Patent Publication Number: US-2023159902-A1

Title: Alcohol dehydrogenase variants

Description:
PRIORITY CLAIM 
     This application is a continuation of U.S. patent application Ser. No. 16/792,592, filed Feb. 17, 2020, which is a continuation of U.S. patent application Ser. No. 15/027,169, filed Apr. 4, 2016, now U.S. Pat. No. 10,563,180, which claims priority to International Patent Application No. PCT/US14/59135, filed Oct. 3, 2014, which claims the benefit of U.S. Provisional Patent Application No. 61/887,251, filed Oct. 4, 2013, entitled ALCOHOL DEHYDROGENASE VARIANTS, the disclosure of which is incorporated herein by reference. Also, the entire contents of the text file entitled “GN00003US3_Sequence_Listing.XML” created on Nov. 19, 2022, having a size of 222 kilobytes is incorporated herein by reference. 
    
    
     BACKGROUND 
     Alcohol dehydrogenases (ADHs; EC 1.1.1.1) promote the conversion of alcohols to and aldehydes or ketones, typically along with the reduction of nicotinamide adenine dinucleotide (NAD +  to NADH). ADHs are instrumental in the generation of important compounds having aldehyde, ketone, and alcohol groups during biosynthesis of various metabolites. 
     One class of alcohol dehydrogenase is methanol dehydrogenases (MDHs). MDHs, converts methanol (MeOH) to formaldehyde (Fald), may be used in an enzymatic pathway engineered into a microbe to enable MeOH as a sole carbon source or as a co-carbon source with other feed stocks such as, for example, glucose, dextrose, plant biomass or syngas, to produce valuable products. Microorganisms have been reported that metabolize methanol, and in some instances do so via a methanol dehydrogenase, and in even fewer instances produce valuable products. Increasing MDH activity will enable improved use of MeOH, improving MeOH as a sole carbon source, decreasing production costs, decreasing amounts of any more expensive secondary or co-carbon source, e.g. glucose, increasing product yields, and providing faster rate of MeOH use. 
     SUMMARY 
     Generally, presented herein are non-natural NAD + -dependent alcohol dehydrogenases (ADHs) (i.e. engineered enzymes and their encoding-polynucleotides) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its original or unmodified counterpart. Exemplary aspects describe non-natural NAD + -dependent methanol dehydrogenases (MDHs), in particular enzymes of the class EC 1.1.1.244. 
     The ADHs and MDHs have at least one amino acid substitution as compared to its corresponding natural or unmodified alcohol dehydrogenase. By unmodified alcohol dehydrogenase is meant that the ADH or MDH may have been previously engineered (e.g., need not be naturally-occurring), prior to incorporating any modification described herein. Such alcohol dehydrogenases that are starting sequences for incorporating a modification described herein to generate the novel engineered enzyme may be alternatively referred to herein as wild-type, template, starting sequence, natural, naturally-occurring, unmodified, corresponding natural alcohol dehydrogenase, corresponding natural alcohol dehydrogenase without the amino acid substitution, corresponding alcohol dehydrogenase or corresponding alcohol dehydrogenase without the amino acid substitution. Experimental studies described herein demonstrate for the first time that a number of amino acid positions along the length of the amino acid sequence can be substituted to provide a non-natural dehydrogenase having increased substrate conversion. The studies also show that combinations of substitutions (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve, etc.) in an amino acid sequence can also provide even further increased substrate conversion. Provided herein therefore are single and combination variants of a starting or template or corresponding alcohol dehydrogenase, e.g., in particular enzymes of the class EC 1.1.1.244, having increased substrate conversion. 
     Embodiments of the disclosure provide a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase, and capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, as measured relative to a corresponding alcohol dehydrogenase without amino acid substitution. Embodiments of the disclosure also provide a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared a second sequence that is an NAD + -dependent alcohol dehydrogenase, wherein the first and second sequences differ with regards to the at least one amino acid substitution. Embodiments are also directed to engineered cells expressing the non-natural NAD + -dependent alcohol dehydrogenase capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde as described. 
     Some embodiments of the current disclosure are directed to an engineered cell expressing a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution (including single and combination variants). The cells can be used to promote production of a target product or intermediate thereof. For example, the cell may provide either or both an increased amount of reducing equivalents, e.g. 
     NADH, for an increase in a target product or may provide for increased fixation of carbon from formaldehyde and/or acetaldehyde into a target product. Exemplary products include (a) 1,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), (b) butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol, (c) 1,3-butanediol and intermediates thereto, such as 2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol, (d) adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine and levulinic acid and their intermediates, e.g. 4-aminobutyryl-CoA, (e) methacrylic acid (2-methyl-2-propenoic acid) and its esters known collectively as methacrylates, such as methyl methacrylate, methyl methacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their intermediates, (f) 1,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol and glycerol, and their intermediates and (g) succinic acid and intermediates thereto. 
     Embodiments of the engineered cell may further optionally include one or more additional metabolic pathway transgene(s) to further promote production of the target product or intermediate thereof. In exemplary embodiments the cell further comprises one or more methanol metabolic pathway (MMP) transgene(s), such as a formaldehyde dehydrogenase transgene, allowing expression of the encoded pathway enzyme or accessory protein. 
     In exemplary embodiments the cell further comprises a product pathway comprising enzymes (and their encoding polynucleotides) for production of a target product, such as the enzymes described herein for production of 1,4-butanediol from glucose. 
     Other embodiments are directed to compositions including engineered cell, such as cell culture compositions, and also compositions including one or more product(s) produced from the engineered cell. For example, a composition can include a target product or intermediate thereof produced by the cells, where the composition has been purified to remove cells or other components useful for cell culturing. The composition may be treated to enrich or purify the target product or intermediate thereof. 
     Other embodiments of the disclosure are directed to products made from the target product obtained from methods using the engineered cell. Exemplary products include polymers made with target products, such as polymers made from diol target products combined with diacids, including target product succinic acid, such as polybutylene terephthalate (PBT) and polybutylene succinate (PBS) made from 1,4-butanediol polymerized with terephthalic acid or succinic acid respectively. 
     Other embodiments of the disclosure are directed to nucleic acids encoding the non-natural alcohol dehydrogenases with one or more variant amino acids, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs. 
     In other embodiments the disclosure also provides methods for generating non-natural NAD + -dependent alcohol dehydrogenases capable of at least two-fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified (original; template) counterpart. In some embodiments, the method includes steps of (a) identifying a variant amino acid that provides increased conversion in a template sequence, (b) identifying corresponding amino acid position in a target sequence having identify to the template sequence, and (c) changing the amino acid at the corresponding amino acid position in a target sequence to the variant amino acid. The starting template for incorporation of modifications described herein can be a naturally-occurring enzyme sequence or a previously engineered enzyme sequence. 
     In other embodiments, the methods includes steps of (a) identifying an amino acid position in a non-natural NAD + -dependent alcohol dehydrogenases that is not a variant position, (b) providing, in a original template, a variation at an amino acid position that is a non-variant position, and (c) identifying variants from step (b) that provide increased conversion of the substrate. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIGS.  1 A-D  are graphs listing amino acid positions of methanol dehydrogenase (2315A) and the effect of substitution of those positions on enzyme activity. 
         FIG.  2    illustrates a pathway using MDH to produce 1, 4-butanediol (BDO) in organism such as  E. coli.    
         FIGS.  3 A-D  illustrate pathways using certain methanol metabolizing enzymes.  FIG.  3 D  illustrates using enzymes to fix carbon from methanol via formaldehyde assimilation into a product pathway of interest. The “Product Pathway” can be that of 1,4-butanediol as described herein or other product pathway. 
         FIG.  4    is an amino acid sequence alignment of various Fe-dependent alcohol dehydrogenases with  Bacillus  MeDH. 
     
    
    
     DETAILED DESCRIPTION 
     The embodiments of the description described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the description. 
     All publications and patents mentioned herein are hereby incorporated by reference. The publications and patents disclosed herein are provided solely for their disclosure. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any publication and/or patent, including any publication and/or patent cited herein. 
     Generally, the disclosure provides non-natural NAD + -dependent alcohol dehydrogenases (ADHs) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified counterpart. Nucleic acids encoding the non-natural alcohol dehydrogenases, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs are described. 
     Also described are engineered cells expressing a non-natural NAD + -dependent alcohol dehydrogenase, optionally including one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, and/or target product pathway genes; cell culture compositions including the cells; methods for promoting production of the target product or intermediate thereof from the cells; compositions including the target product or intermediate; and products made from the target product or intermediate. 
     The term “non-naturally occurring”, when used in reference to an organism (e.g., microbial) is intended to mean that the organism has at least one genetic alteration not normally found in a naturally occurring organism of the referenced species. Naturally-occurring organisms can be referred to as “wild-type” such as wild type strains of the referenced species. Likewise, a “non-natural” polypeptide or nucleic acid can include at least one genetic alteration not normally found in a naturally-occurring polypeptide or nucleic acid. Naturally-occurring organisms, nucleic acids, and polypeptides can be referred to as “wild-type” or “original” such as wild type strains of the referenced species. Likewise, amino acids found in the wild type organism can be referred to as “original” with regards to any amino acid position. 
     A genetic alteration that makes an organism non-natural can 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 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. 
     An NAD(P)+-dependent methanol dehydrogenase from  Bacillus methanolicus  MGA3 (Genbank Accession number EIJ77596.1, GI number: 387585261; designated herein as MDH 2315, 382 amino acids long; SEQ ID NO: 1), was selected as a template for mutagenesis to identify variants with improved activity. This sequence was selected as it was surprisingly found to be very active on methanol, typically nearly twice as active as other alcohol dehydrogenases tested, and used NAD+ and thus able to regenerate NADH that can be useful to enzymes in target pathways. MDH 2315 is reported in the literature as an NAD(P)-dependent methanol dehydrogenase from  Bacillus methanolicus  MGA3 and its sequence was described in Brautaset et al., “Plasmid-Dependent Methylotrophy in Thermotolerant  Bacillus methanolicus ”, Journal of Bacteriology, vol. 186, pp1229-1238 (2004). It is also referred to as MDH MGA3 in WO2013/110797 to Brautaset and MDH “M” in Krog et al., “Methylotrophic  Bacillus methanolicus  Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Mathanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties”, PLOS ONE, pp. 1-11, (2013), which report additional wild-type  Bacillus  MDHs. 
     MDH 2315A was expressed in  E. coli  and a library of variants was generated by error prone PCR (the epPCR Library). Specifically, MDH 2315A was expressed in  E. coli  and subjected to saturation mutagenesis at 375 of 382 positions to generate a library of all single point substitutions of the common 20 amino acids (the NNK library). 
     In the primary screen, the NNK Library was screened by assaying individual colony cell extracts for MeOH to Fald conversion. The library was screened for variants that had greater than 2-fold activity over wild-type (defined as a positive). Such a library can contain variants with multiple mutations, deletions, etc. However, variants with single point substitutions were identified. In that primary screen about 150 positions gave colonies whose extracts had activity reliably 2-fold greater than wild-type. Of those, 21 positions gave colonies with activity reliably greater than 4-fold wild-type. 
     The primary screen of the NNK library identified about 10 positions where 90% or more of the colonies were inactive, suggesting that any amino acid other than wild-type at those positions led to enzyme inactivity (or rapid degradation). About 30 positions were identified in which less than 5% of the colonies were inactive. Many positions were identified in which no positives were observed but the total number of inactives was less than 90%. Many positions were identified in which the total number of positives and inactives were less than 95%. 
     Secondary in vitro assays in triplicate and sequencing were done on all positives greater than 4-fold, and many greater than 2-fold activity. At 40 positions, a total of 90 substitutions were determined to be responsible for the activity increase either greater than 2-fold or in many cases greater than 4-fold, and up to 10-fold. In many positions, only a single specific amino acid gave improvement, in other positions several amino acid substitutions gave improvement (e.g., at 213 only N213D, but at 121 both G121D, G121V, etc). 
     Secondary screening of the epPCR library revealed about 20 positions whose substitutions gave activity greater than 3-fold wild-type. Sequencing of these colonies revealed the specific substitutions at each position, for example, N213D (nomenclature: N is original or unmodified amino acid at position 213; D is the substitution). At these 20 positions, a total of 30 substitutions provided greater than 3-fold wild-type activity of which about 15 variants provided greater than 4-fold activity. 
     Some of the colonies whose MDH variant sequence were identified were also assayed in vivo by measuring MeOH conversion to formaldehyde in a strain background in which genes for use of formaldehyde were inactivated, thus enabling accumulation of formaldehyde. Generally, all MDH variants that were positive (greater than wild-type) in in vitro screening also enabled greater than wild-type activity in vivo, although the in vitro to in vivo activities did not always correlate exactly. 
     At some positions the frequency of a particular substitution in the total population of colonies at that position in a library was zero or less than statistically expected. Thus some substitutions at some positions may not have been present or present and not detected. 
     Table 1 lists amino acids mutations with respect to SEQ ID NO: 1, providing greater than two fold activity when present as single mutations. While not to be bound by theory, as depicted in  FIG.  1   , functional features associated with amino acid positions of methanol dehydrogenase designated 2315A and corresponding positions in other methanol dehydrogenase described herein include: NADH cofactor binding correlating with positions 38D, 39A, 40F, 70D, 97G, 98S, 137T, 138T, 141T, 142G, 143S, 145T, 146T, 147S, 148L, 149A, 150V, 161P, 162V, 163I; activation site correlating with positions 95G, 97G, 98S; and substrate correlating with positions 145T, 146T, 147S, 148L, 149A, 150V, 161P, 162V, 163I, 253F, 258L, 266H, 359D, 360V, 361C. 
     As can be seen from  FIG.  1    depicting site saturation mutagenesis, numerous amino acid positons of methanol dehydrogenase designated 2315A are tolerant to substitution (indicated by a percentage of colonies having greater than 2-fold (designated “hits”) or from 0.2 to less than 2-fold activity of wild-type enzyme) and others less tolerant (indicated by percentage of colonies where enzyme activity was less than 20% of wild-type enzyme activity (designated “dead”). For example, 168T and 270G are very tolerant to change, where substitutions at 168T generally had little effect on activity, whereas substitutions at 270G predominantly improved activity. 
     Art known methods can be used for the testing the enzymatic activity of alcohol dehydrogenases, and such methods can be used to test activity of alcohol dehydrogenase (ADH) variant enzymes as well. As a general matter, a reaction composition including the alcohol dehydrogenase (ADH) variant, an alcohol (substrate) and NAD (cofactor) can be converted to a dehydrogenated product. For example, conversion of ethanol is shown as follows: 
       Ethanol+β-NAD   Alcohol Dehydrogenase   &gt;Acetaldehyde+β-NADH
 
     Reaction can be carried out at a desired temperature, such as 25° C., and pH, such as pH 7. 
     The ADH variant can be defined in terms of its enzymatic activity with one unit of enzyme converting 1.0 μmole of alcohol to dehydrogenated product per minute at pH 8.8@25° C. See, for example, Kagi, J. H. R. and Vallee, B. L. (1960)  Journal of Biological Chemistry  235, 3188-3192 
     Of particular interest herein is conversion of methanol to formaldehyde to regenerate NADH. This conversion can be followed by either or both conversion to formate or fixation of the formaldehyde carbon into target product. The formate can be either or both converted to CO 2  or have its carbon fixed into target product, such as by conversion back to formaldehyde. See the attached figures. 
     A representative in vivo assay was developed to determine the activity of methanol dehydrogenase variants in organisms is reported in U.S. application Ser. No. 13/975,678. This assay relies on the detection of formaldehyde (Palp), thus measuring the forward activity of the enzyme (oxidation of methanol). To this end, a strain comprising a BDOP and lacking frmA, frmB, frmR was created using Lambda Red recombinase technology (Datsenko and Wanner,  Proc. Natl. Acad. Sci. USA,  6 97(12): 6640-5 (2000). Plasmids expressing methanol dehydrogenases were transformed into the strain, then grown to saturation in LB medium+antibiotic at 37° C. with shaking. Transformation of the strain with an empty vector served as a negative control. Cultures were adjusted by O.D. and then diluted 1:10 into M9 medium+0.5% glucose+antibiotic and cultured at 37° C. with shaking for 6-8 hours until late log phase. Methanol was added to 2% v/v and the cultures were further incubated for 30 min. with shaking at 37° C. Cultures were spun down and the supernatant was assayed for formaldehyde produced using DETECTX Formaldehyde Detection kit (Arbor Assays; Ann Arbor, Mich.) according to manufacturer&#39;s instructions. The frmA, frmB, frmR deletions resulted in the native formaldehyde utilization pathway to be deleted, which enables the formation of formaldehyde that can be used to detect methanol dehydrogenase activity in the organism. These genes are deleted in this case solely to facilitate measurement of methanol conversion by preventing loss of the measured analyte, formaldehyde. 
     Enzymatic kinetic assays were done for 10 single point variants, and that 2-10 fold improvements in activity were reflected in 2-10 fold improvements in Km, Vmax or both. Co-factor binding nor substrate or product on-off rates were not measured. 
     Table 7 shows enzymology data from various wild type ADH proteins. Tables 8 and 9 show data for wild type and variant enzymes, with Table 8 showing activity using either methanol or 1,4-butanediol, and Table 9 showing 1,4-butanediol-dependent steady-state kinetic parameters for wild-type and variant methanol dehydrogenase. 
     Results of the mutagenesis procedures and rationale design and screening of the positives (by “positive” is meant a sequence modified as described herein having at least a two (2) fold increase in activity compared to the unmodified template sequence) revealed a number of amino acid variants along the MDH protein 2315A template for use in the invention. Positives showing greater than two fold increase in activity are shown in Table 1, and listed as follows: S11T, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M. These changes, their positions in SEQ ID NO: 1, and their corresponding positions in other template sequences are described further in the tables and elsewhere herein. 
     Of more interest are positives showing greater than two fold increase in activity as single mutations shown in Table 1, and listed as follows: D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S, C361R. These changes, their positions in SEQ ID NO: 1, and their corresponding positions in other template sequences are described further in the tables and elsewhere herein. 
     Results of the rationale design mutagenesis procedures and the other library generation procedures described herein and screening of the positive revealed a number of combination amino acid variants along the MDH protein 2315A template. Positives showing greater than two fold increase in activity are shown in Tables 2-4, and listed as variations in the following sets: (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (j) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G; (1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) N112K, S147R, P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K, S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao) V150I, P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat) V150I, P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I, V360G; (aaw) S11T, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) N117Y, T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R, E126G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161S, and (aaaap) N112R/P161S. 
     Also identified were positions in SEQ ID NO:1 that are generally intolerant to substitution, including P8, V132, E177, A207, T208, Q246, 1264, P285, E308, Y339, A340, A353, T362, R367, P369, D373, and 1377; where original amino acids are of particular interest at these positions in SEQ ID NO:1 and their corresponding positions in other templates as described herein. 
     Also identified were positions in SEQ ID NO:1 that are generally very tolerant to substitution, including E56, E78, E86, T2, N4, E237, E240, E327, E341, E347, P9, V18, R24, T44, L46, K52, D60, A62, F64, A67, P71, A72, D73, T74, V80, K84, Q85, I106, V109, R115, I116, N117, V122, K127, 1135, T136, S154, A155, R156, P161, V162, 1163, P169, T170, V171, V174, L178, M179, A184, G185, L186, A189, T190, A194, A198, Y202, V203, T211, F214, 1216, Q217, K220, L221, N223, Y225, A229, Y244, A245, M248, A252, N254, H266, G270, Y272, 1278, M284, H286, V287, N291, 1293, A294, R298, H301, I302, L305, N309, A311, G312, T315, A317, R321, V324, I329, S332, S337, M342, K345, K354, N355, A356, Y357, T370, A375, and I378 in SEQ ID NO:1 and their corresponding positions in other templates; where it was observed that typically all amino acids, except for occasionally 1 or 2, were neutral, beneficial, or less than an 80% decrease to activity at these positions. Of these, D60, I378, L46, A62, A67, N117, K52, I135, Y202, 1216, M248, N291, M342, T2, E240, T44, I116, V203, S332, and thus their corresponding positions in other templates, were tolerant to all changes. 
     Positions for additional interest for substitution to increase activity include 23T, 81D, 141T, 174V, 189A, 332S, 372Q and 379K and their corresponding positions in other templates. 
     Embodiments of the disclosure are also directed to the preparation and use of other alcohol dehydrogenase (ADH) variant enzymes including methanol dehydrogenases variant enzymes, based on the single and combination amino acid variants identified in and with respect to the MDH protein 2315A template. In these embodiments generation of non-natural NAD + -dependent alcohol dehydrogenases (that are not 2315A) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its original unmodified counterpart can be based on information of those MDH 2315A variants that provided increased activity. 
     In some embodiments, the method for preparing other ADH variants includes steps of (a) identifying a variant amino acid that provides increased conversion when present in the 2315A template sequence, (b) identifying a corresponding amino acid position in a target sequence (i.e., other ADH or MDH sequence) having identity to the template sequence, and (c) changing the amino acid at the corresponding amino acid position in the target sequence to the variant amino acid. For example, the D at position 38 of 2315A when substituted with N provides greater than two fold increase in activity compared to the unmodified 2315A sequence. A position corresponding to D38 is identified in the new target ADH or MDH template sequence, e.g., it may be D38 or D39 appropriate, and replaced with N to generate the new non-naturally-occurring ADH or MDH. 
     In some cases an “ortholog” of the NAD(P)-dependent methanol dehydrogenase from  Bacillus methanolicus  MGA3 (2315A), SEQ ID NO: 1, is first identified. 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. 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 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. 
     Genes sharing a desired amount of identify (e.g., 45%, 50%, 55%, or 60% or greater) to the NAD(P)-dependent methanol dehydrogenase from  Bacillus methanolicus  MGA3 (2315A), including orthologs and paralogs, 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. 
     Computational approaches to sequence alignment and generation of sequence identity include global alignments and local alignments. Global alignment uses global optimization to forces alignment to span the entire length of all query sequences. Local alignments, by contrast, identify regions of similarity within long sequences that are often widely divergent overall. For understanding the identity of a target sequence to the  Bacillus methanolicus  MGA3 (2315A) template a global alignment can be used. Optionally, amino terminal and/or carboxy-terminal sequences of the target sequence that share little or no identify with the template sequence can be excluded for a global alignment and generation of an identify score. 
     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, 45% 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%). 
     Pairwise global sequence alignment was carried out for each of the template polypeptides with SEQ ID No. 1 (2315A) as the reference. The alignment was performed using the Needleman-Wunsch algorithm (Needleman, S. &amp; Wunsch, C. A general method applicable to the search for similarities in the amino acid sequence of two proteins J. Mol. Biol, 1970, 48, 443-453) implemented through the BALIGN tool (balign.sourceforge.net). Default parameters were used for the alignment and BLOSUM62 was used as the scoring matrix. 
     Table 10 provides target polypeptides details and alignment to SEQ ID NO. 1 (2315A). These sequences represent target sequences in which one or more amino acid variations, based on the variant amino acids in the  Bacillus methanolicus  MGA3 (2315A) variants showing increased conversion, can be made. For example, as a general matter, this process can involve steps of aligning the template 2315A sequence to a target sequence, such as any sequence listed in Table 10. Next a position of the amino acid substitution/variant (or set of substitutions) in the template 2315A sequence providing the increased conversion of methanol or ethanol is identified. The amino acid alignment at the substitution/variant position is inspected to identify what amino acid position in the target sequence corresponds to that of the template 2315A sequence. Preferred target sequences for substitution with the amino acid variants based on the 2315A variants are highlighted. 
     In some cases the original amino acid and its position on the template 2315A sequence will precisely correlate with the original amino acid and position on the target. In other cases the original amino acid and its position on the template 2315A sequence will correlate with the original amino acid, but its position on the target will not be in the corresponding template position. However, the corresponding amino acid on the target can be a predetermined distance from the position on the template, such as within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions from the template position. In other cases the original amino acid on the template 2315A sequence will not precisely correlate with the original amino acid on the target. However one can understand what the corresponding amino acid on the target sequence is based on the general location of the amino acid on the template and the sequence of amino acids in the vicinity of the target amino acid. For example, amino acids in the vicinity of the target amino acids may be viewed as a “sequence motif” having a certain amount of identity or similarity to between the template and target sequences. 
     In some cases, it can be useful to use the Basic Local Alignment Search Tool (BLAST) algorithm to understand the sequence identity between an amino acid motif in a template sequence and a target sequence. Therefore, in preferred modes of practice, BLAST is used to identify or understand the identity of a shorter stretch of amino acids (e.g. a sequence motif) between a template and a target protein. BLAST finds similar sequences using a heuristic method that approximates the Smith-Waterman algorithm by locating short matches between the two sequences. The (BLAST) algorithm can identify library sequences that resemble the query sequence above a certain threshold. 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. 
     In view of and following the teachings herein, using methods known in the art such as sequence alignment and 3D modeling, the “corresponding positions and amino acids” for substitution in template polypeptides other than SEQ ID NO: 1 are readily determined. Table 11 indicates for each template the corresponding positions for substitution to improve polypeptide activity, from which each original amino acid, its location and substitution is specifically contemplated as if expressly listed. For example, using the 385 amino acid template polypeptide of EIJ83020.1, GI:387590701 (SEQ ID NO:7), from  Bacillus methanolicus  MGA3, having 61% global identity and 79% similarity to SEQ ID NO:1, the formula R 1 XR 2  is directly and unambiguously derived, is evident, and is contemplated, as if expressly listed herein and is from which the group of positions for R 1 XR 2  are readily envisioned as: D41, E63, P74, N91, S102, G106, A110, L111, V112, K118, 1119, H120, G124, V125, D126, V127, S128, K129, E130, P131, M132, V134, S146, T149, T152, 1153, K160, V161, V166, D167, Q270, G273, K348, N358, A363, C364. This is readily derived as evident in the following that depicts the amino acid position for each amino acid for substitution (corresponding to those of SEQ ID NO: 1 and accepting the corresponding substitution). The above approach applies to obtain a resulting R 1 XR 2  formula for each template polypeptide herein, as well as for polypeptides sharing identity thereto as described herein. 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 GI: 
                 MTNTQSAFFMPSVNLFGAGSVNEVGTRLAD 
               
               
                 387590701 
                 LGVKKALLVT   D41   AGLHGLGLSEKISSIIR 
               
               
                   
                 AAGV   E63   VSIFPKAEPN   P74   TDKNVAEGLE 
               
               
                   
                 AYNAE   N91   CDSIVTLGGGS   S102   HDA   G106     
               
               
                   
                 KAI   A110L111V112   AANGG   K118I119H1     
               
               
                   
                     20   DYEG 124 V 125 D 126 V 127 S 128 K 129 E 
               
               
                   
                   130 P 131 M 132 V 134 PLIAINTTAGTGS 14   
               
               
                   
                   6 ELT 149 KFT 152 I 153 ITDTER   K160V16     
               
               
                   
                     1   KMAI   V166D167   KHVTPTLSINDPELMVG 
               
               
                   
                 MPPSLTAATGLDALTHAIEAYVSTGATPIT 
               
               
                   
                 DALAIQAIKIISKYLPRAVANGKDIEAREQ 
               
               
                   
                 MAFAQSLAGMAFNNAGLGYVHAIAH   Q270   L 
               
               
                   
                 G   G273   FYNFPHGVCNAVLLPYVCRFNLISK 
               
               
                   
                 VERYAEIAAFLGENVDGLSTYDAAEKAIKA 
               
               
                   
                 IERMAKDLNIPKGFKELGAK 348 EEDIETL 
               
               
                   
                 AK   N358   AMKD   A363C364   ALTNPRKPKLEE 
               
               
                   
                 VIQIIKNAM 
               
               
                   
               
            
           
         
       
     
     In another example, the amino acids and positions for substitution R 1 XR 2  in 387 amino acid template polypeptide YP_002138168.1 GI:197117741 (SEQ ID NO:17) from  Geobacter bemidjiensis  Bem with 52% global identity and 71% similarity to SEQ ID NO:1 are: D43, S65, P76, G92, S104, A108, G112, M113, V114, H120, 1121, R122, G126, V127, N128, K129, T130, T131, K132, P133, M134, P135, S148, T151, C154, I155, H162, V163, V168, D169, Q272, G275, K350, N360, A365, C366. The following, readily derivable from the tables herein, indicates these positions. 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 GI: 
                 MALGEQTYGFYIPTVSLMGIGSAKETGGQI 
               
               
                 197117741 
                 KALGASKALIVT   D43   KGLSAMGVADKIKSQ 
               
               
                   
                 VEEAGV   S65   AVIFDGAEPN P76 TDINVHDG 
               
               
                   
                 VKVYQDN   G92   CDAIISLGGGS   S104   HDC   A1     
               
               
                   
                     08   KGI   G112M113V114   IGNGG   H120I121     
               
               
                   
                     R122   DLE   G126V127N128K129T130T13     
               
               
                   
                     1K132P133M134P135   AFVAINTTAGTA   S     
               
               
                   
                     148   EM   T151   RF   C154I155   ITNTDT   H162V     
               
               
                   
                     163   KMAI   V168D169   WRCTPNVAINDPLLM 
               
               
                   
                 VGKPAALTAATGMD ALTHA VEAYVSTIA 
               
               
                   
                 TPITDACAIKAIELIAEFLSKAVANGEDLE 
               
               
                   
                 ARDKMAYAEYLAGMAFNNASLGYVHSMAH   Q     
               
               
                   
                     272   LG   G275   FYNLPHGVCNAILLPAVSQYN 
               
               
                   
                 LIACPKRFADIAKALG   E   NIDGLSVTEAGQK 
               
               
                   
                 AIDRIRTLSASIGIPTGLKALNV   K350   EAD 
               
               
                   
                 LTIMAE   N360   AKKD   A365C366   QFTNPRKA 
               
               
                   
                 TLEQVVQIFKDAM 
               
               
                   
               
            
           
         
       
     
     Table 11 provides amino acid sequences of target polypeptides, having underlined target amino acids for substitution with the variant amino acids generated in the  Bacillus methanolicus  MGA3 (2315A) variants. It is understood that upon replacement of amino acid in the target sequence (with a variant amino acid from the corresponding location in the 2315A variant), the substituted target sequence can be considered a “template sequence,” useful in some embodiments for the further screening of polypeptides sequences for substitution. 
     Table 11 also illustrates a consensus of the templates of 60% or better identity to SEQ ID NO: 1 with positions for substitution indicated by underlining. Non-underlined positions are not required for substitution and, in embodiments, remain constant (identical across all templates). These positions can be tolerant to change by selection from at least amongst the wild-type alternatives indicated at a specific position, and tolerant sites for substitution with the substitutions at the variant amino acid positions. 
     Site-directed mutagenesis or sequence alteration (e.g., site-specific mutagenesis or oligonucleotide-directed) can be used to make specific changes to a target alcohol dehydrogenase DNA sequence to provide a variant DNA sequence encoding alcohol dehydrogenase with the desired amino acid substitution. As a general matter, an oligonucleotide having a sequence that provides a codon encoding the variant amino acid is used. Alternatively, artificial gene sequence of the entire coding region of the variant alcohol dehydrogenase DNA sequence can be performed as preferred alcohol dehydrogenases targeted for substitution are generally less than 400 amino acids long. 
     Exemplary techniques using mutagenic oligonucleotides for generation of a variant ADH sequence include the Kunkel method which may utilize an ADH gene sequence placed into a phagemid. The phagemid in  E. coli  produces ADH ssDNA which is template for mutagenesis using an oligonucleotide which is primer extended on the template. 
     Depending on the restriction enzyme sites flanking a location of interest in the ADH DNA, cassette mutagenesis may be used to create a variant sequence of interest. For cassette mutagenesis, a DNA fragment is synthesized inserted into a plasmid, cleaved with a restriction enzyme, and then subsequently ligated to a pair of complementary oligonucleotides containing the ADH variant mutation The restriction fragments of the plasmid and oligonucleotide can be ligated to one another. 
     Another technique that can be used to generate the variant ADH sequence is PCR site directed mutagensis. Mutageneic oligonucleotide primers are used to introduce the desired mutation and to provide a PCR fragment carrying the mutated sequence. Additional oligonucleotides may be used to extend the ends of the mutated fragment to provide restriction sites suitable for restriction enzyme digestion and insertion into the gene. 
     Commercial kits for site-directed mutagenesis techniques are also available. For example, the Quikchange™ kit uses complementary mutagenic primers to PCR amplify a gene region using a high-fidelity non-strand-displacing DNA polymerase such as pfu polymerase. The reaction generates a nicked, circular DNA which is relaxed. The template DNA is eliminated by enzymatic digestion with a restriction enzyme such as DpnI which is specific for methylated DNA. 
     An expression vector or vectors can be constructed to include one or more variant ADH 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. 
     The term “exogenous” 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 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, 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 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. 
     Exogenous variant ADH-encoding nucleic acid sequences 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. Optionally, 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. 
     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. 
     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. 
     In some aspects the ADH variant gene is introduced into a cell with a gene disruption. 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. 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. 
     A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, microorganisms may have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein. 
     The microorganisms provided herein 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. 
     A variety of microorganism may be suitable for the incorporating the variant ADH, optionally with one or more other transgenes Such organisms 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 are reported in U.S. application Ser. No. 13/975,678 (filed Aug. 26, 2013), which is incorporated herein by reference, and 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. 
     In certain embodiments, suitable organisms include  Acinetobacter baumannii  Naval-82,  Acinetobacter  sp. ADP 1,  Acinetobacter  sp. strain M-1,  Actinobacillus succinogenes  130Z,  Allochromatium vinosum  DSM 180 , Amycolatopsis methanolica, Arabidopsis thaliana, Atopobium parvulum  DSM 20469,  Azotobacter vinelandii  DJ,  Bacillus alcalophilus  ATCC 27647,  Bacillus azotoformans  LMG 9581,  Bacillus coagulans  36D1,  Bacillus megaterium, Bacillus methanolicus  MGA3,  Bacillus methanolicus  PB1,  Bacillus methanolicus  PB-1,  Bacillus selenitireducens  MLS10,  Bacillus smithii, Bacillus subtilis, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis  E264, Burkholderiales bacterium Joshi 001, Butyrate-producing bacterium L2-50,  Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Carboxydothermus hydrogenoformans, Carboxydothermus hydrogenoformans  Z-2901,  Caulobacter  sp. AP07 , Chloroflexus aggregans  DSM 9485 , Chloroflexus aurantiacus  J-10-fl,  Citrobacter freundii, Citrobacter koseri  ATCC BAA-895,  Citrobacter youngae, Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicum  ATCC 824,  Clostridium acidurici, Clostridium aminobutyricum, Clostridium asparagiforme  DSM 15981,  Clostridium beijerinckii, Clostridium beijerinckii  NCIMB 8052,  Clostridium bolteae  ATCC BAA-613,  Clostridium carboxidivorans  P7,  Clostridium cellulovorans  743B,  Clostridium difficile, Clostridium hiranonis  DSM 13275,  Clostridium hylemonae  DSM 15053,  Clostridium kluyveri, Clostridium kluyveri  DSM 555,  Clostridium ljungdahli, Clostridium ljungdahlii  DSM 13528,  Clostridium methylpentosum  DSM 5476,  Clostridium pasteurianum, Clostridium pasteurianum  DSM 525,  Clostridium perfringens, Clostridium perfringens  ATCC 13124,  Clostridium perfringens  str. 13,  Clostridium phytofermentans  ISDg,  Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum  NI-4,  Clostridium tetani, Corynebacterium glutamicum  ATCC 14067,  Corynebacterium glutamicum  R,  Corynebacterium  sp. U-96,  Corynebacterium variabile, Cupriavidus  necator N-1, Cyanobium PCC7001 , Desulfatibacillum alkenivorans  AK-01 , Desulfitobacterium hafniense, Desulfitobacterium metallireducens  DSM 15288,  Desulfotomaculum reducens  MI-1,  Desulfovibrio africanus  str. Walvis Bay,  Desulfovibrio fructosovorans  JJ,  Desulfovibrio vulgaris  str. Hildenborough,  Desulfovibrio vulgaris  str. Miyazaki F,  Dictyostelium discoideum  AX4,  Escherichia coli, Escherichia coli  K-12,  Escherichia coli  K-12 MG1655,  Eubacterium hallii  DSM 3353,  Flavobacterium frigoris, Fusobacterium nucleatum  subsp. polymorphum ATCC 10953,  Geobacillus  sp. Y4. IMC1,  Geobacillus themodenitrificans  NG80-2 , Geobacter bemidjiensis  Bem,  Geobacter sulfurreducens, Geobacter sulfurreducens  PCA,  Geobacillus stearothermophilus  DSM 2334,  Haemophilus influenzae, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacter thermophilus  TK-6,  Hyphomicrobium denitrificans  ATCC 51888,  Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniae  subsp.  pneumoniae  MGH 78578,  Lactobacillus brevis  ATCC 367,  Leuconostoc mesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Mesorhizobium loti  MAFF303099 , Metallosphaera sedula, Methanosarcina acetivorans, Methanosarcina acetivorans  C2A,  Methanosarcina barkeri, Methanosarcina mazei  Tuc01,  Methylobacter marinus, Methylobacterium extorquens, Methylobacterium extorquens  AM1,  Methylococcus capsulatas, Methylomonas aminofaciens, Moorella thermoacetica, Mycobacter  sp. strain 1 DSM 3803,  Mycobacterium avium  subsp. paratuberculosis K-10,  Mycobacterium bovis  BCG,  Mycobacterium gastri, Mycobacterium marinum  M,  Mycobacterium smegmatis, Mycobacterium smegmatis  MC2 155,  Mycobacterium tuberculosis, Nitrosopumilus salaria  BD31 , Nitrososphaera gargensis  Ga9.2,  Nocardia farcinica  IFM 10152,  Nocardia iowensis  (sp. NRRL 5646),  Nostoc  sp. PCC 7120 , Ogataea angusta, Ogataea parapolymorpha  DL-1 ( Hansenula polymorpha  DL-1),  Paenibacillus peoriae  KCTC 3763,  Paracoccus denitrificans, Penicillium chrysogenum, Photobacterium profundum  3TCK,  Phytofermentans  ISDg,  Pichia pastoris, Picrophilus torridus  DSM9790,  Porphyromonas gingivalis, Porphyromonas gingivalis  W83,  Pseudomonas aeruginosa  PA01,  Pseudomonas denitrificans, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas  sp,  Pseudomonas syringae  pv.  syringae  B728a,  Pyrobaculum islandicum  DSM 4184,  Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii  OT3 , Ralstonia eutropha, Ralstonia eutropha  H16,  Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides  ATCC 17025,  Rhodopseudomonas palustris, Rhodopseudomonas palustris  CGA009,  Rhodopseudomonas palustris  DX-1,  Rhodospirillum rubrum, Rhodospirillum rubrum  ATCC 11170 , Ruminococcus obeum  ATCC 29174,  Saccharomyces cerevisiae, Saccharomyces cerevisiae  S288c,  Salmonella enterica, Salmonella enterica  subsp.  enterica  serovar  Typhimurium  str. LT2,  Salmonella enterica typhimurium, Salmonella typhimurium, Schizosaccharomyces pombe, Sebaldella termitidis  ATCC 33386,  Shewanella oneidensis  MR-1,  Sinorhizobium meliloti  1021,  Streptomyces coelicolor, Streptomyces griseus  subsp.  griseus  NBRC 13350,  Sulfolobus acidocalarius, Sulfolobus solfataricus  P-2 , Synechocystis  str. PCC 6803 , Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacter  sp. X514,  Thermococcus kodakaraensis, Thermococcus litoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotoga maritima, Thiocapsa roseopersicina, Tolumonas auensis  DSM 9187,  Trichomonas vaginalis  G3,  Trypanosoma brucei, Tsukamurella paurometabola  DSM 20162,  Vibrio cholera, Vibrio harveyi  ATCC BAA-1116 , Xanthobacter autotrophicus  Py2,  Yersinia intermedia , or  Zea mays.    
     In some aspects the variant ADH gene is introduced into a cell engineered with increased of levels of 1,4-butanediol (BDO) or hydroxylbutyrate (4-HB) 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. 
     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 BDO or 4-HB biosynthetic pathway as well as other known biosynthetic pathways for 1,3-butanediol (13BDO), butadiene, 6-amino caproic acid (GACA), hexamethyldiamine (HMDA), adipic acid or derivatives thereof, croytl alcohol, methyl vinyl carbinol, 3-buten-1-ol, succinic acid or derivatives thereof, n-propanol, isopropanol, propylene, methacrylic acid or derivatives thereof, methanol metabolic and/or formaldehyde assimilation 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 various target products including 1,3-butanediol (13BDO), 1, 4-butanediol (BDO), 4-HB, butadiene, 6-amino caproic acid (GACA), hexamethyldiamine (HMDA), adipic acid or derivatives thereof, croytl alcohol, methyl vinyl carbinol, 3-buten-1-ol, succinic acid or derivatives thereof, n-propanol, isopropanol, propylene, methacrylic acid or derivatives thereof, 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. 
     Therefore, the engineered cell including the non-natural NAD + -dependent alcohol dehydrogenase, can include one or more genetic alterations, such as inserted transgenes, deletions, attenuation, mutations, etc., desired to increase levels of one or more intermediates or a product thereof, and include those genetic modifications as described in U.S. application Ser. No. 13/975,678 (filed Aug. 26, 2013), which is incorporated herein by reference. 
     Exemplary alcohol metabolic pathway gene(s), such as described in U.S. application Ser. No. 13/975,678, encode a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EM11), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16), any or more can be coexpressed with the non-natural NAD + -dependent alcohol dehydrogenase in the engineered cell. 
     Other exemplary alcohol metabolic pathway gene(s), such as described in U.S. application Ser. No. 13/975,678, encode an alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-14B) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A), any or more can be coexpressed with the non-natural NAD + -dependent alcohol dehydrogenase in the engineered cell. 
     Target products obtained from, and product pathways suitable for producing in, host cells expressing the engineered NAD+-dependent methanol or ethanol dehydrogenases described herein include the following. Of particular interest are a target product obtained using pyruvate and acetyl-CoA as entry point or precursor to its product pathway(s), in part because the methanol metabolic pathway using the novel enzymes enables fixing the carbon of methanol into pathways to pyruvate and acetyl-CoA. Target products include (a) 1,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), (b) butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol, (c) 1,3-butanediol and intermediates thereto, such as 2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol, (d) adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine and levulinic acid and their intermediates, e.g. 4-aminobutyryl-CoA, (e) methacrylic acid (2-methyl-2-propenoic acid) and its esters known collectively as methacrylates, such as methyl methacrylate, methyl methacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their intermediates, (f) 1,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol and glycerol, and their intermediates and (g) succinic acid and intermediates thereto. 
     1,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), are target products that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2008115840A2 published 25 Sep. 2008 entitled Compositions and Methods for the Biosynthesis of 1, 4-Butanediol and Its Precursors; WO2010141780A1 published 9 Dec. 2010 entitled Process of Separating Components of A Fermentation Broth; WO2010141920A2 published 9 Dec. 2010 entitled Microorganisms for the Production of 1, 4-Butanediol and Related Methods; WO2010030711A2 published 18 Mar. 2010 entitled Microorganisms for the Production of 1, 4-Butanediol; WO2010071697A1 published 24 Jun. 2010 Microorganisms and Methods for Conversion of Syngas and Other Carbon Sources to Useful Products; WO2009094485A1 published 30 Jul. 2009 Methods and Organisms for Utilizing Synthesis Gas or Other Gaseous Carbon Sources and Methanol; WO2009023493A1 published 19 Feb. 2009 entitled Methods and Organisms for the Growth-Coupled Production of 1, 4-Butanediol; WO2008115840A2 published 25 Sep. 2008 entitled Compositions and Methods for the Biosynthesis of 1,4-Butanediol and Its Precursors; and International Application No. PCT/US13/56725 filed 27 Aug. 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing 1,4-Butanediol Related Thereto. 
     Butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol, are target products that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described in the following documents. In addition to direct fermentation to produce butadiene, 1,3-butanediol, 1,4-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol can be separated, purified (for any use), and then dehydrated to butadiene in a second step involving metal-based catalysis. Suitable product pathways and enzymes, methods for screening and methods for isolating are found in the following documents, incorporated herein by reference: WO2011140171A2 published 10 Nov. 2011 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2012018624A2 published 9 Feb. 2012 entitled Microorganisms and Methods for the Biosynthesis of Aromatics, 2,4-Pentadienoate and 1,3-Butadiene; WO2011140171A2 published 10 Nov. 2011 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013040383A1 published 21 Mar. 2013 entitled Microorganisms and Methods for Producing Alkenes; WO2012177710A1 published 27 Dec. 2012 entitled Microorganisms for Producing Butadiene and Methods Related thereto; WO2012106516A1 published 9 Aug. 2012 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013028519A1 published 28 Feb. 2013 entitled Microorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and Related Alcohols; and U.S. Ser. No. 61/799,255 filed 15 Mar. 2013. 
     1,3-butanediol and intermediates thereto, such as 2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol, are target products that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2011071682A1 published 16 Jun. 2011 entitled Methods and Organisms for Converting Synthesis Gas or Other Gaseous Carbon Sources and Methanol to 1, 3-Butanediol; WO2011031897A published 17 Mar. 2011 entitled Microorganisms and Methods for the Co-Production of Isopropanol with Primary Alcohols, Diols and Acids; WO2010127319A2 published 4 Nov. 2010 entitled Organisms for the Production of 1,3-Butanediol; WO2013071226A1 published 16 May 2013 entitled Eukaryotic Organisms and Methods for Increasing the Availability of Cytosolic Acetyl-CoA, and for Producing 1,3-Butanediol; WO2013028519A1 published 28 Feb. 2013 entitled Microorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and Related Alcohols; WO2013036764A1 published 14 Mar. 2013 entitled Eukaryotic Organisms and Methods for Producing 1,3-Butanediol; WO2013012975A1 published 24 Jan. 2013 entitled Methods for Increasing Product Yields; WO2012177619A2 published 27 Dec. 2012 entitled Microorganisms for Producing 1, 3-Butanediol and Methods Related Thereto; and U.S. Ser. No. 61/799,255 filed 15 Mar. 2013. 
     Adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine and levulinic acid, and their intermediates, e.g. 4-aminobutyryl-CoA, are target products, useful for example for making nylon polymers, that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2010129936A1 published 11 Nov. 2010 entitled Microorganisms and Methods for the Biosynthesis of Adipate, Hexamethylenediamine and 6-Aminocaproic Acid; WO2013012975A1 published 24 Jan. 2013 entitled Methods for Increasing Product Yields; WO2012177721A1 published 27 Dec. 2012 entitled Microorganisms for Producing 6-Aminocaproic Acid; WO2012099621A1 published 26 Jul. 2012 entitled Methods for Increasing Product Yields; and application U.S. Ser. No. 61/766,620 filed 19 Feb. 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing Adipate, 6-Aminocaproate, Hexamethylenediamine or Caprolactam Related Thereto. 
     Methacrylic acid (2-methyl-2-propenoic acid; used in the preparation of its esters known collectively as methacrylates, such as methyl methacrylate, which is used most notably in the manufacture of polymers), methacrylate ester such as methyl methacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their intermediates are target products, useful for example for making polymers, that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2012135789A2 published 4 Oct. 2012 entitled Microorganisms for Producing Methacrylic Acid and Methacrylate Esters and Methods Related Thereto; WO2009135074A2 published 5 Nov. 2009 entitled Microorganisms for the Production of Methacrylic Acid; and application U.S. Ser. No. 61/766,660 filed 19 Feb. 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing 3-Hydroxyisobutyate or Methacrylic Acid Related Thereto. 
     1,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol and glycerol, and their intermediates are target products, useful for example for making polymers, that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: WO2009111672A1 published 9 Nov. 2009 entitled Primary Alcohol Producing Organisms; WO2011031897A1 17 Mar. 2011 entitled Microorganisms and Methods for the Co-Production of Isopropanol with Primary Alcohols, Diols and Acids; WO2012177599A2 published 27 Dec. 2012 entitled Microorganisms for Producing N-Propanol 1, 3-Propanediol, 1, 2-Propanediol or Glycerol and Methods Related Thereto; and application U.S. Ser. No. 61/766,635 filed 19 Feb. 2013 entitled Microorganisms an Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing 1,2-Propanediol, n-Propanol, 1,3-Propanediol, or Glycerol Related Thereto. 
     Succinic acid and intermediates thereto (useful to produce products including polymers, e.g. PBS, 1,4-butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, and detergents) are target products that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described herein as well as in the following documents. Suitable product pathways and enzymes, methods for screening and methods for isolating are found herein as well as in the following documents, incorporated herein by reference: EP1937821A2 published 2 Jul. 2008 entitled Methods and Organisms for the Growth-Coupled Production of Succinate; and application U.S. Ser. No. 61/766,635 filed 19 Feb. 2013 entitled Microorganisms and Methods for Enhancing the Availability of Reducing Equivalents in the Presence of Methanol, and for Producing Succinate Related Thereto. 
     Target products obtained from, and product pathways suitable for producing in, host cells co-expressing the engineered NAD+-dependent methanol or ethanol dehydrogenases described herein include the following. 
     Butadiene and intermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol, are target products that can be made by co-expressing the novel alcohol dehydrogenases described herein with a product pathway described in the following documents. In addition to direct fermentation to produce butadiene, 1,3-butanediol, 1,4-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol can be separated, purified (for any use), and then dehydrated to butadiene in a second step involving metal-based catalysis. Suitable product pathways and enzymes, methods for screening and methods for isolating are found in: WO2011140171A2 published 10 Nov. 2011 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2012018624A2 published 9 Feb. 2012 entitled Microorganisms and Methods for the Biosynthesis of Aromatics, 2, 4-Pentadienoate and 1, 3-Butadiene; 02011140171A2 published 10 Nov. 2011 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013040383A1 published 21 Mar. 2013 entitled Microorganisms and Methods for Producing Alkenes; WO2012177710A1 published 27 Dec. 2012 entitled Microorganisms for Producing Butadiene and Methods Related thereto; WO2012106516A1 published 9 Aug. 2012 entitled Microorganisms and Methods for the Biosynthesis of Butadiene; WO2013028519A1 published 28 Feb. 2013 entitled Microorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and Related Alcohols; and U.S. Ser. No. 61/799,255 filed 15 Mar. 2013. 
     In some embodiments, the disclosure provides organisms comprising a MDH variant and that are engineered to improve the availability of reducing equivalents or utilizing formaldehyde resulting from methanol via a formaldehyde assimilation pathway (FAB), which can be used for the production of target product molecules. 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 other biosynthetic pathways. 
     BDO is a valuable chemical for the production of high performance polymers, solvents, and fine chemicals. It is the basis for producing other high value chemicals such as tetrahydrofuran (THF) and gamma-butyrolactone (GBL). The value chain is comprised of three main segments including: (1) polymers, (2) THF derivatives, and (3) GBL derivatives. In the case of polymers, BDO is a comonomer for polybutylene terephthalate (PBT) production. PBT is a medium performance engineering thermoplastic used in automotive, electrical, water systems, and small appliance applications. Conversion to THF, and subsequently to polytetramethylene ether glycol (PTMEG), provides an intermediate used to manufacture spandex products such as LYCRA® fibers. PTMEG is also combined with BDO in the production of specialty polyester ethers (COPE). COPEs are high modulus elastomers with excellent mechanical properties and oil/environmental resistance, allowing them to operate at high and low temperature extremes. PTMEG and BDO also make thermoplastic polyurethanes processed on standard thermoplastic extrusion, calendaring, and molding equipment, and are characterized by their outstanding toughness and abrasion resistance. The GBL produced from BDO provides the feedstock for making pyrrolidones, as well as serving the agrochemical market. The pyrrolidones are used as high performance solvents for extraction processes of increasing use, including for example, in the electronics industry and in pharmaceutical production. Accordingly, provided herein is bioderived BDO produced according to the methods described herein and biobased products comprising or obtained using the bioderived BDO. 
     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.  3   a   . The reducing equivalents produced by the metabolism of methanol can then be used to power the glucose to BDO production pathways, for example, as shown in  FIG.  2   . 
     IN  FIG.  2   , the organism comprises at least one exogenous nucleic acid encoding a BDOPE expressed in a sufficient amount to produce BDO. In certain embodiments, the BDOPE is selected from the group consisting of a succinyl-CoA transferase (EB1) or a succinyl-CoA synthetase (EB2A) (or succinyl-CoA ligase); a succinyl-CoA reductase (aldehyde forming) (EB3); a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4); a 4-HB kinase (EB5); a phosphotrans-4-hydroxybutyrylase (EB6); a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7); a 1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9); a succinyl-CoA reductase (alcohol forming) (EB10); a 4-hydroxybutyryl-CoA transferase (EB11) or a 4-hydroxybutyryl-CoA synthetase (EB12); a 4-HB reductase (EB13); a 4-hydroxybutyryl-phosphate reductase (EB14); and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15). 
     Enzymes, genes and methods for engineering pathways from succinate and succinyl-CoA to various products, such as BDO, into a microorganism, are now known in the art (see, e.g., U.S. Publ. No. 2011/0201089). A set of BDOPEs represents a group of enzymes that can convert succinate to BDO as shown in  FIG.  2   . The additional reducing equivalents obtained from the MDH pathway, as disclosed herein, improve the yields of all these products when utilizing carbohydrate-based feedstock. For example, BDO can be produced from succinyl-CoA via previously disclosed pathways (see for example, Burk et al., WO 2008/115840). Exemplary enzymes for the conversion succinyl-CoA to BDO include EB3 ( FIG.  2   , Step B), EB4 ( FIG.  2   , Step C), EB5 ( FIG.  2   , Step D), EB6 ( FIG.  2   , Step E), EB7 ( FIG.  2   , Step F), EB8 ( FIG.  2   , Step G), EB10 ( FIG.  1   , Step I), EB11 ( FIG.  2   , Step J), EB12 ( FIG.  2   , Step J), EB14 ( FIG.  2   , Step L), EB13 ( FIG.  2   , Step K), and EB15 ( FIG.  2   , Step M). EB9 ( FIG.  2   , Step H) can be additionally useful in converting succinate directly to the BDOP intermediate, succinate semialdehyde. 
     The maximum theoretical yield of BDO 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 BDO 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 BDO from glucose at 2 mol BDO 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 succinate and 4-HB 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 other embodiments, the organism having a MDH protein, either alone or in combination with a BDOP, as provided herein, may further comprises a formaldehyde assimilation pathway (FAP) 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 certain embodiments, the organism further comprises a FAP, wherein said organism comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme (FAPE) expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass. In one embodiment, the FAPE is expressed in a sufficient amount to produce an intermediate of glycolysis. In another embodiment, the FAPE is expressed in a sufficient amount to produce an intermediate of a metabolic pathway that can be used in the formation of biomass. In some of the embodiments, the FAP comprises a hexulose-6-phosphate (H6P) synthase (EF1), a 6-phospho-3-hexuloisomerase (EF2), a dihydroxyacetone (DHA) synthase (EF3) or a DHA kinase (EF4). In one embodiment, the FAP comprises an EF1 and an EF2. In one embodiment, the intermediate is a H6P, a fructose-6-phosphate (F6P), or a combination thereof. In other embodiments, the FAP comprises an EF3 or an EF4. In one embodiment, the intermediate is a DHA, a DHA phosphate, or a combination thereof. In certain embodiments, the organism comprises two exogenous nucleic acids, each encoding a FAPE. 
     Also provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  3 A , step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. One exemplary FAP that can utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  3 A ) is shown in  FIG.  3   b   , which involves condensation of formaldehyde and D-ribulose-5-phosphate to form H6P by EF1 ( FIG.  3   b   , 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 F6P by EF2 ( FIG.  3   b   , 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.  3   a   ) is shown in  FIG.  3   c    and proceeds through DHA. EF3 is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of DHA and G3P, which is an intermediate in glycolysis ( FIG.  3   c   , step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase ( FIG.  3   c   , step B). DHAP can be assimilated into glycolysis and several other pathways. Rather than converting formaldehyde to formate and on to CO 2  off-gassed, the pathways provided in  FIGS.  3   b  and  3   c    show that carbon is assimilated, going into the final product. 
     Thus, in one embodiment, an organism having a MDH protein, either alone or in combination with a BDOP, as provided herein, further comprises a FAP 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 embodiments, the FAP comprises 3A or 3B, wherein 3A is an EF1 and 3B is an EF2 In other embodiments, the FAP comprises 4A or 4B, wherein 4A is an EF3 and 4B is an EF4. In certain embodiments, provided herein is a organism having a MDH protein, wherein said organism comprises at least one exogenous nucleic acid encoding an EM9 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 organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In other embodiments, the organism comprises at least one exogenous nucleic acid encoding an EM9 expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a FAP. 
     In certain embodiments, the organism further comprises at least one exogenous nucleic acid encoding a FAPE expressed in a sufficient amount to produce an intermediate of glycolysis. In certain embodiments, the FAPE is selected from the group consisting of an EF1, an EF2, an EF3 and an EF4. 
     Exemplary enzymes suitable for the reactions described herein to metabolize methanol for either or both reducing equivalents or carbon include the following, with respect to  FIG.  3 A , particularly as regards to Steps J, L, I, G, H, M, N and O. 
     FIG.  3 , Step G—Formate Hydrogen Lyase (EM15) 
     An EM15 enzyme can be employed to convert formate to carbon dioxide and hydrogen. An exemplary EM15 enzyme can be found in  Escherichia coli . The  E. coli  EM15 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 EM15 activity (Soini et al.,  Microb. Cell Fact.  7:26 (2008)). Various hydrogenase 3, EM8 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 
               
               
                   
               
            
           
         
       
     
     An EM15 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 
                   
               
               
                 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 EM15 systems have been found in  Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacterium formicicum  (Vardar-Schara et al.,  Microbial Biotechnology  1:107-125 (2008)). 
     FIG.  3 , Step H—Hydrogenase (EM16) 
     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 “02-tolerant” EM16 (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 EM16 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 EM16 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 EM16 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 EM16 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 EM16 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. EM16 activity in  E. coli  is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the EM16 complexes (Jacobi et al.,  Arch. Microbiol  158:444-451 (1992); Rangarajan et al.,  J Bacteriol.  190:1447-1458 (2008)). The  M. thermoacetica  and  Clostridium ljungdahli  EM16s are suitable for a host that lacks sufficient endogenous EM16 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 EM16 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  EM16 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 EM16 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, EM16 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 H2 (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 
                 AAC45123 
                 1498748 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 (CooS) 
               
               
                 CooC 
                 AAC45124 
                 1498749 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooT 
                 AAC45125 
                 1498750 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CooJ 
                 AAC45126 
                 1498751 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 CODH-I 
                 YP_360644 
                 78043418 
                 
                   Carboxydothermus 
                 
               
               
                 (CooS-I) 
                   
                   
                 
                   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 EM16 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-45]-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 (Rnffi) 
                 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 (NfinB) 
                 YP_360812.1 
                 78044266 
                 
                   Carboxydothermus hydrogenoformans 
                 
               
               
                 CLJU_c37220 
                 YP_003781850.1 
                 300856866 
                 
                   Clostridium ljungdahlii 
                 
               
               
                 (NfnAB) 
               
               
                   
               
            
           
         
       
     
     FIG.  3 , Step I—Formate Dehydrogenase (EM8) 
     Formate dehydrogenase (FDH; EM8) 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 EM16s (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 EM8 while the beta subunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol (2008)). Another set of genes encoding EM8 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)). EM8s 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 EM8 from  Ralstonia eutropha  reduces NAD +  (fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998). 
     Several EM8 enzymes have been identified that have higher specificity for NADP as the cofactor as compared to NAD. This enzyme has been deemed as the NADP-dependent formate dehydrogenase and has been reported from 5 species of the  Burkholderia cepacia  complex. It was tested and verified in multiple strains of  Burkholderia multivorans, Burkholderia stabilis, Burkholderia pyrrocinia , and  Burkholderia cenocepacia  (Hatrongjit et al.,  Enzyme and Microbial Tech.,  46: 557-561 (2010)). The enzyme from  Burkholderia stabilis  has been characterized and the apparent K m  of the enzyme were reported to be 55.5 mM, 0.16 mM and 1.43 mM for formate, NADP, and NAD respectively. More gene candidates can be identified using sequence homology of proteins deposited in Public databases such as NCBI, JGI and the metagenomic databases. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                 GI 
                   
               
               
                 Protein 
                 GenBank ID 
                 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, 
                 EIJ82879.1 
                 387590560 
                   Bacillus methanolicus  MGA3 
               
               
                 MGA3_06625 
               
               
                 fdhA, PB1_11719 
                 ZP_10131761.1 
                 387929084 
                   Bacillus methanolicus  PB1 
               
               
                 fdhD, 
                 EIJ82880.1 
                 387590561 
                   Bacillus methanolicus  MGA3 
               
               
                 MGA3_06630 
               
               
                 fdhD, PB1_11724 
                 ZP_10131762.1 
                 387929085 
                   Bacillus methanolicus  PB1 
               
               
                 fdh 
                 ACF35003.1 
                 194220249 
                 
                   Burkholderia stabilis 
                 
               
               
                 fdh 
                 ACF35004.1 
                 194220251 
                 
                   Burkholderia pyrrocinia 
                 
               
               
                 fdh 
                 ACF35002.1 
                 194220247 
                 
                   Burkholderia cenocepacia 
                 
               
               
                 fdh 
                 ACF35001.1 
                 194220245 
                 
                   Burkholderia multivorans 
                 
               
               
                 fdh 
                 ACF35000.1 
                 194220243 
                 
                   Burkholderia cepacia 
                 
               
               
                 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 
               
               
                 fdsG 
                 YP_725156.1 
                 113866667 
                 
                   Ralstonia eutropha 
                 
               
               
                 fdsB 
                 YP_725157.1 
                 113866668 
                 
                   Ralstonia eutropha 
                 
               
               
                 fdsA 
                 YP_725158.1 
                 113866669 
                 
                   Ralstonia eutropha 
                 
               
               
                 fdsC 
                 YP_725159.1 
                 113866670 
                 
                   Ralstonia eutropha 
                 
               
               
                 fdsD 
                 YP_725160.1 
                 113866671 
                 
                   Ralstonia eutropha 
                 
               
               
                   
               
            
           
         
       
     
     FIG.  3 , Step J—Methanol Dehydrogenase (EM9) 
     NAD+ dependent EM9 enzymes (EC 1.1.1.244) catalyze the conversion of methanol and NAD+ to formaldehyde and NADH. See the present invention as described herein. 
     FIG.  3 , Step L—Formaldehyde Dehydrogenase (EM11) 
     Oxidation of formaldehyde to formate is catalyzed by EM11. A NAD+ dependent EM11 enzyme is encoded by fdhA of  Pseudomonas putida  (Ito et al, J Bacteriol 176: 2483-2491 (1994)). Additional EM11 enzymes include the NAD+ and glutathione independent EM11 from  Hyphomicrobium zavarzinii  (Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione dependent EM11 of  Pichia pastoris  (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent EM11 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 EM11 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 EM12 (EC 4.4.1.22), EM13 (EC 1.1.1.284) and EM14 (EC 3.1.2.12). 
     FIG.  3 , Step M—Spontaneous or S-(hydroxymethyl)glutathione Synthase (EM12) 
     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 EM13, 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.  3 , Step N—Glutathione-Dependent Formaldehyde Dehydrogenase (EM13) 
     EM13 (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 
                 YP_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.  3 , Step O—S-formylglutathione Hydrolase (EM14) 
     EM14 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 
                 
               
               
                   
               
            
           
         
       
     
     Exemplary enzymes for the methods of using formaldehyde produced from the oxidation of methanol in the formation of intermediates of central metabolic pathways for the formation of target product or biomass are further described, particularly with respect to  FIGS.  3 B and  3 C . 
     Provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (see, e.g.,  FIG.  3   , step J) in the formation of intermediates of certain central metabolic pathways that can be used for the formation of biomass. Exemplary MMPs for enhancing the availability of reducing equivalents, as well as the producing formaldehyde from methanol (step J), are provided in  FIG.  3   . 
     One exemplary pathway that can utilize formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  3   ) is shown in  FIG.  3 B , which involves condensation of formaldehyde and D-ribulose-5-phosphate to form H6P by EF1 ( FIG.  3 B , 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 F6P by EF2 ( FIG.  3 B , 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.  3   ) is shown in  FIG.  3 C  and proceeds through DHA. EF3 is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of DHA and G3P, which is an intermediate in glycolysis ( FIG.  3 C , step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase ( FIG.  3 C , step B). DHAP can be assimilated into glycolysis and several other pathways. 
       FIG.  3 B , Steps A and B—Hexulose-6-phosphate synthase (EF1) (Step A) and 6-Phospho-3-Hexuloisomerase (EF2) (Step B) 
     Both of the EF1 and EF2 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, Appl Microbiol Biotechnol. 76:439-445). In some organisms, these two enzymes naturally exist as a fused version that is bifunctional. 
     Exemplary candidate genes for H6P 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 EF2 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.  3 C, Step A—Dihydroxyacetone Synthase (EF3) 
     Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol (e.g., as provided in  FIG.  3   ) is shown in  FIG.  3 C  and proceeds through DHA. EF3 is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of DHA and G3P, which is an intermediate in glycolysis ( FIG.  3 C , step A). The DHA obtained from DHA synthase is then further phosphorylated to form DHA phosphate by a DHA kinase ( FIG.  3 C , step B). DHAP can be assimilated into glycolysis and several other pathways. 
     The EF3 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 EF3 (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.  3 C, 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. EF4 has been purified from  Ogataea angusta  to homogeneity (Bystrkh, 1983, Biokhimiia, 48(10):1611-6). The enzyme, which phosphorylates DHA 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 fruendii  (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. pnuemoniae  is different from glycerol kinase in several respects. While both enzymes can phosphorylate DHA, 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 DHA (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 DHA 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 EF4 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 
                 
               
               
                   
               
            
           
         
       
     
     Suitable purification and/or assays to test, e.g., for the production of BDO 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 BDO or other target molecules may 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, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration. All of the above methods are well known in the art. 
     Examples of target molecule isolation processes include distillation for 13BDO, 14BDO, butadiene, methyl vinyl carbinol, 3-buten-1-ol, n-propanol, isopropanol, propylene, and crotyl alcohol; crystallization for GACA (alternatively it can be converted to caprolactam and then purified via distillation as a final step), HMDA, adipic acid or derivatives thereof, succinic acid or derivatives thereof, or any of crystallization, distillation, or extraction for methacrylic acid or derivatives thereof. 
     Target molecules such as 13BDO, 14BDO, butadiene, methyl vinyl carbinol n-propanol, isopropanol, propylene, crotyl alcohol; 3-buten-1-ol, 6ACA, HMDA, adipic acid or derivatives thereof, succinic acid or derivatives thereof, or methacrylic acid or derivatives thereof are chemicals used in commercial and industrial applications. In some embodiments, BDO and/or 4-HB are used in various commercial and industrial applications. Non-limiting examples of such applications include production of plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like. Moreover, BDO and/or 4-HB are also used as a raw material in the production of a wide range of products including plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like. 
     Accordingly, in some embodiments, provided are biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, comprising one or more bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof produced by an organism provided herein 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 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 disclosure. 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 disclosure provides plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, comprising bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof, wherein the bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof includes all or part of the BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof used in the production of plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like. Thus, in some aspects, the disclosure provides a biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, 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 BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof as disclosed herein. Additionally, in some aspects, the disclosure provides a biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, wherein the BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof used in its production is a combination of bioderived and petroleum derived BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof. For example, a biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, can be produced using 50% bioderived BDO and/or 4-HB and 50% petroleum derived BDO and/or 4-HB 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 plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, using the bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof of the disclosure are well known in the art. 
     In one embodiment, the product is a plastic. In one embodiment, the product is an elastic fiber. In one embodiment, the product is a polyurethane. In one embodiment, the product is a polyester. In one embodiment, the product is a polyhydroxyalkanoate. In one embodiment, the product is a poly-4-HB. In one embodiment, the product is a co-polymer of poly-4-HB. In one embodiment, the product is a poly(tetramethylene ether) glycol. In one embodiment, the product is a polyurethane-polyurea copolymer. In one embodiment, the product is a spandex. In one embodiment, the product is an elastane. In one embodiment, the product is a Lycra™. In one embodiment, the product is a nylon. 
     In some embodiments, provided herein is a culture medium comprising bioderived BDO. In some embodiments, the bioderived BDO is produced by culturing an organism having a MDH protein and BDOP, as provided herein. In certain embodiments, the bioderived BDO 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 organism having a MDH protein and BDOP. 
     In other embodiments, provided herein is a bioderived BDO. In some embodiments, the bioderived BDO is produced by culturing an organism having a MDH protein and BDOP, as provided herein. n some embodiments, the bioderived BDO 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 BDO is a component of culture medium. 
     In certain embodiments, provided herein is a composition comprising a bioderived BDO provided herein, for example, a bioderived BDO produced by culturing an organism having a MDH protein and BDOP, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived BDO. In certain embodiments, the compound other than said bioderived BDO is a trace amount of a cellular portion of an organism having a MDH protein and a BDOP, as provided herein. 
     In some embodiments, provided herein is a biobased product comprising a bioderived BDO provided herein. In certain embodiments, the biobased product is a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer, spandex, elastane, Lycra™, or nylon. In certain embodiments, the biobased product comprises at least 5% bioderived BDO. In certain embodiments, the biobased product is (i) a polymer, THF or a THF derivative, or GBL or a GBL derivative; (ii) a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer, spandex, elastane, Lycra™, or nylon; (iii) a polymer, a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or—filled or mineral-reinforced or—filled; (iv) a polymer, wherein the polymer comprises polybutylene terephthalate (PBT); (v) a polymer, wherein the polymer comprises PBT and the biobased product is a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or—filled or mineral-reinforced or—filled; (vi) a THF or a THF derivative, wherein the THF derivative is polytetramethylene ether glycol (PTMEG), a polyester ether (COPE) or a thermoplastic polyurethane; (viii) a THF derivative, wherein the THF derivative comprises a fiber; or (ix) a GBL or a GBL derivative, wherein the GBL derivative is a pyrrolidone. In certain embodiments, the biobased product comprises at least 10% bioderived BDO. In some embodiments, the biobased product comprises at least 20% bioderived BDO. In other embodiments, the biobased product comprises at least 30% bioderived BDO. In some embodiments, the biobased product comprises at least 40% bioderived BDO. In other embodiments, the biobased product comprises at least 50% bioderived BDO. In one embodiment, the biobased product comprises a portion of said bioderived BDO 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-BDO 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 BDO. In other embodiments, provided herein is a method for producing a polymer, comprising chemically or enzymatically converting the bioderived BDO to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived BDO, or a cell lysate or culture supernatant thereof. 
     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 BDO and/or 4-HB or any BDO and/or 4-HB 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 BDO and/or 4-HB or BDO and/or 4-HB pathway intermediate, or for side products generated in reactions diverging away from a BDO and/or 4-HB pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens. The same holds true for the MMPs and FAPs, as well as intermediates thereof, provided herein. 
     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/12C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the  14 C/12C 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/12C 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 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, provided are BDO and/or 4-HB or a BDO and/or 4-HB pathway intermediate thereof 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 BDO and/or 4-HB or a BDO and/or 4-HB 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, provided is BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereof that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereof 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, provided is BDO and/or 4-HB or a BDO and/or 4-HB 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 disclosure relates, in part, to biologically produced BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof as disclosed herein, and to the products derived therefrom, wherein the BDO and/or 4-HB or a BDO and/or 4-HB 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, provided are a bioderived BDO and/or 4-HB or a bioderived BDO and/or 4-HB 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 BDO and/or 4-HB or a bioderived BDO and/or 4-HB 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 BDO and/or 4-HB, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. Also provided are plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG)(also referred to as PTMO, polytetramethylene oxide) and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, 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 plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, are generated directly from or in combination with bioderived BDO and/or 4-HB or a bioderived BDO and/or 4-HB intermediate thereof as disclosed herein. 
     Those skilled in the art will understand that an organism can be engineered that secretes the biosynthesized compounds when grown on a carbon source such as a methanol alone or combined with other carbohydrates. Such compounds include, for example, BDO and any of the intermediate metabolites in the BDOP. 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 BDO biosynthetic pathways. Accordingly, provided herein is an organism that produces and/or secretes BDO when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the BDOP when grown on a carbohydrate or other carbon source. The BDO producing microbial organisms provided herein can initiate synthesis from an intermediate. The same holds true for intermediates in the formaldehyde assimilation. 
     In one embodiment, the carbon source is methanol or formate. In certain embodiments, methanol is used as a carbon source. In one embodiment, the carbon source is methanol or formate. In other embodiments, formate is used as a carbon source. In specific embodiments, methanol is used as a carbon source in the organisms 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 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. 
     1. An engineered cell either (a) expressing a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered cell expressing the corresponding alcohol dehydrogenase without amino acid substitution or (b) expressing a first sequence that is a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered cell expressing a second sequence that is a non-natural NAD + -dependent alcohol dehydrogenase, wherein the first and second sequences differ with regards to the at least one amino acid substitution. 
     2. The engineered cell of embodiment 1 further comprising one or more metabolic pathway transgene(s) encoding a protein of a metabolic pathway that promotes production of a target product or intermediate thereof. 
     3. The engineered cell of embodiments 1 or 2, wherein expression of the non-natural alcohol dehydrogenase provides an increased amount of reducing equivalents for an increase in a target product and/or for increased fixation of carbon from the formaldehyde into a target product. 
     4. The engineered cell of embodiment any of the previous embodiments further comprising a transgene encoding an enzyme to convert the formaldehyde to formate thereby generating reducing equivalents useful to product the target product and/or able to fix carbon of formate into the target product. 
     5. The engineered cell of any of the previous embodiments wherein the target product is selected from the group consisting of a diol, 1,4-butadiol, 1,3-butadiol, butadiene, succinate, adipate, HMDA, 6-aminocaproic acid (GACA), or an intermediate compound thereof. 
     6. The engineered cell of any of the previous embodiments further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EM11), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16). 
     7. The engineered cell of any of the previous embodiments further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A). 
     8. A composition comprising the cell of any of the previous embodiments, or a cell extract thereof. 
     9. The composition of embodiment 8 wherein the composition is a cell culture composition, optionally comprising a target product or intermediate thereof. 
     10. A cell culture composition comprising a target product or intermediate thereof produced by the cell of any of the previous embodiments. 
     11. A composition comprising a target product or intermediate thereof produced by the cell of any of the previous embodiments, optionally comprising cell debris and/or residual culture medium. 
     12. The composition of embodiment 11 comprising target product or intermediate thereof. which is at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or 99.9% pure in the composition. 
     13. The composition of embodiment 11 or 12 comprising a detectable trace amount of a nucleic acid encoding the non-natural NAD + -dependent alcohol dehydrogenase, or a detectable trace amount of a metabolic pathway intermediate or product not produced in the corresponding original cell absent expression of the non-natural NAD+-dependent alcohol dehydrogenase. 
     14. The composition of embodiment 11 wherein the metabolic pathway intermediate or product is 4-hydroxybutyrate (4-HB) and 1,3 propanediol (1,3-PDO). 
     15. The composition of embodiment 14 comprising an amount of 1,4-butanediol or 1,3-butanediol in the range of 70-90% (vol/vol) and an amount of water in the range of 10-30% (vol/vol). 
     16. A method for increasing the conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, comprising a step of (a) culturing an engineered cell expressing a NAD + -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase in a culture medium comprising methanol or ethanol, where in said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to an engineered cell expressing the corresponding alcohol dehydrogenase without amino acid substitution. 
     17. A method for increasing the conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, comprising a step of (a) providing a reaction composition having a pH in the range of 6-8, the composition comprising a NAD + -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and methanol or ethanol, where in the composition said NAD + -dependent non-natural alcohol dehydrogenase provides at least two fold greater conversion of methanol or ethanol to a formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution. 
     18. A nucleic acid encoding a NAD + -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase capable, when expressed in a cell, of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution. 
     19. An expression construct comprising the nucleic acid of embodiment 18. 
     20. A NAD + -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase capable, when expressed in a cell, of at least two fold greater conversion of a methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution. 
     21. The subject matter of any of the previous embodiments wherein the methanol is converted to formaldehyde. 
     22. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase is capable of at least three fold greater, of at least four fold, of at least five fold, of at least six fold, of at least seven fold, at least 8 fold, at least 9 fold, at least 10 fold, or at least 11 fold, conversion of methanol or ethanol to a formaldehyde or acetaldehyde, respectively, in vivo, as compared to the corresponding alcohol dehydrogenase without amino acid substitution. 
     23. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase is capable of at least three fold greater, of at least four fold, of at least five fold, of at least six fold, of at least seven fold, at least 8 fold, at least 9 fold, at least 10 fold, or at least 11 fold, conversion of methanol or ethanol to a formaldehyde or acetaldehyde, respectively, in vitro, as compared to the corresponding alcohol dehydrogenase without amino acid substitution. 
     24. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase is capable of an increase in conversion of methanol or ethanol to a formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution, in the range of two fold to twelve fold greater, in the range of two fold to eleven fold greater, in the range of two fold to ten fold greater, in the range of two fold to nine fold greater, in the range of two fold to eight fold greater, in the range of two fold to seven fold greater, in the range of two fold to six fold greater, in the range of two fold to five fold greater, or in the range of two fold to four fold greater. 
     25. A NAD + -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase having a catalytic efficiency (k cat /K m ) for the conversion of methanol to formaldehyde of 8.6×10 −4  or greater. 
     26. The subject matter of any of the previous embodiments comprising an activator protein that is an Act Nudix hydrolase. 
     27. A method of producing a target product or its intermediate comprising culturing the engineered cell of embodiment any of embodiments 1-7 in a culture medium comprising methanol or ethanol to produce the target product (TP) or its intermediate (INT). 
     28. The method of embodiment 27 further comprising a step of isolating or purifying target product (TP) or its intermediate (INT). 
     29. The method of embodiment 28 wherein the step of isolating or purifying comprises one or more of continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration. 
     30. The method of embodiment 29 selected from the group consisting of: (a) target product: 1,4-butanediol, purification: distillation; (b) target product: 1,3-butanediol, purification:distillation; (c) target product:Butadiene, purification:distillation; (d) target product:6-AminoCaproic Acid, purification:crystallization, (a) target product: caprolactam, purification:distillation as a final step; (e) target product: hexamethylenediame (HMDA), purification:crystallization; (f) target product: Adipic acid, purification: crystallization (adipic acid crystals); (g) target product: Crotyl alcohol, purification:distillation (h) target product: methyl vinyl carbinol, purification:distillation; (i) target product:succinic acid-crystallization (succinic acid crystals); (j) target product:n-propanol, purification:distillation; (k) target product: isopropanol, purification:distillation; (1) target product:propylene, purification:distillation; (m) target product: methacrylic acid, purification:crystallization, distillation, or extraction (n) target product: methylmethacrylate (MMA) or another ester, purification: distillation or crystallization. 
     31. The method of any of embodiments 31 wherein the step of isolating or purifying further comprises distillation. 
     32. The method of embodiments 28-31 wherein the target product is a diol. 
     33. The method of embodiments 28-32 wherein the target product is a diol is 1,4-butanediol or 1,3-butanediol. 
     34. The method of embodiments 28-32 comprising purifying the target product to at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or 99.9% purity in a composition. 
     35. A method of preparing a polymer comprising obtaining a target product produced by the engineered cell of any of embodiments 1-7 or method of any of the embodiments 27-34 and polymerizing the target product, optionally with one or more other monomeric compounds, to provide a polymeric product. 
     36. The method of embodiment 35 further comprising a step of isolating or purifying the polymeric product. 
     37. The method of embodiments 35 or 36 comprising purifying the polymer product to at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or 99.9% purity in a composition. 
     38. A polymer prepared according to the method of any of embodiments 35-37. 
     39. The polymer of embodiment 38 which is a homopolymer or copolymer. 
     40. The polymer of embodiment 39 that is selected from the group consisting of polybutylene terephthalate (PBT) and polybutylene succinate (PBS). 
     41. A composition comprising a polymer blend comprising the polymer of any ones of embodiments 38-40. 
     42. An article comprising the polymer or composition any one of embodiments 38-41. 
     43. The article of embodiment 42 which is a plastic article. 
     44. The article of embodiment 17d or 17e which is molded, extruded, or shaped from the polymer or composition any one of embodiments 41-43. 
     45. A biobased product comprising target product produced by the engineered cell of any of embodiments 1-7 or the polymer of any ones of embodiments 38-40 wherein said biobased product is
         (i) a polymer, THF or a THF derivative, or GBL or a GBL derivative;   (ii) a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer, spandex, elastane, Lycra™, or nylon;   (iii) a polymer, a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or—filled or mineral-reinforced or—filled;   (iv) a polymer, wherein the polymer comprises polybutylene terephthalate (PBT);   (v) a polymer, wherein the polymer comprises PBT and the biobased product is a resin, a fiber, a bead, a granule, a pellet, a chip, a plastic, a polyester, a thermoplastic polyester, a molded article, an injection-molded article, an injection-molded part, an automotive part, an extrusion resin, an electrical part and a casing; and optionally where the biobased product is reinforced or filled and further where the biobased product is glass-reinforced or—filled or mineral-reinforced or—filled;   (vi) a THF or a THF derivative, wherein the THF derivative is polytetramethylene ether glycol (PTMEG), a polyester ether (COPE) or a thermoplastic polyurethane;   (viii) a THF derivative, wherein the THF derivative comprises a fiber; or   (ix) a GBL or a GBL derivative, wherein the GBL derivative is a pyrrolidone;       

     wherein said biobased product optionally comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived BDO; and/or 
     wherein said biobased product optionally comprises a portion of said bioderived BDO as a repeating unit. 
     46. A molded product obtained by molding the biobased product of embodiment 10. 
     47. A process for producing the biobased product of embodiment 45, comprising chemically reacting said bioderived BDO with itself or another compound in a reaction that produces said biobased product. 
     48. A polymer comprising or obtained by converting the bioderived BDO of embodiment 45. 
     49. A method for producing a polymer, comprising chemically of enzymatically converting the bioderived BDO of embodiment 45 to the polymer. 
     50. A composition comprising the bioderived BDO of embodiment 45, or a cell lysate or culture supernatant thereof. 
     51. A method of producing an intermediate of glycolysis and/or an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing the engineered cell of any one of embodiments 1-7 under conditions and for a sufficient period of time to produce the intermediate, and optionally wherein the intermediate is consumed to provide a reducing equivalent or to incorporate into BDO or target product. 
     52. The method of embodiment 51, wherein the organism is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source. 
     53. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to an NAD + -dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO:1 (MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1, EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1, YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP_004681552.1, AGF87161, YP_002138168.1, YP_359772.1, YP_001343716.1, ZP_16224338.1, AAC45651.1, YP_007491369.1, YP_002434746, YP_005052855, NP 561852.1, YP_001447544, YP_001113612.1, YP_011618, ZP_01220157.1, YP_003990729.1, ZP_07335453.1, NP 717107, YP_003310546.1, ZP_10241531.1, YP_001337153.1, YP_026233.1, YP_694908, YP_725376.1, YP_001663549, EKC54576, YP_001126968.1 or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to the template. 
     54. The subject matter of any of the previous embodiments said template is selected from the group consisting of EIJ77596.1, EIJ78397.1, EFI69743.1, YP_001699778.1, YP_002138168.1, YP_359772.1, YP_005052855, NP 561852.1, YP_001447544, ZP_01220157.1, YP_003990729.1, ZP_10241531.1, and YP_026233.1. 
     55. The subject matter of any of the previous embodiments wherein the alcohol dehydrogenase is a methanol dehydrogenase. 
     56. The subject matter of embodiment 55 wherein the methanol dehydrogenase is from bacteria. 
     57. The subject matter of embodiment 56 wherein the methanol dehydrogenase is from  Bacillus.    
     58. The subject matter of embodiment 57 wherein the methanol dehydrogenase is from  Bacillus methanolicus  MGA3 or  Bacillus methanolicus  PB1. 
     59. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and wherein the dehydrogenase comprises one or more amino acid substitutions based on formula: R′XR 2 , where R 1  is an original amino acid at position X of the template, and R 2  is the variant amino acid that replaces R 1  at a position on the template corresponding to X, wherein XR 2  is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M or group consisting of (b) 38N, 60E, 71I, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D, 117Q, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 125C, 125G, 125V, 125W, 126G, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H, 158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K, 360R, 360S, and 361R. 
     60. The subject matter of embodiment 59 wherein XR 2  is selected from the group consisting of 107S, 121D, 123D, 123I, 123L, 123R, 123Y, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 143T, 146N, 149L, 149M, 149T, 149V, 158E, 158H, 158K, 158W, 267H, 270M, 270S, 270Y, 355D, 360G, 360K, 360R, and 360S 
     61. The subject matter of embodiment 60 wherein R 1 XR 2  is selected from the group consisting of (a) S11T, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S, and C361R. 
     62. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises a original amino acid at all positions that are not substituted at amino acid position numbers of group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361. 
     63. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises a original amino acid at all positions that are not amino acid position numbers 107, 121, 123, 129, 130, 143, 146, 149,158, 267, 270, 355, 360. 
     64. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, amino acid substitutions selected from the group consisting of: (a) S11T, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S and C361R. 
     65. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a set of amino acid substitutions selected from the group consisting of (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (j) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G; (1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) N112K, S147R, P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K, S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao) V150I, P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat) V150I, P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I, V360G; (aaw) S11T, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) N117Y, T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R, E126G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161S, and (aaaap) N112R/P161S. 
     66. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TNA and VTNAF (SEQ ID NO: 79). 
     67. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of VEV and GVEVA (SEQ ID NO: 80). 
     68. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD (SEQ ID NO: 82). 
     69. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of EKC and QEKCD (SEQ ID NO: 83). 70. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of STH and GSTHD (SEQ ID NO: 84). 
     71. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TVK and DTVKA (SEQ ID NO: 85). 
     72. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of SLV, GVV, GWV, GLY, ISLVA (SEQ ID NO: 86), IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA (SEQ ID NO: 89) 73. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of HIN, RFN, RID, RIQ, GHIND (SEQ ID NO: 90), GRFND (SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93). 
     74. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQ ID NO: 95), LVNSVEKPVV (SEQ ID NO: 96), MVNSVEKPVV (SEQ ID NO: 97), RVNSVEKPVV (SEQ ID NO: 98), SVNSVEKPVV (SEQ ID NO: 99), TVNSVEKPVV (SEQ ID NO: 100), VVNSVEKPVV (SEQ ID NO: 101), WVNSVEKPVV (SEQ ID NO: 102), YVNSVEKPVV (SEQ ID NO: 103), GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPVV (SEQ ID NO: 105), GVISVEKPVV (SEQ ID NO: 106), GVLSVEKPVV (SEQ ID NO: 107), GVRSVEKPVV (SEQ ID NO: 108), GVYSVEKPVV (SEQ ID NO: 109). GVNIVEKPVV (SEQ ID NO: 110), GVNLVEKPVV (SEQ ID NO: 111), GVNSCEKPVV (SEQ ID NO: 112), GVNSGEKPVV (SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO: 114), GVNSVGKPVV (SEQ ID NO: 115), GVNSVECPVV (SEQ ID NO: 116), GVNSVERPVV (SEQ ID NO: 117), GVNSVEKAVV (SEQ ID NO: 118). GVNSVEKRVV (SEQ ID NO: 119). GVNSVEKSVV (SEQ ID NO: 120). GVNSVEKPAV (SEQ ID NO: 121). GVNSVEKPMV (SEQ ID NO: 122). GVNSVEKPPV (SEQ ID NO: 123). GVNSVEKPSV (SEQ ID NO: 124). GVNSVEKPVF (SEQ ID NO: 125). GVNSVEKPVI (SEQ ID NO: 126), and GVNSVEKPVY (SEQ ID NO: 127). 
     75. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TETT (SEQ ID NO: 128), SETN (SEQ ID NO: 129), GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131). 
     76. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of LLVI (SEQ ID NO: 132), LMVI (SEQ ID NO: 133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO: 135), and LAAI (SEQ ID NO: 136). 
     77. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of NVKMPVID (SEQ ID NO: 137), KEKMPVID (SEQ ID NO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140), KWKMPVID (SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQ ID NO: 143). 
     78. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO: 145), QVGS (SEQ ID NO: 146), and QVGY (SEQ ID NO: 147). 
     79. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of VEE and GVEEE (SEQ ID NO: 148). 
     80. The subject matter of any of the previous embodiments wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ ID NO: 150), NAYEDKC (SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), and NAYEDSC (SEQ ID NO: 153), and NAYEDVR (SEQ ID NO: 154). 
     81. A nucleic acid encoding the non-natural alcohol dehydrogenase of any of embodiments 53-80. 
     82. A non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to an NAD + -dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO:1 (MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1, EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1, YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP_004681552.1, AGF87161, YP_002138168.1, YP_359772.1, YP_001343716.1, ZP_16224338.1, AAC45651.1, YP_007491369.1, YP_002434746, YP_005052855, NP_561852.1, YP_001447544, YP_001113612.1, YP_011618, ZP_01220157.1, YP_003990729.1, ZP_07335453.1, NP_717107, YP_003310546.1, ZP_10241531.1, YP_001337153.1, YP_026233.1, YP_694908, YP_725376.1, YP_001663549, EKC54576, YP_001126968.1 or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to the template. 
     83. A non-natural alcohol dehydrogenase of embodiment 82 wherein said template is selected from the group consisting of EIJ77596.1, EIJ78397.1, EFI69743.1, YP_001699778.1, YP_002138168.1, YP_359772.1, YP_005052855, NP_561852.1, YP_001447544, ZP_01220157.1, YP_003990729.1, ZP_10241531.1, and YP_026233.1. 
     84. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase has a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and wherein the dehydrogenase comprises one or more amino acid substitutions based on formula: R′XR 2 , where R 1  is a original amino acid at position X of the template, and R 2  is the variant amino acid that replaces R 1  at a position on the template corresponding to X, wherein XR 2  is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M or the group consisting of (b) 38N, 60E, 71I, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D, 117Q, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 125C, 125G, 125V, 125W, 126G, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H, 158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K, 360R, 360S, and 361R. 
     85. A non-natural alcohol dehydrogenase of embodiment 82 wherein XR 2  is selected from the group consisting of 107S, 121D, 123D, 123I, 123L, 123R, 123Y, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 143T, 146N, 149L, 149M, 149T, 149V, 158E, 158H, 158K, 158W, 267H, 270M, 270S, 270Y, 355D, 360G, 360K, 360R, and 360S 
     86. A non-natural alcohol dehydrogenase of embodiment 82 wherein R 1 XR 2  is selected from the group consisting of (a) S11T, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M; or the group consisting of (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S, and C361R. 
     87. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises an original amino acid at all positions that are not substituted at amino acid position numbers of group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361. 
     88. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of embodiment 53, and comprises an original amino acid at all positions that are not amino acid position numbers 107, 121, 123, 129, 130, 143, 146, 149,158, 267, 270, 355, 360. 
     89. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions selected from the group consisting of: (a) S11T, D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or the group consisting of (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R, V360S and C361R. 
     90. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a set of amino acid substitutions selected from the group consisting of (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (j) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G; (1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) N112K, S147R, P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K, S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao) V150I, P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat) V150I, P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I, V360G; (aaw) S11T, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) N117Y, T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R, E126G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161S, and (aaaap) N112R/P161S. 
     91. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TNA and VTNAF (SEQ ID NO: 79). 
     92. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of VEV and GVEVA (SEQ ID NO: 80). 
     93. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD (SEQ ID NO: 82). 
     94. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of EKC and QEKCD (SEQ ID NO: 83). 
     95. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of STH and GSTHD (SEQ ID NO: 84). 
     96. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TVK and DTVKA (SEQ ID NO: 85). 
     97. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of SLV, GVV, GWV, GLY, ISLVA (SEQ ID NO: 86), IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA (SEQ ID NO: 89). 
     98. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of HIN, RFN, RID, RIQ, GHIND (SEQ ID NO: 90), GRFND (SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93). 
     99. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQ ID NO: 95), LVNSVEKPVV (SEQ ID NO: 96), MVNSVEKPVV (SEQ ID NO: 97), RVNSVEKPVV (SEQ ID NO: 98), SVNSVEKPVV (SEQ ID NO: 99), TVNSVEKPVV (SEQ ID NO: 100), VVNSVEKPVV (SEQ ID NO: 101), WVNSVEKPVV (SEQ ID NO: 102), YVNSVEKPVV (SEQ ID NO: 103), GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPVV (SEQ ID NO: 105), GVISVEKPVV (SEQ ID NO: 106), GVLSVEKPVV (SEQ ID NO: 107), GVRSVEKPVV (SEQ ID NO: 108), GVYSVEKPVV (SEQ ID NO: 109). GVNIVEKPVV (SEQ ID NO: 110), GVNLVEKPVV (SEQ ID NO: 111), GVNSCEKPVV (SEQ ID NO: 112), GVNSGEKPVV (SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO: 114), GVNSVGKPVV (SEQ ID NO: 115), GVNSVECPVV (SEQ ID NO: 116), GVNSVERPVV (SEQ ID NO: 117), GVNSVEKAVV (SEQ ID NO: 118). GVNSVEKRVV (SEQ ID NO: 119). GVNSVEKSVV (SEQ ID NO: 120). GVNSVEKPAV (SEQ ID NO: 121). GVNSVEKPMV (SEQ ID NO: 122). GVNSVEKPPV (SEQ ID NO: 123). GVNSVEKPSV (SEQ ID NO: 124). GVNSVEKPVF (SEQ ID NO: 125). GVNSVEKPVI (SEQ ID NO: 126), and GVNSVEKPVY (SEQ ID NO: 127). 
     100. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of TETT (SEQ ID NO: 128), SETN (SEQ ID NO: 129), GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131). 
     101. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of LLVI (SEQ ID NO: 132), LMVI (SEQ ID NO: 133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO: 135), and LAAI (SEQ ID NO: 136). 
     102. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of NVKMPVID (SEQ ID NO: 137), KEKMPVID (SEQ ID NO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140), KWKMPVID (SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQ ID NO: 143). 
     103. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO: 145), QVGS (SEQ ID NO: 146), and QVGY (SEQ ID NO: 147). 
     104. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of VEE and GVEEE (SEQ ID NO: 148). 
     105. A non-natural alcohol dehydrogenase of embodiment 82 wherein the non-natural alcohol dehydrogenase comprises a sequence motif selected from the group consisting of DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ ID NO: 150), NAYEDKC (SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), and NAYEDSC (SEQ ID NO: 153), and NAYEDVR (SEQ ID NO: 154). 
     106. A nucleic acid encoding the non-natural alcohol dehydrogenase of any of embodiments 82-105. 
     107. An expression construct comprising the nucleic acid of 106. 
     108. An engineered cell comprising the nucleic acid or expression construct of embodiments 106 or 107. 
     109. The engineered cell of embodiment 108 further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EM11), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), a hydrogenase (EM16). 
     110. The engineered cell of embodiment 108 further comprising one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A). 
     111. The engineered cell of embodiment 108-110 which is bacteria. 
     112. The transgenic bacteria of embodiment 111 which is  Bacillus.    
     113. A method for increasing the conversion of a methanol or ethanol to a dehydrogenated product of the alcohol comprising a step of (a) culturing the engineered cell of any of embodiments 108-112 in a culture medium comprising a methanol or ethanol, where in said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to a dehydrogenated product of the alcohol, as compared to an engineered cell expressing a corresponding alcohol dehydrogenase without amino acid substitution. 
     114. A method for increasing the conversion of a methanol or ethanol to a dehydrogenated product of the alcohol comprising a step of (a) providing a reaction composition having a pH in the range of 6-8, the composition comprising a non-natural alcohol dehydrogenase of any of embodiments 82-105, where in the composition said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to a dehydrogenated product of the alcohol, as compared to an engineered cell expressing a corresponding alcohol dehydrogenase without amino acid substitution. 
     115. A method of providing a diol comprising culturing the engineered cell of any of embodiments 108-112 in a culture medium comprising a methanol or ethanol to provide the diol. 
     116. The method of embodiment 115 wherein the diol is 1,4 butanediol. 
     117. A method of preparing a polymer comprising obtaining a monomer product produced by the engineered cell or method of any of embodiments 108-116 and polymerizing the monomer to provide a polymeric product. 
     118. A polymer prepared according to the method of embodiment 117. 
     119. A method of screening for a non-natural alcohol dehydrogenase having increased activity, optionally at least 2 fold, optionally at least 4 fold or greater activity, compared to its unmodified counterpart, comprising (1) creating one or more non-natural alcohol dehydrogenases selected from SEQ ID NO:1 and non-natural alcohol dehydrogenases having a sequence identity of 45% or greater to SEQ ID NO:1 having a substitution at a position other than an amino acid position selected from group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361, or a position corresponding thereto, (2) assay the created enzyme for the activity and (3) selecting those having increased activity, optionally at least 2 fold, optionally at least 4 fold, or greater activity compared to the unmodified counterpart. 
     EXAMPLES 
     Assay for Testing Activity of Methanol Dehydrogenase In Vitro 
     A high-throughput screening assay was used to evaluate lysates for methanol dehydrogenase (MeDH) oxidation activity of methanol and other alcohol substrates. Lysates were prepared by a commercial chemical reagent from  Escherichia coli  cells that contained a plasmid harboring a MeDH library variant and an integrated chromosomal copy of the activator protein. An aliquot of the lysate was applied to a 384-well assay plate. To initiate the alcohol oxidation reaction, a substrate-buffer mix (pH 7.6 or pH 8.5) containing 0.5 M methanol or other alcohol, 0.5 mM NAD, 5 mM MgCl 2 , 10 □M 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS), &amp; 1 mM 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was added. Initial rates were monitored via absorbance at 560 nm. MeDH variants that showed higher activity than the wild-type control were evaluated for further characterization. 
     Formaldehyde Assay 
     A strain lacking frmA, frmB, frmR (the genes responsible for formaldehyde utilization in  E. coli ) was created using Lambda Red recombinase technology. Plasmids expressing methanol dehydrogenases were transformed into the strain, then grown to saturation in LB medium+antibiotic at 37° C. with shaking. Cultures were adjusted by OD and then diluted 1:10 into M9 medium+0.5% glucose+antibiotic and cultured at 37° C. with shaking for 6-8h until late log phase. Methanol was added to 2% v/v and the cultures were further incubated for 30 min with shaking at 37° C. Cultures were spun down and the supernatant was assayed for formaldehyde produced using DetectX Formaldehyde Detection kit from Arbor Assays, MI according to manufacturer&#39;s instructions. 
     Formate Assay 
     The assay was developed to evaluate in vivo activity of methanol dehydrogenases by measuring the formate production in a host strain containing the first two steps of the MeOH pathway but lacking formate dehydrogenases (hycE, fdnGHI, fdoGHI, fdhF) that convert formate to CO 2 . Plasmids expressing methanol dehdyrogenases were transformed into this strain. Strains were inoculated from colonies or glycerol stocks in LB+antibiotics in 96-deep well plates. The plates were sealed with breathable culture films and shaken at 37° C. at 800 rpm. Overnight cultures were centrifuged at 5250 rpm for 10 minutes to pellet the cells. Cells were resuspended in 1 ml M9 medium in plates that were sealed with breathable culture films and shaken at 37 degree at 800 rpm. Samples were taken for a time course study and formate concentrations were measured using the formate kit based on instructions provided by the manufacturer. 
     Assay for Purification of Methanol Dehydrogenases and for Characterizing their Activity 
     Cells expressing methanol dehydrogenase are cultured at 37° C. in LB containing 2 mM MgSO 4 . Once harvested, cells are lysed in BugBuster Protein Extraction Reagent (Novagen) supplemented with 15 kU/mL lysozyme (Novagen), 25 U/mL bezonase (Novagen), 1× Pierce Protease Inhibitors (Thermo Scientific), 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride, and 2 mM MgSO 4 . Lysates are clarified via centrifugation and purified on a 5 mL StrepTrap HP column (GE Healthcare Life Sciences). The column is prepared in and washed with 100 mM MOPS pH 7.5, 0.2 M NaCl2, 2 mM MgSO 4 , 0.5 mM TCEP (buffer A). The purified proteins are eluted with buffer A containing 0.3 mg/mL desthiobiotin. 
     DNA 2.0 Gene Synthesis 
     Methanol dehydrogenase gene candidates were synthesized after optimizing for codon usage by DNA 2.0 (Welch et al., PloS One 2009, 4(9):e7002, Design parameters to control synthetic gene expression in  Escherichia coli ). 
     In Vivo Labeled Assay for Conversion of Methanol to CO 2    
     Strains with functional reductive TCA branch and pyruvate formate lyase deletion were grown aerobically in LB medium overnight, followed by inoculation of M9 high-seed media containing IPTG and aerobic growth for 4 hrs. These strains had methanol dehydrogenase/ACT pairs in the presence and absence of formaldehyde dehydrogenase or formate dehydrogenase. At this time, strains were pelleted, resuspended in fresh M9 medium high-seed media containing 2%  13 CH 3 OH, and sealed in anaerobic vials. Head space was replaced with nitrogen and strains grown for 40 hours at 37° C. Following growth headspace was analyzed for 13-CO 2 . Media was examined for residual methanol as well as BDO and byproducts. 
     All constructs expressing MeDH mutants and MeDH/ACT pairs grew to slightly lower ODs than strains containing empty vector controls. This is likely due to the high expression of these constructs. 
     Description of the NAD-Dependent Methanol Dehydrogenase/Activator Protein, its Expression and Use 
     Sequence analysis of the NADH-dependent methanol dehydrogenase from  Bacillus methanolicus  places the enzyme in the alcohol dehydrogenase family III. It does not contain any tryptophan residues, resulting in a low extinction coefficient (18,500 M −1 , cm −1 ) and should be detected on SDS gels by Coomassie staining. 
     The enzyme has been characterized as a multisubunit complex built from 43 kDa subunits containing one Zn and 1-2 Mg atoms per subunit. Electron microscopy and sedimentation studies determined it to be a decamer, in which two rings with five-fold symmetry are stacked on top of each other (Vonck et al., J. Biol. Chem. 266, p. 3949-3954, 1991). It is described to contain a tightly but not covalently bound cofactor and requires exogenous NAD +  as e − -acceptor to measure activity in vitro. A strong increase (10-40-fold) of in vitro activity was observed in the presence of an activator protein (Act), which is a homodimer (21 kDa subunits) and contains one Zn and one Mg atom per subunit. 
     The mechanism of the activation was investigated by Kloosterman et al. (J. Biol. Chem. 277, p. 34785-34792, 2002), showing that Act is a Nudix hydrolase and Hektor et al. (J. Biol. Chem. 277, p. 46966-46973, 2002), demonstrating that mutation of residue S97 to G or T in MeDH changes activation characteristics along with the affinity for the cofactor. While mutation of residues G15 and D88 had no significant impact, a role of residue G13 for stability as well as of residues G95, D100, and K103 for the activity is suggested. Both papers together propose a hypothesis in which Act cleaves MeDH-bound NAD + . MeDH retains AMP bound and enters an activated cycle with increased turnover. 
     The stoichiometric ratio between Act and MeDH is not well defined in the literature. Kloosterman et al. (J. Biol. Chem. 277, p. 34785-34792, 2002) determine the ratio of dimeric Act to decameric MeDH for full in vitro activation to be 10:1. In contrast, Arfman et al. (J. Biol. Chem. 266, 3955-3960, 1991) determined a ratio of 3:1 in vitro for maximum and a 1:6 ratio for significant activation, but observe a high sensitivity to dilution. Based on expression of both proteins in  Bacillus , the authors estimate the ratio in vivo to be around 1:17.5. In vitro experiments with purified activator protein (2317A) and methanol dehydrogenase (2315A) have showed the ratio of “act” to methanol dehydrogenase to be 10:1. This in vitro test was done with 5 M methanol, 2 mM NAD and 10 uM methanol dehydrogenase 2315A at pH 7.4. 
     The sequence of the activator protein (SEQ ID NO: 157) from  Bacillus methanolicus  MGA3 (locus tag: MGA3_09170, GI number: 387591061, Accession number: EIJ83380) used in the assays is shown below: 
     
       
         
           
               
               
            
               
                   
                 MGKLFEEKTIKTEQIFSGRVVKLQVDDVELPNGQTSKRE 
               
               
                   
               
               
                   
                 IVRHPGAVAVIAITNENKIVMVEQYRKPLEKSIVEIPAG 
               
               
                   
               
               
                   
                 KLEKGEDPRITALRELEEETGYECEQMEWLISFATSPGF 
               
               
                   
               
               
                   
                 ADEIIHIYVAKGLSKKENAAGLDEDEFVDLIELTLDEAL 
               
               
                   
               
               
                   
                 QYIKEQRIYDSKTVIAVQYLQLQEALKNK. 
               
            
           
         
       
     
     2315 Stability Assay and Data 
     The thermostability of methanol dehydrogenase 2315A and the corresponding activator protein 2317A were assessed and melting temperatures were found to be 62 and 75° C., respectively. The melting temperatures were measured using a Protein thermal shift assay from Applied biosystems. The assay provides relative thermal stabilities (melting temperatures) of purified proteins. It relies on a proprietary fluorescent dye that binds to hydrophobic regions of denatured proteins upon heating in the RT-PCR machine. The relative melting temperature is calculated from the slope of the fluorescence signal peak. 
     Current Promoter and Plasmid for Overexpression 
     Methanol dehydrogenase 2315 was expressed with several constitutive and inducible promoters of varying strengths. The figure below shows the expression levels of two MeDH variants when expressed under three promoters: p119, p104 and p107. The two variants that were expressed were 2315L and 2315B. 2315B was a mutant constituted based on a mutation S97G identified from Hektor et al (ibid). 
     MDH Protein Concentrations 
     Methanol dehydrogenase is a very soluble protein. SDS-PAGE analysis of soluble proteins from lysates of  E. coli  strains expressing different variants of the WT 2315A are shown. Specifically, the left panel shows the gel run on the lysates and the right panel shows the gel run on supernatant for the WT enzyme 2315A, compared with the variants 2315L and R, a variant from Hektor et al. called 2315B, and an empty vector. 
     The cells were lysed using Bugbuster as described previously. The amount of protein was quantified using the Image Lab 3.0 software from BioRad. The WT protein was estimated to be ˜27% of the total protein. 
     Background on Plasmids and Promoters 
     Vector backbones were obtained from Dr. Rolf Lutz of Expressys (www.expressys.de). The vectors and strains are based on the pZ Expression System developed by Dr. Rolf Lutz and Prof. Hermann Bujard (Lutz, R. &amp; Bujard, H. Independent and tight regulation of transcriptional units in  Escherichia coli  via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements.  Nucleic Acids Res  25, 1203-1210 (1997)). Art available promoters P119, p104, p107, p119 provided varying levels of enzyme expression as desired. Vectors obtained were pZE13luc, pZA33luc, pZS*13luc and pZE22luc and contained the luciferase gene as a stuffer fragment. To replace the luciferase stuffer fragment with a lacZ-alpha fragment flanked by appropriate restriction enzyme sites, the luciferase stuffer fragment was first removed from each vector by digestion with EcoRI and XbaI. The lacZ-alpha fragment was PCR amplified from pUC19 with the following primers: 
     
       
         
           
               
               
            
               
                   
                 lacZalpha-RI 
               
               
                   
                 (SEQ ID NO: 155) 
               
               
                   
                 5′GACGAATTCGCTAGCAAGAGGAGAAGTCGA 
               
               
                   
               
               
                   
                 CATGTCCAATTCACTGGCCGTCGTTTTAC3′ 
               
               
                   
               
               
                   
                 lacZalpha 3′BB 
               
               
                   
                 (SEQ ID NO: 156) 
               
               
                   
                 5′-GACCCTAGGAAGCTTTCTAGAGTCGACCT 
               
               
                   
               
               
                   
                 ATGCGGCATCAGAGCAGA-3′ 
               
            
           
         
       
     
     This generated a fragment with a 5′ end of EcoRI site, NheI site, a Ribosomal Binding Site, a SalI site and the start codon. The 3′ end of the fragment contained the stop codon, XbaI, HindIII, and AvrII sites. The PCR product was digested with EcoRI and AvrII and ligated into the base vectors digested with EcoRI and XbaI (XbaI and AvrII have compatible ends and generate a non-site). Because NheI and XbaI restriction enzyme sites generate compatible ends that can be ligated together (but generate a site after ligation that is not digested by either enzyme), the genes cloned into the vectors could be “Biobricked” together (openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly, this method enables joining an unlimited number of genes into the vector using the same 2 restriction sites (as long as the sites do not appear internal to the genes), because the sites between the genes are destroyed after each addition. Initially, expression was low from these vectors, and they were subsequently modified using the Phusion® Site-Directed Mutagenesis Kit (NEB, Ipswich, Mass., USA) to insert the spacer sequence AATTAA between the EcoRI and NheI sites. This eliminated a putative stem loop structure in the RNA that bound the RBS and start codon. 
     All vectors have the pZ designation followed by letters and numbers indicating the origin of replication, antibiotic resistance marker and promoter/regulatory unit. The origin of replication is the second letter and is denoted by E for ColE1, A for p15A and S for pSC101 (as well as a lower copy number version of pSC101 designated S*)—based origins. The first number represents the antibiotic resistance marker (1 for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final number defines the promoter that regulated the gene of interest (1 for PLtetO-1, 2 for PLlacO-1 and 3 for PA1lacO-1) and each of these promoters became activated by its corresponding inducer molecule (pLtetO can be induced by tetracycline; pLlacO-1 and pA1lacO-1 can be induced by IPTG). Three base vectors, pZS*13S, pZA33S and pZE13S, were then designed and constructed to serve as “inducible” plasmid vectors. 
     In addition to the “inducible” promoters mentioned above, a set of “constitutive” promoters were sampled from the Registry (partsregistry.org). Each of these “constitutive” promoters was then introduced into the pZS*13S vector backbone to replace the pA1lacO-1 inducible promoter via Sequence and Ligation Independent Cloning (SLIC) method described by Li &amp; Eledge (Nature Methods 2007, 4:251-256). Of these sampled “constitutive” promoters (p100, p104, p105, p107, p108, p 111, p115 &amp; p119), experiments were carried out to establish an order of promoter strength that was verified by protein expression levels. For the work discussed here, we employed both “inducible” and “constitutive” plasmid vectors, modified for the biobricks and SLIC insertions as discussed above. To further fine-tune protein expression levels of some overly expressed proteins, ribosomal binding site (RBS) in between promoter and gene coding sequence was modified accordingly using the RBS calculator (salis.psu.edu/software). 
     Mutagenesis Techniques—Error Prone-PCR EpPCR (Pritchard et al.,  J Theor. Biol.  234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn 2+  ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance. This method can generate multiple mutations in a single gene simultaneously, which can be useful to screen a larger number of potential variants having a desired activity. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method, for example, using robotics, is useful to identify those with desirable characteristics. 
     Mutagenesis Techniques—Site Saturation Mutagenesis 
     In Site Saturation Mutagenesis, the starting materials are a supercoiled 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)). Primers carrying the mutation of interest, anneal to the same sequence on opposite strands of DNA. The mutation is typically in the middle of the primer and flanked on each side by approximately 20 nucleotides of correct sequence. The sequence in the primer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A, C). After extension, DpnI is used to digest dam-methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (that is, one codon). The technique facilitates the generation of all possible replacements at a single-site with no nonsense codons and results in equal to near-equal representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The usefulness of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme. 
     Combinatorial Mutants 
     MeDH Structure Model and Structures for Comparison 
     To design a library of mutations for improving the catalytic rates of 2315, genes of several MeDHs as well as various Fe-dependent ADH genes were aligned. The described structure/function relationships for the Fe-dependent ADHs (40-47% sequence identity to MeDHs) were used to identify regions of functional importance within the MeDH sequence. An alignment of the identified regions is presented in  FIG.  4   . 
     Blast search using the MeDH sequence against the PDB structure database found several structures of Fe-dependent alcohol dehydrogenases with sequence identities between 40 and 47%. 
     Similarities are spread out over the whole length of the protein. Given this similarity to known structures, the MeDH sequence was used to generate a 3-dimensional model using the web-based iTasser structure prediction tool (Roy et al, Nature Protocols, 5: 725-738 (2010)). The following three structures were specifically used for alignment and comparison with the MeDH from  Bacillus:    
     3OX4: ADH-2 from  Zymomonas mobilis  (sequence identity 47%) 
     1RRM: Lactaldehyde reductase from  Escherichia coli  (FucO, sequence identity 43%) 
     3BFJ:1,3-PDO oxidoreductase from  Klebsiella pneumoniae  (DhaT, sequence identity 47%) 
     The  Zymomonas  enzyme was crystallized with bound cofactor and the structure was well analyzed including the annotation of certain amino acid residues for metal, cofactor and proposed substrate binding (EtOH modeled into structure, Moon et al., J. Mol. Biol. 407, p 413-424, 2011). Like the Lactaldehyde reductase (Montella et al., J. Bact. 187, p. 4957-4966, 2005) from  E. coli , the  Zymomonas  ADH is a homodimer. In contrast, the  Klebsiella  enzyme (Marcal et al., J. Bact. 191, p. 1143-1151, 2009) was found to be a decamer with a structure that resembles the MeDH appearance in electron microscopy studies. 
     Sequence comparison shows that all four coordination residues of the Fe-dependent ADHs are conserved in the MeDH structure. Two of these four residues are in a histidine-rich sequence (residues 258-290) suggested by Hektor et al. (J. Biol. Chem. 277, p. 46966-46973, 2002) as a putative metal binding site. As Fe and Zn share very similar binding characteristics, the same amino acids responsible for the Fe-binding in the ADHs may coordinate the Zn-atom of MeDH. From the alignment, the following four amino acids are likely to constitute the metal binding site in MeDH (numbering transferred to Genomatica gene ID 2315): D193, H197, H262, H276 
     Amino acids considered important for cofactor binding in the  Zymomonas  ADH are mostly conserved in the MeDH sequence. The respective residues are listed below for Genomatica gene ID 2315. If the respective amino acid differs, the  Zymomonas  ADH residue is noted in parentheses: D38, D70(N), G97, S98, T137, T138, T146, L148(F), L178 
     To cast a wide net, residues in a distance of 8 Å or less from the Cl atom of propanediol were identified and are listed together with the annotated  Zymomonas  residues below. Residue numbers were transferred to Genomatica gene ID 2315 and the respective amino acids in the original protein are given in parentheses. 
     From  Zymomonas  ADH-2: L148(F149), V150(I151), P161(A162), F253(F254), L258(L259), H266(H267), D359(D360), V360(A361), C361(C362) 
     From  E. coli  FucO (≤8 Å distance): T141(T144), G142(A145), S143(A146), L148(N151), A149(Y152), H266(H267) 
     Mapping of the suggested mutagenesis sites onto the structure model of MeDH shows that the residues selected as target sites line the entrance to the active site of the monomer. Positions 253, 258, 266, and 359 were found to be strictly conserved, suggesting that they are more likely to be essential for function and were therefore eliminated from the list of residues identified for mutations. 
     For the remaining five residues, amino acids for substitution were selected based on their occurrence in related sequences. Only for position 148 which was annotated with having a role in limiting the substrate size as well as positioning the nicotinamide ring in the active site, a full panel of amino acids (NNK) is proposed. Narrowing down the pool of substitutions in the other four positions made it possible to include additional target sites while maintaining a reasonable library size. The following three sites were added based on their proposed function and location in Fe-dependent ADHs: D70, T145, S147. 
     When comparing the variation for the respective positions in a sequence alignment it was noted that one of the residues is homolog to residue 160 in an in house tested alcohol dehydrogenase. As a P160G mutation increased the activity of this alcohol dehydrogenase, a glycine was added to the list of substitutions in the respective MeDH position. The table below summarizes the final list of targeted residues and substitutions (positions based on gene 2315A): 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Position 
                 amino acids 
                 Variants 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 70 
                 D, N 
                 2 
               
               
                 145 
                 T, M 
                 2 
               
               
                 147 
                 S, R 
                 2 
               
               
                 148 
                 NNK 
                 20 
               
               
                 150 
                 V, I 
                 2 
               
               
                 161 
                 P, A, V, G 
                 4 
               
               
                 360 
                 V, A, G 
                 3 
               
               
                 361 
                 C, N 
                 2 
               
               
                   
                 Total 
                 7680 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Amino acids mutations with respect to SEQ ID NO: 1, providing greater than two fold activity when present as single mutations: 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 In Vitro Activity 
                   
                 Wild-type Amino 
                 Position and 
                 Position 
                 Top Hits (at 
                 x = Top 16 
               
               
                   
                 (Secondary Screen; 
                 In Vivo Formaldehyde 
                 Acid and Position 
                 Substitution 
                 with 
                 least 3x in 
                 for initial 
               
               
                   
                 (Average of triplicate 
                 Activity (fold over 
                 with respect 
                 with respect 
                 respect 
                 vitro + 2x 
                 combinatorial 
               
               
                 Mutation 
                 assays) 
                 wild-type) 
                 to 2315 
                 to 2315 
                 to 2315 
                 in vivo) 
                 library 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 D60E 
                 5.8 
                 0.6 
                 D60 
                 60E 
                 60 
                   
                   
               
               
                 N87K 
                 3.0 
                 0.6 
                 N87 
                 87K 
                 87 
               
               
                 S99T 
                 3.3 
                 0.9 
                 S99 
                 99T 
                 99 
               
               
                 A103V 
                 3.1 
                 1.9 
                 A103 
                 103V 
                 103 
               
               
                 V109Y 
                 2.0 
                 4.3 
                 V109 
                 109Y 
                 109 
               
               
                 R115H 
                 2.6 
                 3.9 
                 R115 
                 115H 
                 115 
               
               
                 I116F 
                 3.4 
                 1.5 
                 I116 
                 116F 
                 116 
               
               
                 N117D 
                 2.6 
                 2.0 
                 N117 
                 117D 
                 117 
               
               
                 G121D 
                 2.7 
                 2.0 
                 G121 
                 121D 
                 121 
                   
                 x 
               
               
                 G121E 
                 2.5 
                 1.6 
                 G121 
                 121E 
                 121 
               
               
                 G121L 
                 2.7 
                 1.3 
                 G121 
                 121L 
                 121 
               
               
                 G121M 
                 2.6 
                 1.8 
                 G121 
                 121M 
                 121 
               
               
                 G121R 
                 2.8 
                 1.7 
                 G121 
                 121R 
                 121 
               
               
                 G121S 
                 2.8 
                 2.6 
                 G121 
                 121S 
                 121 
                   
                 x 
               
               
                 G121T 
                 2.7 
                   
                 G121 
                 121T 
                 121 
               
               
                 G121V 
                 3.9 
                 1.5 
                 G121 
                 121V 
                 121 
               
               
                 G121W 
                 2.5 
                 1.7 
                 G121 
                 121W 
                 121 
               
               
                 G121Y 
                 3.1 
                 1.5 
                 G121 
                 121Y 
                 121 
               
               
                 V122P 
                 2.5 
                 2.3 
                 V122 
                 122P 
                 122 
               
               
                 N123L 
                 2.7 
                 1.8 
                 N123 
                 123L 
                 123 
               
               
                 N123R 
                 2.7 
                 1.6 
                 N123 
                 123R 
                 123 
               
               
                 N123Y 
                 3.0 
                 1.8 
                 N123 
                 123Y 
                 123 
               
               
                 S124I 
                 3.4 
                 0.9 
                 S124 
                 124I 
                 124 
               
               
                 S124L 
                 2.4 
                 1.2 
                 S124 
                 124L 
                 124 
               
               
                 V125C 
                 2.6 
                 3.2 
                 V125 
                 125C 
                 125 
               
               
                 V125G 
                 2.6 
                 3.3 
                 V125 
                 125G 
                 125 
               
               
                 V125W 
                 2.7 
                 3.9 
                 V125 
                 125W 
                 125 
               
               
                 E126G 
                 4.0 
                 0.6 
                 E126 
                 126G 
                 126 
               
               
                 K127C 
                 2.6 
                 3.9 
                 K127 
                 127C 
                 127 
               
               
                 K127R 
                 2.5 
                 3.3 
                 K127 
                 127R 
                 127 
               
               
                 P128A 
                 2.3 
                 3.0 
                 P128 
                 128A 
                 128 
               
               
                 P128R 
                 2.4 
                 3.3 
                 P128 
                 128R 
                 128 
               
               
                 V129A 
                 3.7 
                 1.9 
                 V129 
                 129A 
                 129 
               
               
                 V129M 
                 4.7 
                 1.4 
                 V129 
                 129M 
                 129 
                   
                 X 
               
               
                 V129P 
                 2.8 
                 1.3 
                 V129 
                 129P 
                 129 
               
               
                 V129S 
                 3.0 
                 1.5 
                 V129 
                 129S 
                 129 
               
               
                 V130F 
                 2.1 
                 1.4 
                 V130 
                 130F 
                 130 
               
               
                 V130Y 
                 2.0 
                 2.0 
                 V130 
                 130Y 
                 130 
               
               
                 A134T 
                 4.4 
                 0.0 
                 A134 
                 134T 
                 134 
               
               
                 A149T 
                 5.9 
                 0.8 
                 A149 
                 149T 
                 149 
               
               
                 V150A 
                 3.0 
                 1.2 
                 V150 
                 150A 
                 150 
               
               
                 K157N 
                 3.6 
                 0.8 
                 K157 
                 157N 
                 157 
               
               
                 V158E 
                 2.6 
                 4.3 
                 V158 
                 158E 
                 158 
                   
                 x 
               
               
                 V158H 
                 2.2 
                 2.8 
                 V158 
                 158H 
                 158 
               
               
                 V158K 
                 2.0 
                 2.6 
                 V158 
                 158K 
                 158 
               
               
                 V158W 
                 2.5 
                 4.0 
                 V158 
                 158W 
                 158 
               
               
                 D164N 
                 3.7 
                 0.7 
                 D164 
                 164N 
                 164 
               
               
                 G270S 
                 2.8 
                 2.9 
                 G270 
                 270S 
                 270 
                   
                 x 
               
               
                 K345E 
                 4.1 
                 0.6 
                 K345 
                 345E 
                 345 
               
               
                 N355D 
                 3.3 
                 1.7 
                 N355 
                 355D 
                 355 
                   
                 x 
               
               
                 C361R 
                 3.0 
                 0.8 
                 C361 
                 361R 
                 361 
               
               
                 D38N 
                 5.4 
                 7.3 
                 D38 
                 38N 
                 38 
                 + 
               
               
                 P71I 
                 7.6 
                 2.5 
                 P71 
                 71I 
                 71 
                 + 
               
               
                 P71V 
                 6.8 
                 3.5 
                 P71 
                 71V 
                 71 
                 + 
               
               
                 G107S 
                 5.1 
                 2.5 
                 G107 
                 107S 
                 107 
                 + 
                 x 
               
               
                 L108V 
                 6.4 
                 3.9 
                 L108 
                 108V 
                 108 
                 + 
               
               
                 L108W 
                 7.4 
                 4.8 
                 L108 
                 108W 
                 108 
                 + 
               
               
                 N117Q 
                 3.3 
                 4.3 
                 N117 
                 117Q 
                 117 
                 + 
               
               
                 N123D 
                 3.0 
                 2.0 
                 N123 
                 123D 
                 123 
                 + 
                 x 
               
               
                 N123I 
                 3.1 
                 2.5 
                 N123 
                 123I 
                 123 
                 + 
                 X 
               
               
                 P128S 
                 3.1 
                 3.7 
                 P128 
                 128S 
                 128 
                 + 
               
               
                 V130I 
                 3.4 
                 2.3 
                 V130 
                 130I 
                 130 
                 + 
                 X 
               
               
                 S143T 
                 3.8 
                 2.4 
                 S143 
                 143T 
                 143 
                 + 
                 X 
               
               
                 T146N 
                 3.4 
                 2.0 
                 T146 
                 146N 
                 146 
                 + 
                 x 
               
               
                 A149L 
                 4.8 
                 3.0 
                 A149 
                 149L 
                 149 
                 + 
               
               
                 A149M 
                 4.6 
                 2.7 
                 A149 
                 149M 
                 149 
                 + 
               
               
                 A149V 
                 4.9 
                 2.9 
                 A149 
                 149V 
                 149 
                 + 
                 x 
               
               
                 I163Q 
                 4.4 
                 2.1 
                 I163 
                 163Q 
                 163 
                 + 
               
               
                 Q267H 
                 6.3 
                 4.3 
                 Q267 
                 267H 
                 267 
                 + 
                 X 
               
               
                 G270M 
                 4.3 
                 4.2 
                 G270 
                 270M 
                 270 
                 + 
               
               
                 G270Y 
                 4.2 
                 4.2 
                 G270 
                 270Y 
                 270 
                 + 
               
               
                 V360G 
                 4.9 
                 2.1 
                 V360 
                 360G 
                 360 
                 + 
                 x 
               
               
                 V360K 
                 4.6 
                 2.6 
                 V360 
                 360K 
                 360 
                 + 
               
               
                 V360R 
                 4.6 
                 3.4 
                 V360 
                 360R 
                 360 
                 + 
                 x 
               
               
                 V360S 
                 4.5 
                 3.5 
                 V360 
                 360S 
                 360 
                 + 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Additional combination mutations with respect to 
               
               
                 SEQ ID NO: 1 (generated from rationale design) 
               
            
           
           
               
               
               
            
               
                   
                 In Vitro Activity 
                   
               
               
                   
                 (fald generation; 
                 In Vivo Activity 
               
               
                   
                 average of 
                 (formaldehyde 
               
               
                 Mutations 
                 triplicates) 
                 generation) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 D70N, L148G, P161G, V360A 
                 6.2 
                 2.92 
               
               
                 D70N, L148G, V360A, C361N 
                 6.9 
                 3.02 
               
               
                 D70N, L148V, V150I, P161A, 
                 6.3 
                 3.91 
               
               
                 V360G 
               
               
                 D70N, L148V, V360G 
                 6.6 
                 3.95 
               
               
                 D70N, P161A, V360A 
                 6.2 
                 3.93 
               
               
                 D70N, P161V, V360G, C361N 
                 5.6 
                 3.95 
               
               
                 D70N, V150I, P161A, V360A 
                 6.1 
                 3.69 
               
               
                 D70N, V150I, P161V, V360G, 
                 6.0 
                 3.35 
               
               
                 C361N 
               
               
                 E48D, L148V, P161A, V360A 
                 7.7 
                 5.79 
               
               
                 L148G, P161A, V360A, C361N 
                 5.7 
                 2.42 
               
               
                 L148G, P161A, V360G 
                 5.6 
                 3.0 
               
               
                 L148G, P161A, V360G, C361N 
                 6.1 
                 3.00 
               
               
                 L148G, P161G, V360A 
                 7.4 
                 3.0 
               
               
                 L148G, P161G, V360G, C361N 
                 6.8 
                 2.50 
               
               
                 L148G, V360A, C361N 
                 6.3 
                 3.80 
               
               
                 L148G, V360G, C361N 
                 6.6 
                 3.67 
               
               
                 L148I, P161G, V360G 
                 5.5 
                 3.89 
               
               
                 L148I, P161V, V360G 
                 7.1 
                 5.51 
               
               
                 L148T, V150I, V360A 
                 5.7 
                 6.92 
               
               
                 L148T, V360G 
                 5.6 
                 6.26 
               
               
                 L148V, P161A, V360A 
                 7.5 
                 5.7 
               
               
                 L148V, V150I, P161A, V360A 
                 6.4 
                 5.7 
               
               
                 L148V, V150I, P161A, V360A, 
                 6.1 
                 1.97 
               
               
                 C361N 
               
               
                 L148V, V150I, P161A, V360G 
                 8.2 
                 5.27 
               
               
                 L148V, V150I, P161A, V360G, 
                 6.6 
                 4.23 
               
               
                 C361N 
               
               
                 L148V, V150I, P161A, V360G, 
                 5.9 
                 3.80 
               
               
                 C361N 
               
               
                 L148V, V150I, P161G, V360A 
                 6.6 
                 5.14 
               
               
                 L148V, V150I, P161V, V360G, 
                 7.3 
                 4.07 
               
               
                 C361N 
               
               
                 L148W, P161A, V360A, C361N 
                 5.7 
                 1.06 
               
               
                 N112K, S147R, P161A, V360A 
                 9.7 
                 5.40 
               
               
                 P161A, Q217K, V360A, C361N 
                 6.7 
                 4.15 
               
               
                 P161A, V360A, C361N 
                 8.0 
                 3.58 
               
               
                 P161A, V360G 
                 7.4 
                 4.8 
               
               
                 P161V, E358G, V360G 
                 6.0 
                 4.96 
               
               
                 P161V, V360A, C361N 
                 9.7 
                 5.2 
               
               
                 P161V, V360G 
                 6.4 
                 4.0 
               
               
                 P65Q, L148G, V150I, P161A, 
                 5.5 
                 2.32 
               
               
                 V360G, C361N 
               
               
                 S147R, L148A, V150I, P161A, 
                 5.6 
                 4.07 
               
               
                 V360G 
               
               
                 S147R, L148F, V150I, P161G, 
                 6.7 
                 6.0 
               
               
                 V360G 
               
               
                 S147R, L148V, P161G, V360A 
                 6.3 
                 4.4 
               
               
                 S147R, L148V, P161V, V360G 
                 8.9 
                 6.15 
               
               
                 S147R, L148V, V150I, P161A, 
                 5.8 
                 3.10 
               
               
                 C361N 
               
               
                 S147R, L148V, V150I, P161G, 
                 6.7 
                 4.17 
               
               
                 V360G 
               
               
                 S147R, P161A, V360A 
                 6.3 
                 5.53 
               
               
                 S147R, P161A, V360A, C361N 
                 6.6 
                 3.6 
               
               
                 S147R, P161A, V360G 
                 5.7 
                 5.22 
               
               
                 S147R, P161V, V360G 
                 10.0 
                 5.84 
               
               
                 S147R, P161V, V360G, C361N 
                 5.7 
                 2.11 
               
               
                 S147R, V150I, P161V, V360A 
                 5.7 
                 5.13 
               
               
                 S147R, V150I, V360A, C361N 
                 5.6 
                 4.66 
               
               
                 T145M, L148I, V360G 
                 8.0 
                 3.48 
               
               
                 V150I, I302V, V360G, C361N 
                 7.8 
                 4.01 
               
               
                 V150I, P161A, C361N 
                 5.8 
                 3.42 
               
               
                 V150I, P161G, V360A, C361N 
                 5.6 
                 2.88 
               
               
                 V150I, P161G, V360G 
                 5.6 
                 4.28 
               
               
                 V150I, P161G, V360G, C361N 
                 7.1 
                 3.26 
               
               
                 V150I, P161V, C361N 
                 5.7 
                 3.9 
               
               
                 V150I, P161V, K354R, V360A, 
                 8.8 
                 4.24 
               
               
                 C361N 
               
               
                 V150I, P161V, V360A, C361N 
                 6.0 
                 5.04 
               
               
                 V150I, P161V, V360G, C361N 
                 5.6 
                 4.9 
               
               
                 V150I, V360A, C361N 
                 6.4 
                 4.43 
               
               
                 V150I, V360G 
                 6.8 
                 6.79 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Additional combination mutations with respect 
               
               
                 to SEQ ID NO: 1 (generated from epPCR) 
               
            
           
           
               
               
               
            
               
                   
                   
                 In Vitro Assay-Average of 
               
               
                   
                 Mutations 
                 Triplicates 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 S11T, T74S, G269S, V344A 
                 5.75 
               
               
                   
                 K84R, I163T 
                 5.38 
               
               
                   
                 V122A, I163N 
                 5.01 
               
               
                   
                 G107S, F333L 
                 4.55 
               
               
                   
                 V129M, T152M, G343D 
                 4.45 
               
               
                   
                 I63F, N355K 
                 4.44 
               
               
                   
                 G107S, F333L 
                 4.42 
               
               
                   
                 E86K, S99T, A149V 
                 4.41 
               
               
                   
                 N53I, V158E 
                 4.38 
               
               
                   
                 N355I, K379M 
                 4.30 
               
               
                   
                 H42Q, G107S 
                 4.08 
               
               
                   
                 Q120H, I163N 
                 4.06 
               
               
                   
                 A149V, I323M 
                 4.04 
               
               
                   
                 G107S, F333L 
                 3.69 
               
               
                   
                 D164G, K181R 
                 3.68 
               
               
                   
                 A155V, R298H, N355D 
                 3.66 
               
               
                   
                 N123D, E165G 
                 3.65 
               
               
                   
                 I163F, L186M 
                 3.65 
               
               
                   
                 G121A, T296S 
                 3.63 
               
               
                   
                 I94V, S99P, N123I 
                 3.62 
               
               
                   
                 E126V, V129M, V344G 
                 3.60 
               
               
                   
                 Q120R, S143T 
                 3.58 
               
               
                   
                 G256C, A316V 
                 3.56 
               
               
                   
                 P161Q, G312V 
                 3.52 
               
               
                   
                 L226M, A300T, V360A 
                 3.49 
               
               
                   
                 S337C, E350K, N355D, Q363K 
                 3.43 
               
               
                   
                 D81G, V158E 
                 3.42 
               
               
                   
                 I106L, N117Y, E126V 
                 3.40 
               
               
                   
                 G107S, G121D 
                 3.36 
               
               
                   
                 V61A, V158E 
                 3.31 
               
               
                   
                 N53I, V158E 
                 3.28 
               
               
                   
                 N117Y, T190S 
                 3.16 
               
               
                   
                 S124R, I199V 
                 3.13 
               
               
                   
                 K354M, C361R 
                 2.97 
               
               
                   
                 A184T, C361R 
                 2.86 
               
               
                   
                 E56K, Q267H 
                 2.85 
               
               
                   
                 S124R, E126G 
                 2.79 
               
               
                   
                 T190A, N355K 
                 2.77 
               
               
                   
                 P71T, F333L 
                 2.75 
               
               
                   
                 G107S, F333L 
                 2.74 
               
               
                   
                 N123I, P336L 
                 2.71 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Additional combination mutations with respect to SEQ ID NO: 1: 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 in vivo 
               
               
                   
                   
                 Average 
                 Formaldehyde 
               
               
                   
                 Mutation 
                 Secondary 
                 assay 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 D38D/A149V 
                 5.1 
                 3.0 
               
               
                   
                 D38N/V163V 
                 6.6 
                 9.9 
               
               
                   
                 D73D/L108V 
                 6.8 
                 3.9 
               
               
                   
                 G121R/P161S 
                 4.4 
                 2.8 
               
               
                   
                 G121R/P161S 
                 3.9 
                 2.8 
               
               
                   
                 G121R/P161S 
                 4.1 
                 2.9 
               
               
                   
                 N112R/P161S 
                 7.8 
                 3.2 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Putative motifs and roles of the amino acid 
               
               
                 positions with respect to SEQ ID NO: 1. 
               
            
           
           
               
               
               
               
               
            
               
                 Wild-type 
                   
                   
                   
                   
               
               
                 Sequence 
                 From 
                 To 
                 Length 
                 Rationale 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 DAF 
                 38 
                 40 
                 3 
                 NADH binding 
               
               
                 D 
                 70 
                 70 
                 1 
                 NADH binding 
               
               
                 G 
                 95 
                 95 
                 1 
                 Activation 
               
               
                 GS 
                 97 
                 98 
                 2 
                 NADH &amp; Activation 
               
               
                 TT 
                 137 
                 138 
                 2 
                 NADH binding 
               
               
                 TGS 
                 141 
                 143 
                 3 
                 NADH binding 
               
               
                 TTSLAV 
                 145 
                 150 
                 6 
                 NADH &amp; Substrate binding 
               
               
                 PVI 
                 161 
                 163 
                 3 
                 Substrate &amp; NADH (956 Gtp) 
               
               
                 L 
                 178 
                 178 
                 1 
                 NADH binding 
               
               
                 A 
                 201 
                 201 
                 1 
                 956 gTp 
               
               
                 F 
                 253 
                 253 
                 1 
                 Substrate binding 
               
               
                 L 
                 258 
                 258 
                 1 
                 Substrate binding 
               
               
                 H 
                 266 
                 266 
                 1 
                 Substrate binding 
               
               
                 G 
                 270 
                 270 
                 1 
                 956 gtP 
               
               
                 DVC 
                 359 
                 361 
                 3 
                 Substrate binding 
               
            
           
           
               
               
               
            
               
                   
                 30 
                 Total 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 In vivo assays showing formaldehyde (HCHO) 
               
               
                 production by various NNOMO comprising a 
               
               
                 plasmid expressing a methanol dehydrogenase 
               
            
           
           
               
               
               
            
               
                   
                 Accession number 
                 HCHO (μM) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 Experiment 1 
                   
               
               
                   
                 EIJ77596.1 
                 &gt;50 
               
               
                   
                 EIJ83020.1 
                 &gt;20 
               
               
                   
                 EIJ80770.1 
                 &gt;50 
               
               
                   
                 ZP_10132907.1 
                 &gt;20 
               
               
                   
                 ZP_10132325.1 
                 &gt;20 
               
               
                   
                 ZP_10131932.1 
                 &gt;50 
               
               
                   
                 ZP_07048751.1 
                 &gt;50 
               
               
                   
                 YP_001699778.1 
                 &gt;50 
               
               
                   
                 YP_004681552.1 
                 &gt;10 
               
               
                   
                 ZP_10819291.1 
                 &lt;1 
               
               
                   
                 Empty vector 
                 2.33 
               
               
                   
                 Experiment 2 
               
               
                   
                 EIJ77596.1 
                 &gt;50 
               
               
                   
                 NP_00659.2 
                 &gt;50 
               
               
                   
                 YP_004758576.1 
                 &gt;20 
               
               
                   
                 ZP_09352758.1 
                 &gt;50 
               
               
                   
                 ZP_10129817.1 
                 &gt;20 
               
               
                   
                 YP_001139613.1 
                 &gt;20 
               
               
                   
                 NP_014555.1 
                 &gt;10 
               
               
                   
                 WP_007139094.1 
                 &gt;10 
               
               
                   
                 NP_343875.1 
                 &gt;1 
               
               
                   
                 YP_006863258 
                 &gt;1 
               
               
                   
                 NP_394301.1 
                 &gt;1 
               
               
                   
                 ZP_10750164.1 
                 &gt;1 
               
               
                   
                 YP_023929.1 
                 &gt;1 
               
               
                   
                 ZP_08977641.1 
                 &lt;1 
               
               
                   
                 ZP_10117398.1 
                 &lt;1 
               
               
                   
                 YP_004108045.1 
                 &lt;1 
               
               
                   
                 ZP_09753449.1 
                 &lt;1 
               
               
                   
                 Empty vector 
                 0.17 
               
               
                   
                 Experiment 3 
               
               
                   
                 EIJ77596.1 
                 &gt;50 
               
               
                   
                 NP_561852 
                 &gt;50 
               
               
                   
                 YP_002138168 
                 &gt;50 
               
               
                   
                 YP_026233.1 
                 &gt;50 
               
               
                   
                 YP_001447544 
                 &gt;50 
               
               
                   
                 Metalibrary 
                 &gt;50 
               
               
                   
                 YP_359772 
                 &gt;50 
               
               
                   
                 ZP_01220157.1 
                 &gt;50 
               
               
                   
                 ZP_07335453.1 
                 &gt;20 
               
               
                   
                 YP_001337153 
                 &gt;20 
               
               
                   
                 YP_694908 
                 &gt;20 
               
               
                   
                 NP_717107 
                 &gt;20 
               
               
                   
                 AAC45651 
                 &gt;10 
               
               
                   
                 ZP_11313277.1 
                 &gt;10 
               
               
                   
                 ZP_16224338.1 
                 &gt;10 
               
               
                   
                 YP_001113612 
                 &gt;10 
               
               
                   
                 YP_004860127 
                 &gt;10 
               
               
                   
                 YP_003310546 
                 &gt;10 
               
               
                   
                 YP_001343716 
                 &gt;10 
               
               
                   
                 NP_717107 
                 &gt;10 
               
               
                   
                 YP_002434746 
                 &gt;10 
               
               
                   
                 Empty vector 
                 0.11 
               
               
                   
                 Experiment 4 
               
               
                   
                 EIJ77596.1 
                 &gt;50 
               
               
                   
                 ZP_10241531.1 
                 &gt;50 
               
               
                   
                 YP_005052855 
                 &gt;50 
               
               
                   
                 ZP_10132907.1 
                 &gt;50 
               
               
                   
                 NP_617528 
                 &gt;50 
               
               
                   
                 NP_617528 
                 &gt;50 
               
               
                   
                 ZP_08977641.1 
                 &gt;20 
               
               
                   
                 YP_237055 
                 &gt;20 
               
               
                   
                 Empty vector 
                 &lt;20 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Wild-type enzymology 
               
            
           
           
               
               
               
               
            
               
                   
                 Methanol 
                 Ethanol 
                 EtOH/MeOH 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 k cat   
                 K M   
                 k cat /K M   
                 k cat   
                 K M   
                 k cat /K M   
                 k cat   
                 k cat /K M   
               
               
                   
                 (s −1 ) 
                 (mM) 
                 (s −1  mM −1 ) 
                 (s −1 ) 
                 (mM) 
                 (s −1  mM −1 ) 
                 (s −1 ) 
                 (s −1  mM −1 ) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 MeDH  B. methanolicus  (2315A + 
                 0.03 
                 70 
                 4.3 × 10 −4   
                 0.16 
                 209 
                 7.7 × 10 −4   
                 5.3 
                 1.7 
               
               
                 2317A) 
               
               
                 Human ADHB1 (2479B) 
                 0.27 
                 290 
                 9.3 × 10 −4   
                 2.85 
                 1 
                 2.85 
                 11 
                 3061 
               
               
                 
                   Corynebacterium glutamicum 
                 
                 0.7 
                 3 
                 0.23  
                 4.8 
                 6.8 
                 0.71 
                 7 
                 3 
               
               
                 (2496B) 
               
               
                 
                   Geobacillus 
                 
                 0.06 
                 20 
                 0.003 
                 1.3 
                 82 
                 0.016 
                 22 
                 5 
               
               
                   stearothermophilus  (2480B) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 
                   Saccharomyces cerevisiae 
                 
                 Not available 
                 340 
                 17 
                 20 
                 N a 
                 N a 
               
               
                 (2497B) 
               
               
                 
                   Flavobacterium frigidimaris 
                 
                 Not available 
                 27 
                 0.17 
                 158 
                 N a 
                 N a 
               
               
                 (2499B) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   Escherichia coli  (58) 
                 0.047 
                 2500 
                 1.9 × 10 −5   
                 1.5 
                 115 
                 0.013 
                 32 
                 699 
               
               
                   Clostridium perfringens  (2430) 
                 0.009 
                 84 
                 1.1 × 10 −4   
                 0.73 
                 33 
                 0.022 
                 91 
                 232 
               
               
                   Geobacter bemijiensis  (2449) 
                 0.022 
                 88 
                 2.5 × 10 −4   
                 0.95 
                 72 
                 0.013 
                 43 
                 53 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Wild type and variant enzymology 
               
            
           
           
               
               
               
               
            
               
                   
                 k cat , 
                 K M , 
                 k cat /K M , 
               
               
                 Variant 
                 min −1   
                 mM 
                 M −1  min −1   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Wild-type 
                 4.9 
                 95 
                 53 
               
               
                 V360R 
                 12 
                 280 
                 43 
               
               
                 V360G 
                 22 
                 130 
                 170 
               
               
                 S147R, L148F, V150I, P161G, V360G 
                 4.4 
                 370 
                 12 
               
               
                 P161V, V360A, C361N 
                 12 
                 180 
                 70 
               
               
                 S147R, P161A, V360G 
                 9.9 
                 180 
                 55 
               
               
                 S147R, P161V, V360G 
                 8.7 
                 190 
                 47 
               
               
                 N112K, S147R, P161A, V360A 
                 4.2 
                 210 
                 20 
               
               
                 A149V 
                 7.2 
                 410 
                 18 
               
               
                   
               
               
                 Activity using methanol was determined. 
               
               
                   a Assays were performed at pH 7.6, 37° C. in the presence of 2 mM NAD. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Wild type and variant enzymology 
               
            
           
           
               
               
               
               
            
               
                   
                 k cat , 
                 K M , 
                 k cat /K M , 
               
               
                 Variant 
                 min −1   
                 mM 
                 M −1  min −1   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Wild-type 
                 3.8 
                 310 
                 13 
               
               
                 V360R 
                 3.7 
                 210 
                 18 
               
               
                 V360G 
                 3.2 
                 280 
                 12 
               
               
                 S147R, L148F, V150I, P161G, V360G 
                 5.3 
                 240 
                 23 
               
               
                 P161V, V360A, C361N 
                 52 
                 110 
                 500 
               
               
                 S147R, P161A, V360G 
                 15 
                 190 
                 76 
               
               
                 S147R, P161V, V360G 
                 15 
                 280 
                 55 
               
               
                 N112K, S147R, P161A, V360A 
                 7.8 
                 120 
                 66 
               
               
                 A149V 
                 2.8 
                 460 
                 62 
               
               
                   
               
               
                 1,4-Butanediol-dependent steady-state kinetic parameters for wild-type and variant methanol dehydrogenase. a   
               
               
                   a Assays were performed at pH 7.6, 37° C. in the presence of 2 mM NAD. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Substitution templates 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 AA/NA 
                   
                   
                   
                   
                 % 
                   
                   
               
               
                 Tested 
                 SEQ ID 
                   
                   
                   
                 AA 
                 Identity 
                 % 
                 # 
               
               
                 Activity 
                 NO: 
                 GenBankID 
                 GI No. 
                 Organism 
                 length 
                 (global) 
                 Similarity 
                 gap 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 +++ 
                 1/2 
                 EIJ77596.1 
                 387585261 
                 
                   Bacillus 
                 
                 382 
                 100 
                 100 
                 0 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 MGA3 
               
               
                 n.d 
                 3/4 
                 AAA22593.1 
                 143175 
                 
                   Bacillus 
                 
                 381 
                 97 
                 99 
                 0 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 C1 
               
               
                 + 
                 5/6 
                 EIJ77618.1 
                 387585284 
                 
                   Bacillus 
                 
                 383 
                 93 
                 96 
                 0 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 PB1 
               
               
                 + 
                 7/8 
                 EIJ78790.1 
                 387586466 
                 
                   Bacillus 
                 
                 383 
                 90 
                 93 
                 0 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 PB1 
               
               
                 + 
                  9/10 
                 EIJ80770.1 
                 387588449 
                 
                   Bacillus 
                 
                 385 
                 62 
                 79 
                 1 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 MGA3 
               
               
                 ++ 
                 11/12 
                 EIJ78397.1 
                 387586073 
                 
                   Bacillus 
                 
                 385 
                 61 
                 78 
                 1 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 PB1 
               
               
                 + 
                 13/14 
                 EIJ83020.1 
                 387590701 
                 
                   Bacillus 
                 
                 385 
                 61 
                 79 
                 1 
               
               
                   
                   
                   
                   
                 
                   methanolicus 
                 
               
               
                   
                   
                   
                   
                 MGA3 
               
               
                 ++ 
                 15/16 
                 EFI69743.1 
                 298729190 
                 
                   Lysinibacillus 
                 
                 401 
                 56 
                 74 
                 5 
               
               
                   
                   
                   
                   
                 
                   fusiformis 
                 
               
               
                 + 
                 17/18 
                 YP_004860127.1 
                 347752562 
                 
                   Bacillus 
                 
                 386 
                 56 
                 76 
                 1 
               
               
                   
                   
                   
                   
                 
                   coagulans 
                 
               
               
                   
                   
                   
                   
                 36D1 
               
               
                 ++ 
                 19/20 
                 YP_001699778.1 
                 169829620 
                 
                   Lysinibacillus 
                 
                 402 
                 54 
                 73 
                 5 
               
               
                   
                   
                   
                   
                 
                   sphaericus 
                 
               
               
                 + 
                 21/22 
                 ZP_11313277.1 
                 410459529 
                 
                   Bacillus 
                 
                 386 
                 54 
                 73 
                 1 
               
               
                   
                   
                   
                   
                 
                   azotoformans 
                 
               
               
                   
                   
                   
                   
                 LMG 9581 
               
               
                 n.d 
                 23/24 
                 ZP_05587334.1 
                 257139072 
                 
                   Burkholderia 
                 
                 390 
                 54 
                 70 
                 2 
               
               
                   
                   
                   
                   
                 
                   thailandensis 
                 
               
               
                   
                   
                   
                   
                 E264 
               
               
                 + 
                 25/26 
                 YP_004681552.1 
                 339322658 
                 
                   Cupriavidus 
                 
                 390 
                 53 
                 70 
                 2 
               
               
                   
                   
                   
                   
                   necator  N-1 
               
               
                 n.d 
                 27/28 
                 AGF87161 
                 451936849 
                 uncultured 
                 393 
                 53 
                 71 
                 3 
               
               
                   
                   
                   
                   
                 organism 
               
               
                 ++ 
                 29/30 
                 YP_002138168.1 
                 197117741 
                 
                   Geobacter 
                 
                 387 
                 52 
                 71 
                 1 
               
               
                   
                   
                   
                   
                 
                   bemidjiensis 
                 
               
               
                   
                   
                   
                   
                 Bem 
               
               
                 ++ 
                 31/32 
                 YP_359772.1 
                 78043360 
                 
                   Carboxydothermus 
                 
                 383 
                 52 
                 72 
                 0 
               
               
                   
                   
                   
                   
                 
                   hydrogenoformans 
                 
               
               
                   
                   
                   
                   
                 Z-2901 
               
               
                 + 
                 33/34 
                 YP_001343716.1 
                 152978087 
                 
                   Actinobacillus 
                 
                 385 
                 51 
                 71 
                 1 
               
               
                   
                   
                   
                   
                 
                   succinogenes 
                 
               
               
                   
                   
                   
                   
                 130Z 
               
               
                 + 
                 35/36 
                 ZP_16224338.1 
                 421788018 
                 
                   Acinetobacter 
                 
                 390 
                 51 
                 70 
                 2 
               
               
                   
                   
                   
                   
                 
                   baumannii 
                 
               
               
                   
                   
                   
                   
                 Naval-82 
               
               
                 + 
                 37/38 
                 AAC45651.1 
                 2393887 
                 
                   Clostridium 
                 
                 385 
                 51 
                 69 
                 1 
               
               
                   
                   
                   
                   
                 
                   pasteurianum 
                 
               
               
                   
                   
                   
                   
                 DSM 525 
               
               
                 n.d 
                 39/40 
                 YP_007491369.1 
                 452211255 
                 
                   Methanosarcina 
                 
                 386 
                 51 
                 71 
                 1 
               
               
                   
                   
                   
                   
                 
                   mazei 
                 
               
               
                   
                   
                   
                   
                 Tuc01 
               
               
                 n.d 
                 41/42 
                 YP_002434746 
                 218885425 
                 
                   Desulfovibrio 
                 
                 393 
                 50 
                 70 
                 3 
               
               
                   
                   
                   
                   
                   vulgaris  str. 
               
               
                   
                   
                   
                   
                 ‘Miyazaki F’ 
               
               
                 ++ 
                 43/44 
                 YP_005052855 
                 374301216 
                 
                   Desulfovibrio 
                 
                 393 
                 49 
                 70 
                 3 
               
               
                   
                   
                   
                   
                   africanus  str. 
               
               
                   
                   
                   
                   
                 Walvis Bay 
               
               
                 ++ 
                 45/46 
                 NP_561852.1 
                 18309918 
                 
                   Clostridium 
                 
                 385 
                 49 
                 68 
                 1 
               
               
                   
                   
                   
                   
                 
                   perfringens 
                 
               
               
                   
                   
                   
                   
                 str. 13 
               
               
                 ++ 
                 47/48 
                 YP_001447544 
                 156976638 
                 
                   Vibrio 
                 
                 382 
                 49 
                 69 
                 0 
               
               
                   
                   
                   
                   
                 
                   campbellii 
                 
               
               
                   
                   
                   
                   
                 ATCC BAA- 
               
               
                   
                   
                   
                   
                 1116 
               
               
                 + 
                 49/50 
                 YP_001113612.1 
                 134300116 
                 
                   Desulfotomaculum 
                 
                 388 
                 49 
                 70 
                 2 
               
               
                   
                   
                   
                   
                   reducens  MI-1 
               
               
                 n.d 
                 51/52 
                 YP_011618 
                 46580810 
                 
                   Desulfovibrio 
                 
                 393 
                 49 
                 70 
                 3 
               
               
                   
                   
                   
                   
                   vulgaris  str. 
               
               
                   
                   
                   
                   
                 Hildenborough 
               
               
                 ++ 
                 53/54 
                 ZP_01220157.1 
                 90412151 
                 
                   Photobacterium 
                 
                 382 
                 48 
                 69 
                 0 
               
               
                   
                   
                   
                   
                 
                   profundum 
                 
               
               
                   
                   
                   
                   
                 3TCK 
               
               
                 ++ 
                 55/56 
                 YP_003990729.1 
                 312112413 
                 
                   Geobacillus 
                 
                 384 
                 48 
                 67 
                 1 
               
               
                   
                   
                   
                   
                 sp. Y4.1MC1 
               
               
                 + 
                 57/58 
                 ZP_07335453.1 
                 303249216 
                 
                   Desulfovibrio 
                 
                 393 
                 48 
                 69 
                 3 
               
               
                   
                   
                   
                   
                 
                   fructosovorans 
                 
               
               
                   
                   
                   
                   
                 JJ 
               
               
                 + 
                 59/60 
                 NP_717107 
                 24373064 
                 
                   Shewanella 
                 
                 382 
                 48 
                 66 
                 0 
               
               
                   
                   
                   
                   
                 
                   oneidensis 
                 
               
               
                   
                   
                   
                   
                 MR-1 
               
               
                 + 
                 61/62 
                 YP_003310546.1 
                 269122369 
                 
                   Sebaldella 
                 
                 384 
                 48 
                 68 
                 1 
               
               
                   
                   
                   
                   
                 
                   termitidis 
                 
               
               
                   
                   
                   
                   
                 ATCC 33386 
               
               
                 ++ 
                 63/64 
                 ZP_10241531.1 
                 390456003 
                 
                   Paenibacillus 
                 
                 384 
                 47 
                 67 
                 1 
               
               
                   
                   
                   
                   
                 
                   peoriae 
                 
               
               
                   
                   
                   
                   
                 KCTC 3763 
               
               
                 + 
                 65/66 
                 YP_001337153.1 
                 152972007 
                 
                   Klebsiella 
                 
                 387 
                 47 
                 67 
                 1 
               
               
                   
                   
                   
                   
                 
                   pneumoniae 
                 
               
               
                   
                   
                   
                   
                 subsp. 
               
               
                   
                   
                   
                   
                 
                   pneumoniae 
                 
               
               
                   
                   
                   
                   
                 MGH 78578 
               
               
                 ++ 
                 67/68 
                 YP_026233.1 
                 49176377 
                 
                   Escherichia 
                 
                 383 
                 46 
                 64 
                 0 
               
               
                   
                   
                   
                   
                 
                   coli 
                 
               
               
                 + 
                 69/70 
                 YP_694908 
                 110799824 
                 
                   Clostridium 
                 
                 382 
                 46 
                 69 
                 0 
               
               
                   
                   
                   
                   
                 
                   perfringens 
                 
               
               
                   
                   
                   
                   
                 ATCC 13124 
               
               
                 n.d 
                 71/72 
                 YP_725376.1 
                 113866887 
                 
                   Ralstonia 
                 
                 366 
                 46 
                 60 
                 15 
               
               
                   
                   
                   
                   
                   eutropha  H16 
               
               
                 n.d 
                 73/74 
                 YP_001663549 
                 167040564 
                 
                   Thermoanaerobacter 
                 
                 389 
                 45 
                 68 
                 2 
               
               
                   
                   
                   
                   
                 sp. X514 
               
               
                 n.d 
                 75/76 
                 EKC54576 
                 406526935 
                 human gut 
                 384 
                 37 
                 55 
                 3 
               
               
                   
                   
                   
                   
                 metagenome 
               
               
                 n.d 
                 77/78 
                 YP_001126968.1 
                 138896515 
                 
                   Geobacillus 
                 
                 387 
                 27 
                 44 
                 7 
               
               
                   
                   
                   
                   
                 
                   themodenitrificans 
                 
               
               
                   
                   
                   
                   
                 NG80-2 
               
               
                   
               
               
                 Percent identity is given based on global alignment to SEQ ID NO: 1. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Template Polypeptides 
               
               
                 Sequences of Template Polypeptides indicating 
               
               
                 exemplary substitutions, their positions 
               
               
                 and their corresponding positions in 
               
               
                 other template sequences: 
               
            
           
           
               
               
               
            
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 GI No. 
                 Protein Sequence 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 387585261 
                 MTTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVT   D   AFLHSTG 
               
               
                   
                   
                 LSEEVAKNIREAGV   D   VAIFPKAQPD   P   ADTQVHEGVDVFKQE   N   C 
               
               
                   
                   
                 DSLVSIGGGS   S   HDTA   K   AI   GLV   AANGG   RIN   DYQG   VNSVEKPVV   P 
               
               
                   
                   
                 WAITTTAGTG   S   ET   T   SL   AV   ITDSAR   KV   KMPV   ID   EKITPTVAIVDPE 
               
               
                   
                   
                 LMVKKPAGLTIATGMDALSHAIEAYVAKGATPVTDAFAIQAMK 
               
               
                   
                   
                 LINEYLPKAVANGEDIEAREKMAYAQYMAGVAFNNGGLGLVH 
               
               
                   
                   
                 SISH   Q   VG   G   VYKLQHGICNSVNMPHVCAFNLIAKTERFAHIAELL 
               
               
                   
                   
                 GENVAGLSTAAAAERAIVALERINKSFGIPSGYAEMGV   K   EEDIEL 
               
               
                   
                   
                 LAK   N   AYED   VC   TQSNPRVPTVQDIAQIIKNAM 
               
               
                   
               
               
                 3 
                 143175 
                 MTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVT   D   AFLHSTGL 
               
               
                   
                   
                 SEEVAKNIREAGL   D   VAIFPKAQPD   P   ADTQVHEGVDVFKQE   N   CD 
               
               
                   
                   
                 ALVSIGGGS   S   HDT   A   KAI   GLV   AANGG   RIN   DYQ   GVNSVEKPVV   PV 
               
               
                   
                   
                 VAITTTAGTGSE   T   TSL   AV   ITDSAR   KV   KMPV   ID   EKITPTVAIVDPEL 
               
               
                   
                   
                 MVKKPAGLTIATGMDALSHAIEAYVAKGATPVTDAFAIQAMKL 
               
               
                   
                   
                 INEYLPKAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLVHSI 
               
               
                   
                   
                 SH   Q   VG   G   VYKLQHGICNSVNMPHVCAFNLIAKTERFAHIAELLGE 
               
               
                   
                   
                 NVSGLSTAAAAERAIVALERYNKNFGIPSGYAEMGV   K   EEDIELL 
               
               
                   
                   
                 AK   N   AFED   VC   TQSNPRVATVQDIAQIIKNAL 
               
               
                   
               
               
                 5 
                 387585284 
                 MTQRNFFIPPASVIGRGAVKEVGTRLKQIGATKALIVT   D   AFLHGT 
               
               
                   
                   
                 GLSEEVAKNIREAGL   D   AVIFPKAQPD   P   ADTQVHEGVDIFKQE   K   C 
               
               
                   
                   
                 DALVSIGGGSSHDTAKAIGLVAANGGRINDYQGVNSVEKPVVP 
               
               
                   
                   
                 VVAITTTAGTG   S   ET   T   SL   AV   ITDSAR   KV   KMPV   ID   EKITPTVAIVDPE 
               
               
                   
                   
                 LMVKKPAGLTIATGMDALSHAIEAYVAKRATPVTDAFAIQAMK 
               
               
                   
                   
                 LINEYLPRAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLVHS 
               
               
                   
                   
                 ISH   Q   VG   G   VYKLQHGICNSVNMPHVCQFNLIARTERFAHIAELLG 
               
               
                   
                   
                 ENVSGLSTASAAERAIVALQRYNKNFGIPSGYAEMGV   K   EEDIEL 
               
               
                   
                   
                 LAN   N   AYQD   VC   TLDNPRVPTVQDIAQIIKNAL 
               
               
                   
               
               
                 7 
                 387586466 
                 MTKTKFFIPSSTVFGRGAVKEVGARLKAIGATKALIVT   D   AFLHST 
               
               
                   
                   
                 GLSEEVAKNIREAGL   D   VVIFPKAQPD   P   ADTQVHEGVEVFKQE   K   C 
               
               
                   
                   
                 DALVSIGGGS   S   HDTAKGI   GLV   AANGG   RIN   DYQ   GVNSVEKQVV   P 
               
               
                   
                   
                 QIAITTTAGTG   S   ET   T   SL   AV   ITDSAR   KV   KMPV   ID   EKITPTVAIVDPE 
               
               
                   
                   
                 LMVKKPAGLTIATGMDALSHAIEAYVAKRATPVTDAFAIQAMK 
               
               
                   
                   
                 LINEYLPKAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLVH 
               
               
                   
                   
                 SISH   Q   VG   G   VYKLQHGICNSVVMPHVCQFNLIARTERFAHIAELL 
               
               
                   
                   
                 GENVSGLSTASAAERTIAALERYNRNFGIPSGYKAMGV   K   EEDIE 
               
               
                   
                   
                 LLAN   N   AMQD   VC   TLDNPRVPTVQDIQQIIKNAL 
               
               
                   
               
               
                 9 
                 387588449 
                 MKNTQSAFYMPSVNLFGAGSVNEVGTRLAGLGVKKALLVT   D   A 
               
               
                   
                   
                 GLHSLGLSEKIAGIIREAGV   E   VAIFPKAEPN   P   TDKNVAEGLEAYN 
               
               
                   
                   
                 AE   N   CDSIVTLGGGS   S   HDA   G   KAI   ALV   AANGG   TIH   DYE   GVDVSKK     
               
               
                   
                   
                     PMV   PLIAINTTAGTG   S   EL   T   KF   TI   ITDTER   KV   KMAI   VD   KHVTPTLSI 
               
               
                   
                   
                 NDPELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQ 
               
               
                   
                   
                 AIKIISKYLPRAVANGKDIEAREQMAFAQSLAGMAFNNAGLGYV 
               
               
                   
                   
                 HAIAH   Q   LG   G   FYNFPHGVCNAILLPHVCRFNLISKVERYAEIAAFL 
               
               
                   
                   
                 GENVDGLSTYEAAEKAIKAIERMARDLNIPKGFKELGA   K   EEDIET 
               
               
                   
                   
                 LAK   N   AMND   AC   ALTNPRKPKLEEVIQIIKNAM 
               
               
                   
               
               
                 11 
                 387586073 
                 MTNTQSIFYIPSVNLFGPGSVNEVGTRLAGLGVKKALLVT   D   AGL 
               
               
                   
                   
                 HGLGLSEKIASIIREAGV   E   VLIFPKAEPN   P   TDKNVAEGLEVYNAE 
               
               
                   
                   
                     N   CDSIVTLGGGS   S   HDA   G   KGI   ALV   AANGG   TIY   DYE   GVDKSKKP     
               
               
                   
                   
                     MV   PLIAINTTAGTG   S   EL   T   RF   TI   ITDTER   KV   KMAI   VD   KHVTPTLSIN 
               
               
                   
                   
                 DPELMVGMPPSLTAATGLDALTHAIEAYVSTAATPITDALAIQAI 
               
               
                   
                   
                 KIISKYLPRAFANGKDMEAREQMAFAQSLAGMAFNNASLGYVH 
               
               
                   
                   
                 AIAH   Q   FG   G   FYNFPHGVCNAILLPHVCRFNLISKVERFAEIAALLG 
               
               
                   
                   
                 ENVAGLSTREAAEKGIKAIERMAKDLNIPRGFKELGA   K   EEDIVTL 
               
               
                   
                   
                 AE   N   AMKD   AT   ALTNPRKPKLEEVIQIIKNAM 
               
               
                   
               
               
                 13 
                 387590701 
                 MTNTQSAFFMPSVNLFGAGSVNEVGTRLADLGVKKALLVT   D   AG 
               
               
                   
                   
                 LHGLGLSEKISSIIRAAG   V   EVSIFPKAEPNPTDKNVAEGLEAYNA 
               
               
                   
                   
                 E   N   CDSIVTLGGGS   S   HDA   G   KAI   ALV   AANGG   KIH   DYE   GVDVSKEP     
               
               
                   
                   
                     MV   PLIAINTTAGTG   S   EL   T   KF   TI   ITDTER   KV   KMAI   VD   KHVTPTLSIN 
               
               
                   
                   
                 DPELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAI 
               
               
                   
                   
                 KIISKYLPRAVANGKDIEAREQMAFAQSLAGMAFNNAGLGYVH 
               
               
                   
                   
                 AIAH   Q   LG   G   FYNFPHGVCNAVLLPYVCRFNLISKVERYAEIAAFL 
               
               
                   
                   
                 GENVDGLSTYDAAEKAIKAIERMAKDLNIPKGFKELGA   K   EEDIE 
               
               
                   
                   
                 TLAK   N   AMKD   AC   ALTNPRKPKLEEVIQIIKNAM 
               
               
                   
               
               
                 15 
                 298729190 
                 MSDVLKQFVMPKTNLFGPGAIQEVGKRLNDLEVKKTLIVT   D   EGL 
               
               
                   
                   
                 HKLGLSEQIANIITAAGI   D   VAIFPKAEPN   P   TDQNIEDGISVYHAE   N     
               
               
                   
                   
                 CDSIVSLGGGS   A   HDA   A   KGI   GLI   ASNGG   RIH   DYE   GVDKSQNPLV   P 
               
               
                   
                   
                 LIAINTTAGTA   S   EM   T   RF   TI   ITDTAR   KV   KMAI   VD   KHVTPLLSINDPE 
               
               
                   
                   
                 LMIGLPPALTAATGVDALTHAIESFVSTNATPITDACAEKVLQLIP 
               
               
                   
                   
                 EYLPRAYANGADIEAREQMVYAQFLAGMAFNNASLGYVHAIA 
               
               
                   
                   
                 H   Q   LG   G   FYNLPHGVCNAILLPHVCRFNVTARTERFARIAELLGEN 
               
               
                   
                   
                 VEGLSKRDAAEKAITAIEKLSQDLNIPSGFRELGA   K   DEDIEILAK   N     
               
               
                   
                   
                 ALLD   VC   AETNPRKATLEDIKQIITNAMGPIVKKEESLEAVALS 
               
               
                   
               
               
                 17 
                 347752562 
                 MLTGLRTDFQMPSVNLFGQGTAEEIGNRLKNLGCRRPLIVT   D   EG 
               
               
                   
                   
                 LHQLGYSEKIAAYIKEAGL   E   VAIYPKAEPNPTDKNVEDGLKTYH 
               
               
                   
                   
                 EE   N   CDSIVSLGGGS   A   HDC   A   KGI   GLV   AANGG   KIH   DYE   GLDRSEK     
               
               
                   
                   
                     PMV   PLVAINTTAGTA   S   EM   T   KF   TI   ITDTSR   KV   KMAI   VD   KHVTPVL 
               
               
                   
                   
                 SINDPLLMVGMPPSLTAATGLDALTHAVEAYVSTAATPVTDAC 
               
               
                   
                   
                 AIKAIQIIPQYLPKAVANGNDMEAREQMVYAQYLAGMAFNNAS 
               
               
                   
                   
                 LGYVHAIAH   Q   FG   G   FYNLPHGVCNAILLPHVCRFNLIARKERFAEI 
               
               
                   
                   
                 AVALGEKTDSLSVDEAAEKAITAIERLAAQLNIPKGFKELGA   K   EE 
               
               
                   
                   
                 DIEILAQ   H   AMQD   AC   AATNPRKPTQKEVEAIIKAAM 
               
               
                   
               
               
                 19 
                 169829620 
                 MSDVLKQFVMPKKNLFGPGAIQEVGKHLNDLEVKKTLIVT   D   EG 
               
               
                   
                   
                 LHKLGLSEQIANIITAAGI   D   VAIFPKAEPN   P   TDQNIEDGIADYHAE 
               
               
                   
                   
                     S   CDSIVSLGGGS   A   HDA   A   KGI   GLI   ASNGG   RIQ   DYE   GVDKSQNPLV     
               
               
                   
                   
                 PLIAINTTAGTA   S   EM   T   RF   TI   ITDTAR   KV   KMAI   VD   KHVTPLLSINDS 
               
               
                   
                   
                 ELMIGLPPALTAATGVDALTHAIESFVSTNATPITDACAEKVLQL 
               
               
                   
                   
                 VPEFLPRAYANGADLEAREQMVYAQFLAGMAFNNASLGYVHAI 
               
               
                   
                   
                 AH   Q   LG   G   YYNLPHGVCNAILLPHVCRFNVTARTERFARIAELLGE 
               
               
                   
                   
                 NVTGLSKRDAAEKAISAIEKLSKDLNIPSGFRELGA   K   DEDIEILAK 
               
               
                   
                   
                     N   AMLD   VC   AETNPRKATLDDIKQIITNAMGPIVKKEESLEAVAAL 
               
               
                   
                   
                 s 
               
               
                   
               
               
                 21 
                 410459529 
                 MANQKVYGFFMPTVNLMGVGAVNEAGPRIKALGCNKSLLVT   D     
               
               
                   
                   
                 KGLSKMGVAEEIANIIGQAGV   E   VSIFDGAEPN   P   TDLNVEAGLKQ 
               
               
                   
                   
                 YRE   LG   CDSIISLGGGSSHDCAKGIGLVASNGGTIHDYEGVDMSK 
               
               
                   
                   
                 EPMIPLVAINTTAGTA   S   EM   T   RF   CI   ITDTSR   KI   KMAI   VD   KHTTPLIS 
               
               
                   
                   
                 INDPILTVKMPAGLTAATGMDALTHAIEAYVSTDATPITDACAL 
               
               
                   
                   
                 QTIRLVSQNLRAAVANGEDIDARNNMCYAQFLGGMAFNNASLG 
               
               
                   
                   
                 YVHAIAH   Q   LG   G   FYNLPHGVCNAVLLPHVERFNLIAKPERFVDIAI 
               
               
                   
                   
                 ALGENVSGLPTRAAAEIALTAIETLAKDVGIPGSLTELGV   K   EEDIP 
               
               
                   
                   
                 LLAE   N   AMRD   AC   SFTNPRKATLDDVQGMIRAAL 
               
               
                   
               
               
                 23 
                 257139072 
                 MSYLNIAQRTDSFFIPCVTLIGPGCARETGVRAKSLGAKKALIVT 
               
               
                   
                   
                     D   AGLHKMGLSEIVAGHIRDAGL   Q   AVIFAGAEPN   P   TDVNVHDGV 
               
               
                   
                   
                 ERFQRE   G   CDFIVSLGGGS   S   HDC   A   KGI   GLV   TAGGG   HIR   DYE   GIDK     
               
               
                   
                   
                     STVPMT   PLISINTTAGTA   A   EM   T   RF   CI   ITNSSN   HV   KMAI   VD   WRCTP 
               
               
                   
                   
                 LIAIDDPCLMVAMPPALTAATGMDALTHAVEAYVSTAATPITDA 
               
               
                   
                   
                 CAEKAIALIGEWLPKAVANGESMEARAAMCYAQYLAGMAFNN 
               
               
                   
                   
                 ASLGYVHAMAH   Q   LG   G   FYNLPHGVCNAILLPHVCEFNLIAAPERF 
               
               
                   
                   
                 ATIASLLGVNTAGSSTVDAARAGHAAIPRLSASIGIPAGLAALGV 
               
               
                   
                   
                     R   VEDHEVMAS   N   AQKD   AC   MLTNPRKATLAQVIAIFAAAM 
               
               
                   
               
               
                 25 
                 339322658 
                 MTHLNIANRVDSFFIPCVTLFGPGCARETGARARSLGARKALIVT 
               
               
                   
                   
                     D   AGLHKMGLSEVVAGHIREAGL   Q   AVIFPGAEPN   P   TDVNVHDGV 
               
               
                   
                   
                 KLFERE   E   CDFIVSLGGGS   S   HDC   A   KGI   GLV   TAGGG   HIR   DYE   GIDK     
               
               
                   
                   
                     STVPMT   PLISINTTAGTA   A   EM   T   RF   CI   ITNSSN   HV   KMAI   VD   WRCTP 
               
               
                   
                   
                 LIAIDDPSLMVAMPPALTAATGMDALTHAIEAYVSTAATPITDA 
               
               
                   
                   
                 CAEKAIVLIAEWLPKAVANGDSMEARAAMCYAQYLAGMAFNN 
               
               
                   
                   
                 ASLGYVHAMAH   Q   LG   G   FYNLPHGVCNAILLPHVSEFNLIAAPERY 
               
               
                   
                   
                 ARIAELLGENIGGLSAHDAAKAAVSAIRTLSTSIGIPAGLAGLGV 
               
               
                   
                   
                     K   ADDHEVMAS   N   AQKD   AC   MLTNPRKATLAQVMAIFAAAM 
               
               
                   
               
               
                 27 
                 451936849 
                 MSLVNYLQLADRTDGFFIPSVTLVGPGCVKEVGPRAKMLGAKR 
               
               
                   
                   
                 ALIVT   D   AGLHKMGLSQEIADLLRSEGI   D   SVIFAGAEPN   P   TDINVH 
               
               
                   
                   
                 DGVKVYQKE   K   CDFIVSLGGG   S   SHDC   A   KGI   GLV   TAGGG   HIR   DYE 
               
               
                   
                   
                     GVDKSKVPMT   PLIAINTTAGTA   S   EM   T   RF   CI   ITNTDT   HV   KMAI   VD     
               
               
                   
                   
                 WRCTPLVAIDDPRLMVKMPPALTAATGMDALTHAVEAYVSTA 
               
               
                   
                   
                 ATPITDTCAEKAIELIGQWLPKAVANGDWMEARAAMCYAQYL 
               
               
                   
                   
                 AGMAFNNASLGYVHAMAH   Q   LG   G   FYNLPHGVCNAILLPHVCQF 
               
               
                   
                   
                 NLIAATERYARIAALLGVDTSGMETREAALAAIAAIKELSSSIGIP 
               
               
                   
                   
                 RGLSELGV   K   AADHKVMAE   N   AQKD   AC   MLTNPRKATLEQVIGIFE 
               
               
                   
                   
                 AAM 
               
               
                   
               
               
                 29 
                 197117741 
                 MALGEQTYGFYIPTVSLMGIGSAKETGGQIKALGASKALIVT   D   K 
               
               
                   
                   
                 GLSAMGVADKIKSQVEEAGV   S   AVIFDGAEPN   P   TDINVHDGVKV 
               
               
                   
                   
                 YQDN   G   CDAIISLGGGS   S   HDC   A   KGI   GMV   IGNGG   HIR   DLE   GVNKT     
               
               
                   
                   
                     TKPMP   AFVAINTTAGTA   S   EM   T   RF   CI   ITNTDT   HV   KMAI   VD   WRCTP 
               
               
                   
                   
                 NVAINDPLLMVGKPAALTAATGMDALTHAVEAYVSTIATPITDA 
               
               
                   
                   
                 CAIKAIELIAEFLSKAVANGEDLEARDKMAYAEYLAGMAFNNA 
               
               
                   
                   
                 SLGYVHSMAH   Q   LG   G   FYNLPHGVCNAILLPAVSQYNLIACPKRFA 
               
               
                   
                   
                 DIAKALGENIDGLSVTEAGQKAIDRIRTLSASIGIPTGLKALNV   K   E 
               
               
                   
                   
                 ADLTIMAE   N   AKKD   AC   QFTNPRKATLEQVVQIFKDAM 
               
               
                   
               
               
                 31 
                 78043360 
                 MKTYRFYMPPVSLMGIGCLKEAGEEIKKLGFKKALIVT   D   KVLVK 
               
               
                   
                   
                 IGLVNKLTEILDNEGI   E   YVIFDETKPN   P   TVKNVEDGLKMLKEN   N   C 
               
               
                   
                   
                 DFLISFGGGS   P   HDC   A   KGI   GLV   ATNGG   SIK   DYE   GVNKSAKPML   P 
               
               
                   
                   
                 LVAVNTTAGTA   S   EM   T   RF   SI   ITDEDR   HV   KMAI   VD   WHVTPIMAVN 
               
               
                   
                   
                 DPELMVEMPKALTAATGMDALTHAIEAYVSIDATPVTDAAALK 
               
               
                   
                   
                 AIELIFKYLKRAVENGKDIEARDKMAYAEYLAGVAFNNAGLGY 
               
               
                   
                   
                 VHAMAH   Q   LG   G   FYDLPHGVCNAVLLPHVQAYNLQVVPERFIDIA 
               
               
                   
                   
                 KAMGINVENLTAKEAGEKVLEAIKNLSREIGIPSGLKELGV   K   EED 
               
               
                   
                   
                 LKTLAE   N   ALKD   AC   GFTNPKQASLDDIIRIFKEAM 
               
               
                   
               
               
                 33 
                 152978087 
                 MSTYYFLPTRNVFGENAVEEVGTLMKSLGGNNPLIVT   D   AFLAK 
               
               
                   
                   
                 NGMADQLAAVLSNAGL   K   PVIFGGAEPN   P   TDKNVEEGIVFYNEH 
               
               
                   
                   
                     G   CDSIISLGGGS   S   HDC   A   KGI   GLI   ASNGG   RIQ   DYE   GVDRSHNAMV     
               
               
                   
                   
                 PLMAVNTTAGTA   S   EI   T   RF   CI   ITDTAR   KV   KMAI   VD   WRITPQIAVND 
               
               
                   
                   
                 PLLMKGMPPSLTAATGMDALTHAIEAYVSTAANPLTDAAALMA 
               
               
                   
                   
                 ITMIQQYLPKAVANGDYMKARDKMAYAQYLAGIAFNNASLGY 
               
               
                   
                   
                 VHAMAH   Q   LG   G   FYNLPHGVCNAILLPYVEEFNLIGNLNRFRDIAK 
               
               
                   
                   
                 AMGENIDGLCTDDAALKAIGAIRRLSKQVGIPANLQLLGVKPED 
               
               
                   
                   
                 FDVMAE   N   AMKDVCMLTNPRKATKQQVIEIFQRAYDGD 
               
               
                   
               
               
                 35 
                 421788018 
                 MAFKNIADQTNGFYIPCVSLFGPGCAKEIGTKAQNLGAKKALIV 
               
               
                   
                   
                 T   D   EGLFKFGVADLIASYLTEAGV   A   SHIFPGAEPN   P   TDINVHNGVN 
               
               
                   
                   
                 AYNEN   G   CDFIVSLGGGS   S   HDC   A   KGI   GLV   TAGGG   HIR   DYE   GIDK     
               
               
                   
                   
                     SKVPMT   PLIAVNTTAGTA   S   EM   T   RF   CI   ITNTDT   HV   KMAI   VD   WRCT 
               
               
                   
                   
                 PLIAIDDPKLMIAKPAGLTAATGMDALTHAVEAYVSTAANPITD 
               
               
                   
                   
                 ACAEKAITMISQWLQPAVANGENIEARDAMSYAQYLAGMAFN 
               
               
                   
                   
                 NASLGYVHAMAH   Q   LG   G   FYNLPHGVCNAILLPHVCEFNLIACPD 
               
               
                   
                   
                 RYAKIAELMGVNTHGLTVTEAAYAAIDAIRKLSSLIGIPSGLTEL 
               
               
                   
                   
                 GV   K   TEDLAVMAE   N   AQKD   AC   MLTNPRKANHAQVVEIFKAAL 
               
               
                   
               
               
                 37 
                 2393887 
                 MRMYDFLAPNVNFMGAGAIKLVGERCKILGGKKALIVT   D   KFLR 
               
               
                   
                   
                 NMEDGAVAQTVKYIKEAGI   D   VAFYDDVEPN   P   KDTNVRDGLKV 
               
               
                   
                   
                 YRKE   N   CDLIVTVGGGS   S   HDC   G   KGI   GIA   ATHEG   DLY   DYA   GIETL     
               
               
                   
                   
                     TNPLP   PIVAVNTTAGTG   S   EV   T   RH   CV   ITNTKT   KI   KFVI   VS   WRNLPL 
               
               
                   
                   
                 VSINDPILMIKKPAGLTAATGMDALTHAIESYVSKDANPVTDAL 
               
               
                   
                   
                 AIQAIKLIANNLRQAVALGENLEARENMAYASLLAGMAFNNAN 
               
               
                   
                   
                 LGYVHAMAH   Q   LG   G   LYDMAHGVANAMLLPHVERYNLISNPKKF 
               
               
                   
                   
                 ADIAEFMGENIEGLSVMEAAEKAIDAMFRLSKDVGIPASLKEMG 
               
               
                   
                   
                 V   N   EGDFEYMAK   M   ALKD   GN   AFSNPRKGNEKDIVKIFREAF 
               
               
                   
               
               
                 39 
                 452211255 
                 MIEKMTYTYLNPKIALMGPGCVNGIGTHAKDLGGTKALIVS   G   KS 
               
               
                   
                   
                 RHGKELAADIRRILERAGI   E   AAIFPGADPN   P   TDTSVMEGADIYRK 
               
               
                   
                   
                 E   N   CNMIVAVGGGS   P   MDC   A   KAI   GIV   VYNGG   RIN   DYE   GVGKVTR     
               
               
                   
                   
                     GIP   PLITVNTTAGTA   S   EM   T   SF   TI   ITDTER   HI   KMAI   VD   PRITPDVAV 
               
               
                   
                   
                 NDPELMVSMPPALTAATGMDALTHAVEAYVSTMATPTTDAAAI 
               
               
                   
                   
                 KAIELISKYLPEAVLHGEDIRARDMMAHAEYLAGIAFNNASLGY 
               
               
                   
                   
                 VHSMAH   Q   LG   G   FYDLPHGVCNAILLPYVEMYNKQVCPERFADIA 
               
               
                   
                   
                 KAMGEKVEGLSPEEAADKAIEAIKKLAAEIGIPSGLKELGAREED 
               
               
                   
                   
                 LELLAE   N   AMQD   VC   RLTNPRELSKEDIIEIYRKAL 
               
               
                   
               
               
                 41 
                 218885425 
                 MAVQEQVYGFFIPSVTLIGIGASKAIPEKIKALGGSKPLIVT   D   MGI 
               
               
                   
                   
                 VKAGILKQITDLLDAAKM   A   YSVYDETIPN   P   TDDNVHKGVEVYK 
               
               
                   
                   
                 KN   K   CDSLITLGGGS   S   HDC   G   KGI   GLV   IANGG   KIH   DFE   GVDKSFKP     
               
               
                   
                   
                     MP   PYVAVNTTAGTASEMTRFCIITDTSRKVKMAIVDWRVTPSIA 
               
               
                   
                   
                 LDDPLLMMGMPPALTAATGMDALTHAVEAYVSTIATPMTDAC 
               
               
                   
                   
                 AEQAITLIATFLRRAVANGRDIEARERMCFAQYLAGMAFNNASL 
               
               
                   
                   
                 GHVHAMAH   Q   LG   G   FYDLPHGECNAILLPHVSQFNLIAKLDRFARI 
               
               
                   
                   
                 AELMGENISGLSVRDAAEKAICAIKRLSADVGIPAGLVALGKRY 
               
               
                   
                   
                 GKDV   K   AKDIAIMTK   N   AQKD   AC   GLTNPRCPTDADVAAIYEAAM 
               
               
                   
               
               
                 43 
                 374301216 
                 MAVREQVYGFFIPSVTLIGIGASKEIPNKIRDLGGKKPLIVT   D   QGI 
               
               
                   
                   
                 VKAGILKMITDHMDKAGM   Q   YSVYDKTIPN   P   TDNNVAEGVEVY 
               
               
                   
                   
                 KKE   G   CDSLITLGGGS   S   HDC   G   KGV   GLV   VSNGG   KIH   DYE   GVDKST     
               
               
                   
                   
                     KPLP   PYVAVNTTAGTA   S   EM   T   RF   CI   ITDTSR   KV   KMAI   VD   WRVTP 
               
               
                   
                   
                 GIALDDPLLMVGMPPALTAATGMDALTHAVEAYVSTIATPMTD 
               
               
                   
                   
                 ACAEKAISLIFTFLRRATANGQDIEAREGMCFAQYLAGMAFNNA 
               
               
                   
                   
                 SLGHVHAMAH   Q   LG   G   FYDLPHGECNAILLPHVEKYNLIAKVERF 
               
               
                   
                   
                 GKMAEIMGENIQGMSPRAAAEKCLDAIRQLSQDVGIPSGLIELG 
               
               
                   
                   
                 KRYGKNV   K   KEDIDTMTG   N   AQKD   AC   GFTNPRCPSDKDVKAIYEA 
               
               
                   
                   
                 AL 
               
               
                   
               
               
                 45 
                 18309918 
                 MRMYDYLVPSVNFMGANSISVVGERCKILGGKKALIVT   D   KFLR 
               
               
                   
                   
                 GLKGGAVELTEKYLKEAGI   E   VAYYDGVEPN   P   KDTNVKDGLKIF 
               
               
                   
                   
                 QDE   N   CDMIVTVGGGS   S   HDC   G   KGI   GIA   ATHEG   DLY   DYA   GIETLT     
               
               
                   
                   
                     NPLP   PIVAVNTTAGTA   S   EV   T   RH   CV   ITNTKT   KV   KFVI   VS   WRNLPL 
               
               
                   
                   
                 VSINDPMLMVGKPAGLTAATGMDALTHAVEAYVSKDANPVTD 
               
               
                   
                   
                 AAAIQAIKLISSNLRQAVALGENLVARENMAYGSLLAGMAFNN 
               
               
                   
                   
                 ANLGYVHAMAH   Q   LG   G   LYDMPHGVANAMLLPHVCKYNLISNP 
               
               
                   
                   
                 QKFADIAEFMGENIEGLSVMDAAQKAIDAMFRLSTDIGIPAKLR 
               
               
                   
                   
                 DMGV   K   EEDFGYMAE   M   ALKD   GN   AFSNPRKGNERDIVEIFKAAF 
               
               
                   
               
               
                 47 
                 156976638 
                 MTSAFFIPTVNLMGAGCLKDATDSIQSQGFKKGLIVT   D   KILNQIG 
               
               
                   
                   
                 VVKQVQDLLAERDV   E   TVVFDGTQPN   P   TISNVNDGLALLTDN   E   C 
               
               
                   
                   
                 DFVISLGGGS   P   HDC   A   KGI   ALV   ASNGG   KIA   DYE   GVDQSAKPMM   P 
               
               
                   
                   
                 LIAINTTAGTA   S   EM   T   RF   CI   ITDEER   HI   KMAI   VD   KHTTPLISVNDPE 
               
               
                   
                   
                 LMLAKPASLTAATGMDALTHAIEAYVSIAATPITDAVAIKAIELI 
               
               
                   
                   
                 QAYLRTAVKNGEDLEAREQMAYAQFMAGMAFNNASLGYVHA 
               
               
                   
                   
                 MAH   Q   LG   G   FYDLPHGVCNAILLPHVQRYNAQVCPERLRDVAKA 
               
               
                   
                   
                 MGVNVEDMSAEAGAAAAIDAIVTLAKDVGIPAGIKELGA   K   LEDI 
               
               
                   
                   
                 PTLAD   N   ALKD   AC   GFTNPKQATHEEISKIFEEAM 
               
               
                   
               
               
                 49 
                 134300116 
                 MTVGEQVFGYFIPTVNLMGVGAHKEIPDQVKVLGGSNVLIVT   D     
               
               
                   
                   
                 AFLGRPGGMADDIKGMLEAENI   K   VTIYAGAEPN   P   TDVNVHDGL 
               
               
                   
                   
                 KVYQ   E   CGADMILSLGGGS   S   HDC   A   KGI   GIV   ATNGG   NIR   DYE   GIN     
               
               
                   
                   
                     KSSKAMP   PFIAVNTTAGTA   S   EM   T   RF   CI   ITNTSN   HV   KMAI   VD   WRC 
               
               
                   
                   
                 TPNIAINDPLLMAGMPPALTAATGMDALTHAIEAYVSVAATPVT 
               
               
                   
                   
                 DSAALMAIKLISQYLRAAVANGENMEARDKMAYAEFLGGMAF 
               
               
                   
                   
                 NNASLGYVHAMAH   Q   LG   G   FYNLPHGVCNAILLPHVEAFNLIACP 
               
               
                   
                   
                 ERFVDIAVAMGENVEGLSVRDAADKALSAIRKLSADVGIPAGLT 
               
               
                   
                   
                 ELGV   K   EEDLKTMAE   N   AMKD   AC   ALTNPRKATLNDIVGIYKTAL 
               
               
                   
               
               
                 51 
                 46580810 
                 MAVQEQVYGFFIPRVTLIGIGASKAIPEKIKALGGSKPLIVT   D   MGI 
               
               
                   
                   
                 VKAGILKQITDLLDAAKM   A   YSVYDETIPN   P   TDDNVHKGVDVYK 
               
               
                   
                   
                 KN   K   CDSLITLGGGS   S   HDC   G   KGI   GLV   VANGG   KIH   DFE   GVDKSTQ     
               
               
                   
                   
                     RMP   PYLAVNTTAGTA   S   EM   T   RF   CI   ITDTSR   KV   KMAI   VD   WRVTPNI 
               
               
                   
                   
                 ALDDPLLMLGMPPALTAATGMDALTHAVEAYVSTIATPMTDAC 
               
               
                   
                   
                 AEQAITLIATFLRRAVANGQDLEARERMCFAQYLAGMAFNNAS 
               
               
                   
                   
                 LGHVHAMAH   Q   LG   G   FYDLPHGECNAILLPHVSKFNLIAKLDRYA 
               
               
                   
                   
                 RIAQLMGENIAGLSTREAAERAISAIKCLSTDVGIPAGLVALGKR 
               
               
                   
                   
                 YGKDV   K   AADIAIMTK   N   AQKD   AC   GLTNPRCPTDADVAAIYEAAL 
               
               
                   
               
               
                 53 
                 90412151 
                 MSSAFFIPSVNLMGAGCLTEAADAVKAHGFKKALIVT   D   KVLNQI 
               
               
                   
                   
                 GVVKQVVDLLAERN   V   EAVVFDGTQPN   P   TMGNVEAGLALLKAN 
               
               
                   
                   
                 ECDFVISLGGGS   P   HDC   A   KGI   ALV   ASNGG   SIS   DYE   GVDVSAKPQL     
               
               
                   
                   
                 PLVAINTTAGTA   S   EM   T   RF   CI   ITDEAR   HI   KMAI   VD   KNTTPLMSVND 
               
               
                   
                   
                 PELMLAKPASLTAATGMDALTHAIEAYVSTAATPITDAVAIKAM 
               
               
                   
                   
                 ELIQAHLRTAVNDGQNLEAREQMAYAQFMAGMAFNNASLGYV 
               
               
                   
                   
                 HAMAH   Q   LG   G   FYDLPHGVCNAVLLPHVQRYNAKVCPERLRDVA 
               
               
                   
                   
                 KAMGVNVEAMTADQGADAALEAIQVLSKDVGIPAGLKDLGA   K     
               
               
                   
                   
                 NEDISILAD   N   ALKD   AC   GFTNPKQATHEEISEIFAAAM 
               
               
                   
               
               
                 55 
                 312112413 
                 MSNAHVFYVPSTNLMGRGCLAKVGPFIKEFGFKKALVVT   D   KFL 
               
               
                   
                   
                 HKSGIAGKVLAVLDEIGV   N   YVVYDDVKPN   P   TTKNVYAGADLFK 
               
               
                   
                   
                 KN   E   CDFLVSVGGGS   P   QDT   A   KAI   GLY   VTNGG   DIR   DYE   GVNKTK     
               
               
                   
                   
                     NKSV   PIVAVNTTAGTS   S   EF   T   IN   YV   ITDEER   NV   KMVM   VD   KNSLVT 
               
               
                   
                   
                 ISVNDPELMVDKPAALTAATGMDALTHAIEAVVTPGSYTVTDA 
               
               
                   
                   
                 TALAAIEIIFNYLPRAVKNGHDIEAREQMAYAMFLVGIAFNNAG 
               
               
                   
                   
                 LGMVHAMAH   Q   LG   G   MYDLPHGVCNAMLLPIVERENAKRDPRKF 
               
               
                   
                   
                 RAIAKAAGIDVTGKTDEQCAEEVIEAIKALSREIGIPSKLSELGV   D     
               
               
                   
                   
                 EVDLEKLAN   N   ALKD   AC   APGNPFQPTKEEVISMFKEIL 
               
               
                   
               
               
                 57 
                 303249216 
                 MAVREQVYGFFIPSVTLIGIGAAKQIPEKIKALGGTKPLIVT   D   KGV 
               
               
                   
                   
                 VKVGVCKMITDLLDAAGM   K   YHIYDETIPN   P   TDENVHKGVEVYK 
               
               
                   
                   
                 KE   G   CDSLITLGGGS   S   HDC   G   KGI   GLV   ISNGG   KIH   DYE   GVDKSSKP     
               
               
                   
                   
                     FM   PYLAVNTTAGTA   S   EM   T   RF   CI   ITDLSR   HV   KMAI   VD   WRVTPHIA 
               
               
                   
                   
                 IDDPVLMVGMPPALTASTGMDALTHAVEAFVSTIANPMTDACAI 
               
               
                   
                   
                 EAIKLIFKYLRKAVANGQDMEAREGMCFAEYLAGMAFNNASLG 
               
               
                   
                   
                 HVHAMAH   Q   LG   G   FYDLPHGECNAILLPHVESYNLIAKVEKFAEM 
               
               
                   
                   
                 AKIMGENIEGMAPRDAAELCLKAIRQLSVDVGIPAGLVELGKRY 
               
               
                   
                   
                 GKDV   K   AADIPTMTG   N   AQKD   AC   GLTNPRCPTDKDVAAIYTAAL 
               
               
                   
               
               
                 59 
                 24373064 
                 MAAKFFIPSVNVLGKGAVDDAIGDIKTLGFKRALIVT   D   KPLVNIG 
               
               
                   
                   
                 LVGEVAEKLGQNGI   T   STVFDGVQPN   P   TVGNVEAGLALLKANQC 
               
               
                   
                   
                 DFVISLGGGS   P   HDC   A   KGI   ALV   ATNGG   SIK   DYE   GLDKSTKPQL   PL 
               
               
                   
                   
                 VAINTTAGTA   S   EM   T   RF   CI   ITDEAR   HI   KMAI   VD   KHTTPILSVNDPE 
               
               
                   
                   
                 LMLKKPASLTAATGMDALTHAVEAYVSIAANPITDACAIKAIELI 
               
               
                   
                   
                 QGNLVNAVKQGQDIEAREQMAYAQFLAGMAFNNASLGYVHA 
               
               
                   
                   
                 MAH   Q   LG   G   FYDLPHGVCNALLLPHVQEYNAKVVPHRLKDIAKA 
               
               
                   
                   
                 MGVDVAKMTDEQGAAAAITAIKTLSVAVNIPENLTLLGV   K   AEDI 
               
               
                   
                   
                 PTLAD   N   ALKD   AC   GFTNPKQATHAEICQIFTNAL 
               
               
                   
               
               
                 61 
                 269122369 
                 MKVSRRIYWPAVTLIGPGCVKEIGGDIKDLGLKKALVVT   D   NVLV 
               
               
                   
                   
                 KIGVVKKVTDVLDESGI   N   YVVVDDIQPN   P   TMKNIHDGLNTYKSE 
               
               
                   
                   
                     N   CDFVISIGGGS   P   QDA   G   KAI   GLL   ATNGG   EIK   DYE   GINMSKHKS     
               
               
                   
                   
                     V   PIIAINTTAGTA   S   EV   T   IN   YV   ITNEDT   HI   KMVM   VD   KNCLASIAVS 
               
               
                   
                   
                 DPELMTGKPADLTAATGMDALTHAIEAYVSTGAYELTDVLALE 
               
               
                   
                   
                 AVKLIGESLEDAVKDGNNIEARSKMAYASYIAGMSFNNAGLGY 
               
               
                   
                   
                 VHSMAH   Q   LG   G   FYNLPHGVCNAILLPHVEKFNSANTGDKLRKVA 
               
               
                   
                   
                 EILGENVEGLSVEEANAKAIEAIMKLSERVGIPKGLKELGV   K   EED 
               
               
                   
                   
                 FKVMAE   N   ALKD   VC   AGTNPREVTLEDTIALYKEAL 
               
               
                   
               
               
                 63 
                 390456003 
                 MTGTSKFMMPGMSLMGSGALADAGTEIGKLGYTNALIVT   D   KPL 
               
               
                   
                   
                 VDIGIVKKVTSVLESINV   K   SVVYSGTQPN   P   TVTNVNEGLELLSQS 
               
               
                   
                   
                     K   CDFIISLGGGS   P   HDCAKGIALLASNGGQIGDYE   GVDKSTKPSF     
               
               
                   
                   
                 PLIAINTTAGTASEMTMFCIITDEERHIKMAIVDNHTTPLIAVNDP 
               
               
                   
                   
                 DLMMAMPKSLTAATGMDALTHSIEAYVSTNATPITDACAIKAIE 
               
               
                   
                   
                 LIRDNLARAVDDGNDVEARSQMAYAEFLAGMAFNNAGLGFVH 
               
               
                   
                   
                 AMAH   Q   LG   G   FYNLPHGVCNAILLPHVERYNAKASAERLTDIARA 
               
               
                   
                   
                 LGENTDGVTPEQGANLALQAIEKLAKRVNIPSGLEELGV   K   REDF 
               
               
                   
                   
                 TVLAA   N   ALKD   AC   GVTNPVQPTQQEVIAIFEQAM 
               
               
                   
               
               
                 65 
                 152972007 
                 MSYRMFDYLVPNVNFFGPNAISVVGERCQLLGGKKALLVT   D   KG 
               
               
                   
                   
                 LRAIKDGAVDKTLHYLREAGI   E   VAIFDGVEPN   P   KDTNVRDGLAV 
               
               
                   
                   
                 FRRE   Q   CDIIVTVGGGS   P   HDCGKGI   GIA   ATHEG   DLY   QYA   GIETLT     
               
               
                   
                   
                     NPLP   PIVAVNTTAGTA   S   EV   T   RH   CV   LTNTET   KV   KFVI   VS   WRNLPS 
               
               
                   
                   
                 VSINDPLLMIGKPAALTAATGMDALTHAVEAYISKDANPVTDAA 
               
               
                   
                   
                 AMQAIRLIARNLRQAVALGSNLQARENMAYASLLAGMAFNNA 
               
               
                   
                   
                 NLGYVHAMAH   Q   LG   G   FYLYDMPHGVANAVLLPHVARYNLIANPEK 
               
               
                   
                   
                 FADIAELMGENITGLSTLDAAEKAIAAITRLSMDIGIPQHLRDLGV 
               
               
                   
                   
                     K   EADFPYMAE   M   ALKD   GN   AFSNPRKGNEQEIAAIFRQAF 
               
               
                   
               
               
                 67 
                 49176377 
                 MAASTFFIPSVNVIGADSLTDAMNMMADYGFTRTLIVT   D   NMLT 
               
               
                   
                   
                 KLGMAGDVQKALEERNI   F   SVIYDGTQPN   P   TTENVAAGLKLLKE 
               
               
                   
                   
                 N   N   CDSVISLGGGS   P   HDC   A   KGI   ALV   AANGG   DIR   DYE   GVDRSAKP     
               
               
                   
                   
                     QL   PMIAINTTAGTA   S   EM   T   RF   CI   ITDEAR   HI   KMAI   VD   KHVTPLLSV 
               
               
                   
                   
                 NDSSLMIGMPKSLTAATGMDALTHAIEAYVSIAATPITDACALK 
               
               
                   
                   
                 AVTMIAENLPLAVEDGSNAKAREAMAYAQFLAGMAFNNASLG 
               
               
                   
                   
                 YVHAMAH   Q   LG   G   LYNLPHGVCNAVLLPHVQVFNSKVAAARLRD 
               
               
                   
                   
                 CAAAMGVNVTGKNDAEGAEACINAIRELAKKVDIPAGLRDLNV 
               
               
                   
                   
                     K   EEDFAVLAT   N   ALKD   AC   GFTNPIQATHEEIVAIYRAAM 
               
               
                   
               
               
                 69 
                 110799824 
                 MSYKFFMPAISLMGADCLKDAGDQVGELGFKKALIVT   D   KVLGQ 
               
               
                   
                   
                 IGIVKKVTDVLDNKNI   E   YAIYDETKPN   P   TVKNVNDGLALLKEK   E     
               
               
                   
                   
                 CDFVISLGGGS   A   HDC   A   KGI   ALL   ATNGG   EIK   DYE   GVDKSKKPQL     
               
               
                   
                   
                 PMVGINTTAGTG   S   EM   T   LF   AI   ITDEER   HI   KMAL   VD   KHLTPIIAVND 
               
               
                   
                   
                 PILMLAMPKSLTAATGMDALTHAIEAYVSTAATPITDACAEKAIE 
               
               
                   
                   
                 LISNYLVNAVENGQDVEARDMMAYAEYLAGMAFNNASLGYVH 
               
               
                   
                   
                 AMAH   Q   LG   G   FYNLPHGVCNAILLPHVQEYNKSTSASRLAKIAKI 
               
               
                   
                   
                 MGGNIEGLTDEQGADLCIDMIKSLSQTIGIPEGLGVLGVKESDFE 
               
               
                   
                   
                 TLAT   N   ALND   AC   SLTNPRKGNLEEVIAIFKKAM 
               
               
                   
               
               
                 71 
                 113866887 
                 MRARPARAPKRKAQERPSSSRMPACTRWGYPKPSRGTSARQGF 
               
               
                   
                   
                     R   PLIFPGAEPN   P   TDVNVHDGVKLFEQE   G   CDFIVSLGGGSSHDC   A     
               
               
                   
                   
                 KGI   GLV   TAGGG   HIR   DYE   GIDKSTVPMT   PLISINTTAGTA   A   EM   T   R 
               
               
                   
                   
                 F   CI   ITNSSN   HV   KMAI   VD   WRCTPLIAIDDPRLMVAMPPALTAATG 
               
               
                   
                   
                 MDALTHAVEAYVSTAATPITDACAEKAIALIGEWLPKAVANGN 
               
               
                   
                   
                 SLEARAAMCYAQYLAGMAFNNASLGYVHAMAH   Q   LG   G   LYNLP 
               
               
                   
                   
                 HGVCNAILLPHVSEFNLIAAPERFAKIAELLGENVASLSTSDAAK 
               
               
                   
                   
                 AAISAIRALAASIGIPAGLASLGV   K   AEDHEVMAH   N   AQKD   AC   MLT 
               
               
                   
                   
                 NPRRATTAQVIAIFAAAM 
               
               
                   
               
               
                 73 
                 167040564 
                 MKIFKFHMPPINLIGVGCLKDVGREIKKLGFKKGIIVT   D   KVLVRA 
               
               
                   
                   
                 GLVNNVISVLEEEGI   E   YVVFDETKPN   P   TIKNVTNGLKLLIEN   K   CD 
               
               
                   
                   
                 FIISCGGGS   A   HDC   A   KGI   GLI   AKEKN   FID   EVE   RLDKVK   CGGWNS   A     
               
               
                   
                   
                     LLL   PLVAINTTAGTG   S   EV   T   KF   AI   ITDEEK   RI   KMPI   VD   WRITPLIAV 
               
               
                   
                   
                 NDPLLMIGMPKSLTAASGMDALTHAIEAYISIDANPFTDALALK 
               
               
                   
                   
                 AIEIIFNYLKRAVENGNDIEAREKMAYAEFLAGIAFNNAGLGYV 
               
               
                   
                   
                 HAMAH   Q   LG   G   FYDLPHGVCNAVLLPHVLEYNLEAVQNKLIYIAK 
               
               
                   
                   
                 AMGIDVDKLTTKEIGGKIIESINQLSQEIGIPSRLKELGV   K   EEDIKE 
               
               
                   
                   
                 LSQ   N   ALKD   VC   GFTNPKKATLEDIINIFKSAM 
               
               
                   
               
               
                 75 
                 406526935 
                 MGNRIILNGTSYFGRGARENVITELRNRNFTKALVVT   D   KNLLDA 
               
               
                   
                   
                 HVTNLVTDVLDKNDFSYQIYSDIKPNPTTLNVQEGVTFCRNSKA 
               
               
                   
                   
                 DVIIAVGGGS   A   ID   TA   KAI   SII   MTNPEHFDVISLD   GAVETKNAGM   P 
               
               
                   
                   
                 IIALPTTAGTA   A   EV   T   IN   YV   ITNPVG   PK   KMVC   VD   PHDIPIVAIIDQD 
               
               
                   
                   
                 LMEKMPKSLAASTGMDALTHAMEGYTTKAAWLMTDMFHLNA 
               
               
                   
                   
                 MALIYKNLEKAVNLKDRDAIDNVGYGQYIAGMGFSNVGLGIVH 
               
               
                   
                   
                 SMAH   S   LG   A   FFDTPHGLANALLLPHVLKFNGKICPDLFRNMGRA 
               
               
                   
                   
                 MGLDMDNLTDDEAVDKVVDAVRSLAIKIGIPQTLKEIGI   K   KEDL 
               
               
                   
                   
                 PMLAH   Q   AIDD   VC   TAGNPRNVTEQDILALYQEAYE 
               
               
                   
               
               
                 77 
                 138896515 
                 MQNFTFRNPTKLIFGRGQIEQLKEEVPKYGKKVLLVYGGGSIKR 
               
               
                   
                   
                 NGLYDEVMSLLTDIGA   E   VVELPGVEPN   P   RLSTVKKGVDICRREG 
               
               
                   
                   
                 IEFLLAVGGGS   V   IDC   T   KAI   AAG   AKFDG   DPW   EFI   TKKATVTEAL   P 
               
               
                   
                   
                 FGTVLTLAATG   S   EM   N   AG   SV   ITNWET   KE   KYGW   GS   PVTFPQFSILD 
               
               
                   
                   
                 PTYTMTVPKDHTVYGIVDMMSHVFEQYFHHTPNTPLQDRMCEA 
               
               
                   
                   
                 VLKTVIEAAPKLVDDLENYELRETIMYSGTIALNGFLQMGVRGD 
               
               
                   
                   
                 WATHDIE   H   AVS   A   VYDIPHAGGLAILFPNWMKHVLDENVSRFAQ 
               
               
                   
                   
                 LAVRVFDVDPTGKTERDVALEGIERLRAFWSSLGAPSRLADYGI 
               
               
                   
                   
                     G   EENLELMADKAMAFGEFGRFKTLNRDDVLAILRASL 
               
               
                   
               
               
                   
                 consensus 
                 M(T,K)(N,—)(T,—)(Q,K)(S,T,R)(N,A,I,K)F(F,Y)(I,M)P(P,S)(A,V,S)(N,S, 
               
               
                   
                   
                 T)(V,L)(F,D)G(R,A,P)G(A,S)V(K,N)EVG(T,A)RL(K,A)(Q,G,D,A)(I,L) 
               
               
                   
                   
                 G(A,V)(K,T)KAL(I,I)VT   D   A(F,G)LH(G, S)(T,I)GLSE(E,K)(V,T)(A, S) 
               
               
                   
                   
                 (K,S,G)((N,I)IR(E,A)AG(V,L)(   D , E   )V,A)A,V,S,L)IFPKA(Q,E)P(D,N)   P     
               
               
                   
                   
                 (A,T)D(T,K)(Q,N)V(H,A)EG(V,L)(D,E)(V,A,D)(F,Y)(K,N)(Q,A)E(   N , K     
               
               
                   
                   
                 )CD(S,A)((L,T)V(S,T)L,L)GGGSSHD(T,A)(   G , A   )K(A,G)I(   G , A   )   LV   AA 
               
               
                   
                   
                 NGG(   R , T , K ) I ( N , H , Y   )DY(Q,E)   GV ( N , D )( S , V , K ) V ,)(, K )( K , E ) P , Q )(   
               
               
                   
                   
                     M , V ) V   P(L,V,Q)(T,V)AI(N,T)TTAGTG   S   E(T,L)   T   (S,K,R)(F,L) ( A , T )( V ,   
               
               
                   
                   
                     I ) ITD(S,T)(A,E)R   KV   KM(P,A)V,D(   I , V ) D   (E,K)H,K)LV)TPT(V,L)(A,S) 
               
               
                   
                   
                 I(V,N)DPELMV(K,G)(K,M)P(P,A)(G,S)LT(I,A)ATG(M,L)DAL(S, 
               
               
                   
                   
                 T)HAIEAYV(A,S)(K,T)(G,R,A)ATP(V,I)TDA(F,L)AIQA(M,I)K(L,T)I 
               
               
                   
                   
                 (N,S)(E,K)YLP(R,K)A(V,F)ANG(E,K)D(I,M)EARE(Q,A,K)MA(Y,F) 
               
               
                   
                   
                 AQ(Y,S)(M,L)AG(M,V)AFNN(G,A)(G,S)LG(Y,L)VH(S,A)I(S,A)H   Q     
               
               
                   
                   
                 (V,L,F)G   G   (F,V)Y(K,N)(F,L)(P,Q)HG(I,V)CN(S,A)(V,I)(N,L,V)(M,L)P 
               
               
                   
                   
                 (H,Y)VC(R,Q,A)FNLI(A,S)(K,R)(T,V)ER(F,Y)A(H,E)IA(E,A)(L,F)L 
               
               
                   
                   
                 GENV(S,A,D)GLST(A,Y,R)(S,A,E,D)AAE(R,K)(A,T,G)I(K,V,A)A(L, 
               
               
                   
                   
                 T)(E,Q)R(M,Y,D)(N,A)(K,R)(D,N,S)(F,L)(G,N)IP(S,K,R)G(Y,F)(K,A) 
               
               
                   
                   
                 E,A)(M,L)G(V,A)   K   EEDI(E,V)(L,T)LA(K,N,E)   N   A(M,Y,F)(Q,N,K,E)D 
               
               
                   
                   
                   ( V , A )( C , T ) (T,A)(L,Q)(T,S,D)NPR(V,K)(P,A)(T,K)(V,L)(Q,E)(D,E)(I, 
               
               
                   
                   
                 V)(A,I,Q)QIIKNA(M,L),