Production of methionine by condensation of C1-derived formyl-coA with 3-hydroxypropionaldehyde derived from multiple substrates

Abstract

The present invention provides a method for producing liquid methionine or methionine, and particularly provides a method for producing liquid methionine or methionine by condensation reaction of 3-hydroxypropionaldehyde and formyl-CoA derived from a C1 substrate. The present invention also provides a genetically engineered microorganism for the production of liquid methionine or methionine.

Description

Technical Field

[0001] This invention generally relates to a method for producing methionine, and more particularly to a method for producing methionine by condensation of C1-derived formyl-CoA with 3-hydroxypropionaldehyde derived from various substrates. This invention also relates to genetically modified microorganisms for producing methionine or liquid methionine. Invention Overview The present invention provides the following exemplary embodiments.

[0003] Implementation Method 1. A method for producing 2,4-dihydroxy-butyryl-CoA, comprising contacting formyl-CoA with 3-hydroxypropionaldehyde in the presence of a TPP-dependent enzyme (selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxaloyl-CoA decarboxylase or benzaldehyde lyase).

[0004] Implementation Method 2. According to the method of Implementation Method 1, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0005] Implementation Method 3. According to the method of Implementation Method 2, the TPP-dependent enzyme is selected from the following table: Implementation Method 4. The method according to Implementation Method 1 further includes generating formyl-CoA by contacting a one-carbon (C1) substrate selected from formaldehyde or formic acid with an enzyme.

[0006] Implementation Method 5. According to the method of Implementation Method 4, wherein the C1 substrate is formaldehyde and the enzyme is acyl-CoA reductase.

[0007] Implementation Method 6. According to the method of Implementation Method 4, wherein the C1 substrate is formic acid, and the enzyme is selected from: (i) Acyl-CoA transferase that catalyzes the conversion of formic acid to formyl-CoA; (ii) Acyl-CoA synthase that catalyzes the conversion of formic acid to formyl-CoA; or (iii) Formate kinase that catalyzes the conversion of formic acid to formyl phosphate and a phosphoformyltransferase that catalyzes the conversion of formyl phosphate to formyl-CoA.

[0008] Implementation Method 7. According to the method of Implementation Method 5, wherein the acyl-CoA reductase is selected from: Implementation Method 8. According to the method of Implementation Method 6, wherein the formate kinase and phosphoformyltransferase pair is selected from the following combinations: Implementation Method 9. According to the method of Implementation Method 6, wherein the acyl-CoA transferase is selected from: Implementation Method 10. According to the method of Implementation Method 6, wherein the acyl-CoA synthase is selected from: Implementation Method 11. The method according to Implementation Method 5 further includes generating formaldehyde by contacting methanol with methanol dehydrogenase or methanol oxidase.

[0009] Embodiment 12. According to the method of Embodiment 11, wherein the methanol dehydrogenase or methanol oxidase is selected from: Implementation Method 13. The method according to Implementation Method 11 or 12 further includes generating methanol by contacting methane with methane monooxygenase.

[0010] Embodiment 14. According to the method of Embodiment 13, wherein the methane monooxygenase is selected from: Embodiment 15. The method according to any one of Embodiments 1 to 14 further includes generating 3-hydroxypropanal from glycerol.

[0011] Embodiment 16. The method according to any one of Embodiments 1 to 14 further includes generating 3-hydroxypropanal from ethylene glycol and a one-carbon compound.

[0012] Implementation Method 17. The method according to Implementation Method 16 further includes generating glycolaldehyde by contacting ethylene glycol with an alcohol dehydrogenase.

[0013] Embodiment 18. The method according to Embodiment 17, wherein the alcohol dehydrogenase is selected from: Implementation Method 19. The method according to Implementation Methods 16 to 18 further includes condensing ethanolaldehyde and formyl-CoA to generate glyceryl-CoA using 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0014] Implementation 20. The method according to any one of Implementations 16 to 19 further includes generating glyceraldehyde from glyceryl-CoA using acyl-CoA reductase.

[0015] Implementation Method 21. The method according to any one of Implementation Methods 16 to 20 further includes using an alcohol dehydrogenase to generate glycerol from glyceraldehyde.

[0016] Embodiment 22. The method according to Embodiment 21, wherein the alcohol dehydrogenase is selected from: Implementation Method 23. The method according to any one of Implementation Methods 1 to 16 further includes generating 3-hydroxypropanal by means of a one-carbon compound selected from, but not limited to, formic acid, formaldehyde, or methanol.

[0017] Implementation Method 24. The method according to any one of Implementation Methods 1 to 23 further includes generating formyl-CoA through the interconversion of one-carbon compounds.

[0018] Implementation Method 24. The method according to any one of Implementation Methods 1 to 24 further includes generating formaldehyde through the interconversion of one-carbon compounds.

[0019] Implementation Method 26. The method according to any one of Implementation Methods 1 to 25 further includes using a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxaloyl-CoA decarboxylase or benzaldehyde lyase to condense formaldehyde and formyl-CoA to generate glycolyl-CoA.

[0020] Implementation Method 27. The method according to any one of Implementation Methods 1 to 26 further includes using acyl-CoA reductase to generate glycolyl-CoA from glycolaldehyde.

[0021] Implementation 28. A method for producing 3-hydroxypropanal, comprising generating 3-hydroxypropanal by contacting glycerol with a glycerol dehydrating enzyme.

[0022] Embodiment 29. The method according to Embodiment 28, wherein the glycerol dehydrating enzyme is selected from: Implementation Method 30. A method for producing 2,4-dihydroxybutyric acid, comprising: (a) Providing 2,4-dihydroxybutyryl coenzyme A according to any one of embodiments 1 to 27, and (b) Converting 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid by contacting it with either of the following (i) and (iii): (i) Thioesterases or acyl-CoA transferases that catalyze the conversion of 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyrate; (ii) Phosphotransferases that catalyze the conversion of 2,4-dihydroxybutyryl coenzyme A to 2,4-dihydroxybutyryl phosphate, and carboxylic acid kinases that promote the conversion of 2,4-dihydroxybutyryl phosphate to 2,4-dihydroxybutyrate; or (iii) Acyl-CoA reductase that catalyzes the conversion of 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyraldehyde, and aldehyde dehydrogenase that promotes the conversion of 2,4-dihydroxybutyraldehyde to 2,4-dihydroxybutyric acid.

[0023] Embodiment 31. The method according to any one of Embodiments 20, 27 or 30, wherein the acyl-CoA reductase is selected from: Embodiment 32. The method according to Embodiment 30, wherein the aldehyde dehydrogenase is selected from: Example 33. A method for producing 2-keto-4-hydroxybutyric acid, comprising: (a) providing 2,4-dihydroxybutyric acid by any one of Examples 30 to 32; and (b) converting 2,4-dihydroxybutyric acid into 2-keto-4-hydroxybutyric acid by contacting it with a 2-hydroxy acid dehydrogenase / oxidase.

[0024] Embodiment 34. According to the method of Embodiment 33, wherein the 2-hydroxy acid dehydrogenase / oxidase is selected from: Example 35. A method for producing 2-keto-4-methylthiobutyric acid, comprising: (a) providing 2-keto-4-hydroxybutyric acid according to the method of Example 33 or 34; and (b) converting 2-keto-4-hydroxybutyric acid into 2-keto-4-methylthiobutyric acid by chemically condensing it with methanethiol.

[0025] Implementation Method 36. A method for producing L-methionine or D-methionine, comprising: (a) 2-keto-4-methylthiobutyric acid is provided by the method of embodiment 35; and (b) 2-keto-4-methylthiobutyric acid is converted into L-methionine or D-methionine by contacting it with an enzyme selected from amino acid dehydrogenases or methionine / homoserine aminotransferases.

[0026] Embodiment 37. According to the method of Embodiment 36, wherein the amino acid dehydrogenase is selected from: Embodiment 38. The method according to Embodiment 36, wherein the methionine / homoserine aminotransferase is selected from: Implementation Method 39. The method according to any one of Implementation Methods 1 to 38, wherein the enzyme used is isolated from microorganisms.

[0027] Implementation 40. The method according to any one of Implementation 1 to 38, wherein the enzyme used is contained in microorganisms.

[0028] Implementation Method 41. A genetically modified microorganism that provides 2,4-dihydroxybutyryl coenzyme A by any one of Implementation Methods 1 to 26.

[0029] Implementation 42. The microorganism of Implementation 41 is further provided with 2,4-dihydroxybutyric acid by any one of the methods of Implementation 30 to 32.

[0030] Implementation Method 43. The microorganism of Implementation Method 41 further provides 2-keto-4-methylthiobutyric acid by the method of Implementation Method 35 or 36.

[0031] Implementation 44. The microorganism of Implementation 41 further provides L-methionine or D-methionine by any one of Implementations 36 to 38.

[0032] Implementation method 45. The microorganism according to any one of implementation methods 41 to 44, wherein the microorganism is selected from bacteria, yeast or fungi.

[0033] Implementation Method 46. The microorganism according to any one of Implementation Methods 41 to 45, wherein the microorganism is a bacterium, yeast or fungus, including but not limited to Escherichia coli, Bacillus, Pseudomonas, Corynebacterium, Fermentosum, Clostridium, Streptococcus, Rhodococcus, Bacillus thermophilus, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia, Rhodotorula methylbacterium, Candida, Kluyveromyces, Aspergillus, Penicillium, Rhizopus, or Trichoderma.

[0034] The following examples provide further details, any of which can be combined with other details for a patent application. The entirety of this specification should be considered as providing various details that are interchangeable with other details.

[0035] The content of this invention is illustrated by the following non-limiting examples.

[0036] It should be understood that the foregoing overview and the following detailed description are merely examples and explanations, and are not intended to limit the invention. Furthermore, the accompanying drawings, which form part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. Attached Figure Description

[0037] The accompanying drawings referenced in this specification form part of this specification. The features shown in the drawings are merely illustrative of some embodiments of this application, and not all embodiments of this application. Unless the contrary is expressly indicated in the detailed description, the reader of this specification should not infer the opposite.

[0038] Figure 1 This diagram illustrates the overall process of producing methionine via the condensation reaction of 3-hydroxypropionaldehyde with formyl-CoA derived from a C1 substrate. In this diagram, MMO represents methane monooxygenase; MDH represents methanol dehydrogenase; FOK represents formate kinase; PTA represents phosphoformyltransferase; ACS represents acyl-CoA synthase; ACT represents acyl-CoA transferase; ACR represents acyl-CoA reductase; HACS represents 2-hydroxyacyl-CoA synthase; TE represents thioesterase; ALDH represents aldehyde dehydrogenase; 2HADH represents 2-hydroxy acid dehydrogenase; AADH represents amino acid dehydrogenase; MAT represents methionine transaminase; and HAT represents homoserine transaminase.

[0039] Figure 2 This diagram illustrates the route for producing 3-hydroxypropional from glycerol and from ethylene glycol and a one-carbon compound via the FORCE pathway. Wherein, MMO represents methane monooxygenase; MDH represents methanol dehydrogenase; FOK represents formate kinase; PTA represents phosphoformyltransferase; ACS represents acyl-CoA synthase; ACT represents acyl-CoA transferase; ACR represents acyl-CoA reductase; HACS represents 2-hydroxyacyl-CoA synthase; TE represents thioesterase; GDHt represents glycerol dehydratase; and ADH represents alcohol dehydrogenase.

[0040] Figure 3 The pathways for the interconversion of one-carbon compounds are shown. a) Formyl-CoA is produced from one-carbon compounds; b) Formic acid is produced by GSH and MSH-dependent oxidation of formaldehyde; c) Formic acid is produced by H4F and H4MPT (tetrahydromethylenepterin)-dependent oxidation of formaldehyde. Wherein, MMO stands for methane monooxygenase; MDH stands for methanol dehydrogenase; AOD stands for alcohol oxidase; FOK stands for formate kinase; PTA stands for formylphosphotransferase; ACS stands for acyl-CoA synthase; ACT stands for acyl-CoA transferase; ACR stands for acyl-CoA reductase; HACS stands for 2-hydroxyacyl-CoA synthase; TE stands for thioesterase; FLD stands for formaldehyde dehydrogenase; FGH stands for formyl-GS hydrolase; FHC stands for formyltransferase / hydrolase; MTDA stands for methylenetetrahydromethylenepterin dehydrogenase; FCH stands for methylenetetrahydrofolate cyclase; FTFL stands for formatetetrahydrofolate ligase; FAE stands for formaldehyde activator; MCH stands for methylenetetrahydromethylenepterin cyclase. Figure 4 The results of the in vitro detection of 2,4-dihydroxybutyric acid from 3-hydroxypropionaldehyde using purified enzymes are shown.

[0041] Figure 5 The results of the in vivo reaction to generate 2,4-dihydroxybutyric acid from 3-hydroxypropanal are shown.

[0042] Figure 6 The results of the in vivo reaction to generate 2,4-dihydroxybutyric acid from 3-hydroxypropanal are shown.

[0043] Figure 7 The effect of aldehyde dehydrogenase / alcohol dehydrogenase on the consumption of 3-hydroxypropionaldehyde is shown.

[0044] Figure 8 The effect of double knockout aldehyde dehydrogenase on the consumption of 3-hydroxypropionaldehyde is shown.

[0045] Figure 9 The pathway and detection results for the generation of glycerol from formaldehyde are shown. A) Schematic diagram of the generation of glycerol from formaldehyde; B) Generation of glyceric acid from formaldehyde in engineered strains; C) Generation of glycerol from formaldehyde in engineered strains.

[0046] Figure 10 The results of the product detection for the formation of 2,4-dihydroxybutyric acid from glycerol and formic acid are shown.

[0047] Figure 11 The results of identification of liquid methionine chemically synthesized from 2,4-dihydroxybutyric acid are shown.

[0048] Figure 12 The results of screening for 2-hydroxy acid oxidases for converting liquid methionine to 4-methylthio-2-oxobutyric acid are shown.

[0049] Figure 13 The results show the detection results of liquid methionine being converted to methionine by co-expression of AOX and Spo in the strain. ST-MET represents methionine standard, and MET represents methionine.

[0050] In the accompanying drawings, the same reference numerals are used to denote the same or similar parts. Detailed Implementation

[0051] The following detailed description of exemplary embodiments of this application refers to the accompanying drawings, which form a part of this specification. The drawings illustrate specific exemplary embodiments that can be used to implement this application. This specification (including the drawings) has described these embodiments in sufficient detail to enable those skilled in the art to implement this application. Other embodiments of this application may also be employed by those skilled in the art, and logical, mechanical, or other modifications may be made without departing from the spirit or scope of this application. Therefore, the reader of this application should not interpret the following detailed description in a restrictive manner, and the scope of this application is defined only by the appended claims.

[0052] In this application, unless otherwise expressly stated, the singular form includes the plural form. In this application, "or" should be understood as "and / or" unless otherwise indicated. Furthermore, "including" and its variations such as "comprising" and "including" are not limiting. Additionally, the section headings in this specification are for organizational purposes only and should not be construed as limiting the subject matter described. Specific Implementation Example 1: Overview of the production of methionine via the condensation of 3-hydroxypropionaldehyde and formyl-CoA This embodiment demonstrates an implementation method for condensing 3-hydroxypropionaldehyde with formyl-CoA and further derivatizing it into methionine using the C1+ biological platform (see below). Figure 1 This condensation reaction can be performed by HACS (as listed in Table 1, but not limited to), including but not limited to BsmHACS derived from beach sand metagenomics (GenBank accession number: HAK63664.1), derived from dehalogenated cocci ( Dehalococcoidia bacterium The data is from DhcHACS (GenBank accession number: PWB41796.1), sourced from... Chloroflexi bacterium CfhHACS (GenBank accession number: PKN81274.1), etc., catalyze the formation of 2,4-dihydroxybutyryl-CoA. Subsequently, 2,4-dihydroxybutyryl-CoA spontaneously cyclizes to form 2-hydroxybutyrolactone. Alternatively, 2,4-dihydroxybutyryl-CoA may be generated by thioesterases [b, for example, from Escherichia coli or Pseudomonas]. Pseudomonas putida TesB (McMahon, MD and Prather, KLJ Appl. Environ. Microbiol. 80:1042-1050, 2014), coenzyme A transferase (ACT, such as those listed in Table 5 below), or phosphoacyltransferase-acyl kinase (PTA-ACK, such as those listed in Table 5 below) is further converted to 2,4-dihydroxybutyrate. Alternatively, 2,4-dihydroxybutyryl coenzyme A may be derived from... Salmonella typhimuriumAcyl-CoA reductase StEutE (GenBank accession number: P41793) or other acyl-CoA reductases (ACRs) (e.g., ACRs listed in Table 2) catalyze the reduction to 2,4-dihydroxybutyraldehyde. 2,4-Dihydroxybutyraldehyde can then be oxidized to 2,4-dihydroxybutyrate by a suitable aldehyde dehydrogenase (ALDH, as shown in Table 3 below). 2,4-Dihydroxybutyrate is further lactone-esterified to 2-hydroxybutyrolactone by adjusting the pH below 6.

[0054] 2-Hydroxybutyrolactone can chemically condense with methanethiol (CH3SH) to form liquid methionine (2-hydroxy-4-methylthiobutyric acid).

[0055] As described above, liquid methionine is further converted to 4-methylthio-2-oxobutyric acid (or 4-methylthio-2-ketobutyric acid) by 2-hydroxy acid dehydrogenase (2HADH or 2HDH). Alternatively, 2-hydroxybutyrolactone is converted to 2-ketobutyrolactone by 2HDH, and 2-ketobutyrolactone can be chemically converted to 4-methylthio-2-oxobutyric acid. 4-Methylthio-2-oxobutyric acid is converted to L-methionine (as listed in Table 3) by amino acid dehydrogenase (AADH) (Nitta et al., J. Bacteriol. 117:588-592, 1974) or methionine aminotransferase (MAT) (Venos et al., BMC Microbiol. 2004;4:39) (as listed in Table 3). Figure 1 ).

[0056] Simultaneously, 2-hydroxybutyrolactone is converted to 2-ketobutyrolactone under the action of 2HADH / 2HDH. 2-Ketobutyrolactone is further catalyzed by amino acid dehydrogenases (AADH, Nitta et a., J Bacteriol. 1974; 117(2): 588–592) or homoserine transaminases (HAT, Venos et al., BMC Microbiol, 2004; 4, 39) to generate homoserine lactone. Homoserine lactone can be chemically converted to methionine by reacting with methanethiol. Homoserine lactone can also be converted to homoserine by raising the pH to above 8. Figure 1 ) Alternatively, 2,4-dihydroxybutyric acid is first converted to 4-hydroxy-2-oxobutyric acid under the action of 2HADH. Further addition of an amino group to 4-hydroxy-2-oxobutyric acid yields homoserine. Homoserine can be chemically converted to methionine (…). Figure 1 ).

[0057] Example 2: Preparation of 3-hydroxypropanal from glycerol, ethylene glycol and a one-carbon compound This embodiment demonstrates the generation of 3-hydroxypropanal from various substrates, including but not limited to glycerol (C3), ethylene glycol (C2), and methanol (C1). Once generated, 3-hydroxypropanal can further condense with formyl-CoA derived from a one-carbon compound to generate 2,4-dihydroxybutyryl-CoA, and as... Figure 1 As shown, it is ultimately converted into methionine.

[0058] When 3-hydroxypropanal is generated from glycerol, overexpression of glycerol dehydratase (GDHt) is required to convert glycerol to 3-hydroxypropanal. Figure 2 ).

[0059] When 3-hydroxypropanal is generated from ethylene glycol, two rounds of condensation are required for methionine synthesis. Figure 2 Ethylene glycol is first converted to glycolaldehyde by Escherichia coli FucO. Glycoaldehyde can then condense with formyl-CoA to glyceryl-CoA under HACS catalysis, wherein the HACS includes components derived from... Chloroflexi bacterium The CfhHACS (GenBank accession number: PKN81274.1) data is from... Rhodocyclaceae bacterium The RhbHACS (GenBank accession number: MBL8483477.1) data is from... Pseudonocardia sediminis PdsHACS (GenBank accession number: WP_130292058.1), or other HACS variants (e.g., the HACS listed in Table 1). Glyceryl-CoA can be reduced to glyceraldehyde by acyl-CoA reductase ACR, wherein the ACR comprises derived from PdsHACS (GenBank accession number: WP_130292058.1), or other HACS variants (e.g., the HACS listed in Table 1). Salmonella typhimurium StEutE (GenBank accession number: P41793) or other ACR variants (e.g., Table 2). Glyceraldehyde can be derived from E. coli via YqhD, YahK, or from... Trichoderma reesei Glyceraldehyde is further converted to glycerol by glyceraldehyde reductases such as GLD1 (ADH, enzymes listed in Table 3). As mentioned above, glycerol can be converted to 3-hydroxypropionaldehyde by glycerol dehydrating enzymes, said glycerol dehydrating enzymes including those derived from... Klebsiella pneumonia DhaB123 from other microorganisms (such as GDHt listed in Table 3) is then further converted into methionine via the pathway described in Example 1.

[0060] When 3-hydroxypropanal is generated from methanol or other one-carbon compounds, an additional condensation round is required to synthesize glycolal, which is then used for methionine synthesis, compared to using ethylene glycol as a co-substrate. Figure 2 Methanol or other one-carbon compounds are first converted into formaldehyde and formyl-CoA through internal interconversion within the one-carbon compound. Figure 3 Formaldehyde and formyl-CoA condense under the action of HACS to form glycolyl-CoA, wherein the HACS includes components derived from... Chloroflexi bacterium The CfhHACS (GenBank accession number: PKN81274.1) data is from... Rhodocyclaceae bacterium The RhbHACS (GenBank accession number: MBL8483477.1) data is from... Pseudonocardia sediminis PdsHACS (GenBank accession number: WP_130292058.1), or other HACS variants (e.g., the HACS listed in Table 1). Glycol-CoA is further converted to glycolaldehyde by ACR, wherein the ACR includes those derived from... Salmonella typhimurium StEutE (GenBank accession number: P41793) or other ACR variants (e.g., Table 2). Under the action of the aforementioned HACS variants (HACS listed in Table 1), glycolaldehyde can act as a substrate for the next round of condensation with formyl-CoA to generate glyceryl-CoA. Glyceryl-CoA is further converted to glyceraldehyde under the action of ACRs, wherein the ACRs include those derived from... Salmonella typhimurium StEutE (GenBank accession number: P41793) or other ACR variants (e.g., Table 2). Glyceraldehyde can be derived from E. coli via YqhD, YahK, or from... Trichoderma reesei Glyceraldehyde is further converted to glycerol by glyceraldehyde reductases such as GLD1 (ADH, enzymes listed in Table 3). As mentioned above, glycerol can be converted to 3-hydroxypropionaldehyde by glycerol dehydrating enzymes, said glycerol dehydrating enzymes including those derived from... Klebsiella pneumonia DhaB123 from other microorganisms (such as GDHt listed in Table 3) is then further converted into methionine via the pathway described in Example 1.

[0061] Example 3: Interconversion of one-carbon compounds to formyl-CoA Formyl-CoA can be obtained through the interconversion of one-carbon compounds (such as methane, methanol, formaldehyde, and formic acid) (see...). Figure 3 a) These transformations are catalyzed by appropriate enzymes (as listed in Table 4). Methane is widely derived from natural gas, landfills, and agricultural activities. The bio-oxidation of methane to methanol is catalyzed by methane monooxygenase. Methane monooxygenase has been successfully expressed in *E. coli* host. Methylococcus capsulatus Soluble methane monooxygenase (sMMO) (bioRxiv2021.08.05.455234). Subsequently, methanol was converted to NAD+. + It depends on methanol dehydrogenase (MDH) to oxidize to formaldehyde, from Bacillus methanolicus MGA3 (BmMDH) Bacillus stearothermophilus (BsMDH) (Metab.Eng. 39:49-59, 2017) and Cupriavidus necator(CnMDH) (Appl. Microbiol. Biotechnol. 100:4969-4983, 2016), PQQ (pyrroloquinoline quinone)-dependent MDH (MxaFI or XoxF), or oxygen-dependent alcohol oxidase (AOD). When using AOD, catalase (CTA) is required to detoxify hydrogen peroxide (e.g., enzymes listed in Table 4 below).

[0062] Formaldehyde can be directly oxidized to formic acid via four cofactor-dependent oxidation pathways, involving glutathione (GSH), tetrahydrofolate (H4F, THF), tetrahydromethopterin (H4MPT), or thiocyanate (MSH). Formaldehyde spontaneously reacts with glutathione (GSH) to generate S-hydroxymethyl-GS. Subsequently, S-hydroxymethyl-GS is converted to S-formyl-GS under the catalysis of hydroxymethyl-GS dehydrogenase (FrmA). S-formyl-GS is further converted to formic acid by formyl-GS hydrolase (FrmB). Formaldehyde can also spontaneously react with thiocyanate to generate S-hydroxymethylthiocyanate, in the same process as the GSH-dependent pathway, and is subsequently converted to S-formylthiocyanate by MD-FalDH. The product is further hydrolyzed by hydrolytic enzymes to generate formic acid and MSH. Figure 3 b). When H4MPT is used as a cofactor, formaldehyde spontaneously condenses with H4MPT or under the action of formaldehyde activator (FAE) to form methylene-H4MPT; subsequently, methylene-H4MPT is converted to methylene-H4MPT under the action of methylene-tetrahydromethylenepterin dehydrogenase MtdA or MtdB. Methylene-H4MPT is converted to formyl-H4MPT under the action of methylene-H4MPT cyclohydrolase (MCH), and then to formic acid under the action of formyltransferase / hydrolase FHC. Formaldehyde can also spontaneously react with H4F to form methylene-H4F, which is then converted to methylene-H4F upon contact with MtdA, and then to formyl-H4F under the action of methylene-H4F cyclohydrolase. Formyl-H4F can be further converted to formic acid under the action of formic acid-H4F ligase FTFL. Figure 3 c).

[0063] Alternatively, formaldehyde oxidation to formyl-CoA can be catalyzed by various acylated aldehyde dehydrogenase candidate enzymes (Nat. Chem. Biol. 15:900-906, 2019). Formyl-CoA can serve as a substrate for C1 elongation and can also be further converted into CO2 for energy generation. The hydrolysis of formyl-CoA to release formic acid can be catalyzed by two types of enzymes: acyl-CoA transferases (ACT, as listed in Table 5) can catalyze the reversible CoA transfer between various CoA donors (e.g., 4-methylthio-2-hydroxybutyryl-CoA, 2,4-dihydroxybutyryl-CoA, acetyl-CoA, or succinyl-CoA) and formic acid; phosphoryl-transferase-formate kinase (PTA-FOK, as listed in Table 6) can catalyze the reversible phosphorylation of formyl-CoA to formyl phosphate, which is then dephosphorylated to formic acid, generating 1 ATP. Although these reactions are reversible, AMP-formative acyl-CoA synthetase is more conducive to driving formic acid activation to formyl-CoA. Figure 3 a) Example 4: Overview of Gene Manipulation Methods Genes intended for overexpression are cloned into suitable vectors or inserted into chromosomes along with strong synthetic constitutive promoters (e.g., M1-93). If cloned into a vector, these genes are amplified by PCR, with homologous sequences added to both ends using appropriate primers for recombination with the vector backbone. PCR is performed using Phusion polymerase (ThermoScientific, Waltham, MA), and the resulting product serves as the gene insert. Plasmids are linearized with appropriate restriction endonucleases (New England Biolabs, Ipswich, MA, USA) and recombinated with the gene fragment using the In-Fusion HD Eco-Dry cloning system. The mixture is then transformed into Stellar competent cells. Transformants grown on LB agar plates containing the appropriate antibiotics are picked and selected for gene insertion by PCR. Plasmids from PCR-validated transformants are extracted and the gene sequence is further confirmed by DNA sequencing. The confirmed plasmids are then introduced into the host strain via electroporation.

[0064] If chromosome insertion is used, the CRISPR method is employed, and the following options are available: tesB and adhEOther sites can, of course, be used as appropriate. This 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 vector pCas expressing Cas9 and λ-red recombinase was transformed into the host strain. The resulting strain was cultured at 30°C, and L-arabinose was added to induce λ-red recombinase expression. When the OD reached approximately 0.6, competent cells were prepared and transformed to express sgRNA, an N20 spacer targeting this site, and pTargetF (AddGene 62226) for inserting into the template. The template consisted of the gene to be inserted and the M1-93 promoter, along with approximately 500 bp sequences homologous to the upstream and downstream of the target site. It was constructed using Phusion polymerase via overlap PCR, or synthesized by GenScript (Piscataway, NJ) or GeneArt® (Life Technologies, Carlsbad, CA). The N20 spacer replacement method for the pTargetF vector involved reverse PCR using primers with the modified N20 sequence at the 5' end and Phusion polymerase, followed by self-ligation using T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Transformants grown at 30°C on LB agar plates containing spectinomycin and kanamycin (or other suitable antibiotics) were picked and screened for chromosomal inserts by PCR. The insert was amplified from genomic DNA using Phusion polymerase and further confirmed by DNA sequencing. pTargetF can then be removed by IPTG induction, while pCas can be eliminated by culturing at higher temperatures (37-42°C).

[0065] All molecular biology techniques were performed according to standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) or as indicated by the kit instructions. The strains were stored in glycerol tubes at -80°C. LB plates contained 1.5% agar and were supplemented with appropriate antibiotics: ampicillin (100 μg / mL), kanamycin (50 μg / mL), spectinomycin (50 μg / mL), and chloramphenicol (12.5 μg / mL).

[0066] Expression of the selected enzyme variants was achieved by cloning the target gene into pETDuet-1 or pCDFDuet-1 vectors digested with the corresponding restriction enzymes (Novagen, Darmstadt, Germany) using In-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, CA). The linear DNA fragment used for insertion was obtained by PCR of the target open reading frame (if the gene is an endogenous E. coli gene) or by gene synthesis (GeneArt, Life Technologies, Carlsbad, CA). The resulting In-Fusion reaction product was transformed into E. coli Stellar competent cells, and positive clones were further confirmed by DNA sequencing after PCR screening. As hosts for these vectors, MG1655(DE3), BW25113, or ATCC8739-based vectors with formaldehyde oxidation knockout (...) could be used. ∆frmA ), formic acid oxidation ( ∆fdhF ∆fdnG ∆fdoG ) and aldehyde consumption pathways (including adhP, yjgB, yqhD, adhE We knocked out these genes in modified E. coli (e.g., *E. coli*) because we hypothesized that these pathways would compete with or interfere with our target pathway analysis.

[0067] Example 5: Fermentation conditions for in vivo methionine production Fermentation was performed using MOPS basal medium (Neidhardt et al. J. Bacteriol. 119:736-47 (1974)), containing 125 mM MOPS, with Na2HPO4 replacing K2HPO4 (2.8 mM), and supplemented with 20 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract, 100 μM FeSO4, 5 mM (NH4)2SO4, and 30 mM NH4Cl. 55 g / L CaCO3 was added as a pH buffer if necessary. If intracellular lactate synthesis was insufficient and required for the experiment, 20 mM lactate was added. 500 mM methanol was also added, and antibiotics (50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin) were added as needed. All chemical reagents were purchased from Fisher Scientific (Pittsburg, PA) and Sigma-Aldrich (St. Louis, MO).

[0068] Fermentation was carried out in 25 mL Pyrex conical flasks (narrow-mouth / thickened, Corning Inc., Corning, NY) or 96-well plates (2.2 mL, V-bottom, USA Scientific) with an appropriate volume of fermentation medium added and sealed with foam stoppers. Anaerobic fermentation was carried out by completely filling 17.5 mL Hungate tubes with medium and sealing with rubber stoppers. Single clones of the target strain were inoculated into LB culture media containing antibiotics and cultured at 37°C for 14–16 hours as seed culture, with an initial OD600 of approximately 0.05. After inoculation in shake flasks, the flasks were placed in an NBS I24 shaking incubator (200 rpm, 37°C or 30°C). When the OD550 reached ~0.3–0.5, appropriate IPTG (or other inducers) was added for induction. Other fermentations were carried out in a SixFors multi-stage fermentation system (Infors HT) with air or argon purge at 2 NL / hr (standard liters / hour), and temperature (37°C), pH (7.0, adjusted by NaOH and H2SO4), and agitation rate independently controlled. Seed culture, under the same conditions, was induced in 25 mL Pyrex Erlenmeyer flasks, cultured for 4 hours, centrifuged, washed twice, and used for inoculation with an initial volume of 400 mL. Fermentation in the fermenter used the aforementioned fermentation medium containing 40 g / L glucose, supplemented with IPTG and antibiotics. Lactic acid (20 mM) was added at 0, 24, and 48 hours if necessary.

[0069] After fermentation, the supernatant was obtained by centrifugation at 5000 g for 5 minutes using a Beckman Coulter Optima L-80XP centrifuge (Optima L-80XP, Beckman-Coulter). 2 mL of the obtained supernatant was prepared for HPLC analysis.

[0070] Example 6 HPLC Analysis of Methionine Add 0.1 mL of fermentation supernatant to a 15 mL centrifuge tube, then add 0.2 mL of acetonitrile, 0.1 mL of 0.5 mM NaHCO3, and 0.1 mL of 2,4-dinitrofluorobenzene (DNFB) solution (0.2 mL DNFB diluted to 10 mL with acetonitrile). Tighten the cap and vortex vigorously for 30 seconds to ensure thorough mixing. Then derivatize in a 60°C water bath in the dark for 20 min. After cooling in an ice-water bath, stop the reaction by adding 1 mL of 0.5 M phosphate buffer, followed by 1 mL of acetonitrile and 7.5 mL of MiniQ water. Mix well, filter through a 0.22 μm nylon membrane, and transfer to an autosampler vial for HPLC analysis.

[0071] HPLC was performed using a Waters 2695 system equipped with a 996 PDA detector and a Phenomenex Luna C18 column (250 mm × 4.6 mm × 5 μm). The column temperature was maintained at 30°C, the detection wavelength was set to 360 nm, and the injection volume was 10 μL. The mobile phase consisted of MiniQ water (A) and acetonitrile (B) containing 0.1% acetic acid and 0.1% trimethylamine. Separation was achieved at a flow rate of 0.8 mL / min using the following gradient elution: 0 min, 30% acetonitrile (mobile phase B); 15 min, 85% acetonitrile (mobile phase B); 18 min, 85% acetonitrile (mobile phase B); 18.01 min, 30% acetonitrile (mobile phase B); 30 min, 30% acetonitrile (mobile phase B).

[0072] Example 7: 2,4-Dihydroxybutyric acid was generated from 3-hydroxypropionaldehyde and formyl-CoA using a purified enzyme.

[0073] This embodiment demonstrates the generation of 2,4-dihydroxybutyric acid via the condensation reaction of 3-hydroxypropionaldehyde (3-HPA) and formyl-CoA using a purified enzyme. In this embodiment, the ACK-PTA pathway is used to provide formyl-CoA for the condensation reaction.

[0074] The expression of selected enzyme variants was achieved through plasmid-mediated gene expression. Specifically, the target gene was cloned into a pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) vector digested with appropriate restriction endonucleases using In-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, California, USA). The linear DNA fragment used for insertion was obtained through PCR amplification of the target open reading frame (for genes derived from *E. coli*), or through gene synthesis after codon optimization. Gene synthesis was performed by GeneArt (LifeTechnologies, Carlsbad, California, USA). The resulting In-Fusion reaction products were used to transform *E. coli* Stellar cells (Clontech Laboratories, Inc., Mountain View, California, USA), and positive clones were identified by PCR screening, followed by further confirmation by DNA sequencing.

[0075] The expression strain was cultured overnight in LB medium and then inoculated at a rate of 1% (v / v) into 250 mL baffled Erlenmeyer flasks containing 25 mL of TB medium. The cultures were incubated at 30°C and 250 rpm in a track shaker until the OD600 reached 0.4–0.6, at which point 0.1 mM IPTG was added to induce protein expression. Twenty-four hours after inoculation, cells were collected by centrifugation. The resulting cell pellet was washed once with pre-chilled 9 g / L NaCl solution and stored at -80°C for later use. Antibiotics were added as needed, with the following final concentrations: ampicillin (100 µg / L), carbenicillin (50 µg / L), and spectinomycin (50 µg / L).

[0076] The *E. coli* cell pellet containing the His-tagged enzyme required for protein purification was prepared as described above. The frozen cell pellet was resuspended in pre-chilled lysis buffer (50 mM NaPi, pH 7.4, 300 mM NaCl, 10 mM imidazole) to an OD600 of approximately 40, and lysozyme was added to a concentration of 1 mg / mL, along with 250 U of Benzonase nuclease. The pellet was then sonicated on ice using a Branson Sonifier 250 (25% duty cycle, output control 3, 5 min) and centrifuged at 7500 × g for 15 min at 4°C. The supernatant was loaded into a column containing 1 mL of TALON metal affinity resin (Clontech Laboratories, Inc., Mountain View, California, USA) equilibrated with lysis buffer. The column was then washed with 10 mL of lysis buffer, followed by two washes with 20 mL of wash buffer (50 mM NaPi, pH 7.4, 300 mM NaCl, 20 mM imidazole). The target His-tagged protein was eluted 1-2 times with 4 mL of elution buffer (50 mM NaPi, pH 7.4, 300 mM NaCl, 250 mM imidazole). The eluent was collected and loaded into a 10,000 MWCO Amicon ultrafiltration centrifuge (Millipore, Billerica, Massachusetts, USA). The resulting concentrate (approximately 100 µL) was washed twice with 4 mL of 50 mM KPi, pH 7.4 buffer to desalt the protein. Protein concentration was estimated using the Bradford method. The purified protein was aliquoted into 20 µL tubes and stored at -80°C for later use.

[0077] SDS-PAGE electrophoresis was performed using NuPAGE 12% Bis-Tris protein gel, and staining was performed according to the SimplyBlue SafeStain manufacturer's instructions (ThermoFisher Scientific, Waltham, Massachusetts, USA).

[0078] The in vitro purification reaction of the enzyme was carried out in the following system: 50 mM HEPES (pH 7.8), 5 mM ATP, 10 mM MgCl2, 5 mM CoA, 0.2 mM TPP, 2 µM CaoHACS or CfhHACS, 1 µM PTA, 1 µM ACK, 30 mM sodium formate, and 10 mM 3-hydroxypropionaldehyde. Unless otherwise specified, the reaction was incubated at 30°C for 18 hours. The reaction was terminated by adding 1 / 20 of the reaction volume of 10 M NaOH solution. After hydrolysis for 30 minutes, the pH was neutralized by adding 1 / 20 of the reaction volume of 10 N H2SO4 solution. The mixture was then centrifuged at 20817 × g for 15 minutes, and the supernatant was analyzed by HPLC as described below.

[0079] Product and substrate concentrations were quantified using HPLC analysis on a Shimadzu Prominence SIL-20 system (Shimadzu Scientific Instruments, Inc., Columbia, Maryland, USA) equipped with a diode array detector (DAD) and a Supelco Aspera-NH2 column (5 µm × 4.6 mm × 250 mm). Peak separation was optimized under the following conditions: flow rate 1 mL / min, mobile phase 0.1% H3PO4:CAN = 70:30, and column temperature 40°C.

[0080] The results showed that both CaoHACS and CfhHACS could generate 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid) via the condensation reaction of 3-hydroxypropionaldehyde with formyl-CoA. Figure 4 ).

[0081] Example 8: Screening different HACS for the generation of 2,4-dihydroxybutyric acid This embodiment aims to screen for better HACS for the condensation of 3-hydroxypropionaldehyde and formyl-CoA to 2,4-dihydroxybutyric acid. This embodiment tested eight candidate HACS, including ApbHACS (JGI15), CdcHACS (JGI24), DhcHACS (JGI20), MeOX4, CabHACS (JGI23), CaoHACS (JGIH48), CfhHACS (JGIH65), and BbHACS (JGI19). (Source: [Original Source]) Clostridium aminobutyricum Acyl-CoA transferase CaAbfT is used to provide formyl-CoA.

[0082] To compare the ability of HACS for 2,4-dihydroxybutyrate generation in vivo, the plasmid was modified to allow for independent control over different HACS candidate genes and those derived from [specific genes]. Clostridium aminobutyricum Overexpression of the acyl-CoA transferase CaAbfT was performed, with the HACS candidate enzyme expressed on pCDFDuet-1 controlled by an IPTG-induced T7 promoter, and CaAbfT expressed on pETDuet-1 controlled by a coumarate-induced T5 promoter. Formaldehyde oxidation (…) was knocked out. Δ frmA ), formic acid oxidation ( ΔfdhF ΔfdnG ΔfdoG ) and glycolic acid utilization gene ( ΔglcD E. coli MG1655(DE3) engineered bacteria containing genes such as ) were used as host bacteria for these plasmid vectors.

[0083] Unless otherwise specified, in vivo product synthesis was performed on M9 basal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2, and 15 µM thiamine hydrochloride). Cells were initially cultured in 96-well plates containing 0.5 mL of the above medium supplemented with 20 g / L glycerol, 10 g / L tryptone, and 5 g / L yeast extract. Single clones of the desired strain were cultured overnight (14–16 hours) in LB medium containing appropriate antibiotics, followed by inoculation at a 1% inoculum. 100 μg / mL carbenicillin or 100 μg / mL spectinomycin was added as needed. Cultures were incubated at 30°C and 1000 rpm using a digital microplate shaker (shaker) until OD. 600 At approximately 0.4 h, an appropriate amount of IPTG and coumaric acid were added for induction, followed by continued culturing for a total of 24 h.

[0084] The cultured cells were then collected by centrifugation at 4000 rpm and 22°C, washed with the aforementioned basal medium (without any carbon source), and resuspended in 1 mL of the same basal medium supplemented with 10 mM 3-hydroxypropionaldehyde and 30 mM formic acid. Biotransformation was then performed by shaking in a digital microplate shaker at 30°C and 1000 rpm. After culturing at 30°C for 2 hours, the cells were centrifuged to precipitate, and the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0085] The results showed that 6 out of the 8 enzymes tested exhibited condensation activity, with DhcHACS showing higher activity than the other tested enzymes, generating 35 mg / L of 2,4-dihydroxybutyric acid (DHA). Figure 5 ).

[0086] Subsequently, in engineered strain BW25113 del ( glcD, frmA, fdhF, fdoG, fdnG, yqhD, aldB The performance of HACS in the production of 2,4-dihydroxybutyric acid was further evaluated in this study, and the strain exhibited low background consumption of 3-hydroxypropanal. Biotransformation was carried out at 30°C for 21 hours with 10 mM 3-hydroxypropanal and 20 mM formic acid added to the system, achieving a final OD of 20. The results showed that DhcHACS and CaoHACS produced the highest yields of 2,4-dihydroxybutyric acid, at 154 mg / L and 153 mg / L, respectively. CfhHACS and ApbHACS also showed good performance. Figure 6 ).

[0087] Example 9: Construction of a host strain with low background consumption of 3-hydroxypropionaldehyde The purpose of this embodiment is to reduce the background consumption of 3-hydroxypropanal in the host strain by knocking out genes associated with 3-hydroxypropanal background consumption. In BW25113 del ( frmA, fdhF, fdoG, fdnG, glcD In strain (also known as BWΔ5R), the genes related to aldehyde dehydrogenase and alcohol dehydrogenase were knocked out, respectively.

[0088] The newly constructed strain was used to perform biotransformation experiments. After culturing cells in TB medium for 24 hours, they were centrifuged at 4000×g for 10 minutes, the supernatant was discarded, and the cells were washed once with M9 medium. The cells were then resuspended in 1 mL of M9 medium containing 2 mM 3-hydroxypropionaldehyde and shaken at 30°C for 2 hours. The residual 3-hydroxypropionaldehyde content in the supernatant was determined using the previously reported 3-methyl-2-benzothiazolinone hydrazone (MBTH) method.

[0089] The results showed that knockout of aldB had the greatest impact on the consumption of 3-hydroxypropionaldehyde, followed by yiaY, yahK, and other genes. Figure 7 ).

[0090] Further in BW25113 del ( frmA, fdhF, fdoG, fdnG, glcD, aldB Seven other aldehyde dehydrogenases were inactivated in the strain, and the background consumption of 3-hydroxypropanal in the newly constructed strain was assessed using the same method. The results showed that the consumption of 3-hydroxypropanal was further reduced, among which... aldB and aldA The consumption of double knockout strains is the lowest. Figure 8 ).

[0091] Example 10: Production of glycerol from C1 compounds This embodiment uses formaldehyde as an example to demonstrate the production of glycerol using C1 compounds. Figure 9 As shown in Figure A, formaldehyde is converted to formyl-CoA by ACR, which then condenses with formaldehyde to form glycolyl-CoA, and further to glycolaldehyde. Glycoaldehyde then condenses with formyl-CoA to form glyceryl-CoA, which is subsequently converted to glyceraldehyde and finally to glycerol.

[0092] The process was validated on two strains, MJI0301 and MJI0308, both of which are MG1655(DE3) derivatives. MJI0301 is a derivative of MG1655(DE3) del( frmA, fdhF, fdnG, fdoG, glcD The thioesterase gene was knocked out in the background. yciA, tesA, tesB, ybgC, ydiI and fadM The strain was further inactivated with FucO and ackA-pta to obtain MJI0308. MJI0301 and MJI0308 carrying pCDFduet-CfhHACS and pET-PCT5-LmACR were cultured in NBS medium and induced with 75 µM IPTG and 50 µM coumaric acid, respectively. After 24 hours of culture, cells were collected, washed with M9 medium, and resuspended in M9 medium containing 5 mM formaldehyde to achieve a final OD of 20. Biotransformation was performed at 30°C for 1 hour and 3 hours.

[0093] After 3 hours, 686 µM glyceric acid accumulated in MJI0301, and 766 µM glyceric acid was generated in MJI0308. Figure 9 B). Furthermore, MJI0301 and MJI0308 produced 20 µM and 61 µM glycerol, respectively. Figure 9 C).

[0094] Example 11 Production of 2,4-dihydroxybutyric acid from glycerol and formic acid This embodiment demonstrates the process of producing 2,4-dihydroxybutyric acid from glycerol and formic acid. Glycerol is converted to 3-hydroxypropionaldehyde by glycerol dehydrating enzymes, and formic acid is converted to formyl-CoA by CaAbfT. Subsequently, the two condense to form 2,4-dihydroxybutyryl-CoA, which is further converted to 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid). Figure 1 and Figure 2 ).

[0095] This process is performed at BW25113(DE3) del ( frmA, fdhF, fdnG, fdoG, glcD Validation was performed on strains of Klebsiella pneumoniae. Klebsiella peneumoniaeThe glycerol dehydratase, DhcHACS, and CaAbfT of the cells were co-overexpressed. The biotransformation process was the same as described above. Cells were cultured in NBS medium and induced with appropriate concentrations of IPTG and coumaric acid, and biotransformed for 3 hours at a final OD of 20. The results showed that the cells could generate 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid) from glycerol and formic acid. Figure 10 ).

[0096] Example 12 Chemical conversion of 2,4-dihydroxybutyric acid to liquid methionine This example demonstrates the process of producing liquid methionine from 2,4-dihydroxybutyric acid. The pH of the 2,4-dihydroxybutyric acid solution was adjusted to 1 using 1 N H₂SO₄ to convert the 2,4-dihydroxybutyric acid into 2-hydroxybutyrolactone. The 2-hydroxybutyrolactone was then extracted with ethyl acetate and dried for subsequent experiments.

[0097] Dissolving 1 g of 2-hydroxybutyrolactone and 700 mg of potassium methanethiol in 20 mL of dimethylacetamide and heating to 160°C for 1 hour yielded the target product, liquid methionine, with a conversion rate exceeding 99%.

[0098] After cooling the reaction system to 20–30°C, the pH was adjusted to 1 with 1 N sulfuric acid, and the system was diluted with 100 mL of saturated brine. The product was then extracted twice with ethyl acetate to extract the target product into the organic phase. The combined organic phases were dried over sodium sulfate and concentrated to obtain a brown liquid. The reaction product was identified as the same substance as the standard. Figure 11 ).

[0099] Example 13: Conversion of liquid methionine to 4-(meththio)-2-oxobutyric acid via 2-hydroxy acid oxidase / dehydrogenase This embodiment demonstrates the process of generating 4-(meththio)-2-oxobutyric acid from liquid methionine. As previously mentioned, liquid methionine can be further oxidized to 4-(meththio)-2-oxobutyric acid by 2-hydroxy acid oxidase / dehydrogenase. The methionine derived from Arabidopsis thaliana (… Arabidopsis thaliana AOX1 (NP_001327205.1) and AOX2 (NP_001078152.1), HmGOX (NP_060015.1) derived from humans, and HeGOX (ABY61829.1) derived from spinach were cloned into the pCDFDuet vector for enzyme expression.

[0100] This embodiment employs resting cell biotransformation to convert liquid methionine to 4-(methylthio)-2-oxobutyrate. To overexpress 2-hydroxy acid oxidase, a plasmid containing 2-hydroxy acid oxidase was transformed into BL21(DE3), and expression was induced overnight at 25°C. Cells were collected by centrifugation, washed once with 100 mM PBS buffer (pH 7.0), and collected for later use. The collected cells were resuspended in the reaction system to initiate resting cell biotransformation. The reaction system contained 100 mM PBS (pH 7), 0.1% Triton-X100, and 20 mM liquid methionine, achieving a final OD600 of 20. Resting cell biotransformation was performed at 30°C. After 18 hours, cells were precipitated by centrifugation, and the supernatant was analyzed by HPLC.

[0101] The results of resting cell biotransformation showed that all tested 2-hydroxy acid oxidases exhibited catalytic activity towards liquid methionine. Among them, AOX2 showed the best activity under the tested conditions, with a conversion rate as high as 85%, followed by AOX1, with a conversion rate of approximately 50%. Figure 12 ).

[0102] Example 14: Converting liquid methionine to methionine via 2-hydroxy acid oxidase and transaminase This embodiment demonstrates the process of generating methionine from liquid methionine. As previously mentioned, liquid methionine can first be oxidized to 4-(methylthio)-2-oxobutyric acid by 2-hydroxy acid oxidase / dehydrogenase, and then this intermediate is reduced to methionine by aminodehydrogenase / transaminase. Since 2-hydroxy acid oxidase produces hydrogen peroxide during the catalytic process, the hydrogen peroxide derived from... Ureibacillus thermosphaericus The catalase UtCAT (WP_016837596.1) and AOX2 were co-expressed in the vector pCDFDuet-AOX2-RBS-UtCAT. The transaminase Spo was inserted into the pET28a(+) vector.

[0103] For biotransformation, plasmids pCDFDuet-AOX2-RBS-UtCAT and pET28-spo were co-transformed into BL21(DE3) and induced overnight at 25°C. Cells were collected by centrifugation, washed once with 100 mM PBS buffer (pH 7.0), and collected for later use. The collected cells were resuspended in the reaction system to initiate resting cell biotransformation. The reaction system contained 100 mM PBS (pH 7), 0.1% Triton-X100, 0.1 mM PLP, 10 g / L liquid methionine, and 70 mM pentanediamine hydrochloride to achieve a final OD600 of 20. Resting cell biotransformation was performed at 30°C. After 18 hours, cells were precipitated by centrifugation, and the supernatant was analyzed by HPLC. HPLC results showed that 10 g / L liquid methionine could generate 1.4 g / L of methionine (…). Figure 13 ).

[0104] Table 1. 2-Hydroxyacyl-CoA synthase (HACS) for the condensation of 3-hydroxypropionaldehyde and formyl-CoA. Table 2. List of Acyl-CoA Reductases (ACR) Table 3. Enzymes used to convert intermediate products into methionine Notes: 2HADH: 2-hydroxy acid dehydrogenase; GDHt: glycerol dehydratase; ADH: alcohol dehydrogenase; ALDH: aldehyde dehydrogenase; AADH: amino acid dehydrogenase; MAT: methionine aminotransferase Table 4. List of enzymes used for the interconversion of one-carbon compounds to formyl-CoA. Notes: 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.

[0105] Table 5. List of acyl-CoA transferases (ACTs) used for the interconversion of one-carbon compounds to synthesize formyl-CoA. Table 6. List of acyl-CoA kinases (ACK) and phosphoryltransferases (PTA) used for the interconversion of one-carbon compounds to generate formyl-CoA. This document describes various embodiments of the present invention in conjunction with the accompanying drawings. However, it should be understood that various modifications and alterations can be made to the above embodiments without departing from the broad scope of protection defined in the following claims, and other embodiments can also be implemented. Furthermore, other embodiments will be readily apparent to those skilled in the art upon reading this specification and practicing one or more of the embodiments disclosed herein. Therefore, this application and its embodiments are merely illustrative examples, and their true scope and spirit should be determined by the exemplary claims listed below.

Claims

1. A genetically modified microorganism for converting 3-hydroxypropionaldehyde and formyl-CoA into 2,4-dihydroxybutyryl-CoA, characterized in that, The microorganism contains an overexpressed TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxaloyl-CoA decarboxylase, or benzaldehyde lyase.

2. The genetically modified microorganism according to claim 1, characterized in that, It also contains an overexpressed enzyme that catalyzes the conversion of 2,4-dihydroxycoenzyme A to 2,4-dihydroxybutyrate, wherein the enzyme that catalyzes the conversion of 2,4-dihydroxycoenzyme A to 2,4-dihydroxybutyrate is selected from a combination of thioesterase, acyl-CoA transferase, phosphotransacetase and carboxylic acid kinase, or a combination of acyl-CoA reductase and aldehyde dehydrogenase.

3. The genetically modified microorganism according to claim 2, characterized in that, It also contains an overexpressed 2-hydroxy acid dehydrogenase, which catalyzes the conversion of 2,4-dihydroxybutyric acid to 2-keto-4-hydroxybutyric acid.

4. A genetically modified microorganism for converting 2-keto-4-methylthiobutyric acid into L-methionine or D-methionine, characterized in that, This microorganism contains overexpressed amino acid dehydrogenases or methionine transaminases.

5. The genetically modified microorganism according to claim 3, characterized in that, It also contains an overexpressed amino acid dehydrogenase that catalyzes the conversion of 2-keto-4-hydroxybutyrate to L-homoserine or D-homoserine.

6. The genetically modified microorganism according to claim 3, characterized in that, It also contains an overexpressed amino acid transaminase that catalyzes the conversion of 2-keto-4-hydroxybutyric acid to L-homoserine or D-homoserine.

7. The genetically modified microorganism according to any one of claims 1-6, characterized in that, It also contains an overexpressed enzyme selected from: (i) an acylated formaldehyde dehydrogenase or acyl-CoA reductase that converts formaldehyde to formyl-CoA; (ii) a formate kinase that converts formate to formyl phosphate and a phosphoformyltransferase that converts formyl phosphate to formyl-CoA; (iii) an acyl-CoA transferase that converts formate to formyl-CoA; and (iv) an acyl-CoA synthase that converts formate to formyl-CoA.

8. The genetically modified microorganism according to any one of claims 1-7, characterized in that, It can convert 3-hydroxypropionaldehyde and C1 substrate-derived formyl-CoA selected from methane, methanol, formaldehyde or formic acid into products selected from 2,4-dihydroxybutyric acid, 2-hydroxybutyrolactone, liquid methionine, 2-keto-4-methylthiobutyric acid, L-methionine or D-methionine.

9. The genetically modified microorganism according to any one of claims 1-8, characterized in that, The microorganisms are selected from bacteria, yeast, or fungi.

10. The genetically modified microorganism according to any one of claims 1-9, characterized in that, The microorganisms are selected from the genera *Escherichia coli*, *Bacillus*, *Pseudomonas*, *Corynebacterium*, *Fermentosum*, *Clostridium*, *Streptococcus*, *Rhodococcus*, *Bacillus thermophilus*, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Yarlosporium*, *Rhodotorula methylbacterium*, *Candida*, *Kluyveromyces*, *Aspergillus*, *Penicillium*, *Rhizopus*, or *Trichoderma*.

11. The genetically modified microorganism according to any one of claims 1-10, characterized in that, The microorganism in question is an Escherichia coli-derived strain.

12. The genetically modified microorganism according to claim 11, characterized in that, The microorganism is a strain derived from MG1655.

13. The genetically modified microorganism according to claim 12, characterized in that, The microorganism is MG1655 del( frmA, fdhF, fdnG, fdoG, glcD ) Derivative strains.

14. The genetically modified microorganism according to claim 13, characterized in that, The microorganism is yciA, tesA, tesB, ybgC, ydiI and / or fadM Thioesterase knockout strains.

15. The genetically modified microorganism according to claim 14, characterized in that, The microorganism contains fucO or ackA-Pta Knockout derivative strains.

16. The genetically modified microorganism according to claim 11, characterized in that, The microorganism is a strain derived from BW25113 (DE3).

17. The genetically modified microorganism according to claim 16, characterized in that, The microorganism is BW25113del ( frmA, fdhF, fdnG, fdoG, glcD ) Derivative strains.

18. The genetically modified microorganism according to claim 17, characterized in that, The microorganisms contain overexpression of... Klebsiella peneumoniae Glyceryl dehydratase, DhcHACS and / or CaAbfT.

19. The genetically modified microorganism according to claim 11, characterized in that, The microorganism is a BL21(DE3) derived strain.

20. The genetically modified microorganism according to claim 19, characterized in that, The microorganism contains overexpressed UtCAT and / or AOX2.

21. The genetically modified microorganism according to any one of claims 1-20, characterized in that, The microorganism contains aldB Knock out.

22. The genetically modified microorganism according to claim 21, characterized in that, The microorganisms also contain aldA Knock out.

23. A method for preparing 2,4-dihydroxybutyryl coenzyme A, characterized in that, Formicyl-CoA is reacted with 3-hydroxypropionaldehyde in the presence of a TPP-dependent enzyme, wherein the TPP-dependent enzyme is selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase, or benzaldehyde lyase.

24. A method for preparing 2,4-dihydroxybutyric acid, characterized in that, include: (a) Providing 2,4-dihydroxybutyryl-CoA obtained by the method of claim 23; and (b) Converting the 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid by any of the following (i)-(iii): (i) converting the 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyric acid under the action of a thioesterase or an acyl-CoA transferase; (ii) first converting the 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyryl phosphate under the action of a phosphoacyltransferase, and then converting the 2,4-dihydroxybutyryl phosphate to 2,4-dihydroxybutyric acid under the action of a carboxylic acid kinase; (iii) converting the 2,4-dihydroxybutyryl-CoA to 2,4-dihydroxybutyraldehyde under the action of an acyl-CoA reductase, and then converting the 2,4-dihydroxybutyraldehyde to 2,4-dihydroxybutyric acid under the action of an aldehyde dehydrogenase.

25. A method for preparing 2-hydroxybutyrolactone, characterized in that, include: (a) providing 2,4-dihydroxybutyryl-CoA obtained by the method of claim 23; and (b) spontaneously lactonizing 2,4-dihydroxybutyryl-CoA to 2-hydroxybutyrolactone; or (c) Providing 2,4-dihydroxybutyric acid obtained by the method of claim 24; and (d) Adjust the pH to below 6 to drive the lactonization of 2,4-dihydroxybutyrate to 2-hydroxybutyrolactone.

26. A method for preparing 2-ketobutyrolactone, characterized in that, include: (a) Providing 2-hydroxybutyrolactone obtained by the method of claim 25; (b) 2-hydroxybutyrolactone is converted to 2-ketobutyrolactone by 2-hydroxy acid dehydrogenase.

27. A method for preparing 2-hydroxy-4-methylthiobutyric acid, characterized in that, include: (a) Providing 2-hydroxybutyrolactone obtained by the method of claim 25; (b) Condensing 2-hydroxybutyrolactone with methanethiol to form 2-hydroxy-4-methylthiobutyric acid.

28. A method for preparing 2-keto-4-methylthiobutyric acid, characterized in that, include: (a1) Provides 2-hydroxy-4-methylthiobutyric acid obtained by the method of claim 27; And (b1) under the action of 2-hydroxy acid dehydrogenase, 2-hydroxy-4-methylthiobutyric acid is converted to 2-keto-4-methylthiobutyric acid; or (a2) Providing 2-ketobutyrolactone obtained by the method of claim 26; and (b2) condensing 2-ketobutyrolactone with methanethiol to form 2-keto-4-methylthiobutyric acid.

29. A method for preparing L-methionine or D-methionine, characterized in that, include: (a) Providing 2-keto-4-methylthiobutyric acid obtained by the method of claim 28; (b) 2-keto-4-methylthiobutyric acid is converted to L-methionine or D-methionine by amino acid dehydrogenase or methionine transaminase.

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