Patent Publication Number: US-9890400-B2

Title: Method of production of 2,4-dihydroxybutyric acid

Description:
The present invention relates to a novel method of production of 2,4-dihydroxybutyric acid from malate and/or glyoxylate and/or succinyl-CoA by the implementation of a synthetic pathway that converts malate and/or glyoxylate and/or succinyl-CoA into malylCoA, malylCoA into malate-4-semialdehyde, and then converting said malate-4-semialdehyde into 2,4-dihydroxybutyric acid (2,4-DHB). 
     The carboxylic acids cited within the present application are equally named under their salt (e.g. 2,4-dihydroxyburyrate) or acid forms (e.g. 2,4-dihydroxybutyric acid). 
     2,4-dihydroxybutyric acid (equally 2,4-DHB or DHB) is a compound of considerable economic interest. DHB can be readily converted into α-hydroxy-γ-butyrolactone in aqueous media by adjusting the appropriate pH. α-hydroxy-γ-butyrolactone is a prominent precursor for the production of the methionine substitute, 2-hydroxy-4-(methylthio)-butyrate (HMTB), (US 2009/318715) which has a large market in animal nutrition. At present, α-hydroxy-γ-butyrolactone is derived from γ-butyrolactone by a multi-stage process that implies halogenation of the γ-butyrolactone in position α, and subsequent substitution of the halogen atom by a hydroxyl group in alkaline medium (US 2009/318715). 
     From growing oil prices, the need for the production of DHB from renewable resources arises. Microorganisms are capable of transforming biomass-derived raw material, e.g. sugars or organic acids, into a large variety of different chemical compounds (Werpy &amp; Petersen, 2004). With the growing body of biochemical and genomic information, it is possible to modify microorganisms such that they overproduce naturally occurring metabolic intermediates with high yield and productivity (Bailey, 1991). Optimization of production microorganisms often requires rational engineering of metabolic networks which ensures, among others, overexpression of enzymes required for the biosynthesis of the metabolite of interest, and alleviation of product feedback inhibition. Another possibility is the implementation of novel enzymatic systems that catalyze the production of a metabolite of interest. 
     Metabolic engineering approaches and enzymatic catalyses require detailed knowledge of the biochemistry and regulation of the metabolic pathway leading to the metabolite of interest. In the case of 2,4-DHB production, this information is not available. Only few studies report the occurrence of 2,4-DHB in patients with succinic semialdehyde dehydrogenase deficiency (Shinka et al., 2002) without, however, identifying enzymatic reactions implicated in DHB production. The zymotic or enzymatic production of 2,4-DHB, therefore, requires (i) the identification of a thermodynamically feasible pathway which transforms an accessible precursor into 2,4-DHB, (ii) the identification or construction of enzymes that are capable to catalyze individual reaction steps in the pathway and (iii) the functional expression of the pathway enzymes in an appropriate production organism. 
     The present invention has as an objective to satisfy these needs. 
     Accordingly, one object of the present invention is a method of producing 2,4-DHB comprising a first step of converting malate and/or glyoxylate and/or succinyl-CoA in malyl-CoA, a second step of converting malyl-CoA in malate-4-semialdehyde and, a third step of converting malate-4-semialdehyde in 2,4-DHB. 
     Accordingly, one object of the present invention is a method of producing 2,4-DHB which comprises a first reaction (see  FIG. 1 ), wherein malate is converted into malyl-CoA by the action of an enzyme which possesses malyl-CoA synthetase activity [1.1], and/or wherein succinyl-CoA is converted into malyl-CoA by the action of an enzyme having a succinyl-CoA:(L)-malate CoA transferase activity [1.2], and/or wherein glyoxylate is converted into malyl-CoA by the action of an enzyme which possesses malyl-CoA lyase activity [1.3]. In the second reaction [2], malyl-CoA is converted into malate-4-semialdehyde by the action of an enzyme which possesses malyl-CoA reductase activity. In the third reaction [3], malate-4-semialdehyde is converted into DHB by the action of an enzyme which possesses DHB dehydrogenase activity. More precisely, reaction [3] is catalysed by an enzyme bearing malate-4-semialdehyde reductase activity in the biosynthetic sense of the pathway. 
     Within another aspect of the invention, the first step of the method of producing 2,4-DHB involves an enzyme having malyl-CoA synthetase (equally named malate thiokinase or malate-coenzyme A ligase (ADP forming), EC 6.2.1.9), succinyl-CoA:(L)-malate CoA transferase (EC 2.8.3.-), or malyl-CoA lyase (EC 4.1.3.24) activity that transforms malate, succinyl-CoA, or glyoxylate, respectively, into malyl-CoA. 
     Said enzymes have been identified in methylotrophic bacteria that employ the serine cycle for fixation of formaldehyde (Chistoserdova et al., 2009; Smejkalová et al., 2010; Vuilleumier et al., 2009), in bacteria that rely on acetate assimilation pathways that are independent from the glyoxylate cycle and isocitrate lyase activity (Meister et al., 2005), and in bacteria that employ the 3-hydroxypropionate CO2-fixation cycle for autotrophic growth (Zarzycki et al., 2009). 
     Proteins sharing homology with the above enzymes are also another aspect of the invention such as functional variants or functional fragments. 
     Malyl-CoA synthetase consists of two subunits, MtkA and MtkB. Therefore, according to the invention, proteins comprising a malyl-CoA synthetase activity designate all polypeptides having at least 30% of identity with the protein sequences of the malyl-CoA synthetase subunits MtkA and MtkB of  M. petroleiphilum  (YP 001022444 and YP 001022445)  Methylobacter extorquens  (YP002962851 and YP 002962852) or two subunits SucC and SucD of  M. capsulatus  (YP 114179 and YP 114180), preferentially at least 50% and more preferentially 70% of identity. 
     Malyl-CoA lyase is a homohexamer and found in bacteria that do not employ the glyoxylate cycle for acetate assimilation (Meister et al., 2005). Therefore, according to the invention, proteins comprising a malyl-CoA lyase activity designate all polypeptides having at least 30% of identity with the protein sequences of the malyl-CoA lyase, Mcl, of  Methylobacter extorquens, Rhodobacter capsulatus , or  Streptomyces coelicolor , preferentially at least 50 and more preferentially 70% of identity. 
     In a further aspect of the invention, the malyl-CoA lyase of the invention is represented by SEQ ID No. 1 or by any variant thereof. 
     Succinyl-CoA:(L)-malate CoA transferase consists of two subunits, SmtA and SmtB (Zarzycki et al., 2009)(Friedmann et al., 2006). Therefore, according to the invention, proteins having a succinyl-CoA:(L)-malate CoA transferase activity designate all polypeptides having at least 30% of identity with the protein sequences of the succinyl-CoA:(L)-malate CoA transferase subunits SmtA and SmtB of  Chloroflexus aurantiacus  (represented by SEQ ID No. 191 and SEQ ID No. 193 or encoded by SEQ ID No. 192 and SEQ ID No. 194), preferentially at least 50% and more preferentially 70% of identity. 
     More generally, within the meaning of the invention the identity between two protein sequences can be determined by methods well known by the skilled man in the art. Examples of such methods are the use of the CLUSTALW (Larkin et al., 2007) software (with the default parameters indicated on the website) or the BLAST alignment program (with the default parameters indicated on the website). 
     The term functional variant encompasses enzymes that may present substantial sequence modifications when compared to the sequences specifically described within the present application but that still retain the original enzymatic activity. 
     The term functional fragment, according to the invention, means that the sequence of the enzyme may comprise less amino acids than the original one but said truncated enzyme still retains the original enzymatic activity. 
     Improvement of said enzymes can be obtained by at least one mutation, said mutation(s) improving the activity and/or substrate affinity of the mutated enzyme for malate, succinyl-CoA, or glyoxylate respectively. 
     Within the present invention, the expression “improve the activity and/or substrate affinity” means that the enzyme before mutation was either
         unable to use the substrate, and/or   synthesized the product of the reaction at a maximum specific rate at least three times lower, and/or   had an affinity for malate, succinyl-CoA or glyoxylate, malyl-CoA or malate-4-semialdehyde that was at least two more preferably three times lower.       

     The malyl-CoA synthetase and the malyl-CoA lyase activities can be measured by the enzymatic tests described by (Smejkalová et al., 2010) or (Meister et al., 2005), respectively. The succinyl-CoA:(L)-malate CoA transferase activity can be measured as described by (Friedmann et al., 2006) 
     Within a still further aspect, the second step of the method of producing 2,4-DHB according to the invention involves an enzyme having malyl-CoA reductase activity characterized in that it transforms malyl-CoA into malate-4-semialdehyde. 
     Said enzyme can be identified among enzymes having malonyl-CoA reductase, a succinyl-CoA reductase or reported 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, cinnamoyl-CoA reductase, or acetaldehyde dehydrogenase activity, or they can be obtained by modification of said enzymes. 
     Malonyl-CoA reductase (EC 1.2.1.75) and succinyl-CoA reductase (EC 1.2.1.76) were found in bacteria that possess a modified 3-hydroxypropionate cycle for carbon dioxide fixation (Alber et al., 2006; Kockelkorn &amp; Fuchs, 2009), and in bacteria that employ an anaerobic succinate degradation pathway (Seedorf et al., 2008; Söhling &amp; Gottschalk, 1993). HMG-CoA reductase (EC 1.1.1.38, EC 1.1.1.88) is part of the biosynthetic pathway of isoprenoids in eukaryotes and some bacteria. Cinnamoyl-CoA reductase (EC 1.2.1.44), is an enzyme implicated in lignin biosynthesis (Kawasaki et al., 2006). Acetaldehyde dehydrogenase (EC 1.2.1.10) is found in a large variety of bacteria and catalyses the entry into the ethanol producing pathway or the detoxification of acetaldehyde. 
     Within a further aspect of the invention, the malyl-CoA reductase is represented by ID No. 7, or SEQ ID No.10 or by any functional variant thereof or any functional fragment thereof. 
     Therefore, according to the invention, proteins having a malonyl-CoA reductase activity designate all polypeptides having at least 30% of identity with the protein sequences of the  Sulfolobus tokodaii  malonyl-CoA reductase, Mcr (SEQ ID No. 7). Preferentially they have at least 50% and more preferentially 70% of identity. 
     The malonyl-CoA reductase of  Chloroflexus auranthiacus  (SEQ ID No.189 encoded by SEQ ID No. 190) constitutes another aspect of the invention. Polypeptides having at least 30% of identity with the protein sequences of  Chloroflexus auranthiacus  are also part of the invention. Preferentially they have at least 50% and more preferentially 70% of identity. 
     Therefore, according to the invention, proteins having a succinyl-CoA reductase activity designate all polypeptides having at least 30% of identity with the protein sequences of the  Porphyromonas gingivalis  succinyl-CoA reductase, SucD (SEQ ID No. 10), or with the bifunctional  S. tokodaii  malonyl-CoA and succinyl-CoA reductase, Mcr (SEQ ID No. 7). Preferentially they have at least 50% and more preferentially 70% of identity. 
     The malyl-CoA reductase activity can be measured by the enzymatic test described in Example 2 (see “Enzymatic assay”). 
     This enzyme activity can be improved by at least one mutation of an enzyme, said mutation(s) improving the activity and/or substrate affinity of the mutated enzyme for malyl-CoA or decreasing its activity on the natural substrate. 
     The present invention also encompasses modified malyl-CoA reductase having improved activities. 
     The malyl-CoA reductase according to the invention corresponds in a specific aspect to  Sulfolobus tokodaii  malonyl-CoA reductase comprising at least one mutation when compared to the wild type enzyme in at least one of the positions P111, L152, T154, L202, G203, D204, Y206, D207, K209, T210, T238, T239, D295, R318, wherein the naturally occurring amino acid in said positions is replaced by anyone of the other 19 naturally existing proteinogenic amino acids, that is by either alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. 
     Within another aspect, the third step of the method of producing 2,4-DHB according to the invention involves a DHB dehydrogenase characterized in that it transforms malate-4-semialdehyde into 2,4-DHB, said enzyme bearing malate-4-semialdehyde reductase activity in the biosynthetic sense of the pathway. 
     Candidate DHB dehydrogenase enzymes that potentially already possess DHB dehydrogenase activity can be chosen from the class of beta-hydroxyacid dehydrogenases that act on C3, C4, or C5 compounds. 
     According to a still further aspect of the invention, said DHB dehydrogenase enzymes can be structurally and mechanistically related to β-hydroxyacid dehydrogenases such as tartronate semialdehyde reductases, succinate semialdehyde reductases, 4-hydroxybutyrate dehydrogenases, malonate semialdehyde reductases, methylbutyraldehyde reductases, zinc-type alcohol dehydrogenases, L-threonine-3-dehydrogenases, cinnamyl alcohol dehydrogenases, alcohol dehydrogenases, or homoserine dehydrogenases. 
     The present invention also deals with the use of a methylbutyraldehyde reductase, or of a succinic semialdehyde reductase (equally named as 4-hydroxybutyrate dehydrogenase), or of an alcohol dehydrogenase, to transform malate-4-semialdehyde in 2,4-DHB. 
     In another specific aspect of the invention, the DHB dehydrogenase corresponds to methylbutyraldehyde reductase (Ypr1) of  S. cerevisiae , the succinic semialdehyde reductase of  M. sedula , the 4-hydroxybutyrate dehydrogenase (4hbd) of  P. gingivalis , or to the alcohol dehydrogenase (YqhD) of  Escherichia coli.    
     In specific embodiments, said methylbutyraldehyde reductase is represented by SEQ ID No. 14, said succinic semialdehyde reductase is represented by SEQ ID No. 16, said 4-hydroxybutyrate dehydrogenase is represented by SEQ ID No. 187, said alcohol dehydrogenase is represented by SEQ ID No. 185. The DHB dehydrogenase activity can be measured by the enzymatic test described in Example 3 (see “Enzymatic assay”). 
     The affinity of DHB dehydrogenase for malate-4-semi aldehyde can be increased by at least one mutation of an enzyme, said mutation(s) increasing the activity and/or substrate affinity of the mutated enzyme for malate-4-semialdehyde, and/or reducing the activity or affinity for its natural substrate by at least factor 2. 
     The DHB dehydrogenase according to the invention corresponds in a specific aspect to  M. sedula  succinic semialdehyde reductase (SEQ ID No. 16) comprising at least one mutation when compared to the wild type enzyme in at least one of the positions S40, N43, H39 T49, F85, Q108, L281 and N305 wherein the naturally occurring amino acid in said positions is replaced by anyone of the other 19 naturally existing proteinogenic amino acids, that is by either alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. 
     As demonstrated in a non-exclusive example, the affinity of  M. sedula  succinic semialdehyde reductase for (L)-malate-4-semialdehyde was increased by introducing the double mutation H39R N43H by site-directed mutagenesis, as represented by SEQ ID No. 36. Simple mutants H39R (SEQ ID No. 32) and N43H (SEQ ID No. 34) are also encompassed by the present invention (Example 5). 
     DHB dehydrogenase can be used to transform malate-4-semialdehyde into 2,4-DHB, which constitutes a further aspect of the invention. 
     The nucleic acid sequence of genes can be adapted to the codon usage of the host organism thereby increasing the production of the heterologously expressed proteins. This constitutes a further aspect of the invention. 
     The synthesis of a synthetic gene coding for  M. sedula  succinic semialdehyde reductase H39R N43H whose nucleotide sequence was optimized for the expression of said enzyme in  E. coli  as represented by SEQ ID No. 38 is a further aspect of the invention. 
     In a still further aspect, the present invention also deals with nucleic acids, and more particularly with isolated nucleic acid sequences encoding malyl-CoA synthetase. 
     In a still further aspect, the present invention deals with isolated nucleic acid sequences encoding malyl-CoA lyase and more specifically by SEQ ID No.2. 
     In a still further aspect, the present invention deals with isolated nucleic acid sequences encoding malyl-CoA reductase and more specifically by SEQ ID No. 8, SEQ ID No.11, or SEQ ID No.190. 
     In a still further aspect, the present invention also deals with isolated nucleic acid sequences encoding a DHB dehydrogenase as described above. 
     In another aspect, said nucleic acid is represented by SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 37, SEQ ID No. 186 or SEQ ID No. 188. 
     In accordance with this invention, a “nucleic acid sequence” refers to a DNA or RNA molecule in single or double stranded form, preferably a DNA molecule. An “isolated DNA”, as used herein, refers to a DNA which is not naturally-occurring or no longer in the natural environment wherein it was originally present, e.g., a DNA coding sequence associated with other regulatory elements in a chimeric gene, a DNA transferred into another host cell, or an artificial, synthetically-made DNA sequence having a different nucleotide sequence compared to any naturally-occurring DNA sequence. 
     The present invention also relates to a chimeric gene comprising, functionally linked to one another, at least one promoter which is functional in a host organism, a polynucleotide encoding anyone of the malyl-CoA synthetase or malyl-CoA lyase, malyl-CoA reductase, malonyl-CoA reductase, succinyl-CoA reductase or DHB dehydrogenase activities as defined according to the invention, and a terminator element that is functional in the same host organism. The various elements which a chimeric gene may contain are, firstly, elements regulating transcription, translation and maturation of proteins, such as a promoter, a sequence encoding a signal peptide or a transit peptide, or a terminator element constituting a polyadenylation signal and, secondly, a polynucleotide encoding a protein. The expression “functionally linked to one another” means that said elements of the chimeric gene are linked to one another in such a way that the function of one of these elements is affected by that of another. By way of example, a promoter is functionally linked to a coding sequence when it is capable of affecting the expression of said coding sequence. The construction of the chimeric gene according to the invention and the assembly of its various elements can be carried out using techniques well known to those skilled in the art. The choice of the regulatory elements constituting the chimeric gene depends essentially on the host organism in which they must function, and those skilled in the art are capable of selecting regulatory elements which are functional in a given host organism. The term “functional” is intended to mean capable of functioning in a given host organism. 
     The promoters which the chimeric gene according to the invention may contain are either constitutive or inducible. By way of example, the promoters used for expression in bacteria may be chosen from the promoters mentioned below. For expression in  Escherichia coli  mention may be made of the lac, trp, lpp, phoA, recA, araBAD, prou, cst-I, tetA, cadA, nar, tac, trc, lpp-lac, Psyn, cspA, PL, PL-9G-50, PR-PL, T7, [lambda]PL-PT7, T3-lac, T5-lac, T4 gene 32, nprM-lac, VHb and the protein A promoters or else the Ptrp promoter (WO 99/64607). For expression in Gram-positive bacteria such as  Corynebacteria  or  Streptomyces , mention may be made of the PtipA or PS1 and PS2 (FR91/09870) promoters or those described in application EP0629699A2. For expression in yeasts and fungi, mention may be made of the  K. lactis  PLAC4 promoters or the  K. lactis  Ppgk promoter (patent application FR 91/05294), the  Trichoderma  tef1 or cbh1 promoter (WO 94/04673), the  Penicillium  his, csl or apf promoter (WO 00/68401) and the  Aspergillus  gla promoter. 
     According to the invention, the chimeric gene may also comprise other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators (enhancers). 
     As such, the chimeric gene of the invention comprises, in a specific embodiment at least, in the direction of transcription, functionally linked, a promoter regulatory sequence which is functional in a host organism, a nucleic acid sequence encoding the malyl-CoA synthetase, and/or the succinyl-CoA:(L)-malate-CoA transferase, and/or the malyl-CoA lyase, the malyl-CoA reductase and the DHB dehydrogenase of the invention and a terminator regulatory sequence which is functional in said host organism. 
     The present invention also relates to a cloning and/or expression vector comprising a chimeric gene according to the invention or a nucleic acid sequence of the invention. The vector according to the invention is of use for transforming a host organism and expressing in this organism anyone of the malyl-CoA synthetase, and/or the succinyl-CoA:(L)-malate CoA transferase, and/or the malyl-CoA lyase, the malyl-CoA reductase and/or DHB dehydrogenase. This vector may be a plasmid, a cosmid, a bacteriophage or a virus. Preferentially, the transformation vector according to the invention is a plasmid. Generally, the main qualities of this vector should be an ability to maintain itself and to self-replicate in the cells of the host organism, in particular by virtue of the presence of an origin of replication, and to express anyone of the malyl-CoA synthetase, and/or the succinyl-CoA:(L)-malate CoA transferase and/or the malyl-CoA lyase, the malyl-CoA reductase and/or DHB dehydrogenase therein. For the purpose of stable transformation of a host organism, the vector may also integrate into the genome. The choice of such a vector, and also the techniques of insertion of the chimeric gene according to the invention into this vector and are part of the general knowledge of those skilled in the art. Advantageously, the vector used in the present invention also contains, in addition to the chimeric gene according to the invention, a chimeric gene encoding a selectable marker. This selectable marker makes it possible to select the host organisms which are effectively transformed, i.e. those which incorporated the vector. According to a particular embodiment of the invention, the host organism to be transformed is a bacterium, a yeast, a fungus. Among the selectable markers which can be used, mention may be made of markers containing genes for resistance to antibiotics, such as, for example, the hygromycin phosphotransferase gene. Other markers may be genes to complement an auxotrophy, such as the pyrA, pyrB, pyrG, pyr4, arg4, argB and trpC genes, the molybdopterin synthase gene or that of acetamidase. Mention may also be made of genes encoding readily identifiable enzymes such as the GUS enzyme, or genes encoding pigments or enzymes regulating the production of pigments in the transformed cells. Such selectable marker genes are in particular described in patent applications WO 91/02071, WO 95/06128, WO 96/38567 and WO 97/04103. 
     The present invention also relates to transformed host organisms containing at least one chimeric gene according to the invention, either integrated into their genome or carried on an extra-chromosomal genetic element, for example a plasmid. In a more specific aspect of the invention, the transformed host organism comprises a nucleic acid of the invention encoding a polypeptide having malyl-CoA synthetase activity, and/or succinyl-CoA:(L)-malate-CoA transferase and/or malyl-CoA lyase activity or a chimeric gene comprising a nucleic acid encoding a polypeptide having malyl-CoA synthetase activity, and/or succinyl-CoA:(L)-malate CoA transferase and/or a malyl-CoA lyase activity or an expression vector comprising a nucleic acid encoding a polypeptide having malyl-CoA synthetase activity, and/or succinyl-CoA:(L)-malate CoA transferase and/or a malyl-CoA lyase activity, and/or a nucleic acid encoding a polypeptide having malyl-CoA reductase activity, or a chimeric gene comprising a nucleic acid encoding a polypeptide having malyl-CoA reductase activity or an expression vector comprising a nucleic acid encoding a polypeptide having malyl-CoA reductase activity, and/or a nucleic acid encoding a polypeptide having DHB dehydrogenase activity, a chimeric gene comprising a nucleic acid encoding a polypeptide having DHB dehydrogenase activity or an expression vector comprising a nucleic acid encoding a polypeptide having DHB dehydrogenase activity. 
     The activity of heterologously enzymes in the host organism is often limited by their poor solubility and the formation of inclusion bodies. Therefore, the present invention also relates to chimeric proteins in that a functional enzyme is physically fused to another protein or peptide (equally named fusion protein) in order to increase the activity of said enzyme upon expression in the host organism. Such fusion proteins are known in the art and are commonly selected among the following non-exclusive examples: maltose binding protein, Mbp, thioredoxin, ThrX, glutathione-S-transferase, Gst, transcription termination factor, NusA. 
     The term “host organism” is intended to mean any lower monocellular organism into which the chimeric gene(s), nucleic acid(s) or vector(s) according to the invention may be introduced in order to produce 2,4-DHB. Preferably, the host organism is a microorganism, in particular a fungus, for example of the  Penicillium, Aspergillus  and more particularly  Aspergillus flavus, Chrysosporium  or  Trichoderma  genus, a yeast, in particular of the Saccharomycetaceae, Pichiaceae or Schizosaccharomycetaceae, most preferentially  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces marxianus , or  Pichia jadinii, Pichia stipitis  or  Pichia pastoris , a bacterium, preferentially selected among Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, Streptococcaceae, Methylobacteriacae, and Corynebacteriaceae, most preferentially  Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Clostridium acetobutylicum, Methylobacterium extorquens , or  Lactococcus lactis.    
     The host organism can be a host organism that naturally overproduces malate or succinate from sugars such as glucose or a host organism that was engineered to overproduce malate or succinate from sugars such as glucose and in which all potential membrane transporters that facilitate export of organic acids, such as malate, pyruvate, succinate, and fumarate have been deleted. The host organism can be an organism that was engineered to overproduce DHB and in which membrane transporters that facilitate export of organic acids such as malate, pyruvate, succinate, and fumarate have been deleted. Examples of permeases that facilitate export of malate and other organic acids are Mae1 from  Schizosaccharomyces pombe  (Camarasa et al., 2001) (Grobler et al., 1995), DctA from  Bacillus subtilis  (Groeneveld et al., 2010), Dct 1-4 from coil, Jen1 from  S. cerevisiae  (Akita et al., 2000). For an expert, it will be possible to identify candidate permeases in  E. coli  based on sequence identity. These constructions will serve to keep malate and other organic acids inside the cell to make them available for DHB production. 
     The expression “transformed host organism” is intended to mean a host organism which has incorporated into its genome, or in an extra chromosomal genetic element, for example a plasmid, at least one chimeric gene according to the invention, and consequently produces any one of malyl-CoA synthetase, malyl-CoA lyase, malyl-CoA reductase and/or DHB dehydrogenase in cell interior or in a culture medium. To obtain the host organisms according to the invention, those skilled in the art may use one of the many known transformation methods. 
     One of these methods consists in bringing the cells of the host organisms to be transformed into contact with polyethylene glycol (PEG) and with the vectors according to the invention. Electroporation is another method, which consists in subjecting the cells to be transformed and the vectors of the invention to an electric field. Another method consists in directly injecting the vectors into the cells or the tissues by microinjection. The “biolistic” method may be used. It consists in bombarding cells or tissues with particles onto which the vectors of the invention are adsorbed (U.S. Pat. No. 4,945,050). 
     Several methods for transforming bacteria are described in the literature for  Escherichia coli  and other Gram-negative bacteria. Conjugation may also be used. For Gram-positive bacteria, electroporation may be used, and also protoplast transformation, in particular for bacteria of the  Streptomyces  genus. 
     Several methods for transforming fungi are also described in the literature. Protoplast transformation with PEG is described for  Aspergillus  in EP 0260762, and an adaptation of this method to the species  Penicillium funiculosum  is described in WO 00/36120. Transformation by restriction enzyme mediated integration, or REMI, is also known, as is protoplast transformation using bacteria of the  Agrobacterium  genus. Techniques for transforming yeasts are also described in the literature, 
     In a further aspect, the invention deals with a process of production of 2,4-DHB comprising the step of cultivating a transformed microorganism of the invention. 
     For the production of DHB various carbohydrates could be utilized individually or as a mixture such as glucose, fructose, sucrose, molasses, maltose, blackstrap molasses, starch hydrolysate (glucose, oligosaccharides), lactose, maltose, starch and starch hydrolysates, cellulose, cellulose hydrolysate, glycerol, acetate and certain hydrocarbons, oils and fats such as soy bean oil, sunflower oil, groundnut oil and coconut oil as well as fatty acids such as e.g. palmitic acid, stearic acid and linoleic acid. Those substances may be used individually or as mixtures. 
     Various sources of nitrogen could be utilized individually or as mixtures for the commercial and pilot scale production, including inorganic compounds such as gaseous and aqueous ammonia, ammonium salts of inorganic or organic acids such as ammonium sulphate, ammonium nitrate, ammonium phosphate, ammonium chloride, ammonium acetate and ammonium carbonate. Alternatively, natural nitrogen containing organic materials like soybean-hydrolysate, soy protein HCl-hydrolysate (total nitrogen of about 7%), soy bean meal, soybean cake hydrolysate, corn steep liquor, casein hydrolysate, yeast extract, meat extract, malt extract, urea, peptones and amino acids may also be utilized 
     The production process can be carried out under aerobic, anaerobic, and oxygen limited conditions. It can be carried out as a fed-batch process or a batch process. 
     Said production of 2,4-DHB can be made by cultivating the host organism in media where malate (or another organic acid such as pyruvate, succinate, or fumarate) was added alone or together with another carbon source that ensures growth. Malate (and other organic acids) can be added either directly, or by designing a two-stage fermentation process where malate (or other organic acids) is produced in a first process stage by a malate-overproducing microorganism, and 2,4-DHB production is realised in the following stage by a host organism according to the invention. 
     Product separation and purification is very important factor enormously affecting overall process efficiency and product costs. Methods for product recovery commonly comprise the steps cell separation, as well as product purification, concentration and drying, respectively. 
     Cell Separation 
     Ultra filtration and centrifugation can be used to separate cells from the fermentation medium. Cell separation from fermentation media is often complicated by high medium viscosity. Therefore, we can add additives such as mineral acids or alkali salts, or heating of the culture broth to optimize cell separation. 
     Product Recovery 
     A variety of ion-exchange chromatographic methods can be applied for the separation of DHB either before or after biomass removal. They include the use of primary cation exchange resins that facilitate separation of products according to their isoelectric point. Typically, the resin is charged with the solution, and retained product is eluted separately following increase of pH (e.g. by adding ammonium hydroxide) in the eluent. Another possibility is the use of ion-exchange chromatography using fixed or simulated moving bed resins. Different chromatographic steps may have to be combined in order to attain adequate product purity. Those purification methods are more economical compared with a costly crystallization step, also providing additional advantages and flexibility regarding the form of final product. 
     Product Concentration and Drying 
     The purification process can also comprises a drying step which may involve any suitable drying means such as a spray granulator, spray dryer, drum dryer, rotary dryer, and tunnel dryer. Concentrated DHB solutions can be obtained by heating fermentation broths under reduced pressure by steam at 130° C. using a multipurpose concentrator or thin film evaporator. 
     Efficient production of DHB can be ensured by optimizing carbon flux repartitioning in the metabolic network of the host organism and by ensuring sufficient NADPH and ATP supply for the three enzymes of the DHB pathway. Channeling of carbon flux into a desired metabolic pathway and supply of NAD(P)H cofactor is commonly facilitated by deleting or alleviating competing natural fermentative pathways. Nonexclusive examples are
         the optimization of malate production in  S. cerevisiae  by impeding the formation of ethanol (by the deletion of pyruvate decarboxylases (Zelle et al., 2008)(Zelle et al., 2010).   the optimization of succinate or malate production in  E. coli  by impeding the formation of lactate (e.g. deletion of IdhA), the formation of acetate (e.g. deletion of pta, ackA), the formation of ethanol (e.g. deletion of adhE), the formation of formate (e.g. deletion of pflB, focA), the oxidation of pyruvate (e.g. deletion of poxB), the degradation of malate (deletion of maeB and scfA), the formation of succinate (e.g. deletion of frdBC), the formation of methylglyoxal (deletion of mgsA) (Jantama et al., 2008a) (Jantama et al., 2008b) (Lin et al., 2005) (Zhang et al., 2011) (Sanchez et al., 2005).   the deletion of phosphoglucose isomerase, pgi, to channel carbon flux across the pentose phosphate pathway thereby increasing NADPH availability for biosynthetic reactions (Auriol et al., 2011).       

     Another possibility to increase carbon flux and ATP supply for the production of organic acids is the engineering of the phosphoenolpyruvate (PEP)/pyruvate/oxaloacetate branch node (reviewed in (Sauer &amp; Eikmanns, 2005)). Nonexclusive examples for metabolic engineering strategies that ensure the increase of carbon flux from phosphoenolpyruvate to oxaloacetate are:
         the optimization of malate production in  S. cerevisiae  by impeding the function of pyruvate kinase and increasing the activity of PEP carboxykinase (Zelle et al., 2010).   the optimization of succinate production in  E. coli  by increasing the activity of natural or heterologously expressed PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase (Millard et al., 1996) (Sanchez et al., 2005) (Zhang et al., 2011).       

     Another possibility to increase carbon flux and ATP supply for the production of organic acids in  E. coli  and other bacteria employing the PEP-consuming phosphotransferase system (PTS) for the initial phosphorylation step of glucose is the deletion of essential components of the PTS system (for example ptsI or ptsG) (Lin et al., 2005) (Zhang et al., 2009). To ensure further glucose uptake in mutants carrying deleterious mutations of the PTS system, the activity of alternative glucose uptake systems (e.g. GalP) has to be ensured. 
     Another possibility to increase carbon flux into the desired pathways for the production of organic acids is the engineering of the citric acid and glyoxylate cycle. Non-exclusive examples are
         the optimization of succinic acid production in  E. coli  by increasing the activity of isocitrate lyase (deletion of transcriptional repressor iclR) (Lin of al., 2005) (Sanchez et al., 2005) (Lin et al., 2005; Sanchez et al., 2005a).   the optimization of succinic acid production by the deletion of isocitrate dehydrogenase, and/or succinate dehydrogenase (Lin et al., 2005).       

     Another possibility to increase the availability of malate, glyoxylate and acetyl-CoA, which are the substrates of the entry reactions into the DHB-producing pathways, is the attenuation of aspartate transaminase (aspC, tyrB), fumarase (fumABC), fumarate reductase (frdBC), malate synthase (aceB) and glyoxylate reductase (ghrAB) enzymes. 
     In another metabolic setting it is possible to produce the 2,4-DHB precursor malate exclusively via the Krebs cycle and the glyoxylate shunt. This setting requires deletion of the cytosolic and membrane bound malate dehydrogenases, mdh and mqo, respectively. The approach largely avoids potential leakage of carbon flux into aspartate and its derivatives. 
     Another possibility to increase carbon flux into the desired pathways for the production of 2,4-DHB is the expression of appropriate pyruvate dehydrogenases and citrate synthases in the production organism. Natural pyruvate dehydrogenase and citrate synthase of  E. coli  are inhibited by high intracellular NADH concentrations rendering these enzymes less active under anaerobic conditions. In  E. coli , the expression of a pyruvate dehydrogenase mutant that is insensitive to NADH resulted in the overproduction of acetyl-CoA under anaerobic conditions and modified carbon flux repartitioning between the fermentative end-products (acetate, lactate, ethanol, formate, and pyruvate) (Wang et al., 2010). The heterologous expression of the  Bacillus subtilis  citrate synthase which is insensitive to NADH increased succinic acid production in engineered  E. coli  strains (Sanchez et al., 2005). In combination with the above described mutations, the use of the appropriate pyruvate dehydrogenases and citrate synthases (NADH sensitive or insensitive) enables the tuning of carbon flux repartitioning between glyoxylate and citric acid cycle reactions and fermentative pathways under anaerobic and aerobic conditions. 
     Another possibility to increase carbon flux through the DHB pathway is the deletion of enzymatic reactions that may degrade the pathway intermediates malyl-CoA, or 4-malate semialdehyde. Candidate enzymes that may degrade malate semialdehyde are succinic semialdehyde dehydrogenases (sad, gabD), and other dehydrogenases that are able to oxidize short and medium carbon chain molecules with terminal aldehyde groups. Furthermore, it is known that malyl-CoA may be degraded by citrate synthase. 
     Another possibility to increase 2,4-DHB productivity of the host organism is the deletion of metabolic reactions that degrade 2,4-DHB. 2,4-DHB is a competitive inhibitor of malic enzyme, thus, having comparatively high affinity for the active site of this enzyme (Rognstad &amp; Katz, 1979). Therefore, 2,4-DHB may be recognized by other enzymes and potentially degraded. These enzymes can be identified and deleted from the host organism. 
     When 2,4-DHB production is based on addition of malate or other organic acids, the 2,4-DHB-producing microorganisms should functionally express a membrane transport protein that facilitates uptake of malate (or other organic acids such as pyruvate, succinate, etc). 
     The transformed host organisms of the invention may further contain an additional pathway of synthesizing 2,4-DHB, said host organism comprises at least one chimeric gene, either integrated into their genome or carried on an extra-chromosomal genetic element, for example a plasmid encoding a malate kinase or a chimeric gene comprising a nucleic acid encoding a malate kinase or an expression vector comprising a nucleic acid encoding a malate kinase, and/or a nucleic acid encoding a malate semialdehyde dehydrogenase, or a chimeric gene comprising a nucleic acid encoding a malate semialdehyde dehydrogenase or an expression vector comprising a nucleic acid encoding a malate semialdehyde dehydrogenase, and/or a nucleic acid encoding a DHB dehydrogenase, a chimeric gene comprising a nucleic acid encoding a DHB dehydrogenase or an expression vector comprising a nucleic acid encoding a DHB dehydrogenase. Said enzymes are described in the International patent application WO 2012/056318. 
     The following examples illustrate the invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 : (i) Reaction scheme that describes the conversion of (L)-malate, succinyl-CoA, or glyoxylate into (L)-2,4-dihydroxybutyrate (2,4-DHB). 
         FIG. 2 : Figure shows (top graph) activity on malate semialdehyde, (middle graph) activity on succinic semialdehyde, (lower graph) changes of enzyme specificity compared to the wild-type enzyme expressed as the logarithm of the ratio of mutant activity on malate semialdehyde and succinic semialdehyde over the ratio of wild type activity on malate semialdehyde and succinic semialdehyde. (positive values indicate changes of specificity in favour of malate semialdehyde). 
         FIG. 3 : Chromatograms showing the presence of 2,4-DHB after incubation of 2 mM acetyl-CoA, 2 mM glyoxylate, and 2 mM NADPH, with different combinations of DHB pathway enzymes (Reaction 1: malyl-CoA lyase (150 μg/mL Me-Mcl), malyl-CoA reductase (100 μg/mL St-Mcr), and malate semialdehyde reductase (100 μg/mL Ms-SSAred H39R N43H); Reaction 2:same as reaction 1 but using 100 μg/mL Pg-SucD as malyl-CoA reductase; Control 1: same as reaction 1 but without malyl-CoA reductase; Control 2: same as reaction 1 but without malate semialdehyde reductase.) 
     
    
    
     EXAMPLES 
     Example 1: Demonstration of Malyl-CoA Lyase Activity 
     Construction of plasmids containing wild-type genes coding for malyl-CoA lyase: The DNA sequences of the mcl genes coding for malyl-CoA lyase in  M. extorquens  (Arps et al., 1993) and  Rhodobacter capsulatus  (Meister et al., 2005) were optimized for the expression in  Escherichia coli  using the GENEius software (Eurofins). The optimized sequences were synthesized by Eurofins MWG OPERON® adding NheI and EcoRI restriction sites upstream of the start codon and downstream of the stop codon of mcl, respectively, which allowed direct cloning of the synthesized DNA fragments into the pET28a+ vector (Novagen) using T4 DNA ligase (Biolabs). Ligation products were transformed into  E. coli  DH5α cells, amplified, and the plasmids pET28-Mex-mcl (expressing the malyl-CoA lyase from  M. extorquens ) and pET28-Rca-mcl (expressing the malyl-CoA lyase from  R. capsulatus ) were isolated using standard genetic protocols (Sambrook et al., 1989). NCBI and Integrated Genomics references of the utilized mcl protein sequences, and the references for the corresponding natural and synthetic DNA sequences are listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 References to proteins from different organisms having annotated 
               
               
                 malyl-CoA lyase activity, and references to natural 
               
               
                 and optimized DNA sequences. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 NCBI/Integrated 
                 Natural 
                 Optimized 
               
               
                   
                   
                 Genomics accession 
                 DNA 
                 DNA 
               
               
                 Organism 
                 Protein 
                 number 
                 sequence 
                 sequence 
               
               
                   
               
               
                 
                   M. extorquens 
                 
                 Mcl 
                 YP 002962854 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 SEQ ID 
                   
                 No. 2 
                 No. 3 
               
               
                   
                 No. 1 
               
               
                   
               
            
           
         
       
     
     Expression of Enzymes: 
       E. coli  BL21 (DE3) cells were transformed with the appropriate plasmids using standard genetic protocols (Sambrook et al., 1989). Enzymes with an N-terminal hexa-His tag were expressed in 250 mL LB cultures that were inoculated from an overnight culture at OD 600  of 0.1 and grown to OD 600  of 0.6 before protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. After 3 h of protein expression, cells were harvested by centrifugation at 13000 g for 10 min and the supernatant is discarded. Cell pellets were stored at −20° C. until further analysis. Growth and protein expression were carried out at 37° C. Culture media contained 50 μg/mL kanamycin. 
     Purification of Enzymes: 
     Frozen cell pellets of expression cultures were resuspended in 0.5 mL of breakage buffer (50 mM Hepes, 300 mM NaCl, pH 7.5) and broken open by four successive rounds of sonication (sonication interval: 20 sec, power output: 30%, sonicator: Bioblock Scientific, VibraCell™ 72437). Cell debris were removed by centrifuging the crude extracts for 15 min at 4° C. at 13000 g and retaining the clear supernatant. RNA and DNA were removed from the extracts by adding 15 mg/mL streptomycin (Sigma), centrifuging the samples at 13000 g for 10 min at 4° C. and retaining the supernatant. Clear protein extract was incubated for 20 min at room temperature (1 h at 4° C.) with 0.3 (0.75 mL) (bed volume) of Talon™ Cobalt affinity resin (Clontech). The suspension was centrifuged at 700 g in a table top centrifuge and supernatant was removed. The resin was washed with 10 bed volumes of wash buffer (50 mM Hepes, 300 mM NaCl, 15 mM Imidazole, pH 7.5) before proteins were eluted with 0.5 mL of elution buffer (50 mM Hepes, 300 mM NaCl, 200 mM Imidazole, pH 7.5). Purity of eluted enzymes was verified by SDS-PAGE analysis. Protein concentrations were estimated with the method of Bradford. 
     Enzymatic Assays: 
     Malyl-CoA lyase activity was assayed using a method adapted from (Meister et al., 2005). Malyl-CoA synthesis by malyl-CoA lyase was coupled to the citrate synthase-catalyzed release of coenzyme A which was monitored by its spontaneous reaction with DTNB. 
     Reaction Scheme
 
acetyl-CoA+glyoxylate→(L)-malyl-CoA  Malyl-CoA lyase:
 
(L)-malyl-CoA→(L)-malate+Coenzyme A  Citrate synthase:
 
coenzyme A+DTNB→CoA-DTNB disulfide  Spontaneous:
 
     The reaction mixture according to Assay 1 contained 50 mM MOPS/KOH (pH 7.5), 0.25 mM DTNB, 5 mM MgCl 2 , 1 mM acetyl-CoA, 20 U/mL citrate synthetase (all products from Sigma), and appropriate amounts of purified malyl-CoA lyase or cell extract. Reactions were started by adding 10 mM glyoxylate. Enzymatic assays were carried out at 37° C. in 96-well flat bottomed microtiter plates in a final volume of 250 μL. The reactions were followed by the characteristic absorption of DNTB at 412 nm (ε DNTB+CoA =13.6 mM −1  cm −1 ) in a microplate reader (BioRad 680XR). 
     Purified malyl-CoA lyase from  M. extorquens  characterized had a Vmax of 36 μmol/(min mg prot), and a Km on glyoxylate of 0.5 mM. 
     Example 2: Demonstration of Malyl-CoA Reductase Activity 
     Construction of plasmids containing wild-type genes coding for malonyl-CoA reductase and succinyl-CoA reductase: The DNA sequence of the mcr gene coding for malyl-CoA reductase in  Sulfolobus tokodaii  str 7 (Alber et al., 2006) was optimized for the expression in  Escherichia coli  using the GENEius software (Eurofins). The optimized mcr sequence, and the natural DNA sequence of the sucD gene coding for succinyl-CoA reductase in  Porphyromonas gingivalis  W83 were synthesized by Eurofins MWG OPERON® adding NheI and EcoRI restriction sites upstream of the start codon and downstream of the stop codon of mcr, respectively, which allowed direct cloning of the synthesized DNA fragments into the pET28a+ vector (Novagen) using T4 DNA ligase (Biolabs). Ligation products were transformed into  E. coli  DH5α cells, amplified, and the plasmids, pET28-St-mcr (expressing the malonyl-CoA reductase from  S. tokodaii ), and pET28-Pgi-sucD (expressing the succinyl-CoA reductase from  P. gingivalis ), were isolated using standard genetic protocols (Sambrook et al., 1989). NCBI references of the utilized mcr and sucD protein sequences, and the references for the corresponding natural and synthetic DNA sequences are listed in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 References to proteins from different organisms having annotated 
               
               
                 malyl-CoA reductase or succinyl-CoA reductase activity, and 
               
               
                 references to natural and optimized DNA sequences. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 NCBI/Integrated 
                 Natural 
                 Optimized 
               
               
                   
                   
                 Genomics 
                 DNA 
                 DNA 
               
               
                 Organism 
                 Protein 
                 accession number 
                 sequence 
                 sequence 
               
               
                   
               
               
                 
                   S. tokodaii 
                 
                 St-Mcr 
                 NP 378167 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 SEQ ID No. 7 
                   
                 No. 8 
                 No. 9 
               
               
                 
                   P. gingivalis 
                 
                 Pg-SucD 
                 AAQ65862 
                 SEQ ID 
               
               
                   
                 SEQ ID No. 10 
                   
                 No. 11 
               
               
                   
               
            
           
         
       
     
     Expression and purification of Pg-SucD was carried out as described in Example 1 using plasmid pET28-Pgi-sucD. 
     The St-mcr gene was amplified from plasmid pET28-St-mcr using primers 5′-TATAATGAGCTCGTTTAACTTTAAGAAGGAGATATACCATGATTCTGATGC GCCGT-3′(SEQ ID No. 12) and 5′-TATAATGGATCCCTCGAATTCTTACTTCTC-3′ (SEQ ID No. 13) which added a SacI and a BamHI restriction site upstream of the start codon and downstream of the stop codon, respectively. The PCR fragment was ligated into the pACT3 expression vector using the SacI and BamHI restriction sites. The resulting plasmid pACT3-St-Mcr was transformed into strain  E. coli  MG1655. The resulting expression strain was cultivated on mineral medium at 37° C. One liter mineral medium contained 20 g glucose, 18 g Na 2 HPO 4 *12 H 2 O, 3 g KH 2 PO 4 , 0.5 g NaCl, 2 g NH 4 Cl, 0.5 g MgSO 4 *7 H 2 O, 0.015 CaCl 2 *2 H 2 O, 1 mL of 0.06 mol/L FeCl 3  stock solution prepared in 100 times diluted concentrated HCl, 2 mL of 10 mM thiamine HCl stock solution, 20 g MOPS, 50 μg kanamycin sulphate (and 25 μg chloramphenicol when necessary), and 1 mL of trace element solution (containing per liter: 0.04 g Na 2 EDTA*2H 2 O, 0.18 g CoCl 2 *6 H 2 O, ZnSO4*7 H 2 O, 0.04 g Na 2 MoO4*2 H 2 O, 0.01 g H 3 BO 3 , 0.12 g MnSO 4 *H 2 O, 0.12 g CuCl 2 *H2O.). Medium pH was adjusted to 7 and medium was filter-sterilized. 
     When the exponentially growing culture reached an OD (600 nm) of 0.6, 1 mM IPTG was added and cultures were incubated at 20° C. during 14 h before harvesting the cells by centrifugation (13000×g, 10 min). After discarding the supernatant cell pellets were stored at −20° C. 
     To purify St-Mcr, frozen cell pellets of expression cultures were resuspended in 0.5 mL of breakage buffer (50 mM Hepes, 300 mM NaCl, pH 7.5) and broken open by four successive rounds of sonication (sonication interval: 20 sec, power output: 30%, sonicator: Bioblock Scientific, VibraCell™ 72437). Cell debris were removed by centrifuging the crude extracts for 15 min at 4° C. at 13000×g and retaining the clear supernatant. Native proteins of  E. coli  were removed by heat precipitation at 85° C. during 30 min followed by centrifugation at 13000×g. Purity of the protein preparations was analysed by SDS-page analysis which showed only one band corresponding to the expected size of the St-Mcr protein. 
     Enzymatic Assays: 
     Malyl-CoA reductase activity was assayed in the reductive and in the oxidative sense of the reaction employing Assay 1 or Assay 2, respectively. 
     Assay 1 (Reaction Scheme):
 
glyoxylate+acetyl-CoA→malyl-CoA+acetate  Malyl-CoA lyase:
 
(L)-Malyl-CoA+NADPH→(L)-Malate semialdehyde+Coenzyme A+NADP  Malyl-CoA reductase:
 
Assay 2 (Reaction Scheme):
 
(L)-Malate semialdehyde+Coenzyme A+NADP→(L)-Malyl-CoA+NADPH
 
The reaction mixture according to Assay 1 contained 50 mM MOPS/KOH (pH 7.5), 10 mM glyoxylate, 4 mM acetyl-CoA, 5 mM MgCl 2 , 0.25 mM NADPH (all products from Sigma), 5 U/mL of malyl-CoA lyase, and appropriate amounts of purified malyl-CoA reductase or cell extract. Reactions were started by adding glyoxylate. Enzymatic assays were carried out at 37° C. in 96-well flat bottomed microtiter plates in a final volume of 250 μL. The reactions were followed by the characteristic absorption of NADPH at 340 nm (ε NADPH =6.22 mM −1  cm −1 ) in a microplate reader (BioRad 680XR).
 
     The reaction mixture according to assay 2 contained 200 mM HEPES (pH 9), 5 mM MgCl 2 , 1 mM NADP, 0.5 mM coenzyme A (all products from Sigma), and appropriate amounts of purified malyl-CoA reductase. Reactions were started by adding 5 mM (L)-malate semialdehyde. Enzymatic assays were carried out at 37° C. in 96-well flat bottomed microtiter plates in a final volume of 250 μL. The reactions were followed by the characteristic absorption of NADPH at 340 nm (ε NADPH =6.22 mM −1  cm −1 ) in a microplate reader (BioRad 680XR). Unstable malate semialdehyde was produced freshly prior to the enzymatic tests by the deprotection of the stable malate semialdehyde derivative 2-[(4S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl]acetaldehyde (DMODA) (provided by Activation®). Malate semialdehyde was obtained by dissolving appropriate amounts of DMODA in 2 M hydrochloric acid, short heating of the suspension to boiling temperature, and leaving the hot suspension for 15 min at room temperature. The released acetone was evaporated at 35° C. and 50 mbar in a rotary evaporator. The pH of the malate semialdehyde solution was fixed at 3.5 using sodium bicarbonate. 
     Results listed in Tables 3 and 4 demonstrate malyl-CoA reductase activity for malonyl-CoA reductase, Mcr, of  S. tokodaii  and succinyl-CoA reductase, SucD, of  P. gingivalis . 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Kinetic parameters for the reductive sense of reaction (malonyl-CoA 
               
               
                 reductase and succinyl-CoA reductase activities were estimated by directly 
               
               
                 adding the substrates malonyl-CoA or succinyl-CoA to the reaction mixture). 
               
            
           
           
               
               
            
               
                   
                 Substrate 
               
            
           
           
               
               
               
               
            
               
                   
                 Malonyl-CoA 
                 Succinyl-CoA 
                 Malyl-CoA 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Vmax 
                 Km 
                 Vmax 
                 Km 
                 Vmax 
                 Km 
               
               
                 Enzyme 
                 [μmol/(min mg)] 
                 [mM] 
                 [μmol/(min mg)] 
                 [mM] 
                 [μmol/(min mg)] 
                 [mM] 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 St-Mcr 
                 0.67 ± 0.15 
                 nd 
                 0.98 ± 0.17 
                 0.2 
                 0.24 ± 0.045 
                 nd 
               
               
                 Pg-SucD 
                 nd 
                 nd 
                 1 
                 1 
                 0.025 
                 nd 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Kinetic parameters for the oxidative sense of reaction 
               
            
           
           
               
               
            
               
                   
                 Substrate 
               
            
           
           
               
               
               
            
               
                   
                 Succinic semialdehyde 
                 Malate semialdehyde 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vmax 
                 Km 
                 Vmax 
                 Km 
               
               
                 Enzyme 
                 [μmol/(min mg)] 
                 [mM] 
                 [μmol/(min mg)] 
                 [mM] 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 St-Mcr 
                 1.7 
                 1.15 
                 0.1 
                 0.25 
               
               
                 Pg-SucD 
                 4 
                 nd 
                 0.007 
                 nd 
               
               
                   
               
            
           
         
       
     
     Example 3: Demonstration of DHB Dehydrogenase Activity 
     To identify a suitable 2,4 DHB dehydrogenase, beta-hydroxyacid dehydrogenases from different biological sources were tested for their ability to reduce malate semialdehyde. Among the tested enzymes were the methylbutyraldehyde reductase from  Saccharomyces cerevisiae, Ypr 1 (Ford &amp; Ellis, 2002) (SEQ ID No.14), the 4-hydroxybutyrate dehydrogenase, 4hbdh, of  P. gingivalis  (SEQ ID No.187), the alcohol dehydrogenase, YqhD, of  E; coli  (SEQ ID no. 185), and the succinic semialdehyde reductase, Ms-Ssr, from  Metallosphaera sedula  (Kockelkorn &amp; Fuchs, 2009) (SEQ ID No. 16). The genes YPR1, 4hbdh, yqhD, and Ms-SSR were amplified using primers listed in Table 5 and cloned into vector pET28 (restriction enzymes see Table 5) yielding plasmids pET28-Sce-YPR1, pET28-Pgi-4-hbdh, pET28-Eco-yqhd and pET28-Mse-SSR, respectively. The proteins were expressed and purified as described in Example 1. 
                     TABLE 5                  Primers and restriction enzymes        used to clone       candidate beta-hydroxyacid        dehydrogenases                                         Re-           Acces-       stric-           sion       tion       Enzyme   No   Primer 5′-3′   enzymes               YPR1   GI:   TATAATGCTAGCATGCCTGC   NheI           6320576   TACGTTAAAGAA                    (SEQ ID No. 18)                   TATAATGAGCTCTCATTGGA   SacI               AAATTGGGAAGG                    (SEQ ID No. 18)                   YqhD   GI:   TATAATGAATTCTTAGCGGG   EcoRI           16130909   CGG                   CTTCGTATATACGGCGGCTG                   ACA                   (SEQ ID No. 20)   NheI               TATCGTGCTAGCATGAACAA                   CTTTAATCTGCACA                   (SEQ ID No. 21)                   4hbdh   GI:   TATAATGGATCCTTAGTAGA   BamHI           188994588   GTCTTCTGTAG                    (SEQ ID No. 22)                   TATAATCATATGCAACTTTT   NdeI               CAAACTC                    (SEQ ID No. 23)                   Ms-SSR   GI:   TATAATGCTAGCATGAAAGC   NheI           146304190   TGCAGTACTTCA                    (SEQ ID No. 24)                   TATAATGAATTCTTACGGGA   EcoRI               TTATGAGACTTC                    (SEQ ID No. 25)                    
Test for Malate Semialdehyde Reductase Activity:
 
(L)-Malate semialdehyde+NAD(P)H→(L)-2,4-dihydroxybutyric acid+NAD(P)  Reaction scheme:
 
     The assay mixture contained 200 mM Hepes (pH 7.5), 50 mM KCl, 5 mM MgCl 2 , 0.24 mM NADH or NADPH, and appropriate amounts of purified enzyme or cell extract. Reactions were started by adding 10 mM (L)-malate semialdehyde (malate semialdehyde was prepared freshly for each test, see Example 3). Enzymatic assays were carried out at 30° C. in 96-well flat bottomed microtiter plates in a final volume of 250 μL. The reactions were followed by the characteristic absorption of NAD(P)H at 340 nm (ε NADPH =6.22 mM −1  cm −1 ) in a microplate reader (BioRad 680XR). Results are listed in Table 6. 
                     TABLE 6                  Reducing activity of selected beta-hydroxyacid dehydrogenases on       malate semialdehyde (Results represent the average of at least two       independent experiments).                                             Activity   Activity                   on malate   on malate                   semialdehyde   semialdehyde                   (cofactor   (cofactor                   NADH)   NADPH)               Reported   [μmol/(min *   [μmol/(min *       Enzyme   Origin   function   mg_prot)]   mg_prot)]               Ms-SSR     M.     Succinic   4.9   4.9       (SEQ     sedula     semialdehyde       ID       reductase       No. 16)       YqhD     E. coli     Alcohol   nd   1.2       (SEQ       dehydrogenase       ID No       185)       4hbdh     P.     4-hydroxy-   33   nd       (SEQ     gingivalis     butyrate       ID No       dehydrogenase       187)       YPR1     S.     Methyl-   nd    0.19       (SEQ     cerevisiae     butyraldehyde       ID No.       reductase       14)                    
The succinic semialdehyde dehydrogenase from  M. sedula  and the methylbutyraldehyde reductase from  S. cerevisiae  have malate semialdehyde reductase activity. The Km of Ms-SSR for malate semialdehyde was 4 mM.
 
     Example 4: Rational Construction of an Improved Malyl-CoA Reductase Enzyme 
     Site-directed mutagenesis was carried out using the oligonucleotide pairs listed in Table 7 and the pET28-Sto-mcr plasmid as the template. Point mutations to change the amino acid sequences were introduced by PCR (Phusion 1U, HF buffer 20% (v/v), dNTPs 2.5 mM, direct and reverse primers 1 μM each, template plasmid 200 ng, water). When possible, plasmids created by PCR contained new restriction sites (introduced using silent mutations) in addition to the functional mutation to facilitate identification of mutated clones. The PCR products were digested by DpnI at 37° C. for 2×2 h to remove template DNA, and transformed into NEB DH5-α competent  E. coli  cells (NEB). The mutated plasmids were identified by restriction site analysis and verified to carry the desired mutations by DNA sequencing. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Primer pairs used to mutate the mcr 
               
               
                 gene of  S. tokodaii . 
               
            
           
           
               
               
               
            
               
                   
                 Mutation 
                 Primer 5′-3′ 
               
               
                   
                   
               
               
                   
                 Tyr206 
                 Forward 
               
               
                   
                   
                 CATTCTGCCTTTAGGGGACGGCNNKGACGCCAAAACG 
               
               
                   
                   
                 (SEQ ID No. 26) 
               
               
                   
                   
               
               
                   
                   
                 Revers 
               
               
                   
                   
                 CGTTTTGGCGTCMNNGCCGTCCCCTAAAGGCAGAATG 
               
               
                   
                   
                 (SEQ ID No. 27) 
               
               
                   
                   
               
            
           
         
       
     
     The impact of the genetic modifications of St-Mcr was tested in the oxidative sense of the reaction using Assay 3 described in Example 2.  FIG. 3  shows that replacing the natural Tyr206 by other amino acids decreases the activity on the natural substrate, succinic semialdehyde, causing at the same time an increased or at least constant activity on malate semialdehyde. Thus, replacing Tyr206 by appropriate amino acid residues provides a selective advantage regarding the specificity of Mcr for the DHB pathway intermediate. 
     Preferred amino acid residues in position 206 are therefore phenylalanine, histidine, isoleucine, lysine, methionine, glycine, asparagine, proline, arginine, glutamine, leucine, serine, tryptophane, and threonine. 
     The protein wherein the Tyrosine 206 is replaced by a Proline residue is represented by SEQ ID No. 202. 
     Example 5: Rational Construction of an Improved DHB Dehydrogenase 
     Site-directed mutagenesis was carried out using the oligonucleotide pairs listed in Table 6 and the pET28-Mse-SSR plasmid as the template. Point mutations to change the amino acid sequences were introduced by PCR (Phusion 1U, HF buffer 20% (v/v), dNTPs 2.5 mM, direct and reverse primers 1 μM each, template plasmid 200 ng, water). When possible, plasmids created by PCR contained new restriction sites (introduced using silent mutations) in addition to the functional mutation to facilitate identification of mutated clones. The PCR products were digested by DpnI at 37° C. for 2×2 h to remove template DNA, and transformed into NEB DH5-α competent  E. coli  cells (NEB). The mutated plasmids were identified by restriction site analysis and verified to carry the desired mutations by DNA sequencing. Table 8 summarizes kinetic parameters of the mutants. The results demonstrate that the double mutant Ms-SSR H39R N43H (SEQ ID No.38) has improved affinity for malate semialdehyde when compared to the wild type enzyme. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Primer pairs used to mutate  M. sedula  succinic 
               
               
                 semialdehyde reductase (Ms-SSR) 
               
            
           
           
               
               
               
            
               
                 Muta- 
                   
                 Restriction 
               
               
                 tion 
                 Primer 5′-3′ 
                 enzymes 
               
               
                   
               
               
                 H39R 
                 gtcaaggcaaccggtctctgtcg 
                 NheI 
               
               
                   
                 ctccgacgtcaatg (SEQ ID No. 28) 
                   
               
               
                   
                 cattgacgtcggagcgacagaga 
                   
               
               
                   
                 ccggttgccttgac (SEQ ID No. 29) 
                   
               
               
                   
               
               
                 N43H 
                 ggctctgtcactccgacgtacat 
                 NheI 
               
               
                   
                 gtctttgaggggaaaac (SEQ ID No. 30) 
                   
               
               
                   
                 gttttcccctcaaagacatgtac 
                   
               
               
                   
                 gtcggagtgacagagcc (SEQ ID No. 31) 
               
               
                   
               
            
           
         
       
     
                     TABLE 9                  Summary of kinetic parameters of  M. sedula  succinic semialdehyde       reductase (Ms-SSR) mutants (Results represent the average       of at least two independent experiments).                                     Maximum activity   Km           Mutant   [μmol/(min * mg prot )]   [mmol/L]                       Wild type (SEQ ID No.   4.9   4           16)           H39R (SEQ ID No. 32)   1.7   1           N43H (SEQ ID No. 34)   4.3   5           H39R N43H (SEQ ID   4.7   1           No. 36)                        
The corresponding nucleic sequences are represented by SEQ ID No. 17, SEQ ID No. 33, SEQ ID No. 35 and SEQ ID No. 37.
 
     The coding sequence of  M. sedula  succinic semialdehyde reductase including the mutations H39R and N43H was optimized for maximum expression in  E. coli , using the GeneOptimizer® software. The synthetic gene was produced by GeneArt® Gene Synthesis (Invitrogen Life Technologie). NheI and EcoRI restriction sites were introduced upstream of the start codon and downstream of the stop codon, respectively, allowing direct cloning into pET28a+(Novagen). 
     The resulting pET28-Mse-DHB-Dh-H39R_N43H-opt plasmid was isolated and shown by DNA sequencing to contain the full-length  M. sedula  SSR H39R N43H gene having the correct sequence (SEQ ID No.38). 
     Example 6: Demonstration of In Vitro Production of DHB by the Synthetic Malyl-CoA Pathway 
     The enzymes malyl-CoA lyase (Me-Mcl), malyl-CoA reductase (St-Mcr or Pg-SucD), and DHB dehydrogenase (Ms-SSA-red H 39 N N43H) were expressed and purified as described in Examples 1, 2, and 3. 
     Production of DHB by the pathway comprising malyl-CoA lyase, malyl-CoA reductase, and DHB dehydrogenase was demonstrated in vitro by adding 2 mM glyoxylate to a reaction mixture that contained 50 mM Hepes (pH 7.5), 2 mM acetyl-CoA, 2 mM NADPH, 100 μg/mL DHB dehydrogenase, 150 μg/mL malyl-CoA lyase, and 100 μg/mL malyl-CoA reductase (which was either St-Mcr (reaction 1), or Pg-SucD (reaction 2)). 
     Control reactions contained all components but were lacking either DHB dehydrogenase (Control 1) or malyl-CoA reductase (Control 2). After 120 min of incubation at 37° C. the DHB content in the reaction mixture was analysed by gas chromatography [GCMS-QP2010 Ultra Shimadzu; equipped with a FID detector (FID-2010 Plus Shimadzu); autosampler AOC20s (Shimadzu); splitless injector AOC20i (Shimadzu) (230° C.); column: Zebron ZB-FFAP, 30 m×0.25 mm, d f  0.25 μm; and liner: Tapered focus Liner5×95×3.4 mm (SGE). Carrier gas was hydrogen at a total flow rate of 25 mL/min. Flame ionization was carried out using an air-hydrogen mixture (flow rates were 300 mL/min and 30 mL/min, respectively). Detector temperature was 240° C. Injected sample volume was 1 μL. Temperature program is provided in Table 10.
 
Chromatograms showing presence of DHB in the reactions containing all pathway enzymes and absence of DHB in samples containing only two out of three pathway enzymes are shown in  FIG. 2 .
 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Temperature program for GC analysis of reaction mixtures 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Column temperature 
                 Hold 
                 Gradient 
                 Runtime 
               
               
                   
                 [° C.] 
                 [min] 
                 [° C./min] 
                 [min] 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 90 
                 0 
                 0 
                 0 
               
               
                   
                 115 
                 1.8 
                 30 
                 2.63 
               
               
                   
                 170 
                 1 
                 4 
                 17.38 
               
               
                   
                 230 
                 3 
                 50 
                 21.58 
               
               
                   
                   
               
            
           
         
       
     
     Example 7: Construction of Optimized DHB Producer Strains 
     Construction of a Plasmid for Simultaneous Expression of Malyl-CoA Synthetase, Malyl-CoA Reductase, and DHB-Dehydrogenase: 
     The coding sequence of the malyl-CoA lyase from  M. extorquens , Me-mcl, was amplified from plasmid pET28-Mex-mcl using the high fidelity polymerase Phusion (Fermentas) and the forward and reverse primers 5′-TCACACAGGAAACAGAATTC GAGCTC GGTAATGTCGTTTACCCTGATTCAG CAAGCGACT-3′ (SEQ ID No. 39) and 5′-GGTATATCTCCTTCTTAAAGTTAAACTTATTTGCCGCCCATTGCATCCGCTT TCTG-3′ (SEQ ID No. 40) which contained restriction sites for SacI upstream of the start codon (underlined). The coding sequence of the malonyl-CoA reductase from  S. tokodaii , St-mcr, was amplified from plasmid pET28-Sto-mcr using the forward and reverse primers 5′-GTTTAACTTTAAGAAGGAGATATACCATGATTCTGATGCGCCGTACCCTGA AAGCG-3′ (SEQ ID No. 41) and 5′-GGTATATCTCCTTCTTAAAGTTAAACTTACTTCTCGATGTAGCCTTTCTCCA CGAG-3′ (SEQ ID No. 42) which contained restriction sites for BamHI downstream of the stop codon. The plasmid pET28-Mse-DHB-Dh-H39R_N43H-opt (Example 5) was used as the template to amplify the optimized coding sequence of the succinic semialdehyde reductase H39R N43H from  M. sedula  using the forward and reverse primers 5′-GTTTAACTTTAAGAAGGAGATATACCATGAAAGCAGCAGTTCTGCATACCT ATAAAGAACCGCTGAGCAT-3′ (SEQ ID No. 43) and 5′-ATGCCTGCAGGTCGACTCTAGA GGATCC TTACGGAATAATCAGGCTACGA ATTGCTTC-3′ (SEQ ID No. 44) that introduced a BamHI restriction site downstream of the stop codon (underlined). 
     The forward primers for St-mcr and the succinic semialdehyde reductase H39R N43H from  M. sedula  contained a rbs motif. The three genes were simultaneously cloned into the pACT3 expression vector by homologous recombination using the In-Fusion cloning kit (Clontech). 
     The resulting and pACT3-MCL-DHB (SEQ ID No. 45) plasmid was isolated and shown by DNA sequencing to have the correct sequence. 
     Construction of a Plasmid for Simultaneous Expression of Malyl-CoA Synthetase, Malyl-CoA Reductase, and DHB-Dehydrogenase: 
     The DNA sequences coding for the two protein subunits of malyl-CoA synthetase, mtkA (YP_00296285) and mtkB (YP_002962852), from  Methylobacterium extorquens  AM1 were optimized for the expression in  Escherichia coli  using the GENEius software (Eurofins). The optimized DNA sequences of the subunit were physically linked by the DNA sequence naturally occurring between the mtkA and mtkB genes in  M. Extorquens  genome (CGAACGGGGGAGGAATCACGCC, SEQ ID No. 46). The resulting DNA fragment, ‘mtkA gene-linker DNA-mtkB gene’, was synthesized by Eurofins MWG OPERON® and subcloned into pET28b expression vector using NheI and EcoRI restriction enzymes. The resulting DNA plasmid pET28-Mex-mtkAB (SEQ ID No. 47) was used to simultaneously amplify the two codons optimized genes encoding malyl-CoA synthetase from  M. extorquens , Me-mtkA and Me-mtkB using the high fidelity polymerase Phusion (Fermentas) and the forward and reverse primers 5′-CAGGAAACAGAATTC GAGCTC GGTAATGGATGTGCACGAATATCAGGCGA AAGAACTGCT-3′ (SEQ ID No. 48) and 5′-TACGGCGCATCAGAATCATtacgccgcacgtgctaacacatcggcaac-3′ (SEQ ID No. 49) which contained restriction sites for SacI upstream of the start codon (underlined). The coding sequence of the malonyl-CoA reductase from  S. tokodaii , St-mcr, was amplified from plasmid pET28-Sto-mcr using the forward and reverse primers 5′-GGCGTAATGATTCTGATGCGCCGTACCCTGAAAGCG-3′ (SEQ ID No. 50) and 5′-CTGCTGCTTTCATTACTTCTCGATGTAGCCTTTCTCCACGAG-3′ (SEQ ID No. 51) which contained restriction sites for BamHI downstream of the stop codon. The plasmid pET28-Mse-DHB-Dh-H39R_N43H-opt (Example 5) was used as the template to amplify the optimized coding sequence of the succinic semialdehyde reductase H39R N43H from  M. sedula  using the forward and reverse primers 5′-TACATCGAGAAGTAATGAAAGCAGCAGTTCTGCATACCTATAAAGAAC-3′ (SEQ ID No. 52) and 5′-CCTGCAGGTCGACTCTAGAGGATCCTTACGGAATAATCAGGCTACGAATT GCTTCAC-3′ (SEQ ID No. 53) that introduced a BamHI restriction site downstream of the stop codon (underlined). 
     The three genes were simultaneously cloned into the pEXT20 expression vector by homologous recombination using the In-Fusion cloning kit (Clonetch). 
     The resulting pEXT20-MCS-DHB (SEQ ID No.54) plasmid was isolated and shown by DNA sequencing to have the correct sequence. 
     Construction of Plasmids for Overexpression of Phosphoenolpyruvate (PEP) Carboxykinase, PEP Carboxylase, Pyruvate Kinase, Pyruvate Carboxylase, Isocitrate Lyase Enzymes and the Galactose Symporter Permease: 
     The plasmid pACT3-pck harbouring the PEP carboxykinase encoding pck gene of  E. coli  was constructed by amplifying the pck coding sequence using genomic DNA from  E. coli  MG1655 as the template and the forward and reverse primers, respectively,  5′ TATAATCCCGGGATGCGCGTTAACAATGGTTTGACC 3′  (SEQ ID No. 56 and  5′ TATAATTCTAGATTACAGTTTCGGACCAGCCG 3′  (SEQ ID No. 57). The DNA fragment was digested with XmaI and XbaI, ligated into the corresponding sites of the pACT3 expression vector (Dykxhoorn et al., 1996) using T4 DNA ligase (Biolabs), and transformed into  E. coli  DH5α cells. The transformants were selected on solid LB medium containing chloramphenicol (25 μg/mL). The resulting plasmid was isolated and correct insertion of the pck gene was verified by sequencing. Plasmids pACT3-aceA, pACT3-ppc, pACT3-galP, pACT3-pck and pACT3-pyc harbouring, respectively, aceA, ppc, galP, or pykA (all  E. coli ) or pck from  Lactococcus lactis  were constructed analogously using the primers listed in Table 11. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Primers used for construction of plasmids 
               
               
                 for gene overexpression. 
               
            
           
           
               
               
               
               
            
               
                 Gene 
                 Primer 
                 Linker 
                 Sequence 
               
               
                   
               
               
                 Ec_ 
                 Ec_pck_ 
                 XmaI 
                 tataat cccggg atgcgcgttaa 
               
               
                 pck 
                 clon_for 
                   
                 caatggtttgacc 
               
               
                   
                   
                   
                 (SEQ ID No. 57) 
               
               
                   
                 Ec_pck_ 
                 XbaI 
                 tataat tctaga ttacagtttcg 
               
               
                   
                 clon_rev 
                   
                 gaccagccg 
               
               
                   
                   
                   
                 (SEQ ID No. 58) 
               
               
                   
               
               
                 Ec_ 
                 Ec_ppc_ 
                 XmaI 
                 tataat cccggg atgaacgaaca 
               
               
                 ppc 
                 clon_for 
                   
                 atattcc 
               
               
                   
                   
                   
                 (SEQ ID No. 59) 
               
               
                   
                 Ec_ppc_ 
                 XbaI 
                 tataat tctaga ttagccggtat 
               
               
                   
                 clon_rev 
                   
                 tacgcat 
               
               
                   
                   
                   
                 (SEQ ID No. 60) 
               
               
                   
               
               
                 Ec_ 
                 Ec_pykA_ 
                 XmaI 
                 tataat cccggg atgtccagaag 
               
               
                 pykA 
                 clon_for 
                   
                 gcttcgcagaaca 
               
               
                   
                   
                   
                 (SEQ ID No. 61) 
               
               
                   
                 Ec_pykA_ 
                 XbaI 
                 tataat tctaga ttactctaccg 
               
               
                   
                 clon_rev 
                   
                 ttaaaatac 
               
               
                   
                   
                   
                 (SEQ ID No. 62) 
               
               
                   
               
               
                 Ec_ 
                 Ec_aceA_ 
                 XmaI 
                 tataat cccggg atgaaaacccg 
               
               
                 aceA 
                 clon_for 
                   
                 tacacaacaaatt 
               
               
                   
                   
                   
                 (SEQ ID No. 63) 
               
               
                   
                 Ec_aceA_ 
                 XbaI 
                 tataat tctaga ttagaactgcg 
               
               
                   
                 clon_rev 
                   
                 attcttcag 
               
               
                   
                   
                   
                 (SEQ ID No. 64) 
               
               
                   
               
               
                 Ll_ 
                 Ll_pycA_ 
                 XmaI 
                 tataat cccggg atgaaaaaact 
               
               
                 pycA 
                 clon_for 
                   
                 actcgtcgccaat 
               
               
                   
                   
                   
                 (SEQ ID No. 65) 
               
               
                   
                 Ll_pycA_ 
                 XbaI 
                 tataatt ctagat taattaattt 
               
               
                   
                 clon_rev 
                   
                 cgattaaca 
               
               
                   
                   
                   
                 (SEQ ID No. 66) 
               
               
                   
               
               
                 Ec_ 
                 Ec_galP_ 
                 XmaI 
                 tataat cccggg atgcctgacgc 
               
               
                 galP 
                 clon_for 
                   
                 taaaaaacaggggcggt 
               
               
                   
                   
                   
                 (SEQ ID No. 67) 
               
               
                   
                 Ec_galP_ 
                 XbaI 
                 tataat tctaga ttaatcgtgag 
               
               
                   
                 clon_rev 
                   
                 cgcctatttc 
               
               
                   
                   
                   
                 (SEQ ID No. 68) 
               
               
                   
               
               
                 Restriction sites used for cloning into pACT3 are underlined 
               
            
           
         
       
     
     It is understood that removal of the lacI gene from the backbone of the above described plasmids along with the genomic deletion of lacI in the host strain may render protein expression from above described plasmids constitutive. 
     Construction of Strains with Optimized Carbon Flux Repartitioning for DHB Production 
     Several genes were disrupted in  E. coli  strain MG1655 in order to optimise carbon flux repartitioning and cofactor supply for DHB production. Gene deletions were carried out using either the lambda red recombinase method according to Datsenko et al. (Datsenko &amp; Wanner, 2000), or the phage transduction method adapted from Miller (Miller, 1992). 
     Protocol for introduction of gene deletions using the lambda red recombinase method: the deletion cassettes were prepared by PCR using high fidelity polymerase Phusion™ (Finnzymes), and the FRT-flanked kanamycin resistance gene (kan) of plasmid pKD4 as the template (Datsenko &amp; Wanner, 2000). Sense primers contained sequences corresponding to the 5′-end of each targeted gene (underlined) followed by 20 bp corresponding to the FRT-kan-FRT cassette of pKD4. Anti-sense primers contained sequences corresponding to the 3′-end region of each targeted gene (underlined) followed by 20 bp corresponding to the cassette. The primers are described in Table 12. PCR products were digested with DpnI and purified prior to transformation. 
       E. coli  MG1655 strain was rendered electro-competent by growing the cells to an OD 600  of 0.6 in LB liquid medium at 37° C., concentrating the cells 100-fold, and washing them twice with ice-cold 10% glycerol. The cells were transformed with plasmid pKD46 (Datsenko &amp; Wanner, 2000) by electroporation (2.5 kV, 200 Ω, 25 μF, in 2 mm gap cuvettes). Transformants were selected at 30° C. on ampicillin (100 μg/mL) LB solid medium. 
     Disruption cassettes were transformed into electro-competent  E. coli  strains harbouring the lambda Red recombinase-expressing plasmid pKD46. The cells were grown at 30° C. in liquid SOB medium containing ampicillin (100 μg/mL). The lambda red recombinase system was induced by adding 10 mM arabinose when OD 600  of the cultures reached 0.1. Cells were further grown to an OD 600  of 0.6 before they were harvested by centrifugation, washed twice with ice-cold 10% glycerol, and transformed with the disruption cassette by electroporation. After an overnight phenotypic expression at 30° C. in LB liquid medium, cells were plated on solid LB medium containing 25 μg/mL kanamycin. Transformants were selected after cultivation at 30° C. 
     The gene replacement was verified by colony PCR using Crimson Taq polymerase (NEB). A first reaction was carried out with the flanking locus-specific primers (see Table 12) to verify simultaneous loss of the parental fragment and gain of the new mutant specific fragment. Two additional reactions were done by using one locus-specific primer together with one of the corresponding primers k1rev, or k2for (see Table 12) that align within the FRT-kanamycin resistance cassette (sense locus primer/k1rev and k2for/reverse locus primer). 
     The resistance gene (FRT-kan-FRT) was subsequently excised from the chromosome using the FLP recombinase-harbouring plasmid pCP20 (Cherepanov &amp; Wackernagel, 1995) leaving a scar region containing one FRT site. pCP20 is an ampicillin and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP recombinase synthesis. Kanamycin resistant mutants were transformed with pCP20, and ampicillin-resistant transformants were selected at 30° C. Transformants were then grown on solid LB medium at 37° C. and tested for loss of all antibiotic resistances. Excision of the FRT-kanamycin cassette was analysed by colony PCR using crimson Taq polymerase and the flanking locus-specific primers (Table 12). Multiple deletions were obtained by repeating the above described steps. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Primers used for gene disruptions. 
               
            
           
           
               
               
               
            
               
                 Gene 
                 Primer 
                 Sequence 
               
               
                   
               
               
                 ldhA 
                 Δ_ldhA_ 
                   gaaggttgcgcctacactaagcatagttgttgatga gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 69) 
               
               
                   
                 Δ_ldhA_ 
                   ttaaaccagttcgttcgggcaggtttcgccttttt catgggaattagccatggtcc 
               
               
                   
                 rev 
                 SEQ ID No. 70) 
               
               
                   
               
               
                 adhE 
                 Δ_adhE_ 
                   atggctgttactaatgtcgctgaacttaacgcactcgtagagcgt gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 71) 
               
               
                   
                 Δ_adhE_ 
                   ttaagcggattttttcgcttttttctcagctttagccggagcagc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 72) 
               
               
                   
               
               
                 ackA 
                 Δ_ackA_ 
                   atgtcgagtaagttagtactggttctgaactgcggtagttcttca gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 73) 
               
               
                   
                 Δ_ackA_ 
                   tcaggcagtcaggcggctcgcgtcttgcgcgataaccagttcttc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 74) 
               
               
                   
               
               
                 focA- 
                 Δ_focA- 
                   ttactccgtatttgcataaaaaccatgcgagttacgggcctataa gtgtaggctggagctgcttc 
               
               
                 pfIB 
                 pfIB_for 
                 (SEQ ID No. 75) 
               
               
                   
                 Δ_focA- 
                   atagattgagtgaaggtacgagtaataacgtcctgctgctgttct catatgaatatcctccttag 
               
               
                   
                 pfIB_rev 
                 (SEQ ID No. 76) 
               
               
                   
               
               
                 pta 
                 Δ_pta_ 
                   gtgtcccgtattattatgctgatccctaccggaaccagcgtcggt gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 77) 
               
               
                   
                 Δ_pta_ 
                   ttactgctgctgtgcagactgaatcgcagtcagcgcgatggtgta catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 78) 
               
               
                   
               
               
                 poxB 
                 Δ_poxB_ 
                   atgaaacaaacggttgcagcttatatcgccaaaacactcgaatcg gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 79) 
               
               
                   
                 Δ_poxB_ 
                   ttaccttagccagtttgttttcgccagttcgatcacttcatcacc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 80) 
               
               
                   
               
               
                 sad 
                 Δ_sad_ 
                   atgaccattactccggcaactcatgcaatttcgataaatcctgcc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 81) 
               
               
                   
                 Δ_sad_ 
                   tcagatccggtctttccacaccgtctggatattacagaattcgtg catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 82) 
               
               
                   
               
               
                 gabD 
                 Δ_gabD_ 
                   atgaaacttaacgacagtaacttattccgccagcaggcgttgatt gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 83) 
               
               
                   
                 Δ_gabD_ 
                   ttaaagaccgatgcacatatatttgatttctaagtaatcttcgat catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 847) 
               
               
                   
               
               
                 gadA 
                 Δ_gadA_ 
                   atggaccagaagctgttaacggatttccgctcagaactactcgat gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 85) 
               
               
                   
                 Δ_gadA_ 
                   tcaggtgtgtttaaagctgttctgctgggcaataccctgcagttt catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 86) 
               
               
                   
               
               
                 gadB 
                 Δ_gadB_ 
                   atggataagaagcaagtaacggatttaaggtcggaactactcgat gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 87) 
               
               
                   
                 Δ_gadB_ 
                   tcaggtatgtttaaagctgttctgttgggcaataccctgcagttt catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 88) 
               
               
                   
               
               
                 gadC 
                 Δ_gadC_ 
                   atggctacatcagtacagacaggtaaagctaagcagctcacatta gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 89) 
               
               
                   
                 Δ_gadC_ 
                   ttagtgtttcttgtcattcatcacaatatagtgtggtgaacgtgc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 90) 
               
               
                   
               
               
                 sfcA 
                 Δ_sfcA_ 
                   atggaaccaaaaacaaaaaaacagcgttcgctttatatcccttac gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 91) 
               
               
                   
                 Δ_sfcA_ 
                   ttagatggaggtacggcggtagtcgcggtattcggcttgccagaa catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 92) 
               
               
                   
               
               
                 maeB 
                 Δ_maeB_ 
                   atggatgaccagttaaaacaaagtgcacttgatttccatgaattt gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 93) 
               
               
                   
                 Δ_maeB_ 
                   ttacagcggttgggtttgcgcttctaccacggccagcgccaccat catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 94) 
               
               
                   
               
               
                 ppc 
                 Δ_ppc_ 
                   atgaacgaacaatattccgcattgcgtagtaatgtcagtatgctc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 95) 
               
               
                   
                 Δ_ppc_ 
                   ttagccggtattacgcatacctgccgcaatcccggcaatagtgac catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 96) 
               
               
                   
               
               
                 pykA 
                 Δ_pykA_ 
                   atgtccagaaggcttcgcagaacaaaaatcgttaccacgttaggc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 97) 
               
               
                   
                 Δ_pykA_ 
                   ttactctaccgttaaaatacgcgtggtattagtagaacccacggt catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 98) 
               
               
                   
               
               
                 pykF 
                 Δ_pykF_ 
                   atgaaaaagaccaaaattgtttgcaccatcggaccgaaaaccgaa gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 99) 
               
               
                   
                 Δ_pykF_ 
                   ttacaggacgtgaacagatgcggtgttagtagtgccgctcggtac catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 100) 
               
               
                   
               
               
                 mgsA 
                 Δ_mgsA_ 
                   atggaactgacgactcgcactttacctgcgcggaaacatattgcg gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 101) 
               
               
                   
                 Δ_mgsA_ 
                   ttacttcagacggtccgcgagataacgctgataatcggggatcag catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 102) 
               
               
                   
               
               
                 icIR 
                 Δ_icIR_ 
                   atggtcgcacccattcccgcgaaacgcggcagaaaacccgccgtt gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 103) 
               
               
                   
                 Δ_icIR_ 
                   tcagcgcattccaccgtacgccagcgtcacttccttcgccgcttt catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 104) 
               
               
                   
               
               
                 icd 
                 Δ_icd_ 
                   atggaaagtaaagtagttgttccggcacaaggcaagaagatcacc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 105) 
               
               
                   
                 Δ_icd_ 
                   ttacatgttttcgatgatcgcgtcaccaaactctgaacatttcag catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 106) 
               
               
                   
               
               
                 sucA 
                 Δ_sucA_ 
                   atgcagaacagcgctttgaaagcctggttggactcttcttacctc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 107) 
               
               
                   
                 Δ_sucA_ 
                   ttattcgacgttcagcgcgtcattaaccagatcttgttgctgttt catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 108) 
               
               
                   
               
               
                 sucB 
                 Δ_sucB_ 
                   atgagtagcgtagatattctggtccctgacctgcctgaatccgta gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 109) 
               
               
                   
                 Δ_sucB_ 
                   ctacacgtccagcagcagacgcgtcggatcttccagcaactcttt catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 110) 
               
               
                   
               
               
                 frdA 
                 Δ_frdA_ 
                   gtgcaaacctttcaagccgatcttgccattgtaggcgccggtggc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 111) 
               
               
                   
                 Δ_frdA_ 
                   tcagccattcgccttctcclicttattggctgcttccgccttatc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 112) 
               
               
                   
               
               
                 frdB 
                 Δ_frdB_ 
                   atggctgagatgaaaaacctgaaaattgaggtggtgcgctataac gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 113) 
               
               
                   
                 Δ_frdB_ 
                   ttagcgtggtttcagggtcgcgataagaaagtctttcgaactttc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 114) 
               
               
                   
               
               
                 frdC 
                 Δ_frdC_ 
                   atgacgactaaacgtaaaccgtatgtacggccaatgacgtccacc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 115) 
               
               
                   
                 Δ_frdC_ 
                   ttaccagtacagggcaacaaacaggattacgatggtggcaaccac catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 116) 
               
               
                   
               
               
                 frdD 
                 Δ_frdD_ 
                   atgattaatccaaatccaaagcgttctgacgaaccggtattctgg gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 117) 
               
               
                   
                 Δ_frdD_ 
                   ttagattgtaacgacaccaatcagcgtgacaactgtcaggatag ccatatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 118) 
               
               
                   
               
               
                 ptsl 
                 Δ_ptsl_ 
                   atgatttcaggcattttagcatccccgggtatcgctttcggtaaa gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 119) 
               
               
                   
                 Δ_ptsl_ 
                   ttagcagattgttttttcttcaatgaacttgttaaccagcgtcat catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 120) 
               
               
                   
               
               
                 ptsG 
                 Δ_ptsG_ 
                   atgtttaagaatgcatttgctaacctgcaaaaggtcggtaaatcg gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 121) 
               
               
                   
                 Δ_ptsG_ 
                   ttagtggttacggatgtactcatccatctcggttttcaggttatc catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 122) 
               
               
                   
               
               
                 lacl 
                 Δ_lacl_ 
                   gtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 123) 
               
               
                   
                 Δ_lacl_ 
                   tcactgcccgctttccagtcgggaaacctgtcgtgccagctgcat catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 124) 
               
               
                   
               
               
                 lldD 
                 Δ_lldD_ 
                   atgattatttccgcagccagcgattatcgcgccgcagcgcaacgc gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 125) 
               
               
                   
                 Δ_lldD_ 
                   ctatgccgcattccctttcgccatgggagccagtgccgcaggcaa catatgaatatcctccttag  
               
               
                   
                 rev 
                 (SEQ ID No. 126) 
               
               
                   
               
               
                 pgi 
                 Δ_pgi_ 
                   atgaaaaacatcaatccaacgcagaccgctgcctggcaggcacta gtgtaggctggagctgcttc 
               
               
                   
                 for 
                 (SEQ ID No. 127) 
               
               
                   
                 Δ_pgi_ 
                   ttaaccgcgccacgctttatagcggttaatcagaccattggtcga catatgaatatcctccttag 
               
               
                   
                 rev 
                 (SEQ ID No. 128) 
               
               
                   
               
               
                 Sequences homologous to target genes are underlined 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Primer pairs used for verification 
               
               
                 of gene disruptions 
               
            
           
           
               
               
            
               
                 Deleted 
                 Sequence (5′-3′) 
               
            
           
           
               
               
               
            
               
                 gene 
                 Forward primer 
                 Reverse Primer 
               
               
                   
               
               
                 K2 for/ 
                 cggtgccctga 
                 cagtcatagcc 
               
               
                 k1 rev 
                 atgaactgc 
                 gaatagcct 
               
               
                   
                 (SEQ ID No. 129) 
                 (SEQ ID No. 130) 
               
               
                   
               
               
                 ldhA 
                 atacgtgtccc 
                 tacacatcccg 
               
               
                   
                 gagcggtag 
                 ccatcagca 
               
               
                   
                 (SEQ ID No. 131) 
                 (SEQ ID No. 132) 
               
               
                   
               
               
                 adhE 
                 gaagtaaacgg 
                 agaagtggcata 
               
               
                   
                 gaaaatcaa 
                 agaaaacg 
               
               
                   
                 (SEQ ID No. 133) 
                 (SEQ ID No. 134) 
               
               
                   
               
               
                 ackA 
                 ccattggctga 
                 gttccattgca 
               
               
                   
                 aaattacgc 
                 cggatcacg 
               
               
                   
                 (SEQ ID No. 135) 
                 (SEQ ID No. 136) 
               
               
                   
               
               
                 focA_ 
                 atgccgtagaa 
                 tgttggtgcgca 
               
               
                 pflB 
                 gccgccagt 
                 gctcgaag 
               
               
                   
                 (SEQ ID No. 137) 
                 (SEQ ID No. 138) 
               
               
                   
               
               
                 pta 
                 gcaaatctggt 
                 tcccttgcacaa 
               
               
                   
                 ttcatcaac 
                 aacaaagt 
               
               
                   
                 (SEQ ID No. 139) 
                 (SEQ ID No. 140) 
               
               
                   
               
               
                 poxB 
                 ggatttggtt 
                 agcattaacgg 
               
               
                   
                 ctcgcataat 
                 tagggtcgt 
               
               
                   
                 (SEQ ID No. 141) 
                 (SEQ ID No. 142) 
               
               
                   
               
               
                 sad 
                 gctgattctcg 
                 aaaaacgttct 
               
               
                   
                 cgaataaac 
                 tgcgcgtct 
               
               
                   
                 (SEQ ID No. 143) 
                 (SEQ ID No. 144) 
               
               
                   
               
               
                 gabD 
                 tctgtttgtca 
                 aagccagcacc 
               
               
                   
                 ccaccccgc 
                 tggaagcag 
               
               
                   
                 (SEQ ID No. 145) 
                 (SEQ ID No. 146) 
               
               
                   
               
               
                 gadA 
                 aagagctgccg 
                 gccgccctctt 
               
               
                   
                 caggaggat 
                 aagtcaaat 
               
               
                   
                 (SEQ ID No. 147) 
                 (SEQ ID No. 148) 
               
               
                   
               
               
                 gadB 
                 ggattttagca 
                 cctaatagcag 
               
               
                   
                 atattcgct 
                 gaagaagac 
               
               
                   
                 (SEQ ID No. 149) 
                 (SEQ ID No. 150) 
               
               
                   
               
               
                 gadC 
                 gctgaactgt 
                 ggcgtgctttt 
               
               
                   
                 tgctggaaga 
                 acaactaca 
               
               
                   
                 (SEQ ID No. 151) 
                 (SEQ ID No. 152) 
               
               
                   
               
               
                 sfcA 
                 tagtaaataa 
                 tcagtgagcgc 
               
               
                   
                 cccaaccggc 
                 agtgtttta 
               
               
                   
                 (SEQ ID No. 153) 
                 (SEQ ID No. 154) 
               
               
                   
               
               
                 maeB 
                 attaatggtga 
                 tgctttttttt 
               
               
                   
                 gagtttgga 
                 attattcgc 
               
               
                   
                 (SEQ ID No. 155) 
                 (SEQ ID No. 156) 
               
               
                   
               
               
                 ppc 
                 gctttataaa 
                 gtaacgacaat 
               
               
                   
                 agacgacgaa 
                 tccttaagg 
               
               
                   
                 (SEQ ID No. 157) 
                 (SEQ ID No. 158) 
               
               
                   
               
               
                 pykA 
                 tttatatgccc 
                 atctgttagag 
               
               
                   
                 atggtttct 
                 gcggatgat 
               
               
                   
                 (SEQ ID No. 159) 
                 (SEQ ID No. 160) 
               
               
                   
               
               
                 pykF 
                 ctggaacgtt 
                 ccagtttagt 
               
               
                   
                 aaatctttga 
                 agctttcatt 
               
               
                   
                 (SEQ ID No. 161) 
                 (SEQ ID No. 162) 
               
               
                   
               
               
                 iclR 
                 gatttgttcaacat 
                 tgcgattaac 
               
               
                   
                 taactcatcgg 
                 agacaccctt 
               
               
                   
                 (SEQ ID No. 163) 
                 (SEQ ID No. 164) 
               
               
                   
               
               
                 mgsA 
                 tctcaggtgct 
                 tatggaagagg 
               
               
                   
                 cacagaaca 
                 cgctactgc 
               
               
                   
                 (SEQ ID No. 165) 
                 (SEQ ID No. 166) 
               
               
                   
               
               
                 icd 
                 cgacctgctgc 
                 tgaacgctaag 
               
               
                   
                 ataaacacc 
                 gtgattgca 
               
               
                   
                 (SEQ ID No. 167) 
                 (SEQ ID No. 168) 
               
               
                   
               
               
                 sucA 
                 acgtagacaa 
                 catcacgtacg 
               
               
                   
                 gagctcgcaa 
                 actgcgtcg 
               
               
                   
                 (SEQ ID No. 169) 
                 (SEQ ID No. 170) 
               
               
                   
               
               
                 sucB 
                 tgcaactttg 
                 tatcgcttccg 
               
               
                   
                 tgctgagcaa 
                 ggcattgtc 
               
               
                   
                 (SEQ ID No. 171) 
                 (SEQ ID No. 172) 
               
               
                   
               
               
                 frdA 
                 aaatcgatctcgt 
                 aggaaccacaa 
               
               
                   
                 caaatttcagac 
                 atcgccata 
               
               
                   
                 (SEQ ID No. 173) 
                 (SEQ ID No. 174) 
               
               
                   
               
               
                 frdB 
                 gacgtgaaga 
                 agttcaatgc 
               
               
                   
                 ttactacgct 
                 tgaaccacac 
               
               
                   
                 (SEQ ID No. 175) 
                 (SEQ ID No. 176) 
               
               
                   
               
               
                 frdC 
                 tagccgcgaccac 
                 cagcgcatcac 
               
               
                   
                 ggtaagaaggag 
                 ccggaaaca 
               
               
                   
                 (SEQ ID No. 177) 
                 SEQ ID No. 178) 
               
               
                   
               
               
                 frdD 
                 atcgtgatca 
                 ttaccctgat 
               
               
                   
                 ttaacctgat 
                 aaattaccgc 
               
               
                   
                 (SEQ ID No. 179) 
                 (SEQ ID No. 180) 
               
               
                   
               
               
                 lacI 
                 gaatctggtg 
                 tcttcgctat 
               
               
                   
                 tatatggcga 
                 tacgccagct 
               
               
                   
                 (SEQ ID No. 181) 
                 (SEQ ID No. 182) 
               
               
                   
               
               
                 lldD 
                 cgtcagcgga 
                 gcggaatttct 
               
               
                   
                 tgtatctggt 
                 ggttcgtaa 
               
               
                   
                 (SEQ ID No. 183) 
                 (SEQ ID No. 184) 
               
               
                   
               
               
                 pgi 
                 ttgtcaacga 
                 aaaaatgccg 
               
               
                   
                 tggggtcatg 
                 acataacgtc 
               
               
                   
                 (SEQ ID No. 195) 
                 (SEQ ID No. 196) 
               
               
                   
               
               
                 ptsG 
                 ccatccgttga 
                 tggtgttaact 
               
               
                   
                 atgagtttt 
                 ggcaaaatc 
               
               
                   
                 (SEQ ID No. 197) 
                 (SEQ ID No. 198) 
               
               
                   
               
               
                 ptsI 
                 gtgacttccaa 
                 ccgttggtttg 
               
               
                   
                 cggcaaaag 
                 atagcaata 
               
               
                   
                 (SEQ ID No. 199) 
                 (SEQ ID No. 200) 
               
               
                   
               
            
           
         
       
     
     Protocol for introduction of gene deletions using the phage transduction method: strains carrying the desired single deletions were obtained from the Keio collection (Baba et al., 2006). Phage lysates of single deletion mutants were prepared by inoculating 10 mL of LB medium containing 50 μg/mL kanamycin, 2 g/L glucose, and 5 mM CaCl 2  with 100 μL of overnight precultures. Following an incubation of 1 h at 37° C., 200 μL of phage lysate prepared from the wild-type MG1655 strain were added, and cultures were incubated for another 2-3 h until cell lysis had completed. After addition of 200 μL chloroform, cell preparations were first vigorously vortexed and then centrifuged for 10 min at 4500×g. The clear lysate was recovered and stored at 4° C. 
     The receptor strain was prepared for phage transduction by an overnight cultivation at 37° C. in LB medium. A volume of 1.5 mL of the preculture was centrifuged at 1500×g for 10 min. The supernatant was discarded and the cell pellet was resuspended in 600 μL of a solution containing 10 mM MgSO 4  and 5 mM CaCl 2 . The transduction was carried out by mixing 100 μL of the solution containing the receptor strain with 100 μL of lysate and incubating this mixture at 30° C. for 30 min. Thereafter, 100 μL of a 1M sodium citrate solution were added followed by vigorous vortexing. After addition of 1 mL LB medium, the cell suspension was incubated at 37° C. for 1 h before spreading the cells on LB agar dishes containing 50 μg/mL kanamycin. Clones able to grow in presence of the antibiotic were confirmed by colony PCR to contain the desired deletion using the primers listed in Table 13. After the introduction of each gene deletion, the antibiotic marker was removed as described above following the method of (Cherepanov &amp; Wackernagel, 1995) 
     The plasmids co-expressing malyl-CoA synthetase, malyl-CoA reductase, and DHB dehydrogenase (pEXT20-MCS-DHB or pACT3-MCS-DHB); or plasmids co-expressing malyl-CoA lyase, malyl-CoA reductase, and DHB dehydrogenase (pEXT20-MCL-DHB or pACT3-MCL-DHB); or the empty control plasmids (pEXT20 or pACT3) were transformed alone or together with one of the plasmids pACT3-aceA, pACT3-ppc, pACT3-galP, pACT3-pck or pACT3-pyc into the optimized host strains. Transformants containing both a plasmid expressing the DHB-pathway enzymes, and a plasmid expressing an anaplerotic enzyme were selected on solid LB medium containing chloramphenicol (25 μg/mL) and kanamycin (50 μg/mL). Non-exclusive examples of constructed strains are listed in Table 14. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Examples of strains constructed for DHB production 
               
            
           
           
               
               
            
               
                 Strain 
                 Relevant Genotype 
               
               
                   
               
               
                 MG1655 
                 Wild-type 
               
               
                 ECE50 
                 pEXT20-MCS-DHB 
               
               
                 ECE51 
                 pACT3-MCS-DHB 
               
               
                 ECE52 
                 pEXT20-MCL-DHB 
               
               
                 ECE53 
                 pACT3-MCL-DHB 
               
               
                 ECE54 
                 ΔldhA ΔadhE Δpta-ack ΔpflB 
               
               
                   
                 pEXT20-MCS-DHB 
               
               
                 ECE55 
                 ΔldhA ΔadhE Δpta-ack ΔpflB 
               
               
                   
                 pACT3-MCS-DHB 
               
               
                 ECE56 
                 ΔldhA ΔadhE Δpta-ack ΔpflB 
               
               
                   
                 pACT3-MCS-DHB, pACT3-ppc 
               
               
                 ECE57 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB 
               
               
                   
                 pEXT20-MCS-DHB 
               
               
                 ECE58 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB 
               
               
                   
                 pACT3-MCS-DHB 
               
               
                 ECE59 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-ppc 
               
               
                 ECE60 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA 
               
               
                   
                 pEXT20-MCS-DHB 
               
               
                 ECE61 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA 
               
               
                   
                 pACT3-MCS-DHB 
               
               
                 ECE62 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-ppc 
               
               
                 ECE63 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA Δpts 
               
               
                   
                 pEXT20-MCS-DHB 
               
               
                 ECE64 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA Δpts 
               
               
                   
                 pACT3-MCS-DHB 
               
               
                 ECE65 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA Δpts 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-ppc 
               
               
                 ECE66 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA Δpts 
               
               
                   
                 ΔfrdBC pEXT20-MCS-DHB 
               
               
                 ECE67 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA Δpts 
               
               
                   
                 ΔfrdBC pACT3-MCS-DHB 
               
               
                 ECE68 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA Δpts 
               
               
                   
                 ΔfrdBC pEXT20-MCS-DHB, pACT3-ppc 
               
               
                 ECE69 
                 Δpta ΔiclR ΔaceB 
               
               
                   
                 pACT3-MCL-DHB 
               
               
                 ECE70 
                 Δpta ΔiclR ΔaceB 
               
               
                 ECE71 
                 Δpta ΔiclR ΔaceB ΔadhE 
               
               
                   
                 pACT3-MCL-DHB 
               
               
                 ECE72 
                 Δpta ΔiclR ΔaceB ΔadhE 
               
               
                 ECE73 
                 ΔldhA ΔadhE Δpta-ack ΔpoxB ΔmaeB ΔsfcA Δmdh Δmqo 
               
               
                   
                 ΔiclR ΔaceB pEXT20-MCS-DHB, pACT3-MCL-DHB 
               
               
                 ECE74 
                 ΔldhA ΔadhE Δpta-ack ΔpoxB ΔmaeB ΔsfcA Δmdh Δmqo 
               
               
                   
                 ΔiclR ΔaceB Δpts 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-MCL-DHB 
               
               
                 ECE75 
                 ΔldhA ΔadhE Δpta-ack ΔpoxB ΔmaeB ΔsfcA Δmdh Δmqo 
               
               
                   
                 ΔiclR ΔaceB Δpts Δpgi 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-MCL-DHB 
               
               
                 ECE76 
                 ΔldhA ΔadhE Δpta-ack ΔpoxB ΔmaeB ΔsfcA Δmdh Δmqo 
               
               
                   
                 ΔiclR ΔaceB Δpgi 
               
               
                   
                 pEXT20-MCS-DHB, pACT3 MCL-DHB 
               
               
                 ECE77 
                 ΔldhA ΔadhE Δpta-ack ΔpoxB ΔmaeB ΔsfcA Δmdh Δmqo 
               
               
                   
                 ΔiclR ΔaceB ΔghrAB 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-MCL-DHB 
               
               
                 ECE79 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA aspC 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-ppc 
               
               
                 ECE80 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA ΔaspC 
               
               
                   
                 ΔiclR ΔaceB pEXT20-MCS-DHB, pACT3-ppc 
               
               
                 ECE81 
                 ΔldhA ΔadhE Δpta-ack ΔpflB ΔpoxB ΔmaeB ΔsfcA ΔaspC 
               
               
                   
                 ΔiclR ΔaceB ΔghrAB 
               
               
                   
                 pEXT20-MCS-DHB, pACT3-ppc 
               
               
                   
               
            
           
         
       
     
     Example 8: Demonstration of Zymotic Production of DHB by the Synthetic Malyl-CoA Pathway 
     Strains and Cultivation Conditions: 
     Experiments were carried out using strain ECE69 which expressed the DHB pathway from plasmid pACT3-MCL-DHB represented by SEQ ID No. 203 (the wild-type Mcr enzyme was replaced by the Mcr Tyr206Pro mutant in this experiment) and the isogenic control strain ECE70 containing the empty plasmid pACT3. All cultivations were carried out at 37° C. on an Infors rotary shaker running at 170 rpm. Overnight cultures (3 mL medium in test tube) were inoculated from glycerol stocks and used to adjust an initial OD 600  of 0.05 in 100 mL growth cultures cultivated in 500 mL shake flasks. IPTG was added at a concentration of 1 mmol/L when OD 600  of the growth cultures reached 1. The composition of the growth mineral medium is provided in Example 2. 
     Estimation of DHB Concentration by LC-MS/MS Analyses: 
     DHB was quantified using LC-MS: Liquid anion exchange chromatography was performed on an ICS-3000 system from Dionex (Sunnyvale, USA) equipped with an automatic eluent (KOH) generator system (RFIC, Dionex), and an autosampler (AS50, Dionex) holding the samples at 4° C. Analytes were separated on an IonPac AS11 HC (250×2 mm, Dionex) column protected by an AG11 HC (50×2 mm, Dionex) pre-column. Column temperature was held at 25° C., flow rate was fixed at 0.25 mL/min, and analytes were eluted applying the KOH gradient described earlier (Groussac E, Ortiz M &amp; Francois J (2000) Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection.  Enzyme. Microb. Technol.  26, 715-723). Injected sample volume was 15 μL. For background reduction, an ASRS ultra II (2 mm, external water mode, 75 mA) anion suppressor was used. Analytes were quantified a mass-sensitive detector (MSQ Plus, Thermo) running in ESI mode (split was ⅓, nitrogen pressure was 90 psi, capillary voltage was 3.5 kV, probe temperature was 450° C.). 
     Results: 
     After 24 h of cultivation the supernatant of strains ECE69 and ECE70 contained 0.05 mM DHB and 0 mM DHB, respectively, demonstrating DHB production via the synthetic pathway. 
     REFERENCES 
     
         
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