacrylic acid. MAA is then esterified with methanol to MMA. 
     The conventional production process, using the acetone cyanohydrin route, involves the conversion of hydrogen cyanide (HCN) and acetone to acetone cyanohydrin, which then undergoes acid assisted hydrolysis and esterification with methanol to give MMA. Difficulties in handling potentially deadly HCN along with the high costs of byproduct disposal (1.2 tons of ammonium bisulfate are formed per ton of MMA) have sparked a great deal of research aimed at cleaner and more economical processes. A number of new processes have been commercialized over the last two decades and many more are close to commercialization. The Asahi “Direct Metha” route, which involves the oxidation of isobutylene to methacrolein, which is then mixed with methanol, oxidized with air, and esterified to MMA, has been described as an economical process. 
     Other than MMA polymers, the other major product of this industry is crude methacrylic acid, which accounts for about 20 percent of the total production of MMA. Crude MAA is processed into butyl methacrylates and/or “glacial” MAA, which is highly purified crude MAA. Glacial MAA can be used directly as a comonomer in various polymers and is also used to make a variety of small volume methacrylates. On the other hand, MAA can also be converted into MMA via esterification with methanol. 
     There exists a need for the development of methods for effectively producing commercial quantities of compounds, such as 3-hydroxyisobutyrate and MAA. 
     Accordingly, provided herein is bioderived 3-hydroxyisobutyrate produced according to the methods described herein and biobased products comprising or obtained using the bioderived 3-hydroxyisobutyrate. The biobased product can comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived 3-hydroxyisobutyrate. The biobased product can comprises a portion of said bioderived 3-hydroxyisobutyrate as a repeating unit. The biobased product can be a molded product obtained by molding the biobased product. 
     Also provided herein is bioderived MAA produced according to the methods described herein and biobased products comprising or obtained using the bioderived MAA. The biobased product can comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived MAA. The biobased product can comprises a portion of said bioderived MAA as a repeating unit. The biobased product can be a molded product obtained by molding the biobased product. 
     In numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents to byproducts. Methanol is a relatively inexpensive organic feedstock that can be used to generate reducing equivalents by employing one or more methanol metabolic enzymes as shown in  FIG.  1   . The reducing equivalents produced by the metabolism of methanol by one or more of the methanol metabolic pathways can then be used to power the glucose to 3-hydroxyisobutyrate or MAA production pathways, for example, as shown in  FIG.  2   . 
     The product yields per C-mol of substrate of microbial cells synthesizing reduced fermentation products such as 3-hydroxyisobutyrate and MAA are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing equivalents, or electrons, can be extracted from methanol using one or more of the enzymes described in  FIG.  1   . The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H 2 , or water, respectively. Reduced ferredoxin, reduced quinones and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway, reductive TCA cycle, or product pathway enzymes. 
     Specific examples of how additional redox availability from methanol can improve the yield of reduced products such as 3-hydroxyisobutyrate or MAA are shown. 
     The maximum theoretical yield of 3-hydroxyisobutyrate or MAA via the pathway shown in  FIG.  2    supplemented with the reactions of the oxidative TCA cycle (e.g., citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase) is 1.09 mol/mol. 
       1C 6 H 12 O 6 →1.09C 4 H 10 O 2 +1.64CO 2 +0.55H 2 O
 
     When both feedstocks of sugar and methanol are available, the methanol can be utilized to generate reducing equivalents by employing one or more of the enzymes shown in  FIG.  1   . The reducing equivalents generated from methanol can be utilized to power the glucose to 3-hydroxyisobutyrate or MAA production pathways, e.g., as shown in  FIG.  2   . Theoretically, all carbons in glucose will be conserved, thus resulting in a maximal theoretical yield to produce 3-hydroxyisobutyrate from glucose at 2 mol 3-hydroxyisobutyrate per mol of glucose under either aerobic or anaerobic conditions as shown in  FIG.  2   : 
       10CH 3 OH+3C 6 H 12 O 6 =6C 4 H 10 O 2 +8H 2 O+4CO 2    
     In a similar manner, the maximum theoretical yields of MAA can reach 2 mol/mol glucose using the reactions shown in  FIGS.  1  and  2   . 
       C 6 H 12 O 6 +0.667CH 3 OH+1.333CO 2 →2C 4 H 6 O 4 +1.333H 2 O
 
       C 6 H 12 O 6 +2CH 3 OH→2C 4 H 8 O 3 +2H 2 O
 
     In a first aspect, provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In certain embodiments, the methanol metabolic pathway comprises one or more enzymes selected from the group consisting of a methanol methyltransferase; a methylenetetrahydrofolate reductase; a methylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolate cyclohydrolase; a formyltetrahydrofolate deformylase; a formyltetrahydrofolate synthetase; a formate hydrogen lyase; a hydrogenase; a formate dehydrogenase; a methanol dehydrogenase; a formaldehyde activating enzyme; a formaldehyde dehydrogenase; a S-(hydroxymethyl)glutathione synthase; a glutathione-dependent formaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Such organisms advantageously allow for the production of reducing equivalents, which can then be used by the organism for the production of 3-hydroxyisobutyrate or MAA using any one of the 3-hydroxyisobutyrate or MAA pathways provided herein. 
     In certain embodiments, the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is a methylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolate dehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is a formyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolate synthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is a formate dehydrogenase; 1J is a methanol dehydrogenase; 1K is a formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1M is a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependent formaldehyde dehydrogenase; and 1O is S-formylglutathione hydrolase. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     In one embodiment, the methanol metabolic pathway comprises 1A. In another embodiment, the methanol metabolic pathway comprises 1B. In another embodiment, the methanol metabolic pathway comprises 1C. In yet another embodiment, the methanol metabolic pathway comprises 1D. In one embodiment, the methanol metabolic pathway comprises 1E. In another embodiment, the methanol metabolic pathway comprises 1F. In another embodiment, the methanol metabolic pathway comprises 1G. In yet another embodiment, the methanol metabolic pathway comprises 1H. In one embodiment, the methanol metabolic pathway comprises 1I. In another embodiment, the methanol metabolic pathway comprises 1J. In another embodiment, the methanol metabolic pathway comprises 1K. In yet another embodiment, the methanol metabolic pathway comprises 1L. In yet another embodiment, the methanol metabolic pathway comprises 1M. In another embodiment, the methanol metabolic pathway comprises 1N. In yet another embodiment, the methanol metabolic pathway comprises 1O. Any combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen methanol metabolic pathway enzymes 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O is also contemplated. 
     In some embodiments, the methanol metabolic pathway is a methanol metabolic pathway depicted in  FIG.  1   . 
     In one aspect, provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said methanol metabolic pathway comprises: (i) 1A and 1B, (ii) 1J; or (iii) 1J and 1K. In one embodiment, the methanol metabolic pathway comprises 1A and 1B. In another embodiment, the methanol metabolic pathway comprises 1J. In one embodiment, the methanol metabolic pathway comprises 1J and 1K. In certain embodiments, the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E. In some embodiments. the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F. In some embodiments, the methanol metabolic pathway comprises 1J, 1C, 1D and 1E. In one embodiment, the methanol metabolic pathway comprises 1J, 1C, 1D and 1F. In another embodiment, the methanol metabolic pathway comprises 1J and 1L. In yet another embodiment, the methanol metabolic pathway comprises 1J, 1M, 1N and 1O. In certain embodiments, the methanol metabolic pathway comprises 1J, 1N and 1O. In some embodiments, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E. In one embodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     In certain embodiments, the methanol metabolic pathway comprises 1I. In certain embodiments, the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I. In some embodiments. the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I. In some embodiments, the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I. In one embodiment, the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I. In another embodiment, the methanol metabolic pathway comprises 1J, 1L and 1I. In yet another embodiment, the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I. In certain embodiments, the methanol metabolic pathway comprises 1J, 1N, 1O and 1I. In some embodiments, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I. In one embodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     In certain embodiments, the methanol metabolic pathway comprises 1G. In certain embodiments, the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G. In some embodiments. the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G. In some embodiments, the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G. In one embodiment, the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G. In another embodiment, the methanol metabolic pathway comprises 1J, 1L and 1G. In yet another embodiment, the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G. In certain embodiments, the methanol metabolic pathway comprises 1J, 1N, 1O and 1G. In some embodiments, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G. In one embodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     In certain embodiments, the methanol metabolic pathway comprises 1G and 1H. In certain embodiments, the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H. In some embodiments. the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H. In some embodiments, the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H. In one embodiment, the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H. In another embodiment, the methanol metabolic pathway comprises 1J, 1L, 1G and 1H. In yet another embodiment, the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H. In certain embodiments, the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H. In some embodiments, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H. In one embodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     In certain embodiments, the formation of 5-hydroxymethylglutathione from formaldehyde is spontaneous (see, e.g.,  FIG.  1   , step M). In some embodiments, the formation of 5-hydroxymethylglutathione from formaldehyde is catalyzed by a S-(hydroxymethyl)glutathione synthase (see, e.g.,  FIG.  1   , step M). In certain embodiments, the formation of methylene-THF from formaldehyde is spontaneous (see, e.g.,  FIG.  1   , step K). In certain embodiments, the formation of methylene-THF from formaldehyde is catalyzed by a formaldehyde activating enzyme (see, e.g.,  FIG.  1   , step K). 
     In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises three exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises four exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises five exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises six exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. 
     Any non-naturally occurring eukaryotic organism comprising a methanol metabolic pathway and engineered to comprise a methanol metabolic pathway enzyme, such as those provided herein, can be engineered to further comprise one or more 3-hydroxyisobutyrate or MAA pathway enzymes. 
     In one embodiment, the non-naturally occurring microbial organism further comprises a 3-hydroxyisobutyrate pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate pathway enzyme expressed in a sufficient amount to produce 3-hydroxyisobutyrate. In certain embodiments, the 3-hydroxyisobutyrate pathway enzyme is selected from the group consisting of succinyl-CoA transferase, ligase, or synthetase; methylmalonyl-CoA mutase; methylmalonyl-CoA epimerase; methylmalonyl-CoA reductase (aldehyde forming); methylmalonate semialdehyde reductase; and methylmalonyl-CoA reductase (alcohol forming). 
     In another embodiment, the non-naturally occurring microbial organism further comprises a MAA pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a MAA pathway enzyme expressed in a sufficient amount to produce MAA. In certain embodiments, the MAA pathway enzyme is selected from the group consisting of succinyl-CoA transferase, ligase, or synthetase; methylmalonyl-CoA mutase; methylmalonyl-CoA epimerase; methylmalonyl-CoA reductase (aldehyde forming); methylmalonate semialdehyde reductase; 3-hydroxyisobutyrate dehydratase; and methylmalonyl-CoA reductase (alcohol forming). 
     In some embodiments, the non-naturally occurring microbial organisms having a 3-hydroxyisobutyrate pathway include a set of 3-hydroxyisobutyrate pathway enzymes. In other embodiments, the non-naturally occurring microbial organisms having a MAA pathway include a set of MAA pathway enzymes. 
     Enzymes, genes and methods for engineering pathways from succinate or succinyl-CoA to various products, such as 3-hydroxyisobutyrate or MAA, into a microorganism, are now known in the art, as are enzymes for the conversion of glucose to phosphoenolpyruvate (PEP), phosphoenolpyruvate to oxaloacetate, oxaloacetate to malate, malate to fumarate, and fumarate to succinate (see, e.g., U.S. Publ. No. 2011/0201089, which is herein incorporated by reference in its entirety). A set of 3-hydroxyisobutyrate or MAA pathway enzymes represents a group of enzymes that can convert succinate to 3-hydroxyisobutyrate or MAA, respectively, as shown in  FIG.  2   . The additional reducing equivalents obtained from the methanol metabolic pathways, as disclosed herein, improve the yields of all these products when utilizing carbohydrate-based feedstock. 
     Exemplary enzymes for the conversion of succinate to 3-hydroxyisobutyrate include succinyl-CoA transferase, ligase, or synthetase ( FIG.  2   , step A); methylmalonyl-CoA mutase ( FIG.  2   , step B); methylmalonyl-CoA epimerase ( FIG.  2   , step C); methylmalonyl-CoA reductase (aldehyde forming) ( FIG.  2   , step D); methylmalonate semialdehyde reductase ( FIG.  2   , step E); and methylmalonyl-CoA reductase (alcohol forming) ( FIG.  2   , step G). 
     In one aspect, provided herein is a non-naturally occurring microbial organism, comprising (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a 3-hydroxyisobutyrate pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate pathway enzyme expressed in a sufficient amount to produce 3-hydroxyisobutyrate. In one embodiment, the at least one exogenous nucleic acid encoding the methanol metabolic pathway enzyme enhances the availability of reducing equivalents in the presence of methanol in a sufficient amount to increase the amount of 3-hydroxyisobutyrate produced by the non-naturally microbial organism. In some embodiments, the methanol metabolic pathway comprises any of the various combinations of methanol metabolic pathway enzymes described above or elsewhere herein. 
     In certain embodiments, (1) the methanol metabolic pathway comprises: 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is a methylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolate dehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is a formyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolate synthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is a formate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneous or formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependent formaldehyde dehydrogenase and 1O is S-formylglutathione hydrolase; and (2) the 3-hydroxyisobutyrate pathway comprises 2A, 2B, 2C, 2D, 2E or 2G, or any combination thereof, wherein 2A is a succinyl-CoA transferase, ligase, or synthetase; 2B is a methylmalonyl-CoA mutase; 2C is a methylmalonyl-CoA epimerase; 2D is a methylmalonyl-CoA reductase (aldehyde forming); 2E is a methylmalonate semialdehyde reductase; and 2G is a methylmalonyl-CoA reductase (alcohol forming). In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, the 3-hydroxyisobutyrate pathway comprises 2A. In another embodiment, the 3-hydroxyisobutyrate pathway comprises 2B. In an embodiment, the 3-hydroxyisobutyrate pathway comprises 2C. In another embodiment, the 3-hydroxyisobutyrate pathway comprises 2D. In another embodiment, the 3-hydroxyisobutyrate pathway comprises 2E. In another embodiment, the 3-hydroxyisobutyrate pathway comprises 2G. Any combination of two, three, four, five or six 3-hydroxyisobutyrate pathway enzymes 2A, 2B, 2C, 2D, 2E and 2G is also contemplated. 
     In some embodiments, the methanol metabolic pathway is a methanol metabolic pathway depicted in  FIG.  1   , and the 3-hydroxyisobutyrate pathway is a 3-hydroxyisobutyrate pathway depicted in  FIG.  2   . 
     Exemplary sets of 3-hydroxyisobutyrate pathway enzymes to convert succinate to 3-hydroxyisobutyrate, according to  FIG.  2   , include (i) 2A, 2B, 2C, 2D and 2E; (ii) 2A, 2B, 2D and 2E; and (iii) 2A, 2B and 2G. 
     In one embodiment, (1) the methanol metabolic pathway comprises 1A and 1B; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2C, 2D and 2E. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, (1) the methanol metabolic pathway comprises 1A and 1B; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B, 2D and 2E. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, (1) the methanol metabolic pathway comprises 1A and 1B; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In another embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the 3-hydroxybutyrate pathway comprises 2A, 2B and 2G. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, the non-naturally occurring microbial organism comprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1J and 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and 1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J, 1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and 1I; 1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I; 1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D, 1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A, 1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J, 1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1E and 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and 1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C, 1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N, 1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) a 3-hydroxyisobutyrate pathway. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     Any methanol metabolic pathway provided herein can be combined with any 3-hydroxyisobutyrate pathway provided herein. 
     Exemplary enzymes for the conversion of succinate to MAA include succinyl-CoA transferase, ligase, or synthetase ( FIG.  2   , step A); methylmalonyl-CoA mutase ( FIG.  2   , step B); methylmalonyl-CoA epimerase ( FIG.  2   , step C); methylmalonyl-CoA reductase (aldehyde forming) ( FIG.  2   , step D); methylmalonate semialdehyde reductase ( FIG.  2   , step E); 3-hydroxyisobutyrate dehydratase ( FIG.  2   , step F); and methylmalonyl-CoA reductase (alcohol forming) ( FIG.  2   , step G). 
     In one aspect, provided herein is a non-naturally occurring microbial organism, comprising (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) a MAA pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a MAA pathway enzyme expressed in a sufficient amount to produce MAA. In one embodiment, the at least one exogenous nucleic acid encoding the methanol metabolic pathway enzyme enhances the availability of reducing equivalents in the presence of methanol in a sufficient amount to increase the amount of MAA produced by the non-naturally microbial organism. In some embodiments, the methanol metabolic pathway comprises any of the various combinations of methanol metabolic pathway enzymes described above or elsewhere herein. 
     In certain embodiments, (1) the methanol metabolic pathway comprises: 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is a methylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolate dehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is a formyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolate synthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is a formate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneous or formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependent formaldehyde dehydrogenase and 10 is S-formylglutathione hydrolase; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E, 2F or 2G, or any combination thereof, wherein 2A is a succinyl-CoA transferase, ligase, or synthetase; 2B is a methylmalonyl-CoA mutase; 2C is a methylmalonyl-CoA epimerase; 2D is a methylmalonyl-CoA reductase (aldehyde forming); 2E is a methylmalonate semialdehyde reductase; 2F is a 3-hydroxyisobutyrate dehydratase; and 2G is a methylmalonyl-CoA reductase (alcohol forming). In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, the MAA pathway comprises 2A. In another embodiment, the MAA pathway comprises 2B. In an embodiment, the MAA pathway comprises 2C. In another embodiment, the MAA pathway comprises 2D. In another embodiment, the MAA pathway comprises 2E. In another embodiment, the MAA pathway comprises 2F. In another embodiment, the MAA pathway comprises 2G. Any combination of two, three, four, five, six or seven MAA pathway enzymes 2A, 2B, 2C, 2D, 2E, 2F and 2G is also contemplated. 
     In some embodiments, the methanol metabolic pathway is a methanol metabolic pathway depicted in  FIG.  1   , and the MAA pathway is a MAA pathway depicted in  FIG.  2   . 
     Exemplary sets of MAA pathway enzymes to convert succinate to MAA, according to  FIG.  2   , include (i) 2A, 2B, 2C, 2D, 2E and 2F; (ii) 2A, 2B, 2D, 2E and 2F; and (iii) 2A, 2B, 2G and 2F. 
     In one embodiment, (1) the methanol metabolic pathway comprises 1A and 1B; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2C, 2D, 2E and 2F. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, (1) the methanol metabolic pathway comprises 1A and 1B; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2D, 2E and 2F. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, (1) the methanol metabolic pathway comprises 1A and 1B; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the MAA pathway comprises 2A, 2B, 2G and 2F. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a succinyl-CoA transferase. In other embodiments, 2A is a succinyl-CoA ligase. In certain embodiments, 2A is a succinyl-CoA synthetase. 
     In one embodiment, the non-naturally occurring microbial organism comprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1J and 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and 1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J, 1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 11; 1A, 1B, 1C, 1D, 1E and 1I; 1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I; 1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D, 1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A, 1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J, 1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1E and 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and 1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C, 1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N, 1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) a MAA pathway. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. 
     Any methanol metabolic pathway provided herein can be combined with any MAA pathway provided herein. 
     Also provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  1   , step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. One exemplary formaldehyde assimilation pathway that can utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  1   ) is shown in  FIG.  3   , which involves condensation of formaldehyde and D-ribulose-5-phosphate to form hexulose-6-phosphate (h6p) by hexulose-6-phosphate synthase ( FIG.  3   , step A). The enzyme can use Mg2+ or Mn2+ for maximal activity, although other metal ions are useful, and even non-metal-ion-dependent mechanisms are contemplated. H6p is converted into fructose-6-phosphate by 6-phospho-3-hexuloisomerase ( FIG.  3   , step B). Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  1   ) is shown in  FIG.  4    and proceeds through dihydroxyacetone. Dihydroxyacetone synthase is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis ( FIG.  4   , step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase ( FIG.  4   , step B). DHAP can be assimilated into glycolysis and several other pathways. Rather than converting formaldehyde to formate and on to CO2 off-gassed, the pathways provided in  FIGS.  3  and  4    show that carbon is assimilated, going into the final product. 
     Thus, in one embodiment, an organism having a methanol metabolic pathway, either alone or in combination with a 3-hydroxyisobutyrate or MAA pathway, as provided herein, further comprises a formaldehyde assimilation pathway that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In some of embodiments, the formaldehyde assimilation pathway comprises 3A or 3B, wherein 3A is a hexulose-6-phosphate synthase and 3B is a 6-phospho-3-hexuloisomerase In other embodiments, the formaldehyde assimilation pathway comprises 4A or 4B, wherein 4A is a dihydroxyacetone synthase and 4B is a dihydroxyacetone kinase. 
     In certain embodiments, provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol dehydrogenase (1J) expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a formaldehyde assimilation pathway. In certain embodiments, the organism further comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used, for example, in the formation of biomass. In certain embodiments, the formaldehyde assimilation pathway enzyme is selected from the group consisting of a hexulose-6-phosphate synthase (3A), 6-phospho-3-hexuloisomerase (3B), dihydroxyacetone synthase (4A) and dihydroxyacetone kinase (4B). 
     In one aspect, provided herein is a non-naturally occurring microbial organism, comprising (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde; and (2) a formaldehyde assimilation pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used, for example, in the formation of biomass. In specific embodiments, the methanol metabolic pathway comprises a methanol dehydrogenase (1J). In certain embodiments, the formaldehyde assimilation pathway enzyme is 3A, and the intermediate is a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof. In other embodiments, the formaldehyde assimilation pathway enzyme is 3B, and the intermediate is a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof. In yet other embodiments, the formaldehyde assimilation pathway enzyme is 3A and 3B, and the intermediate is a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof. In some embodiments, the formaldehyde assimilation pathway enzyme is 4A, and the intermediate is a dihydroxyacetone (DHA), a dihydroxyacetone phosphate, or a combination thereof. In other embodiments, the formaldehyde assimilation pathway enzyme is 4B, and the intermediate is a DHA, a dihydroxyacetone phosphate, or a combination thereof. In yet other embodiments, the formaldehyde assimilation pathway enzyme is 4A and 4B, and the intermediate is a DHA, a dihydroxyacetone phosphate, or a combination thereof. In one embodiment, the at least one exogenous nucleic acid encoding the methanol metabolic pathway enzyme, in the presence of methanol, sufficiently enhances the availability of reducing equivalents and sufficiently increases formaldehyde assimilation to increase the production of 3-hydroxyisobutyrate, MAA or other products described herein by the non-naturally microbial organism. In some embodiments, the methanol metabolic pathway comprises any of the various combinations of methanol metabolic pathway enzymes described above or elsewhere herein. 
     In certain embodiments, (1) the methanol metabolic pathway comprises: 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is a methylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolate dehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is a formyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolate synthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is a formate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneous or formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependent formaldehyde dehydrogenase and 10 is S-formylglutathione hydrolase; and (2) the formaldehyde assimilation pathway comprises 3A, 3B or a combination thereof, wherein 3A is a hexulose-6-phosphate synthase, and 3B is a 6-phospho-3-hexuloisomerase. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, the intermediate is a hexulose-6-phosphate. In other embodiments, the intermediate is a fructose-6-phosphate. In yet other embodiments, the intermediate is a hexulose-6-phosphate and a fructose-6-phosphate. 
     In one embodiment, the formaldehyde assimilation pathway comprises 3A. In another embodiment, the formaldehyde assimilation pathway comprises 3B. In one embodiment, the formaldehyde assimilation pathway comprises 3A and 3B. 
     In some embodiments, the methanol metabolic pathway is a methanol metabolic pathway depicted in  FIG.  1   , and a formaldehyde assimilation pathway depicted in  FIG.  3   . An exemplary set of formaldehyde assimilation pathway enzymes to convert D-ribulose-5-phosphate and formaldehyde to fructose-6-phosphate (via hexulose-6-phosphate) according to  FIG.  3    include 3A and 3B. 
     In a specific embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In other embodiments, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In some embodiments, the intermediate is a hexulose-6-phosphate. In other embodiments, the intermediate is a fructose-6-phosphate. In yet other embodiments, the intermediate is a hexulose-6-phosphate and a fructose-6-phosphate. 
     In certain embodiments, (1) the methanol metabolic pathway comprises: 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is a methylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolate dehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is a formyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolate synthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is a formate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneous or formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependent formaldehyde dehydrogenase and 1O is S-formylglutathione hydrolase; and (2) the formaldehyde assimilation pathway comprises 4A, 4B or a combination thereof, wherein 4A is a dihydroxyacetone synthase and 4B is a dihydroxyacetone kinase. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. In some embodiments, the intermediate is a DHA. In other embodiments, the intermediate is a dihydroxyacetone phosphate. In yet other embodiments, the intermediate is a DHA and a dihydroxyacetone phosphate. 
     In one embodiment, the formaldehyde assimilation pathway comprises 4A. In another embodiment, the formaldehyde assimilation pathway comprises 4B. In one embodiment, the formaldehyde assimilation pathway comprises 4A and 4B. 
     In some embodiments, the methanol metabolic pathway is a methanol metabolic pathway depicted in  FIG.  1   , and a formaldehyde assimilation pathway depicted in  FIG.  4   . An exemplary set of formaldehyde assimilation pathway enzymes to convert xyulose-5-phosphate and formaldehyde to dihydroxyacetone-phosphate (via DHA) according to  FIG.  4    include 4A and 4B. 
     In a specific embodiment, (1) the methanol metabolic pathway comprises 1J; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In other embodiments, (1) the methanol metabolic pathway comprises 1J and 1K; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In another embodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In another embodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In yet another embodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In certain embodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B. In some embodiments, 1K is spontaneous. In other embodiments, 1K is a formaldehyde activating enzyme. In some embodiments, 1M is spontaneous. In some embodiments, the intermediate is a DHA. In other embodiments, the intermediate is a dihydroxyacetone phosphate. In yet other embodiments, the intermediate is a DHA and a dihydroxyacetone phosphate. 
     Any methanol metabolic pathway provided herein can be combined with any formaldehyde assimilation pathway provided herein. In addition, any methanol metabolic pathway provided herein can be combined with any 3-hydroxyisobutyrate or MAA pathway and any formaldehyde pathway provided herein. 
     Also provided herein are methods of producing formaldehyde comprising culturing a non-naturally occurring microbial organism having a methanol metabolic pathway provided herein. In some embodiments, the methanol metabolic pathway comprises 1J. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. In specific embodiments, the formaldehyde is an intermediate that is consumed (assimilated) in the production of 3-hydroxyisobutyrate, MAA and other products described herein. 
     Also provided herein are methods of producing an intermediate of glycolysis and/or a metabolic pathway that can be used, for example, in the formation of biomass, comprising culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and a formaldehyde assimilation pathway, as provided herein, under conditions and for a sufficient period of time to produce the intermediate. In some embodiments, the intermediate is a hexulose-6-phosphate. In other embodiments, the intermediate is a fructose-6-phosphate. In yet other embodiments, the intermediate is a hexulose-6-phosphate and a fructose-6-phosphate. In some embodiments, the intermediate is a DHA. In other embodiments, the intermediate is a dihydroxyacetone phosphate. In yet other embodiments, the intermediate is a DHA and a dihydroxyacetone phosphate. In some embodiments, the methanol metabolic pathway comprises 1J. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. Such biomass can also be used in methods of producing any of the products, such as the biobased products, provided elsewhere herein. 
     In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In some embodiments, the organism comprises two exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In some embodiments, the organism comprises three exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In some embodiments, the organism comprises four exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In other embodiments, the organism comprises five exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In some embodiments, the organism comprises six exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In some embodiments, the organism comprises seven exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme. In certain embodiments, the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate or MAA pathway enzyme; and the organism further comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises two exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises three exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises further four exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises five exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises six exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. 
     In some embodiments, the organism comprises two or more exogenous nucleic acids, each encoding a formaldehyde assimilation pathway enzyme. In some embodiments, the organism comprises two exogenous nucleic acids, each encoding a formaldehyde assimilation pathway enzyme. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a formaldehyde assimilation pathway enzyme; and the organism further comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises two exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises three exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism comprises further four exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises five exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises six exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. In certain embodiments, the organism further comprises seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. 
     In some embodiments, the at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme is a heterologous nucleic acid. In other embodiments, the at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme is a heterologous nucleic acid. In other embodiments, the at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate or MAA pathway enzyme is a heterologous nucleic acid. In certain embodiments, the at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme is a heterologous nucleic acid, and the at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate or MAA pathway enzyme is a heterologous nucleic acid. In other embodiments, the at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme is a heterologous nucleic acid, and the at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme is a heterologous nucleic acid. 
     In certain embodiments, the organism is in a substantially anaerobic culture medium. 
     It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the figures, including the pathways of  FIGS.  1 ,  2 ,  3  and  4   , can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. Non-limiting examples of such intermediate or products are 3-hydroxyisobutyrate or MAA. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring eukaryotic organism that produces a 3-hydroxyisobutyrate or MAA pathway intermediate can be utilized to produce the intermediate as a desired product. 
     In certain embodiments, a non-naturally occurring microbial organism comprising a methanol metabolic pathway and a 3-hydroxyisobutyrate or MAA pathway provided herein, further comprises one or more gene disruptions. In certain embodiments, the one or more gene disruptions confer increased production of 3-hydroxyisobutyrate or MAA in the organism. In other embodiments, a non-naturally occurring microbial organism comprising a methanol metabolic pathway and a formaldehyde assimilation pathway provided herein, further comprises one or more gene disruptions. In some embodiments, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 , amino acids, or any combination thereof, by said microbial organism. In one embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of ethanol. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of glycerol. In other embodiments, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of acetate. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of lactate. In one embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of formate. In another embodiment, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of CO 2 . In other embodiments, the gene disruption is in an endogenous gene encoding a protein and/or enzyme involved in native production of amino acids by said microbial organism. In some embodiments, the protein or enzyme is a pyruvate decarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactate dehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate:quinone oxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, a lactate dehydrogenase, a pyruvate dehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, a monocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase, a pyruvate kinase, or any combination thereof. In certain embodiments, the organism comprises from one to twenty-five gene disruptions. In other embodiments, the organism comprises from one to twenty gene disruptions. In some embodiments, the organism comprises from one to fifteen gene disruptions. In other embodiments, the organism comprises from one to ten gene disruptions. In some embodiments, the organism comprises from one to five gene disruptions. In certain embodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 gene disruptions or more. 
     In other embodiments, a non-naturally occurring microbial organism comprising a methanol metabolic pathway and a 3-hydroxyisobutyrate or MAA pathway provided herein, further comprises one or more endogenous proteins or enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2  and/or amino acids by said microbial organism, wherein said one or more endogenous proteins or enzymes has attenuated protein or enzyme activity and/or expression levels. In some embodiments, a non-naturally occurring microbial organism comprising a methanol metabolic pathway and a formaldehyde assimilation pathway provided herein, further comprises one or more endogenous proteins or enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO2 and/or amino acids by said microbial organism, wherein said one or more endogenous proteins or enzymes has attenuated protein or enzyme activity and/or expression levels. In one embodiment the endogenous protein or enzyme is a pyruvate decarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactate dehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate:quinone oxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, a lactate dehydrogenase, a pyruvate dehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, a monocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase, a pyruvate kinase, or any combination thereof. 
     Each of the non-naturally occurring alterations provided herein result in increased production and an enhanced level of 3-hydroxyisobutyrate or MAA, for example, during the exponential growth phase of the microbial organism, compared to a strain that does not contain such metabolic alterations, under appropriate culture conditions. Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution. 
     Given the teachings and guidance provided herein, those skilled in the art will understand that to introduce a metabolic alteration, such as attenuation of an enzyme, it can be necessary to disrupt the catalytic activity of the one or more enzymes involved in the reaction. Alternatively, a metabolic alteration can include disrupting expression of a regulatory protein or cofactor necessary for enzyme activity or maximal activity. Furthermore, genetic loss of a cofactor necessary for an enzymatic reaction can also have the same effect as a disruption of the gene encoding the enzyme. Disruption can occur by a variety of methods including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity, disruption can occur by a genetic alteration that reduces or eliminates the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits required to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction of the invention. Such other functions are well known to those skilled in the art. Similarly, a target enzyme activity can be reduced or eliminated by disrupting expression of a protein or enzyme that modifies and/or activates the target enzyme, for example, a molecule required to convert an apoenzyme to a holoenzyme. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the invention can be disrupted so long as the targeted reaction is reduced or eliminated. 
     Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis. Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods of the invention and incorporated into the non-naturally occurring microbial organisms of the invention in order to achieve the increased production of fatty alcohol, fatty aldehyde or fatty acid or growth-coupled product production. 
     Given the teachings and guidance provided herein, those skilled in the art also will understand that enzymatic activity or expression can be attenuated using well known methods. Reduction of the activity or amount of an enzyme can mimic complete disruption if the reduction causes activity of the enzyme to fall below a critical level that is normally required for the pathway to function. Reduction of enzymatic activity by various techniques rather than disruption can be important for an organism&#39;s viability. Methods of reducing enzymatic activity that result in similar or identical effects of a gene disruption include, but are not limited to: reducing gene transcription or translation; destabilizing mRNA, protein or catalytic RNA; and mutating a gene that affects enzyme kinetics. Natural or imposed regulatory controls can also accomplish enzyme attenuation including: promoter replacement; loss or alteration of transcription factors; introduction of inhibitory RNAs or peptides such as siRNA, antisense RNA, RNA or peptide/small-molecule binding aptamers, ribozymes, aptazymes and riboswitches; and addition of drugs and other chemicals that reduce or disrupt enzymatic activity such as gene splicing. 
     One of ordinary skill in the art will also recognize that attenuation of an enzyme can be done at various levels. For example, at the gene level, mutations causing a partial or complete null phenotype or epistatic genetic effects that mask the activity of a gene product can be used to attenuate an enzyme. At the gene expression level, methods for attenuation include: coupling transcription to an endogenous or exogenous inducer such as IPTG, then adding low or 0 levels of inducer during the production phase; introducing or modifying positive or negative regulators; modify histone acetylation/deacetylation in region where gene is integrated; introducing a transposition to disrupt a promoter or a regulatory gene; flipping of a transposable element or promoter region; deleting one allele resulting in loss of heterozygosity in a diploid organism; introducing nucleic acids that increase RNA degradation; or in bacteria, for example, introduction of a tmRNA tag, which can lead to RNA degradation and ribosomal stalling. At the translational level, attenuation can include: introducing rare codons to limit translation; introducing RNA interference molecules that block translation; modifying regions outside the coding sequence, such as introducing secondary structure into UTR regions to block translation or reduce efficiency of translation; adding RNAase sites for rapid transcript degradation; introducing antisense RNA oligomers or antisense transcripts; introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches; or introducing translational regulatory elements involving RNA structure that can prevent or reduce translation that can be controlled by the presence or absence of small molecules. At the level of enzyme localization and/or longevity, enzyme attenuation can include: adding a degradation tag for faster protein turnover; or adding a localization tag that results in the enzyme being localized to a compartment where it would not be able to react normally. At the level of post-translational regulation, enzyme attenuation can include: increasing intracellular concentration of known inhibitors; or modifying post-translational modified sites. At the level of enzyme activity, enzyme attenuation can include: adding endogenous or exogenous inhibitor, such as a target-specific drug, to reduce enzyme activity; limiting availability of essential cofactors, such as B12, for an enzyme that require it; chelating a metal ion that is required for activity; or introducing a dominant negative mutation. 
     In some embodiments, microaerobic designs can be used based on the growth-coupled formation of the desired product. To examine this, production cones can be constructed for each strategy by first maximizing and, subsequently minimizing the product yields at different rates of biomass formation feasible in the network. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the product in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs are given a low priority. 
     The 3-hydroxyisobutyrate- or MAA-production strategies provided herein can be disrupted to increase production of 3-hydroxyisobutyrate or MAA. Accordingly, also provided herein is a non-naturally occurring microbial organism having metabolic modifications coupling 3-hydroxyisobutyrate or MAA production to growth of the organism, where the metabolic modifications includes disruption of one or more genes selected from the genes encoding proteins and/or enzymes provided herein. 
     Each of the strains can be supplemented with additional deletions if it is determined that the strain designs do not sufficiently increase the production of 3-hydroxyisobutyrate or MAA and/or couple the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such activities can also be knocked out. However, gene deletions provided herein allow the construction of strains exhibiting high-yield production of 3-hydroxyisobutyrate or MAA, including growth-coupled production of 3-hydroxyisobutyrate or MAA. 
     In another aspect, provided herein is a method for producing 3-hydroxyisobutyrate or MAA, comprising culturing any one of the non-naturally occurring microbial organisms comprising a methanol metabolic pathway and an 3-hydroxyisobutyrate or MAA pathway provided herein under conditions and for a sufficient period of time to produce 3-hydroxyisobutyrate or MAA. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium. 
     In one embodiment, provided herein are methods for producing 3-hydroxyisobutyrate, comprising culturing an organism provided herein (e.g., a non-naturally occurring microbial organisms comprising a methanol metabolic pathway and an 3-hydroxyisobutyrate pathway) under conditions and for a sufficient period of time to produce 3-hydroxyisobutyrate. In some embodiments, the method comprises culturing, for a sufficient period of time to produce 3-hydroxyisobutyrate, a non-naturally occurring microbial organism, comprising (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) an 3-hydroxyisobutyrate pathway, comprising at least one exogenous nucleic acid encoding an 3-hydroxyisobutyrate pathway enzyme expressed in a sufficient amount to produce 3-hydroxyisobutyrate. 
     In another embodiment, provided herein are methods for producing MAA, comprising culturing an organism provided herein (e.g., a non-naturally occurring microbial organisms comprising a methanol metabolic pathway and an MAA pathway) under conditions and for a sufficient period of time to produce MAA. In some embodiments, the method comprises culturing, for a sufficient period of time to produce MAA, a non-naturally occurring microbial organism, comprising (1) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol; and (2) an MAA pathway, comprising at least one exogenous nucleic acid encoding an MAA pathway enzyme expressed in a sufficient amount to produce MAA. 
     In certain embodiments of the methods provided herein, the organism further comprises at least one nucleic acid encoding a 3-hydroxyisobutyrate or MAA pathway enzyme expressed in a sufficient amount to produce 3-hydroxyisobutyrate or MAA. In some embodiments, the nucleic acid is an exogenous nucleic acid. In other embodiments, the nucleic acid is an endogenous nucleic acid. In some embodiments, the organism further comprises one or more gene disruptions provided herein that confer increased production of 3-hydroxyisobutyrate or MAA in the organism. In certain embodiments, the one or more gene disruptions occurs in an endogenous gene encoding a protein or enzyme involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2  and/or amino acids by said microbial organism. In other embodiments, the organism further comprises one or more endogenous proteins or enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2  and/or amino acids by said microbial organism, wherein said one or more endogenous proteins or enzymes has attenuated protein or enzyme activity and/or expression levels. In certain embodiments, the organism is a Crabtree positive, eukaryotic organism, and the organism is cultured in a culture medium comprising glucose. In certain embodiments, the organism comprises from one to twenty-five gene disruptions. In other embodiments, the organism comprises from one to twenty gene disruptions. In some embodiments, the organism comprises from one to fifteen gene disruptions. In other embodiments, the organism comprises from one to ten gene disruptions. In some embodiments, the organism comprises from one to five gene disruptions. In certain embodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 gene disruptions or more. 
     In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation and/or methanol metabolic pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product. By way of example, in  FIG.  1   , the substrate of 1J is methanol, and the product is formaldehyde; the substrate of 1L is formaldehyde, and the product is formate; and so forth. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, provided herein are non-naturally occurring microbial organisms containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a methanol metabolic pathway, such as that shown in  FIG.  1   ; a 3-hydroxyisobutyrate or MAA pathway, such as that shown in  FIG.  2   ; and/or a formaldehyde assimilation pathway, such as that shown in  FIG.  3  or  4   . 
     While generally described herein as a microbial organism that contains a 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation pathway, and/or a methanol metabolic pathway, it is understood that provided herein are also non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation pathway, and/or a methanol metabolic pathway enzyme expressed in a sufficient amount to produce an intermediate of a 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation pathway, and/or a methanol metabolic pathway intermediate. For example, as disclosed herein, a 3-hydroxyisobutyrate or MAA pathway is exemplified in  FIG.  2   . Therefore, in addition to a microbial organism containing a 3-hydroxyisobutyrate or MAA pathway that produces 3-hydroxyisobutyrate or MAA, also provided herein is a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate or MAA pathway enzyme, where the microbial organism produces a 3-hydroxyisobutyrate or MAA pathway intermediate. 
     In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in 3-hydroxyisobutyrate or MAA or any 3-hydroxyisobutyrate or MAA pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as “uptake sources.” Uptake sources can provide isotopic enrichment for any atom present in the product 3-hydroxyisobutyrate or MAA and/or a 3-hydroxyisobutyrate or MAA pathway intermediate, or for side products generated in reactions diverging away from a 3-hydroxyisobutyrate or MAA pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens. 
     In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon-13, and carbon-14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can selected to alter the nitrogen-14 and nitrogen-15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios. 
     In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target isotopic ratio of an uptake source can be obtained by selecting a desired origin of the uptake source as found in nature For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon-14, or an environmental or atmospheric carbon source, such as CO 2 , which can possess a larger amount of carbon-14 than its petroleum-derived counterpart. 
     Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC) and/or high performance liquid chromatography (HPLC). 
     The unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 10 12  carbon atoms in the earth&#39;s atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( 14 N). Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called “Suess effect”. 
     Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like. 
     In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product&#39;s biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective Apr. 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein. 
     The biobased content of a compound is estimated by the ratio of carbon-14 ( 14 C) to carbon-12 ( 12 C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm=(S-B)/(M-B), where B, S and M represent the  14 C/ 12 C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the  14 C/ 12 C ratio of a sample from “Modern.” Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to δ 13 C VPDB =−19 per mil (Olsson,  The use of Oxalic acid as a Standard.  in,  Radiocarbon Variations and Absolute Chronology,  Nobel Symposium, 12th Proc., John Wiley &amp; Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to δ 13 C VPDB =−19 per mil. This is equivalent to an absolute (AD 1950)  14 C/ 12 C ratio of 1.176±0.010×10 −12  (Karlen et al.,  Arkiv Geoftsik,  4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C 12  over C 13  over C 14 , and these corrections are reflected as a Fm corrected for δ 13 . 
     An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980&#39;s, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann,  Radiocarbon,  25(2):519-527 (1983)). A Fm=0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm=100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a “modern” source includes biobased sources. 
     As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a “pre-bomb” standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old. 
     ASTM D6866 quantifies the biobased content relative to the material&#39;s total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50% water would be considered to have a Biobased Content=100% (50% organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content=66.7% (75% organic content but only 50% of the product is biobased). In another example, a product that is 50% organic carbon and is a petroleum-based product would be considered to have a Biobased Content=0% (50% organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content. 
     Applications of carbon-14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al.,  Nuclear Instruments and Methods in Physics Research B,  172:281-287 (2000)). For example, carbon-14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al.,  Green Chemistry,  13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable BDO and renewable terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011). 
     Accordingly, in some embodiments, the present invention provides 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects, the 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate thereof can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is CO 2 . In some embodiments, the present invention provides 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate thereof, that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the a 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides a 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate thereof, that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon-12, carbon-13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources. 
     Further, the present invention relates to biologically produced 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate thereof, as disclosed herein, and to the products derived therefrom, wherein the a 3-hydroxyisobutyrate or MAA, or an intermediate thereof, has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the CO 2  that occurs in the environment. For example, in some aspects the invention provides bioderived 3-hydroxyisobutyrate or MAA, or an intermediate thereof, having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO 2  that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO 2  that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived 3-hydroxyisobutyrate or MAA, or an intermediate thereof, as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of 3-hydroxyisobutyrate or MAA, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO 2  that occurs in the environment, wherein the polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, are generated directly from or in combination with bioderived 3-hydroxyisobutyrate or MAA or a bioderived intermediate thereof, as disclosed herein. 
     3-hydroxyisobutyrate and MAA, as well as intermediates thereof, are chemicals used in commercial and industrial applications. Non-limiting examples of such applications include production of polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like. Moreover, 3-hydroxyisobutyrate and MAA are also used as a raw material in the production of a wide range of products including polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like. Accordingly, in some embodiments, the invention provides biobased polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, comprising one or more of bioderived 3-hydroxyisobutyrate or MAA, or a bioderived intermediate thereof, produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein. 
     As used herein, the term “bioderived” means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term “biobased” means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock. 
     In some embodiments, the invention provides polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, comprising bioderived 3-hydroxyisobutyrate or MAA, or a bioderived intermediate thereof, wherein the bioderived 3-hydroxyisobutyrate or MAA, or bioderived intermediate thereof, includes all or part of the a 3-hydroxyisobutyrate or MAA, or an intermediate thereof, used in the production of polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like. Thus, in some aspects, the invention provides a biobased polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived 3-hydroxyisobutyrate or MAA, or a bioderived 3-hydroxyisobutyrate or MAA intermediate, as disclosed herein. Additionally, in some aspects, the invention provides biobased polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, wherein the a 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate, used in its production is a combination of bioderived and petroleum derived 3-hydroxyisobutyrate or MAA, or a 3-hydroxyisobutyrate or MAA intermediate thereof. For example, biobased polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA, and the like, can be produced using 50% bioderived 3-hydroxyisobutyrate or MAA and 50% petroleum derived 3-hydroxyisobutyrate or MAA or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing polymers, co-polymers, plastics, methacrylates (e.g., a methyl methacrylate or a butyl methacrylate), glacial MAA aids, food and oral medicinal coatings/products, and the like, using the bioderived 3-hydroxyisobutyrate or MAA, or a bioderived 3-hydroxyisobutyrate or MAA intermediate thereof, of the invention are well known in the art. 
     In one embodiment, the product is a polymer. In one embodiment, the product is a polymer. In one embodiment, the product is a co-polymer. In one embodiment, the product is a plastic. In one embodiment, the product is a methacrylate. In one embodiment, the product is a methyl methacrylate. In one embodiment, the product is a butyl methacrylate. In one embodiment, the product is a glacial MAA. 
     In some embodiments, provided herein is a culture medium comprising bioderived 3-hydroxyisobutyrate. In some embodiments, the bioderived 3-hydroxyisobutyrate is produced by culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and 3-hydroxyisobutyrate pathway, as provided herein. In certain embodiments, the bioderived 3-hydroxyisobutyrate has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In one embodiment, the culture medium is separated from a non-naturally occurring microbial organism having a methanol metabolic pathway and 3-hydroxyisobutyrate pathway. 
     In other embodiments, provided herein is a bioderived 3-hydroxyisobutyrate. In some embodiments, the bioderived 3-hydroxyisobutyrate is produced by culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and 3-hydroxyisobutyrate pathway, as provided herein. In certain embodiments, the bioderived 3-hydroxyisobutyrate has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In some embodiments, the bioderived 3-hydroxyisobutyrate has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%. In certain embodiments, the bioderived 3-hydroxyisobutyrate is a component of culture medium. 
     In certain embodiments, provided herein is a composition comprising a bioderived 3-hydroxyisobutyrate provided herein, for example, a bioderived 3-hydroxyisobutyrate produced by culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and 3-hydroxyisobutyrate pathway, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived 3-hydroxyisobutyrate. In certain embodiments, the compound other than said bioderived 3-hydroxyisobutyrate is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a methanol metabolic pathway and a 3-hydroxyisobutyrate pathway, as provided herein. 
     In some embodiments, provided herein is a biobased product comprising a bioderived 3-hydroxyisobutyrate provided herein. In certain embodiments, the biobased product is a polymer, co-polymer, plastic, methacrylate, methyl methacrylate, butyl methacrylate, or glacial MAA. In certain embodiments, the biobased product comprises at least 5% bioderived 3-hydroxyisobutyrate. In certain embodiments, the biobased product comprises at least 10% bioderived 3-hydroxyisobutyrate. In some embodiments, the biobased product comprises at least 20% bioderived 3-hydroxyisobutyrate. In other embodiments, the biobased product comprises at least 30% bioderived 3-hydroxyisobutyrate. In some embodiments, the biobased product comprises at least 40% bioderived 3-hydroxyisobutyrate. In other embodiments, the biobased product comprises at least 50% bioderived 3-hydroxyisobutyrate. In one embodiment, the biobased product comprises a portion of said bioderived 3-hydroxyisobutyrate as a repeating unit. In another embodiment, provided herein is a molded product obtained by molding the biobased product provided herein. In other embodiments, provided herein is a process for producing a biobased product provided herein, comprising chemically reacting said bioderived 3-hydroxyisobutyrate with itself or another compound in a reaction that produces said biobased product. In certain embodiments, provided herein is a polymer comprising or obtained by converting the bioderived 3-hydroxyisobutyrate. In other embodiments, provided herein is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived 3-hydroxyisobutyrate to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived 3-hydroxyisobutyrate, or a cell lysate or culture supernatant thereof. 
     In some embodiments, provided herein is a culture medium comprising bioderived MAA. In some embodiments, the bioderived MAA is produced by culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and MAA pathway, as provided herein. In certain embodiments, the bioderived MAA has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In one embodiment, the culture medium is separated from a non-naturally occurring microbial organism having a methanol metabolic pathway and MAA pathway. 
     In other embodiments, provided herein is a bioderived MAA. In some embodiments, the bioderived MAA is produced by culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and MAA pathway, as provided herein. In certain embodiments, the bioderived MAA has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. In some embodiments, the bioderived MAA has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%. In certain embodiments, the bioderived MAA is a component of culture medium. 
     In certain embodiments, provided herein is a composition comprising a bioderived MAA provided herein, for example, a bioderived MAA produced by culturing a non-naturally occurring microbial organism having a methanol metabolic pathway and MAA pathway, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived MAA. In certain embodiments, the compound other than said bioderived MAA is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a methanol metabolic pathway and a MAA pathway, as provided herein. 
     In some embodiments, provided herein is a biobased product comprising a bioderived MAA provided herein. In certain embodiments, the biobased product is a polymer, co-polymer, plastic, methacrylate, methyl methacrylate, butyl methacrylate, or glacial MAA. In certain embodiments, the biobased product comprises at least 5% bioderived MAA. In certain embodiments, the biobased product comprises at least 10% bioderived MAA. In some embodiments, the biobased product comprises at least 20% bioderived MAA. In other embodiments, the biobased product comprises at least 30% bioderived MAA. In some embodiments, the biobased product comprises at least 40% bioderived MAA. In other embodiments, the biobased product comprises at least 50% bioderived MAA. In one embodiment, the biobased product comprises a portion of said bioderived MAA as a repeating unit. In another embodiment, provided herein is a molded product obtained by molding the biobased product provided herein. In other embodiments, provided herein is a process for producing a biobased product provided herein, comprising chemically reacting said bioderived—MAA with itself or another compound in a reaction that produces said biobased product. In certain embodiments, provided herein is a polymer comprising or obtained by converting the bioderived MAA. In other embodiments, provided herein is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived MAA to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived MAA, or a cell lysate or culture supernatant thereof. 
     The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction and that reference to any of these metabolic constitutes also references the gene or genes encoding the enzymes that catalyze the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes, or a protein associated with the reaction, as well as the reactants and products of the reaction. 
     Microbial organisms generally lack the capacity to synthesize 3-hydroxyisobutyrate or MAA, and therefore any of the compounds disclosed herein to be within the 3-hydroxyisobutyrate or MAA family of compounds, or otherwise known by those in the art to be within the 3-hydroxyisobutyrate or MAA family of compounds. Moreover, organisms having all of the requisite metabolic enzymatic capabilities are not known to produce 3-hydroxyisobutyrate or MAA from the enzymes described and biochemical pathways exemplified herein. In contrast, the non-naturally occurring microbial organisms of the invention can generate 3-hydroxyisobutyrate or MAA as a product, as well as intermediates thereof. The biosynthesis of 3-hydroxyisobutyrate or MAA, as well as intermediates thereof, is particularly useful in chemical synthesis of 3-hydroxyisobutyrate or MAA family of compounds, it also allows for the further biosynthesis of 3-hydroxyisobutyrate or MAA family compounds and avoids altogether chemical synthesis procedures. 
     The non-naturally occurring microbial organisms of the invention that can produce 3-hydroxyisobutyrate or MAA are produced by ensuring that a host microbial organism includes functional capabilities for the complete biochemical synthesis of at least one 3-hydroxyisobutyrate or MAA biosynthetic pathway of the invention. Ensuring at least one requisite 3-hydroxyisobutyrate or MAA biosynthetic pathway confers 3-hydroxyisobutyrate or MAA biosynthesis capability onto the host microbial organism. 
     The organisms and methods are described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction. 
     The non-naturally occurring microbial organisms described herein can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more methanol metabolic, formaldehyde assimilation, and/or 3-hydroxyisobutyrate or MAA biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular methanol metabolic, formaldehyde assimilation, and/or 3-hydroxyisobutyrate or MAA biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired metabolic, assimilation, or biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve 3-hydroxyisobutyrate or MAA biosynthesis and/or methanol metabolism. Thus, a non-naturally occurring microbial organism described herein can be produced by introducing exogenous enzyme or protein activities to obtain a desired metabolic pathway and/or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as 3-hydroxyisobutyrate or MAA. 
     Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from  Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens,  and  Pseudomonas putida.  Exemplary yeasts or fungi include species selected from  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae,  and the like.  E. coli  is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as  Saccharomyces cerevisiae.  It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product. 
     Depending on the 3-hydroxyisobutyrate or MAA biosynthetic, methanol metabolic and/or formaldehyde assimilation pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms provided herein will include at least one exogenously expressed 3-hydroxyisobutyrate or MAA, formaldehyde assimilation and/or methanol metabolic pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more 3-hydroxyisobutyrate or MAA biosynthetic pathways, formaldehyde assimilation pathways and/or methanol metabolic pathways. For example, 3-hydroxyisobutyrate or MAA biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a 3-hydroxyisobutyrate or MAA pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of 3-hydroxyisobutyrate or MAA can be included. The same holds true for the methanol metabolic pathways and formaldehyde assimilation pathways provided herein. 
     Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation pathway, and methanol metabolic pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, nine, or up to all nucleic acids encoding the enzymes or proteins constituting a methanol metabolic pathway, formaldehyde assimilation pathway, and/or 3-hydroxyisobutyrate or MAA biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize 3-hydroxyisobutyrate or MAA biosynthesis, formaldehyde assimilation, and/or methanol metabolism or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the 3-hydroxyisobutyrate or MAA pathway precursors. 
     Generally, a host microbial organism is selected such that it produces the precursor of a 3-hydroxyisobutyrate or MAA pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a 3-hydroxyisobutyrate or MAA pathway. 
     In some embodiments, a non-naturally occurring microbial organism provided herein is generated from a host that contains the enzymatic capability to synthesize 3-hydroxyisobutyrate or MAA, assimilate formaldehyde and/or metabolize methanol. In this specific embodiment it can be useful to increase the synthesis or accumulation of a 3-hydroxyisobutyrate or MAA pathway product, formaldehyde assimilation pathway product and/or methanol metabolic pathway product (e.g., reducing equivalents and/or formaldehyde) to, for example, drive 3-hydroxyisobutyrate or MAA pathway reactions toward 3-hydroxyisobutyrate or MAA production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described 3-hydroxyisobutyrate or MAA, formaldehyde assimilation and/or methanol metabolic pathway enzymes or proteins. Over expression the enzyme(s) and/or protein(s) of the 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation, and/or methanol metabolic pathway can occur, for example, through exogenous expression of the endogenous gene(s), or through exogenous expression of the heterologous gene(s). Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms, for example, producing 3-hydroxyisobutyrate or MAA through overexpression of one, two, three, four, five, six, seven, eight, up to all nucleic acids encoding 3-hydroxyisobutyrate or MAA biosynthetic pathway, and/or methanol metabolic pathway enzymes or proteins. Naturally occurring organisms can also be readily generated to be non-naturally occurring microbial organisms, for example, assimilating formaldehyde, through overexpression of one, two, three, four, five, six, seven, eight, up to all nucleic acids encoding formaldehyde assimilation pathway, and/or methanol metabolic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the 3-hydroxyisobutyrate or MAA, formaldehyde assimilation and/or methanol metabolic pathway biosynthetic pathway. 
     In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene&#39;s promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism. 
     It is understood that, in methods provided herein, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism provided herein. The nucleic acids can be introduced so as to confer, for example, a 3-hydroxyisobutyrate or MAA biosynthetic, formaldehyde assimilation and/or methanol metabolic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer 3-hydroxyisobutyrate or MAA biosynthetic, formaldehyde assimilation and/or methanol metabolic capability. For example, a non-naturally occurring microbial organism having a 3-hydroxyisobutyrate or MAA biosynthetic pathway, formaldehyde assimilation pathway and/or methanol metabolic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway, formaldehyde assimilation pathway and/or metabolic pathway can be included in a non-naturally occurring microbial organism provided herein. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway, formaldehyde assimilation pathway and/or metabolic pathway can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway, formaldehyde assimilation pathway and/or metabolic pathway results in production of the corresponding desired product. Similarly, any combination of four or more enzymes or proteins of a biosynthetic pathway, formaldehyde assimilation pathway and/or methanol metabolic pathway as disclosed herein can be included in a non-naturally occurring microbial organism provided herein, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic, assimilation and/or metabolic pathway results in production of the corresponding desired product. 
     In addition to the metabolism of methanol, assimilation of formaldehyde, and biosynthesis of 3-hydroxyisobutyrate or MAA, as described herein, the non-naturally occurring microbial organisms and methods provided also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce 3-hydroxyisobutyrate or MAA, other than use of the 3-hydroxyisobutyrate or MAA producers is through addition of another microbial organism capable of converting a 3-hydroxyisobutyrate or MAA pathway intermediate to 3-hydroxyisobutyrate or MAA. One such procedure includes, for example, the fermentation of a microbial organism that produces a 3-hydroxyisobutyrate or MAA pathway intermediate. The 3-hydroxyisobutyrate or MAA pathway intermediate can then be used as a substrate for a second microbial organism that converts the 3-hydroxyisobutyrate or MAA pathway intermediate to 3-hydroxyisobutyrate or MAA. The 3-hydroxyisobutyrate or MAA pathway intermediate can be added directly to another culture of the second organism or the original culture of the 3-hydroxyisobutyrate or MAA pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. 
     In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, 3-hydroxyisobutyrate or MAA. In these embodiments, biosynthetic pathways for a desired product can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of 3-hydroxyisobutyrate or MAA can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, 3-hydroxyisobutyrate or MAA also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a 3-hydroxyisobutyrate or MAA intermediate and the second microbial organism converts the intermediate to 3-hydroxyisobutyrate or MAA. 
     Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce 3-hydroxyisobutyrate or MAA and/or metabolize methanol. 
     Sources of encoding nucleic acids for a 3-hydroxyisobutyrate or MAA, formaldehyde assimilation, or methanol metabolic pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example,  Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas  species, including  Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter  species, including  Acinetobacter calcoaceticus  and  Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum,  marine gamma proteobacterium, butyrate producing bacterium,  Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis  MC2 155,  Mycobacterium avium  subsp.  paratuberculosis  K-10,  Mycobacterium marinum  M,  Tsukamurella paurometabola  DSM 20162, Cyanobium PCC7001,  Dictyostelium discoideum  AX4, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite 3-hydroxyisobutyrate or MAA biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of 3-hydroxyisobutyrate or MAA, metabolism of methanol and/or assimilation of formaldehyde described herein with reference to a particular organism such as  E. coli  can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms. 
     In some instances, such as when an alternative 3-hydroxyisobutyrate or MAA biosynthetic, formaldehyde assimilation and/or methanol metabolic pathway exists in an unrelated species, 3-hydroxyisobutyrate or MAA biosynthesis, formaldehyde assimilation and/or methanol metabolism can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods provided herein can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize 3-hydroxyisobutyrate or MAA, assimilate formaldehyde, and/or metabolize methanol. 
     Methods for constructing and testing the expression levels of a non-naturally occurring 3-hydroxyisobutyrate- or MAA-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al.,  Current Protocols in Molecular Biology,  John Wiley and Sons, Baltimore, Md. (1999). 
     Exogenous nucleic acid sequences involved in a pathway for metabolism of methanol, assimilation of formaldehyde and/or production of 3-hydroxyisobutyrate or MAA can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in  E. coli  or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in  E. coli  (Hoffmeister et al.,  J. Biol. Chem.  280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins. 
     An expression vector or vectors can be constructed to include one or more 3-hydroxyisobutyrate or MAA biosynthetic, formaldehyde assimilation and/or methanol metabolic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms provided include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein. 
     Suitable purification and/or assays to test, e.g., for the production of 3-hydroxyisobutyrate or MAA can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al.,  Biotechnol. Bioeng.  90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. 
     The 3-hydroxyisobutyrate or MAA can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art. 
     Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products, or intermediates thereof. For example, the 3-hydroxyisobutyrate or MAA producers can be cultured for the biosynthetic production of 3-hydroxyisobutyrate or MAA. Accordingly, in some embodiments, the invention provides culture medium having a 3-hydroxyisobutyrate or MAA, formaldehyde assimilation and/or methanol metabolic pathway intermediate described herein. In some aspects, the culture medium can also be separated from the non-naturally occurring microbial organisms provided herein that produced the 3-hydroxyisobutyrate or MAA, formaldehyde assimilation and/or methanol metabolic pathway intermediate. Methods for separating a microbial organism from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like. 
     In certain embodiments, for example, for the production of the production of 3-hydroxyisobutyrate or MAA, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in U.S. Publ. No. 2009/0047719. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high 3-hydroxyisobutyrate or MAA yields. 
     If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. 
     The growth medium, can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch; or glycerol, alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source is a sugar-containing biomass. In some embodiments, the sugar is glucose. In one embodiment, the sugar is xylose. In another embodiment, the sugar is arabinose. In one embodiment, the sugar is galactose. In another embodiment, the sugar is fructose. In other embodiments, the sugar is sucrose. In one embodiment, the sugar is starch. In certain embodiments, the carbon source is glycerol. In some embodiments, the carbon source is crude glycerol. In one embodiment, the carbon source is crude glycerol without treatment. In other embodiments, the carbon source is glycerol and glucose. In another embodiment, the carbon source is methanol and glycerol. In one embodiment, the carbon source is carbon dioxide. In one embodiment, the carbon source is formate. In one embodiment, the carbon source is methane. In one embodiment, the carbon source is methanol. In one embodiment, the carbon source is chemoelectro-generated carbon (see, e.g., Liao et al. (2012) Science 335:1596). In one embodiment, the chemoelectro-generated carbon is methanol. In one embodiment, the chemoelectro-generated carbon is formate. In one embodiment, the chemoelectro-generated carbon is formate and methanol. In one embodiment, the carbon source is a sugar and methanol. In another embodiment, the carbon source is a sugar and glycerol. In other embodiments, the carbon source is a sugar and crude glycerol. In yet other embodiments, the carbon source is a sugar and crude glycerol without treatment. In one embodiment, the carbon source is a sugar-containing biomass and methanol. In another embodiment, the carbon source is a sugar-containing biomass and glycerol. In other embodiments, the carbon source is a sugar-containing biomass and crude glycerol. In yet other embodiments, the carbon source is a sugar-containing biomass and crude glycerol without treatment. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms provided herein for the production of 3-hydroxyisobutyrate or MAA, and other pathway intermediates. 
     In one embodiment, the carbon source is glycerol. In certain embodiments, the glycerol carbon source is crude glycerol or crude glycerol without further treatment. In a further embodiment, the carbon source comprises glycerol or crude glycerol, and also sugar or a sugar-containing biomass, such as glucose. In a specific embodiment, the concentration of glycerol in the fermentation broth is maintained by feeding crude glycerol, or a mixture of crude glycerol and sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass. In certain other embodiments of the ratios provided above, the glycerol is a crude glycerol or a crude glycerol without further treatment. In other embodiments of the ratios provided above, the sugar is a sugar-containing biomass, and the glycerol is a crude glycerol or a crude glycerol without further treatment. 
     Crude glycerol can be a by-product produced in the production of biodiesel, and can be used for fermentation without any further treatment. Biodiesel production methods include (1) a chemical method wherein the glycerol-group of vegetable oils or animal oils is substituted by low-carbon alcohols such as methanol or ethanol to produce a corresponding fatty acid methyl esters or fatty acid ethyl esters by transesterification in the presence of acidic or basic catalysts; (2) a biological method where biological enzymes or cells are used to catalyze transesterification reaction and the corresponding fatty acid methyl esters or fatty acid ethyl esters are produced; and (3) a supercritical method, wherein transesterification reaction is carried out in a supercritical solvent system without any catalysts. The chemical composition of crude glycerol can vary with the process used to produce biodiesel, the transesterification efficiency, recovery efficiency of the biodiesel, other impurities in the feedstock, and whether methanol and catalysts were recovered. For example, the chemical compositions of eleven crude glycerol collected from seven Australian biodiesel producers reported that glycerol content ranged between 38% and 96%, with some samples including more than 14% methanol and 29% ash. In certain embodiments, the crude glycerol comprises from 5% to 99% glycerol. In some embodiments, the crude glycerol comprises from 10% to 90% glycerol. In some embodiments, the crude glycerol comprises from 10% to 80% glycerol. In some embodiments, the crude glycerol comprises from 10% to 70% glycerol. In some embodiments, the crude glycerol comprises from 10% to 60% glycerol. In some embodiments, the crude glycerol comprises from 10% to 50% glycerol. In some embodiments, the crude glycerol comprises from 10% to 40% glycerol. In some embodiments, the crude glycerol comprises from 10% to 30% glycerol. In some embodiments, the crude glycerol comprises from 10% to 20% glycerol. In some embodiments, the crude glycerol comprises from 80% to 90% glycerol. In some embodiments, the crude glycerol comprises from 70% to 90% glycerol. In some embodiments, the crude glycerol comprises from 60% to 90% glycerol. In some embodiments, the crude glycerol comprises from 50% to 90% glycerol. In some embodiments, the crude glycerol comprises from 40% to 90% glycerol. In some embodiments, the crude glycerol comprises from 30% to 90% glycerol. In some embodiments, the crude glycerol comprises from 20% to 90% glycerol. In some embodiments, the crude glycerol comprises from 20% to 40% glycerol. In some embodiments, the crude glycerol comprises from 40% to 60% glycerol. In some embodiments, the crude glycerol comprises from 60% to 80% glycerol. In some embodiments, the crude glycerol comprises from 50% to 70% glycerol. In one embodiment, the glycerol comprises 5% glycerol. In one embodiment, the glycerol comprises 10% glycerol. In one embodiment, the glycerol comprises 15% glycerol. In one embodiment, the glycerol comprises 20% glycerol. In one embodiment, the glycerol comprises 25% glycerol. In one embodiment, the glycerol comprises 30% glycerol. In one embodiment, the glycerol comprises 35% glycerol. In one embodiment, the glycerol comprises 40% glycerol. In one embodiment, the glycerol comprises 45% glycerol. In one embodiment, the glycerol comprises 50% glycerol. In one embodiment, the glycerol comprises 55% glycerol. In one embodiment, the glycerol comprises 60% glycerol. In one embodiment, the glycerol comprises 65% glycerol. In one embodiment, the glycerol comprises 70% glycerol. In one embodiment, the glycerol comprises 75% glycerol. In one embodiment, the glycerol comprises 80% glycerol. In one embodiment, the glycerol comprises 85% glycerol. In one embodiment, the glycerol comprises 90% glycerol. In one embodiment, the glycerol comprises 95% glycerol. In one embodiment, the glycerol comprises 99% glycerol. 
     In one embodiment, the carbon source is methanol or formate. In certain embodiments, methanol is used as a carbon source in the formaldehyde assimilation pathways provided herein. In one embodiment, the carbon source is methanol or formate. In other embodiments, formate is used as a carbon source in the formaldehyde assimilation pathways provided herein. In specific embodiments, methanol is used as a carbon source in the methanol metabolic pathways provided herein, either alone or in combination with the product pathways provided herein. 
     In one embodiment, the carbon source comprises methanol, and sugar (e.g., glucose) or a sugar-containing biomass. In another embodiment, the carbon source comprises formate, and sugar (e.g., glucose) or a sugar-containing biomass. In one embodiment, the carbon source comprises methanol, formate, and sugar (e.g., glucose) or a sugar-containing biomass. In specific embodiments, the methanol or formate, or both, in the fermentation feed is provided as a mixture with sugar (e.g., glucose) or sugar-comprising biomass. In certain embodiments, sugar is provided for sufficient strain growth. 
     In certain embodiments, the carbon source comprises methanol and a sugar (e.g., glucose). In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass. 
     In certain embodiments, the carbon source comprises formate and a sugar (e.g., glucose). In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass. 
     In certain embodiments, the carbon source comprises a mixture of methanol and formate, and a sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass. 
     Given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds when grown on a carbon source such as a carbohydrate. Such compounds include, for example, 3-hydroxyisobutyrate or MAA and any of the intermediate metabolites in the 3-hydroxyisobutyrate or MAA pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the 3-hydroxyisobutyrate or MAA biosynthetic pathways. Accordingly, provided herein is a non-naturally occurring microbial organism that produces and/or secretes 3-hydroxyisobutyrate or MAA when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the 3-hydroxyisobutyrate or MAA pathway when grown on a carbohydrate or other carbon source. The 3-hydroxyisobutyrate or MAA-producing microbial organisms provided herein can initiate synthesis from an intermediate. The same holds true for intermediates in the formaldehyde assimilation and methanol metabolic pathways. 
     The non-naturally occurring microbial organisms provided herein are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a 3-hydroxyisobutyrate or MAA biosynthetic pathway and/or methanol metabolic pathway enzyme or protein in sufficient amounts to produce 3-hydroxyisobutyrate or MAA. It is understood that the microbial organisms are cultured under conditions sufficient to produce 3-hydroxyisobutyrate or MAA. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms can achieve biosynthesis of 3-hydroxyisobutyrate or MAA, resulting in intracellular concentrations between about 0.1-500 mM or more. Generally, the intracellular concentration of 3-hydroxyisobutyrate or MAA is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms provided herein. 
     In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. Publ. No. 2009/0047719. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the 3-hydroxyisobutyrate or MAA producers can synthesize 3-hydroxyisobutyrate or MAA at intracellular concentrations of 5-100 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, 3-hydroxyisobutyrate or MAA can produce 3-hydroxyisobutyrate or MAA intracellularly and/or secrete the product into the culture medium. 
     Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. Under anaerobic conditions, the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N2/CO2 mixture, argon, helium, and the like. As the cells grow and utilize the carbon source, additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients. The temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C., but the temperature can be maintained at a higher or lower temperature depending on the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermenter, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the fermenation process. 
     In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and/or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermenter is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and/or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired. Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like), standard continuous distillation methods, and the like, or other methods well known in the art. 
     In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of 3-hydroxyisobutyrate or MAA can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms provided herein can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethyl slfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM. 
     The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions. 
     As described herein, one exemplary growth condition for achieving biosynthesis of 3-hydroxyisobutyrate or MAA, as well as other pathway intermediates, includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms provided can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N 2 /CO 2  mixture or other suitable non-oxygen gas or gases. 
     The culture conditions described herein can be scaled up and grown continuously for manufacturing of 3-hydroxyisobutyrate or MAA. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of 3-hydroxyisobutyrate or MAA. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of 3-hydroxyisobutyrate or MAA will include culturing a non-naturally occurring 3-hydroxyisobutyrate or MAA producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can be included, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms provided can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose. 
     Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of 3-hydroxyisobutyrate or MAA can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art. 
     In addition to the above fermentation procedures using the 3-hydroxyisobutyrate or MAA producers for continuous production of substantial quantities of 3-hydroxyisobutyrate or MAA, the 3-hydroxyisobutyrate or MAA producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired. 
     To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 3-hydroxyisobutyrate or MAA. 
     One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al.,  Biotechnol. Bioeng.  84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product. 
     Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. Publ. No. 2002/0168654, International Patent Application No. PCT/US02/00660, and U.S. Publ. No. 2009/0047719. 
     Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. Publ. No. 2003/0233218, and International Patent Application No. PCT/US03/18838. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components. 
     These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. 
     Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art. 
     The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes. 
     Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur. 
     To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al.,  Biotechnol. Prog.  17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®. 
     The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion. 
     As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism&#39;s ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al.,  Biotechnol. Bioeng.  84:647-657 (2003)). 
     An in silico stoichiometric model of  E. coli  metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above. 
     As disclosed herein, a nucleic acid encoding a desired activity of a 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation pathway, and/or methanol metabolic pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a 3-hydroxyisobutyrate or MAA pathway, formaldehyde assimilation, or methanol metabolic pathway enzyme or protein to increase production of 3-hydroxyisobutyrate or MAA; formaldehyde, and/or reducing equivalents. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator. 
     One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, &gt;10 4 ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al.,  Biomol. Eng.  22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax.  Biomol. Eng.  22:1-9 (2005).; and Sen et al.,  Appl Biochem.Biotechnol  143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K m ), including broadening substrate binding to include non-natural substrates; inhibition (K i ), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen. 
     A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a 3-hydroxyisobutyrate or MAA pathway and/or a methanol metabolic enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al.,  J. Theor. Biol.  234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al.,  Nucleic Acids Res.  32:e145 (2004); and Fujii et al.,  Nat. Protocolsl : 2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer,  Proc. Natl. Acad. Sci. U.S.A.  91:10747-10751 (1994); and Stemmer,  Nature  370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al.,  Nat. Biotechnol.  16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al.,  Nucleic Acids Res.  26:681-683 (1998)). 
     Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al,  Nucleic Acids Res.  27:e18 (1999); and Volkov et al.,  Methods Enzymol.  328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al.,  Nat. Biotechnol.  19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al.,  J. Molec. Catalysis  26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs,  Methods Mol. Biol.  352:191-204 (2007); Bergquist et al.,  Biomol. Eng.  22:63-72 (2005); Gibbs et al.,  Gene  271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al.,  Proc. Natl. Acad. Sci. U.S.A.  96:3562-3567 (1999); and Ostermeier et al.,  Nat. Biotechnol.  17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al.,  Nucleic Acids Res.  29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al.,  Proc. Natl. Acad. Sci. U.S.A.  98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al.,  Biomol. Eng.  22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of “universal” bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al.,  Biotechnol. J.  3:74-82 (2008); Wong et al.,  Nucleic Acids Res.  32:e26 (2004); and Wong et al.,  Anal. Biochem.  341:187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode “all genetic diversity in targets” and allows a very high diversity for the shuffled progeny (Ness et al.,  Nat. Biotechnol.  20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al.,  Nucleic Acids Res.  33:e117 (2005)). 
     Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al.,  Nat. Biotechnol.  19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al.,  Methods Enzymol.  388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al.  Methods Enzymol.  208:564-586 (1991); and Reidhaar-Olson et al.  Science  241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al.,  Angew. Chem. Int. Ed Engl.  40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al.,  Appl. Environ. Microbiol.  67:3645-3649 (2001)); Low et al.,  J. Mol. Biol.  260:359-3680 (1996)). 
     Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al.,  Proc. Natl. Acad. Sci. U.S.A.  102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al.,  Proc. Natl. Acad. Sci. U.S.A.  99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego Calif.), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al.,  Nat. Protocols  2:891-903 (2007); and Reetz et al.,  Angew. Chem. Int. Ed Engl.  45:7745-7751 (2006)). 
     Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein. 
     Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. 
     It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. 
     4. EXAMPLES 
     4.1 Example I 
     Production of Reducing Equivalents Via a Methanol Metabolic Pathway 
     Exemplary methanol metabolic pathways are provided in  FIG.  1   . 
     FIG.  1 , Step A—Methanol Methyltransferase 
     A complex of 3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC, perform the desired methanol methyltransferase activity (Sauer et al.,  Eur. J. Biochem.  243:670-677 (1997); Naidu and Ragsdale,  J. Bacteriol.  183:3276-3281 (2001); Tallant and Krzycki,  J. Biol. Chem.  276:4485-4493 (2001); Tallant and Krzycki,  J. Bacteriol.  179:6902-6911 (1997); Tallant and Krzycki,  J. Bacteriol.  178:1295-1301 (1996); Ragsdale, S. W.,  Crit. Rev. Biochem. Mol. Biol.  39:165-195 (2004)). 
     MtaB is a zinc protein that can catalyze the transfer of a methyl group from methanol to MtaC, a corrinoid protein. Exemplary genes encoding MtaB and MtaC can be found in methanogenic archaea such as  Methanosarcina barkeri  (Maeder et al.,  J. Bacteriol.  188:7922-7931 (2006) and  Methanosarcina acetivorans  (Galagan et al.,  Genome Res.  12:532-542 (2002), as well as the acetogen,  Moorella thermoacetica  (Das et al.,  Proteins  67:167-176 (2007). In general, the MtaB and MtaC genes are adjacent to one another on the chromosome as their activities are tightly interdependent. The protein sequences of various MtaB and MtaC encoding genes in  M. barkeri, M. acetivorans,  and  M. thermoaceticum  can be identified by their following GenBank accession numbers. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 MtaB1 
                 YP_304299 
                 73668284 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaC1 
                 YP_304298 
                 73668283 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaB2 
                 YP_307082 
                 73671067 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaC2 
                 YP_307081 
                 73671066 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaB3 
                 YP_304612 
                 73668597 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaC3 
                 YP_304611 
                 73668596 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaB1 
                 NP_615421 
                 20089346 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaB1 
                 NP_615422 
                 20089347 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaB2 
                 NP_619254 
                 20093179 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaC2 
                 NP_619253 
                 20093178 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaB3 
                 NP_616549 
                 20090474 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaC3 
                 NP_616550 
                 20090475 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaB 
                 YP_430066 
                 83590057 
                 
                   Moorella thermoacetica 
                 
               
               
                 MtaC 
                 YP_430065 
                 83590056 
                 
                   Moorella thermoacetica 
                 
               
               
                 MtaA 
                 YP_430064 
                 83590056 
                 
                   Moorella thermoacetica 
                 
               
               
                   
               
            
           
         
       
     
     The MtaB1 and MtaC1 genes, YP_304299 and YP_304298, from  M. barkeri  were cloned into  E. coli  and sequenced (Sauer et al.,  Eur. J. Biochem.  243:670-677 (1997)). The crystal structure of this methanol-cobalamin methyltransferase complex is also available (Hagemeier et al.,  Proc. Natl. Acad. Sci. U.S.A.  103:18917-18922 (2006)). The MtaB genes, YP_307082 and YP_304612, in  M. barkeri  were identified by sequence homology to YP_304299. In general, homology searches are an effective means of identifying methanol methyltransferases because MtaB encoding genes show little or no similarity to methyltransferases that act on alternative substrates such as trimethylamine, dimethylamine, monomethylamine, or dimethylsulfide. The MtaC genes, YP_307081 and YP_304611 were identified based on their proximity to the MtaB genes and also their homology to YP_304298. The three sets of MtaB and MtaC genes from  M. acetivorans  have been genetically, physiologically, and biochemically characterized (Pritchett and Metcalf,  Mol. Microbiol.  56:1183-1194 (2005)). Mutant strains lacking two of the sets were able to grow on methanol, whereas a strain lacking all three sets of MtaB and MtaC genes sets could not grow on methanol. This suggests that each set of genes plays a role in methanol utilization. The  M. thermoacetica  MtaB gene was identified based on homology to the methanogenic MtaB genes and also by its adjacent chromosomal proximity to the methanol-induced corrinoid protein, MtaC, which has been crystallized (Zhou et al.,  Acta Crystallogr. Sect. F. Struct. Biol. Cyrst. Commun.  61:537-540 (2005) and further characterized by Northern hybridization and Western Blotting ((Das et al.,  Proteins  67:167-176 (2007)). 
     MtaA is zinc protein that catalyzes the transfer of the methyl group from MtaC to either Coenzyme M in methanogens or methyltetrahydrofolate in acetogens. MtaA can also utilize methylcobalamin as the methyl donor. Exemplary genes encoding MtaA can be found in methanogenic archaea such as  Methanosarcina barkeri  (Maeder et al.,  J. Bacteriol.  188:7922-7931 (2006) and  Methanosarcina acetivorans  (Galagan et al.,  Genome Res.  12:532-542 (2002), as well as the acetogen,  Moorella thermoacetica  ((Das et al.,  Proteins  67:167-176 (2007)). In general, MtaA proteins that catalyze the transfer of the methyl group from CH 3 -MtaC are difficult to identify bioinformatically as they share similarity to other corrinoid protein methyltransferases and are not oriented adjacent to the MtaB and MtaC genes on the chromosomes. Nevertheless, a number of MtaA encoding genes have been characterized. The protein sequences of these genes in  M. barkeri  and  M. acetivorans  can be identified by the following GenBank accession numbers. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 MtaA 
                 YP_304602 
                 73668587 
                 
                   Methanosarcina barkeri 
                 
               
               
                 MtaA1 
                 NP_619241 
                 20093166 
                 
                   Methanosarcina acetivorans 
                 
               
               
                 MtaA2 
                 NP_616548 
                 20090473 
                 
                   Methanosarcina acetivorans 
                 
               
               
                   
               
            
           
         
       
     
     The MtaA gene, YP_304602, from  M. barkeri  was cloned, sequenced, and functionally overexpressed in  E. coli  (Harms and Thauer,  Eur. J. Biochem.  235:653-659 (1996)). In  M. acetivorans,  MtaA1 is required for growth on methanol, whereas MtaA2 is dispensable even though methane production from methanol is reduced in MtaA2 mutants (Bose et al.,  J. Bacteriol.  190:4017-4026 (2008)). There are multiple additional MtaA homologs in  M. barkeri  and  M. acetivorans  that are as yet uncharacterized, but may also catalyze corrinoid protein methyltransferase activity. 
     Putative MtaA encoding genes in  M. thermoacetica  were identified by their sequence similarity to the characterized methanogenic MtaA genes. Specifically, three  M. thermoacetica  genes show high homology (&gt;30% sequence identity) to YP_304602 from  M. barkeri.  Unlike methanogenic MtaA proteins that naturally catalyze the transfer of the methyl group from CH 3 -MtaC to Coenzyme M, an  M. thermoacetica  MtaA is likely to transfer the methyl group to methyltetrahydrofolate given the similar roles of methyltetrahydrofolate and Coenzyme M in methanogens and acetogens, respectively. The protein sequences of putative MtaA encoding genes from  M. thermoacetica  can be identified by the following GenBank accession numbers. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 MtaA 
                 YP_430937 
                 83590928 
                 
                   Moorella thermoacetica 
                 
               
               
                 MtaA 
                 YP_431175 
                 83591166 
                 
                   Moorella thermoacetica 
                 
               
               
                 MtaA 
                 YP_430935 
                 83590926 
                 
                   Moorella thermoacetica 
                 
               
               
                 MtaA 
                 YP_430064 
                 83590056 
                 
                   Moorella thermoacetica 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step B—Methylenetetrahydrofolate Reductase 
     The conversion of methyl-THF to methylenetetrahydrofolate is catalyzed by methylenetetrahydrofolate reductase. In  M. thermoacetica,  this enzyme is oxygen-sensitive and contains an iron-sulfur cluster (Clark and Ljungdahl,  J. Biol. Chem.  259:10845-10849 (1984). This enzyme is encoded by metF in  E. coli  (Sheppard et al.,  J. Bacteriol.  181:718-725 (1999) and CHY_1233 in  C. hydrogenoformans  (Wu et al.,  PLoS Genet.  1:e65 (2005). The  M. thermoacetica  genes, and its  C. hydrogenoformans  counterpart, are located near the CODH/ACS gene cluster, separated by putative hydrogenase and heterodisulfide reductase genes. Some additional gene candidates found bioinformatically are listed below. In  Acetobacterium woodii  metF is coupled to the Rnf complex through RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are found in other organisms by blast search. The Rnf complex is known to be a reversible complex (Fuchs (2011) Annu. Rev. Microbiol. 65:631-658). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Moth_1191 
                 YP_430048.1 
                 83590039 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1192 
                 YP_430049.1 
                 83590040 
                 
                   Moorella thermoacetica 
                 
               
               
                 metF 
                 NP_418376.1 
                 16131779 
                 
                   Escherichia coli 
                 
               
               
                 CHY_1233 
                 YP_360071.1 
                 78044792 
                 
                   Carboxydothermus 
                 
               
               
                   
                   
                   
                 
                   hydrogenoformans 
                 
               
               
                 CLJU_c37610 
                 YP_003781889.1 
                 300856905 
                   Clostridium ljungdahlii  DSM 
               
               
                   
                   
                   
                 13528 
               
               
                 DesfrDRAFT_3717 
                 ZP_07335241.1 
                 303248996 
                   Desulfovibrio fructosovorans  JJ 
               
               
                 CcarbDRAFT_2950 
                 ZP_05392950.1 
                 255526026 
                 
                   Clostridium carboxidivorans 
                 
               
               
                   
                   
                   
                 P7 
               
               
                 Ccel74_010100023124 
                 ZP_07633513.1 
                 307691067 
                 
                   Clostridium cellulovorans 
                 
               
               
                   
                   
                   
                 743B 
               
               
                 Cphy_3110 
                 YP_001560205.1 
                 160881237 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                   phytofermentans  ISDg 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Steps C and D—Methylenetetrahydrofolate Dehydrogenase, Methenyltetrahydrofolate Cyclohydrolase 
     In  M. thermoacetica, E. coli,  and  C. hydrogenoformans,  methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase are carried out by the bi-functional gene products of Moth_1516, folD, and CHY_1878, respectively (Pierce et al.,  Environ. Microbiol.  10:2550-2573 (2008); Wu et al.,  PLoS Genet.  1:e65 (2005); D&#39;Ari and Rabinowitz,  J. Biol. Chem.  266:23953-23958 (1991)). A homolog exists in  C. carboxidivorans  P7. Several other organisms also encode for this bifunctional protein as tabulated below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Moth_1516 
                 YP_430368.1 
                 83590359 
                 
                   Moorella thermoacetica 
                 
               
               
                 folD 
                 NP_415062.1 
                 16128513 
                 
                   Escherichia coli 
                 
               
               
                 CHY_1878 
                 YP_360698.1 
                 78044829 
                 
                   Carboxydothermus 
                 
               
               
                   
                   
                   
                 
                   hydrogenoformans 
                 
               
               
                 CcarbDRAFT_2948 
                 ZP_05392948.1 
                 255526024 
                   Clostridium carboxidivorans  P7 
               
               
                 folD 
                 ADK16789.1 
                 300437022 
                   Clostridium ljungdahlii  DSM 13528 
               
               
                 folD-2 
                 NP_951919.1 
                 39995968 
                   Geobacter sulfurreducens  PCA 
               
               
                 folD 
                 YP_725874.1 
                 113867385 
                   Ralstonia eutropha  H16 
               
               
                 folD 
                 NP_348702.1 
                 15895353 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                   acetobutylicum  ATCC 824 
               
               
                 folD 
                 YP_696506.1 
                 110800457 
                 
                   Clostridium perfringens 
                 
               
               
                 MGA3_09460 
                 EIJ83438.1 
                 387591119 
                   Bacillus methanolicus  MGA3 
               
               
                 PB1_14689 
                 ZP_10132349.1 
                 387929672 
                   Bacillus methanolicus  PB1 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step E—Formyltetrahydrofolate Deformylase 
     This enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate (formyl-THF) to THF and formate. In  E. coli,  this enzyme is encoded by purU and has been overproduced, purified, and characterized (Nagy, et al.,  J. Bacteriol.  3:1292-1298 (1995)). Homologs exist in  Corynebacterium  sp. U-96 (Suzuki, et al., Biosci. Biotechnol. Biochem. 69(5):952-956 (2005)),  Corynebacterium glutamicum  ATCC 14067,  Salmonella enterica,  and several additional organisms. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 purU 
                 AAC74314.1 
                 1787483 
                   Escherichia coli  K-12 MG1655 
               
               
                 purU 
                 BAD97821.1 
                 63002616 
                   Corynebacterium  sp. U-96 
               
               
                 purU 
                 EHE84645.1 
                 354511740 
                 
                   Corynebacterium glutamicum 
                 
               
               
                   
                   
                   
                 ATCC 14067 
               
               
                 purU 
                 NP_460715.1 
                 16765100 
                   Salmonella enterica  subsp. 
               
               
                   
                   
                   
                   enterica  serovar  Typhimurium   
               
               
                   
                   
                   
                 str. LT2 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step F—Formyltetrahydrofolate Synthetase 
     Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate at the expense of one ATP. This reaction is catalyzed by the gene product of Moth_0109 in  M. thermoacetica  (O&#39;brien et al.,  Experientia Suppl.  26:249-262 (1976); Lovell et al.,  Arch. Microbiol.  149:280-285 (1988); Lovell et al.,  Biochemistry  29:5687-5694 (1990)), FHS in  Clostridium acidurici  (Whitehead and Rabinowitz,  J. Bacteriol.  167:203-209 (1986); Whitehead and Rabinowitz,  J. Bacteriol.  170:3255-3261 (1988), and CHY_2385 in  C. hydrogenoformans  (Wu et al.,  PLoS Genet.  1:e65 (2005). Homologs exist in  C. carboxidivorans  P7. This enzyme is found in several other organisms as listed below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Moth_0109 
                 YP_428991.1 
                 83588982 
                 
                   Moorella thermoacetica 
                 
               
               
                 CHY_2385 
                 YP_361182.1 
                 78045024 
                 
                   Carboxydothermus 
                 
               
               
                   
                   
                   
                 
                   hydrogenoformans 
                 
               
               
                 FHS 
                 P13419.1 
                 120562 
                 
                   Clostridium acidurici 
                 
               
               
                 CcarbDRAFT_1913 
                 ZP_05391913.1 
                 255524966 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_2946 
                 ZP_05392946.1 
                 255526022 
                   Clostridium carboxidivorans  P7 
               
               
                 Dhaf_0555 
                 ACL18622.1 
                 219536883 
                 
                   Desulfitobacterium hafniense 
                 
               
               
                 Fhs 
                 YP_001393842.1 
                 153953077 
                   Clostridium kluyveri  DSM 555 
               
               
                 Fhs 
                 YP_003781893.1 
                 300856909 
                   Clostridium ljungdahlii  DSM 
               
               
                   
                   
                   
                 13528 
               
               
                 MGA3_08300 
                 EIJ83208.1 
                 387590889 
                   Bacillus methanolicus  MGA3 
               
               
                 PB1_13509 
                 ZP_10132113.1 
                 387929436 
                   Bacillus methanolicus  PB1 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step G—Formate Hydrogen Lyase 
     A formate hydrogen lyase enzyme can be employed to convert formate to carbon dioxide and hydrogen. An exemplary formate hydrogen lyase enzyme can be found in  Escherichia coli.  The  E. coli  formate hydrogen lyase consists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al.,  Appl Microbiol Biotechnol  77:879-890 (2007)). It is activated by the gene product of fhlA. (Maeda et al.,  Appl Microbiol Biotechnol  77:879-890 (2007)). The addition of the trace elements, selenium, nickel and molybdenum, to a fermentation broth has been shown to enhance formate hydrogen lyase activity (Soini et al.,  Microb. Cell Fact.  7:26 (2008)). Various hydrogenase 3, formate dehydrogenase and transcriptional activator genes are shown below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 hycA 
                 NP_417205 
                 16130632 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycB 
                 NP_417204 
                 16130631 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycC 
                 NP_417203 
                 16130630 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycD 
                 NP_417202 
                 16130629 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycE 
                 NP_417201 
                 16130628 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycF 
                 NP_417200 
                 16130627 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycG 
                 NP_417199 
                 16130626 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycH 
                 NP_417198 
                 16130625 
                   Escherichia coli  K-12 MG1655 
               
               
                 hycI 
                 NP_417197 
                 16130624 
                   Escherichia coli  K-12 MG1655 
               
               
                 fdhF 
                 NP_418503 
                 16131905 
                   Escherichia coli  K-12 MG1655 
               
               
                 fhlA 
                 NP_417211 
                 16130638 
                   Escherichia coli  K-12 MG1655 
               
               
                   
               
            
           
         
       
     
     A formate hydrogen lyase enzyme also exists in the hyperthermophilic archaeon,  Thermococcus litoralis  (Takacs et al.,  BMC. Microbiol  8:88 (2008)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 mhyC 
                 ABW05543 
                 157954626 
                 
                   Thermococcus litoralis 
                 
               
               
                 mhyD 
                 ABW05544 
                 157954627 
                 
                   Thermococcus litoralis 
                 
               
               
                 mhyE 
                 ABW05545 
                 157954628 
                 
                   Thermococcus litoralis 
                 
               
               
                 myhF 
                 ABW05546 
                 157954629 
                 
                   Thermococcus litoralis 
                 
               
               
                 myhG 
                 ABW05547 
                 157954630 
                 
                   Thermococcus litoralis 
                 
               
               
                 myhH 
                 ABW05548 
                 157954631 
                 
                   Thermococcus litoralis 
                 
               
               
                 fdhA 
                 AAB94932 
                 2746736 
                 
                   Thermococcus litoralis 
                 
               
               
                 fdhB 
                 AAB94931 
                 157954625 
                 
                   Thermococcus litoralis 
                 
               
               
                   
               
            
           
         
       
     
     Additional formate hydrogen lyase systems have been found in  Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacterium formicicum  (Vardar-Schara et al., Microbial Biotechnology 1:107-125 (2008)). 
     FIG.  1 , Step H—Hydrogenase 
     Hydrogenase enzymes can convert hydrogen gas to protons and transfer electrons to acceptors such as ferredoxins, NAD+, or NADP+.  Ralstonia eutropha  H16 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an “O2-tolerant” hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel,  Biochim. Biophys. Acta,  567,315-324 (1979); Bernhard et al.,  Eur. J. Biochem.  248,179-186 (1997)).  R. eutropha  also contains an O 2 -tolerant soluble hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel,  Biochim. Biophys. Acta  452,66-80 (1976); Burgdorf,  J. Bact.  187(9) 3122-3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms including  Geobacter sulfurreducens  (Coppi,  Microbiology  151,1239-1254 (2005)),  Synechocystis  str. PCC 6803 (Germer,  J. Biol. Chem.,  284(52), 36462-36472 (2009)), and  Thiocapsa roseopersicina  (Rakhely,  Appl. Environ. Microbiol.  70(2) 722-728 (2004)). The  Synechocystis  enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from  Synechocystis  str. PCC 6803 and the accessory genes encoded by the Hyp operon from  Nostoc  sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer,  J. Biol. Chem.  284(52), 36462-36472 (2009)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 HoxF 
                 NP_942727.1 
                 38637753 
                   Ralstonia eutropha  H16 
               
               
                 HoxU 
                 NP_942728.1 
                 38637754 
                   Ralstonia eutropha  H16 
               
               
                 HoxY 
                 NP_942729.1 
                 38637755 
                   Ralstonia eutropha  H16 
               
               
                 HoxH 
                 NP_942730.1 
                 38637756 
                   Ralstonia eutropha  H16 
               
               
                 HoxW 
                 NP_942731.1 
                 38637757 
                   Ralstonia eutropha  H16 
               
               
                 HoxI 
                 NP_942732.1 
                 38637758 
                   Ralstonia eutropha  H16 
               
               
                 HoxE 
                 NP_953767.1 
                 39997816 
                 
                   Geobacter sulfurreducens 
                 
               
               
                 HoxF 
                 NP_953766.1 
                 39997815 
                 
                   Geobacter sulfurreducens 
                 
               
               
                 HoxU 
                 NP_953765.1 
                 39997814 
                 
                   Geobacter sulfurreducens 
                 
               
               
                 HoxY 
                 NP_953764.1 
                 39997813 
                 
                   Geobacter sulfurreducens 
                 
               
               
                 HoxH 
                 NP_953763.1 
                 39997812 
                 
                   Geobacter sulfurreducens 
                 
               
               
                 GSU2717 
                 NP_953762.1 
                 39997811 
                 
                   Geobacter sulfurreducens 
                 
               
               
                 HoxE 
                 NP_441418.1 
                 16330690 
                   Synechocystis  str. PCC 6803 
               
               
                 HoxF 
                 NP_441417.1 
                 16330689 
                   Synechocystis  str. PCC 6803 
               
               
                 Unknown 
                 NP_441416.1 
                 16330688 
                   Synechocystis  str. PCC 6803 
               
               
                 function 
                   
                   
                   
               
               
                 HoxU 
                 NP_441415.1 
                 16330687 
                   Synechocystis  str. PCC 6803 
               
               
                 HoxY 
                 NP_441414.1 
                 16330686 
                   Synechocystis  str. PCC 6803 
               
               
                 Unknown 
                 NP_441413.1 
                 16330685 
                   Synechocystis  str. PCC 6803 
               
               
                 function 
                   
                   
                   
               
               
                 Unknown 
                 NP_441412.1 
                 16330684 
                   Synechocystis  str. PCC 6803 
               
               
                 function 
                   
                   
                   
               
               
                 HoxH 
                 NP_441411.1 
                 16330683 
                   Synechocystis  str. PCC 6803 
               
               
                 HypF 
                 NP_484737.1 
                 17228189 
                   Nostoc  sp. PCC 7120 
               
               
                 HypC 
                 NP_484738.1 
                 17228190 
                   Nostoc  sp. PCC 7120 
               
               
                 HypD 
                 NP_484739.1 
                 17228191 
                   Nostoc  sp. PCC 7120 
               
               
                 Unknown 
                 NP_484740.1 
                 17228192 
                   Nostoc  sp. PCC 7120 
               
               
                 function 
                   
                   
                   
               
               
                 HypE 
                 NP_484741.1 
                 17228193 
                   Nostoc  sp. PCC 7120 
               
               
                 HypA 
                 NP_484742.1 
                 17228194 
                   Nostoc  sp. PCC 7120 
               
               
                 HypB 
                 NP_484743.1 
                 17228195 
                   Nostoc  sp. PCC 7120 
               
               
                 Hox1E 
                 AAP50519.1 
                 37787351 
                 
                   Thiocapsa roseopersicina 
                 
               
               
                 Hox1F 
                 AAP50520.1 
                 37787352 
                 
                   Thiocapsa roseopersicina 
                 
               
               
                 Hox1U 
                 AAP50521.1 
                 37787353 
                 
                   Thiocapsa roseopersicina 
                 
               
               
                 Hox1Y 
                 AAP50522.1 
                 37787354 
                 
                   Thiocapsa roseopersicina 
                 
               
               
                 Hox1H 
                 AAP50523.1 
                 37787355 
                 
                   Thiocapsa roseopersicina 
                 
               
               
                   
               
            
           
         
       
     
     The genomes of  E. coli  and other enteric bacteria encode up to four hydrogenase enzymes (Sawers, G.,  Antonie Van Leeuwenhoek  66:57-88 (1994); Sawers et al.,  J Bacteriol.  164:1324-1331 (1985); Sawers and Boxer,  Eur. J Biochem.  156:265-275 (1986); Sawers et al.,  J Bacteriol.  168:398-404 (1986)). Given the multiplicity of enzyme activities  E. coli  or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. Endogenous hydrogen-lyase enzymes of  E. coli  include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase activity in  E. coli  is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al.,  Arch. Microbiol  158:444-451 (1992); Rangaraj an et al.,  J Bacteriol.  190:1447-1458 (2008)). The  M. thermoacetica  and  Clostridium ljungdahli  hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity.  M. thermoacetica  and  C. ljungdahli  can grow with CO 2  as the exclusive carbon source indicating that reducing equivalents are extracted from H 2  to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L.,  J Bacteriol.  150:702-709 (1982); Drake and Daniel,  Res Microbiol  155:869-883 (2004); Kellum and Drake,  J Bacteriol.  160:466-469 (1984)).  M. thermoacetica  has homologs to several hyp, hyc, and hyf genes from  E. coli.  These protein sequences encoded for by these genes are identified by the following GenBank accession numbers. In addition, several gene clusters encoding hydrogenase functionality are present in  M. thermoacetica  and  C. ljungdahli  (see for example US 2012/0003652). 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                   
                 HypA 
                 NP_417206 
                 16130633 
                 
                   Escherichia coli 
                 
               
               
                   
                 HypB 
                 NP_417207 
                 16130634 
                 
                   Escherichia coli 
                 
               
               
                   
                 HypC 
                 NP_417208 
                 16130635 
                 
                   Escherichia coli 
                 
               
               
                   
                 HypD 
                 NP_417209 
                 16130636 
                 
                   Escherichia coli 
                 
               
               
                   
                 HypE 
                 NP_417210 
                 226524740 
                 
                   Escherichia coli 
                 
               
               
                   
                 HypF 
                 NP_417192 
                 16130619 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycA 
                 NP_417205 
                 16130632 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycB 
                 NP_417204 
                 16130631 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycC 
                 NP_417203 
                 16130630 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycD 
                 NP_417202 
                 16130629 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycE 
                 NP_417201 
                 16130628 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycF 
                 NP_417200 
                 16130627 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycG 
                 NP_417199 
                 16130626 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycH 
                 NP_417198 
                 16130625 
                 
                   Escherichia coli 
                 
               
               
                   
                 HycI 
                 NP_417197 
                 16130624 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfA 
                 NP_416976 
                 90111444 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfB 
                 NP_416977 
                 16130407 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfC 
                 NP_416978 
                 90111445 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfD 
                 NP_416979 
                 16130409 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfE 
                 NP_416980 
                 16130410 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfF 
                 NP_416981 
                 16130411 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfG 
                 NP_416982 
                 16130412 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfH 
                 NP_416983 
                 16130413 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfI 
                 NP_416984 
                 16130414 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfJ 
                 NP_416985 
                 90111446 
                 
                   Escherichia coli 
                 
               
               
                   
                 HyfR 
                 NP_416986 
                 90111447 
                 
                   Escherichia coli 
                 
               
               
                   
               
            
           
         
       
     
     Proteins in  M. thermoacetica  whose genes are homologous to the  E. coli  hydrogenase genes are shown below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Moth_2175 
                 YP_431007 
                 83590998 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2176 
                 YP_431008 
                 83590999 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2177 
                 YP_431009 
                 83591000 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2178 
                 YP_431010 
                 83591001 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2179 
                 YP_431011 
                 83591002 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2180 
                 YP_431012 
                 83591003 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2181 
                 YP_431013 
                 83591004 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2182 
                 YP_431014 
                 83591005 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2183 
                 YP_431015 
                 83591006 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2184 
                 YP_431016 
                 83591007 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2185 
                 YP_431017 
                 83591008 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2186 
                 YP_431018 
                 83591009 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2187 
                 YP_431019 
                 83591010 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2188 
                 YP_431020 
                 83591011 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2189 
                 YP_431021 
                 83591012 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2190 
                 YP_431022 
                 83591013 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2191 
                 YP_431023 
                 83591014 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2192 
                 YP_431024 
                 83591015 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0439 
                 YP_429313 
                 83589304 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0440 
                 YP_429314 
                 83589305 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0441 
                 YP_429315 
                 83589306 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0442 
                 YP_429316 
                 83589307 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0809 
                 YP_429670 
                 83589661 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0810 
                 YP_429671 
                 83589662 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0811 
                 YP_429672 
                 83589663 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0812 
                 YP_429673 
                 83589664 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0814 
                 YP_429674 
                 83589665 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0815 
                 YP_429675 
                 83589666 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_0816 
                 YP_429676 
                 83589667 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1193 
                 YP_430050 
                 83590041 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1194 
                 YP_430051 
                 83590042 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1195 
                 YP_430052 
                 83590043 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1196 
                 YP_430053 
                 83590044 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1717 
                 YP_430562 
                 83590553 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1718 
                 YP_430563 
                 83590554 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1719 
                 YP_430564 
                 83590555 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1883 
                 YP_430726 
                 83590717 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1884 
                 YP_430727 
                 83590718 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1885 
                 YP_430728 
                 83590719 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1886 
                 YP_430729 
                 83590720 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1887 
                 YP_430730 
                 83590721 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1888 
                 YP_430731 
                 83590722 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1452 
                 YP_430305 
                 83590296 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1453 
                 YP_430306 
                 83590297 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1454 
                 YP_430307 
                 83590298 
                 
                   Moorella thermoacetica 
                 
               
               
                   
               
            
           
         
       
     
     Genes encoding hydrogenase enzymes from  C. ljungdahli  are shown below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 CLJU_c20290 
                 ADK15091.1 
                 300435324 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c07030 
                 ADK13773.1 
                 300434006 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c07040 
                 ADK13774.1 
                 300434007 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c07050 
                 ADK13775.1 
                 300434008 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c07060 
                 ADK13776.1 
                 300434009 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c07070 
                 ADK13777.1 
                 300434010 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c07080 
                 ADK13778.1 
                 300434011 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c14730 
                 ADK14541.1 
                 300434774 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c14720 
                 ADK14540.1 
                 300434773 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c14710 
                 ADK14539.1 
                 300434772 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c14700 
                 ADK14538.1 
                 300434771 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c28670 
                 ADK15915.1 
                 300436148 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c28660 
                 ADK15914.1 
                 300436147 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c28650 
                 ADK15913.1 
                 300436146 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c28640 
                 ADK15912.1 
                 300436145 
                 
                   Clostridium ljungdahli 
                 
               
               
                   
               
            
           
         
       
     
     In some cases, hydrogenase encoding genes are located adjacent to a CODH. In  Rhodospirillum rubrum,  the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H 2 O to CO 2  and H 2  (Fox et al.,  J Bacteriol.  178:6200-6208 (1996)). The CODH-I of  C. hydrogenoformans  and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the  R. rubrum  CODH/hydrogenase gene cluster (Wu et al.,  PLoS Genet.  1:e65 (2005)). The  C. hydrogenoformans  CODH-I was also shown to exhibit intense CO oxidation and CO 2  reduction activities when linked to an electrode (Parkin et al.,  J Am. Chem. Soc.  129:10328-10329 (2007)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 CooL 
                 AAC45118 
                 1515468 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooX 
                 AAC45119 
                 1515469 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooU 
                 AAC45120 
                 1515470 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooH 
                 AAC45121 
                 1498746 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooF 
                 AAC45122 
                 1498747 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CODH (CooS) 
                 AAC45123 
                 1498748 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooC 
                 AAC45124 
                 1498749 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooT 
                 AAC45125 
                 1498750 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooJ 
                 AAC45126 
                 1498751 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CODH-I (CooS-I) 
                 YP_360644 
                 78043418 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooF 
                 YP_360645 
                 78044791 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 HypA 
                 YP_360646 
                 78044340 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooH 
                 YP_360647 
                 78043871 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooU 
                 YP_360648 
                 78044023 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooX 
                 YP_360649 
                 78043124 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooL 
                 YP_360650 
                 78043938 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooK 
                 YP_360651 
                 78044700 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooM 
                 YP_360652 
                 78043942 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooC 
                 YP_360654.1 
                 78043296 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CooA-1 
                 YP_360655.1 
                 78044021 
                   Carboxydothermus _ hydrogenoformans   
               
               
                   
               
            
           
         
       
     
     Some hydrogenase and CODH enzymes transfer electrons to ferredoxins. Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP +  oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The  H. thermophilus  gene fdx1 encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al.,  Extremophiles  14:79-85 (2010)). The ferredoxin associated with the  Sulfolobus solfataricus  2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N-terminal domain shares 93% homology with the zfx ferredoxin from  S. acidocaldarius.  The  E. coli  genome encodes a soluble ferredoxin of unknown physiological function, fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in  Helicobacter pylori  (Mukhopadhyay et al. 2003) and  Campylobacter jejuni  (van Vliet et al. 2001). A 2Fe-2S ferredoxin from  Clostridium pasteurianum  has been cloned and expressed in  E. coli  (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as  Moorella thermoacetica, Clostridium carboxidivorans  P7,  Clostridium ljungdahli  and  Rhodospirillum rubrum  are predicted to encode several ferredoxins, listed below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 fdx1 
                 BAE02673.1 
                 68163284 
                 
                   Hydrogenobacter thermophilus 
                 
               
               
                 M11214.1 
                 AAA83524.1 
                 144806 
                 
                   Clostridium pasteurianum 
                 
               
               
                 Zfx 
                 AAY79867.1 
                 68566938 
                 
                   Sulfolobus acidocalarius 
                 
               
               
                 Fdx 
                 AAC75578.1 
                 1788874 
                 
                   Escherichia coli 
                 
               
               
                 hp_0277 
                 AAD07340.1 
                 2313367 
                 
                   Helicobacter pylori 
                 
               
               
                 fdxA 
                 CAL34484.1 
                 112359698 
                 
                   Campylobacter jejuni 
                 
               
               
                 Moth_0061 
                 ABC18400.1 
                 83571848 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1200 
                 ABC19514.1 
                 83572962 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1888 
                 ABC20188.1 
                 83573636 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2112 
                 ABC20404.1 
                 83573852 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_1037 
                 ABC19351.1 
                 83572799 
                 
                   Moorella thermoacetica 
                 
               
               
                 CcarbDRAFT_4383 
                 ZP_05394383.1 
                 255527515 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_2958 
                 ZP_05392958.1 
                 255526034 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_2281 
                 ZP_05392281.1 
                 255525342 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_5296 
                 ZP_05395295.1 
                 255528511 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_1615 
                 ZP_05391615.1 
                 255524662 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_1304 
                 ZP_05391304.1 
                 255524347 
                   Clostridium carboxidivorans  P7 
               
               
                 cooF 
                 AAG29808.1 
                 11095245 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 fdxN 
                 CAA35699.1 
                 46143 
                 
                   Rhodobacter capsulatus 
                 
               
               
                 Rru_A2264 
                 ABC23064.1 
                 83576513 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 Rru_A1916 
                 ABC22716.1 
                 83576165 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 Rru_A2026 
                 ABC22826.1 
                 83576275 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 cooF 
                 AAC45122.1 
                 1498747 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 fdxN 
                 AAA26460.1 
                 152605 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 Alvin_2884 
                 ADC63789.1 
                 288897953 
                   Allochromatium vinosum  DSM 180 
               
               
                 Fdx 
                 YP_002801146.1 
                 226946073 
                   Azotobacter vinelandii  DJ 
               
               
                 CKL_3790 
                 YP_001397146.1 
                 153956381 
                   Clostridium kluyveri  DSM 555 
               
               
                 fer1 
                 NP_949965.1 
                 39937689 
                   Rhodopseudomonas palustris  CGA009 
               
               
                 Fdx 
                 CAA12251.1 
                 3724172 
                 
                   Thauera aromatica 
                 
               
               
                 CHY_2405 
                 YP_361202.1 
                 78044690 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 Fer 
                 YP_359966.1 
                 78045103 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 Fer 
                 AAC83945.1 
                 1146198 
                 
                   Bacillus subtilis 
                 
               
               
                 fdx1 
                 NP_249053.1 
                 15595559 
                   Pseudomonas aeruginosa  PA01 
               
               
                 yfhL 
                 AP_003148.1 
                 89109368 
                   Escherichia coli  K-12 
               
               
                 CLJU_c00930 
                 ADK13195.1 
                 300433428 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c00010 
                 ADK13115.1 
                 300433348 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c01820 
                 ADK13272.1 
                 300433505 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c17980 
                 ADK14861.1 
                 300435094 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c17970 
                 ADK14860.1 
                 300435093 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c22510 
                 ADK15311.1 
                 300435544 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c26680 
                 ADK15726.1 
                 300435959 
                 
                   Clostridium ljungdahli 
                 
               
               
                 CLJU_c29400 
                 ADK15988.1 
                 300436221 
                 
                   Clostridium ljungdahli 
                 
               
               
                   
               
            
           
         
       
     
     Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins or flavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transfer of electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low-potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al.,  Eur. J. Biochem.  123:563-569 (1982); Fujii et al., 1977). The  Helicobacter pylori  FNR, encoded by HP1164 (fqrB), is coupled to the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in  Campylobacter jejuni  (St Maurice et al.,  J Bacteriol.  189:4764-4773 (2007)). A ferredoxin:NADP+ oxidoreductase enzyme is encoded in the  E. coli  genome by fpr (Bianchi et al. 1993). Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD+. In several organisms, including  E. coli,  this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD+ oxidoreductase of  E. coli,  encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity was detected in cell extracts of  Hydrogenobacter thermophilus,  although a gene with this activity has not yet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in  Clostridium carboxydivorans  P7. The NADH-dependent reduced ferredoxin: NADP oxidoreductase of  C. kluyveri,  encoded by nfnAB, catalyzes the concomitant reduction of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al,  J Bacteriol  192: 5115-5123 (2010)). Finally, the energy-conserving membrane-associated Rnf-type proteins (Seedorf et al,  PNAS  105:2128-2133 (2008); and Herrmann,  J Bacteriol  190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 fqrB 
                 NP_207955.1 
                 15645778 
                 
                   Helicobacter pylori 
                 
               
               
                 fqrB 
                 YP_001482096.1 
                 157414840 
                 
                   Campylobacter jejuni 
                 
               
               
                 RPA3954 
                 CAE29395.1 
                 39650872 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 Fpr 
                 BAH29712.1 
                 225320633 
                 
                   Hydrogenobacter thermophilus 
                 
               
               
                 yumC 
                 NP_391091.2 
                 255767736 
                 
                   Bacillus subtilis 
                 
               
               
                 Fpr 
                 P28861.4 
                 399486 
                 
                   Escherichia coli 
                 
               
               
                 hcaD 
                 AAC75595.1 
                 1788892 
                 
                   Escherichia coli 
                 
               
               
                 LOC100282643 
                 NP_001149023.1 
                 226497434 
                 
                   Zea mays 
                 
               
               
                 NfnA 
                 YP_001393861.1 
                 153953096 
                 
                   Clostridium kluyveri 
                 
               
               
                 NfnB 
                 YP_001393862.1 
                 153953097 
                 
                   Clostridium kluyveri 
                 
               
               
                 CcarbDRAFT_2639 
                 ZP_05392639.1 
                 255525707 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_2638 
                 ZP_05392638.1 
                 255525706 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_2636 
                 ZP_05392636.1 
                 255525704 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_5060 
                 ZP_05395060.1 
                 255528241 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_2450 
                 ZP_05392450.1 
                 255525514 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_1084 
                 ZP_05391084.1 
                 255524124 
                   Clostridium carboxidivorans  P7 
               
               
                 RnfC 
                 EDK33306.1 
                 146346770 
                 
                   Clostridium kluyveri 
                 
               
               
                 RnfD 
                 EDK33307.1 
                 146346771 
                 
                   Clostridium kluyveri 
                 
               
               
                 RnfG 
                 EDK33308.1 
                 146346772 
                 
                   Clostridium kluyveri 
                 
               
               
                 RnfE 
                 EDK33309.1 
                 146346773 
                 
                   Clostridium kluyveri 
                 
               
               
                 RnfA 
                 EDK33310.1 
                 146346774 
                 
                   Clostridium kluyveri 
                 
               
               
                 RnfB 
                 EDK33311.1 
                 146346775 
                 
                   Clostridium kluyveri 
                 
               
               
                 CLJU_c11410 (RnfB) 
                 ADK14209.1 
                 300434442 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 CLJU_c11400 (RnfA) 
                 ADK14208.1 
                 300434441 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 CLJU_c11390 (RnfE) 
                 ADK14207.1 
                 300434440 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 CLJU_c11380 (RnfG) 
                 ADK14206.1 
                 300434439 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 CLJU_c11370 (RnfD) 
                 ADK14205.1 
                 300434438 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 CLJU_c11360 (RnfC) 
                 ADK14204.1 
                 300434437 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 MOTH_1518 (NfnA) 
                 YP_430370.1 
                 83590361 
                 
                   Moorella thermoacetica 
                 
               
               
                 MOTH_1517(NfnB) 
                 YP_430369.1 
                 83590360 
                 
                   Moorella thermoacetica 
                 
               
               
                 CHY_1992 (NfnA) 
                 YP_360811.1 
                 78045020 
                 
                   Carboxydothermus 
                 
               
               
                   
                   
                   
                 
                   hydrogenoformans 
                 
               
               
                 CHY_1993 (NfnB) 
                 YP_360812.1 
                 78044266 
                 
                   Carboxydothermus 
                 
               
               
                   
                   
                   
                 
                   hydrogenoformans 
                 
               
               
                 CLJU_c37220 (NfnAB) 
                 YP_003781850.1 
                 300856866 
                 
                   Clostridium ljungdahlii 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step I—Formate Dehydrogenase 
     Formate dehydrogenase (FDH) catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and hydrogenases (EC 1.1.99.33). FDH enzymes have been characterized from  Moorella thermoacetica  (Andreesen and Ljungdahl,  J Bacteriol  116:867-873 (1973); Li et al.,  J Bacteriol  92:405-412 (1966); Yamamoto et al.,  J Biol Chem.  258:1826-1832 (1983). The loci, Moth_2312 is responsible for encoding the alpha subunit of formate dehydrogenase while the beta subunit is encoded by Moth_2314 (Pierce et al.,  Environ Microbiol  (2008)). Another set of genes encoding formate dehydrogenase activity with a propensity for CO 2  reduction is encoded by Sfum_2703 through Sfum_2706 in  Syntrophobacter fumaroxidans  (de Bok et al.,  Eur J Biochem.  270:2476-2485 (2003)); Reda et al.,  PNAS  105:10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY_0731, CHY_0732, and CHY_0733 in  C. hydrogenoformans  (Wu et al.,  PLoS Genet  1:e65 (2005)). Formate dehydrogenases are also found many additional organisms including  C. carboxidivorans  P7,  Bacillus methanolicus, Burkholderia stabilis, Moorella thermoacetica  ATCC 39073,  Candida boidinii, Candida methylica,  and  Saccharomyces cerevisiae  S288c. The soluble formate dehydrogenase from  Ralstonia eutropha  reduces NAD + (fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998) 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Moth_2312 
                 YP_431142 
                 148283121 
                 
                   Moorella thermoacetica 
                 
               
               
                 Moth_2314 
                 YP_431144 
                 83591135 
                 
                   Moorella thermoacetica 
                 
               
               
                 Sfum_2703 
                 YP_846816.1 
                 116750129 
                 
                   Syntrophobacter fumaroxidans 
                 
               
               
                 Sfum_2704 
                 YP_846817.1 
                 116750130 
                 
                   Syntrophobacter fumaroxidans 
                 
               
               
                 Sfum_2705 
                 YP_846818.1 
                 116750131 
                 
                   Syntrophobacter fumaroxidans 
                 
               
               
                 Sfum_2706 
                 YP_846819.1 
                 116750132 
                 
                   Syntrophobacter fumaroxidans 
                 
               
               
                 CHY_0731 
                 YP_359585.1 
                 78044572 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CHY_0732 
                 YP_359586.1 
                 78044500 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CHY_0733 
                 YP_359587.1 
                 78044647 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CcarbDRAFT_0901 
                 ZP_05390901.1 
                 255523938 
                   Clostridium carboxidivorans  P7 
               
               
                 CcarbDRAFT_4380 
                 ZP_05394380.1 
                 255527512 
                   Clostridium carboxidivorans  P7 
               
               
                 fdhA, MGA3_06625 
                 EIJ82879.1 
                 387590560 
                   Bacillus methanolicus  MGA3 
               
               
                 fdhA, PB1_11719 
                 ZP_10131761.1 
                 387929084 
                   Bacillus methanolicus  PB1 
               
               
                 fdhD, MGA3_06630 
                 EIJ82880.1 
                 387590561 
                   Bacillus methanolicus  MGA3 
               
               
                 fdhD, PB1_11724 
                 ZP_10131762.1 
                 387929085 
                   Bacillus methanolicus  PB1 
               
               
                 fdh 
                 ACF35003. 
                 194220249 
                 
                   Burkholderia stabilis 
                 
               
               
                 FDH1 
                 AAC49766.1 
                 2276465 
                 
                   Candida boidinii 
                 
               
               
                 fdh 
                 CAA57036.1 
                 1181204 
                 
                   Candida methylica 
                 
               
               
                 FDH2 
                 P0CF35.1 
                 294956522 
                   Saccharomyces cerevisiae  S288c 
               
               
                 FDH1 
                 NP_015033.1 
                 6324964 
                   Saccharomyces cerevisiae  S288c 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step J—Methanol Dehydrogenase 
     NAD+ dependent methanol dehydrogenase enzymes (EC 1.1.1.244) catalyze the conversion of methanol and NAD+ to formaldehyde and NADH. An enzyme with this activity was first characterized in  Bacillus methanolicus  (Heggeset, et al.,  Applied and Environmental Microbiology,  78(15):5170-5181 (2012)). This enzyme is zinc and magnesium dependent, and activity of the enzyme is enhanced by the activating enzyme encoded by act (Kloosterman et al,  J Biol Chem  277:34785-92 (2002)). Additional NAD(P)+ dependent enzymes can be identified by sequence homology. Methanol dehydrogenase enzymes utilizing different electron acceptors are also known in the art. Examples include cytochrome dependent enzymes such as mxaIF of the methylotroph  Methylobacterium extorquens  (Nunn et al, Nucl Acid Res 16:7722 (1988)). Methanol dehydrogenase enzymes of methanotrophs such as  Methylococcus capsulatis  function in a complex with methane monooxygenase (MMO) (Myronova et al, Biochem 45:11905-14 (2006)). Methanol can also be oxidized to formaldehyde by alcohol oxidase enzymes such as methanol oxidase (EC 1.1.3.13) of  Candida boidinii  (Sakai et al, Gene 114: 67-73 (1992)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 mdh, MGA3_17392 
                 EIJ77596.1 
                 387585261 
                   Bacillus methanolicus  MGA3 
               
               
                 mdh2, MGA3_07340 
                 EIJ83020.1 
                 387590701 
                   Bacillus methanolicus  MGA3 
               
               
                 mdh3, MGA3_10725 
                 EIJ80770.1 
                 387588449 
                   Bacillus methanolicus  MGA3 
               
               
                 act, MGA3_09170 
                 EIJ83380.1 
                 387591061 
                   Bacillus methanolicus  MGA3 
               
               
                 mdh, PB1_17533 
                 ZP_10132907.1 
                 387930234 
                   Bacillus methanolicus  PB1 
               
               
                 mdh1, PB1_14569 
                 ZP_10132325.1 
                 387929648 
                   Bacillus methanolicus  PB1 
               
               
                 mdh2, PB1_12584 
                 ZP_10131932.1 
                 387929255 
                   Bacillus methanolicus  PB1 
               
               
                 act, PB1_14394 
                 ZP_10132290.1 
                 387929613 
                   Bacillus methanolicus  PB1 
               
               
                 BFZC1_05383 
                 ZP_07048751.1 
                 299535429 
                 
                   Lysinibacillus fusiformis 
                 
               
               
                 BFZC1_20163 
                 ZP_07051637.1 
                 299538354 
                 
                   Lysinibacillus fusiformis 
                 
               
               
                 Bsph_4187 
                 YP_001699778.1 
                 169829620 
                 
                   Lysinibacillus sphaericus 
                 
               
               
                 Bsph_1706 
                 YP_001697432.1 
                 169827274 
                 
                   Lysinibacillus sphaericus 
                 
               
               
                 MCA0299 
                 YP_112833.1 
                 53802410 
                 
                   Methylococcus capsulatis 
                 
               
               
                 MCA0782 
                 YP_113284.1 
                 53804880 
                 
                   Methylococcus capsulatis 
                 
               
               
                 mxaI 
                 YP_002965443.1 
                 240140963 
                 
                   Methylobacterium extorquens 
                 
               
               
                 mxaF 
                 YP_002965446.1 
                 240140966 
                 
                   Methylobacterium extorquens 
                 
               
               
                 AOD1 
                 AAA34321.1 
                 170820 
                 
                   Candida boidinii 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step K—Spontaneous or Formaldehyde Activating Enzyme 
     The conversion of formaldehyde and THF to methylenetetrahydrofolate can occur spontaneously. It is also possible that the rate of this reaction can be enhanced by a formaldehyde activating enzyme. A formaldehyde activating enzyme (Fae) has been identified in  Methylobacterium extorquens  AM1 which catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin (Vorholt, et al.,  J. Bacteriol., 182(23), 6645-6650 (2000)). It is possible that a similar enzyme exists or can be engineered to catalyze the condensation of formaldehyde and tetrahydrofolate to methylenetetrahydrofolate. Homologs exist in several organisms including  Xanthobacter autotrophicus  Py2 and  Hyphomicrobium denitrificans  ATCC 51888. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 MexAM1_META1p1766 
                 Q9FA38.3 
                 17366061 
                 
                   Methylobacterium extorquens 
                 
               
               
                   
                   
                   
                 AM1 
               
               
                 Xaut_0032 
                 YP_001414948.1 
                 154243990 
                   Xanthobacter autotrophicus  Py2 
               
               
                 Hden_1474 
                 YP_003755607.1 
                 300022996 
                 
                   Hyphomicrobium denitrificans 
                 
               
               
                   
                   
                   
                 ATCC 51888 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step L—Formaldehyde Dehydrogenase 
     Oxidation of formaldehyde to formate is catalyzed by formaldehyde dehydrogenase. An NAD+ dependent formaldehyde dehydrogenase enzyme is encoded by fdhA of  Pseudomonas putida  (Ito et al,  J Bacteriol  176: 2483-2491 (1994)). Additional formaldehyde dehydrogenase enzymes include the NAD+ and glutathione independent formaldehyde dehydrogenase from  Hyphomicrobium zavarzinii  (Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione dependent formaldehyde dehydrogenase of  Pichia pastoris  (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent formaldehyde dehydrogenase of  Methylobacter marinus  (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 fdhA 
                 P46154.3 
                 1169603 
                 
                   Pseudomonas putida 
                 
               
               
                 faoA 
                 CAC85637.1 
                 19912992 
                 
                   Hyphomicrobium zavarzinii 
                 
               
               
                 Fld1 
                 CCA39112.1 
                 328352714 
                 
                   Pichia pastoris 
                 
               
               
                 fdh 
                 P47734.2 
                 221222447 
                 
                   Methylobacter marinus 
                 
               
               
                   
               
            
           
         
       
     
     In addition to the formaldehyde dehydrogenase enzymes listed above, alternate enzymes and pathways for converting formaldehyde to formate are known in the art. For example, many organisms employ glutathione-dependent formaldehyde oxidation pathways, in which formaldehyde is converted to formate in three steps via the intermediates S-hydroxymethylglutathione and S-formylglutathione (Vorholt et al,  J Bacteriol  182:6645-50 (2000)). The enzymes of this pathway are S-(hydroxymethyl)glutathione synthase (EC 4.4.1.22), glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) and S-formylglutathione hydrolase (EC 3.1.2.12). 
     FIG.  1 , Step M—Spontaneous or S-(hydroxymethyl)glutathione Synthase 
     While conversion of formaldehyde to S-hydroxymethylglutathione can occur spontaneously in the presence of glutathione, it has been shown by Goenrich et al (Goenrich, et al., J Biol Chem 277(5);3069-72 (2002)) that an enzyme from  Paracoccus denitrificans  can accelerate this spontaneous condensation reaction. The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene encoding it, which was named gfa, is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from  P. denitrificans  are present also in  Rhodobacter sphaeroides, Sinorhizobium meliloti,  and  Mesorhizobium loti.    
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 Gfa 
                 Q51669.3 
                 38257308 
                 
                   Paracoccus denitrificans 
                 
               
               
                 Gfa 
                 ABP71667.1 
                 145557054 
                   Rhodobacter sphaeroides  ATCC 
               
               
                   
                   
                   
                 17025 
               
               
                 Gfa 
                 Q92WX6.1 
                 38257348 
                   Sinorhizobium meliloti  1021 
               
               
                 Gfa 
                 Q98LU4.2 
                 38257349 
                   Mesorhizobium loti  MAFF303099 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step N—Glutathione-Dependent Formaldehyde Dehydrogenase 
     Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) belongs to the family of class III alcohol dehydrogenases. Glutathione and formaldehyde combine non-enzymatically to form hydroxymethylglutathione, the true substrate of the GS-FDH catalyzed reaction. The product, S-formylglutathione, is further metabolized to formic acid. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 frmA 
                 ΥP_488650.1 
                 388476464 
                   Escherichia coli  K-12 MG1655 
               
               
                 SFA1 
                 NP_010113.1 
                 6320033 
                   Saccharomyces cerevisiae  S288c 
               
               
                 flhA 
                 AAC44551.1 
                 1002865 
                 
                   Paracoccus denitrificans 
                 
               
               
                 adhI 
                 AAB09774.1 
                 986949 
                 
                   Rhodobacter sphaeroides 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  1 , Step O—S-Formylglutathione Hydrolase 
     S-formylglutathione hydrolase is a glutathione thiol esterase found in bacteria, plants and animals. It catalyzes conversion of S-formylglutathione to formate and glutathione. The fghA gene of  P. denitrificans  is located in the same operon with gfa and flhA, two genes involved in the oxidation of formaldehyde to formate in this organism. In  E. coli,  FrmB is encoded in an operon with FrmR and FrmA, which are proteins involved in the oxidation of formaldehyde. YeiG of  E. coli  is a promiscuous serine hydrolase; its highest specific activity is with the substrate S-formylglutathione. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 frmB 
                 NP_414889.1 
                 16128340 
                   Escherichia coli  K-12 MG1655 
               
               
                 yeiG 
                 AAC75215.1 
                 1788477 
                   Escherichia coli  K-12 MG1655 
               
               
                 fghA 
                 AAC44554.1 
                 1002868 
                 
                   Paracoccus denitrificans 
                 
               
               
                   
               
            
           
         
       
     
     4.2 Example II 
     Enhanced Yield of 3-hydroxyisobutyrate or MAA from Carbohydrates Using Methanol 
     Exemplary methanol metabolic pathways for enhancing the availability of reducing equivalents are provided in  FIG.  1   . 
     3-hydroxyisobutyrate and/or MAA production can be achieved in a recombinant organism by the pathway shown in  FIG.  2   . For example, MAA and/or 3-hydroxyisobutyric acid can be produced from succinate via a methylmalonyl-CoA intermediate as shown in  FIG.  2   . Exemplary enzymes for the conversion of succinate to MAA or 3-hydroxyisobutyric acid by this route include succinyl-CoA transferase, ligase, or synthetase; methylmalonyl-CoA mutase; methylmalonyl-CoA epimerase; methylmalonyl-CoA reductase (aldehyde forming); methylmalonate semialdehyde reductase; 3-hydroxyisobutyrate dehydratase; and methylmalonyl-CoA reductase (alcohol forming). 
     In this pathway, central metabolic intermediates are first channeled into succinate. For formation of succinate, phosphoenolpyruvate (PEP) is converted into oxaloacetate either via PEP carboxykinase or PEP carboxylase. Alternatively, PEP is converted first to pyruvate by pyruvate kinase and then to oxaloacetate by methylmalonyl-CoA carboxytransferase or pyruvate carboxylase. Oxaloacetate is then converted to succinate by means of the reductive TCA cycle. 
     Succinate is then activated to succinyl-CoA by a succinyl-CoA transferase or synthetase. Methylmalonyl-CoA mutase then forms methylmalonyl-CoA from succinyl-CoA. Methylmalonyl-CoA is then reduced to methylmalonate semialdehyde. Further reduction of methylmalonate semialdehyde yields 3-hydroxyisobutyric acid, which can be secreted as a product or further transformed to MAA via dehydration. 
     Exemplary enzyme candidates for the transformations shown in  FIG.  2    are described below. 
     FIG.  2 , Step A—Succinyl-CoA Transferase, Ligase, or Synthetase 
     The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5). The product of the LSC1 and LSC2 genes of  S. cerevisiae  and the sucC and sucD genes of  E. coli  naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al.,  Biochemistry  24:6245-6252 (1985)). These proteins are identified below: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 LSC1 
                 NP_014785 
                 6324716 
                 
                   Saccharomyces cerevisiae 
                 
               
               
                 LSC2 
                 NP_011760 
                 6321683 
                 
                   Saccharomyces cerevisiae 
                 
               
               
                 sucC 
                 NP_415256.1 
                 16128703 
                 
                   Escherichia coli 
                 
               
               
                 sucD 
                 AAC73823.1 
                 1786949 
                 
                   Escherichia coli 
                 
               
               
                   
               
            
           
         
       
     
     Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate, propionate, vinylacetate, and butyrate, among others. 
     The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product of cat1 of  Clostridium kluyveri  has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and Gottschalk,  J. Bacteriol.  178:871-880 (1996)). In addition, the activity is present in  Trichomonas vaginalis  (van Grinsven et al.,  J Biol Chem.  283:1411-1418 (2008)) and  Trypanosoma brucei  (Riviere et al.,  J. Biol. Chem.  279(44):45337-45346 (2004)). The succinyl-CoA:acetate CoA-transferase from  Acetobacter aceti,  encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al.,  J. Bacteriol.  190(14):4933-4940 (2008)). Similar succinyl-CoA transferase activities are also present in  Trichomonas vaginalis  (van Grinsven et al., supra, 2008),  Trypanosoma brucei  (Riviere et al., supra, 2004) and  Clostridium kluyveri  (Sohling and Gottschalk, supra, 1996). The beta-ketoadipate:succinyl-CoA transferase encoded by pcaI and pcal in  Pseudomonas putida  is yet another candidate (Kaschabek et al.,  J. Bacteriol.  184(1):207-215 (2002)). The aforementioned proteins are identified below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 cat1 
                 P38946.1 
                 729048 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   kluyveri 
                 
               
               
                 TVAG_395550 
                 XP_001330176 
                 123975034 
                 
                   Trichomonas 
                 
               
               
                   
                   
                   
                   vaginalis  G3 
               
               
                 Tb11.02.0290 
                 XP_828352 
                 71754875 
                 
                   Trypanosoma 
                 
               
               
                   
                   
                   
                 
                   brucei 
                 
               
               
                 pcaI 
                 AAN69545.1 
                 24985644 
                 
                   Pseudomonas 
                 
               
               
                   
                   
                   
                 
                   putida 
                 
               
               
                 pcaJ 
                 NP_746082.1 
                 26990657 
                 
                   Pseudomonas 
                 
               
               
                   
                   
                   
                 
                   putida 
                 
               
               
                 aarC 
                 ACD85596.1 
                 189233555 
                 
                   Acetobacter 
                 
               
               
                   
                   
                   
                 
                   aceti 
                 
               
               
                   
               
            
           
         
       
     
     An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in  Helicobacter pylori  (Corthesy-Theulaz et al.,  J. Biol. Chem.  272(41) : 25659-25667 (1997)),  Bacillus subtilis,  and  Homo sapiens  (Fukao et al.,  Genomics  68(2):144-151 (2000); Tanaka et al.,  Mol. Hum. Reprod.  8(1):16-23 (2002)). The aforementioned proteins are identified below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 HPAG1_0676 
                 YP_627417 
                 108563101 
                 
                   Helicobacter pylori 
                 
               
               
                 HPAG1_0677 
                 YP_627418 
                 108563102 
                 
                   Helicobacter pylori 
                 
               
               
                 ScoA 
                 NP_391778 
                 16080950 
                 
                   Bacillus subtilis 
                 
               
               
                 ScoB 
                 NP_391777 
                 16080949 
                 
                   Bacillus subtilis 
                 
               
               
                 OXCT1 
                 NP_000427 
                 4557817 
                 
                   Homo sapiens 
                 
               
               
                 OXCT2 
                 NP_071403 
                 11545841 
                 
                   Homo sapiens 
                 
               
               
                   
               
            
           
         
       
     
     Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoA transferase requires the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a 3-ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA: acetate: CoA transferase. Acetoacetyl-CoA: acetate: CoA transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa. Exemplary enzymes include the gene products of atoAD from  E. coli  (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007), ctfAB from  C. acetobutylicum  (Jojima et al., Appl Microbiol Biotechnol 77:1219-1224 (2008), and ctfAB from  Clostridium saccharoperbutylacetonicum  (Kosaka et al.,  Biosci. Biotechnol Biochem.  71:58-68 (2007)) are shown below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 AtoA 
                 NP_416726.1 
                 2492994 
                 
                   Escherichia coli 
                 
               
               
                 AtoD 
                 NP_416725.1 
                 2492990 
                 
                   Escherichia coli 
                 
               
               
                 CtfA 
                 NP_149326.1 
                 15004866 
                 
                   Clostridium acetobutylicum 
                 
               
               
                 CtfB 
                 NP_149327.1 
                 15004867 
                 
                   Clostridium acetobutylicum 
                 
               
               
                 CtfA 
                 AAP42564.1 
                 31075384 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   saccharoperbutylacetonicum 
                 
               
               
                 CtfB 
                 AAP42565.1 
                 31075385 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   saccharoperbutylacetonicum 
                 
               
               
                   
               
            
           
         
       
     
     Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as  Thauera aromatica  (Leutwein and Heider,  J. Bact.  183(14) 4288-4295 (2001)). Homologs can be found in  Azoarcus  sp. T,  Aromatoleum aromaticum  EbN1, and  Geobacter metallireducens  GS-15. The aforementioned proteins are identified below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 bbsE 
                 AAF89840 
                 9622535 
                 
                   Thauera aromatica 
                 
               
               
                 Bbsf 
                 AAF89841 
                 9622536 
                 
                   Thauera aromatica 
                 
               
               
                 bbsE 
                 AAU45405.1 
                 52421824 
                   Azoarcus  sp. T 
               
               
                 bbsF 
                 AAU45406.1 
                 52421825 
                   Azoarcus  sp. T 
               
               
                 bbsE 
                 YP_158075.1 
                 56476486 
                 
                   Aromatoleum 
                 
               
               
                   
                   
                   
                   aromaticum  EbN1 
               
               
                 bbsF 
                 YP_158074.1 
                 56476485 
                 
                   Aromatoleum 
                 
               
               
                   
                   
                   
                   aromaticum  EbN1 
               
               
                 Gmet_1521 
                 YP_384480.1 
                 78222733 
                 
                   Geobacter 
                 
               
               
                   
                   
                   
                   metallireducens  GS-15 
               
               
                 Gmet_1522 
                 YP_384481.1 
                 78222734 
                 
                   Geobacter 
                 
               
               
                   
                   
                   
                   metallireducens  GS-15 
               
               
                   
               
            
           
         
       
     
     Additionally, ygfH encodes a propionyl CoA: succinate CoA transferase in  E. coli  (Haller et al.,  Biochemistry,  39(16) 4622-4629). Close homologs can be found in, for example,  Citrobacter youngae  ATCC 29220,  Salmonella enterica  subsp.  arizonae serovar,  and  Yersinia intermedia  ATCC 29909. The aforementioned proteins are identified below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 ygfH 
                 NP_417395.1 
                 16130821 
                 
                   Escherichia coli 
                 
               
               
                   
                   
                   
                 str. K-12 substr. 
               
               
                   
                   
                   
                 MG1655 
               
               
                 CIT292_04485 
                 ZP_03838384.1 
                 227334728 
                 
                   Citrobacter youngae 
                 
               
               
                   
                   
                   
                 ATCC 29220 
               
               
                 SARI_04582 
                 ΥP_001573497.1 
                 161506385 
                 
                   Salmonella enterica 
                 
               
               
                   
                   
                   
                 subsp.  arizonae   
               
               
                   
                   
                   
                 
                   serovar 
                 
               
               
                 yinte0001_14430 
                 ZP_04635364.1 
                 238791727 
                 
                   Yersinia intermedia 
                 
               
               
                   
                   
                   
                 ATCC 29909 
               
               
                   
               
            
           
         
       
     
       FIG.  2   , Step B—Methylmalonyl-CoA Mutase 
     Methylmalonyl-CoA mutase (MCM) (EC 5.4.99.2) is a cobalamin-dependent enzyme that converts succinyl-CoA to methylmalonyl-CoA. In  E. coli,  the reversible adenosylcobalamin-dependant mutase participates in a three-step pathway leading to the conversion of succinate to propionate (Haller et al.,  Biochemistry  39:4622-4629 (2000)). Overexpression of the MCM gene candidate along with the deletion of YgfG can be used to prevent the decarboxylation of methylmalonyl-CoA to propionyl-CoA and to maximize the methylmalonyl-CoA available for MAA synthesis. MCM is encoded by genes scpA in Escherichia coli (Bobik and Rasche,  Anal. Bioanal. Chem.  375:344-349 (2003); Haller et al.,  Biochemistry  39:4622-4629 (2000)) and mutA in  Homo sapiens  (Padovani and Banerjee,  Biochemistry  45:9300-9306 (2006)). In several other organisms MCM contains alpha and beta subunits and is encoded by two genes. Exemplary gene candidates encoding the two-subunit protein are  Propionibacterium fredenreichii  sp.  shermani  mutA and mutB (Korotkova and Lidstrom,  J. Biol. Chem.  279:13652-13658 (2004)),  Methylobacterium extorquens  mcmA and mcmB (Korotkova and Lidstrom, supra, 2004), and  Ralstonia eutropha  sbm1 and sbm2 (Peplinski et al.,  Appl. Microbiol. Biotech.  88:1145-59 (2010)). Additional enzyme candidates identified based on high homology to the  E. coli  spcA gene product are also listed below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 scpA 
                 NP_417392.1 
                 16130818 
                 
                   Escherichia coli 
                 
               
               
                   
                   
                   
                 K12 
               
               
                 mutA 
                 P22033.3 
                 67469281 
                 
                   Homo sapiens 
                 
               
               
                 mutA 
                 P11652.3 
                 127549 
                 
                   Propionibacterium 
                 
               
               
                   
                   
                   
                 
                   fredenreichii 
                 
               
               
                   
                   
                   
                 sp.  shermanii   
               
               
                 mutB 
                 P11653.3 
                 127550 
                 
                   Propionibacterium 
                 
               
               
                   
                   
                   
                 
                   fredenreichii 
                 
               
               
                   
                   
                   
                 sp.  shermanii   
               
               
                 mcmA 
                 Q84FZ1 
                 75486201 
                 
                   Methylobacterium 
                 
               
               
                   
                   
                   
                 
                   extorquens 
                 
               
               
                 mcmB 
                 Q6TMA2 
                 75493131 
                 
                   Methylobacterium 
                 
               
               
                   
                   
                   
                 
                   extorquens 
                 
               
               
                 Sbm1 
                 YP_724799.1 
                 113866310 
                 
                   Ralstonia eutropha 
                 
               
               
                   
                   
                   
                 H16 
               
               
                 Sbm2 
                 YP_726418.1 
                 113867929 
                 
                   Ralstonia eutropha 
                 
               
               
                   
                   
                   
                 H16 
               
               
                 sbm 
                 NP_838397.1 
                 30064226 
                 
                   Shigella flexneri 
                 
               
               
                 SARI_04585 
                 ABX24358.1 
                 160867735 
                 
                   Salmonella enterica 
                 
               
               
                 YfreA_01000861 
                 ZP_00830776.1 
                 77975240 
                 
                   Yersinia 
                 
               
               
                   
                   
                   
                 
                   frederiksenii 
                 
               
               
                   
               
            
           
         
       
     
     These sequences can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (for example, BLASTp). The resulting homologue proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into  E. coli  or other suitable host microorganisms to generate production hosts. Additional gene candidates include the following, which were identified based on high homology to the  E. coli  spcA gene product. 
     There further exists evidence that genes adjacent to the methylmalonyl-CoA mutase catalytic genes contribute to maximum activity. For example, it has been demonstrated that the meaB gene from  M. extorquens  forms a complex with methylmalonyl-CoA mutase, stimulates in vitro mutase activity, and possibly protects it from irreversible inactivation (Korotkova and Lidstrom,  J. Biol. Chem.  279:13652-13658 (2004)). The  M. extorquens  meaB gene product is highly similar to the product of the  E. coli  argK gene (BLASTp: 45% identity, e-value: 4e-67), which is adjacent to scpA on the chromosome. No sequence for a meaB homolog in  P. freudenreichii  is catalogued in GenBank. However, the  Propionibacterium acnes  KPA171202 gene product, YP_055310.1, is 51% identical to the  M. extorquens  meaB protein and its gene is also adjacent to the methylmalonyl-CoA mutase gene on the chromosome. A similar gene is encoded by H16_B1839 of  Ralstonia eutropha  H16. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Gene 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 argK 
                 AAC75955.1 
                 1789285 
                 
                   Escherichia coli 
                 
               
               
                   
                   
                   
                 K12 
               
               
                 PPA0597 
                 YP_055310.1 
                 50842083 
                 
                   Propionibacterium 
                 
               
               
                   
                   
                   
                 
                   acnes 
                 
               
               
                 KPA171202 
                 2QM8_B 
                 158430328 
                 
                   Methylobacterium 
                 
               
               
                   
                   
                   
                 
                   extorquens 
                 
               
               
                 H16_B1839 
                 YP_841351.1 
                 116695775 
                 
                   Ralstonia eutropha 
                 
               
               
                   
                   
                   
                 H16 
               
               
                   
               
            
           
         
       
     
       E. coli  can synthesize adenosylcobalamin, a necessary cofactor for this reaction, only when supplied with the intermediates cobinamide or cobalamin (Lawrence and Roth.  J. Bacteriol.  177:6371-6380 (1995); Lawrence and Roth,  Genetics  142:11-24 (1996)). Alternatively, the ability to synthesize cobalamins de novo has been conferred upon  E. coli  following the expression of heterologous genes (Raux et al.,  J. Bacteriol.  178:753-767 (1996)). 
     Alternatively, isobutyryl-CoA mutase (ICM) (EC 5.4.99.13) could catalyze the proposed transformation shown in FIG., step B. ICM is a cobalamin-dependent methylmutase in the MCM family that reversibly rearranges the carbon backbone of butyryl-CoA into isobutyryl-CoA (Ratnatilleke et al.,  J. Biol. Chem.  274:31679-31685 (1999)). A recent study of a novel ICM in  Methylibium petroleiphilum,  along with previous work, provides evidence that changing a single amino acid near the active site alters the substrate specificity of the enzyme (Ratnatilleke et al.,  J. Biol. Chem.  274:31679-31685 (1999); Rohwerder et al.,  Appl. Environ. Microbiol.  72:4128-4135. (2006)). This indicates that, if a native enzyme is unable to catalyze or exhibits low activity for the conversion of 4HB-CoA to 3HIB-CoA, the enzyme can be rationally engineered to increase this activity. Exemplary ICM genes encoding homodimeric enzymes include icmA in  Streptomyces coelicolor  A3 (Alhapel et al.,  Proc. Natl. Acad. Sci. USA  103:12341-12346 (2006)) and Mpe_B0541 in  Methylibium petroleiphilum  PM1 (Ratnatilleke et al.,  J. Biol. Chem.  274:31679-31685 (1999); Rohwerder et al.,  Appl. Environ. Microbiol.  72:4128-4135 (2006)). Genes encoding heterodimeric enzymes include icm and icmB in  Streptomyces cinnamonensis  (Ratnatilleke et al.,  J. Biol. Chem.  274:31679-31685 (1999); Vrijbloed et al.,  J. Bacteriol.  181:5600-5605. (1999); Zerbe-Burkhardt et al.,  J Biol. Chem.  273:6508-6517 (1998)). Enzymes encoded by icmA and icmB genes in  Streptomyces avermitilis  MA-4680 show high sequence similarity to known ICMs. These genes/proteins are identified below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Gene 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 icmA 
                 CAB40912.1 
                 4585853 
                 
                   Streptomyces 
                 
               
               
                   
                   
                   
                   coelicolor  A3(2) 
               
               
                 Mpe_B0541 
                 YP_001023546.1 
                 124263076 
                 
                   Methylibium 
                 
               
               
                   
                   
                   
                   petroleiphilum  PM1 
               
               
                 icm 
                 AAC08713.1 
                 3002492 
                 
                   Streptomyces 
                 
               
               
                   
                   
                   
                 
                   cinnamonensis 
                 
               
               
                 icmB 
                 CAB59633.1 
                 6137077 
                 
                   Streptomyces 
                 
               
               
                   
                   
                   
                 
                   cinnamonensis 
                 
               
               
                 icmA 
                 NP_824008.1 
                 29829374 
                 
                   Streptomyces 
                 
               
               
                   
                   
                   
                 
                   avermitilis 
                 
               
               
                 icmB 
                 NP_824637.1 
                 29830003 
                 
                   Streptomyces 
                 
               
               
                   
                   
                   
                 
                   avermitilis 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  2 , Step C—Methylmalonyl-CoA Epimerase 
     Methylmalonyl-CoA epimerase (MMCE) is the enzyme that interconverts (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA. MMCE is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Methylmalonyl-CoA epimerase activity is not believed to be encoded in the  E. coli  genome (Boynton et al.,  J. Bacteriol. 178:3015-3024 (1996)), but is present in other organisms such as  Homo sapiens  (YqjC) (Fuller and Leadlay,.  Biochem. J.  213:643-650 (1983)),  Rattus norvegicus  (Mcee) (Bobik and Rasche,  J. Biol. Chem.  276:37194-37198 (2001)),  Propionibacterium shermanii  (AF454511) (Fuller. and Leadlay,  Biochem. J.  213:643-650 (1983); Haller et al.,  Biochemistry  39:4622-4629 (2000); McCarthy et al.,.  Structure  9:637-646.2001)) and  Caenorhabditis elegans  (mmce) (Kuhnl et al.,  FEBS J.  272:1465-1477 (2005)). An additional gene candidate, AE016877 in  Bacillus cereus,  has high sequence homology to other characterized enzymes. This enzymatic step may or may not be necessary depending upon the stereospecificity of the enzyme or enzymes used for the conversion of methylmalonyl-CoA to 3-hydroxyisobutyrate (steps 3-4 in  FIG.  3   ). These genes/proteins are described below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Gene 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 YqjC 
                 NP_390273 
                 255767522 
                 
                   Bacillus subtilis 
                 
               
               
                 MCEE 
                 Q96PE7.1 
                 50401130 
                 
                   Homo sapiens 
                 
               
               
                 Mcee_predicted 
                 NP_001099811.1 
                 157821869 
                 
                   Rattus norvegicus 
                 
               
               
                 AF454511 
                 AAL57846.1 
                 18042135 
                 
                   Propionibacterium 
                 
               
               
                   
                   
                   
                   fredenreichii  sp. 
               
               
                   
                   
                   
                 
                   shermanii 
                 
               
               
                 mmce 
                 AAT92095.1 
                 51011368 
                 
                   Caenorhabditis 
                 
               
               
                   
                   
                   
                 
                   elegans 
                 
               
               
                 AE016877 
                 AAP08811.1 
                 29895524 
                 
                   Bacillus cereus 
                 
               
               
                   
                   
                   
                 ATCC 14579 
               
               
                   
               
            
           
         
       
     
     FIG.  2 , Step D—Methylmalonyl-CoA Reductase (Aldehyde Forming) 
     The reduction of methylmalonyl-CoA to its corresponding aldehyde, methylmalonate semialdehyde, is catalyzed by a CoA-dependent aldehyde dehydrogenase (EC 1.2.1.-). Conversion of methylmalonyl-CoA to methylmalonic semialdehyde, is accomplished by a CoA-dependent aldehyde dehydrogenase. An enzyme encoded by a malonyl-CoA reductase gene from  Sulfolobus tokodaii  (Alber et. al.,  J. Bacteriol.  188(24):8551-8559 (2006)), has been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208). A similar enzyme exists in  Metallosphaera sedula  (Alber et. al.,  J. Bacteriol.  188(24):8551-8559 (2006)). Several additional CoA dehydrogenases are capable also of reducing an acyl-CoA to its corresponding aldehyde. The reduction of methylmalonyl-CoA to its corresponding aldehyde, methylmalonate semialdehyde, is catalyzed by a CoA-dependent aldehyde dehydrogenase. Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoA reductase and butyryl-CoA reductase. Exemplary fatty acyl-CoA reductase enzymes are encoded by acr1 of  Acinetobacter calcoaceticus  (Reiser and Somerville.  J. Bacteriol.  179:2969-2975 (1997)), and  Acinetobacter  sp. M-1 fatty acyl-CoA reductase (Ishige et al.,  Appl. Environ. Microbiol.  68:1192-1195 (2002)). Also known is a CoA- and NADP-dependent succinate semialdehyde dehydrogenase (also referred to as succinyl-CoA reductase) encoded by the sucD gene in  Clostridium kluyveri  (Sohling and Gottschalk,  J. Bacteriol.  178:871-880 (1996); Sohling and Gottschalk,  J. Bacteriol.  178:871-880 (1996)) and sucD of  P. gingivalis  (Takahashi,  J. Bacteriol  182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea including Metallosphaera sedula (Berg et al.,  Science  318:1782-1786 (2007)) and  Thermoproteus neutrophilus  (Ramos-Vera et al.,  J Bacteriol,  191:4286-4297 (2009)). The  M. sedula  enzyme, encoded by Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The  T. neutrophilus  enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase in  Pseudomonas  sp, encoded by bphG, is also a good candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, formaldehyde and the branched-chain compound isobutyraldehyde (Powlowski et al.,  J. Bacteriol.  175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in  Leuconostoc mesenteroides  has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya,  J. Gen. Appl. Microbiol.  18:43-55 (1972); and Koo et al.,  Biotechnol Lett.  27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as  Clostridium saccharoperbutylacetonicum  (Kosaka et al.,  Biosci Biotechnol Biochem.,  71:58-68 (2007)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 acr1 
                 YP_047869.1 
                 50086359 
                 
                   Acinetobacter 
                 
               
               
                   
                   
                   
                 
                   calcoaceticus 
                 
               
               
                 acr1 
                 AAC45217 
                 1684886 
                 
                   Acinetobacter 
                 
               
               
                   
                   
                   
                 
                   baylyi 
                 
               
               
                 acr1 
                 BAB85476.1 
                 18857901 
                 
                   Acinetobacter 
                 
               
               
                   
                   
                   
                 sp. Strain M-1 
               
               
                 MSED_0709 
                 YP_001190808.1 
                 146303492 
                 
                   Metallosphaera 
                 
               
               
                   
                   
                   
                 
                   sedula 
                 
               
               
                 Tneu_0421 
                   
                   
                 
                   Thermoproteus 
                 
               
               
                   
                   
                   
                 
                   neutrophilus 
                 
               
               
                 sucD 
                 P38947.1 
                 172046062 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   kluyveri 
                 
               
               
                 sucD 
                 NP_904963.1 
                 34540484 
                 
                   Porphyromonas 
                 
               
               
                   
                   
                   
                 
                   gingivalis 
                 
               
               
                 bphG 
                 BAA03892.1 
                 425213 
                   Pseudomonas  sp 
               
               
                 adhE 
                 AAV66076.1 
                 55818563 
                 
                   Leuconostoc 
                 
               
               
                   
                   
                   
                 
                   mesenteroides 
                 
               
               
                 bld 
                 AAP42563.1 
                 31075383 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                   saccharoper - 
               
               
                   
                   
                   
                 
                   butylacetonicum 
                 
               
               
                   
               
            
           
         
       
     
     An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg,  Science  318:1782-1786 (2007); and Thauer,  Science  318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in  Metallosphaera  and  Sulfolobus  sp. (Alber et al.,  J. Bacteriol.  188:8551-8559 (2006); and Hugler,  J. Bacteriol.  184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in  Metallosphaera sedula  (Alber et al.,  J. Bacteriol.  188:8551-8559 (2006); and Berg,  Science  318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from  Sulfolobus tokodaii  was cloned and heterologously expressed in  E. coli  (Alber et al.,  J. Bacteriol  188:8551-8559 (2006). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from  Chloroflexus aurantiacus,  there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including  Sulfolobus solfataricus  and  Sulfolobus acidocaldarius  and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from  Clostridium beijerinckii  (Toth,  Appl. Environ. Microbiol.  65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of  Salmonella typhimurium  and  E. coli  (Toth,  Appl. Environ. Microbiol.  65:4973-4980 (1999). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Gene 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 Msed_0709 
                 YP_001190808.1 
                 146303492 
                 
                   Metallosphaera sedula 
                 
               
               
                 mcr 
                 NP_378167.1 
                 15922498 
                 
                   Sulfolobus tokodaii 
                 
               
               
                 asd-2 
                 NP_343563.1 
                 15898958 
                 
                   Sulfolobus solfataricus 
                 
               
               
                 Saci_2370 
                 YP_256941.1 
                 70608071 
                 
                   Sulfolobus 
                 
               
               
                   
                   
                   
                 
                   acidocaldarius 
                 
               
               
                 Ald 
                 AAT66436 
                 49473535 
                 
                   Clostridium beijerinckii 
                 
               
               
                 eutE 
                 AAA80209 
                 687645 
                 
                   Salmonella typhimurium 
                 
               
               
                 eutE 
                 P77445 
                 2498347 
                 
                   Escherichia coli 
                 
               
               
                   
               
            
           
         
       
     
     A bifunctional enzyme with acyl-CoA reductase and alcohol dehydrogenase activity can directly convert methylmalonyl-CoA to 3-hydroxyisobutyrate. Exemplary bifunctional oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (for example, adhE from  E. coli  (Kessler et al.,  FEBS.Lett.  281:59-63 (1991))) and butyryl-CoA to butanol (for example, adhE2 from  C. acetobutylicum  (Fontaine et al.,  J. Bacteriol.  184:821-830 (2002)). The  C. acetobutylicum  enzymes encoded by bdh I and bdh II (Walter, et al.,  J. Bacteriol.  174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in  Leuconostoc mesenteroides  has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al.,  J. Gen. Appl. Microbiol.  18:43-55 (1972); Koo et al.,  Biotechnol Lett,  27:505-510 (2005)). Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH-dependent enzyme with this activity has characterized in  Chloroflexus aurantiacus  where it participates in the 3-hydroxypropionate cycle (Hugler et al.,  J Bacteriol,  184:2404-2410 (2002); Strauss et al.,  Eur J Biochem,  215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., supra). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms may have similar pathways (Klatt et al.,  Env Microbiol,  9:2067-2078 (2007)). Enzyme candidates in other organisms including  Roseiflexus castenholzii, Erythrobacter  sp. NAP 1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 adhE 
                 NP_415757.1 
                 16129202 
                 
                   Escherichia coli 
                 
               
               
                 adhE2 
                 AAK09379.1 
                 12958626 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   acetobutylicum 
                 
               
               
                 adhE 
                 AAV66076.1 
                 55818563 
                 
                   Leuconostoc 
                 
               
               
                   
                   
                   
                 
                   mesenteroides 
                 
               
               
                 bdh I 
                 NP_349892.1 
                 15896543 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   acetobutylicum 
                 
               
               
                 bdh II 
                 NP_349891.1 
                 15896542 
                 
                   Clostridium 
                 
               
               
                   
                   
                   
                 
                   acetobutylicum 
                 
               
               
                 mcr 
                 AAS20429.1 
                 42561982 
                 
                   Chloroflexus 
                 
               
               
                   
                   
                   
                 
                   aurantiacus 
                 
               
               
                 Rcas_2929 
                 YP_001433009.1 
                 156742880 
                 
                   Roseiflexus 
                 
               
               
                   
                   
                   
                 
                   castenholzii 
                 
               
               
                 NAP1_02720 
                 ZP_01039179.1 
                 85708113 
                 
                   Erythrobacter 
                 
               
               
                   
                   
                   
                 sp. NAP1 
               
               
                 MGP2080_00535 
                 ZP_01626393.1 
                 119504313 
                 marine gamma 
               
               
                   
                   
                   
                 proteobacterium 
               
               
                   
                   
                   
                 HTCC2080 
               
               
                   
               
            
           
         
       
     
     FIG.  2 , Step E—Methylmalonate Semialdehyde Reductase 
     The reduction of methylmalonate semialdehyde to 3-hydroxyisobutyrate is catalyzed by methylmalonate semialdehyde reductase or 3-hydroxyisobutyrate dehydrogenase. This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from  Thermus thermophilus  HB8 has been structurally characterized (Lokanath et al.,  J. Mol. Biol.  352:905-917 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning and Pollitt,  Biochem. J.  231:481-484 (1985)). Additional genes encoding this enzyme include 3hidh in  Homo sapiens  (Hawes et al.,  Methods Enzymol.  324:218-228 (2000)) and  Oryctolagus cuniculus  (Chowdhury et al.,  Biosci. Biotechnol. Biochem.  60:2043-2047 (1996); Hawes et al.,  Methods Enzymol.  324:218-228 (2000)), mmsb in  Pseudomonas aeruginosa,  and dhat in  Pseudomonas putida  (Aberhart and Hsu  J Chem. Soc. [Perkin  1] 6:1404-1406 (1979); Chowdhury et al.,  Biosci. Biotechnol. Biochem.  67:438-441 (2003); Chowdhury et al.,  Biosci. Biotechnol. Biochem.  60:2043-2047 (1996)). Several 3-hydroxyisobutyrate dehydrogenase enzymes have been characterized in the reductive direction, including mmsB from  Pseudomonas aeruginosa  (Gokarn et al., U.S. Pat. No. 7,393,676 (2008)) and mmsB from  Pseudomonas putida.    
                                         Protein   GenBank ID   GI Number   Organism                  P84067   P84067   75345323     Thermus thermophilus         3hidh   P31937.2   12643395     Homo sapiens         3hidh   P32185.1   416872     Oryctolagus cuniculus         mmsB   NP_746775.1   26991350     Pseudomonas putida         mmsB   P28811.1   127211     Pseudomonas aeruginosa         dhat   Q59477.1   2842618     Pseudomonas putida                        FIG.  2   , Step F—3-Hydroxyisobutyrate dehydratase
 
     The dehydration of 3-hydroxyisobutyrate to methylacrylic acid is catalyzed by an enzyme with 3-hydroxyisobutyrate dehydratase activity (EC 4.2.1.-). The final step involves the dehydration of 3-hydroxyisobutyrate to methacrylic acid. The dehydration of 3-hydroxyisobutyrate to methylacrylic acid is catalyzed by an enzyme with 3-hydroxyisobutyrate dehydratase activity. Although no direct evidence for this specific enzymatic transformation has been identified, most dehydratases catalyze the α,β-elimination of water, which involves activation of the α-hydrogen by an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the β-position (Buckel and Barker,  J Bacteriol.  117:1248-1260 (1974); Martins et al,  Proc. Natl. Acad. Sci. USA  101:15645-15649 (2004)). This is the exact type of transformation proposed for the final step in the methacrylate pathway. In addition, the proposed transformation is highly similar to the 2-(hydroxymethyl)glutarate dehydratase of  Eubacterium barkeri,  which can catalyze the conversion of 2-hydroxymethyl glutarate to 2-methylene glutarate. This enzyme has been studied in the context of nicotinate catabolism and is encoded by hmd (Alhapel et al.,  Proc. Natl. Acad. Sci. USA  103:12341-12346 (2006)). Similar enzymes with high sequence homology are found in  Bacteroides capillosus, Anaerotruncus colihominis,  and  Natranaerobius thermophilius.  Several enzymes are known to catalyze the alpha, beta elimination of hydroxyl groups. Exemplary enzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-), fumarase (EC 4.2.1.2), 2-keto-4-pentenoate dehydratase (EC 4.2.1.80), citramalate hydrolyase and dimethylmaleate hydratase. 
     2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for its role in nicontinate catabolism in  Eubacterium barkeri  (formerly  Clostridium barkeri ) (Alhapel et al.,  Proc Natl Acad Sci USA  103:12341-12346 (2006)). Similar enzymes with high sequence homology are found in  Bacteroides capillosus, Anaerotruncus colihominis,  and  Natranaerobius thermophilius.  These enzymes are also homologous to the α- and β-subunits of [4Fe-4S]-containing bacterial serine dehydratases, for example,  E. coli  enzymes encoded by tdcG, sdhB, and sdaA). An enzyme with similar functionality in  E. barkeri  is dimethylmaleate hydratase, a reversible Fe2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB (Alhapel et al.,  Proc Natl Acad Sci USA  103:12341-6 (2006); Kollmann-Koch et al.,  Hoppe Seylers. Z. Physiol Chem.  365:847-857 (1984)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 hmd 
                 ABC88407.1 
                 86278275 
                 
                   Eubacterium 
                 
               
               
                   
                   
                   
                 
                   barkeri 
                 
               
               
                 BACCAP_02294 
                 ZP_02036683.1 
                 154498305 
                 
                   Bacteroides 
                 
               
               
                   
                   
                   
                 
                   capillosus 
                 
               
               
                 ANACOL_02527 
                 ZP_02443222.1 
                 167771169 
                 
                   Anaerotruncus 
                 
               
               
                   
                   
                   
                 
                   colihominis 
                 
               
               
                 NtherDRAFT_2368 
                 ZP_02852366.1 
                 169192667 
                 
                   Natranaerobius 
                 
               
               
                   
                   
                   
                 
                   thermophilus 
                 
               
               
                 dmdA 
                 ABC88408 
                 86278276 
                 
                   Eubacterium 
                 
               
               
                   
                   
                   
                 
                   barkeri 
                 
               
               
                 dmdB 
                 ABC88409 
                 86278277 
                 
                   Eubacterium 
                 
               
               
                   
                   
                   
                 
                   barkeri 
                 
               
               
                   
               
            
           
         
       
     
     Fumarate hydratase enzymes, which naturally catalyze the reversible hydration of fumarate to malate. Although the ability of fumarate hydratase to react on branched substrates with 3-oxobutanol as a substrate has not been described, a wealth of structural information is available for this enzyme and other researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver,  Acta Crystallogr. D Biol. Crystallogr.  61:1395-1401 (2005)).  E. coli  has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al.,  J. Bacteriol.  183:461-467 (2001); Woods et al.,  Biochem. Biophys. Acta  954:14-26 (1988); Guest et al.,  J Gen Microbiol  131:2971-2984 (1985)). Exemplary enzyme candidates include those encoded by fumC from  Escherichia coli  (Estevez et al.,  Protein Sci.  11:1552-1557 (2002); Hong and Lee,  Biotechnol. Bioprocess Eng.  9:252-255 (2004); Rose and Weaver,  Proc. Natl. Acad. Sci. USA  101:3393-3397 (2004)), and enzymes found in  Campylobacter jejuni  (Smith et al.,  Int. J. Biochem. Cell Biol.  31:961-975 (1999)),  Thermus thermophilus  (Mizobata et al.,  Arch. Biochem. Biophys.  355:49-55 (1998)), and  Rattus norvegicus  (Kobayashi et al.,  J. Biochem.  89:1923-1931 (1981)). Similar enzymes with high sequence homology include fum1 from  Arabidopsis thaliana  and fumC from  Corynebacterium glutamicum.  The MmcBC fumarase from  Pelotomaculum thermopropionicum  is another class of fumarase with two subunits (Shimoyama et al.,  FEMS Microbiol Lett.  270:207-213 (2007)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 fumA 
                 NP_416129.1 
                 16129570 
                 
                   Escherichia coli 
                 
               
               
                 fumB 
                 NP_418546.1 
                 16131948 
                 
                   Escherichia coli 
                 
               
               
                 fumC 
                 NP_416128.1 
                 16129569 
                 
                   Escherichia coli 
                 
               
               
                 fumC 
                 O69294 
                 9789756 
                 
                   Campylobacter jejuni 
                 
               
               
                 fumC 
                 P84127 
                 75427690 
                 
                   Thermus thermophilus 
                 
               
               
                 fumH 
                 P14408 
                 120605 
                 
                   Rattus norvegicus 
                 
               
               
                 fum1 
                 P93033 
                 39931311 
                 
                   Arabidopsis thaliana 
                 
               
               
                 fumC 
                 Q8NRN8 
                 39931596 
                 
                   Corynebacterium 
                 
               
               
                   
                   
                   
                 
                   glutamicum 
                 
               
               
                 MmcB 
                 YP_001211906 
                 147677691 
                 
                   Pelotomaculum 
                 
               
               
                   
                   
                   
                 
                   thermopropionicum 
                 
               
               
                 MmcC 
                 YP_001211907 
                 147677692 
                 
                   Pelotomaculum 
                 
               
               
                   
                   
                   
                 
                   thermopropionicum 
                 
               
               
                   
               
            
           
         
       
     
     Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate is catalyzed by 4-hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This enzyme participates in aromatic degradation pathways and is typically co-transcribed with a gene encoding an enzyme with 4-hydroxy-2-oxovalerate aldolase activity. Exemplary gene products are encoded by mhpD of  E. coli  (Ferrandez et al.,  J Bacteriol.  179:2573-2581 (1997); Pollard et al.,  Eur J Biochem.  251:98-106 (1998)), todG and cmtF of  Pseudomonas putida  (Lau et al.,  Gene  146:7-13 (1994); Eaton,  J Bacteriol.  178:1351-1362 (1996)), cnbE of  Comamonas  sp. CNB-1 (Ma et al.,  Appl Environ Microbiol  73:4477-4483 (2007)) and mhpD of  Burkholderia xenovorans  (Wang et al.,  FEBS J  272:966-974 (2005)). A closely related enzyme, 2-oxohepta-4-ene-1,7-dioate hydratase, participates in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (Burks et al.,  J. Am. Chem. Soc.  120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in  E. coli  C (Roper et al.,  Gene  156:47-51 (1995); Izumi et al.,  J Mol. Biol.  370:899-911 (2007)) and  E. coli  W (Prieto et al.,  J Bacteriol.  178:111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in  Klebsiella pneumonia  (91% identity, eval=2e-138) and  Salmonella enterica  (91% identity, eval=4e-138), among others. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 GenBank 
                   
                   
               
               
                 Gene 
                 Accession No. 
                 GI No. 
                 Organism 
               
               
                   
               
             
            
               
                 mhpD 
                 AAC73453.2 
                 87081722 
                 
                   Escherichia coli 
                 
               
               
                 cmtF 
                 AAB62293.1 
                 1263188 
                 
                   Pseudomonas putida 
                 
               
               
                 todG 
                 AAA61942.1 
                 485738 
                 
                   Pseudomonas putida 
                 
               
               
                 cnbE 
                 YP_001967714.1 
                 190572008 
                   Comamonas  sp. CNB-1 
               
               
                 mhpD 
                 Q13VU0 
                 123358582 
                 
                   Burkholderia xenovorans 
                 
               
               
                 hpcG 
                 CAA57202.1 
                 556840 
                   Escherichia coli  C 
               
               
                 hpaH 
                 CAA86044.1 
                 757830 
                   Escherichia coli  W 
               
               
                 hpaH 
                 ABR80130.1 
                 150958100 
                 
                   Klebsiella pneumoniae 
                 
               
               
                 Sari_01896 
                 ABX21779.1 
                 160865156 
                 
                   Salmonella enterica 
                 
               
               
                   
               
            
           
         
       
     
     Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in  Methanocaldococcus jannaschii  in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al.,  J Bacteriol.  189:4391-4400 (2007)). This enzyme activity was also detected in  Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus  where it is thought to participate in glutamate degradation (Kato et al.,  Arch. Microbiol  168:457-463 (1997)). The  M. jannaschii  protein sequence does not bear significant homology to genes in these organisms. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 leuD 
                 Q58673.1 
                 3122345 
                 
                   Methanocaldococcus jannaschii 
                 
               
               
                   
               
            
           
         
       
     
     Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe 2+ -dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in  Eubacterium barkeri  (Alhapel et al., supra; Kollmann-Koch et al.,  Hoppe Seylers. Z. Physiol Chem.  365:847-857 (1984)). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 dmdA 
                 ABC88408 
                 86278276 
                 
                   Eubacterium barkeri 
                 
               
               
                 dmdB 
                 ABC88409.1 
                 86278277 
                 
                   Eubacterium barkeri 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  2 , Step G—Methylmalonyl-CoA Reductase (Alcohol Forming) 
     Referring to  FIG.  2   , step G can involve a combined Alcohol/Aldehyde dehydrogenase (EC 1.2.1.-). Methylmalonyl-CoA can be reduced to 3-hydroxyisobutyrate in one step by a multifunctional enzyme with dual acyl-CoA reductase and alcohol dehydrogenase activity. Although the direct conversion of methylmalonyl-CoA to 3-hydroxyisobutyrate has not been reported, this reaction is similar to the common conversions such as acetyl-CoA to ethanol and butyryl-CoA to butanol, which are catalyzed by CoA-dependant enzymes with both alcohol and aldehyde dehydrogenase activities. Gene candidates include the  E. coli  adhE (Kessler et al.,  FEBS Lett.  281:59-63 (1991)) and  C. acetobutylicum  bdh I and bdh II (Walter, et al.,  J. Bacteriol.  174:7149-7158 (1992)), which can reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in  Leuconostoc mesenteroides  has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al.,  J. Gen. Appl. Microbiol.  18:43-55 (1972); Koo et al.,  Biotechnol. Lett.  27:505-510 (2005)). An additional candidate enzyme for converting methylmalonyl-CoA directly to 3-hydroxyisobutyrate is encoded by a malonyl-CoA reductase from  Chlorgflexus aurantiacus  (flugler, et al.,  J. Bacteriol.  184(9):2404-2410 (2002). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI Number 
                 Organism 
               
               
                   
               
             
            
               
                 mcr 
                 YP_001636209.1 
                 163848165 
                 
                   Chloroflexus aurantiacus 
                 
               
               
                 adhE 
                 NP_415757.1 
                 16129202 
                 
                   Escherichia coli 
                 
               
               
                 bdh I 
                 NP_349892.1 
                 15896543 
                 
                   Clostridium acetobutylicum 
                 
               
               
                 bdh II 
                 NP_349891.1 
                 15896542 
                 
                   Clostridium acetobutylicum 
                 
               
               
                 adhE 
                 AAV66076.1 
                 55818563 
                 
                   Leuconostoc mesenteroides 
                 
               
               
                   
               
            
           
         
       
     
     4.3 Example III 
     Methods of Using Formaldehyde Produced from the Oxidation of Methanol in the Formation of Intermediates of Central Metabolic Pathways for the Formation of Biomass 
     Provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (see, e.g.,  FIG.  1   , step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. Exemplary methanol metabolic pathways for enhancing the availability of reducing equivalents, as well as the producing formaldehyde from methanol (step J), are provided in  FIG.  1   . 
     One exemplary pathway that can utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  1   ) is shown in  FIG.  3   , which involves condensation of formaldehyde and D-ribulose-5-phosphate to form hexulose-6-phosphate (h6p) by hexulose-6-phosphate synthase ( FIG.  3   , step A). The enzyme can use Mg 2+  or Mn 2+  for maximal activity, although other metal ions are useful, and even non-metal-ion-dependent mechanisms are contemplated. H6p is converted into fructose-6-phosphate by 6-phospho-3-hexuloisomerase ( FIG.  3   , step B). 
     Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  1   ) is shown in  FIG.  4    and proceeds through dihydroxyacetone. Dihydroxyacetone synthase is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis ( FIG.  4   , step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase ( FIG.  4   , step B). DHAP can be assimilated into glycolysis and several other pathways. 
       FIG.  3   , Steps A and B—Hexulose-6-phosphate synthase (Step A) and 6-phospho hexuloisomerase (Step B) 
     Both of the hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase enzymes are found in several organisms, including methanotrops and methylotrophs where they have been purified (Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition, these enzymes have been reported in heterotrophs such as  Bacillus subtilis  also where they are reported to be involved in formaldehyde detoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al., 1999. J Bac 181(23):7154-60. Genes for these two enzymes from the methylotrophic bacterium  Mycobacterium gastri  MB19 have been fused and  E. coli  strains harboring the hps-phi construct showed more efficient utilization of formaldehyde (Orita et al., 2007, Appi Microbiol Biotechnol. 76:439-445). In some organisms, these two enzymes naturally exist as a fused version that is bifunctional. 
     Exemplary candidate genes for hexulose-6-phopshate synthase are: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Hps 
                 AAR39392.1 
                 40074227 
                   Bacillus methanolicus  MGA3 
               
               
                 Hps 
                 EIJ81375.1 
                 387589055 
                   Bacillus methanolicus  PB1 
               
               
                 RmpA 
                 BAA83096.1 
                 5706381 
                 
                   Methylomonas aminofaciens 
                 
               
               
                 RmpA 
                 BAA90546.1 
                 6899861 
                 
                   Mycobacterium gastri 
                 
               
               
                 YckG 
                 BAA08980.1 
                 1805418 
                 
                   Bacillus subtilis 
                 
               
               
                   
               
            
           
         
       
     
     Exemplary gene candidates for 6-phospho-3-hexuloisomerase are: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 Phi 
                 AAR39393.1 
                 40074228 
                   Bacillus methanolicus  MGA3 
               
               
                 Phi 
                 EIJ81376.1 
                 387589056 
                   Bacillus methanolicus  PB1 
               
               
                 Phi 
                 BAA83098.1 
                 5706383 
                 
                   Methylomonas aminofaciens 
                 
               
               
                 RmpB 
                 BAA90545.1 
                 6899860 
                 
                   Mycobacterium gastri 
                 
               
               
                   
               
            
           
         
       
     
     Candidates for enzymes where both of these functions have been fused into a single open reading frame include the following. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 PH1938 
                 NP_143767.1 
                 14591680 
                   Pyrococcus horikoshii  OT3 
               
               
                 PF0220 
                 NP_577949.1 
                 18976592 
                 
                   Pyrococcus furiosus 
                 
               
               
                 TK0475 
                 YP_182888.1 
                 57640410 
                 
                   Thermococcus kodakaraensis 
                 
               
               
                   
                 NP_127388.1 
                 14521911 
                 
                   Pyrococcus abyssi 
                 
               
               
                 MCA2738 
                 YP_115138.1 
                 53803128 
                 
                   Methylococcus capsulatas 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  4 , Step A—Dihydroxyacetone Synthase 
     Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  1   ) is shown in  FIG.  4    and proceeds through dihydroxyacetone. Dihydroxyacetone synthase is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis ( FIG.  4   , step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase ( FIG.  4   , step B). DHAP can be assimilated into glycolysis and several other pathways. 
     The dihydroxyacetone synthase enzyme in  Candida boidinii  uses thiamine pyrophosphate and Mg 2+  as cofactors and is localized in the peroxisome. The enzyme from the methanol-growing carboxydobacterium,  Mycobacter  sp. strain JC1 DSM 3803, was also found to have DHA synthase and kinase activities (Ro et al., 1997, JBac 179(19):6041-7). DHA synthase from this organism also has similar cofactor requirements as the enzyme from  C. boidinii.  The K m s for formaldehyde and xylulose 5-phosphate were reported to be 1.86 mM and 33.3 microM, respectively. Several other mycobacteria, excluding only  Mycobacterium tuberculosis,  can use methanol as the sole source of carbon and energy and are reported to use dihydroxyacetone synthase (Part et al., 2003, JBac 185(1):142-7. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 DAS1 
                 AAC83349.1 
                 3978466 
                 
                   Candida boidinii 
                 
               
               
                 HPODL_2613 
                 EFW95760.1 
                 320581540 
                 
                   Ogataea 
                 
               
               
                   
                   
                   
                   parapolymorpha  DL-1 
               
               
                   
                   
                   
                 ( Hansenula   
               
               
                   
                   
                   
                   polymorpha  DL-1) 
               
               
                   
                 AAG12171.2 
                 18497328 
                   Mycobacter  sp. 
               
               
                   
                   
                   
                 strain JC1 DSM 3803 
               
               
                   
               
            
           
         
       
     
     FIG.  4 , Step B—Dihydroxyacetone (DHA) Kinase 
     DHA obtained from DHA synthase is further phosphorylated to form DHA phosphate by a DHA kinase. DHAP can be assimilated into glycolysis and several other pathways. Dihydroxyacetone kinase has been purified from  Ogataea angusta  to homogeneity (Bystrkh, 1983, Biokhimiia, 48(10):1611-6). The enzyme, which phosphorylates dihydroxyacetone and, to a lesser degree, glyceraldehyde, is a homodimeric protein of 139 kDa. ATP is the preferred phosphate group donor for the enzyme. When ITP, GTP, CTP and UTP are used, the activity drops to about 30%. In several organisms such as  Klebsiella pneumoniae  and  Citrobacter freundii  (Daniel et al., 1995, JBac 177(15):4392-40), DHA is formed as a result of oxidation of glycerol and is converted into DHAP by the kinase DHA kinase of  K. pneumoniae  has been characterized (Jonathan et al, 1984, JBac 160(1):55-60). It is very specific for DHA, with a K m  of 4 μM, and has two apparent K m  values for ATP, one at 25 to 35 μM, and the other at 200 to 300 μM. DHA can also be phosphorylated by glycerol kinases but the DHA kinase from  K. puemoniae  is different from glycerol kinase in several respects. While both enzymes can phosphorylate dihydroxyacetone, DHA kinase does not phosphorylate glycerol, neither is it inhibited by fructose-1,6-diphosphate. In  Saccharomyces cerevisiae,  DHA kinases (I and II) are involved in rescuing the cells from toxic effects of dihydroxyacetone (Molin et al., 2003, J Biol Chem. 17; 278(3):1415-23). 
     In  Escherichia coli,  DHA kinase is composed of the three subunits DhaK, DhaL, and DhaM and it functions similarly to a phosphotransferase system (PTS) in that it utilizes phosphoenolpyruvate as a phosphoryl donor (Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not being involved in transport. The phosphorylation reaction requires the presence of the EI and HPr proteins of the PTS system. The DhaM subunit is phosphorylated at multiple sites. DhaK contains the substrate binding site (Garcia-Alles et al., 2004, 43(41):13037-45; Siebold et al., 2003, PNAS. 100(14):8188-92). The K M  for dihydroxyacetone for the  E. coli  enzyme has been reported to be 6 μM. The K subunit is similar to the N-terminal half of ATP-dependent dihydroxyacetone kinase of  Citrobacter freundii  and eukaryotes. 
     Exemplary DHA kinase gene candidates for this step are: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Protein 
                 GenBank ID 
                 GI number 
                 Organism 
               
               
                   
               
             
            
               
                 DAK1 
                 P54838.1 
                 1706391 
                 
                   Saccharomyces 
                 
               
               
                   
                   
                   
                   cerevisiae  S288c 
               
               
                 DAK2 
                 P43550.1 
                 1169289 
                 
                   Saccharomyces 
                 
               
               
                   
                   
                   
                   cerevisiae  S288c 
               
               
                 D186_20916 
                 ZP_16280678.1 
                 421847542 
                 
                   Citrobacter freundii 
                 
               
               
                 DAK2 
                 ZP_18488498.1 
                 425085405 
                 
                   Klebsiella pneumoniae 
                 
               
               
                 DAK 
                 AAC27705.1 
                 3171001 
                 
                   Ogataea angusta 
                 
               
               
                 DhaK 
                 NP_415718.6 
                 162135900 
                 
                   Escherichia coli 
                 
               
               
                 DhaL 
                 NP_415717.1 
                 16129162 
                 
                   Escherichia coli 
                 
               
               
                 DhaM 
                 NP_415716.4 
                 226524708 
                 
                   Escherichia coli 
                 
               
               
                   
               
            
           
         
       
     
     4.4 Example III 
     Methods for Handling Anaerobic Cultures 
     This example describes methods used in handling anaerobic cultures. 
     A. Anaerobic chamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne Calif.; MBraun, Newburyport Mass.). Conditions included an O 2  concentration of 1 ppm or less and 1 atm pure N 2 . In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an O 2  electrode (such as Teledyne; City of Industry Calif.). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume &gt;5 mL were sparged with pure N 2  prior to introduction into the chamber. Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to changes in oxygen levels. The chamber&#39;s pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve. 
     The anaerobic chambers achieved levels of O 2  that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions. In an alternative anaerobic chamber configuration, platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler. Instead of using instrument-based O 2  monitoring, test strips can be used instead. 
     B. Anaerobic microbiology. Serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with nitrogen for ˜30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, N.J.) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave. The sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle. Prior to addition of reducing agents, the bottles are equilibrated for 30-60 minutes with nitrogen (or CO depending upon use). A reducing agent such as a 100×150 mM sodium sulfide, 200 mM cysteine-HCl is added. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B12 (10 μM cyanocobalamin), nickel chloride (NiCl 2 , 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and ferrous ammonium sulfate (final concentration needed is 100 μM—made as 100-1000× stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pA1-lacO1 promoter in the vectors was performed by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs. 
     Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once all medium components and cells are added, the bottles are incubated in an incubator in room air but with continuous nitrogen sparging into the bottles. 
     Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples and embodiments provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.