Production of methionine via condensation of methional and c1-derived formyl-coa

Abstract

A method for producing desmeninol or methionine, in particular, a method for producing desmeninol or methionine via condensation of methional and C1-derived formyl-CoA was provided. Genetically modified microorganism used for producing desmeninol or methionine was also provided. The key enzymes were engineered to improve their performance on desmeninol or methionine production.

Description

PRODUCTION OF METHIONINE VIA CONDENSATION OFMETHIONAL AND Cl -DERIVED FORMYL-COATECHNICAL FIELD

[0001] The present disclosure generally relates to a method for producing methionine, in particular, a method for producing methionine via condensation of methional and Cl -derived formyl-CoA. The present disclosure also relates to genetically modified microorganism used for producing methionine or desmeninol.SUMMARY OF THE INVENTION

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

[0003] Embodiment 1 . A method for the production of 2-hydroxy-4-(mefhy1fhio)butyry1-CoA comprising contacting formyl-CoA and methional 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 acylating formaldehyde dehydrogenase / acyl-CoA 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 acylating formaldehyde dehydrogenase / 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:room 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.

[0014] Embodiment 12. The method of Embodiment 11, wherein the methanol dehydrogenase 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 monooxygenases.

[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 methional from acrolein.

[0018] Embodiment 16. The method of Embodiment 15, further comprising generating acrolein from propene.

[0019] Embodiment 17. A method for the production of 2-hydroxy-4-(methylthio)butyric acid comprising:(a) providing 2-hydroxy-4-(methylthio)butyryl-CoA obtained by the method of claim 8; and(b) converting 2-hydroxy-4-(methylthio)butyryl-CoA to 2-hydroxy-4-(methylthio)butyric acid by contacting 2-hydroxy-4-(methylthio)butyryl-CoA with any one of (i) and (iii):(i) a thioesterase or an acyl-CoA transferase catalyzing the conversion of 2-hydroxy-4- (methylthio)butyryl-CoA to 2-hydroxy-4-(methylthio)butyric acid;(ii) a phosphotransacylase catalyzing the conversion of 2-hydroxy-4- (methylthio)butyryl-CoA to 2-hydroxy-4-(methylthio)butyry1-phosphate and a carboxylate kinase catalyzing the conversion of 2-hydroxy-4-(methylthio)butyryl- phosphate to 2-hydroxy-4-(methylthio)butyric acid; or(iii) an acyl-CoA reductase catalyzing the conversion of 2-hydroxy-4- (methylthio)butyryl-CoA to 4-(methylthio)-2-hydroxy-butanal and an aldehyde dehydrogenase catalyzing the conversion of 4-(methylthio)-2-hydroxy-butanal to 2- hydroxy-4-(methy1thio)butyric acid. roo2o] Embodiment 18. The method of Embodiment 17, wherein the acyl-CoA reductase is selected from:

[0021] Embodiment 19. The method of Embodiment 17, wherein the aldehyde dehydrogenase is:

[0022] Embodiment 20. A method for the production of 2-keto-4-(methylthio)butyric acid comprising:(a) providing 2-hydroxy-4-(methy1thio)butyric acid by the method of any one ofEmbodiments 17 to 19; and(b) 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.

[0023] Embodiment 21. The method of Embodiment 20, wherein the 2-hydroxy acid dehydrogenase is selected from:

[0024] Embodiment 22. 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 20 or 21 ; and(b) converting 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine by contacting 2-keto-4-(methyl thio) butyric acid with an enzyme selected from an amino acid dehydrogenase or a methionine aminotransferase.

[0025] Embodiment 23. The method of Embodiment 22, wherein the amino acid dehydrogenase is selected from:

[0026] Embodiment 24. The method of Embodiment 22, wherein the methionine aminotransferase is selected from:

[0027] Embodiment 25. The method of any one of Embodiments 1 to 24, wherein the enzyme used in the method is isolated from a microorganism.

[0028] Embodiment 26. The method of any one of Embodiments 1 to 24, wherein the enzyme used in the method is contained in a microorganism.

[0029] Embodiment 27. A genetically modified microorganism providing 2-hydroxy-4- (methylthio)butyryl-CoA by the method of any one of Embodiments 1 to 16.

[0030] Embodiment 28. The microorganism of Embodiment 27, further providing 2-hydroxy-4- (methylthio)butyric acid by the method of any one of Embodiments 17 to 19.

[0031] Embodiment 29. The microorganism of Embodiment 28, further providing 2-keto-4- (methylthio)butyric acid by the method of Embodiment 20 or 21 .

[0032] Embodiment 30. The microorganism of Embodiment 28, further providing L-methionine or D-methionine by the method of any one of Embodiments 22 to 24.

[0033] Also provided herein is use of the genetically modified microorganisms disclosed herein in the production of desmeninol or methionine.

[0034] Enzymes

[0035] Also provided herein is an enzyme, wherein the enzyme is a 2-hydroxyacyl-CoA synthase selected from Table 1 .

[0036] Also provided herein is an enzyme, wherein the enzyme is a 2-hydroxyacyl-CoA synthase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table1, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is a 2-hydroxyacyl-CoA synthase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 1. In some embodiments, the enzyme is a 2-hydroxyacyl-CoA synthase comprising an amino acid sequence having no more than 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 1.

[0037] Also provided herein is an enzyme, wherein the enzyme is a variant of the enzyme of claim 14 or 15, comprising at least one amino acid substitution at position L563, P557A, S556, R553, T552, Y549, L548, H365, R392, D395, T393, V396, Q366, G559, Y474, P543, A92, N93, C94, V28, S103, C104, G112, W95, K151, A152, K164, A36Y, F46, P31, G48, A58, V71, C72, 198, Y88, N66, K67, T158, V30, S150, W62, V81 , S85, V75, L501 , N504, N469, N536, T371 , Q520, F511, K525, L512, M538, S56, G86, E222, E318, V375, Q524, V23, R45, D135, E137, F511, or S533, wherein the substitution modulates e.g., enhances or reduces) the enzyme activity of 2- hydroxy-4-(methylthio)butyryl-CoA production and / or desmeninol production. In some embodiments, the substitution enhances the enzyme activity of 2-hydroxy-4-(methylthio)butyryl- CoA

[0038] Also provided herein is an enzyme, wherein the enzyme is an acylating formaldehyde dehydrogen ase / A cyl -Co A reductase selected from Table 2.

[0039] Also provided herein is an enzyme, wherein the enzyme is an Acyl-CoA reductase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table2, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is an Acyl-CoA reductase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 2. In some embodiments, the enzyme is an Acyl-CoA reductase comprising an amino acid sequence having no more than 1 , 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 2.

[0040] Also provided herein is an enzyme, wherein the enzyme is a variant of the enzyme of claim 17 or 18, comprising at least one amino acid substitution at position 1250, L418, L405, F254, F76, 172, A252, L343, S253, N295, A410, A404, 1408, T411, T447, or D69, wherein the substitution modulates (e.g., enhances or reduces) the enzyme activity of formyl-CoA production and / or 4-(methylthio)-2-hydroxy-butanal production. In some embodiments, the substitution enhances the enzyme activity of formyl-CoA production and / or 4-(methylthio)-2 -hydroxy-butanal production.

[0041] Also provided herein is an enzyme, wherein the enzyme is a 2-hydroxy acid dehydrogenase / oxidase selected from Table 3.

[0042] Also provided herein is an enzyme, wherein the enzyme is a 2-hydroxy acid dehydrogenase / oxidase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 3, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is a 2-hydroxy acid dehydrogenase / oxidase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 3. In some embodiments, the enzyme is a 2-hydroxy acid dehydrogenase / oxidase comprising an amino acid sequence having no more than 1 , , 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 3.

[0043] Also provided herein is an enzyme, wherein the enzyme is an amino acid dehydrogenase selected from Table 3.

[0044] Also provided herein is an enzyme, wherein the enzyme is an amino acid dehydrogenase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 3, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is an amino acid dehydrogenase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 3. In some embodiments, the enzyme is an amino acid dehydrogenase comprising an amino acid sequence having no more than 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 3.

[0045] Also provided herein is an enzyme, wherein the enzyme is a methionine transaminase selected from Table 3.

[0046] Also provided herein is an enzyme, wherein the enzyme is a methionine transaminase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 3, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is a methionine transaminase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 3. In some embodiments, the enzyme is a methionine transaminase comprising an amino acid sequencehaving no more than 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 3.

[0047] Also provided herein is an enzyme, wherein the enzyme is a methanol dehydrogenase selected from Table 4.

[0048] Also provided herein is an enzyme, wherein the enzyme is a methanol dehydrogenase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 4, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is a methanol dehydrogenase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 4. In some embodiments, the enzyme is a methanol dehydrogenase comprising an amino acid sequence having no more than 1 , 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 4.

[0049] Also provided herein is an enzyme, wherein the enzyme is an acyl-CoA synthase selected from Table 4.

[0050] Also provided herein is an enzyme, wherein the enzyme is an acyl-CoA synthase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table4, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is an acyl-CoA synthase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 4. In some embodiments, the enzyme is an acyl-CoA synthase comprising an amino acid sequence having no more than 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 4.

[0051] Also provided herein is an enzyme, wherein the enzyme is an acyl-CoA transferase selected from Table 5.

[0052] Also provided herein is an enzyme, wherein the enzyme is an acyl-CoA transferase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table5, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is an acyl-CoA transferase comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 5. In some embodiments, the enzyme is an acyl-CoA transferase comprising an amino acid sequence havingno more than 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 5.

[0053] Also provided herein is an enzyme, wherein the enzyme is an acyl-CoA kinases (ACK) or a phosphoacyltransferases (PTA) selected from Table 6.

[0054] Also provided herein is an enzyme, wherein the enzyme is an acyl-CoA kinases (ACK) or a phosphoacyltransferases (PTA) comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 6, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the enzyme is an acyl-CoA kinases (ACK) or a phosphoacyltransferases (PTA) comprising an amino acid sequence having at least 90%, 95%, 98% or 99% sequence identity to an amino acid sequence as set forth in Table 6. In some embodiments, the enzyme is an acyl-CoA kinases (ACK) or a phosphoacyltransferases (PTA) comprising an amino acid sequence having no more than 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50 or 60 amino acid difference from an amino acid sequence as set forth in Table 6.

[0055] In some embodiments, the enzyme provided herein comprises at least one, two, three or more amino acid substitution. In some embodiments, the substitution modulates (e.g., enhances or reduces) the enzyme activity of a reaction or method described above. The enzyme activity may be evaluated with any methods or parameters known in the art, for example, by the yield of products, purity of products, reaction time, consumption of starting materials.

[0056] Also provided herein is use of the enzyme or a variant thereof disclosed herein in the production of desmeninol or methionine. Also provided herein is use of the enzyme or a variant thereof disclosed herein in the production of 2-hydroxy-4-(methylthio)butyryl-CoA, desmeninol or methionine.

[0057] In some embodiments, the genetically modified microorganisms disclosed herein comprises the enzyme or a variant thereof disclosed herein. In some embodiments, the enzyme is overexpressed.

[0058] The following description of examples provides additional details, any one of which can be subject to patenting in combination with any other. The specification in its entirety is to be treated as providing a variety of details that can be used interchangeablely with other details.

[0059] The present disclosure is illustrated by the following non-limiting examples.

[0060] 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

[0061] 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.

[0062] Figure 1 is an overview illustrating production of methionine from condensation of methional 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; ALDH: aldehyde dehydrogenase; 2HADH: 2-hydroxyacid dehydrogenase; AADH: amino acid dehydrogenase; MAT: methionine aminotransferase

[0063] Figure 2 is HACS screening results for methional-Cl condensation reaction represented by desmeninol productivity.

[0064] Figure 3 is a scheme illustrating Formyl-CoA production from Cl substrates.

[0065] Figure 4 is a comparison of formyl-CoA generation routes for desmeninol production in vivo. (A) MG 1655 as chassis strain, inducer matrix of different plasmids combination, compare desmeninol flux; (B) BW25113 as chassis strain, inducer matrix of different plasmids combination, compare desmeninol flux.

[0066] Figure 5 is the demonstration of desmeninol production in vitro. (A) desmeninol production of eight HACS; (B) HPLC results of in vitro assays of different HACS. The desired product was pointed with black frame.

[0067] Figure 6 is comparison of different HACS for desmeninol production through inducer matrix of different HACS and LmACR .

[0068] Figure 7 is a scheme illustrating the mechanism of high-throughput screening method for desmeninol quantification.

[0069] Figure 8 is the activity pockets of Cphyl l78. Ref: Insight into Coenzyme A cofactor binding and the mechanism of acyltransfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

[0070] Figure 9 is determination of residues in the LmACR substrate channel

[0071] Figure 10 is sequence alignment of different ACRs

[0072] Figure 11 is the activity of engineered ACR variants (1stround) towards formaldehyde and methional.

[0073] Figure 12. The activity of engineered ACR variants (2ndround) towards formaldehyde and methional.

[0074] Figure 13 is an overview of HACS protein engineering strategies.

[0075] Figure 14 is molecular docking of methional with CdcHACS.

[0076] Figure 15 is identification of key residues of CdcHACS through alanine scanning.

[0077] Figure 16 is screening of saturation mutation library of Q366.

[0078] Figure 17 demonstrated the screening of saturation mutation library of R392, V396, Y474 and Y549

[0079] Figure 18 demonstrated the screening of the combinatorial mutation of 1st shell

[0080] Figure 19 is the first round mutation of CdcHACS 2nd shell.

[0081] Figure 20 is the first round mutation of CdcHACS 3rd shell.

[0082] Figure 21 is the second round mutation of CdcHACS 3rd shell.

[0083] Figure 22 demonstrated the screening of 2-hydroxyacid oxidase for conversion of 4- (methylthio)-2-oxobutyric acid to 4-(methylthio)-2-oxobutyric acid

[0084] Figure 23 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.

[0085] The same reference numbers will be used throughout the drawings to refer to the same or like parts.DETAILED DESCRIPTION OF THE INVENTION

[0086] 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 embodiments of 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.

[0087] 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.

[0088] EXAMPLES

[0089] EXAMPLE 1: OVERVIEW OF METHIONINE PRODUCTION THROUGH CONDENSATION OF METHIONAL AND FORMYL-COA

[0090] This Example demonstrates the implementation of methionine production through Cl+bio platform by condensation of methional and formyl-CoA and followed by derivation to methionine (Figure 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 (GeneBank accession: HAK63664.1), DhcHACS from Dehalococcoidia bacterium (GeneBank accession: PWB41796.1), CfhHACS from Chloroflexi bacterium (GeneBank accession: PKN81274.1), to generate 4-methylthio-2-hydroxybutyryl-CoA. 4-methylthio-2-hydroxybutyryl-CoA is further converted to 4-methylthio-2-hydroxybutyrate (desmeninol, liquid methionine) 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. 80:1042-1050 (2014)). Alternatively, 4-methylthio-2- hydroxybutyryl-CoA can be reduced to 4-methylthio-2-hydroxybutanal by an acyl-CoA reductase such as Clostridium beijerinckii ALD (Kim, S., et al. J. Ind. Microbiol. Biotechnol. 42:465-475 (2015)) (e.g., ACR set forth in Table 2 below). 4-methylthio-2-hydroxybutanal can be further oxidized to desmeninol by a suitable aldehyde dehydrogenase such as E. coli AldA or AldB (Baldoma and Aguilar, J Biol Chem. 1987, 262(29): 13991-6). Desmeninol is then converted to 4- methylthio-2-oxobutyrate by 2-hydroxy acid dehydrogenase (2HADH) (Matelska et al., BMC Evol Biol. 2018;l 8(1): 199), it was then finally converted to L-methionine by amino aciddehydrogenase (AADH) (Nitta et a., J Bacteriol. 1974; 117(2): 588-592) or methionine aminotransferase (MAT) (Venos et al., BMC Microbiol, 2004; 4, 39) (e.g., enzymes set forth in Table 3 below).

[0091] Methional can be chemically produced from propene. Propene was firstly oxidized with O2 to generate acrolein (see, e.g., W02005053839), and then condensed with CH3SH to form methional (see, e.g., W02006015684). Alternatively, methional can be chemically produced from glycerol. Glycerol was firstly dehydrated to form 3-hydroxyaldehyde, and then further dehydrated to generate acrolein (Manfro., et al., Catalysis for Sustainable Energy, 2013 (1): 60-70). As mentioned above, acrolein can be condensed with CH3SH to form methional.

[0092] .EXAMPLE 2. INTERCONVERSION OF ONE-CARBON COMPOUNDS TO GENERATE FORMYL-COA

[0093] Formyl-CoA can be provided through the interconversion of Cl compounds including methane, methanol, formaldehyde, formate (Figure 1 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) have been expressed and characterized in E. coli. Formaldehyde can be directly oxidized to formic acid catalyzed by E. coli formaldehyde detoxification system (frmA). 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 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 reactionsare considered fully reversible, AMP-forming acyl-CoA synthetases are favored toward formate activation to formyl-CoA.

[0094] EXAMPLE 3: OVERVIEW OF METHODS USDED FOR GENETIC MANIPULATION

[0095] 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 Tn-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.

[0096] 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 k-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 othersuitable 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 IPTG induction, and pCas can be cured through growth under higher temperature like 37-42°C.F0097] 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), spectmomycin (50 pg / mL) and chloramphenicol (12.5 pg / mL).

[0098] 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 were created 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. As a host for these vectors, an engineered strain of E. coli based on MG1655(DE3), BW25113 or ATCC8739 with knockouts for formaldehyde (A / rmA), formate l \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.

[0099] EXAMPLE 4: FERMENTATION CONDITIONS USED FOR IN VIVO PRODUCTION OF METHIONINE

[0100] 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 pM FeSO4, 5 mM (NH4hSO4, 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 and50 jig / mL kanamycin) are included when appropriate. All chemicals are obtained from Fisher Scientific Co. (Pittsburg, PA) and Sigma-Aldrich Co. (St. Louis, MO).

[0101] 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 ODmo 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 (Infers 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 fermentation media with 40 g / L glucose and appropriate IPTG and antibiotics. If required, lactic acid (20 mM) is added at 0, 24, and 48 hours.

[0102] After the fermentation, the supernatant obtained through 5000 g, 5 min centrifuge in an Optima L-80XP Ultracentrifuge (Beckman-Coulter, Schaumburg, IL) of 2 mL culture is prepared for HPLC analysis.

[0103] EXAMPLE 5. HPLC ANALYSIS OF METHIONINE

[0104] 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 wasadded. 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.

[0105] 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).

[0106] EXAMPLE 6: USING METHIONAL AS SUBSTRATE FOR CONDENSATION WITH FORMYL-COA

[0107] The purpose of this example is to demonstrate the implementation of using 3- (methylthio)propionic acid (R=CH3SCH2) as substrate for condensation with formyl-CoA (Fig 2).

[0108] Cl elongation is initiated by formyl-CoA production through energy and formyl-CoA generation steps. 2-HydroxyacyLCoA synthase (HACS) condenses methional and formyl-CoA to produce 2-hydroxy-4-(methylthio)butyryl-CoA. 2-hydroxy-4-(methylthio)butyryLCoA is converted to desmeninol by acyl-CoA thioesterase.

[0109] To implement the Cl elongation pathway for desmeninol production in vivo, we engineered vectors 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, we used an engineered strain of E. coli based on MG1655(DE3) with knockouts for formaldehyde (A / rmA) and formate (J\fdhF AfdnG AfdoG) oxidation as well as for glycolate utilization (NglcD).

[0110] In vivo product synthesis was conducted using M9 minimal media (6.78 g / L Na2HPC>4, 3 g / L KH2PO4, 1 g / L NH4CI, 0.5 g / L NaCl, 2 mM MgSCL, 100 pM CaCh, 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 yeastextract. 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.

[0111] 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. 20 mM methional and 20 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 3 hour, the cells were pelleted by centrifugation and the media was analyzed using HPLC.

[0112] The result indicates that CfhHACS is the best candidate under the given experimental conditions reaching up to 43 pM / hr per OD600 (Fig 2B, Table 7).

[0113] EXAMPLE 7: COMPARISON OF DIFFERENT PATHWAYS THAT PRODUCE FORMYL-COA

[0114] The purpose of this example is to compare the three routes for formyl-CoA generation (Fig 3). HACS condenses methional and formyl-CoA to form desmeninoyl-CoA, which can then be hydrolyzed to desmeninol via native termination enzymes.

[0115] Combine different formyl-CoA pathways with CdcHACS expressed in strain MG 1655 and BW25113 that harboring pET-PCT5-CaAbfT+ pCDFDuet-CdcHACS, pET-PCT5-FOK+ pCDFDuet-CdcHACS and pET-PCT5-LmACR+ pCDFDuet-CdcHACS.

[0116] TB medium was used for growth and protein expression. 1% of overnight seed culture was inoculated into TB medium with appropriate antibiotics. After 3 h shaking, IPTG and cumate was added to overexpress proteins at 30 °C for another 21 h.

[0117] Cells from the above pre-cultures were then centrifuged (4000 rpm, 22°C), washed with the M9 minimal media without any carbon source, and resuspended with 1 mL of above minimalmedia containing indicated amounts of substrate. 10 mM methional, 2.5 mM formaldehyde, or 50 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 1 hour, the cells were pelleted by centrifugation and the media was analyzed using HPLC. roiis] The result indicates that CoA transferase using CaAbfT route has better performance than the other two routes in MG 1655 strain, the optimal inducer concentration is 10 pM IPTG and 50 pM cumate. FOK route is the next with optimal inducer concentration at 50 pM IPTG and 50 pM cumate. LmACR route has the worst performance, the optimal inducer concentration is 25 pM IPTG and 100 pM cumate (Fig 4A). In BW25113, LmACR route has the best performance when it was induced by 50 pM IPTG and 50 pM cumate. Followed by CoA transferase route (CaAbfT) and FOK route is the worst (Fig 4B).

[0119] EXAMPLE 8: ESTABLISHING HIGH THROUGHPUT PLATFORM FOR SCREENING HACS VARIANTS FOR DESMENINOL PRODUCTION

[0120] The purpose of this example is to demonstrate high throughput platform for screening 2- hydroxyacyl-CoA synthase (HACS) variants in vivo. We used desmeninol productivity per cell density (pM glycolate / OD600) as indicator of HACS activity. Desmeninol can be produced from methional and formaldehyde in the presence of active HACS variant and acyl-CoA reductase from Listeria monocytogenes (LmACR) as mentioned above. LmACR is shown to be capable of catalyzing oxidation reaction from formaldehyde to formyl-CoA (Chou, A., et al. Nat. Chem. Biol. 15:900-906 (2019)). HACS condenses methional and formyl-CoA to form 4-methy1thio-2- hydroxybutyryl-CoA, which can then be hydrolyzed to glycolate via native thioesterase activities (Fig 2A).

[0121] To prototype glycolate production pathway from formaldehyde in vivo, we constructed vectors overexpress various HACS candidates under control of the IPTG-inducible T7 promoter in pCDFDuet-1 and LmACR under control of a cumate-inducible T5 promoter in pETDuet-1, respectively. As a host for these vectors, we used an engineered strain of E. coli based on BW25113(DE3) with knockouts for formaldehyde (A / rmA) and formate (AfdhF AfdnG AfdoG) oxidation as well as for glycol ate utilization (J\glcD), which we expected could compete or interfere with the analysis of our pathway.

[0122] In vivo product synthesis was conducted using M9 minimal media (6.78 g / L Na2HPC>4, 3 g / L KH2PO4, 1 g / L NH4CI, 0.5 g / L NaCl, 2 mM MgSO4, 100 pM CaCl2, and 15 pM thiamine- HC1) unless otherwise stated. Cells were initially grown in 96-deep well plates (USA Scientific, Ocala, FL) containing 0.2 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 hrs) 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 (IPTG)) were added. Plates were incubated for a total of 24 hrs postinoculation.

[0123] 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. 5 mM formaldehyde and 10 mM methional 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 1 hour, the cells were pelleted by centrifugation and the supernatant analyzed by HPLC or chromatographic method as described below. Cell pellets harvested after bioconversion were resuspended to 20OD in B-PER® Bacterial Protein Extraction Reagent (Thermo Fisher) supplemented with 0.1 mg / mL chicken egg white lysozyme (Fisher) and 5 U / mL Benzonase® nuclease (Sigma) for cell lysis. After incubation in room temperature for 15 minutes, 100 pL of each cell lysate was transferred to 1.5 mL microcentrifuge tubes for centrifugation at 15,000xg for 5 minutes. The soluble cell lysates obtained from the supernatant were analyzed using SDS-PAGE. Relative HACS expression was estimated by band area in the protein gel image.

[0124] Quantification of product and substrate concentrations (formic acid, formaldehyde, methional and desmeninol) were determined via HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a refractive index detector and an HPX-87H organic acid column (Bio-Rad, Hercules, CA) with operating conditions to optimize peak separation (0.3 ml / min flowrate, 30 mM H2SO4 mobile phase, columntemperature 42°C). For library screening, desmeninol was first quantified via chromatographic method.

[0125] EXAMPLE 9: SCREENING OF DIFFERENT HACS FOR DESMENINOL PRODUCTION

[0126] The purpose of this example is to screen better HACS for desmeninol production. Eight HACS candidates from previous study was tested here, including ApbHACS(JGI15), CdcHACS(JGI24), DhcHACS(JGI20), MeOX4, CabHACS(JGI23), CaoHACS(JGIH48), CfhHACS(JGIH65), BbHACS(JGI19).

[0127] Construct plasmid pCDFDuet-6His-HACS to express the eight HACS in BL21(DE3). Purify the HACS proteins according to the steps in the instruction manual of the Beyotime Purification Kit. And protein concentration was determined by BCA method.

[0128] We used desmeninol production (mM) as an indicator for HACS activity. In this pathway, formyl-CoA is generated by formic acid (formate) catalyzed by the acyl-CoA transferase from Clostridium aminohutyricum (CaAbfT). The reaction system containing 100 mM KPi pH 6.9, 10 mM MgCh, 2 mM acetyl-CoA, 150 pM ThDP, 20 mM formate, 1 pM CaAbfT, 2 pMHACS, and 20 mM methional. The assays were run at 30°C for 18 h. The reaction was stopped by adding 4 pL of 10 M NaOH. The samples were then applied for HPLC analysis.

[0129] The result indicates that, 7 of the 8 HACS was able to produce desmeninol (Fig 5).

[0130] We chose CdcHACS(JGI24), CaoHACS(JGIH48) and CfhHACS(JGIH65), combine with LmACR, with HACS under control of the IPTG-inducible T7 promoter in pCDFDuet-1 and LmACR under control of a cumate-inducible T5 promoter in pETDuet-1 and transformed them into the engineered strain of E. coli based on BW25113 with knockouts for formaldehyde (A / rmA) and formate (,\fdhF AfdnG kfdoG) oxidation as well as for glycolate utilization ( \glcl)}.

[0131] In vivo product synthesis was conducted using TB media (6.78 g / L NaiHPOr, 3 g / L KH2PO4, 1 g / L NH4CI, 0.5 g / L NaCl, 2 mM MgSO4, 100 pM CaCl2, and 15 pM thiamine-HCl) unless otherwise stated. Cells were initially grown in 96-deep well plates containing 0.5 mL of the above media. A single colony of the desired strain was cultivated overnight (14-16 hrs) in LBmedium with appropriate antibiotics and used as the inoculum (1%). Antibiotics (100 pg / mL ampicillin, 100 pg / mL streptomycin) were included when appropriate. Cultures were then incubated at 30°C and 1000 rpm in a Digital Microplate Shaker until an ODeoo of ~0.4 was reached, at which point appropriate amounts of inducer(s) (isopropyl P-D-l -thiogalactopyranoside (1PTG) and cumate) were added. Plates were incubated for a total of 24 hrs post-inoculation.

[0132] Cells from the above pre-cultures were then centrifuged (4000 rpm, 22°C), washed with the M9 minimal media without any carbon source, and resuspended with 1 mL of M9 minimal media containing indicated amounts of carbon source. 5 mM formaldehyde and 10 mM methional was added at 0 hr and were incubated at 30°C and 1000 rpm in Digital Microplate Shaker. The cells were harvested after 1 hour and the supernatant analyzed by HPLC as described in EXAMPLE 5.

[0133] The result indicates that, JGI24 performs the best among these three HACS under optimal inducer concentrations (relative gene expressions), based on desmeninol productivity (pM / ODeoo) in 1 hour (Fig 6).

[0134] EXAMPLE 10: DESMENINOL QUANTIFICATION THROUGH HIGH THROUGHPUT DETECT METHOD

[0135] This example established a high throughput chromatographic method for desmeninol quantification. This was achieved by using the oxidase.

[0136] In this method, desmeninol oxidase from Aerococcus viridans (AvLOX) is used to catalyze the oxidation of 2-hydroxyacid to produce 2-oxoacid and hydrogen peroxide (H2O2). Then, ABTS (A10016 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 5= 98%) reagent is used as a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H2O2 in a 1:1 stoichiometric ratio to produce ABTS+, a brightly fluorescent and strongly absorbing reaction product (excitation / emission maxima -420 nm) (Fig. 7). By measuring the reading of ABTS+using microplate reader, we can calculate the concentration of the desmeninol in the samples.

[0137] High-throughput method was conducted using 1 XM9 solution, reaction system (200 pL) includes: 50 pL 32 mM ABTS, 20 pL 36 U / mL HRP, 30 pL 40 U / rnL AvLOX, 100 pL sample.Measure absorbance at 420 nm every 1 min for 30 min , The mean value of the linear interval of the obtained curve was selected as the corresponding value to calculate the desmeninol concentration.

[0138] EXAMPLE 11: IDENTIFICATION OF LMACR CATALYTIC POCKETS AND MOLECULAR DOCKING

[0139] This example is to identify the important residues can be selected for protein engineering to improve the ACR activity.

[0140] The activity pockets of Cphyll78 from C. phytofermentans are shown in Fig 8 (Insight into Coenzyme A cofactor binding and the mechanism of acyltransfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans), and LmACRs were sequence aligned with Cphyl l 78.

[0141] Next, methional was molecularly docked with LmACR(PDB: 3K9D) and residues within 5 A of the substrate were selected for the next step of analysis (Fig 9).

[0142] Several ACRs from different sources that have been reported in the literature were selected for sequence alignment. These ACRs prefer substrates of different chain lengths and the study of residues in their catalytic pockets provides insights into residues associated with substrate selectivity (Fig 10).

[0143] EXAMPLE 12: DESIGNING MUTANTS AND TESTING ACTIVITY OF ACR VARIANTS

[0144] Based on the results of the sequence alignment, non-highly conserved residues were selected for mutation to larger residues (preferably of the same type) to narrow the substrate binding pocket.

[0145] Kinetics or crude enzyme activity on formaldehyde or other aldehyde were determined in reactions containing 100 mM Kpi buffer pH 7.8, 2.5 rnM NAD+, 2 rnM CoA, 0.25 pM ACR (Crude enzyme: 0.5 mg / mL), and 1 mM to 40 mM aldehyde. The reaction rate was represented by the NADH generation rate through monitoring the absorption at 340 nm.

[0146] Mutant I250F slightly reduces the activity towards formaldehyde with significantly decreased activity towards methional, and this residue can be selected as an engineering target to improve substrate specificity. Mutations in L405 and L418 slightly increased activity for formaldehyde (Fig 11). The ACR variants were characterized using purified enzymes. Table 8 shows the corresponding kinetic parameters, L405F has a higher Kcat value, which was used as a template for the next round engineering.

[0147] Crude enzyme assays were used to evaluate the LmACR variants activity. BW A 14(BW25113(DE3) A(adhP, yjgB, yahK, glcD, frmA, fdhF, fdoG, fdnG, yqhD, aldA, aldB, puuC, patD, gshA) strain was used to overexpress LmACR variants. After expressing the LmACR variants, the cells were washed once with M9 and then resuspended to a final OD600 of 20 and lysed with Thermo Scientific B-PER solution, after centrifugation, the total protein concentration was measured using BCA method. The crude enzyme reaction was performed in 50 mM MOPS buffer pH 7.8 containing 2.5 mM NAD+, 2 mM CoA, Crude enzyme 0.5 mg / mL, and 2.5 mM / 5 mM formaldehyde. The reaction rate was represented by the NADH generation rate through monitoring the absorption at 340 nm.

[0148] The results are shown in Figure 12, all three mutants L405F-D69H, I72T, and S253C, showed increased activity for all three substrates, whereas A252S / T and A404S showed increased activity to formaldehyde but decreased activity for methional.

[0149] Meanwhile, the LmACR variants were characterized using purified enzymes same as mentioned above. Kinetics on formaldehyde were determined in reactions containing 100 mM MOPS buffer pH 7.8, 2.5 mM NAD+, 2 mM CoA, 0.25 pM ACR, and 1 mM to 50 mM aldehyde. The reaction rate was represented by the NADH generation rate through monitoring the absorption at 340 nm. The results in Table 8 show that mutants A252S / T, S252C, A404S have lower Km to formaldehyde, A252S, D69H show an increase of Kcat. This implies that these sites can be combinatorically mutated possibly to further improve catalytic efficiency.

[0150] EXAMPLE 13: OVERVIEW OF PROTEIN ENGINEERING OF CDCHACS TOHAVE BETTER ACTIVITY TOWARDS TO METHIONAL

[0151] This example is to show the general strategy for protein engineering of CdcHACS to have better activity towards methional for desmeninol production. Protein engineering of the enzyme CdcHACS through three distinct strategies: protein modeling and alanine scanning, functional analysis using HotSpot Wizard, and co-evolution analysis with EVcouplings. The engineering efforts have led to the identification of several beneficial mutations that enhance the enzyme's activity and stability.

[0152] This was done in a combinatorial way from the inner space to surface of the protein. We “separate” it into 4 shells of the protein to do the engineering work in parallel. The one we called 1stshell is closest to the substrate binding area, molecular docking with alanine scanning was done to identify the key residues. We reshaped the pocket between the 1stand 2ndshell. Meanwhile, channel engineering, loop engineering, surface engineering and also co-evolutionary engineering was done accordingly (Fig. 13).

[0153] EXAMPLE 14: IDENTIFICATION OF KEY RESIDUES OF CDCHACS THROUGH MOLECULAR DOCKING

[0154] This example is to identification of key residues through alanine screening. The crystal structure of CdcHACS was obtained by modelling from AlphaFold2 based on its amino acid sequence. It was docked with substrates TPP, formyl-CoA, and 3-(Methylthio)propionaldehyde (Fig. 14) and residues within 5 A of the substrate were selected for alanine scanning to identify the key residues. Oxalyl-CoA Decarboxylase(PDB ID: 2JI6) with known crystal structure was used as a reference for molecular docking.

[0155] Based on the docking results, key amino acid residues around the substrate-binding site (L563, P557, S556, R553, T552, Y549, L548, H365, R392, D395, T393, V396, Q366, G559, Y474, P543) were subjected to alanine scanning. Mutation H365A showed increased activity. Mutations at Q366A, V396A, and D395A resulted in a significant activity decrease (over 50% for V396A and D395A, and over 70% for Q366A compared to wild-type CdcHACS) (Fig. 15). So, Q366, H365, V396, R392, Y549, and Y474 were chosen as the target for saturation mutation.

[0156] The method was used to build the Q366 saturation mutation library, and then it was screened using the method mentioned above in example 8. The one has better performance thanwild type was picked and sequenced. Re-screening of these positive clones was done and Q366N, Q366D, and Q366R has better performance than others (Fig. 16).

[0157] The saturation mutation library of H365, V396, R392, Y549, and Y474 was screened using the same method and led to several advantageous mutants: H365A, H365F, H365P, H365S, V396I, V396M, Y549N, Y549D, Y549E, Y549Q, Y549S, Y549T, Y549R, Y549K, R392S, R392C, R392M, R392Q with about 20% improvement (Fig. 17).

[0158] The one has better performance than wild type was combined to generate combinatorial mutants and 50% improvement was observed (Fig. 18).

[0159] EXAMPLE 15: IDENTIFICATION OF THE HOTSPOTS OF CDCHACS THROUGH FUNCTIONAL ANALYSIS WITH HOTSPOT WIZARD

[0160] This example is to identify the hotspots of CdcHACS to improve the CdcHACS activities. Bioinformatics analysis were conducted by considering of reshaping the pocket, channel engineering, loop engineering, and surface engineering. Functional analysis identified key residues A92, N93, C94, V28, S103, G112, W95, KI 51 , A152, leading to mutants A92Q, A92M, A92L, C94S, N93D / C94S, V28P / C94S, C94S / S103Q, C94S / C104V, C94S / G112D, W95I, N93D / W95I, C94S / W95I, K151H, A152S, A152H. Residue correlate analysis identified key residues G112, C104, N93, K164, A36, F46, P31, G48, V28, leading to mutants G112D, C104V / G112D, N93D / G112D, G112D / K164D, A36Y / F46H, P31A / G48L, V28P / G48L, G48L / G112D. Stability analysis identified key residues A58, V71, C72, 198, Y88, N66, K67, T158V, V30, P31, S150, W62, V81, S85, V75, N93, leading to mutants A58M, V71I, V71C, C72V, I98V, Y88F, Y88A, N66S, N66T, K67G, T158V, V30G, V30G / P31 A, S150A, W62Y, V81 T / S85T, V75T / N93D (Table 9). Site-directed mutation was applied for the first round of the selected targets, and the mutants were screened as mentioned above.

[0161] The HACS variants were screened using the high throughput method described in example 8 and the desmeninol was quantified using the chromatography method mentioned in example 10. Some of the mutants that have higher desmeninol flux were further verified using HPLC method.

[0162] For the first-round mutation of CdcHACS 2ndshell, although mutations at N93 and C94 did not increase activity, they decreased protein expression levels, suggesting an increase inspecific activity. Saturation mutagenesis led to promising variants N93F, N93R, N93H, N93S, N93C, N93M, C94Y, C94D, C94Q, C94H, C94A, M276P, M276C, M276D, M276A, M276L, C94NDT / R154NDT, N93NDT / C94NDT. V30G / P31 A, N93D / C94S, and A36Y / F46H almost lose their activity on methional, and the activity of W59I and P31 A / G48L dropped significantly (Fig. 19).F0163] EXAMPLE 16: PROTEIN ENGINEERING OF CDCHACS 3RDTHROUGH COEVOLUTION ANALYSIS WITH EVCOUPLINGS

[0164] This example is to engineer CdcHACS considering the co-evolutionary impact on protein activity. Based on structural information and synergistic evolution rules in nature, we designed eleven sets double-sites saturation mutation (Table 10), NNK method was used to build these double mutation libraries. The high throughput HACS screening method described in example 9 was used to evaluate these CdcHACS variants. The NNK library of T371 / Q520, L512 / M538, V375 / Q524, D135 / E137, V23 / R45 showed a diverse activity on methional (Fig. 20), most of E222 / E318 mutants lost their activity on methional, and there is no activity towards to methional in almost all mutants derived from F511 / S533, S56 / G86, and N469 / N533 (Fig. 20)

[0165] According to the first-round screening results and sequencing analysis, NDT methods were used to build the libraries for second round screening to identify key amino acids. The variants were screened in M9 and also NBS medium, most of these variants has similar or higher activity than wild type CdcHACS with up to 1.75 x fold improvement compared to WT (Fig. 21).

[0166] EXAMPLE 17: CONVERSION OF DESMENINOL TO 4-(METHYLTHIO)-2- OXOBUTYRIC ACID BY 2-HYDROXYACID OXIDASE / DEHYDROGENASE

[0167] 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.

[0168] The conversion of desmeninol to 4-(methylthio)-2-oxobutyric acid was conducted in a resting cell bioconversion format. To overexpress 2 -hydroxy acid 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.

[0169] The resting cells bioconversion shows all tested 2-hydroxyacid oxidases have activity on desmeninol, A0X2 shows the best activity under tested conditions with up to 85% conversion rate, followed by A0X1 with 50% conversion rate (Fig. 22).

[0170] EXAMPLE 18: CONVERSION OF DESMENINOL TO METHIONINE BY 2- HYDROXYACID OXIDASE AND TRANSAMINASE.

[0171] 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-(mefhylthio)-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(+).

[0172] 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 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 wasapplied for HPLC analysis. As HPLC shows 1.4 g / L of methionine was produced from 10 g / L desmeninol (Fig. 23).Table 1. List of 2-hydroxyacyl-CoA synthase (HACS) used for the condensation of methional and formyl-CoATable 2. List of acyl-CoA reductase (ACR)Table 3. List of enzymes used for the conversion of intermediate to methionineNote: 2HADH: 2-hydroxyacid dehydrogenase; ALDH: aldehyde dehydrogenase; AADH: amino acid dehydrogenase; MAT: methionine aminotransferaseTable 4. List of enzymes used for the interconversion of one-carbon compounds to generate formyl-CoANote: sMMO: soluble methane monooxygenase; pMMO: Particulate methane monooxygenase; MDH: methanol dehydrogenase; ALDH: aldehyde dehydrogenase; ACS: acyl-CoA synthase.Table 5. List of acyl-CoA transferases (ACT) variants used for the interconversion of one- carbon compounds to generate formyl-CoATable 6. List of List of acyl-CoA kinases (ACK) and phosphoacyltransferases (PT A) variants (JGIK) variants used for the interconversion of one -carbon compounds to generate formyl- CoAVarious 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.Table 7. Activities of HACS variants on different aldehydes for 2-hydroxyacid production.Numbers present product flux in pM / OD / h.Table 8. Kinetic parameters of high performing ACR variantsTable 9. Mutation targets of CdcHACS 2ndshell1st round nurtadon of CdcHACS 2nd shellTable 10. Mutation targets of CdcHACS 3rdshell

Claims

Claims1. A genetically modified microorganism for converting methional and formyl-CoA to 2- hydroxy-4-(methylthio)butyryl-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-hydroxy-4-(methylthio)butyryl-CoA to 2-hydroxy-4- (methylthio)butyric 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-hydroxy-4-(methylthio)butyric acid to 2-keto-4-(methylthio)butyric acid.

4. The genetically modified microorganism of claim 3, further comprising an overexpressed amino acid dehydrogenase catalyzing the conversion of 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine.

5. The genetically modified microorganism of claim 3, further comprising an overexpressed methionine aminotransferase catalyzing the conversion of 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine.

6. The genetically modified microorganisms of any one of claims 1-5, further comprising an overexpressed enzyme selected from the group consisting of:(i) an methanol dehydrogenase / oxidase converting methanol to formaldehyde(ii) an acylating formaldehyde dehydrogenase / Acyl-CoA reductase converting formaldehyde to formyl-CoA;(iii) a formate kinase converting formate to formyl -phosphate and a phosphate formyltransferase converting formyl-phosphate to formyl-CoA;(iv) an acyl-CoA transferase converting formate to formyl-CoA; and(v) an acyl-CoA synthase converting formate to formyl-CoA.

7. The genetically modified microorganisms of any one of claims 1-6, capable of converting methional and a Cl substrate selected from methane, methanol, formaldehyde or formate to a product selected from desmeninol, 2-keto-4-(methylthio)butyric acid, L-methionine or D- methionine.

8. The genetically modified microorganisms of any one of claims 1-7, wherein the microorganisms are selected from the group consisting of bacteria, yeast and fungi.

9. The genetically modified microorganisms of any one of claims 1-8, wherein the microorganisms are Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebacterium sp., Zymonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccha.romyc.es sp., Pichia sp., Yarrowia sp., Methylorubrum. sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. Trichoderma sp..

10. A method for the production of 2-hydroxy-4-(methylthio)butyryl-CoA comprising contacting formyl-CoA and methional with a TPP-dependent enzyme selected from 2- hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

11. A method for the production of 2-hydroxy-4-(methylthio)butyric acid comprising:(a) providing 2-hydroxy-4-(methylthio)butyryl-CoA obtained by the method of claim 8; and(b) converting 2-hydroxy-4-(methylthio)butyryl-CoA to 2-hydroxy-4-(methylthio)butyric acid by contacting 2-hydroxy-4-(methylthio)butyryl-CoA with any one of (i) and (iii):(i) a thioesterase or an acyl-CoA transferase catalyzing the conversion of 2-hydroxy-4- (methylthio)butyryl-CoA to 2-hydroxy-4-(methylthio)butyric acid;(ii) a phosphotransacylase catalyzing the conversion of 2-hydroxy-4- (methylthio)butyryl-CoA to 2-hydroxy-4-(methylthio)butyryl-phosphate and a carboxylate kinase catalyzing the conversion of 2-hydroxy-4-(methylthio)butyryl- phosphate to 2-hydroxy-4-(methylthio)butyric acid; or(iii) an acyl-CoA reductase catalyzing the conversion of 2-hydroxy-4-(methylthio)butyryl-CoA to 4-(methylthio)-2-hydroxy-butanal and an aldehydedehydrogenase catalyzing the conversion of 4-(methylthio)-2-hydroxy-butanal to 2- hydroxy-4-(methylthio)butyric acid.

12. A method for the production of 2-keto-4-(methylthio)butyric acid comprising:(a) providing 2-hydroxy-4-(mefhy1thio)butyric acid obtained by the method of claim 9; and(b) 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.

13. 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 10; and(b) converting 2-keto-4-(methylthio)butyric acid to L-methionine or D-methionine by contacting 2-keto-4-(methyl thio) butyric acid with an enzyme selected from an amino acid dehydrogenase or a methionine aminotransferase.

14. An enzyme, wherein the enzyme is a 2-hydroxyacyl-CoA synthase selected from Table 1.

15. An enzyme, wherein the enzyme is a 2-hydroxyacyl-CoA synthase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 1, or an amino acid sequence having at least 90% sequence identity thereto.

16. An enzyme, wherein the enzyme is a variant of the enzyme of claim 14 or 15, comprising at least one amino acid substitution at position L563, P557A, S556, R553, T552, Y549, L548, H365, R392, D395, T393, V396, Q366, G559, Y474, P543, A92, N93, C94, V28, S103, C104, G112, W95, K151, A152, K164, A36Y, F46, P31, G48, A58, V71, C72, 198, Y88, N66, K67, T158, V30, S150, W62, V81, S85, V75, L501, N504, N469, N536, T371, Q520, F511, K525, L512, M538, S56, G86, E222, E318, V375, Q524, V23, R45, D135, E137, F511, or S533, wherein the substitution modulates (e.g., enhances or reduces) the enzyme activity of 2-hydroxy-4-(methylthio)butyryl-CoA production and / or desmeninol production.

17. An enzyme, wherein the enzyme is an acylating formaldehyde dehydrogenase / Acyl-CoA reductase selected from Table 2.

18. An enzyme, wherein the enzyme is an AcyLCoA reductase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 2, or an amino acid sequence having at least 90% sequence identity thereto.

19. An enzyme, wherein the enzyme is a variant of the enzyme of claim 17 or 18, comprising at least one amino acid substitution at position 1250, L418, L405, F254, F76, 172, A252, L343, S253, N295, A410, A404, 1408, T411, T447, or D69, wherein the substitution modulates (e.g., enhances or reduces) the enzyme activity of formyl-CoA production and / or 4-(methylthio)-2-hydroxy -butanal production.

20. An enzyme, wherein the enzyme is a 2-hydroxy acid dehydrogenase / oxidase selected from Table 3.

21. An enzyme, wherein the enzyme is a 2-hydroxy acid dehydrogenase / oxidase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 3, or an amino acid sequence having at least 90% sequence identity thereto.

22. An enzyme, wherein the enzyme is an amino acid dehydrogenase selected from Table 3.

23. An enzyme, wherein the enzyme is an amino acid dehydrogenase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 3, or an amino acid sequence having at least 90% sequence identity thereto.

24. An enzyme, wherein the enzyme is a methionine transaminase selected from Table 3.

25. An enzyme, wherein the enzyme is a methionine transaminase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 3, or an amino acid sequence having at least 90% sequence identity thereto.

26. An enzyme, wherein the enzyme is a methanol dehydrogenase selected from Table 4.

27. An enzyme, wherein the enzyme is a methanol dehydrogenase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 4, or an amino acid sequence having at least 90% sequence identity thereto.

28. An enzyme, wherein the enzyme is an acyl-CoA synthase selected from Table 4.

29. An enzyme, wherein the enzyme is an acyl-CoA synthase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 4, or an amino acid sequence having at least 90% sequence identity thereto.

30. An enzyme, wherein the enzyme is an acyl-CoA transferase selected from Table 5.

31. An enzyme, wherein the enzyme is an acyl- CoA transferase comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 5, or an amino acid sequence having at least 90% sequence identity thereto.

32. An enzyme, wherein the enzyme is an acyl-CoA kinases (ACK) or a phosphoacyltransferases (PTA) selected from Table 6.

33. An enzyme, wherein the enzyme is an acyl-CoA kinases (ACK) or a phosphoacyl transferases (PTA) comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 6, or an amino acid sequence having at least 90% sequence identity thereto.

34. Use of the enzyme of any one of claims 14-34 in the production of desmeninol or methionine.

35. Use of the enzyme of any one of claims 14-19 in the production of 2-hydroxy-4- (methylthio)butyryl-CoA, desmeninol or methionine.

36. The genetically modified microorganisms of any one of claims 1 -9, comprising the enzyme of any one of claims 14-34, optionally the enzyme is overexpressed.

37. Use of the genetically modified microorganisms of any one of claims 1-9 and 36 in the production of desmeninol or methionine.

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