Production of methacrylic acid and methyl-methacrylate via condensation of acetone and formyl-coa

Abstract

This invention provides a non-naturally occurring genetically modified microbial organism that has been engineered to produce methacrylic acid or methyl-methacrylate using a pathway which enables the condensation of formyl-CoA with acetone. This invention also provides a method for the production of methacrylic acid or methyl-methacrylate using a pathway which enables the condensation of formyl-CoA with acetone. This invention additionally provides a method to produce acetone via a) condensation of formyl-CoA and C2 feedstock-derived acetaldehyde form lactoyl-CoA and conversion to acetone, b) conversion of feedstocks to form acetyl-CoA which can be condensed with another acetyl-CoA and decarboxylated to form acetone. and c) conversion of ethylene glycol to acetaldehyde or acetyl-CoA for acetone production where the ethylene glycol generated through condensation of formaldehyde and formyl-CoA which can be derived from other C1 compounds such as methanol, formaldehyde and formate. Further, this invention provides a method for methacrylic acid or methyl-methacrylate production solely from common feedstocks (e.g. sugars or glycerol) through the conversion of feedstock into pyruvate and then to generate acetyl-CoA and formate, of which two acetyl-CoAs are condensed and decarboxylated to form acetone, while the formate molecules provide the formyl-CoA and methanol required for condensation with acetone and esterification to form methyl-methacrylate respectively.

Description

PRODUCTION OF METHACRYLIC ACID AND METHYL-METHACRYLATE VIA CONDENSATION OF ACETONE AND FORMYL-COATECHNICAL FIELD

[0001] The present disclosure generally relates to a method for producing methacrylic acid (MAA) or methyl-methacrylate (MMA), in particular, this disclosure describes several methods for the production of MAA or MMA. Firstly, the present disclosure provides a method for the production of MAA or MMA via condensation of acetone and Cl-derived formyl-CoA. Secondly, the present disclosure provides a method for producing MAA or MMA via condensation of acetone and C 1 -derived formyl-CoA, with the acetone itself being produced via condensation of acetaldehyde and C1-derived formyl-CoA. Thirdly, the present disclosure also provides a method for producing MAA or MMA via condensation of acetone and Cl-derived formyl-CoA, with the acetone being produced via the iterative condensation of Cl-derived formyl-CoA with formaldehyde and acetaldehyde. Fourthly, the present disclosure also provides a method for producing MAA or MMA via the condensation of two acetyl-CoA molecules followed by decarboxylation to form acetone, whereupon the acetone is condensed with formyl-CoA as described previously. The present disclosure also describes a method for generating these two acetyl-CoA molecules from feedstock (e.g. glucose, sugars, glycerol, fatty acid) along with the Cl required for the condensation with acetone as well as for esterification to form methyl-methacrylate. Lastly, the present disclosure also describes a method for generating acetyl-CoA from acetaldehyde which can also itself be produced via condensation of formaldehyde and C1-derived formyl-CoA. The present disclosure also relates to genetically modified microorganisms used for producing MAA and MMA.SUMMARY OF THE INVENTION

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

[0003] In an aspect, provided herein is a method for the production of MAA or MMA through the condensation of acetone and Cl-derived formyl-CoA.

[0004] Embodiment 1. A method for the production of MAA or MMA from acetone and Cl compounds such as methane, methanol, formaldehyde or formic acid comprising al least one of the following:a. production of acetone from two carbon compound (such as ethylene glycol, acetate, ethanol) derived acetaldehyde via condensation of acetaldehyde with Cl-derived formyl-CoA for example in the methods described in any one of Embodiments 21-29;b. production of acetone via condensation of two acetyl-CoA followed by decarboxylation, for example in the methods described in any one of Embodiments 30-35;c. production of acetaldehyde via condensation of formaldehyde with C1-derived formyl-CoA, for example in the methods described in any one of Embodiments 36-44;d. production of acetyl-CoA from common feedstocks (e.g. sugars, glycerol or fatty acid) via splitting of pyruvate into acetyl-CoA and formate by pyruvate formate lyase, providing the acetyl-CoA formed in step (d) for acetone production, for example in the method described in Embodiment 1 step (b) as well as providing the formate formed in step (d) as or for production of a Cl moiety required for production of MAA or MMA, for example in the methods described in Embodiment 1 step (a) and / or Embodiments 45-57; ande. production of acetyl-CoA via condensation of formaldehyde with Cl-derived formyl-CoA, for example in the methods described in any one of the Embodiments 58-59.

[0005] Embodiment 2. The methods of Embodiment 1, comprising generating 2-hydroxyisobutyryl-CoA by contacting acetone and formyl-CoA with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase (HACS), 2-hydroxyacyl-CoA lyase (HACL), oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0006] Embodiment 3. The method of Embodiment 2, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0007] Embodiment 4. The method of Embodiment 3, wherein the TPP-dependent enzyme is selected from Table 2.

[0008] Embodiment 5. The method of Embodiment 2, wherein 2-hydroxyisobutyryl-CoA is converted to MAA and / or MMA by one or more of the following:i. Conversion of 2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyric acid followed by a chemical dehydration to form MAA;ii. Conversion of 2-hydroxyisobutyryl-CoA to methacrylyl-CoA followed by an enzymatic hydrolysis conversion to form MAA;Iiii. Conversion of 2-hydroxyisobutyryl-CoA to MAA, for example through the methods described in Embodiment 5 step (i) or step (ii), followed by a chemical esterification to form MMA; iv. Conversion of 2 -hydroxyisobuty ryl-CoA to methaciylyl-CoA followed by an enzymatic esterification with methanol to form MMA; and v. Conversion of 2-hydroxyisobutyryl-CoA to methyl-2-hydroxyisobutanoate followed by a chemical dehydration to form MMA.

[0009] Embodiment 6. The method of Embodiment 5, wherein in step (i) the 2-hydroxyisobutyryl-CoA is converted to 2-hydroxyisobutyric acid by contacting the 2-hydroxyisobutyryl-CoA with a thioesterase (TE), acyl-CoA transferase (ACT), phosphotransacylase-acid kinase (ACK-PTA), or acyl-CoA reductase-aldehyde dehydrogenase (ACR-ALDH).

[0010] Embodiment 7. The method of Embodiment 5, wherein step (i) further comprises chemical dehydration of 2-hydroxyisobutyric acid to MAA.

[0011] Embodiment 8. The method of Embodiment 5, wherein in step (ii), step (iii), or step (iv) the 2 -hydroxyisobuty ryl-CoA is converted to methacrylyl-CoA by contacting the 2-hydroxyisobutyryl-CoA with a 2-hydroxyacyl-CoA dehydratase (HACD).

[0012] Embodiment 9. The method of Embodiment 8, wherein the 2-hydroxyacyl-CoA dehydratase is selected from Table 8.

[0013] Embodiment 10. The method of Embodiment 5, wherein step (ii) or step (iv) further comprises conversion of methacrylyl-CoA to MAA by contacting methacrylyl-CoA with a thioesterase (TE), acyl-CoA transferase (ACT), phosphotransacylase-acid kinase (ACK-PTA), or acyl-CoA reductase-aldehyde dehydrogenase (ACR-ALDH).

[0014] Embodiment 11. The method of Embodiment 6 and Embodiment 10, wherein the thioesterase is selected from Table 8.

[0015] Embodiment 12. The method of Embodiment 6 and Embodiment 10, wherein the phosphotransacylase-acid kinase is selected from Table 10

[0016] Embodiment 13. The method of Embodiment 6 and Embodiment 10, wherein the acyl-CoA reductase is selected from Table 3.

[0017] Embodiment 14. The method of Embodiment 6 and Embodiment 10, wherein the acyl-CoA transferase is selected from Table 9.

[0018] Embodiment 15. The method of Embodiment 5, wherein in step (iii) the 2 -hydroxyisobuty ryl-CoA is converted to MAA by methods described in any one of Embodiments 6-14 and the MAA is converted to MMA by a chemical esterification with methanol.

[0019] Embodiment 16. The method of Embodiment 8 and Embodiment 9, wherein step (iv) further comprises conversion of methacrylyl-CoA and methanol to MMA by contacting the methacrylyl-CoA and methanol with an ester synthase (AAT).

[0020] Embodiment 17. The method of Embodiment 16, wherein the ester synthase is selected from Table 8.

[0021] Embodiment 18. The method of Embodiment 5, wherein in step (v) the 2-hydroxyisobutyryl-CoA and methanol is converted to methyl-2-hydroxyisobutanoate / methyl-2-hydroxyisobutyrate by contacting it with an ester synthase (AAT).

[0022] Embodiment 19. The method of Embodiment 18, wherein the ester synthase is selected from Table 8.

[0023] Embodiment 20. The method of Embodiment 18, wherein step (v) further comprises conversion of the methyl-2-hydroxyisobutanoate to MMA via a chemical dehydration.

[0024] Embodiment 21. The method of Embodiment 1, wherein step (a) comprises condensation of acetaldehyde and formyl-Co A to form lactoyl-CoA by contacting the acetaldehyde and formyl-CoA with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0025] Embodiment 22. The method of Embodiment 21, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0026] Embodiment 23. The method of Embodiment 22, wherein the TPP-dependent enzyme is selected from Table 2.

[0027] Embodiment 24. The method of Embodiment 21, wherein step (a) further comprises conversion of lactoyl-CoA to 2-hydroxypropionaldehyde / lactaldehyde by contacting the lactoyl-CoA with an acyl-CoA reductase (ACR).

[0028] Embodiment 25. The method of Embodiment 24, wherein the acyl-CoA reductase is selected from Table 3.

[0029] Embodiment 26. The method of Embodiment 24, wherein step (a) further comprises conversion of 2-hydroxypropionaldehyde to 1,2-propanediol by contacting the 2 -hydroxypropionaldehyde w ith an alcohol dehydrogenase (ADH).

[0030] Embodiment 27. The method of Embodiment 26, wherein the alcohol dehydrogenase is selected from Table 4.

[0031] Embodiment 28. The method of Embodiment 26, wherein step (a) further comprises conversion of 1,2-propanediol to acetone by contacting the 1,2-propanediol with a diol dehydratase (DDR).

[0032] Embodiment 29. The method of Embodiment 28, wherein the diol dehydratase is selected from Table 5.

[0033] Embodiment 30. The method of Embodiment 1, wherein step (b) comprises condensation of two acetyl-CoA molecules to form acetoacetyl-CoA by contacting the two acetyl-CoA molecules with a thiolase (THL).

[0034] Embodiment 31. The method of Embodiment 30, wherein the thiolase is selected from Table 6.

[0035] Embodiment 32. The method of Embodiment 30, wherein step (b) further comprises conversion of acetoacetyl-CoA to acetoacetate by contacting the acetoacetyl-CoA with a thioesterase (TE).

[0036] Embodiment 33. The method of Embodiment 32, wherein the thioesterase is selected from Table 8.

[0037] Embodiment 34. The method of Embodiment 32, wherein step (b) further comprises decarboxylation of acetoacetate to acetone by contacting the decarboxylation with an acetoacetate decarboxylase (AAD).

[0038] Embodiment 35. The method of Embodiment 34, wherein the acetoacetate decarboxylase is selected from Table 7.

[0039] Embodiment 36. The method of Embodiment 1, wherein step (c) comprises condensation of formaldehyde and formyl-CoA to form glycolyl-CoA by contacting the formaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0040] Embodiment 37. The method of Embodiment 36, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0041] Embodiment 38. The method of Embodiment 36, wherein the TPP-dependent enzyme is selected from Table 2.

[0042] Embodiment 39. The method of Embodiment 36, wherein step (c) further comprises conversion of glycolyl-CoA to glycolaldehyde by contacting the glycolyl-CoA with an acyl-CoA reductase.

[0043] Embodiment 40. The method of Embodiment 39, wherein the acyl-CoA reductase is selected from Table 3.

[0044] Embodiment 41. The method of Embodiment 39, wherein step (c) further comprises conversion of glycolaldehyde to ethylene glycol by contacting the glycolaldehyde with an alcohol dehydrogenase.

[0045] Embodiment 42. The method of Embodiment 41, wherein the alcohol dehydrogenase is selected from Table 4.

[0046] Embodiment 43. The method of Embodiment 41, wherein step (c) further comprises conversion of ethylene glycol to acetaldehyde by contacting the ethylene glycol with a diol dehydratase.

[0047] Embodiment 44. The method of Embodiment 43, wherein the diol dehydratase is selected from Table 5.

[0048] Embodiment 45. The method of Embodiment 1, wherein step (d) comprises conversion of common feedstocks to pyruvate through native metabolic pathways such as the Embden-Meyerhoff-Pamas, the pentose phosphate pathway or the Entner-Doudoroff pathway.

[0049] Embodiment 46. The method of Embodiment 45, wherein step (d) further comprises conversion of pyruvate to acetyl-Co A and formate by contacting the pyruvate with pyruvate formate lyase (PFL).

[0050] Embodiment 47. The method of Embodiment 46, wherein step (d) further comprises conversion of formate and acetoacetyl-CoA to formyl-CoA and acetoacetate by contacting the formate and acetoacetyl-CoA with an acyl-CoA transferase (ACT). In this embodiment of this pathway, this could replace the need for the overexpression of a thioesterase as described in Embodiment 32.

[0051] Embodiment 48. The method of Embodiment 47, wherein the acyl-CoA transferase is selected from Table 9.

[0052] Embodiment 49. The method of Embodiment 47, wherein step (d) further comprises conversion of formate to formylphosphate by contacting the formate with a formate kinase (FOK).

[0053] Embodiment 50. The method of Embodiment 49, wherein the formate kinase (ACK / FOK) is selected from Table 10.

[0054] Embodiment 51. The method of Embodiment 49, wherein step (d) further comprises conversion of formyl-phosphate to formyl-CoA by contacting the formyl-phosphate with a phosphate acety ltransferase (PT A).

[0055] Embodiment 52. The method of Embodiment 51, wherein the phosphate acetyltransferase is selected from Table 10.

[0056] Embodiment 53. The method of Embodiment 51, wherein step (d) further comprises conversion of formyl-CoA to formaldehyde by contacting the formyl-CoA with an acyl-CoA reductase (ACR).

[0057] Embodiment 54. The method of Embodiment 53, wherein the acyl-CoA reductase is selected from Table 3.

[0058] Embodiment 55. The method of Embodiment 53, wherein step (d) further comprises conversion of formaldehyde to methanol by contacting the formaldehyde with a methanol dehydrogenase (MDH).

[0059] Embodiment 56. The method of Embodiment 55, wherein the methanol dehydrogenase is selected from Table 1.

[0060] Embodiment 57. The method of Embodiment 1, wherein step (e) comprises conversion of acetaldehyde to acetyl-CoA by contacting the acetaldehyde with an acyl-CoA reductase (ACR).

[0061] Embodiment 58. The method of Embodiment 57, wherein the acyl-CoA reductase is selected from Table 3.

[0062] Embodiment 59. The method of any one of the Embodiments 1 to 58, wherein one or more enzyme or each enzyme used is isolated from a microorganism.

[0063] Embodiment 60. The method of any one of the Embodiments 1 to 58, wherein one or more enzyme or each enzyme used in the method is contained in a microorganism.

[0064] Embodiment 61. A genetically modified microorganism providing MAA or MMA by the method of present disclosure, for example a method described in any one of Embodiments 2-60.

[0065] Embodiment 62. A genetically modified microorganism providing acetone by the method of present disclosure, for example a method described in any one of Embodiments 21-35

[0066] Embodiment 63. A genetically modified microorganism providing acetaldehyde by the method of present disclosure, for example a method described in any one of Embodiments 36-44.

[0067] Embodiment 64. A genetically modified microorganisms providing acetyl-CoA by the method of present disclosure, for example a method described in any one of Embodiments 45-59.

[0068] Embodiment 65. A genetically modified microorganism providing 2-hydroxyisobutyrate by the method of present disclosure, for example a method described in any one of Embodiments 5-9.

[0069] Embodiment 66. A genetically modified microorganism providing methyl-2-hydroxyisobutyrate by the method of present disclosure, for example a method described in any one of Embodiments 18-19.

[0070] Embodiment 67. The microorganism of any one of Embodiments 61-65, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

[0071] Embodiment 68. The microorganism of any one of tire Embodiments 61-66, wherein the microorganism is bacteria, yeast or fungi, including but not limited to Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebactenum sp., Zymomonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccharomyc.es sp., Pichia sp., Yarrowia sp., Methyiorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp., and Trichoderma sp.

[0072] Embodiment El. A method of producing methacrylic acid (MAA) and / or methyl-methacrylate (MMA) comprising at least one of the following steps:(a) condensation of acetone and formyl-CoA to form 2 -hydroxy isobutyryl -Co A: (b) conversion of the 2-hydroxyisobutyryl-CoA to MAA via:bi. conversion of 2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyric acid followed by a chemical dehydration to MAA; and / or bii. conversion of 2-hydroxyisobutyryl-CoA to methaciylyl-CoA followed by conversion to MAA; (c) conversion of the 2-hydroxyisobutyryl-Co A to MMA via:ci. conversion of MAA to MMA by chemical esterification with methanol of MAA formed in step (bi) and / or step (bii); cii. conversion of 2-hydroxyisobutyryl-CoA to methacrylyl-CoA followed by conversion to MMA; and / or ciii. conversion of 2-hydroxyisobutyryl-CoA to methyl-2-hydroxyisobutanoate followed by chemical dehydration to MMA; (d) generation of the formyl-CoA from one carbon substrates selected from methane, methanol, formaldehyde, or formic acid; and (e) generation of the acetone from glucose, glycerol, acetate, ethanol, ethylene glycol, fatty acids, or Cl compounds selected from methane, methanol, formaldehyde or formate.

[0073] Embodiment E2. The method of Embodiment El, further comprising the production of acetone from ethylene glycol through thiolase:(a) converting the ethylene glycol to acetaldehyde by contacting the ethylene glycol with a diol-dchydratasc; (b) converting the two acetaldehyde to two acetyl-CoA by contacting the two acetaldehyde with acetaldehyde dehydrogenase (acetylating) (ACDH) or also called acetyl-CoA reductase (ACR); (c) converting the two acetyl-CoA to acetoacetyl-CoA by contacting the two acetyl-CoA with thiolase; (d) converting the acetoacetyl-CoA and formate to acetoacetate and formyl-CoA by contacting the acetoacetyl-CoA and formate with acyl-CoA transferase; and (e) converting the acetoacetate to acetone by contacting the acetoacetate with acetoacetate decarboxylase.

[0074] Embodiment E3. The method of Embodiment El, further comprising the production of acetone from ethylene glycol through 2-hydroxyacyl-CoA synthase:(a) converting the ethylene glycol to acetaldehyde by contacting the ethylene glycol with a diol-dehydratase;(b) condensation of acetaldehyde and formyl-CoA to form lactoyl-CoA by contacting the acetaldehyde and formyl- CoA with a TPP-dependent enzyme selected from 2-hydroxyacyl-Co A synthase, 2-hydroxyacyl-Co A lyase, oxalyl- CoA decarboxylase or benzaldehyde lyase;(c) converting the lactoyl-CoA to 2-hydroxypropionaldehyde by contacting the lactoyl-CoA with an acyl-CoA reductase;(d) converting the 2-hydroxypropionaldehyde to 1,2 -propanediol by contacting the 2-hydroxypropionaldehyde with an alcohol dehydrogenase; and(c) converting the 1,2-propanediol to acetone by contacting the 1,2-propanediol with a diol-dehydratase.

[0075] Embodiment E4. The method of Embodiment El or 2, further comprising the production of ethylene glycol from one carbon compounds:(a) converting one carbon compound to formaldehyde or formyl -CoA(b) converting between formaldehyde and formyl-CoA by contacting with acyl-CoA reductase;(c) condensation of formaldehyde and formyl-CoA to form glycolyl-CoA by contacting the formaldehyde and formyl-CoA with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-Co A lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase;(d) converting the glycolyl-CoA to glycolaldehyde by contacting the glycolyl-CoA with an acyl-CoA reductase;(e) converting glycolaldeh de to ethylene glycol by contacting the glycolaldehyde with gly colaldehyde oxidoreductase;

[0076] Embodiment E5. The method of Embodiment E4, further comprising the production of acetone from a common feedstock:(a) conversion of a common feedstock (e.g., glycerol, sugars, fatty acid, acetate or ethanol) to two pyruvate through a native metabolic pathway selected from the Embden-Meyerhof-Pamas pathway, the pentose phosphate pathway or the Entner- Doudoroff pathway;(b) converting the two pyruvate to two acetyl-CoA and two formate by contacting the two pyruvate with pyruvate formate lyase;(c) converting the two acetyl-CoA to acetoacetyl-CoA by contacting the two acelyl-CoA with thiolase;(d) converting the acetoacetyl-CoA and formate to acetoacetate and formyl-CoA by contacting the acetoacetyl-CoA and formate with acyl-CoA transferase; and(e) converting the acetoacetate to acetone by contacting the acetoacetate with acetoacetate decarboxylase.

[0077] Embodiment E6. The method of any one of Embodiments El-5, wherein the methacrylic acid production from 2-hydroxyisobutyrate comprising:a) dissolving 2-hydroxyisobutyric acid in mineral oil or other solvents;b) subjecting the solution to a dehydration reaction in the presence of a catalyst selected from the group consisting of zeolites, aluminosilicates, or heteropoly acids (e.g., amberlyst 15 sulfonic acid resin); andc) maintaining the reaction at a temperature of about 180 °C.

[0078] Embodiment E7. The method of any one of Embodiments El -6, wherein tire methyl methacrylate production from methacrylic acid comprising:a) mixing methacry lie acid and methanol in mineral oil or other solvents;b) subjecting the solution to a dehydration reaction in the presence of a catalyst selected from the group consisting of zeolites, aluminosilicates, or heteropoly acids (e.g., amberlyst 15 sulfonic acid resin),c) adding a polymerization inhibitor selected from 4-methoxyphenol (MEHQ) or hydroquinone (HQ) (e.g., monomethyl ether of hydroquinone); andd) maintaining the reaction at a temperature of 80 °C with reflux.

[0079] Embodiment E8. The method of any one of Embodiments El-7, wherein the methyl methacrylate production from methyl-2-hydroxyisobutyrate comprising:a) mixing methyl-2 -hydroxyisobutyrate with 1% hydroquinone; andb) subjecting the solution to a dehydration reaction in the presence of a catalyst selected from the group consisting of zeolites, aluminosilicates, or heteropoly acids (e.g., amberlyst 15 sulfonic acid resin).

[0080] Embodiment E9. The method of Embodiment E8., further comprising, after mixing methyl-2 -hydroxyisobutyrate with hydroquinone, maintaining the reaction mixture at a temperature of 120 °C.

[0081] Embodiment E10. The method of Embodiment E8., further comprising, after mixing methyl-2-hydroxyisobutyrate with hydroquinone, adding phosphorus pentoxide after the reaction mixture was cooled down to 0 °C, and optionally further heating up the reaction mixture and maintaining the reaction at 75 °C.

[0082] Also provided herein is an enzyme, selected from:(A) a 2-hydroxyacy-CoA synthase (HACS), wherein:the enzyme is selected from Table 2;the enzyme comprise 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;the enzyme is a 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase derived from microorganisms including but not limited to ApbHACS from Alphaproteobacteria bacterium (Genbank accession: HAK63664.1), DhcHACS from Dehalococcoidia bacterium (Genbank accession: PWB41796.1), CfhHACS from Chloroflexi bacterium (Genbank accession: PKN8I274.1), and PspHACS from Pseudonocardia sp. (Genbank accession: OJY48151.1); orthe enzyme is JGIH12 / PspHACS, BsmHACS, AcHACL or JGI2;(B) an acyl-CoA reductase (ACR), wherein:the enzyme is selected from Table 3;the enzyme is an ACR 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; orthe enzyme is LmACR or StEutE;(C) an alcohol dehydrogenase (ADH), wherein:the enzyme is selected from Table 4;the enzyme comprises 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; orthe enzyme is derived from microorganisms including but not limited to EcDkgB (GenBank accession: NC 000913), EcYahK (GenBank accession: P75691), EcFucO (GenBank accession: P0A9S1), and EcYqhD (GenBank accession: Q46856) derived from Escherichia coli.(D) a diol dehydratase (DDR), wherein:the enzyme is selected from Table 5; orthe enzyme is a DDR comprises 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;(E) a methanol dehydrogenase (MDH) or alcohol oxidase (AOD), wherein:the enzyme is selected from Table 1;the enzyme comprises 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; orthe enzyme is CnMDH or BmMDH;(F) an acyl-CoA synthase (ACS), wherein:the enzyme is selected from Table 1; orthe enzyme comprises 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;(G) an acyl-CoA transferase (ACT), wherein:the enzyme is selected from Table 9; orthe enzyme comprises an amino acid sequence selected from any amino acid sequence as set forth in Table 9, or an amino acid sequence having at least 90% sequence identity thereto; or(H) a combination of acyl-CoA kinase (ACK) and phosphoacyltransferase (PTA), wherein:the enzyme is selected from Table 10; orthe enzyme comprises an amino acid sequence selected from any amino acid sequence as set forth in Table 10 or an amino acid sequence having at least 90% sequence identity thereto.

[0083] Also provided herein are the genetically modified microorganisms provided herein, comprising at least one of the enzymes provided herein.

[0084] Also provided herein is the method provided herein (e g., the method of any one of Embodiments El -10), comprising at least one of the enz mes provided herein or the genetically modified microorganisms provided herein.

[0085] Also provided herein is use of the genetically modified microorganisms provided herein or the enzymes provided herein in the method provided herein (e.g., the method of any one of Embodiments El -10).

[0086] 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 interchangeably with other details.

[0087] The present disclosure is illustrated by the following non-limiting examples.

[0088] 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

[0089] 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.

[0090] Figure 1 is a scheme illustrating the potential routes of production of MAA or MMA from acetone, ethylene glycol (C2), formaldehyde, formate, methanol (Cl) and Cl -derived formyl-CoA.

[0091] MMO: methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; FOK: formate kinase; PTA: phosphate formyltransferase; ACS: acyl-CoA synthase; ACT: acyl-CoA transferase; ACR: acyl-CoA reductase; HACS: 2-hydroxyacyl-CoA synthase; HACD: 2-hydroxyacyl-CoA dehydratase; TE: thioesterase; ACT: acyl-CoA transferase; ACK: acid kinase; ALDH: aldehyde dehydrogenase.

[0092] Figure 2 shows the result of screening of 2-hydroxyacyl-CoA synthase for tire condensation of acetone and formyl-CoA. A) is a scheme illustrating the route for 2-hydroxyisobutyrate production from acetone and formate used for screening; B) is the HPLC chromatograph overlay of 2-hydroxyisobutyrate standard and samples; C) is the 2-hydroxyisobutyrate flux of screened HACS variants.

[0093] HACS: 2-hydroxyacyl-CoA synthase; CaAbfT: acyl-CoA transferase from Clostridium aminobutyricum.

[0094] Figure 3 shows the results of biosynthesis of 2-hydroxyisobutyrate from acetone and one-carbon compounds using growing cells.

[0095] A) shows the production of 2-hydroxyisobutyrate from acetone and formate; B) shows the production of 2-hydroxy isobutyrate from acetone and (parajformaldehyde; C) shows the production of 2-hydroxyisobutyrate from acetone and methanol

[0096] HACS: 2-hydroxyacyl-CoA synthase; CaAbfT: acyl-CoA transferase from Clostridium aminobutyricum; MDH: methanol dehydrogenase; FrmAB: S-(hydroxymethyl)glutathione dehydrogenase (FrmA), S-formylglutathione hydrolase (FrmB)

[0097] Figure 4 shows the results of acetone production from common feedstock.

[0098] A) is a scheme illustrating tire route for acetone production from common feedstocks through acetoacetate; B) result showing acetone production from glucose using growing cells; C) result showing acetone production from acetate using resting cells; D) result showing acetone production from acetate using resting cells.

[0099] Figure 5 shows the result of acetone production from two carbon and one carbon compound.

[0100] A) is a scheme illustrating the route for acetone production from two carbon (ethylene glycol, ethanol, or acetate) and one carbon compound; B) is a scheme illustrating the protocol of 1,2 -propanediol production from acetaldehyde and formate; C) is the result showing lactate production from acetaldehyde and formate with different HACSs; D) is the result showing 1,2-propanediol production from acetaldehyde and formate.

[0101] MMO: methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; FOK: formate kinase; PTA: phosphate formyltransferase; ACS: acyl-CoA synthase; ACT: acyl-CoA transferase; ACR: acyl-CoA reductase; HACS: 2-hydroxyacyl-CoA synthase; ACT: acyl-CoA transferase; ACK: acid kinase; DDR: diol dehydratase; ADH: alcohol dehydrogenase.

[0102] Figure 6 is a scheme illustrating the potential routes of production of acetone (and even methacrylate and methyl methacrylate) from one carbon compound.

[0103] Figure 7 shows the result of ethylene glycol production from formaldehyde.

[0104] A) is a scheme illustrating the route for ethylene glycol from formaldehyde used in this example; B) is the result of evaluating acyl-CoA reductase for ethylene glycol production; C) is the result of evaluating glycolaldehyde oxidoreductase.

[0105] Figure 8 shows the results of 2-hydroxyisobutyrate production from acetate and methanol.

[0106] A) is a scheme illustrating the route for 2-hydroxyisobutyrate production from acetate and methanol used for this example; B) is a result showing 2-hydroxyisobutyrate production from acetate and methanol through different acid activation enz mes: C) is the result showing 2-hydroxyisobutyrate production from glucose and formate using AbfT to activate formate.

[0107] AbfT: acyl-CoA transferase from Clostridium aminobutyricum. ACS: acyl-CoA synthase; FOK-PTA: formate kinasephosphate formyltransferase;

[0108] Figure 9 is a scheme illustrating the production of MMA from glucose wherein the acetone, formyl-CoA and methanol intermediates required are produced from the glucose molecule.

[0109] PFL: pyruvate formate lyase; THL: thiolase; FOK: formate kinase; PTA: phosphate formyltransferase; ACR: acyl-CoA reductase; MDH: methanol dehydrogenase; ACT: acyl-CoA transferase; AAD: acetoacetyl decarboxylase; HACS: 2-hydroxyacyl-CoA synthase; FPR: formyl phosphate reductase; 2HIB-DH: 2-hydroxyisobutyryl-CoA dehydratase; AAT: alcohol acyl transferase

[0110] Figure 10 shows the result of 2-hydroxyisobutyrate production from acetone and formate in a bioreactor using growing cells.

[0111] Figure 11 shows the result of methyl-2-hydroxyisobutyrate production from acetone, formate and methanol.

[0112] A) is the HPLC chromatograph of methyl-2-hydroxyisobutyrate standard and sample; B) is the inducer matrix for methyl-2-hydroxyisobutyratc production from acetone, formate and methanol.

[0113] Figure 12 shows the methacrylate production from 2-hydroxyisobutyrate

[0114] A) is a scheme illustrating the chemical production of methacrylate from 2-hydroxyisobutyrate; B) is the HPLC result of residual 2-hydroxyisobutyrate after reaction; D) is the HPLC result of produced methacrylate.

[0115] Figure 13 shows the methyl methacrylate production from methacrylate.

[0116] A) is a scheme illustrating the production of methyl-methacrylate from methacrylate chemically; B) and C) are the HPLC result of methyl methacrylate from 10 g (B) and 30 g (C) methacrylate.

[0117] Figure 14 is a scheme illustrating tire production of methyl-methacrylate from methyl-2-hydroxyisobutyrate chemically using amberlyst 15 sulfonic acid resin.

[0118] A) is a scheme illustrating the production condition of methyl-methacrylate from methyl-2-hydroxyisobutyrate chemically; B) is the HPLC result of methyl methacry late from mcthyl-2-liydroxyisobiilyrale.

[0119] Figure 15 is a scheme illustrating the production of methyl-methacrylate from methyl-2-hydroxyisobutyrate chemically using P₂O₅.

[0120] A) is a scheme illustrating the production condition of methyl-methacrylate from methyl-2-hydroxyisobutyrate chemically; B) is the GC result of methyl methacrylate from methyl-2-hydroxyisobutyrate (M-2HTB).DETAILED DESCRIPTION OF THE INVENTION

[0121] 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.

[0122] 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.EXAMPLES

[0123] EXAMPLE 1: OVERVIEW OF METHACRYLIC ACID AND METHYL-METHACRYLATE PRODUCTION THROUGH CONDENSATION OF ACETONE AND FORMYL-COA

[0124] This Example demonstrates the implementation of methacrylic acid and methyl-methacrylate production through the Cl+Bio platform by condensation of acetone and formyl-CoA and followed by derivation to methacrylic acid or methylmethacrylate (Figure 1 below). The condensation can be catalyzed by HACS (e.g., HACS set forth in Table 2 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), PspHACS from Pseudonocardia sp. (Genbank accession: OJY48151.1), to generate 2-hydroxyisobutyryl-CoA. 2-hydroxyisobutyryl-CoA can be further converted to methacrylic acid or methyl-methacrylate through a combination of dehydration, CoA hydrolysis and / or esterification steps which can be performed in any order with a set of intermediates of 2 -hydroxy isobutyrate, methacrylyl-CoA, or methyl-2-hydroxyisobutyrate (Figure 1).

[0125] In one possible route, 2-hydroxyisobiityiyl-CoA can be converted to 2-liydroxyisobiityratc through the action of a thioesterase (such as TesB from E. colt or Pseudomonas putida (McMahon, M. D. and Prather, K. L. J. Appl. Environ. Microbiol.2014, 80:1042-1050)), CoA transferase (ACT, e.g., set forth in Table 9 below), or phosphotransacylase-acid kinase (PTA-ACK, e.g., set forth in Table 10 below). Alternatively, 2-hydroxy isobutyryl-CoA is reduced to 2-hydroxyisobutyraldehyde by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GeneBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 3 below), followed by oxidation of 2-hydroxyisobutyraldehyde to 2-hydroxyisobutyrate with the help of aldehyde dehydrogenase (ALDH). 2-hydroxyisobutyrate can be further converted to methacrylic acid and methyl-methacrylate chemically.

[0126] Alternatively, 2-hydroxyisobutyryl-CoA can be converted to mcthacrylyl-CoA through the action of 2-hydroxyacyl-CoA dehydratase (HACD, e.g., set forth in Table 8 below). Methacrylyl-CoA can be converted to methaciylic acid through the action of a thiocstcrasc (such as TesB from E. coli or Pseudomonas putida (McMahon, M. D. and Prather, K. L. J. Appl. Environ. Microbiol.2014, 80:1042-1050)), CoA transferase (ACT, e.g., set forth in Table 9 below), or phosphotransacylase-acid kinase (PTA-ACK, e.g., set forth in Table 10 below). Alternatively, mcthacrylyl-CoA is reduced to mcthacrylaldchydc by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GeneBank accession: P41793) or other ACR variants (ACR, e g., set forth in Table 3 below), followed by oxidation of methacrylaldehyde to methacrylic acid with the help of aldehyde dehydrogenase (ALDH). Methacrylic acid can be further converted to methyl -methacr late chemically (Figure 13).

[0127] Alternatively, 2-hydroxyisobutyryl-CoA can be converted to nicthyl-2-liydroxyisobiilyratc can be obtained by transesterification with the help of ac ltransferase (AAT, e.g., set forth in Table 8). Methy 1-2 -hydroxy isobuty rate further converted to methyl-methacrylate chemically (Figure 14).

[0128] Acetone can be produced from common feedstocks (e.g. sugars, glycerol), fatty acid, two carbon compound (e.g. acetate, ethanol, ethylene glycol) through acetoacetate or 1,2 -propanediol as described below.

[0129] EXAMPLE 2: THE STANDARD PROTOCOL OF RESTING CELLS BIOCONVERSION FOR ENZYME PERFORMANCE EVALUATION

[0130] The purpose of this example is to establish a standard protocol for resting cells bioconversion to evaluate enzymes activity. We engineered vectors to independently control expression of HACS variants and the formyl-CoA generation genes (LmACR or CaAbfT), with HACS under control of the IPTG-induciblc T7 promoter in pCDFDuct-1 and LmACR or CaAbfT under control of a cumate-inducible T5 promoter in pETDuet-1 (Figure 2A). As a host for these vectors, we used an engineered strain of E. coll based on MG1655(DE3) with knockouts for formaldehyde (A / rn / ,4) and formate (EfdhF kfdnG AfdoG) oxidation as well as for glycolate utilization (Ag / cD), which we expected could compete or interfere with the analysis of our pathway.

[0131] In vivo product synthesis was conducted using M9 minimal media (6.78 g / L Na₂HPO₄, 3 g / L KH2PO4, 1 g / L NH4C1, 0.5 g / L NaCl, 2 mM MgSO₄, 100 μM CaCl₂, and 15 μM thiamine-HCl) 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 lirs) in LB medium with appropriate antibiotics and used as the inoculum (1%). Antibiotics (100 pg / mL carbenicillin, 100 μg / 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 β-D-1-thiogalactopyranoside (IPTG) and cumate) were added. Plates were incubated for a total of 24 hrs.

[0132] 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 100 mM acetone and 20 mM formate. Cells were incubated at 30°C and 1000 rpm in Digital Microplate Shaker (Fisher Scientific). The cells were harvested after 3 hours by centrifugation and the supernatant analyzed by HPLC or GC-MS.

[0133] Cell pellets harvested after bioconversion were resuspended to 20 OD in B-PER® Bacterial Protein Extraction Reagent (Thermo Fisher) supplemented with 0.1 mg / mL chicken egg white lysozyme (Fisher) and 5 U / mL Bcnzonasc® nuclease (Sigma) for cell lysis. After incubation in room temperahire for 15 minutes, 100 μL of each cell lysate was transferred to 1.5 mL microcentrifuge tubes for centrifugation at 15,000 xg 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.

[0134] Quantification of product and substrate concentrations (formic acid, formaldehyde, and glycolic acid) 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, C A) with operating conditions to optimize peak separation (0.3 ml / niin flowrate, 30 mM H₂SO₄ mobile phase, column temperature 42°C). Compound identification and analysis was performed by GC-MS using an Agilent 7890B Series Custom Gas Chromatography system equipped with a 5977B Inert PlusMass Selective Detector Turbo El Bundle (for identification) and an Agilent HP-5-ms capillary column (0.25 mm internal diameter, 0.25 pm film thickness, 30 m length).

[0135] EXAMPLE 3: PRODUCTION OF 2-HYDROXYISOBUTYRATE FROM ACETONE AND ONE-CARBON COMPOUNDS

[0136] This example demonstrates the 2-hydroxyisobutyrate production from acetone and formyl-CoA derived from formate using resting cells. When 2-hydroxyisobutyrate is produced, it can be further converted to methacrylic acid or methyl-methacrylate.

[0137] Acetone can be directly condensed with formyl-CoA to generate 2-hydroxyisobutyryl-CoA (Figure 4A below). The condensation of acetone and formyl-CoA can be catalyzed by HACS (c.g., HACS set forth in Table 2 below), including but not limited to beach sand metagenome BsmHACS (Genbank accession: HAK63664. 1), DhcHACS from Dehalococcoidia bacterium (Genbank accession: PWB41796.1), CfhHACS from Chloroflexi bacterium (Genbank accession: PKN81274.1), PspHACS from Pseudonocardia sp. (Genbank accession: OJ Y48151.1 ). 2-hydroxyisobutyryl-CoA is subsequently converted to 2-hydroxyisobutyrate as described in Example 1.

[0138] The whole-cell bioconversion of 2-hydroxyisobutyrate production from acetone and formate was carried out to demonstrate the feasibility of this pathway. This was tested in E. coli strain MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD) expressing several functional enzymes acyl-CoA transferase from Clostridium aminobutyricum (AbfT), 2-hydroxyacyl-CoA synthases (Figure 2A). The E. coli cells were cultured in LB medium with 2% glycerol at 37 °C for 4 hours with a shaking speed of 1000 rpm. Subsequently, (lie cells were transferred into NBS medium with 2% glycerol at 30 °C for 16 h with a shaking speed of 1000 rpm. The cells were then transferred into NBS medium with 1.5% glucose for 4 h, subsequently, IPTG with the final concentration of 0.05 mM was added, and the culture was further incubated at 30 °C for 24 hours to induce protein expression. The cells were centrifuged at 4,000 rpm for 8 minutes, and the supernatant was discarded. 1 mL M9 wash buffer was added to the 2 mL deep well plate, and the mixture was thoroughly mixed. This washing step was repeated twice. The cells were resuspended in the M9 wash buffer. Acetone and formate were initially added to the reaction at final concentrations of 100 rnM and 20 mM, respectively. The mixture was incubated at 30 °C for 3 hours with a shaking speed of 1000 rpm. The sample of the reaction mixture was collected for HPLC analysis. The 2-hydroxyisobutyrate was identified and quantified by HPLC (Figure 2B), indicating the pathway for 2-hydroxyisobutyrate from acetone and formate is reliable. Through the in vivo bioconversion, the strain overexpressing JGIH12 / PspHACS has the best performance with a flux of 110 pM / OD / h 2-hydroxyisobutyrate in 3 hours (Figure 2C below).

[0139] EXAMPLE 4. PRODUCTION OF 2-HYDROXYISOBUTYRATE FROM ACETONE AND ONE-CARBON COMPOUNDS USING GROWING CELLS

[0140] This example demonstrates the 2-hydroxyisobutyrate production from acetone and formyl-CoA derived from one-carbon compounds including, but not limited to methanol, formaldehyde or formate using growing cells.

[0141] When acetone and formate were used as the feedstock, it has the same route as described above. Formate was first activated to formyl-CoA by acyl-CoA transferase (ACT, e.g. AbfT set forth in Table 9), and then condensed with acetone with the help of HACS to generate 2-hydroxyisobutyryl-CoA, it finally converted to 2-hydroxyisobutyrate by thioesterase. When (para)formaldehyde was used as the formyl-CoA source, (para)formaldehyde will slowly release to be formaldehyde and converted to formate through the native detoxification system including FrmA and FrmB in E. coli, and then further converted to formyl-CoA for condensation with acetone to produce 2-liydrox isobutyrate. When methanol was used as the formyl-CoA source, methanol dehydrogenase from Bacillus methanolicus MGA3(BmMDH or from Cupriavidus necator (CnMDH) was introduced to convert methanol to formaldehyde and then converted to formyl-CoA for condensation to produce 2-hydroxyisobutyrate.

[0142] This was tested in E. coli strain MG1655(DE3) del (fdhF, fdnG, fdoG) expressing BsmHACS or HACL from Actinomycetospora chiangmaiensis (AcHACL). The E. coli cells were cultured in LB medium with 2% glycerol at 37 °C for 4 hours with a shaking speed of 1000 rpm. Subsequently, the cells were transferred into NBS medium with 0.5% glucose at 30 °C for 16 h with a shaking speed of 200 rpm. The cells were then transferred into NBS medium with 0.5% glucose for 4 h, subsequently, IPTG with the final concentration of 0.05 M was added, acetone and one-carbon compound were added immediately after induction, the culture was further incubated at 30 °C for 48 hours for 2-hydroxyisobutyrate. The sample of the reaction mixture was collected by centrifugation for HPLC analysis. The 2-hydroxyisobutyrate was identified and quantified by HPLC.

[0143] The results showed that BsmHACS has better performance than AcHACL, CnMDH has better performance than BmMDH. Up to 3 mM 2-hydroxyisobutyrate was successfully produced from acetone with formate, paraformaldehyde and methanol (Figure 3).

[0144] EXAMPLE 5. PRODUCTION OF ACETONE FROM COMMON FEEDSTOCKS

[0145] This example demonstrates the acetone production from common feedstocks, including but not limited to glucose, glycerol and other sugars. Acetone can be used for methacrylic acid and methyl-methacrylate production. This example demonstrates amethod of acetone production from acetyl -Co A which can be derived from traditional carbon feedstocks such as glucose or glycerol through central metabolism, from other C2 compounds such as acetate, ethanol or ethylene glycol, or produced from Example 1.

[0146] The common feedstocks were first converted to acetyl-CoA through central metabolism including glycolysis and / or glycerol utilization pathways. When ethylene glycol is used as the feedstock, it was first dehydrated to generate acetaldehyde by dehydratase (DDR1, set forth in Table). Acetaldehyde is then converted to acetyl-CoA by ACR (e.g. set forth in Table 3). When acetate was is used, acetate is first activated to acetyl-CoA by acetyl-CoA synthase (ACS), acyl-CoA transferase (ACT) or ACK-PTA. Two acetyl-Co As are condensed to generate acetoacetyl-CoA via the action of an overexpressed thiolase, acetoacetyl-CoA is further converted to acetoacetate by an overexpressed thioesterase or acyl-CoA transferase, and finally converted to acetone by an overexpressed acetoacetate decarboxylase (Figure 4A).

[0147] In this example, we used glucose, glycerol and acetate as representative feedstocks for the generation of acetone through acetyl-CoA. To produce acetone in E. coli, thiolase ThlA, acetoacetate decarboxylase ADC from Clostridium acetobutylicum and acyl-CoA transferase AtoDA from E. coli were co-overexpressed in E. coli MG1655(DE3) del (frmA, fdhF, fdnG. fdoG, glcD). The A coll cells were cultured in LB medium with 2% glycerol at 37 °C for 4 hours with a shaking speed of 200 rpm. Subsequently, the cells were transferred into NBS medium with 0.5% glucose at 30 °C for 16 h with a shaking speed of 200 rpm. The cells were then transferred into NBS medium with 1% to 3% glucose for 4 h, subsequently, cumate was added to induce the protein expression with the final concentration of 0.04 mM to 0.4 mM. Acetone accumulation was quantified by HPLC. When glucose was used as the feedstock, up to 140 mM acetone was produced when the cells were induced with 0.2 mM cumate with 30 g / L glucose after 5 days fermentation (Figure 4B). When acetate was used as the feedstock, acetone producing module from acetyl-CoA was cooverexpressed with acetate activation enzymes, including acetyl-CoA synthase (ACS, set forth in Table 1), acyl-CoA transferase (ACT, set forth in Table 9) or ACK-PTA (set forth in Table 10).100 mM acetate was added to the medium as the feedstock, up to 20-25 mM acetone was produced in all tested strains (Figure 4C). When glycerol was used as the feedstock, we further explored the relative protein expression of acetone producing module by using orthogonal promoters (SH Lee et al., Metabolic engineering 82, 262-273), CaADC under vanillate inducible promoter pV, AtoDA under 2,4-diacetylphophloroglucinol (DAPG) inducible promoter pP, CaThlA or AtoB under the cumate inducible promoter pC. Inducer matrix was performed, and 4.5 mM acetone was produced from glycerol in 24 h fermentation when the cells were over-expressed with 50 pM cumate, 50 pM vanilate and 10 or 50 M DAPG. The strain harboring thiolase CaThlA has better performance than the one has EcAtoB (Figure 4D).

[0148] EXAMPLE 6. PRODUCTION OF ACETONE FROM ETHYLENE GLYCOL AND FORMYL-COA FROM ONE CARBON COMPOUNDS.

[0149] This example is to demonstrate acetone production from ethylene glycol and formyl-CoA from one carbon compounds.

[0150] In this design, two carbon compounds are first converted to acetaldehyde. Ethylene glycol was converted to acetaldehyde by dehydratase (DDR1, set forth in Table 5) as reported (Chou et al., Nature Metabolism 3 (10), 1385-1399), ethanol can be converted to acetaldehyde by alcohol dehydrogenase (Table 4), acetate needs to be activated to acetyl-CoA by acetyl-CoA synthase or through ACK-PTA, acetyl-CoA can further converted to acetaldehyde by acyl-CoA reductase (Table 3).

[0151] Acetaldehyde is condensed with formyl-CoA, which can be generated as described in Example 1, through the activity of an overexpressed 2-hydroxyacyl-CoA synthase (Table 2) to form lactoyl-CoA which is then converted to 2- hydroxypropionaldehyde / lactaldehyde through the activity of an overexpressed acyl-CoA reductase (Table 3). 2- hydroxypropionaldehyde is then converted to 1,2 -propanediol through contact with an overexpressed alcohol dehydrogenase (Table 4), and finally 1,2 -propanediol is converted to acetone by contacting it with a diol dehydratase (Table 5). Alternatively, acetaldehyde is converted to acetyl-CoA, two acetyl-CoAs condensed together to generate acetoacetyl-CoA by thiolase, acetoacetyl-CoA is further converted to acetoacetate by thiocstcrasc or acyl-CoA transferase, and finally converted to acetone by acetoacetate decarboxylase (Figure 5 A). And then the acetone can be used for methacrylic acid and methyl methacrylate production as Example 1 described.

[0152] This can be done using the same protocol as described in Example 2 in E. coli strain MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD) expressing the functional enzymes mentioned above.

[0153] This was also demonstrated using purified enzymes. BL21 (DE3) harbouring plasmid to overexpress enzymes AbfT, HAGS, RpPduP, EcFucO were first cultured in LB and induced with IPTG to obtain overexpressed enzymes. Cells were lysed using sonication and purified using Ni-NTA agarose column. Firstly, purified enzymes were used for in vitro assay to screen HACS for acetaldehyde and formate. 20 mM formate, 2 pM CaAbfT and 2 mM acetyl-CoA were added into the reaction system, after react 2 min at 30 °C, 1 pM HACS was added and react for another 3 min, NaOH was added to hydrolysis the acy 1-CoA. pH was adjusted to pH 7 with HC1 and samples were analysed by HPLC. Amount the tested HACS, JGI23 was found to have better performance than other HACSs (Figure 5C). JG123 was then used to produce 1,2-propanediol from acetaldehyde and formate. 20 mM formate.2 uM CaAbfT and 2 mM acetyl-CoA were added into the reaction system, after reacting for 2 min at 30 °C, 1 pM HACS was added and react for another 3 min for lactoyl-CoA production. 1 pM of RpPduP and EcFucO were further added to convert lactoyl-CoA to 1,2-propanediol, and react for another 7 min (Figure 5B). 0.35 mM 1,2-propanediol was produced in 7 min with a flux of 4.1 mM / h (Figure 5D).

[0154] EXAMPLE 7. PRODUCTION OF ACETONE FROM ONE CARBON COMPOUNDS.

[0155] This example is to demonstrate acetone production from one carbon compounds, including but not limited to methanol, formaldehyde, formate.

[0156] When acetone is produced from methanol or other Cl compounds, the Cl compounds arc first converted to formaldehyde and formyl-CoA as illustrated in Figure 1 with enzymes shown in Table 1. Condensation of formaldehyde andformyl-CoA occurs through the activity of ovcrcxprcsscd 2-hydroxyacyl-CoA synthase (Table 2), forming glycolyl-CoA. Glycolyl-CoA is converted to glycolaldehyde through the activity of an overexpressed acyl-CoA reductase (Table 3), which can then be converted to ethylene glycol through the activity of an ovcrcxprcsscd alcohol dehydrogenase (Table 4). Subsequently, ethylene glycol is converted to acetaldehyde through the activity of an overexpressed diol dehydratase (Table 5)

[0157] The acetaldehyde can then be converted to acetone for either MAA or MMA production through two potential routes. Firstly, it could be further condensed with one additional formyl-CoAs generated from Cl compounds to first form lactoyl-CoA and then 1,2-propanediol which can finally be converted to acetone as presented in Figure 6 below. Alternatively, it can be converted to acetyl-CoA which can be condensed with a second acetyl-CoA to produce acetoacetate, which can be decarboxylated to generate acetone as presented in Figure 6 below.

[0158] The whole-cell bioconversion of acetone production from one carbon compound was tested in E. coli strain MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD) expressing several functional enzymes. The E. coli cells were cultured in LB medium with 2% glycerol at 37 °C for 4 hours with a shaking speed of 1000 rpm. Subsequently, the cells were transferred into NBS medium with 2% glycerol at 30 °C for 16 h with a shaking speed of 1000 rpm. The cells were then transferred into NBS medium with 1.5% glucose for 4 h, subsequently, IPTG with the final concentration of 0.05 mM was added, and the culture was further incubated at 30 °C for 24 hours to induce protein expression. The cells were centrifuged at 4,000 rpm for 8 minutes, and the supernatant was discarded. 1 mL M9 wash buffer was added to the 2 mL deep well plate, and the mixture was thoroughly mixed. This washing step was repeated twice. The cells were resuspended in the M9 wash buffer. One carbon compounds were initially added to the reaction at final concentrations of 100 mM. The mixture was incubated at 30 °C for 3 hours with a shaking speed of 1000 rpm. The sample of the reaction mixture was collected for HPLC analy sis. The acetone was identified and quantified by HPLC.

[0159] EXAMPLE 8. PRODUCTION OF ETHYLENE GLYCOL FROM ONE CARBON COMPOUNDS.

[0160] This example is to demonstrate the ethylene glycol for acetone production can be generated from one carbon compounds, including but not limited to methanol, formaldehyde, formate. One carbon compounds can interconvert to formaldehyde and formyl-CoA as presented in Figure 1. Here, formaldehyde was selected as an example for ethylene glycol production.

[0161] Formaldehyde is converted to formyl-CoA by acyl-CoA reductase (ACR, set forth in Table 3), the HACS is used to condense formaldehy de with formyl-CoA forming glycol l-CoA. Glycolyl-CoA can be further reduced to glycolaldehyde via acyl-CoA reductase. In this case the same enzyme (LmACR) used for reduction of formyl-CoA to formaldehyde can catalyze reduction of glycolyl-CoA to glycolaldehyde (Chou et al. Nat. Metab. 3:1385-1399 (2021)). Glycolaldehyde reduction to ethylene glycol is catalyzed by E. coli fucO. Dehydration of ethylene glycol gives acetaldehyde, catalyzed by PddABC from Klebsiella oxytoca. The resulting acetaldehyde can be fed to the subsequent iteration of Cl elongation or converted to acetyl-CoA for acetone production (Figure 7).

[0162] For ethylene glycol production, glycolaldehyde dehydrogenases aldA, aldB, pUUC, patD were further knocked out in MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD) to eliminate the glycolate production. Different acyl-CoA reductase (ACR) and glycolaldehyde oxidoreductases were tested for ethylene glycol production. The bioconversion was done the same as described above. When 10 mM formaldehyde was used as the substrate. Up to 2.2 mM ethylene glycol was produced from formaldehyde, co-overexpression of LmACR, StEutE and FucO was found to be a good combination for ethylene glycol production (Figure 7).

[0163] EXAMPLE 9. PRODUCTION OF 2-HYDROXYISOBUTYRATE FROM ACETATE AND METHANOL.

[0164] This example is to demonstrate the combination of acetone producing module and further condensation with formyl-CoA to generate 2-hydroxyisobutyrate. Acetate was selected as the feedstock for acetone production, and methanol was used to produce formyl-CoA as an example.

[0165] Acetate is first activated acetyl-CoA and two acetyl-CoAs can be converted to acetone through thiolase as described in example 5, meanwhile, methanol can be converted to formyl-CoA for the further condensation with acetone to produce 2-hydroxyisobutyrate (Figure 8A). This was tested inE. coli AC440 [MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD)\, three acetateactivation routes were introduced as described in example 5. When 50 mM acetate and 500 mM methanol was added to the system, about 0.6-0.8 mM 2-hydroxyisobutyrate was accumulated (Figure 8B).

[0166] EXAMPLE 10. PRODUCTION OF 2-HYDROXYISOBUTYRATE FROM GLUCOSE AND METHANOL.

[0167] This example is to demonstrate the combination of acetone producing module and further condensation with formyl-CoA to generate 2 -hydroxy isobuly rate, glucose was selected as the feedstock for acetone production, and methanol was used to produce formyl-CoA as an example.

[0168] Glucose is first converted to acetyl-CoA through glycolysis and then two acetyl-CoAs can be converted to acetone through thiolase as described in Example 5, meanwhile, methanol can be converted to formyl-CoA for further condensation with acetone to produce 2-hydroxyisobutyrate (Figure 8A). This was tested in E. coli AC440 [MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD)] as described above. An orthogonal inducer system such as described in Example 5 can be used with an inducer matrix, 20 g / L glucose and 20 mM formate were added right after induction, and more than 50 mM 2-hydroxyisobutyrate was produced within 48hours fermentation (Figure 8C), indicating formate was also produced from glucose with limited oxygen during the fermentation.

[0169] EXAMPLE 11: PRODUCTION OF METHYL-METHACRYLATE FROM GLUCOSE

[0170] Here we include a novel design for the production of methyl-methacrylate from common feedstock (e.g. sugars or glycerol) taking advantage of formyl-CoA condensation to achieve a maximum theoretical yield of 83% on a carbon-molar basis (Figure 9).Here glucose was chosen as an example of the feedstock, initially this begins with the metabolism of glucose to two molecules of pyruvate, utilizing native pathways such as the Embden-Meyerhof-Parnas or Entner-Doudoroff pathways. Pyruvate formate lyase splits each molecule of pyruvate into one molecule of acetyl-CoA and one molecule of formate. Similar to the method described in Example 3, both acetyl-CoA molecules are then condensed via an overexpressed thiolase (Table 6) to fonn acetoacetyl-CoA. Acetoacetyl-CoA is then converted to acetoacetate via an overexpressed acyl-CoA transferase (Table 9) which can concurrently convert one of the previously formed formate molecules into a formyl-CoA. Acetoacetate is decarboxylated by an overexpressed acetoacetate decarboxylase (Table 7) to form acetone. Acetone is then condensed with the formyl-CoA to form 2-hydroxyisobutyryl-CoA via an overexpressed HACS (Table 2), which is then converted to mcthacrylyl-CoA via an overexpressed 2-hydroxyisobutyryl-CoA dehydratase. At the same time, the second formate molecule formed from pyruvate can be converted to formyl-phosphate via the activity of an overexpressed or native formate kinase. Formyl-phosphate can be converted to formyl-Co A through the activity of a native or overexpressed phosphate acetyltransferase (PTA), and formyl-CoA can be converted to formaldehyde via an overexpressed acyl-CoA reductase (Table 3). Alternatively, formyl-phosphate can be converted to formaldehyde directly be formyl phosphate reductase (Nattermann et al., Nature Communications 14 (1), 2682). And then formaldehyde can subsequently be converted to a methanol via an overexpressed methanol dehydrogenase (Table 1). Lastly, this methanol can be combined with previously formed methacrylyl-CoA via an overexpressed alcohol acyl transferase (Table 8) to form methyl-methacrylate.

[0171] EXAMPLE 12: PRODUCTION OF 2-HYDROXYISOBUTYRATE FROM ACETONE AND FORMATE IN BIOREACTOR

[0172] This example is to demonstrate the scalability of the design, and 2-hydroxyisobutyrate production from acetone and formate was chosen as an example to do the fermentation in 0.75 L bioreactor.

[0173] Fresh transformed strain AC440 harboring plasmids to overexpress BsmHACS and CaAbfT was used for fermentation. The seed culture was obtained by inoculating 10 colonies into 5 mL of LB medium with antibiotics and cultured at 30 °C, 200 rpm overnight. 4 mL of seed culture was inoculated into 0.75 L bioreactor with 0.4 L M9-LB medium with antibiotics. The fermentation was done at 30 °C, with an air flow of 33 mL / min, the DO was controlled to higher than 40% by adjusting agitation from 200 rpm to 800 rpm if possible. After 2.5 h, inducer was added to induce the protein expression. 100 mM acetone and 20 mM formate were added after induction, the pH was controlled to be lower than pH 7 by adding glucose. 50 mM acetone was manually added when the residual acetone is around 50 mM.

[0174] Due to continuous feeding of glucose, cells were continuing to grow during the fermentation, and up to OD 15 was obtained with a final 2-hydroxyisobutyrate titer of 10 mM (which equals to 1.04 g / L) after 173 h fermentation (Figure 10).

[0175] EXAMPLE 13: PRODUCTION OF METHYL-2-HYDROXYISOBUTYRATE FROM ACETONE, FORMATE AND METHANOL

[0176] This example is to demonstrate methyl-2-hydroxyisobutyrate production from acetone, formate and methanol. Acetone was first condensed with formyl-CoA generated from formate to obtain 2-hydroxyisobutyryl-CoA, methyl-2-hydroxyisobutyrate was then produced through esterification of 2-hydroxyisobutyryl-CoA with methanol by acyltransferase.

[0177] In this example, WS / DGAT was co-overexpressed with HACS and AbfT under the control of T7 or CT5 promoter for esterification. The bioconversion was done the same as described above, inducer matrix was performed to during the cell growthand protein expression stage. 16 mg / L methyl-2-hydroxyisobutyrate was accumulated when 0.025 mM IPTG and 0.2 mM cumate were added for induction, the cells have better performance when WS / DGAT was controlled by T7 promoter (Figure 11).

[0178] EXAMPLE 14: PRODUCTION OF METHACRYLIC ACID FROM 2-HYDROXYISOBUTYRATE CHEMICALLY

[0179] This example is to demonstrate the chemical conversion of 2-hydroxyisobutyrate to methacrylic acid through dehydration.

[0180] 10 g of 2-hydroxyisobutyric acid was dissolved in 20ml mineral oil as the starting material. Amberlyst 15 sulfonic acid resin was added for high-temperature dehydration with an internal temperature of 180 °C, followed by an atmospheric distillation, the samples were then analyzed by HPLC. 3.5 g methacrylic acid (equals to 40% of maximum theoretical yield) was successfullyobtained with a Karl Fischer value of 31.87% (Figure 12).

[0181] EXAMPLE 15: PRODUCTION OF METHYL METHACRYLATE FROM METHACRYLATE AND METHANOL CHEMICALLY

[0182] This example is to demonstrate methyl methacrylate from methacrylate and methanol using chemical processes.

[0183] One equivalent of methacrylate was mixed with 0.9 equivalent of methanol, and diluted with 2 volumes of mineral oil. Two mass equivalents of Amberlyst 15 sulfonic acid resin (>4.7 mmol / g) was added for the chemical conversion. Monomethyl Ether of Hydroquinone (4-methoxyphenol, MEHQ) which is a polymerization inhibitor to prevent polymerization was added to reaction mix. The internal temperature was set to 80 °C, the reaction mix was refluxed for 1-2 hours. When the residual methanol is less than 1%, vacuum concentration was performed by coupling with low -temperature cooling and cold trap collection. The product was further dried by sodium sulfate, samples were analyzed by HPLC after filtration. 7 g methyl methacrylate was obtained from 10 g methacrylate with a yield of 66% when the reflux was only done for 1 hour (Figure 13B). 28.5 g methyl methacrylate was obtained from 30 g methacrylate with a yield of 85% when the reflux time was extended to 2 hours (Figure 13C).

[0184] EXAMPLE 16: PRODUCTION OF METHYL METHACRYLATE FROM METHYL-2-HYDROXYISOBUTYRATE AND METHANOL CHEMICALLY

[0185] This example is to demonstrate methyl methacrylate from methyl-2-hydroxyisobutyrate and methanol using chemical processes.

[0186] 10 g of methyl-2-hydroxyisobutyrate and 1% hydroquinone were mixed with 0.5 mass equivalents of Amberlyst 15 sulfonic acid resin. The mixture was heated up to 120 °C by oil bath, the pressure is maintained at atmospheric pressure for 2 hours, 21.2% purity of MMA was observed by HPLC analysis. The product was distilled out through vacuum distillation, finally 2.5 g MMA was obtained with a Karl Fischer value of 11.7% (Figure 14).

[0187] Alternatively, 4 g of methyl-2 -hydroxyisobutyrate was mixed with 1% hydroquinone, cool the mixture to 0 °C. Add 0.5 equivalents of phosphorus pentoxide (P2O5), allow the temperature to rise to 15-20 °C with a target set to 18 °C, and then cool back down to 0 °C. And then heat the mix up to 75 °C, and maintain it for 3-4 hours. The reaction was monitored by the level of residual methyl-2-hydroxyisobutyrate through GC analysis. The reaction was stopped when all the starting material was less than 5% (0.2 g), and 83% purity of methyl methacrylate was observed through GC analysis with 4% of residual substrate methyl-2-hydroxyisobutyrate. When the 15 g of methyl-2-hydroxyisobutyrate was used as the starting material, 84% purity of methyl methacrylate was observed through GC analysis with 3.3% of residual substrate methyl-2-hydroxyisobutyrate (Figure 15). Vacuum distillation was further performed, while a high degree of polymerization in the reactor happened.

[0188] Table 1. List of enzymes used for the interconversion of one-carbon compounds to generate formyl-CoA Reaction Gene Genbank Accession Number sMMO mmoXYBZCD P18797G1UBD1 pMMO pmoA1A2B1B2Q607G3 BmMDH2 I3E2P9 CnMDH2 F8GNE5 MDHBsMDH P42327 MeMxaF-Mxal P16027, P14775 CbAODl Q00922 PaMOX P04841 PpAOXl P04842 AODPpAOX2 F2R038 PmMODl Q9UVU2 PmMOD2 Q9UVU1 CbCTA Q96VB8 CTA PaPXP9 P30263PpCTAl C4R2S1PmCTAl E0D5F4EcACS P27550BsACS P39062ACS StACSstab Q8ZKF6 PROSSMhACS F2NQX2ArACS A0A0Q7JEV7Note: sMMO: soluble methane monooxygenase; pMMO: particulate methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; ACS: acyl-CoA synthase.

[0189] Table 2. List of 2-hydroxyacyl-CoA synthase (HACS) used for the condensation of 3-hydroxyaldehyde and formyl-CoAGen Bank Accession Gen Bank Accession Gen Bank Accession HACS#Number lifjiioii Number lilliOli Number1 XP 012756082.1 47 MXY78649.1 93 TAJ19927.1 2 TMK01573.1 48 MBA01399.1 94 PKN81274.1 3 PYM26381.1 49 MXX31676.1 95 RLT34960.1 4 EEG70177.1 50 MXV80929.1 96 MBT5775398.1 5 MBH80817.1 51 MBI4083577.1 97 TMD99851.1 6 WP 030891887.1 52 MBK6319978.1 98 MSQ12864.1 7 AGK93615.1 53 MBI5948182.1 99 MBL0714078.1 8 MAX57815.1 54 PFG74273.1 100 WP 114297888.1 9 WP 068916287.1 55 WP 158065972.1 101 MAK25262.1 10 WP 062165271.1 56 MBN9492325.1 102 WP 068138361.1 11 MBB43458.1 57 MBK6663287.1 103 RMG94145.1 12 PCJ72347.1 58 MBT2766664.1 104 MBA4180234.1 13 TMQ19149.1 59 HEM18354.1 105 MBM3723043.1 14 MAX11513.1 60 GBD22648.1 106 ABF11225.1 15 HAK63664.1 61 MBF6599205.1 107 TAL98798.1 16 MBG92919.1 62 MXW00101.1 108 NNN20496.1 17 PZC46201.1 63 MYA07641.1 109 MBP1761901.1 18 MBB84818.1 64 REJ76484.1 110 PPQ43247.1 19 OGA51379.1 65 HDY15625.1 111 MSQ25793.1 20 PWB41796.1 66 MBW2231087.1 112 TMK28344.1 21 MAE93843.1 67 NRA08835.1 113 HIB12002.1 22 OGP60024.1 68 NQZ98823.1 114 WP 179589464.1 23 OWB57166.1 69 MBI3918747.1 115 MXY42918.1 24 KXN72624.1 70 MBI2761137.1 116 WP 184156128.1 25 PVU86112.1 71 MBE0608783.1 117 HET53513.1 26 ORZ16580.1 72 MYA54281.1 118 TMK22624.1 27 XP 005644825.1 73 NRA01576.1 119 MXX66290.1 28 KZV27770.1 74 MBW2623123.1 120 GIS94895.1 29 EJY87672.1 75 MBI5615765.1 121 MBN1557905.1 30 HIG47824.1 76 MSR14309.1 122 MSV30368.1 31 TMD03111.1 77 XP 004342722.2 123 MBN2179295.1 32 MBJ56818.1 78 MSP42197.1 124 TDI90456.1 33 WP 095860310.1 79 TDI61101.1 125 OGN76415.1 34 MBL8483477.1 80 MBO0741576.1 126 WP 102074055.1 35 WP 058697592.1 81 MBO0736096.1 127 PZC47999.1 36 WP 130292058.1 82 MBV9828771.1 128 HHH88785.1 37 WP 207956071.1 83 MAW55136.1 129 OLB93949.1 38 WP 132429652.1 84 MBV38827.1 130 PKB76696.1 39 WP 060575023.1 85 TMJ68231.1 131 HED24197.1 40 WP 068796145.1 86 TMJ64557.1 132 WP 066960443.1 41 OJY48151.1 87 MBV9815528.1 133 WP 169259343.1 42 WP 062397209.1 88 MYH41266.1 134 WP 201494572.1 43 WP 169186431.1 89 MPZ97997.1 135 MBN9621549.1 44 WP 133828190.1 90 MBT5774752.1 136 OZG26106.1 45 MBS0560157.1 91 XP 014714961.1 137 WP 016501746.146 PCJ59575.1 92 TAK78428.1

[0190] Table 3. List of acyl-CoA reductase (ACR)ACR# GenBank Accession Number ACR# GenBank Accession Number 1 ABX41556.1 26 WP 094899923.12 WP 185879480.1 27 WP 152887945.13 WP 051457024.1 28 HAR84250.14 WP 088269124.1 29 MBS5083929.15 WP 087641473.1 30 WP 027296492.16 WP 051541705.1 31 WP 104149274.17 HHY51863.1 32 WP 135035016.18 WP 119112248.1 33 EJO19495.19 WP 114642697.1 34 WP 051245790.110 WP 202656015.1 35 WP 125552613.111 WP 115130814.1 36 HCL03003.112 WP 052127368.1 37 WP 126792036.113 MBN6206692.1 38 NQJ18683.114 WP 051217603.1 39 WP 191507870.115 WP 129009770.1 40 MBR5981728.116 MTK10086.1 41 WP 215633330.117 WP 106009325.1 42 MBP3327663.118 HBB29922.1 43 WP 152889336.119 WP 216437565.1 44 WP 052356672.120 WP 090039956.1 LmACR Q8Y7U121 WP 083963349.1 PtACDH A0A178TYF622 WP 012101452.1 StEutE P4179323 WP 122646076.1 RpPduP Q21A4924 WP 070791575.1 BmACDH I3E34925 WP 010715224.1 CbAld Q716S8

[0191] Table 4. List of alcohol dehydrogenasesADH# Gene Gen Bank 1 EcAdhE P0A9Q7 2 CaAhdE2 Q9ANR5 3 ScALD5 P40047 4 YihU P0A9V8 5 ChnD Q9F7D8 6 LsADH QVQ68835.17 ScADH WP 003971780.18 EcYqhD Q46856 9 EcYahK P7569110 TrGLDl Q0GYU5Note: ADH: alcohol dehydrogenase.

[0192] Table 5. List of diol dehydratasesDDR# Gene Gen Bank Accession Number 1 KpDhaB123 Q59476, 008505, Q59475 2 CbDhaB12 Q8GEZ8, Q8GEZ7 3 CfDhaBCE P45514, A8CH91, A8CH95 4 KoPddABC Q59470, Q59471, Q59472 5 EcPddAB A0A828U7A4, A0A828U8K06 CpDhaBCE 085188, 085189, 085190 7 StPduCDE P37450, 031041, 031042 8 CfPduCDE P0DUM7, P0DUM8, P0DUM99 LrPduCDE A0A0S4NNQ1, A0A0S4NN56, F8KDD810 LrDhaBCE F8DQ36, F8DQ37, F8DQ3811 LdGldCDE KRL62459.1, KRL62460.1, KRL62461.1

[0193] Table 6. List of Thiolases for condensation for acetyl-CoA to acetoacetyl-CoAThiolase# Gen Bank Accession Number Thiolase# GcnBank Accession Number 1 P76461 8 Q51956 2 P45359 9 P14611 3 Q0KBP1 10 P21151 4 P54839 11 Q8VPF1 5 Q9AHY0 12 P45855 6 P41338 13 032177 7 P0C7L2 14 Q9FD718 Q51956 15 Q9R9W0

[0194] Table 7. List of acetoacetate decarboxylases to convert acetoacetate to acetoneAAD# GenBank Accession Number AAD# GenBank Accession Number 1 P23670 16 Q89CN4 2 Q7NSA6 17 A41KF73 B1YXQ0 18 B21JF44 Q1BK36 19 A9AR155 Q98AN6 20 A0B4716 B2T319 21 Q141C97 Q38ZU0 22 B2UIG78 B4EG09 23 Q8XR109 Q98IH6 24 A0A069151810 A0A0A3Y2X2 25 A0A0C4XYU811 A0A0G9H222 26 A0A0K1ZST512 A0A0W0UWH1 27 A0A1A5PUK913 A0A1G7FF76 28 A0A1H3B2N814 A0A3L7A9L4 29 A0A4P8ITE415 A0A4Q8XQC5

[0195] Table 8. List of enzymes used to convert 2-hydroxyisobutyryl-CoA to methacrylic acid or methyl-methacrylate.Reaction Gene Genbank Accession NumberEcYciA P0A8Z0EcTcsB P0AGG2EcYigI P0ADP2TEEcPaaY P77181PpTesB A0A379KHC2EcYpfH P76561BpCaiD_2 A0A1X1PK59CdHadBC Q5U923, Q5U924HACDBcep1808_1524 A4JE28CdHgdB A0A9R0CDB8WS / DGAT NP 179535.1AtfA WP 307002799.1, or WP 004922085.1EaDAcT D6NSS8, A0A221J485AthDGATl NM 127503AAT (EsterBnaDGATl.a JN224473synthase)BtDGATl NP777118YlDGAT1 XP504700CzDGAT1B Cz09g08290.t1PhyBEBTl Q6E593

[0196] Table 9. List of acyl-CoA transferases (ACT) variants used for the interconversion of one-carbon compounds to generate formyl-CoA _ _ _ACT# GenBank Accession Number ACT# GenBank Accession Number 1 NBW24427.1 34 MBW7889249.12 HBE84973.1 35 MBK7676927.13 WP 132821656.1 36 MBV8105822.14 WP 220287672.1 37 MBP7764230.15 WP 121965416.1 38 MSO92582.16 XP 014530103.1 39 MBE9574050.17 2OAS_1 40 MBX9950024.18 WP 084235291.1 41 AEK61848.19 MBS6366228.1 42 MBL8096225.110 MBR5999861.1 43 OPZ27283.111 3D3U_1 44 RYD04206.112 WP 073092544.1 45 MBQ9059638.113 MBR0090292.1 46 2G39_114 MBU4349155.1 47 2NVV_115 NLU48536.1 48 MBQ9530926.116 3EH7_1 49 4EU3_117 MBS0639490.1 50 KAF4531260.118 WP 194298948.1 51 2HJ0_119 3UBM_A 52 SJZ60628.120 WP 203555095.1 53 ALP92681.121 MBC7087538.1 54 GFX41336.122 WP 191390389.1 55 5VIT_123 MBR0127170.1 56 SDR52074.124 WP 206582952.1 57 MBF0160244.125 MBK5252043.1 58 MBF0445994.126 MBR2778738.1 59 HGX18505.11727 MBF8291010.1 60 MBK9387745.128 RLT25587.1 61 MBF0310027.129 HHW62298.1 62 RKY20198.130 NWF83872.1 63 NYH33089.131 NUN70050.1 64 Q9RM8632 MBU2447274.1 65 00664433 P37766 66 WP 206580961.1

[0197] Table 10. List of pairs of acyl-CoA kinases (ACK) and phosphoacyltransferases (PTA) used for the interconversion of one-carbon compounds to generate formyl-CoA _ _ _GenBank Accession Number Gen Bank Accession Num her JG1K# 7(1111700711117 JG1K#ACK iiiiitiiMKiiifiii PTA1 AKJ38693.1 AKJ38694.1 21 MBU2064158.1 MBU2064159.1 2 BCV24779.1 BCV24778.1 22 MBU2501086.1 MBU2501087.1 3 EDM85332.1 EDM85331.1 23 NLA96479.1 NLA96478.1 4 GFI65571.1 GFI65570.1 24 NLI62094.1 NLI62095.1 5 HBG22385.1 HBG22386.1 25 NLM93282.1 NLM93283.1 6 HFV10353.1 HFV10352.1 26 NMA59030.1 NMA59029.1 7 HGG13858.1 HGG13857.1 27 OGI05344.1 OGI05345.1 8 HIX51076.1 HIX51075.1 28 OHB58473.1 OHB58472.1 9 KXK65140.1 KXK65141.1 29 PWM39673.1 PWM39672.1 10 KXL51791.1 KXL51790.1 30 TKJ47541.1 TKJ47542.1 11 MBE5816841.1 MBE5816840.1 31 WP 022744670.1 WP 022744669.1 12 MBE6451999.1 MBE6451998.1 32 WP 023275423.1 WP 023275424.1 13 MBF0205569.1 MBF0205570.1 33 WP 076546120.1 WP 076546119.1 14 MBI4335478.1 MBI4335477.1 34 WP 078810629.1 WP 078810628.1 15 MBN1633386.1 MBN1633385.1 35 WP 099343330.1 WP 099343331.1 16 MBR2784446.1 MBR2784445.1 36 WP 106012460.1 WP 106012461.1 17 MBR3082232.1 MBR3082233.1 37 AAA72042.1 AAA72041.1 18 MBS5449595.1 MBS5449596.1 38 BAG33697.1 BAG33698.1 19 MBS6942200.1 MBS6942201.1 39 A0A0J8DB00 A0A0J8D6J220 MBU0667180.1 MBU0667181.1 40 P0A6A3 P0A9M8

[0198] Various embodiments have been described herein with reference to the accompanying drawings. It will, however, be evident that various modifications and changes may be made thereto, and additional embodiments may be implemented, without departing from the broader scope of the invention as set forth in the claims that follow. Further, other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of one or more embodiments of the invention disclosed herein. It is intended, therefore, that this application and the examples herein be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following listing of exemplary claims.

Claims

Claims1. A genetically modified microorganism capable of producing methacrylic acid (MAA) and / or methyl-methacrylate (MMA) in combination with at least one of the following:(a) condensation of acetone and formyl-CoA to form 2-hydroxyisobutyryl-CoA;(b) conversion of the 2-hydroxyisobutyryl-CoA to MAA via:bi. conversion of 2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyric acid followed by a chemical dehydration to MAA; and / orbii. conversion of 2-hydroxyisobutyryl-CoA to mcthacrylyl-CoA followed by conversion to MAA; (c) conversion of the 2-hydroxyisobutyryl-CoA to MMA via:ci. conversion to MMA by chemical esterification with methanol of MAA formed in step (bi) and / or step (bii);cii. conversion of 2-hydroxyisobutyryl-CoA to methacrylyl-CoA followed by conversion to MMA; and / or ciii. conversion of 2-hydroxyisobutyryl-CoA to methyl-2-hydroxyisobutanoate followed by chemical dehydration to MMA;(d) generation of the formyl-Co A from one carbon substrates selected from methane, methanol, formaldehyde, or formic acid; and(e) generation of the acetone from glucose, glycerol, acetate, ethanol, ethylene glycol, fatty acids, or Cl compounds selected from methane, methanol, formaldehyde or formate.

2. The genetically modified microorganism of claim 1, further 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, catalyzing the conversion of acetone and formyl-CoA to 2-hydroxyisobutyryl-CoA in step (a).

3. The genetically modified microorganism of claim 1 or 2, wherein in step (bi) the 2-hydroxyisobutyryl-CoA is first converted to 2-hydroxyisobutyric acid by an overexpressed thioesterase, acyl-CoA transferase, ACK-PT A, or acyl-CoA reductase-aldehyde dehydrogenase; and the 2-hydroxyisobutyric acid is further converted to MAA by the chemical dehydration.

4. The genetically modified microorganism of any one of claims 1 -3, whereinin step (bii) the 2-hydroxyisobutyryl-CoA is first converted to methacrylyl-CoA by an overexpressed hydroxyacyl-CoA dehydratase, and the methacrylyl-CoA is further converted to methacrylic acid by an overexpressed thioesterase, acyl-CoA transferase, ACK-PTA, or acyl-CoA reductase-aldehyde dehydrogenase; and / orin step (bii) methacrylyl-CoA is first converted to methacrylaldehyde by an overexpressed acyl-CoA reductase, and the methacrylaldehyde is further converted to MAA by an overexpressed aldehyde dehydrogenase.

5. The genetically modified microorganism of any one of claims 1-4, wherein the 2-hydroxyisobutyryl-CoA is first converted to MAA in step (bi) and / or step (bii), and further converted to MMA via the chemical esterification with methanol in step (ci).

6. The genetically modified microorganism of any one of claims 1-5, wherein in steps (cii) the 2-hydroxyisobutyryl-CoA is first converted to methacrylyl-CoA by an overexpressed hydroxyacyl-CoA, and the methacrylyl-CoA and methanol is further converted to MMA by an overexpressed ester synthase.

7. The genetically modified microorganism of any one of claims 1-6, wherein in step (ciii) the 2-hydroxyisobutyryl-CoA and methanol is first converted to methyl-2-hydroxyisobutanoate by an overexpressed ester synthase, and the methyl-2- hydroxyisobutanoate is converted to MMA via chemical dehydration.

8. The genetically modified microorganism of any one of claims 1-7, further comprising:an overexpressed methane monooxy genase cataly zing the conversion of methane to methanol in step (d);an overexpressed enzyme catalyzing the conversion of methanol to formaldehyde in step (d), which is selected from methanol dehydrogenase or alcohol oxidase;an overexpressed enzyme catalyzing the conversion of formaldehyde to formate in step (d), which is selected from the group consisting of:i. a formaldehyde dehydrogenase;ii. a S-(hydroxymethyl) glutathione synthase, S -(hydroxymethyl) glutathione dehydrogenase and S-formylglutathione hydrolase;hi. a mycothiol-dependent formaldehyde dehydrogenase and a hydrolase;iv. a formaldehyde activating enzy me (FAE), a methylene-tetrahydromethanopterin dehydrogenase, a methenyl- tetrahydromethanopterin cyclohydrolase and a formyltransferase / hydrolase complex; andv. a methylene-tetrahydrofolate dehydrogenase, a methenyl-tetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase; and / oran overexpressed enzyme selected from the group consisting of:i. an acylating formaldehyde dehydrogenase converting formaldehyde to formyl-CoA;ii. a formate kinase converting formate to formyl -phosphate and a phosphate formyltransferase converting formylphosphate to formyl-CoA; andhi. an acyl-Co A transferase converting formate to formyl-CoA; and an acyl-Co A synthase converting formate to formyl-CoA9. The genetically modified microorganism of any one of claims 1-8, further comprising a pathway to generate acetone in step (c) via at least one of the following steps:ei. conversion of the Cl compounds to formaldehyde followed by condensation of the formaldehyde and Cl- derived formyl-CoA to form glycolyl-CoA, followed by conversion of the glycolyl-CoA to acetaldehyde; eii. condensation of acetaldehyde and formyl-CoA to generate lactoyl-CoA followed by conversion of the lactoyl-CoA to the acetone; and / oreiii. conversion of acetaldehyde to acetyl-CoA and condensation of two acetyl-CoA to form acetoacetyl-CoA, followed by decarboxylation of the acetoacetyl-CoA to form the acetone.

10. The genetically modified microorganism of claim 9, further comprising an overexpressed TPP -dependent enzy me selected from a 2-hydroxyacyl-CoA synthase, a 2-hydroxyacyl-CoA lyase, an oxalyl-CoA decarboxylase or a benzaldehyde lyase, catalyzing the conversion of formaldehyde and formyl-CoA to form glycolyl-CoA in step (ei).

11. The genetically modified microorganism of claim 9 or 10, further comprising:an overexpressed acyl-CoA reductase which can catalyze a conversion of glycolyl-CoA to glycolaldehyde in step (ei); an overexpressed alcohol dehydrogenase which can catalyze a conversion of glycolaldehyde to ethylene glycol in step (ei); and / oran overexpressed diol-dehy dratase which can catalyze the conversion of ethylene glycol to acetaldehy de in step (ei).

12. The genetically modified microorganism of any one of claims 9-11, further 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, catalyzing the conversion of acetaldehyde and formyl-CoA to lactoyl-CoA in step (eii);an overexpressed acyl-CoA reductase which can catalyze a conversion of lactoyl-CoA to 2-hydroxypropionaldehyde in step (eii);an overexpressed alcohol dehydrogenase which can catalyze a conversion of 2-hydroxypropionaldehyde to 1,2-propanediol in step (eii); and / oran overexpressed diol-dehydratase which can catalyze a conversion of 1,2-propanediol to acetone in step (eii).

13. The genetically modified microorganism of any one of claims 9-12, further comprising:an overexpressed acyl-CoA reductase catalyzing the conversion of acetaldehyde to acetyl-CoA in step (eiii);an overexpressed thiolase catalyzing the condensation of two acetyl-CoA molecules to form an acetoacetyl-CoA in step (ciii);an overexpressed thioesterase catalyzing a conversion of acetoacetyl-CoA to acetoacetate in step (eiii); and / or an overexpressed acetoacetate decarboxylase, catalyzing a decarboxylation of acetoacetate to acetone and carbon dioxide in step (ciii).

14. The genetically modified microorganism of any one of claims 1-13, further comprising a pathway to generate acetone, formyl-CoA and methanol from glucose in step (e) consisting of the following steps:civ. conversion of the glucose to two pyruvate molecules via a native metabolic pathway selected from the Embden-Meyerhoff-Pamas pathway, the Entner-Doudoroff pathway, or the pentose phosphate pathway; ev. conversion of the two pyruvate to two acetyl-CoA and two formate, wherein one of the formate molecules is converted to formyl-CoAfor condensation with acetone, while the other one is reduced to methanol for esterification of MAA;evi. condensation of the two acetyl-CoA molecules to form acetoacetyl-CoA;evii. conversion of the acetoacetyl-CoA and formate to form acetoacetate and formyl-CoA;eviii. conversion of the acetoacetate to acetone via decarboxylation;eix. conversion of formyl-CoA to form formaldehyde; andex. conversion of formaldehyde to methanol.

15. The genetically modified microorganism of claim 14, further comprising:an overexpressed pyruvate formate lyase catalyzing the conversion of pyruvate to acetyl-CoA and formate in step (ev); an overexpressed thiolase catalyzing the condensation of two acetyl-CoA molecules to form acetoacetyl-CoA in step (evi); an overexpressed thioesterase or acyl-CoA transferase catalyzing the conversion of acetoacetyl-CoA to acetoacetate in step (evii) or catalyzing the conversion of a formate to a formyl-CoA;an overexpressed acetoacetate decarboxylase which can catalyze the conversion of acetoacetate to acetone and carbon dioxide in step (eviii);an overexpressed acyl-CoA reductase which can catalyze the conversion of formyl-CoA to formaldehyde in step (eix); and / or an overexpressed methanol dehydrogenasewhich can catalyze the conversion of formaldehyde to methanol in step (ex).

16. The genetically modified microorganisms of any one of claims 1-15, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi, or is selected from the group consisting of Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebacterium sp., Zymonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccharomyces sp., Pichi sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp., and Trichoderma sp..

17. Use of the genetically modified microorganisms of any one of claims 1-16 in a method of producing MAA and / or MMA.

18. A method of producing MAA and / or MMA, comprising:providing the genetically modified microorganisms of any one of claims 1-16;providing a one carbon compound or a common feedstock to the microorganisms;andisolating MAA and / or MMA.

Images