Production of methionine via condensation of c1-derived formyl-COA with 3-hydroxypropionaldehyde derived from various substrates

Abstract

A method for producing methionine, in particular, a method for producing methionine via condensation of 3-hydroxypropionaldehyde and C1-derived formyl-CoA was provided. Genetically modified microorganism used for producing methionine was also provided.

Description

PRODUCTION OF METHIONINE VIA CONDENSATION OFCl-DERIVED FORMYL-COA WITH 3-HYDROXYPROPIONALDEHYDE DERIVED FROM VARIOUSSUBSTRATESTECHNICAL FIELD

[0001] The present disclosure generally relates to a method for producing methionine, in particular, a method for producing methionine via condensation of Cl -derived formyl-CoA with 3-hydroxypropionaldehyde derived from various substrates. The present disclosure also relates to genetically modified microorganism used for producing methionine.SUMMARY OF THE INVENTION

[0002] The present disclosure provides the following exemplary embodiments.

[0003] Embodiment 1. A method for the production of 2,4-dihydroxybutyryl-CoA comprising contacting formyl-CoA and 3-hydroxypropionaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0004] Embodiment 2. The method of Embodiment 1, wherein the TPP-dependent enzyme is 2- hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0005] Embodiment 3. The method of Embodiment 2, wherein the TPP-dependent enzyme is selected from:

[0006] Embodiment 4. The method of Embodiment 1 , further comprising generating formyl-CoA by contacting a one-carbon (Cl) substrate selected from formaldehyde or formate with an enzyme.

[0007] Embodiment 5. The method of Embodiment 4, wherein the Cl substrate is formaldehyde, and the enzyme is an acyl -Co A reductase.

[0008] Embodiment 6. The method of Embodiment 4, wherein the Cl substrate is formate and the enzyme is selected from:(i) an acyl-CoA transferase catalyzing the conversion of formate to formyl-CoA;(ii) an acyl-CoA synthase catalyzing the conversion of formate to formyl-CoA; or(iii) a formate kinase catalyzing the conversion of formate to formyl-phosphate and a phosphate formyltransferase catalyzing the conversion of formyl-phosphate to formyl-CoA.

[0009] Embodiment 7. The method of Embodiment 5, wherein the acyl-CoA reductase is selected from:

[0010] Embodiment 8. The method of Embodiment 6, wherein the formate kinase and the phosphate formyltransferase pair is selected from:

[0011] Embodiment 9. The method of Embodiment 6, wherein the acyl-CoA transferase is selected from:

[0012] Embodiment 10. The method of Embodiment 6, wherein the acyl-CoA synthase is selected from:

[0013] Embodiment 11. The method of Embodiment 5, further comprising generating formaldehyde from methanol by contacting methanol with a methanol dehydrogenase or methanol oxidase.

[0014] Embodiment 12. The method of Embodiment 11, wherein the methanol dehydrogenase or methanol oxidase is selected from:

[0015] Embodiment 13. The method of Embodiment 11 or 12, wherein further comprising generating methanol from methane by contacting methane with a methane monooxygenase.

[0016] Embodiment 14. The method of Embodiment 13, wherein the methane monooxygenases is selected from:

[0017] Embodiment 15. The method of any one of Embodiments 1 to 14, further comprising generating 3-hydroxypropionaldehyde from glycerol.

[0018] Embodiment 16. The method of any one of Embodiments 1 to 14, further comprising generating 3-hydroxypropionaldehyde from ethylene glycol and Cl compound.

[0019] Embodiment 17. The method of Embodiment 16, further comprising generating glycolaldehyde from ethylene glycol by contacting ethylene glycol 1 with an alcohol dehydrogenase.

[0020] Embodiment 18. The method of Embodiment 17, wherein the alcohol dehydrogenase is selected from:

[0021] Embodiment 19. The method of Embodiment 16-18, further comprising generating glyceryl-CoA from glycolaldehyde and formyl-CoA by a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0022] Embodiment 20. The method of Embodiment 16-19, further comprising generating glyceraldehyde from glyceryl-CoA by acyl-CoA reductase

[0023] Embodiment 21. The method of Embodiment 16-20, further comprising generating glycerol from glyceraldehyde by alcohol dehydrogenase

[0024] Embodiment 22. The method of Embodiment 21, wherein the alcohol dehydrogenase is selected from:

[0025] Embodiment 23. The method of any one of Embodiments 1 to 16, further comprising generating 3-hydroxypropionaldehyde from Cl compound, including but not limited to formate, formaldehyde, and methanol.

[0026] Embodiment 24. The method of any one of Embodiments 1 to 23, further comprising generating formyl-CoA from interconversion of C 1 compounds

[0027] Embodiment 25. The method of any one of Embodiments 1 to 24, further comprising generating formaldehyde from interconversion of Cl compounds

[0028] Embodiment 26. The method of any one of Embodiments to 25, further comprising generating glycolyl-CoA from formaldehyde and formyl-CoA by a TPP-dependent enzymeselected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0029] Embodiment 27. The method of any one of Embodiments 1 to 26, further comprising generating glycolaldehyde from glycolyl-CoA by acyl-CoA reductase

[0030] Embodiment 28. A method for the production of 3-hydroxypropionaldehyde comprising converting glycerol to 3-hydroxypropionaldehyde by contacting glycerol with glycerol dehydratase.

[0031] Embodiment 29. The method of Embodiment 28, wherein the glycerol dehydratase is selected from:

[0032] Embodiment 30. A method for the production of 2,4-dihydroxybutyric acid comprising:(a) providing 2,4-dihydroxybutyryl-CoA obtained by the method of Embodiments 1-27; and(b) converting 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid by contacting 2,4- dihydroxybutyryl-CoA with any one of (i) and (iii):(i) a thioesterase or an acyl-CoA transferase catalyzing the conversion of 2,4- dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid;(ii) a phosphotransacylase catalyzing the conversion of 2,4-dihydroxybutyryl-CoA to2,4-dihydroxybutyryl-phosphate and a carboxylate kinase catalyzing the conversion of2.4-dihydroxybutyryl-phosphate to 2,4-dihydroxybutyric acid; or(iii) an acyl-CoA reductase catalyzing the conversion of 2,4-dihydroxybutyryl-CoA to2.4-dihydroxy-butanal and an aldehyde dehydrogenase catalyzing the conversion of 2,4- dihydroxy-butanal to 2,4-dihydroxybutyric acid.

[0033] Embodiment 31 . The method of any one of Embodiments 20, 27, and 30, wherein the acyl- CoA reductase is selected from:

[0034] Embodiment 32. The method of Embodiment 30, wherein the aldehyde dehydrogenase is:

[0035] Embodiment 33. A method for the production of 2-keto-4-hydroxybutyric acid comprising:(a) providing 2,4-dihydroxybutyric acid by the method of any one of Embodiments 30 to 32; and(b) converting 2, 4-dihydroxy -butyric acid to 2-keto-4-hydroxybutyric acid by contacting 2,4-dihydroxybutyric acid with a 2-hydroxy acid dehydrogenase / oxidase.

[0036] Embodiment 34. The method of Embodiment 33, wherein the 2-hydroxy acid dehydrogenase / oxidase is selected from:

[0037] Embodiment 35. A method for the production of 2-keto-4-(methylthio)butyric acid comprising:(a) providing 2-keto-4-hydroxybutyric acid by the method of Embodiment 33 or 34; and(b) converting 2-keto-4-hydroxybutyric acid to 2-keto-4-(methylthio)butyric acid by chemically condense 2-keto-4-hydroxybutyric acid with methanethiol.

[0038] Embodiment 36. A method for the production of L-methionine or D-methionine comprising:(a) providing 2-keto-4-(methylthio)butyric acid by the method of Embodiment 35; and(b) converting 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine by contacting 2-keto-4-(methylthio)butyric acid with an enzyme selected from an amino acid dehydrogenase or a methionine / homoserine aminotransferase.

[0039] Embodiment 37. The method of Embodiment 36, wherein the amino acid dehydrogenase is selected from:

[0040] Embodiment 38. The method of Embodiment 36, wherein the methionine / homoserine aminotransferase is selected from:

[0041] Embodiment 39. Tire method of any one of Embodiments 1 to 38, wherein the enzyme used in the method is isolated from a microorganism.

[0042] Embodiment 40. The method of any one of Embodiments 1 to 38, wherein the enzyme used in the method is contained in a microorganism.

[0043] Embodiment 41. A genetically modified microorganism providing 2,4-dihydroxybutyryl- CoA by the method of any one of Embodiments 1 to 26.

[0044] Embodiment 42. The microorganism of Embodiment 41, further providing 2,4- dihydroxybutyric acid by the method of any one of Embodiments 30 to 32.

[0045] Embodiment 43. The microorganism of Embodiment 41, further providing 2-keto-4- (methylthio)butyric acid by the method of Embodiment 35 or 36.

[0046] Embodiment 44. The microorganism of Embodiment 41, further providing L-methionine or D-methionine by the method of any one of Embodiments 36 to 38.

[0047] Embodiment 45. The microorganism of Embodiments 41 to 44, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

[0048] Embodiment 46. The microorganism of Embodiments 41 to 45, wherein the microorganism is bacteria, yeast or fungi, including but not limited to Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebacterium sp., Zymonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccharomyces sp., Pichia sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. Trichoderma sp.

[0049] The following description of examples provides additional details, any one of which can be subject to patenting in combination with any other. Tire specification in its entirety is to be treated as providing a variety of details that can be used interchangeablely with other details.

[0050] The present disclosure is illustrated by the following non-limiting examples.

[0051] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention. Further, the accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain principles of the invention.BRIEF DESCRIPTION OF DRAWINGS

[0052] The drawings referenced herein form a part of the specification. Features shown in the drawing illustrate only some embodiments of the application, and not of all embodiments of the application, unless the detailed description explicitly indicates otherwise, and readers of the specification should not make implications to the contrary.

[0053] Fig. 1 is a scheme illustrating production of methionine from condensation of 3- hydroxy propion aldehyde and Cl-derived formyl-CoA. MMO: methane monooxygenase; MDH: methanol dehydrogenase; FOK: formate kinase; PTA: phosphate formyl transferase; ACS: acyl- CoA synthase; ACT: acyl-CoA transferase; ACR: acyl-CoA reductase; HACS: 2-hydroxyacyl- CoA synthase; TE: thioesterase; 2HADH: 2-hydroxyacid dehydrogenase; AADH: amino acid dehydrogenase; MAT: methionine aminotransferase; HAT: homoserine aminotransferase.

[0054] Fig. 2 is a scheme illustrating production of 3 -hydroxy propionaldehyde from glycerol, ethylene glycol and Cl compounds through FORCE pathway. MMO: methane monooxygenase; MDH: methanol dehydrogenase; FOK: formate kinase; PTA: phosphate formyltransferase; ACS: acyl-CoA synthase; ACT: acyl-CoA transferase; ACR: acyl-CoA reductase; HACS: 2- hydroxyacyl-CoA synthase; TE: thioesterase; GDHt: Glycerol dehydratase; ADH: alcohol dehydrogenase.

[0055] Fig. 3 is a scheme illustrating interconversion of Cl compounds, a) Generation of formyl- CoA from Cl compounds; b) GSH and MSH-dependent oxidation of formaldehyde to formate; c) FUF and H4MPT-dependent oxidation of formaldehyde to formate MMO: methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; FOK: formate kinase; PTA: phosphate formyltransferase; ACS: acyl-CoA synthase; ACT: acyl-CoA transferase; ACR: acyl-CoA reductase; HACS: 2-hydroxyacyl-CoA synthase; TE: thioesterase; FLD: formaldehyde dehydrogenase; FGH: formyl-GS hydrolase; FHC: formyl transferase / hydrolase; MTDA: methylene-tetrahydromethanopterin dehydrogenases; FCH: methenyl-H4F cyclohydrolase; FTFL:formate-EUF ligase; FAE: formaldehyde activating enzyme; MCH: methenyl-FUMPT cyclohydrolase.

[0056] Fig. 4 is the demonstration of 2,4-dihydroxybutyric acid production from 3- hydroxypropi on aldehyde in vitro using purified enzymes.

[0057] Fig. 5 is the demonstration of 2,4-dihydroxybutyric acid production from 3- hydroxy propion aldehyde in vivo.

[0058] Fig. 6 is the production of 2,4-dihydroxybutyric acid from 3-hydroxypropionaldehyde in vivo.

[0059] Fig. 7 is the effect of aldehyde / alcohol dehydrogenase on 3-hydroxypropinoaldehyde consumption.

[0060] Fig. 8 is the effect of double aldehyde dehydrogenase knockout on 3- hydroxypropinoaldehyde consumption.

[0061] Fig. 9 is the production of glycerol from formaldehyde. A) Scheme of glycerol production from formaldehyde, B) glycerate production from formaldehyde in engineered strains, C) glycerol production from formaldehyde in engineered strains

[0062] Fig. 10 shows 2,4-dihydroxybutyric acid production from glycerol and formate.

[0063] Fig. 11 is the identification of desmeninol produced from 2,4-dihydroxybutyric acid chemically.

[0064] Fig. 12 demonstrated the screening of 2-hydroxyacid oxidase for conversion of desmeninol to 4-(methylthio)-2-oxobutyric acid.

[0065] Fig. 13 demonstrated the production of methionine from desmeninol by co-overexpression of AOX and Spo in vivo. ST-MET indicates methionine standard, and MET means methionine.

[0066] The same reference numbers will be used throughout the drawings to refer to the same or like parts.DETAILED DESCRIPTION OF THE INVENTION

[0067] The following detailed description of exemplary embodiments of the application refers to the accompanying drawings that form a part of the description. The drawings illustrate specific exemplary embodiments in which the application may be practiced. The detailed description, including the drawings, describes these embodiments in sufficient detail to enable those skilled in the art to practice the application. Those skilled in the art may further utilize other embodimentsof the application, and make logical, mechanical, and other changes without departing from the spirit or scope of the application. Readers of the following detailed description should, therefore, not interpret the description in a limiting sense, and only the appended claims define the scope of the embodiment of the application.F0068] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms such as “includes” and “included” is not limiting. Additionally, the section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter described.

[0069] EXAMPLES

[0070] EXAMPLE 1: OVERVIEW OF METHIONINE PRODUCTION THROUGH CONDENSATION OF 3-HYDROXYPROPIONALDEHYDE AND FORMYL-COA

[0071] This Example demonstrates the implementation of methionine production through the Cl+Bio platform by condensation of 3-hydroxypropionaldehyde and formyl-CoA and followed by derivation to methionine (Fig. 1 below). The condensation can be catalyzed by HACS (e.g., HACS set forth in Table 1 below), including but not limited to beach sand metagenome BsmHACS (GenBank accession: HAK63664.1), DhcHACS from Dehalococcoidia bacterium (GenBank accession: PWB41796.1 ), CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), to generate 2,4-dihydroxybutyryl-CoA. 2,4-dihydroxybutyryl-CoA is spontaneously converted to 2-hydroxybutyrolactone. Alternatively, 2,4-dihydroxybutyryl-CoA is further converted to 2,4-dihydroxybutyric acid (also referred to as 2,4-dihydroxybutanoic acid) through the action of a thioesterase (such as TesB from E. coli or Pseudomonas putida (McMahon, M.D. and Prather, K.L.J. Appl. Environ. Microbiol. 2014, 80: 1042-1050)), CoA transferase (ACT, e.g., set forth in Table 5 below), or phosphotransacylase-acid kinase (PTA-ACK, e.g., set forth in Table 6 below). Alternatively, 2,4-dihydroxybutyryl-CoA is reduced to 2,4- dihydroxybutyraldehyde (also referred to as 2,4-dihydroxybutanal) by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below), followed by oxidation of 2,4-dihydroxybutyraldehyde / 2,4- dihydroxybutanal to 2,4-dihydroxybutyrate with the help of aldehyde dehydrogenase (ALDH set forth in Table 3 below). And 2,4-dihydroxybutyrate is lactonized to 2-hydroxybutyrolactone by adjusting the pH to lower than 6.

[0072] 2- hydroxybutyrolactone can be chemically condensed with methanethiol (CHaSH) to form desmeninol. As described above, desmeninol could covert to 4-(methylthio)-2-oxobutanoic acid with the help of 2-hydroxy acid dehydrogenase (2HADH or 2HDH). Alternatively, 2- hydroxybutyrolactone can be converted to 2-ketobutyrolactone with the help of 2HDH, and 2- ketobutyrolactone can be chemically convert to 4-(methylthio)-2-oxobutanoic acid. 4- (methylthio)-2-oxobutanoic acid can be further converted to methionine which catalyzed by amino acid dehydrogenase (AADH) (Nitta et a., J Bacteriol. 1974; 117(2): 588-592) or methionine aminotransferase (MAT) (Venos et al., BMC Microbiol, 2004; 4, 39). (Fig. 1 below; enzymes set forth in Table 3 below)

[0073] In parallel, 2-hydroxybutyrolactone can be reduced to 2-ketobutyrolactone by 2HADH / 2HDH and then generate homoserine lactone which catalyzed by amino acid dehydrogenase (AADH) (Nitta et a., J Bacteriol. 1974; 117(2): 588-592) or homoserine aminotransferase (HAT) (Venos et al., BMC Microbiol, 2004; 4, 39). Homoserine lactone can be converted to methionine chemically together with CH3SH. Homoserine lactone can be converted to homoserine as well by adjusting the pH to higher than pH 8. (Fig. 1)

[0074] Alternatively, 2,4-dihydroxybutyrate is firstly converted to 4-hydroxy-2-oxobutyrate (also referred to as 4-hydroxy-2-oxobutanoic acid) by 2HADH. Followed by adding the amino group to 4-hydroxy-2-oxobutyrate to generate homoserine. Homoserine can be further chemically converted to methionine (Fig. 1).EXAMPLE 2: PRODUCTION OF 3-HYDROXYPROPIONALDEHYDE FROM GLYCEROL, ETHYLENE GLYCOL AND ONE-CARBON COMPOUNDS

[0075] This example demonstrates the 3 -hydroxypropionaldehyde production from different feedstocks, including but not limited to glycerol (C3), ethylene glycol (C2) and methanol (Cl). When 3-hydroxypropionaldehyde is produced, it can be further condensed with formyl-CoA generated from Cl compounds to form 2,4-dihydroxybutyryl-CoA and finally converted to methionine as presented in Fig. 1.

[0076] When 3-hydroxypropionaldehyde is produced from glycerol. Glycerol dehydratase (GDHt) needs to be overexpressed for 3-hydroxypropionaldehyde production from glycerol (Fig. 2).

[0077] When 3-hydroxypropionaldehyde is produced from ethylene glycol, two iterative condensations are needed for methionine production (Fig. 2). Ethylene glycol is firstly converted to glycolaldehyde by E. coli FucO. Glycolaldehyde can be condensed with formyl-CoA to generate glyceryl-CoA, a reaction catalyzed by HACS, including CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), RhbHACS from Rhodocyclaceae bacterium (GenBank accession: MBL8483477.1), PdsHACS from Pseudonocardia sediminis (GenBank accession: WP_ 130292058.1) or other HACS variants (e.g., HACS set forth in Table 1 below). Glyceryl- CoA can be reduced to glyceraldehyde by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Glyceraldehyde was further converted to glycerol by glyceraldehyde reductase (ADH), such as yqhD, yahK from E. coli, GLD1 from Trichoderma reesei (e.g., enzymes set forth in Table 3 below). Glycerol can be converted to 3-hydroxypropionaldehyde as mentioned above by glycerol dehydratase GDHt, dhaB123 from Klebsiella pneumonia or other microorganisms (e.g., GDHt set forth in Table 3 below), and then further convert to methionine through the route described in Example 1.

[0078] When 3-hydroxypropionaldehyde is produced from methanol or other Cl compounds, one more iterative condensation is needed to generate glycolaldehyde for methionine production compare to using ethylene glycol as co-substrate (Fig. 2). Methanol or other Cl compounds were firstly converted to formaldehyde and formyl-CoA through the interconversion module (Fig. 3). Formaldehyde and formyl-CoA condensed together to generate glycolyl-CoA by HACS , including CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), RhbHACS from Rhodocyclaceae bacterium (GenBank accession: MBL8483477.1), PdsHACS from Pseudonocardia sediminis (GenBank accession: WP_130292058.1) or other HACS variants (e.g., HACS set forth in Table 1 below), and further converted to glycolaldehyde with ACR variants from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). And glycolaldehyde can be used as a substrate for next round condensation with formyl-CoA to generate glyceryl-CoA that catalyzed by above mentioned HACS variants (e.g., HACS set forth in Table 1 below). Glyceryl-CoA can be reduced to glyceraldehyde by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Glyceraldehyde was further converted to glycerol by glyceraldehyde reductase (ADH), such as yqhD, yahK fromE. coli, GLD1 from Trichoderma reesei (e.g., enzyme set forth in Table 3 below). Glycerol can be converted to 3-hydroxypropionaldehyde as mentioned above by glycerol dehydratase (GDHt), and then further convert to methionine through the route described in Example 1.

[0079] EXAMPLE 3. INTERCONVERSION OF ONE-CARBON COMPOUNDS TO GENERATE FORMYL-COA

[0080] Formyl-CoA can be provided through the interconversion of Cl compounds including methane, methanol, formaldehyde, formate (Fig. 3a below) with suitable enzymes (e.g., enzymes set forth in Table 4 below). Methane is readily available Cl source from natural gas, landfills, and agriculture. Biological oxidation of methane to methanol is catalyzed by methane monooxygenases. Functional expression of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus is demonstrated in E. coli as a host (bioRxiv 2021 . 08.05.455234). Subsequent oxidation of methanol to formaldehyde can be catalyzed by NAD+-dependent methanol dehydrogenases (MDH), MDH from Bacillus methanolicus MGA3 (BmMDH), Bacillus stearothe rmophilus (BsMDH) (Metab. Eng. 39:49-59, 2017) and Cupriavidus necator (Appl. Microbiol. Biotechnol. 100:4969-4983, 2016), PQQ (pyrroloquinoline quinone)-dependent MDH (MxaFI, or XoxF), or oxygen-dependent alcohol oxidase (AOD). When AOD is used, a catalase (CTA) is used to detoxify of hydrogen peroxide (e.g., enzymes set forth in Table 4 below).

[0081] Formaldehyde can be directly oxidized to formic acid catalyzed by four cofactor-dependent formaldehyde oxidation pathways, involving glutathione (GSH), tetrahydrofolate (H4F, THF), tetrahydromethanopterin (H4MPT), or mycothiol (MSH). Formaldehyde spontaneously reacts with glutathione (GSH) to form S-hydroxymethyl-GS. Hydroxymethyl-GS is then converted to formyl- GS by hydroxymethyl-GS dehydrogenase (FrmA). Formyl-GS is then converted to formate by formyl-GS hydrolase (FrmB). Formaldehyde also spontaneously reacts with mycothiol to form an S-hydroxymethylmycothiol same as GSH-dependent route, and then it was converted to S- formyl mycothiol by MD-FalDH, which is further hydrolyzed to formate and MSH via a hydrolase. (Fig. 3b) When H4MPT was used as the cofactor, formaldehyde condensed with H4MPT to generate mefhylene-FLMPT spontaneously or by formaldehyde activating enzyme (FAE), methylene-H4MPT is then converted to methenyl-H4MPT by methylene-tetrahydromethanopterin dehydrogenases MtdA or MtdB. Methenyl -H4M PT was converted to formyl-H4MPT by methenyl- H4MPT cyclohydrolase (MCH), and then converted to formate with the help of formyltransferase / hydrolase FHC. Formaldehyde spontaneously reacts with H4F to formmethylene-H4F, and then contacting with MtdA it was converted to methenyl -H4F which is then converted to formyl-H4F by methenyl-H4F cyclohydrolase. Formyl-H4F can be further converted to formate by formate-H4F ligase FTFL (Fig. 3c).

[0082] Alternatively, formaldehyde oxidation to formyl-CoA can be catalyzed by various acylating aldehyde dehydrogenase candidates (Nat. Chem. Biol. 15:900-906, 2019). Formyl-CoA can either be fed to the Cl elongation platform or further converted to CO2 to generate energy. Formyl-CoA hydrolysis to formate can be catalyzed by two different families of enzymes. Acyl- CoA transferases (ACT, e.g., set forth in Table 5 below) can catalyze reversible CoA transfer from various CoA donor, such as 4-methylthio-2-hydroxy butyryl-CoA, 2,4-dihydroxy butyryl-CoA, acetyl-CoA or succinyl-CoA to formate. Phosphotransacylase-formate kinase (PTA-FOK, e.g., set forth in Table 6 below) pair catalyze reversible phosphorylation of formyl-CoA to formylphosphate, followed by dephosphorylation to formate generating 1 ATP. While these two reactions are considered fully reversible, AMP-forming acyl-CoA synthetases are favored toward formate activation to formyl-CoA. (Fig. 3a)

[0083] EXAMPLE 4: OVERVIEW OF METHODS USDED FOR GENETIC MANIPULATION

[0084] The genes for overexpression are either cloned into appropriate vectors or inserted into chromosome with strong synthetic constitutive promoter, such as Ml -93. When cloned into vectors, these genes are amplified through PCR using appropriate primers to append homology on each end for recombination into the vector backbone with e.g., Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. Plasmids are linearized by the appropriate restriction enzymes (New England Biolabs, Ipswich, MA, USA) and recombined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system. The mixture is subsequently transformed into Stellar competent cells. Transformants that grow on solid media (LB+Agar) supplemented with the appropriate antibiotic are isolated and screened for the gene insert by PCR. Plasmids from verified transformants are isolated and the sequence of the gene insert is further confirmed by DNA sequencing. The sequence confirmed plasmids are then introduced to host strain through electroporation.

[0085] When inserted into chromosome, CRISPR is used and genetic sites of tesB and adhE are suitable loci, although others could be used. The CRISPR method is based on the method developed by Jiang et al. (Jiang, Y., et al. Appl. Environ. Microbiol. 81 :2506-2514 (2015)). First,the host strain is transformed with plasmid pCas, the vector for expression of Cas9 and X-red recombinase. The resulting strain is grown under 30 °C with L-arabinose for induction of X-red recombinase expression, and when OD reaches ~0.6, competent cells are prepared and transformed with pTargetF (Addgene 62226) expressing sgRNA and N20 spacer targeting the locus and template of insertion of target gene. The template is the inserted gene plus Ml -93 promoter with -500 bp sequences homologous with upstream and downstream of the insertion locus, constructed through overlap PCR with usage of Phusion polymerase or synthesized by GenScript (Piscataway, NJ) or GeneArt® (Life Technologies, Carlsbad, CA). The way to switch N20 spacer of pTargetF plasmid is inverse PCR with the modified N20 sequence hanging at the 5' end of primers with usage of Phusion polymerase and followed by self- ligation with usage of T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Transformants that grow under 30°C on solid media (LB+Agar) supplemented with spectinomycin and kanamycin (or other suitable antibiotic) are isolated and screened for the chromosomal gene insert by PCR. The sequence of the gene insert, which is amplified from genomic DNA through PCR using Phusion polymerase, is further confirmed by DNA sequencing. The pTargetF can then be cured through 1PTG induction, and pCas can be cured through growth under higher temperature like 37-42 °C.

[0086] All molecular biology techniques are performed with standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).) or by manufacturer protocol. Strains are stored in glycerol stocks at -80°C. Plates are prepared using LB medium containing 1.5% agar, and appropriate antibiotics are included at the following concentrations: ampicillin (100 pg / mL), kanamycin (50 pg / mL), spectinomycin (50 pg / mL) and chloramphenicol (12.5 pg / mL).

[0087] Expression of selected enzyme variants was achieved using plasmid-based gene expression by cloning the desired gene(s) into pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes and by utilizing Tn-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, CA). Linear DNA fragments for insertion were created via PCR of the open reading frame of interest (for genes native to E. coll) or by gene synthesis of the codon optimized gene. Genes were synthesized by GeneArt (Life Technologies, Carlsbad, CA). Resulting In-Fusion reaction products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, CA), and clones identified by PCR screening were further confirmed by DNA sequencing. As a host for these vectors, an engineered strain of E. colibased on MG1655(DE3), BW25113 or ATCC8739 with knockouts for formaldehyde (A / rmA), formate ( fdhF AfdnG AfdoG) oxidation and aldehyde consumption (including adhP, yjgB, yqhD, adhE, etc) can be used, which we expected could compete or interfere with the analysis of our pathway.

[0088] EXAMPLE 5: FERMENTATION CONDITIONS USED FOR IN VIVO PRODUCTION OF METHIONINE

[0089] MOPS minimal medium (Neidhardt et al. J. Bacteriol.119736-47 (1974)) with 125 mM MOPS and Na2HPO4 in place of K2HPO4 (2.8 mM), supplemented with 20 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract, 100 LIM FeSO4, 5 mM (NH4)2SO4, and 30 mM NH4CI is used for fermentations. If required, 55 g / L of CaCO3 is also supplemented as pH buffer. 20 mM lactic acid is supplemented, if it is not synthesized intracellularly and needed for the experiment. 500 mM methanol is also supplemented. Antibiotics (50 pg / mL carbenicillin, 50 pg / mL spectinomycin and 50 pg / mL kanamycin) are included when appropriate. All chemicals are obtained from Fisher Scientific Co. (Pittsburg, PA) and Sigma-Aldrich Co. (St. Louis, MO).

[0090] Fermentations are performed in 25 mL Pyrex Erlenmeyer flasks (narrow mouth / heavy duty rim, Corning Inc., Corning, NY) or 96 deep well plates (2.2 mL, v bottom, USA scientific) filled with appropriate volume of fermentation medium and sealed with foam plugs filling the necks. For anaerobic conditions, 17.5 mL Hungate tubes are completely filled with fermentation medium and sealed with tubber septa. A single colony of the desired strain is cultivated overnight (14-16 hrs) in LB medium with appropriate antibiotics and used as the inoculum with initial ODgoo as -0.05. After inoculation, flasks are incubated in a NBS 124 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, NJ) at 200 rpm and 37°C or 30°C. When optical density (550 nm, OD550) reached -0.3-0.5, appropriate concentration of isopropyl beta-D-1 -thiogalactopyranoside (IPTG) (or other suitable inducer) is added for plasmid gene induction. Additional fermentations are conducted in a SixFors multi- fermentation system (Infors HT, Bottmingen, Switzerland) with an air or argon flowrate of 2 N L / hr, independent control of temperature (37 °C), pH (controlled at 7.0 with NaOH and H2SO4), and appropriate stirrer speed. Pre-cultures are grown in 25 mL Pyrex Erlenmeyer flasks as described above and incubated for 4 hours post-induction. An appropriate amount of this pre-culture is centrifuged, washed twice with fresh media, and used for inoculation (400 mL initial volume). The fermentations in bioreactor use described fermentationmedia with 40 g / L glucose and appropriate IPTG and antibiotics. If required, lactic acid (20 mM) is added at 0, 24, and 48 hours.

[0091] After the fermentation, the supernatant obtained through 5000 g, 5 min centrifuge in an Optima L-80XP Ultracentrifuge (Beckman -Coulter, Schaumburg, TL) of 2 mL culture is prepared for HPLC analysis.

[0092] EXAMPLE 6. HPLC ANALYSIS OF METHIONINE

[0093] The supernatant aliquots of 0.1 mL transferred to 15 mL tube, mixed with 0.2 mL of acetonitrile, 0.1 mL of 0.5 mM NaHCOs and 0.1 mL of 2,4-Dinitrofluorobenzene (DNFB) solution (0.2 mL DNFB add acetonitrile to 10 mL), tighten the cap and vigorously vortex for 30 seconds before doing derivatization. The derivatization reaction was carried out on a water bath (60 °C) for 20 min in the dark. After cooling in ice-cold water, 1 mL of 0.5 M phosphate buffer solution was added to terminate the reaction. Another 1 mL acetonitrile and 7.5 mL MiniQ water was added. The resulting solution of DNFB derivatives was then briefly vortexed again and filtered through a 0.22 pm nylon filter into auto-sampler vials for HPLC analysis.

[0094] HPLC analysis is conducted on a Waters 2695 system equipped with 996 photodiode array detector and a Phenomenex Luna Cl 8 column (250mm*4.6mm*5pm). Column temperature was maintained at 30 °C, detector was set at 360 nm, and the injection volume was 10 pL. The mobile phase consisting of 0.1 % acetic acid and 0.1 % trimethylamine in MiniQ water (A), and acetonitrile (B). The separation was achieved using the following gradient conditions with a flow rate of 0.8 mL / min: 0 min, 30% acetonitrile (B); 15 min, 85% acetonitrile (B); 18 min, 85% acetonitrile (B); 18.01 min, 30% acetonitrile (B); 30 min, 30% acetonitrile (B).

[0095] EXAMPLE 7. DEMONSTRATION OF 2,4-DIHYDROXYBUTYRIC ACID PRODUCTION FROM 3-HYDROXYPROPINALDEHYDE AND FORMYL-COA USING PURIFIED ENZYMES.

[0096] This example demonstrates the production of 2,4-dihydroxybutyric acid by condensation of 3-hydroxypropinoaldehyde (3-HPA) and formyl-CoA using purified enzymes. In this example, ACK-PTA route was employed to provide formyl-CoA for condensation.

[0097] Expression of selected enzyme variants was achieved using plasmid-based gene expression by cloning the desired gene(s) into pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes and by utilizing In-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, CA). Linear DNA fragments for insertion werecreated via PCR of the open reading frame of interest (for genes native to E. coli) or by gene synthesis of the codon optimized gene. Genes were synthesized by GeneArt (Life Technologies, Carlsbad, CA). Resulting In-Fusion reaction products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, CA), and clones identified by PCR screening were further confirmed by DNA sequencing.

[0098] Overnight cultures of the expression strains were grown in LB, which was used to inoculate 25 mL TB medium in a 250 mL baffled flask at 1%. The culture was grown at 30°C and 250 rpm in an orbital shaker until ODBOO reached 0.4-0.6, at which point expression was induced with 0.1 mM IPTG. 24 hours post inoculation, cells were harvested by centrifugation. The cell pellets were washed once with cold 9 g / L NaCl solution and stored at -80°C until needed. Antibiotics were included where appropriate at the following concentrations: ampicillin (100 pg / mL), carbenicillin (50 pg / mL), and spectinomycin (50 pg / mL).

[0099] For protein purification, E. coli cell pellets expressing the desired his-tagged enzymes were prepared as described above. The frozen cell pellets were resuspended in cold lysis buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 10 m imidazole) to an approximate OD600 of 40, to which 1 mg / mL of lysozyme and 250 U of Benzonase nuclease was added. The mixture was further treated by sonication on ice using a Branson Sonifier 250 (5 minutes with a 25% duty cycle and output control set at 3), and centrifuged at 7500xg for 15 minutes at 4°C. The supernatant was applied to a chromatography column containing 1 mL TALON metal affinity resin (Clontech Laboratories, Inc., Mountain View, CA), which had been pre-equilibrated with the lysis buffer. The column was then washed first with 10 mL of the lysis buffer and then twice with 20 mL of wash buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 20 mM imidazole). The his-tagged protein of interest was eluted with 1-2 applications of 4 mL elution buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 250 mM imidazole). The eluate was collected and applied to a 10,000 MWCO Amicon ultrafiltration centrifugal device (Millipore, Billerica, MA), and the concentrate (-100 pL) was washed twice with 4 mL of 50 mM KPi pH 7.4 for desalting. Protein concentrations were estimated by the Bradford method. Purified protein was saved in 20 pL aliquots at -80°C until needed.

[0100] SDS-PAGE was performed using NuPAGE 12% Bis-Tris Protein Gels with SDS running buffer and stained with SimplyBlue SafeStain according to manufacturer protocols (ThermoFisher Scientific, Waltham, MA).

[0101] In vitro purified enzyme reaction was done in a reaction containing 50 mM HEPES pH 7.8, 5 mM ATP, 10 mM MgCh, 5 mM CoA, 0.2 mM TPP, 2 pM CaoHACS or CfhHACS, 1 pM PT A, 1 pM ACK, 30 mM sodium formate and 10 mM 3-hydroxypropinoaldehyde. Reactions were incubated at 30°C for 18 hours unless otherwise specified. 1 / 20 of the reaction volume of 10 M NaOH solution was added to terminate the reactions. After 30 min hydrolysis, 1 / 20 of the reaction volume of 10 N H2SO4 was added to neutralize the pH. Samples were centrifuged at 20817xg for 15 minutes and the supernatant analyzed by HPLC as described below.

[0102] Quantification of product and substrate concentrations was performed via HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a DAD and an Supelco aphera-NI L column (5pm*4.6mm*250mm) with operating conditions to optimize peak separation (1 ml / min flowrate, 0.1 % H3P04:CAN=70:30 as the mobile phase, column temperature 40°C).

[0103] The results indicate that both CaoHACS and CfhHACS were able to produce 2,4- dihydroxybutyric acid through the condensation of 3-hydroxypropinoaldehyde and formyl-CoA (Fig. 4)

[0104] EXAMPLE 8: SCREENING OF DIFFERENT HACS FOR 2,4- DIHYDROXYBUTYRIC ACID PRODUCTION

[0105] The purpose of this example is to screen better HACS for 2,4-dihydroxybutyric acid production by condensation of 3-hydroxypropinoaldehyde and formyl-CoA. Eight HACS candidates from previous study were tested here, including ApbHACS (JGI15), DhcHACS (JGI20), CabHACS (JGI23), CdcHACS (JGI24), MeOXC4, CaoHACS (JGIH48), CfhHACS (JGIH65), BbHACS (JGH9). Acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT) was used for formyl-CoA generation.

[0106] To compare HACS for 2,4-dihydroxybutyric acid production in vivo, vectors were engineered to independently control the expression of various HACS candidates and the acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), with HACS under control of the IPTG- inducible T7 promoter in pCDFDuet-1 and CaAbfT under control of a cumate-inducible T5 promoter in pETDuet-1. As a host for these vectors, an engineered strain of E. coli based onMG 1655 (DE3) with knockouts for formaldehyde (AfrmA) and formate (AfdhF AfdnG AfdoG) oxidation as well as for glycolate utilization (AglcD) was used.

[0107] In vivo product synthesis was conducted using M9 minimal media (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4CI, 0.5 g / L NaCl, 2 mM MgSO4, 100 uM CaCl2, and 15 pM thiamine- HC1) unless otherwise stated. Cells were initially grown in 96-deep well plates containing 0.5 mL of the above media further supplemented with 20 g / L glycerol, 10 g / L tryptone, and 5 g / L yeast extract. A single colony of the desired strain was cultivated overnight (14-16 hours) in LB medium with appropriate antibiotics and used as the inoculum (1%). Antibiotics (100 pg / mL carbenicillin, 100 pg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30°C and 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until an OD600 of ~0.4 was reached, at which point appropriate amounts of inducer(s) (isopropyl P-D-1 -thiogalactopyranoside and cumate) were added. Plates were incubated for a total of 24 hours post-inoculation.

[0108] Cells from the above pre-cultures were then centrifuged (4000 rpm, 22°C), washed with the above minimal media without any carbon source, and resuspended with 1 mL of above minimal media containing indicated amounts of carbon source. 10 mM 3-hydroxypropinoaldehyde and 30 mM formate were added at 0 hr and were incubated at 30 °C and 1000 rpm in Digital Microplate Shaker (Fisher Scientific). After incubation at 30°C for 2 hour, the cells were pelleted by centrifugation and the media was analyzed using HPLC.

[0109] The results indicate that 6 out of 8 tested enzymes have condensation activity and DhcHACS has higher activity than other tested enzymes, it produced 35 mg / L 2,4- dihydroxybutyric acid (Fig. 5).

[0110] The HACS performance on 2,4-dihydroxybutyric acid production was further evaluated in an engineered strain BW25113 del (glcD, frmA, fdhF, fdoG, fdnG, yqhD, aldB) that has a lower 3-hydroxypropinoaldehyde background consumption. By adding 10 mM 3- h y drox y propino aldehyde and 20 mM formate to a final OD of 20 for bioconversion at 30 °C for 21 hours, DhcHACS and CaoHACS have the highest 2,4-dihydroxybutyric acid production (154 mg / L and 153 mg / L respectively), CfhHACS and ApbHACS has good performance too (Fig. 6)

[0111] EXAMPLE 9. ENGINEERING OF THE HOST STRAIN TO HAVE A LOWER 3-HYDROXYPROPINOALDEHYDE BACKGROUND CONSUMPTION

[0112] The purpose of this example is to decrease the 3-hydroxypropinoaldehyde background consumption in the host strain by knockout the genes responsible for 3HPA backgroundconsumption. The aldehyde dehydrogenases and alcohol dehydrogenases were deactivated individually in BW25113 del (frmA, fdhF, fdoG, FdnG, glcD) (also referred to as BWA5R).

[0113] A bioconversion with the newly constructed strains was performed as described above. Cells were cultured in TB medium for 24 hours, centrifuged at 4000 x g for 10 min, discarded the broth and washed the cells with M9 once. And then the cells were resuspended in 1 mL M9 with 2 mM 3-hydroxypropinoaldehyde, shaken at 30 °C for 2 hours. The residual 3- hydroxypropinoaldehyde in the supernatant was measured using 3-methyl-2-benzothiazolone hydrazone (MBTH) method as reported before.

[0114] The results indicate that knockout aldB has the highest impact on 3- hydroxypropinoaldehyde consumption, and then yiaY, yahK etc (Fig. 7).

[0115] Seven aldehyde dehydrogenases were further deactivated in BW251 1 del (frmA, fdhF, fdoG, FdnG, glcD, aldB), and the 3-HPA background consumption of newly constructed was evaluated using the same method. The results showed that 3-HPA consumption was further decreased and aldB+aldA knockout has the lowest consumption (Fig. 8).

[0116] EXAMPLE 10. PRODUCTION OF GLYCEROL FROM Cl COMPOUNDS

[0117] This example is to demonstrate the production of glycerol from Cl compounds using formaldehyde as an example. As shown in Fig. 9A, formaldehyde was converted to formyl-CoA by ACR, and then condensed with formaldehyde to generate glycolyl-CoA, it was further converted to glycolaldehyde. Glycolaldehyde is then condensed with formyl-CoA to form glyceryl-CoA, converted to glyceraldehyde and finally converted to glycerol.

[0118] This was tested in two strains MJI0301 and MJI0308 which are MG1655(DE3) derivatives, MJI0301 has thioesterases (yciA, tesA, tesB, ybgC, ydil and fadM) knockout in MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD). FucO and ackA-Pta were further deactivated in MJI0301 to give MJIO3O8. MJI0301 and MJIO3O8 harboring pCDFduet-CfhHACS and pET-PCT5-LmACR were cultured in NBS medium and induced with 75 pM IPTG and 50 pM cumate. After 24 hours of cultivation, cells were harvested and washed with M9 medium, Cells were then resuspended in M9 with 5 mM formaldehyde to a final OD of 20 for bioconversion at 30 °C for 1 hour and 3 hours.

[0119] After 3 hours, 686 pM glycerate was accumulated in MJI0301, 766 pM glycerate was produced in MJI0308 (Fig. 9B). Moreover, 20 pM and 61 pM of glycerol were produced in MJ10301 and MJ10308 respectively (Fig. 9C).

[0120] EXAMPLE 11. PRODUCTION OF 2,4-DIHYDROXYBUTYRIC ACID FROM GLYCEROL AND FORMATE

[0121] This example demonstrates the 2,4-dihydroxybutyric acid production from glycerol and formate. Glycerol was converted to 3-hydroxypropinoaldehyde by glycerol dehydratase and formate was converted to formyl-CoA by CaAbfT, and then they were condensed to produce 2,4- dihydroxybutyryl-CoA and converted to 2,4-dihydroxybutyric acid (Figs. 1 and 2).

[0122] This was tested in BW25113(DE3) del (frmA, fdhF, fdnG, fdoG, glcD). Glycerol dehydratase from Klebsiella peneumoniae, DhcHACS and CaAbfT were co-overexpressed for this purpose. The bioconversion was performed the same as described above. Cells were cultured in NBS and induced with appropriate concentration of IPTG and cumate. Bioconversion was done in a final OD of 20 for 3 hours. The result indicates that the cells can produce 2,4-dihydroxybutyric acid from glycerol and formate (Fig. 10).

[0123] EXAMPLE 12. CHEMICAL CONVERSION OF 2,4-DIHYDROXYBUTYRIC ACID TO DESMENINOL

[0124] This example demonstrates the desmeninol production from 2,4-dihydroxybutyric acid. The pH of 2,4-dihydroxybutyric acid solution was adjusted to 1 by using 1 N H2SO4 to convert 2,4-dihydroxybutyric acid to 2-hydroxybutyrolactone. 2-hydroxybutyrolactone was then extracted by ethyl acetate and dried for further experiments.

[0125] One gram of 2-hydroxybutyrolactone and 700mg of potassium methyl mercaptan were dissolved in 20 ml of dimethylacetamide, heated to 160 °C, reacted for Ih, and the target product desmeninol was detected, and the conversion rate was more than 99%.

[0126] The reaction system was cooled to 20-30 °C, the pH of the system was adjusted to 1 with IN sulfuric acid, diluted with lOOrnL of saturated saline solution, and ethyl acetate was extracted twice to extract the target product to the organic phase. After the organic phases are combined, they are dried with sodium sulfate to concentrate a brown liquid. The reaction product is the same substance as the standard. (Fig. 11).

[0127] EXAMPLE 13: CONVERSION OF DESMENINOL TO 4-(METHYLTHIO)-2- OXOBUTYRIC ACID BY 2-HYDROXYACID OXIDASE / DEHYDROGENASE

[0128] This example demonstrates the 4-(methylthio)-2-oxobutyric acid production from desmeninol. As described above, desmeninol can be further oxidized to 4-(methylthio)-2-oxobutyric acid by 2-hydroxyacid oxidase / dehydrogenase. A0X1 (NP_001327205.1) and A0X2 (NP_001078152.1) from Arabidopsis thaliana, HmGOX (NP_060015.1) fromhuman and HeGOX (ABY61829.1) from spinach were cloned into pCDFDuet for enzyme expression.

[0129] The conversion of desmeninol to 4-(methylthio)-2-oxobutyric acid was conducted in a resting cell bioconversion format. To overexpress 2-hydroxyacid oxidase, the plasmid containing 2-hydroxyacid oxidase was transformed into BL21(DE3) and overexpressed at 25 °C overnight. Cells were harvested by centrifugation, washed once with 100 mM PBS buffer, pH 7.0, and collected for further use. The collected cells were resuspended in a reaction system containing 100 mM PBS pH 7, 0.1% Triton-XlOO, 20 mM desmeninol to a final OD600 of 20 to initiate the resting cells bioconversion. The resting cells bioconversion was conducted at 30°C. After 18h, the cells were pelleted by centrifugation and the supernatant was applied for HPLC analysis.

[0130] The resting cells bioconversion shows all tested 2-hydroxyacid oxidases have activity on desmeninol, AOX2 shows the best activity under tested conditions with up to 85% conversion rate, followed by A0X1 with 50% conversion rate (Fig. 12).

[0131] EXAMPLE 14: CONVERSION OF DESMENINOL TO METHIONINE BY 2- HYDROXYACID OXIDASE AND TRANSAMINASE.

[0132] This example demonstrates the methionine production from desmeninol. As described above, desmeninol can be first oxidized to 4-(methylthio)-2-oxobutyric acid by 2-hydroxyacid oxidase / dehydrogenase. 4-(methylthio)-2-oxobutyric acid is then reduced to methionine by amino dehydrogenase / transaminase. Considering that 2-hydroxyacid oxidase produces hydrogen peroxide during catalysis, catalase (UtCAT, WP_016837596.1 ) from Ureibacillus thermosphaericus was co-overexpressed with AOX2 on the vector pCDFDuet-AOX2-RBS- UtCAT. Transaminases Spo was inserted in pET28a(+).

[0133] To do the bioconversion, plasmid pCDFDuet-AOX2-RBS-UtCAT and pET28-spo were co-transformed into BL21(DE3), and overexpressed at 25 °C overnight. Cells were harvested by centrifugation, washed once with 100 mM PBS buffer, pH 7.0, and collected for further use. The collected cells were resuspended in a reaction system containing 100 mM PBS pH 70.1% Triton- XlOO, 0.1 mM PLP, 10 g / L mM desmeninol, 70 mM Pentylenediamine hydrochloride to a finalOD600 of 20 to initiate the resting cells bioconversion. The resting cells bioconversion was conducted at 30°C. After 18h, the cells were pelleted by centrifugation and the supernatant was applied for HPLC analysis. As HPLC shows 1.4 g / L of methionine was produced from 10 g / L desmeninol (Fig. 13).

[0134] Table 1. List of 2-hydroxyacyl-CoA synthase (HACS) used for the condensation of methional and formyl-CoA

[0135] Table 2. List of acyl-CoA reductase (ACR)

[0136] Table 3. List of enzyme used for the conversion of intermediate to methionine

[0137] Note: 2HADH: 2-hydroxyacid dehydrogenase; GDHt: glycerol dehydratase; ADH: alcohol dehydrogenase; ALDH: aldehyde hydydrogenase; AADH: amino acid dehydrogenase; MAT: methionine aminotransferase

[0138] Table 4. List of enzymes used for the interconversion of one-carbon compounds to generate formyl-CoA

[0139] Note: sMMO: soluble methane monooxygenase; pMMO: Particulate methane monooxygenase; MDH: methanol dehydrogenase; AOD: oxygen-dependent alcohol oxidase; CTA: catalase: ALDH: aldehyde dehydrogenase; ACS: acyl-CoA synthase.

[0140] Table 5. List of acyl-CoA transferases (ACT) variants used for the interconversion of one-carbon compounds to generate formyl-CoA

[0141] Table 6. List of List of acyl-CoA kinases (ACK) and phosphoacyltransferases (PTA) variants (JGIK)variants used for the interconversion of one-carbon compounds to generate formyl-CoA

[0142] Various embodiments have been described herein with reference to the accompanying drawings. It will, however, be evident that various modifications and changes may be made thereto, and additional embodiments may be implemented, without departing from the broader scope of the invention as set forth in the claims that follow. Further, other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of one or more embodiments of the invention disclosed herein. It is intended, therefore, that this application and the examples herein be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following listing of exemplary claims.

Claims

Claims1. A genetically modified microorganism for converting 3-hydroxypropionaldehyde and formyl-CoA to 2,4-dihydroxybutyryl-CoA, comprising an overexpressed TPP-dependent enzyme selected from a 2-hydroxyacyl-CoA synthase, a 2-hydroxyacyl-CoA lyase, an oxalyl-CoA decarboxylase or a benzaldehyde lyase.

2. The genetically modified microorganism of claim 1, further comprising an overexpressed enzyme catalyzing the conversion of 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid, wherein the overexpressed enzyme is selected from thioesterase, acyl-CoA transferase, a phosphotransacylase and a carboxylate kinase or an acyl-CoA reductase and an aldehyde dehydrogenase.

3. The genetically modified microorganism of claim 2, further comprising an overexpressed 2- hydroxy acid dehydrogenase catalyzing the conversion of 2,4-dihydroxybutyric acid to 2- keto-4-hydroxybutyric acid.

4. A genetically modified microorganism for converting 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine, comprising an overexpressed amino acid dehydrogenase, or methionine aminotransferase.

5. The genetically modified microorganism of claim 3, further comprising an overexpressed amino acid dehydrogenase catalyzing the conversion of 2-keto-4-hydroxybutyric acid to L- homoserine or D- homoserine.

6. The genetically modified microorganism of claim 3, further comprising an overexpressed aminotransferase catalyzing the conversion of 2-keto-4-hydroxybutyric acid to L- homoserine or D- homoserine.

7. The genetically modified microorganisms of any one of claims 1-6, further comprising an overexpressed enzyme selected from the group consisting of:(i) an acylating formaldehyde dehydrogenase converting formaldehyde to formyl-CoA;(ii) a formate kinase converting formate to formyl -phosphate and a phosphate formyltransferase converting formyl-phosphate to formyl-CoA;(iii) an acyl-CoA transferase converting formate to formyl-CoA; and(iv) an acyl-CoA synthase converting formate to formyl-CoA.

8. The genetically modified microorganisms of any one of claims 1-7, capable of converting 3- hydroxypropionaldehyde and a Cl substrate selected from methane, methanol, formaldehyde or formate to a product selected from 2,4-dihydroxybutyric acid, hydroxybutyrolactone, desmeninol, 2-keto-4-(methylthio)butyric acid, L-methionine or D-methionine.

9. The genetically modified microorganisms of any one of claims 1-8, wherein the microorganisms are selected from the group consisting of bacteria, yeast and fungi.

10. The genetically modified microorganisms of any one of claims 1-9, wherein the microorganisms are Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebacterium sp., Zymonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccharomyces sp., Pichia sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyc.es sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. Trichoderma sp..

11. The genetically modified microorganisms of any one of claims 1-10, wherein the microorganisms are derivates of Escherichia coli.

12. The genetically modified microorganisms of claim 11, wherein the microorganisms are derivates of MG1655(DE3).

13. The genetically modified microorganisms of claim 12, wherein the microorganisms are derivates of MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD).

14. The genetically modified microorganisms of claim 13, wherein the microorganisms have thioesterases (yciA, tesA, tesB, ybgC, ydil and / or fadM) knockout.

15. The genetically modified microorganisms of claim 14, wherein the microorganisms have FucO and / or ackA-Pta deactivated.

16. The genetically modified microorganisms of claim 11, wherein the microorganisms are derivates of BW25113(DE3).

17. The genetically modified microorganisms of claim 16, wherein the microorganisms are derivates of BW25113(DE3) del (frmA, fdhF, fdnG, fdoG, glcD).

18. The genetically modified microorganisms of claim 17, wherein the microorganisms have dehydratase from Klebsiella peneumoniae. DhcHACS and / or CaAbfT overexpressed.

19. The genetically modified microorganisms of claim 11, wherein the microorganisms are derivates of BL21(DE3).

20. The genetically modified microorganisms of claim 19, wherein the microorganisms have UtCAT and / or AOX2 overexpressed.

21. The genetically modified microorganisms of any one of claims 1 -20, wherein the microorganisms have aldB knockout.

22. The genetically modified microorganisms of claim 21, wherein the microorganisms further have aldA knockout.

23. A method for the production of 2,4-dihydroxybutyryl-CoA comprising contacting formyl- CoA and 3-hydroxypropionaldehyde with a TPP-dependent enzyme selected from 2- hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

24. A method for the production of 2,4-dihydroxybutyric acid comprising:(a) providing 2,4-dihydroxybutyryl-CoA obtained by the method of claim 23; and(b) converting 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid by contacting 2,4- dihydroxybutyryl-CoA with any one of (i) and (iii):(i) a thioesterase or an acyl-CoA transferase catalyzing the conversion of 2,4- dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid;(ii) a phosphotransacylase catalyzing the conversion of 2,4-dihydroxybutyryl-CoA to2,4-dihydroxybutyryl-phosphate and a carboxylate kinase catalyzing the conversion of2.4-dihydroxybutyryl-phosphate to 2,4-dihydroxybutyric acid; or(iii) an acyl-CoA reductase catalyzing the conversion of 2,4-dihydroxybutyryl-CoA to2.4-dihydroxybutanal and an aldehyde dehydrogenase catalyzing the conversion of 4- (methylthio)-2-hydroxy-butanal to 2,4-dihydroxybutyric acid.

25. A method for the production of hydroxybutyrolactone comprising:(a) providing 2,4-dihydroxybutyryl-CoA obtained by the method of claim 23; and(b) lactonization of 2,4-dihydroxybutyryl-CoA to hydroxybutyrolactone spontaneously; or(c) providing 2,4-dihydroxybutyric acid obtained by the method of claim 24; and(d) adjusting pH to lower than 6 driving the lactonization of 2,4-dihydroxybutyric acid to hydroxybutyrolactone.

26. A method for the production of 2-ketobutyrolactone comprising:(a) providing hydroxybutyrolactone obtained by the method of claim 25; and(b) converting hydroxybutyrolactone to 2-ketobutyrolactone by contacting hydroxybutyrolactone with a 2-hydroxy acid dehydrogenase.

27. A method for the production of 2-hydroxy-4-(methylthio)butyric acid (desmeninol) comprising:(a) providing hydroxybutyrolactone obtained by the method of claim 25; and(b) converting hydroxybutyrolactone to desmeninol by condensation of hydroxybutyrolactone with methanethiol.

28. A method for the production of 2-keto-4-(methylthio)butyric acid comprising:(al) providing 2-hydroxy-4-(methylthio)butyric acid (desmeninol) obtained by the method of claim 25; and(bl) converting 2-hydroxy-4-(methylthio)butyric acid to 2-keto-4-(methylthio)butyric acid by contacting 2-hydroxy-4-(methylthio)butyric acid with a 2-hydroxy acid dehydrogenase; or(a2) providing 2-ketobutyrolactone obtained by the method of claim 26; and(b2) converting 2-ketobutyrolactone to 2-keto-4-(methylthio)butyric acid by condensation of 2-ketobutyrolactone with methanethiol.

29. A method for the production of L-methionine or D-methionine comprising:(a) providing 2-keto-4-(methylthio)butyric acid obtained by the method of claim 28; and(b) converting 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine by contacting 2-keto-4-(methylthio)butyric acid with an enzyme selected from an amino acid dehydrogenase or a methionine aminotransferase.

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