WO2025038041A2

WO2025038041A2 - Production of dl-pantoic acid and dl-pantolactone via condensation of hydroxypivaldehyde and c1-derived formyl-coa

Info

Publication number: WO2025038041A2
Application number: PCT/SG2024/050525
Authority: WO
Inventors: Ramon Gonzalez; Natthaporn TAKPHO
Original assignee: Mojia Biotech Pte Ltd
Current assignee: Mojia Biotech Pte Ltd
Priority date: 2023-08-17
Filing date: 2024-08-16
Publication date: 2025-02-20
Anticipated expiration: 2026-02-17
Also published as: WO2025038041A3

Abstract

A method for producing DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL-pantoic acid) and/or DL-3-hydroxy-4,4-dimethyloxolan-2-one (DL-pantolactone), in particular, a method for producing DL-2,4-dihydroxy-3,3-dimethylbutanoic acid and/or DL-3-hydroxy-4,4- dimethyloxolan-2-one via condensation of hydroxypivaldehyde and C1-derived formyl-CoA was provided. Genetically modified microorganism used for producing DL-2,4-dihydroxy-3,3- dimethylbutanoic acid and/or DL-3-hydroxy-4,4-dimethyloxolan-2-one was also provided. The key enzymes were engineered to improve their performance on DL-pantoic acid or DL- pantolactone production.

Description

PRODUCTION OF DL-PANTOIC ACID AND DL-PANTOLACTONE VIA CONDENSATION OF HYDROXYPIVALDEHYDE AND Cl-DERIVED FORMYL-COA

TECHNICAL FIELD

[0001] The present disclosure generally relates to a method for producing DL-2,4-dihydroxy-3,3- dimethylbutanoic acid (DL-pantoic acid) and/or DL-3-hydroxy-4,4-dimethyloxolan-2-one (DL- pantolactone), in particular, a method for producing DL-2,4-dihydroxy-3,3-dimethylbutanoic acid and/or DL-3-hydroxy-4,4-dimethyloxolan-2-one via condensation of hydroxypivaldehyde and C 1 - derived formyl-CoA. The present disclosure also relates to genetically modified microorganism used for producing DL-2,4-dihydroxy-3,3-dimethylbutanoic acid and/or DL-3-hydroxy-4,4- dimethyloxolan-2-one.

SUMMARY OF THE INVENTION

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

[0003] Embodiment 1. A method for the production of DL-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA (DL-Pantoyl-CoA) comprising contacting formyl-CoA and hydroxypivaldehyde with a TPP- dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl- CoA decarboxylase or benzaldehyde lyase, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA is D-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA, L-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA or any mixture thereof.

[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.

[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 c atalyzing 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 formaldehyde dehydrogenase is selected from:

[0010] Embodiment 8. The method of Embodiment 6, wherein the formate kinase (e.g., an acyl- CoA kinase (ACK)) and the phosphate formyltransferase (e.g., a phosphoacyltransferase (PTA)) pair is selected from:

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

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

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

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

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

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

[ Gene | Gen Bank Accession Number |

[0017] Embodiment 15. The method of any one of Embodiments 1 to 14, further comprising generating hydroxypivaldehyde from isobutyraldehyde and formaldehyde.

[0018] Embodiment 16. A method for the production of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid or DL-3-hydroxy-4,4-dimethyloxolan-2-one comprising:

(a) providing DL-2,4-dihydroxy-3,3-dimethy1butanoyl-CoA obtained by the method of any one of Embodiments 1 to 15; and

(b) converting DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3- dimethylbutanoic acid or DL-3-hydroxy-4,4-dimethyloxolan-2-one by any one of (i) to (iv):

(i) a thioesterase or an acyl-CoA transferase catalyzing the conversion of DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanoic acid;

(ii) a phosphoacyltransferase catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanoyl-phosphate and an acyl-CoA kinase catalyzing the conversion of DL-2,4-dihydroxy-3,3-dimethylbutanoyl- phosphate to DL-2,4-dihydroxy-3,3-dimethylbutanoic acid;

(iii) an acyl-CoA reductase catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanal and an aldehyde dehydrogenase catalyzing the conversion of DL-2,4-dihydroxy-3,3-dimethylbutanal to DL-2,4-dihydroxy-3 ,3-dimethylbutanoic acid;

(iv) a lactonization catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-3-hydroxy-4,4-dimethyloxolan-2-one, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is D-2,4-dihydroxy-3,3- dimethylbutanoic acid, L-2,4-dihydroxy-3,3-dimethylbutanoic acid or any mixture thereof, and the DL-3 -hydroxy-4, 4-dimethyloxolan-2-one is D-3-hydroxy-4,4-dimethyloxolan-2-one, L-3- hydroxy-4,4-dimethyloxolan-2-one or any mixture thereof.

[0019] Embodiment 17. The method of Embodiment 16, wherein the acyl-CoA reductase is selected from:

[0020] Embodiment 18. The method of Embodiment 16 or 17, wherein the aldehyde dehydrogenase is seleeted from:

[0021] Embodiment 19. The method of any one of Embodiments 1 to 18, wherein the enzyme used in the method is isolated from a microorganism.

[0022] Embodiment 20. The method of any one of Embodiments 1 to 18, wherein the enzyme used in the method is contained in a microorganism.

[0023] Embodiment 21. A genetically modified microorganism providing DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA by the method of any one of Embodiments 1 to 15.

[0024] Embodiment 22. A genetically modified microorganism providing DL-2,4-dihydroxy-3,3- dimethylbutanoic acid by the method of any one of Embodiments 16 to 18.

[0025] Embodiment 23. A genetically modified microorganism providing DL-3-hydroxy-4,4- dimethyloxolan-2-one by the method of any one of Embodiments 16 to 18.

[0026] Embodiment 24. The microorganism of any one of Embodiments 21 to 23, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

[0027] Embodiment 25. The microorganism of any one of Embodiments 21 to 24, wherein the microorganism is bacteria, yeast or fungi, including but not limited to Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebacterium sp., Zymonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccharomyces sp., Pichia sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. Trichoderma sp. (or Escherichia coli, Bacillus subtilis, Bacillus methanolicus, Pseudomonas putida, Corynebacterium glutamnicum, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Methylorubrum extorquens, etc.)

[0028] Embodiment 25. The method of any one of Embodiments 1 to 15, wherein the DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA is D-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA.

[0029] Embodiment 26. The method of any one of Embodiments 1 to 15, wherein the DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA is L-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA.

[0030] Embodiment 27. The method of any one of Embodiments 1 to 15, wherein the DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA is or a mixture of D-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA and L-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA.

[0031] Embodiment 28. The method of any one of Embodiments 16 to 18, wherein the DL-2,4- dihydroxy-3,3-dimethylbutanoic acid is D-2,4-dihydroxy-3,3-dimethylbutanoic acid.

[0032] Embodiment 29. The method of any one of Embodiments 16 to 18, wherein the DL-2,4- dihydroxy-3,3-dimethy1butanoic acid is L-2,4-dihydroxy-3,3-dimefhylbutanoic acid. [0033] Embodiment 30. The method of any one of Embodiments 16 to 18, wherein the DL-2,4- dihydroxy-3,3-dimethylbutanoic acid is a mixture of D-2,4-dihydroxy-3,3-dimethylbutanoic acid and L-2,4-dihydroxy-3,3-dimethylbutanoic acid.

[0034] Embodiment 31. The method of any one of Embodiments 16 to 18, wherein the DL-3- hydroxy-4,4-dimethyloxolan-2-one is D-3-hydroxy-4,4-dimethyloxolan-2-one.

[0035] Embodiment 32. The method of any one of Embodiments 16 to 18, wherein the DL-3- hydroxy-4,4-dimethyloxolan-2-one is L-3-hydroxy-4,4-dimethyloxolan-2-one.

[0036] Embodiment 33. The method of any one of Embodiments 16 to 18, wherein the DL-3- hydroxy-4,4-dimethyloxolan-2-one is a mixture of D-3 -hydroxy-4, 4-dimethyloxolan-2-one and L- 3-hydroxy-4,4-dimethyloxolan-2-one.

[0037] Embodiment 34. The microorganism of any one of Embodiments 21 , 24 and 25, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is D-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA.

[0038] Embodiment 35. The microorganism of any one of Embodiments 21, 24 and 25, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is L-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA.

[0039] Embodiment 36. The microorganism of any one of Embodiments 21, 24 and 25, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is a mixture of D-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA and L-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA.

[0040] Embodiment 37. The microorganism of any one of Embodiments 22, 24 and 25, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is D-2,4-dihydroxy-3,3-dimethylbutanoic acid.

[0041] Embodiment 38. The microorganism of any one of Embodiments 22, 24 and 25, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is L-2,4-dihydroxy-3, 3 -dimethylbutanoic acid.

[0042] Embodiment 39. The microorganism of any one of Embodiments 22, 24 and 25, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is a mixture of D-2,4-dihydroxy-3,3- dimethylbutanoic acid and L-2,4-dihydroxy-3,3-dimethylbutanoic acid.

[0043] Embodiment 40. The microorganism of any one of Embodiments 23 to 25, wherein the DL-3 -hydroxy -4, 4-dimethyloxolan-2-one is D-3 -hydroxy-4, 4-dimethyloxolan-2-one.

[0044] Embodiment 41. The microorganism of any one of Embodiments 23 to 25, wherein the DL-3 -hydroxy-4, 4-dimethyloxolan-2-one is L-3-hydroxy-4,4-dimethyloxolan-2-one. [0045] Embodiment 42. The microorganism of any one of Embodiments 23 to 25, wherein the DL-3 -hydroxy-4, 4-dimethyloxolan-2-one is a mixture of D-3-hydroxy-4,4-dimethyloxolan-2-one and L-3-hydroxy-4,4-dimethyloxolan-2-one.

[0046] Enzymes

[0047] Provided herein is an enzyme, wherein the enzyme is a 2 -hydroxy acy-CoA synthase (HACS) selected from Table 1.

[0048] Provided herein is an enzyme, wherein the enzyme is a HACS 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. In some embodiments, the enzyme is a HACS 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 HACS 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.

[0049] Provided herein is an enzyme, wherein 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, DhcHACS from Dehalococcoidia bacterium and CfhHACS from Chloroflexi bacterium.

[0050] In some embodiments, the enzyme comprises at least one amino acid substitution at position Y371, N394, T395, T401, D445, S446, G474, 1475, G476, N472, L493, P521, L523, M527, F529, K530, G531, P532, 1534, V535, N536, 1537, K538, 1539, T542, D544, R545, K546, P547, Q548, F550, N55I, W552, H553, G554 or Q549, (e.g., T40IA, Q549A, D445A, I475A, G474A, S446A, T395A, Y371A, D544A, K546A, N472A, L493A, H553L, G554T, N394A, G476A, G476L, G476T, G554A, W552A, N551A, F550A, Q548A, P547A, R545A, T542A, I539A, K538A, I537A, N536A, V535A, I534A, P532A, G531A, K530A, F529A, M527A, L523A or P521 A), wherein the substitution modulates (e.g., enhances or reduces) the enzyme activity of DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA production and/or DL-2,4-dihydroxy-3,3- dimethylbutanoic acid production. In some embodiments, the substitution enhances the enzyme activity of DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA production and/or DL-2,4-dihydroxy- 3,3-dimethylbutanoic acid production.

[0001] In some embodiments, the enzyme comprises at least one amino acid substitution at position N394, T395, T401 , S446, G474, G476, D544, K546, Q549, P521 , H553 or G554, (e.g., N394A, T395A, T401A, S446A, G474A, G476A, G476L, G476T, D544A, K546A, Q549A, P521A or G554T) wherein the substitution enhances the enzyme activity of DL-2,4-dihydroxy- 3,3-dimethylbutanoyl-CoA production and/or DL-2,4-dihydroxy-3,3-dimethylbutanoic acid production. Tn some embodiments, the enzyme is produced by a method comprising introducing a substitution at position N394, T395, T401, S446, G474, G476, D544, K546, Q549, P521, H553, G554, (e.g., of wild-type ApbHACS, such as ApbHACS from Alphaproteobacteria bacterium (Genbank accession: HAK63664.1)) wherein the substitution enhances the enzyme activity towards DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA and DL-2,4-dihydroxy-3,3- dimethylbutanoic acid production. In some embodiments, the enzyme is selected from any enzyme set forth in Tables 7-9.

[0002] Provided herein is an enzyme, wherein the enzyme is an acyl-CoA transferase (ACT) selected from Table 4.

[0003] Provided herein is an enzyme, wherein the enzyme is an ACT 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 an ACT 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 ACT 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 4.

[0004] Provided herein is an enzyme, wherein the enzyme is an acyl-CoA reductase (ACR) selected from Table 2.

[0005] Provided herein is an enzyme, wherein the enzyme is an ACR 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. In some embodiments, the enzyme is an ACR 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 ACR 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.

[0006] Provided herein is an enzyme, wherein the enzyme is a phosphoacyltransferase (PTA) selected from Table 5. [0007] Provided herein is an enzyme, wherein the enzyme is a PTA 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. In some embodiments, the enzyme is a 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 5. In some embodiments, the enzyme is a 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 5.

[0008] Provided herein is an enzyme, wherein the enzyme is an aldehyde dehydrogenase (ALDH) selected from Table 6.

[0009] Provided herein is an enzyme, wherein the enzyme is an ALDH 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 ALDH 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 ALDH 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.

[0010] Provided herein is an enzyme, wherein the enzyme is an acyl-CoA kinase (ACK) selected from Table 5. room Provided herein is an enzyme, wherein the enzyme is an ACK 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. In some embodiments, the enzyme is an ACK 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 ACK 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 5.

[0012] Provided herein is an enzyme, wherein the enzyme is a methanol dehydrogenase (MDH) or alcohol oxidase (AOD) selected from Table 3.

[0013] Provided herein is an enzyme, wherein the enzyme is a MDH or AOD 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 MDH or AOD 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 MDH or AOD 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.

F0014] Provided herein is an enzyme, wherein the enzyme is an acyl-CoA synthase (ACS) selected from Table 3.

[0015] Provided herein is an enzyme, wherein the enzyme is an ACS 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 ACS 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 ACS 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.

[0016] In some embodiments, the enzymes or variants thereof provided herein were engineered to modulate (e.g., improve) their performance on DL-pantoic acid and/or DL-pantolactone production. In some embodiments, the enzymes or variants thereof provided herein comprise at least one amino acid substitution (for example, comparing to a wild type enzyme thereof), wherein the substitution modulates (e.g., enhances or reduces) the enzyme activity of DL-pantoic acid and/or DL-pantolactone production. In some embodiments, the substitution enhances the enzyme activity of DL-pantoic acid and/or DL-pantolactone production.

[0017] Also provided herein is a genetically modified microorganism comprising the enzyme disclosed herein.

[0018] Also provided herein is use of the enzyme disclosed herein in the production of DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA, DL-2,4-dihydroxy-3,3-dimethylbutanoic acid and/or DL- 3-hydroxy-4,4-dimethyloxolan-2-one.

[0019] 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.

[0020] The present disclosure is illustrated by the following non-limiting examples.

[0021] 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

[0022] 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.

[0023] Figure 1 is a scheme illustrating production of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL-pantoic acid) and/or DL-3-hydroxy-4,4-dimethyloxolan-2-one (DL-pantolactone) from condensation of hydroxypivaldehyde and Cl -derived formyl-CoA. MMO: methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; FOK: formate kinase; PTA: phosphoacyl transferase; ACS: acyl-CoA synthase; ACT: acyl-CoA transferase; ACR: acyl- CoA reductase; HACS: 2-hydroxyacyl-CoA synthase; TE: thioesterase; ALDH: aldehyde dehydrogenase; ACK: acyl-CoA kinase.

[0024] Figure 2 is a HPLC chromatogram analysis of cell-free bioconversion samples through the condensation of hydroxypivaldehyde and formyl-CoA catalyzed by different HACS variants where A) DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL-Pantoic acid) and B) DL-3-hydroxy- 4,4-dimethyloxolan-2-one (DL-Pantolactone) after esterfification and ethyl acetate extraction The system is operated by using Phenomenex Luna C18 column (250 mm x4.6 mm, 5 pm) with a 1 % of acetonitrile in 10 mM/L of NaH2PO4 buffer pH 2.5 as a mobile phase. Detection was performed at 214 nm using photodiode array detector and retention times of A) Pantoic acid is 12.3 min and B) Pantolactone is 21.4 min.

[0025] Figure 3 is a scheme illustrating the interconversion of Cl compounds. A) Production of formyl-CoA from Cl compounds; B) GSH- and MSH-dependent oxidation of formaldehyde to formate; C) H4F and H4MPT-dependent oxidation of formaldehyde to formate. MMO: methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; FOK: formate kinase; PTA: phosphate formyltransferase; ACS: acyl-CoA synthase; ACT: acyl-CoA transferase; ACR: acyl-CoA reductase; HACS: 2-hydroxyacyl-CoA synthase; TE: thioesterase; FLD: formaldehyde dehydrogenase; FGH: formyl-GS hydrolase; FHC: formyltransferase/ hydrolase; MTDA: methylene-tetrahydromethanopterin dehydrogenases; FCH: methenyl-H4F cyclohydrolase; FTFL: formate-H4F ligase; FAE: formaldehyde activating; MCH: methenyl-FLiMPT cyclohydrolase enzyme; MCH: methenyl-H4MPT cyclohydrolase.

[0026] Figure 4 is a scheme illustrating the generation of formyl-CoA from formaldehyde and the generation of glycolic acid from the condensation of formaldehyde and formyl-CoA under in vitro system where the reaction is driven by ACR derived from Listeria monocytogenes and different HACS variants.

[0027] Figure 5 is a scheme illustrating the generation of formyl-CoA from formate and the generation of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL-pantoic acid) from condensation of hydroxypiv aldehyde and formyl-CoA under in vitro system where A) shows the reaction driven by ACT and different HACS variants; B) shows the reaction driven by different ACT variants and ApbHACS; C) shows the reaction driven by different ACS variants and ApbHACS; D) shows the reaction driven by a set of PT A- ACK and ApbHACS.

[0028] Figure 6 is a scheme illustrating the generation of formyl-CoA from formate and the generation of DL-2,4-dihydroxy-3,3-dimcthylbiitanoic acid (DL-pantoic acid) from condensation of hydroxypivaldehyde and formyl-CoA under in vivo system where the whole-cell bioconversion is driven by Esherchia coli MG 1655 expressing CaAbfT and ApbHACS.

[0029] Figure 7 is a scheme illustrating the protein engineering approach and improvement of DL- 2, 4-dihydroxy-3, 3 -dimethylbutanoic acid (DL-pantoic acid) production through HACS engineering using cell-free bioconversion in vitro) system.

[0030] The same reference numbers will be used throughout the drawings to refer to the same or like parts.

DETAILED DESCRIPTION OF THE INVENTION

[0031] 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.

[0032] In this application, the use of the singular includes the plural unless specifically stated otherwise. Tn 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.

[0033] The compounds of present disclosure can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. Thus, the compounds of present disclosure and compositions thereof may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the present disclosure are enantiopure compounds. In certain embodiments, mixtures of enantiomers or diastereomers are provided. A stereoisomer may, in some embodiments, be provided substantially free of one or more corresponding stereoisomers, and may also be referred to as “stereochemically enriched”.

[0034] Where a particular enantiomer is preferred, it may, in some embodiments be provided substantially free of the opposite enantiomer, and may also be referred to as “optically enriched”. “Optically enriched”, as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments, the compound is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments, the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer.

[0035] As used herein, the term “DL-2,4-dihydroxy-3,3-dimethylbutanoic acid”, “2,4-dihydroxy- 3,3-dimethylbutanoic acid”, “DL-pantoic acid” or “pantoic acid” includes D-2,4-dihydroxy-3,3- dimethylbutanoic acid (D-pantoic acid), L-2,4-dihydroxy-3,3-dimethylbutanoic acid (L-pantoic acid) and any mixture thereof, e.g., racemic mixtures of enantiomers.

[0036] As used herein, the term “DL-3-hydroxy-4,4-dimethyloxolan-2-one”, “3 -hydroxy-4, 4- dimethyloxolan-2-one”, “DL-pantolactone” or “pantolactone” includes D-3-hydroxy-4,4- dimethyloxolan-2-one (D-pantolactone), L-3-hydroxy-4,4-dimethyloxolan-2-one (L- pantolactone) and any mixture thereof, e.g., racemic mixtures of enantiomers.

[0037] As used herein, the term “DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA”, “2,4- dihydroxy-3,3-dimethy1butanoyl-CoA”, “DL-pantoyl-CoA” or “pantoyl-CoA” includes D-2,4- dihydroxy-3, 3-dimethylbutanoyl-CoA (D-pantoyl-CoA), L-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA (L-pantoyl-CoA) and any mixture thereof, e.g., racemic mixtures of enantiomers.

[0038] As used herein, the term “DL-2,4-dihydroxy-3,3-dimethylbutanal” or “2,4-dihydroxy-3,3- di methyl butan l” includes D-2,4-dihydroxy-3,3-dimefhylbutanal, L-2,4-dihydroxy-3,3- dimethylbutanal and any mixture thereof, e.g., racemic mixtures of enantiomers.

[0039] As use herein, the term “DL-2,4-dihydroxy-3,3-dimethylbutanoyl-phosphate”, “2,4- dihydroxy-3,3-dimethylbutanoyl-phosphate”, “DL-pantoyl-phosphate” or “pantoyl-phosphate” includes D-2,4-dihydroxy-3,3-dimethylbutanoyl-phosphate, L-2,4-dihydroxy-3,3- dimethylbutanoyl-phosphate and any mixture thereof, e.g., racemic mixtures of enantiomers.

[0040] EXAMPLES

[0041] EXAMPLE 1: PRODUCTION OF DL-PANTOIC ACID AND DL- PANTOLACTONE THROUGH CONDENSATION OF HYDROXYPIVALDEHYDE AND FORMYL-COA

[0042] This Example demonstrates the implementation of Cl+Bio™ platform for DL-2,4- dihydroxy-3,3-dimethylbutanoic acid (DL -pantoic acid) and DL-3-hydroxy-4,4-dimethyloxolan- 2-one (DL-pantolactone) production through the condensation of hydroxypivaladehyde and formyl-CoA (Figure 1 below). The concept of the module is to utilize acyloin condensations between formyl-CoA and carbonyl-containing molecules such as aldehyde. These condensation reactions are facilitated by the enzyme 2-hydroxyacyl-CoA lyase (HACL) or 2-hydroxvacyl-CoA svnthase (HACS) selected from HACS variants forth in Table 1 below, including but not limited to BsmHACL or ApbHACS from Alphaproteobacteria bacterium (GenBank accession: HAK63664.1), BbHACS from Betaproteobacteria bacterium (GenBank accession: OGA51379.1), DhcHACS from Dehalococcoidia bacterium (GenBank accession: PWB41796.1 ), CbHACS from Candida boidinii (GenBank accession: OWB57166.1), and CcHACS from Conidiobolus coronatus (GenBank accession: KXN72624.1), to generate DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA. DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is further converted to DL- 2,4-dihydroxy-3,3-dimethylbutanoic acid by the activation of of a thioesterase such as TesB from Escherichia coli or Pseudomonas putida (McMahon, M.D. and Prather, K.L.J. Appl. Environ. Microbiol. 80: 1042-1050 (2014)). The reaction could be driven by acyl-CoA transferase (ACT) such as CaAbfT from Clostridium aminobutyricum (Scherf, U., at al. Appl. Environ. Microbiol. 57(9):2699-2702 (1991)) or a combination of phosphoacyltransferase and acyl-CoA kinase (PTA- ACK) such as a pair of Pta-Ack from Streptomyces mutans (Kim, J.N., et al. Appl Environ Microbiol. 81 ( 15):5015-5025 (2015)) (e.g., ACT set forth in Table 4 and PTA-ACK set forth in Table 5 below). Alternatively, DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA could be reduced to DL-2,4-dihydroxy-3,3-dimethylbutanal by an acyl-CoA reductase such as ALD from Clostridium beijerinckii (Kim, S., et al. J. Ind. Microbiol. Biotechnol. 42:465-475 (2015)) (e.g., ACR set forth in Table 2 below). DL-2,4-dihydroxy-3,3-dimethylbutanal can be further oxidized to DL-2,4- dihydroxy-3,3-dimethylbutanoic acid by a suitable aldehyde dehydrogenase such as AldA or AldB from E. coli (Baldoma and Aguilar, J Biol Chem. 1987, 262(29): 13991-6) (e.g., ALDH set forth in Table 6 below). To produce DL-3-hydroxy-4,4-dimefhyloxo1an-2-one as a final product, DL- 2,4-dihydroxy-3,3-dimethylbutanoyl-CoA can directly convert into DL-3-hydroxy-4,4- dimethyloxolan-2-one through a spontaneous lactonization reaction under acidic conditions. However, to prevent the conversion of DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA to DL-2,4- dihydroxy-3,3-dimethylbutanoic acid by endogenous thioesterase (TE) activity, it is necessary to disrupt the TE function in the host microorganism.

[0043] Cell-free bioconversion was carried out to demonstate the feasibilliy of producing DL-2,4- dihydroxy-3,3-dimethy1butanoic acid and DL-3-hydroxy-4,4-dimethyloxolan-2-one through the condensation of hydroxypivaldehyde and formyl-CoA. The total volume of 100 pL of reaction mixture contains 100 mM of potassium phosphate buffer (pH 6.9), lO mMof MgCh, 150 pM TPP, 4 mM of acetyl-CoA, 20 mM of formaldehyde, 20 mM of hydroxypivaldehyde, 4 M of HACS and 2 pM of CaAbfT. The mixture was incubated at room temperature for 18 h. The reaction was terminated by adding 10 M of NaOH and further incubated for 30 min at room temperature to obtain DL-2,4-dihydroxy-3,3-dimethylbutanoic acid. HC1 was then added to the reaction to neutralized the pH. To obtain DL-3-hydroxy-4,4-dimefhyloxo1an-2-one from the reaction, 10 M HC1 was added to the mixture to adjust the pH to 1.0. Subsequently, an equivalent volume of ethyl acetate was added to concentrate DL-3-hydroxy-4,4-dimethyloxolan-2-one. The results from HPLC analysis demonstrate the detection of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL- pantoic acid) and DL-3-hydroxy-4,4-dimethyloxolan-2-one (DL-pantolactone) resulting from the condensation of hydroxypivaldehyde and formyl-CoA, catalyzed by the selected HACS variants (Figure 2 below). [0044] EXAMPLE 2. INTERCONVERSION OF ONE-CARBON COMPOUNDS TO GENERATE FORMYL-COA

[0045] Formyl-CoA can be provided through the interconversion of Cl compounds including methane, methanol, formaldehyde, formate (Figure 3A below) with suitable enzymes (e.g., enzymes set forth in Table 2 and 3 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. coll as a host (bioRxiv 2021. 08.05.455234). [0046] Methanol can be subsequently oxidized to formaldehyde via the action of oxidoreductase, catalyzed by NAD⁺-dependent methanol dehydrogenase (MDH, e.g. Bacillus methanolicus MGA3 (BmMDH), Bacillus stearothermophilus (BsMDH) (Metab. Eng. 39:49-59, 2017) and Cupriavidus necator (Appl. Microbiol. Biotechnol. 100:4969-4983, 2016)), PQQ (pyrroloquinoline quinone)-dependent methanol dehydrogenase (MxaFI from Methylobacterium extorquens orXoxF from Methylacidiphilum fumariolicum) or oxygen-dependent alcohol oxidase (AOD). When methanol oxidation via oxygen-dependent AOD is employed, a catalase (CTA) is essential to break down hydrogen peroxide into water and oxygen (e.g., enzymes set forth in Table 3 below).

[0047] Formyl-CoA can be generated from direct conversion of formaldehyde by the activation of acyl-CoA reductase (ACR) that catalyzes the reduction of acyl-CoA to the corresponding aldehyde. We previously identified that LmACR from Listeria, monocytogenes is the most active acyl-CoA reductase variant specifically for acylating formaldehyde oxidation (formaldehyde to formyl-CoA) reaction (Chou, A., et al. Nat Metab 3:1385-1399 (2021)) (e.g., ACR set forth in Table 4 below). Formaldehyde can be directly converted to formate via formaldehyde oxidation by four cofactor-dependent formaldehyde oxidation pathways, involving glutathione (GSH), tetrahydrofolate (ELF, THF), tetrahydromethanopterin (H4MPT), or mycothiol (MSH). Alternatively, formaldehyde oxidation to formyl-CoA can be catalyzed by various acylating aldehyde dehydrogenase candidates (Nat. Chem. Biol. 15:900-906, 2019) (Figure 3B and 3C below). Formyl-CoA can either be fed to the Cl elongation platform or further converted to generate energy. The conversion of formyl-CoA from formate can also be achieved through the utilization of different families of enzymes. Acyl-CoA transferases (ACT, e.g., set forth in Table 4 below) catalyze reversible CoA transfer from various CoA donor, such as 2,4-dihydroxy-3,3- dimethylbutanoyl-CoA or acyl-CoA to formate. Alternative route involves a pair of formate kinase and phosphoacyltransferase (FOK-PTA e.g., set forth in Table 5 below) that catalyzes a reversible phosphorylation of formyl-CoA to formyl -phosph ate, followed by dephosphorylation to formate which generates 1 ATP from the reaction. While these two reactions are considered fully reversible, AMP-forming acyl-CoA synthetases are favored toward formate activation to formyl- CoA.

[0048] EXAMPLE 3: OVERVIEW OF METHODS USED FOR GENETIC MANIPULATION

[0049] The genes for overexpression are either cloned into appropriate vectors or inserted into chromosome with strong synthetic constitutive promoter, such as Ml -93. When cloned into vectors, these genes are amplified through PCR using appropriate primers to append homology on each end for recombination into the vector backbone with e.g., Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. Plasmids are linearized by the appropriate restriction enzymes (New England Biolabs, Ipswich, MA, USA) and recombined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system. The mixture is subsequently transformed into Stellar competent cells. Transformants that grow on solid media (LB+Agar) supplemented with the appropriate antibiotic are isolated and screened for the gene insert by PCR. Plasmids from verified transformants are isolated and the sequence of the gene insert is further confirmed by DNA sequencing. The sequence confirmed plasmids are then introduced to host strain through electroporation.

[0050] 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 /.-red recombinase. The resulting strain is grown under 30°C with L-arabinose for induction of /.-red recombinase expression, and when OD reaches ~0.6, competent cells are prepared and transformed with pTargetF (AddGene 62226) expressing sgRNA and N20 spacer targeting the locus and template of insertion of target gene. The template is the inserted gene plus Ml -93 promoter with -500 bp sequences homologous with upstream and downstream of the insertion locus, constructed through overlap PCR with usage of Phusion polymerase or synthesized by GenScript (Piscataway, NJ) or GeneArt® (Life Technologies, Carlsbad, CA). The way to switch N20 spacer of pTargetF plasmid is inverse PCR with the modified N20 sequence hanging at the 5' end of primers with usage of Phusion polymerase and followed by self- ligation with usage of T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Transformants that grow under 30°C on solid media (LB+Agar) supplemented with spectinomycin and kanamycin (or other suitable antibiotic) are isolated and screened for the chromosomal gene insert by PCR. The sequence of the gene insert, which is amplified from genomic DNA through PCR using Phusion polymerase, is further confirmed by DNA sequencing. The pTargetF can then be cured through IPTG induction, and pCas can be cured through growth under higher temperature like 37-42 °C. [0051] All molecular biology techniques are performed with standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).) or by manufacturer protocol. Strains are stored in glycerol stocks at -80°C. Plates are prepared using LB medium containing 1.5% agar, and appropriate antibiotics are included at the following concentrations: ampicillin (100 pg/mL), kanamycin (50 pg/mL), spectinomycin (50 pg/mL) and chloramphenicol (12.5 pg/mL).

[0052] 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. coll) or by gene synthesis of the codon optimized gene. Genes were synthesized by GeneArt (Life Technologies, Carlsbad, CA). Resulting In-Fusion reaction products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, CA), and clones identified by PCR screening were further confirmed by DNA sequencing. As a host for these vectors, an engineered strain of E. coli based on MG1655(DE3), BW25113 or ATCC8739 with knockouts for formaldehyde (A/rmA), formate ( \idhF NfdnG NfdoG) 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.

[0053] EXAMPLE 4: FERMENTATION CONDITIONS USED FOR IN VIVO PRODUCTION OF DL-PANTOIC ACID AND DL-PANTOLACTONE [0054] MOPS minimal medium (Neidhardt et al. J. Bacteriol.119736-47 (1974)) with 125 mM MOPS and NazHPO4 in place of K2HPO4 (2.8 mM), supplemented with 20 g/L glucose, 10 g/L tryptone, 5 g/L yeast extract, 100 LIM FeSO4, 5 mM (NH4)2SO4, and 30 mM NH4CI is used for fermentations. Tf required, 55 g/L of CaCO3 is also supplemented as pH buffer. 20 mM lactic acid is supplemented, if it is not synthesized intracellularly and needed for the experiment. 500 mM methanol is also supplemented. Antibiotics (50 pg/mL carbenicillin, 50 pg/mL spectinomycin and 50 pg/mL kanamycin) are included when appropriate. All chemicals are obtained from Fisher Scientific Co. (Pittsburg, PA) and Sigma-Aldrich Co. (St. Louis, MO).

[0055] 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 ODeoo as -0.05. After inoculation, flasks are incubated in a NBS 124 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, NJ) at 200 rpm and 37°C or 30°C. When optical density (550 nm, OD550) reached -0.3-0.5, appropriate concentration of isopropyl beta-D-1 -thiogalactopyranoside (IPTG) (or other suitable inducer) is added for plasmid gene induction. Additional fermentations are conducted in a SixFors multi- fermentation system (Infors HT, Bottmingen, Switzerland) with an air or argon flowrate of 2 N L/hr, independent control of temperature (37 °C), pH (controlled at 7.0 with NaOH and H2SO4), and appropriate stirrer speed. Pre-cultures are grown in 25 mL Pyrex Erlenmeyer flasks as described above and incubated for 4 hours post-induction. An appropriate amount of this pre-culture is centrifuged, washed twice with fresh media, and used for inoculation (400 mL initial volume). The fermentations in bioreactor use described fermentation media with 40 g/L glucose and appropriate IPTG and antibiotics. Tf required, lactic acid (20 mM) is added at 0, 24, and 48 horns.

[0056] 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.

[0057] EXAMPLE 5. HPLC ANALYSIS OF DL-PANTOIC ACID AND DL- PANTOLACTONE [0058] Reaction mixture from the bioconversion was centrifuged at 12,000 rpm for 2 min. A 100 pL of supernatant was transfer and subjected to HPLC analysis. HPLC analysis was conducted on a Waters 2695 system equipped with Waters 996 photodiode array detector and a Phenomenex Luna C18 column (250 mm*4.6 m, 5 pm). Column temperature was maintained at 40 °C, detector was set at 214 nm, and the injection volume was 10 pL with the flow rate of 0.5 mL/min. The mobile phase consisting of 1 % acetonitrile in a buffer containing 10 mM/L of NaLLPCL in MiniQ water at pH 2.5.

[0059] EXAMPLE 6. FORMYL-COA GENERATION FROM FORMALDEHYDE

[0060] Glycolic acid production is used as the example of formyl-CoA generation from formaldehyde via the activation of acyl-CoA reductase (ACR). Cl elongation is initiated by formyl-CoA production through energy and formyl-CoA generation steps. 2-Hydroxyacyl-CoA synthase (HACS) condenses formaldehyde and formyl-CoA to produce glycolyl-CoA which then be converted into glycolic acid by endogenous thioesterase (endo TE) (Figure 4 below). In this experiment, ACR from Listeria monocytogenes (LmACR) and different HACS variants were expressed in E. coli MG 1655 strain. LmACR and HACS varaints were then extracted, purified and used for cell-free bioconversion. The total volume of 100 pL of cell-free reaction contains 100 mM of potassium phosphate buffer (pH 6.9), 4 mM of NAD⁺, 10 mM of MgCh, 150 pM of TPP, 4 mM of acetyLCoA, 20 mM of formate, 10 mM of hydroxypivaldehyde, 2 pM of HACS and 4 pM of ACR was incubated at room temperature for 4 h. The reaction was terminated by adding 5 pL of 10 M of NaOH and further incubated for 30 min at room temperature to obtain glycolic acid. A 2.5 pL of 10 M HC1 was then added to neutralize the pH. The reaction mixture was centrifuged at 12,000 rpm for 2 minutes and analyzed with HPLC. The presence of glycolic acid from the reaction indicates that formyl-CoA could be generated from formaldehyde via the activation of ACR and glycolic acid was produced through the condensation of formaldehyde and formyl-CoA using different HACS variants ((e.g., HACS set forth in Table 1 below), including but not limited to ApbHACS from Alphaproteobacteria bacterium (Genbank accession: HAK63664.1), DhcHACS from Dehalococcoidia bacterium (Genbank accession: PWB41796.1) and CfhHACS from Chloroflexi bacterium (Genbank accession: PKN81274.1)) (Figure 4 below). [0061] EXAMPLE 7. FORMYL-COA GENERATION FROM FORMATE AND IN VITRO PRODUCTION OF DL-PANTOIC ACID THROUGH CONDENSATION OF HYDROXYPIVALDEHYDE AND FORMYL-COA

[0062] This example is to demonstrate the formyl-CoA generation from formate via different routes for example, via the activation of acyl-CoA transferase (ACT), ACS (acyl-CoA synthase), and PTA-ACK (acyl-CoA kinase and phosphoacyl transferase). Condensation of hydroxypivaldehyde with formyl-CoA by HACS produces DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA (DL-pantoyl-CoA) which then be converted to DL-2,4-dihydroxy-3,3- dimethylbutanoic acid (DL-pantoic acid) by engenous thioesterase (endo TE) (Figure 5 below).

[0063] To demonstrate the formyl-coA generation from formate via ACT together with DL- pantoic acid production from the condensation reaction, CaAbfT from Clostridium aminobutyricum and different HACS variants were expressed in E. coli MG 1655 strain. CaAbfT and HACS varaints were then extracted, purified and used for cell-free bioconversion. The total volume of 100 pL of reaction contains 100 mM of potassium phosphate buffer (pH 6.9), 10 mM of MgC12, 150 u M TPP, 4 mM of acetyl-CoA, 20 mM of formate, 20 mM of hydroxypivaldehyde, 4 pM of HACS and 2 pM of ACT was incubated at room temperature for 18 h. AbfT was added to the reaction to support CoA recycling. The reaction was terminated by adding 10 M of NaOH and further incubated for 30 min at room temperature to obtain DL-2,4-dihydroxy-3,.3- dimethylbutanoic acid. A 2.5 pL of 10 M HC1 was then added to neutralize the pH. The reaction mixture was centrifuged at 12,000 rpm for 2 minutes and analyzed with HPLC. The presence of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid through the condensation of hydroxypivaldehyde and formyl-CoA using different HACS variants ((e.g., HACS set forth in Table 1 below), including but not limited to ApbHACS from Alphaproteobacteria bacterium (Genbank accession: HAK63664.1), DhcHACS from Dehalococcoidia bacterium (Genbank accession: PWB41796.1) and CfhHACS from Chloroflexi bacterium (Genbank accession: PKN81274.1 )) indicates that formyl-CoA could be generated from formate via the activation of ACT (acyl -CoA transferase) ((e.g., ACT set forth in Table 4 below), including but not limited to CaAbfT from Clostridium aminobutyricum, PaACT from Pseudomonas aeruginosa (Genbank accession: 2G39), PcACT from Porphyromonas cangingivalis (Genbank accession: SJZ60628.1) (Figure 5A and 5B below). [0064] Futhermore, we have tested the formyl-CoA generation and DL-pantoic acid production via ACS and ACK-PTA routes. CaAbfT from Clostridium aminobutyricum and different HACS variants were expressed in E. coli MG1655 strain. Different ACS, HACS from Alphaproteobacteria bacterium (ApbHACS) and PT A- ACK from Coriobacteriia bacterium were then extracted, purified and used for cell-free bioconversion. A 1 mL of cell-free bioconversion reaction contains 100 mM of potassium phosphate buffer (pH 6.9), 10 mM of MgCh, 150 pM TPP, 10 mM of CoA, 10 mM of ATP, 50 mM of formate, 20 mM of hydroxypivaldehyde, 5 pM of ApbHACS and 2 pM of ACS or ACK-PTA. The reaction mixture was incubated, terminated, and analyzed in the same manner as mentioned above. The HPLC result showed that 2.7 - 5.8 mM of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL-pantoic acid) was produced from the reaction, confirming the generation of formyl-CoA from formate, catalyzed by different ACS variants ((e.g. ACS set forth in Table 3 below), including but not limited to BsACS from Bacillus subtilis (Genbank accession: P39062), Ec ACS from Esherichia coli (Genbank accession: P39062), MhACS from Marinithermus hydrothermalis (Genbank accession: F2NQX2), ScACS from Saccharomyces cerevisiae (Genbank accession: Q01574) and a set of PTA-ACK ((e.g. PTA-ACK set forth in Table 5 below, including but not limited to CrbPTA-ACK from Coriobacteriia bacterium (Figure 5C and 5D below).

[0065] EXAMPLE 8: IN VIVO PRODUCTION OF DL-PANTOIC THROUGH CONDENSATION OF HYDROXYPIVALDEHYDE AND FORMYL-COA

[0066] This example demonstrates the feasibility of DL-2,4-dihydroxy-3,3-dimefhylbutanoic acid (DL-pantoic acid) production via Cl+Bio™ platfrom using whole-cell bioconversion. We engineered vectors to independently control the expression of ApbHACS and CaAbfT, with ApbHACS 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 genes relevant to formaldehyde (AfrmA) and formate (AfdhF AfdnG AfdoG) oxidation, glycolaic acid utilization (AglcD) and acetyl -Co A-dependent acetylation (APatZ). 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 ampicillin, 100 pg/mL streptomycin) were included when appropriate. Protein expression was conducted by culturing modified E. coli MG1655 expressing CaAbfT and ApbHACS in TB medium (6.78 g/L Na₂HPO4, 3 g/L KH2PO4, 1 g/L NH4CI, 0.5 g/L NaCl, 2 mM MgSCL, 100 pM CaCb, and 15 pM thiamine-HCl). Cultures were then incubated at 30°C and 1000 rpm in a Digital Microplate Shaker until an ODgoo of ~0.4 was reached, at which point appropriate amounts of inducer(s) (isopropyl P-D-l -thiogalactopyranoside (IPTG) and cumate) were added. Plates were incubated for a total of 24 hrs post-inoculation. The total volume of 200 p L of reaction mixture contains a cell suspension with an ODeoo of 20, 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 pM CaCl₂, and 15 pM thiamine-HCl), 20 mM of hydroxypivaldehyde, and 50 mM of formate. The mixture was incubated at 30 °C for 20 h with the shaking speed at 400 rpm. The reaction mixture was centrifuged at 5,000 rpm for 5 mins and subjected to HPLC analysis. The HPLC results showed that DL-2,4-dihydroxy-3,3-dimethylbutanoic acid (DL-pantoic acid) could be produced through the condensation of hydroxypivaldehyde and formyl-CoA derived from formate using whole-cell bioconversion system (Figure 6 below).

[0067] EXAMPLE 9: ENHANCEMENT OF DL-PANTOIC ACID PRODUCTION VIA ENZYME ENGINEERING

[0068] To enhance the production of DL-pantoic acid, ApbHACS has been engineered to improve its substrate specificity and catalytic activity towards the condensation of formyl-CoA and hydroxypivaldehyde. Molecular docking using Discovery Studio has been employed to identify the key residues on the ApbHACS; N394 and G476 located at the substrate binding pocket were selected for analine scanning. In comparison to wild-type ApbHACS, the N394A and G476A variants showed lower formaldehyde consumption and glycolic acid production, indicating improved substrate specificity towards hydroxypivaldehyde. In addition, DL-pantoic acid production using ApbHACS-N349A and ApbHACS-G476A variants was enhanced, confirming the importance of these two residues in pantoic acid production.

[0069] To further improve the performance of ApbHACS, alanine scanning has been conducted at the residues around N394, G476 and the C-terminal of ApbHACS. The results reveal that mutations around the substrate binding pocket mostly reduced the substrate specificity towards formaldehyde (lower glyloic acid production), whereas mutations at T395, G474, S446, D544, and Q549 enhanced pantoic acid production as compared to wild-type ApbHACS but not greater than G476A. In addition, we observed that mutations on the residues D445, N472, and L493 have a negative impact on enzyme activity, therefore, a multiple sequence alignment was conducted to elucidate the function of amino acid residues whose substitution led to zero activity. The results showed that D445, N472, and W552 are conserved across different species, whereas L492, G531, and 1539 are not. However, we do not fully understand the role of these residues in enzyme function. Additionally, we conducted site-directed mutagenesis on the G476 residue to enhance the activity of ApbHACS on pantoic acid production; however, no further improvement was observed. (Figure 7 below)

[0070] Table 1. List of 2-hydroxyacyl-CoA synthase (HACS) used for the condensation of hydroxypivaldehyde and formyl-CoA

[0071] Table 2. List of acyl-CoA reductase (ACR)

[0072] Table 3. List of enzymes used for the interconversion of one-carbon compounds to generate formyl-CoA

[0073] Note: sMMO: soluble methane monooxygenase; pMMO: Particulate methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; CTA: catalase; ACS: acyl-CoA synthase. [0074] Table 4. List of acyl-CoA transferases (ACT) variants used for for the interconversion of one-carbon compounds to generate formyl-CoA

[0075] Table 5. List of acyl-CoA kinase (ACK) and phosphoacyltransferase (PTA) variants (JGIK) variants used for the interconversion of one-carbon compounds to generate formyl- CoA or catalyzing the conversion of DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA to DL- 2,4-dihydroxy-3,3-dimethylbutanoic acid

[0076] Table 6. List of aldehyde dehydrogenase (ALDH)

[0077] 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.

[0078] Table 7. Activities of ApbHACS mutants on formaldehyde for glycolic acid production

[0079] Table 8. Activities of ApbHACS mutants on hydroxypivaldehyde for pantoic acid production

[0080] Table 9. Activities of ApbHACS mutants having a mutation at C-terminal on hydroxypivaldehyde for pantoic acid production

Claims

1. A genetically modified microorganism for the production of DL-2,4-dihydroxy-3,3- dimethylbutanoic acid and DL-3-hydroxy-4,4-dimethyloxolan-2-one , wherein the production comprising converting hydroxypivaldehyde and formyl-CoA to DL-2,4- dihy droxy-3 , 3 -dimethylbutanoyl-Co A, the microorganism comprises a first overexpressed enzyme, wherein the first overexpressed enzyme is a TPP-dependent enzyme selected from a 2-hydroxyacyl-CoA synthase, a 2- hydroxyacyl-CoA lyase, an oxalyl-CoA decarboxylase or a benzaldehyde lyase, and the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is D-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA, L-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA or any mixture thereof.

2. The genetically modified microorganism of claim 1, further comprising a second overexpressed enzyme catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanoic acid, wherein the second overexpressed enzyme is selected from a thioesterase or an acyl-CoA transferase.

3. The genetically modified microorganism of claim 1, wherein the microorganism is capable of converting DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA to DL-3-hydroxy-4,4- dimethyloxolan-2-one.

4. The genetically modified microorganism of claim 3 , further comprising a third overexpressed enzyme catalyzing the conversion of DL-2,4-dihydroxy-3,3-dimethylbutanoy1-CoA to DL- 2,4-dihydroxy-3,3-dimethylbutanal, wherein the third overexpressed enzyme is an acyl-CoA reductase.

5. The genetically modified microorganism of claim 4, further comprising a fourth overexpressed enzyme catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanoyl-phosphate, wherein the fourth overexpressed enzyme is a phosphoacyltransferase.

6. The genetically modified microorganism of claim 5, further comprising a fifth overexpressed enzyme catalyzing the conversion of DL-2,4-dihydroxy-3,3-dimethylbutanal to DL-2,4- dihydroxy-3, 3 -dimethylbutanoic acid, wherein the fifth overexpressed enzyme is an aldehyde dehydrogenase.

7. The genetically modified microorganism of claim 6, further comprising a sixth overexpressed enzyme catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-phosphate to DL-2,4-dihydroxy-3,3-dimethylbutanoic acid, wherein the sixth overexpressed enzyme is an acyl-CoA kinase.

8. The genetically modified microorganisms of any one of claims 1-7, further comprising a seventh overexpressed enzyme catalyzing the conversion of methanol to formaldehyde, wherein the seventh overexpressed enzyme is selected from methanol dehydrogenase or alcohol oxidase.

9. The genetically modified microorganisms of any one of claims 1-8, further comprising an eighth overexpressed enzyme catalyzing the conversion of formaldehyde to formate, wherein the eighth overexpressed enzyme is selected from the group consisting of:

(a) a formaldehyde dehydrogenase;

(b) a S -(hydroxymethyl) glutathione synthase, S-(hydroxymethyl) glutathione dehydrogenase and S -formylglutathione hydrolase;

(c) a mycothiol-dependent formaldehyde dehydrogenase and a hydrolase;

(d) a formaldehyde activating enzyme (FAE), a methylene-tetrahydromethanopterin dehydrogenase, a methenyl-tetrahydromethanopterin cyclohydrolase and a formyl transferase/hydrolase complex; and

(e) a methylene-tetrahydrofolate dehydrogenase, a methenyl-tetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase.

10. The genetically modified microorganism of any one of claims 1-9, further comprising a ninth overexpressed enzyme, wherein the ninth overexpressed enzyme is selected from the group consisting of:

(a) an acylating formaldehyde dehydrogenase converting formaldehyde to formyl-CoA; (b) a formate kinase converting formate to formyl-phosphate and a phosphate formyltransferase converting formyl-phosphate to formyl-CoA;

(c) an acyl-CoA transferase converting formate to formyl-CoA; and

(d) an acyl-CoA synthase converting formate to formyl-CoA.

11. An enzyme, wherein the enzyme is a 2-hydroxyacy-CoA synthase (HACS) selected from Table 1.

12. An enzyme, wherein the enzyme is a HACS 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.

13. An enzyme, wherein 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) and CfhHACS from Chloroflexi bacterium (Genbank accession: PKN81274.1).

14. An enzyme, wherein the enzyme is a variant of the enzyme of any one of claims 11-13 comprising at least one amino acid substitution at position Y371, N394, T395, T401, D445, S446, G474, 1475, G476, N472, L493, P521, L523, M527, F529, K530, G531, P532, 1534, V535, N536, 1537, K538, 1539, T542, D544, R545, K546, P547, Q548, F550, N551 , W552, H553, G554 or Q549, (e.g., T401 A, Q549A, D445A, I475A, G474A, S446A, T395A, Y371A, D544A, K546A, N472A, L493A, H553L, G554T, N394A, G476A, G476L, G476T, G554A, W552A, N551A, F550A, Q548A, P547A, R545A, T542A, I539A, K538A, I537A, N536A, V535A, I534A, P532A, G531A, K530A, F529A, M527A, L523A or P521A) wherein the substitution modulates (e.g., enhances or reduces) the enzyme activity of DL- 2,4-dihydroxy-3,3-dimethylbutanoyl-CoA production and/or DL-2,4-dihydroxy-3,3- di methyl butanoic acid production.

15. The enzyme of claim 14, wherein the enzyme comprises at least one amino acid substitution at position N394, T395, T401, S446, G474, G476, D544, K546, Q549, P521, H553 or G554, (e.g., N394A, T395A, T401A, S446A, G474A, G476A, G476L, G476T, D544A, K546A, Q549A, P521A or G554T) wherein the substitution enhances the enzyme activity of DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA production and/or DL-2,4-dihydroxy-3,3- dimethylbutanoic acid production.

16. The emzyme of claim 14, wherein the enzyme is selected from any enzyme set forth in Tables 7-9.

17. An enzyme, wherein the enzyme is an acyl-CoA transferase (ACT) selected from Table 4.

18. An enzyme, wherein the enzyme is an ACT 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.

19. An enzyme, wherein the enzyme is an acyl-CoA reductase (ACR) selected from Table 2.

20. An enzyme, wherein the enzyme is an ACR 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.

21 . An enzyme, wherein the enzyme is a phosphoacyltransferase (PTA) selected from Table 5.

22. An enzyme, wherein the enzyme is a PTA 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.

23. An enzyme, wherein the enzyme is an aldehyde dehydrogenase (ALDH) selected from Table 6.

24. An enzyme, wherein the enzyme is an ALDH 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.

25. An enzyme, wherein the enzyme is an acyl-CoA (ACK) selected from Table 5.

26. An enzyme, wherein the enzyme is an ACK 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.

27. An enzyme, wherein the enzyme is a methanol dehydrogenase (MDH) or alcohol oxidase (AOD)selected from Table 3.

28. An enzyme, wherein the enzyme is a MDH or AOD 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.

29. An enzyme, wherein the enzyme is an acyl-CoA synthase (ACS) selected from Table 3.

30. An enzyme, wherein the enzyme is an ACS 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.

31. The genetically modified microorganism of any one of claims 1-10, comprising the enzyme of any one of claims 11-30.

32. The genetically modified microorganisms of any one of claims 1-10 and 31, wherein the microorganisms are selected from the group consisting of bacteria, yeast and fungi.

33. The genetically modified microorganisms of any one of claims 1-10 and 31-32, wherein the microorganisms are Escherichia sp., Bacillus sp., Pseudomonas sp., Corynebacterium sp., Zymonas sp., Clostridium sp., Streptococcus sp., Rhodococcus sp., Geobacillus sp., Saccharomyces sp., Pichia sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. Trichoderma sp..

34. The genetically modified microorganism of any one of claims 1-10 and 31-33, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is D-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA.

35. The genetically modified microorganism of any one of claims 1 -10 and 31 -34, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is L-2,4-dihydroxy-3,3-dimethylbutanoyl- CoA.

36. The genetically modified microorganism of any one of claims 1-10 and 31-35, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is a mixture of D-2,4-dihydroxy-3,3- dimethylbutanoyl-Co A and L-2 ,4-dihy droxy-3 , 3-dimethy Ibutanoyl-Co A.

37. The genetically modified microorganism of any one of claims 1-10 and 31-36, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is D-2,4-dihydroxy-3,3-dimethylbutanoic acid.

38. The genetically modified microorganism of any one of claims 1-10 and 31 -37, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is L-2,4-dihydroxy-3,3-dimethylbutanoic acid.

39. The genetically modified microorganism of any one of claims 1-10 and 31-38, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is a mixture of D-2,4-dihydroxy-3,3- dimethylbutanoic acid and L-2,4-dihydroxy-3,3-dimethylbutanoic acid.

40. The genetically modified microorganism of any one of claims 1-10 and 31-39, wherein the DL-3-hydroxy-4,4-dimethyloxolan-2-one is D-3-hydroxy-4,4-dimethyloxolan-2-one.

41. The genetically modified microorganism of any one of claims 1-10 and 31-40, wherein the DL-3-hydroxy-4,4-dimethyloxolan-2-one is L-3-hydroxy-4,4-dimethyloxolan-2-one.

42. The genetically modified microorganism of any one of claims 1-10 and 31-41, wherein the DL-3-hydroxy-4,4-dimethyloxolan-2-one is a mixture of D-3-hydroxy-4,4-dimethyloxolan- 2-one and L-3-hydroxy-4,4-dimethyloxolan-2-one.

43. Use of the enzyme of any one of claims 11-30 in the production of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA, DL-2,4-dihydroxy-3,3-dimethylbutanoic acid and/or DL-3- hydroxy-4,4-dimethyloxolan-2-one.

44. A method of producing the enzyme of claim 15, comprising introducing a substitution at position N394, T395, T401, S446, G474, G476, D544, K546, Q549, P521, H553, G554, (e.g., of wild-type ApbHACS, such as ApbHACS from Alphaproteobacteria bacterium (Genbank accession: HAK63664.1)) wherein the substitution enhances the enzyme activity towards DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA and DL-2,4-dihydroxy-3,3- dimethylbutanoic acid production.

45. A method for the production of DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA comprising contacting formyl-CoA and hydroxypivaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA is D-2,4- dihydroxy-3, 3 -dimethyl butanoyl-CoA, L-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA or any mixture thereof.

46. A method for the production of DL-2,4-dihydroxy-3,3-dimethylbutanoic acid or DL-3- hydroxy-4,4-dimefhyloxo1an-2-one comprising:

(a) providing DL-2,4-dihydroxy-3,3-dimethylbutanoyl-CoA obtained by the method of claim 45 ; and

(i) a thioesterase or an acyl-CoA transferase catalyzing the conversion of DL-2,4- dihydroxy-3,3-dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3- dimethylbutanoic acid;

(ii) a phosphoacyltransferase catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanoyl-phosphate and an acyl-CoA kinase catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-phosphate to DL-2,4-dihydroxy-3,3-dimethylbutanoic acid;

(iii)an acyl-CoA reductase catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-2,4-dihydroxy-3,3-dimethylbutanal and an aldehyde dehydrogenase catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanal to DL-2,4-dihydroxy-3,3-dimethy1butanoic acid;

(iv)a lactonization catalyzing the conversion of DL-2,4-dihydroxy-3,3- dimethylbutanoyl-CoA to DL-3-hydroxy-4,4-dimethyloxolan-2-one, wherein the DL-2,4-dihydroxy-3,3-dimethylbutanoic acid is D-2,4-dihydroxy-3,3- dimethylbutanoic acid, L-2,4-dihydroxy-3,3-dimethylbutanoic acid or any mixture thereof, and the DL-3-hydroxy-4,4-dimethyloxolan-2-one is D-3-hydroxy-4,4-dimethyloxolan-2- one, L-3-hydroxy-4,4-dimethyloxolan-2-one or any mixture thereof.