Production of 1,4-butanediol via condensation of c1-derived formyl-COA and 3-hydroxypropionaldehyde derived from glycerol, ethylene glycol, and c1 feedstocks

Abstract

This invention provides a non-naturally occurring genetically modified microbial organism that has been engineered to produce 2,4-dihydroxybutanal, 1,2,4-butanetriol, 4-hydroxybutanal or 1,4- butanediol using a pathway which enables the condensation of formyl-CoA with 3- hydroxypropionaldehyde derived from glycerol. The modified microbial organism has been further engineered to produce glycerol from ethylene glycol or C1 compounds via condensation of formyl-CoA with glycolaldehyde and formaldehyde. This invention provides a method for the production of 2,4-dihydroxybutanal, 1,2,4-butanetriol, 4-hydroxybutanal or 1,4-butanediol via the condensation of formyl-CoA with 3-hydroxypropionaldehyde. This invention also provides a method for the production of glycerol via the condensation of formyl-CoA and glycolaldehyde. This invention additionally provides a method for the production of glycolaldehyde via the condensation of formyl-CoA and formaldehyde. Further, this invention provides a method for the formation of formyl-CoA from methane, methanol, formaldehyde or formate.

Description

PRODUCTION OF 1,4-BUTANEDIOL VIA CONDENSATION OF Cl-DERIVED FORMYL-COA AND 3-HYDROXYPROPIONALDEHYDE DERIVED FROMGLYCEROL, ETHYLENE GLYCOL, AND Cl FEEDSTOCKSTECHNICAL FIELD

[0001] The present disclosure generally relates to a method for producing 1,4-butanediol, in particular, a method for producing 1,4-butanediol via condensation of Cl -drived formyl-CoA and 3-hydroxypropionaldehyde. Firstly, the present disclosure provides a method for the production of 1 ,4-butanediol via condensation of 3-hydroxypropionaldehyde and Cl -derived formyl-CoA, wherein 3-hydroxypropionaldehyde is derived from the dehydration of glycerol. Secondly, the present disclosure provides a method for the production of glycerol via condensation of glycolaldehyde and Cl -derived formyl-CoA, wherein glycolaldehyde is derived from the oxidation of ethylene glycol. Thirdly, the present disclosure provides a method for producing glycerol via condensation of glycolaldehyde and Cl -derived formyl-CoA, wherein glycolaldehyde is produced from the condensation of formaldehyde and Cl-derived formyl-CoA. Additionally, this disclosure describes the method for generation of formyl-CoA from Cl compounds such as methane, methanol, formaldehyde or formate. The present disclosure also relates to genetically modified microorganism used for producing 1 ,4-butanediol.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 1,4-butanediol from glycerol via the condensation of 3-hydroxypropionaldehyde and formyl-CoA derived from Cl compounds such as methane, methanol, formaldehyde or formate.

[0004] Embodiment 1. A method for the production of 1 ,4-butanediol from glycerol comprising one or more of the following:(a) condensation of 3 -hydroxypropionaldehyde and Cl -derived formyl-CoA, for example by the method described in any one of Embodiments 2-14;(b) production of glycerol from ethylene glycol via condensation of glycolaldehyde with formyl-CoA derived from Cl compounds such as methane, methanol, formaldehyde or formate, for example by the method described in Embodiments 15-36;(c) production of glycerol from Cl compounds such as methane, methanol, formaldehyde or formate through iterative condensation of formyl-CoA with formaldehyde (for example by the method described in Embodiments 37-42) followed by the condensation of glycolaldehyde with formyl-CoA (for example by the method described in Embodiments 15-36).

[0005] Embodiment 2. The method of Embodiment 1 , comprising generating 3- hydroxypropionaldehyde from glycerol by contacting glycerol with a glycerol dehydratase.

[0006] Embodiment 3. The method of Embodiment 2, wherein the glycerol dehydratase is selected from:

[0007] Embodiment 4. A method for producing 1 ,4-butanediol comprising:(a) providing 3 -hydroxypropionaldehyde by the method of Embodiment 2 or 3;(b) forming 2,4-dihydroxybutyryl-CoA via condensation of 3-hydroxypropionaldehyde with Cl -derived formyl-CoA;(c) forming 2,4-dihydroxybutanal from 2,4-dihydroxybutyryl-CoA;(d) forming 1 ,2,4-butanetriol from 2,4-dihydroxybutanal;(e) forming 4-hydroxybutanal from 1,2,4-butanetriol; and(f) forming 1,4-butanediol from 4-hydroxybutanal.

[0008] Embodiment 5. The method of Embodiment 4, wherein 2,4-dihydroxybutyryl-CoA is produced by contacting formyl-CoA and 3-hydroxypropionaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0009] Embodiment 6. The method of Embodiment 5, wherein the TPP-dependent enzyme is 2- 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase, selected from:

[0010] Embodiment 7. The method of Embodiment 4, wherein 2,4-dihydroxybutanal is produced by contacting 2,4-dihydroxybutyryl-CoA with an acyl-CoA reductase.

[0011] Embodiment 8. The method of Embodiment 7, wherein the acyl-CoA reductase is selected from:

[0012] Embodiment 9. The method of Embodiment 4, wherein 1 ,2,4-butanetriol is produced by contacting 2,4-dihydroxybutanal with an alcohol dehydrogenase.

[0013] Embodiment 10. The method of Embodiment 9, wherein the alcohol dehydrogenase is selected from:

[0014] Embodiment 11. The method of Embodiment 4, wherein 4-hydroxybutanal is produced by contacting 1 ,2,4-butanetriol with a diol dehydratase.

[0015] Embodiment 12. The method of Embodiment 11, wherein the diol dehydratase is selected from:

[0016] Embodiment 13. The method of Embodiment 4, wherein 1,4-butanediol is produced by contacting 4-hydroxybutanal with an alcohol dehydrogenase.

[0017] Embodiment 14. The method of Embodiment 13, wherein the alcohol dehydrogenase is selected from:

[0018] In another aspect, provided herein is a method for producing 1 ,4-butanediol via condensation of glycolaldehyde and Cl -derived formyl-CoA.

[0019] Embodiment 15. A method for producing glycerol from glycolaldehyde comprising condensation of glycolaldehyde with formyl-CoA, comprising:(a) forming glyceryl-CoA from glycolaldehyde;(b) forming glyceraldehyde from glyceryl-CoA; and(c) forming glycerol from glyceraldehyde.

[0020] Embodiment 16. The method of embodiment 15, wherein forming glyceryl-CoA comprises contacting glycolaldehyde and formyl-CoA with a TPP-dependent enzyme selected from 2- hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0021] Embodiment 17. The method of embodiment 16, wherein the TPP-dependent enzyme is 2- hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0022] Embodiment 18. The method of embodiment 17, wherein the TPP-dependent enzyme is selected from:

[0023] Embodiment 19. The method of any one of Embodiments 16-18, further comprising forming formyl-CoA from one-carbon (Cl) compound.

[0024] Embodiment 20. The method of Embodiment 19, wherein forming formyl-CoA comprises contacting the one-carbon (Cl) compound selected from formaldehyde or formate with an enzyme.

[0025] Embodiment 21. The method of Embodiment 20, wherein the Cl compound is formaldehyde, and the enzyme is an acyl-CoA reductase.

[0026] Embodiment 22. The method of Embodiment 21, wherein the acyl-CoA reductase is selected from:

[0027] Embodiment 23. The method of Embodiment 20, further comprising generating formaldehyde from methanol by contacting methanol with a methanol dehydrogenase or methanol oxidase.

[0028] Embodiment 24. The method of Embodiment 23, wherein the methanol dehydrogenase or methanol oxidase is selected from:

[0029] Embodiment 25. The method of Embodiment 23 or 24, wherein further comprising generating methanol from methane by contacting methane with a methane monooxygenase.

[0030] Embodiment 26. The method of Embodiment 25, wherein the methane monooxygenases is selected from:GeneGenBank Accession Number

[0031] Embodiment 27. The method of Embodiment 20, wherein the Cl compound is formate and the enzyme is selected from:(i) an acyl-CoA transferase catalyzing the conversion of formate to formyl-CoA;(ii) an acyl-CoA synthase catalyzing the conversion of formate to formyl-CoA; or(iii) a formate kinase catalyzing the conversion of formate to formyl -phosphate and a phosphate formyltransferase catalyzing the conversion of formyl-phosphate to formyl- CoA.

[0032] Embodiment 28. The method of Embodiment 27, wherein the acyl-CoA transferase is selected from:

[0033] Embodiment 29. The method of Embodiment 27, wherein the acyl-CoA synthase is selected from:

[0034] Embodiment 30. Tire method of Embodiment 27, wherein the formate kinase and the phosphate formyltransferase pair is selected from:

[0035] Embodiment 31. The method of any one of Embodiments 15-30, wherein forming glyceraldehyde comprising contacting the glyceryl-CoA with an acyl-CoA reductase catalyzing the conversion of glyceryl-CoA to glyceraldehyde.

[0036] Embodiment 32. The method of Embodiment 31, wherein the acyl-CoA reductase is selected from:

[0037] Embodiment 33. The method of any one of Embodiments 15-32, wherein forming glycerol comprises contacting the glyceraldehyde with an alcohol dehydrogenase.

[0038] Embodiment 34. The method of Embodiment 33, wherein the alcohol dehydrogenase is selected from:

[0039] Embodiment 35. The method of any one of Embodiments 15-34, further comprising forming glycolaldehyde via oxidation of ethylene glycol.

[0040] Embodiment 36. The method of Embodiment 35, wherein forming glycolaldehyde comprises contacting the ethylene glycol with an alcohol dehydrogenase selected from:

[0041] Also provided herein is a method for producing 1,4-butanediol via condensation of glycolaldehyde and Cl -derived formyl-CoA, with the glycolaldehyde being itself produced via condensation of formaldehyde and Cl -derived formyl-CoA; and a method for generation of formyl-CoA from Cl compounds such as methane, methanol, formaldehyde or formate.

[0042] Embodiment 37. A method of producing glycolaldehyde from formaldehyde, comprising forming glycolyl-CoA from formyl-CoA and forming glycolaldehyde from glycolyl-CoA.

[0043] Embodiment 38. The method of Embodiment 37, wherein forming glycolyl-CoA comprises contacting formaldehyde and formyl-CoA (generated by the method described in Embodiments 20-30) with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0044] Embodiment 39. The method of Embodiment 38, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0045] Embodiment 40. The method of Embodiment 38, wherein the TPP-dependent enzyme is selected from:

[0046] Embodiment 41. The method of any one of Embodiments 37-40, wherein forming glycolaldehyde comprises contacting the glycolyl-CoA with an acyl-CoA reductase catalyzing the conversion of glycolyl-CoA to glycolaldehyde.

[0047] Embodiment 42. The method of Embodiment 41, wherein the acyl-CoA reductase is selected from:

[0048] Embodiment 43. A method for producing 1,4-butanediol from Cl compounds such as methane or methanol, comprising forming formaldehyde (for example, by the method described in Embodiments 23-26), forming formyl-CoA (for example, by the method described in Embodiments 20-30), forming glycolaldehyde from formaldehyde (for example, by the method described in Embodiments 37-42), forming glycerol from glycolaldehyde (for example, by the method described in Embodiments 15-36), and the conversion of glycerol to 1,4-butanediol (for example, by the method described in Embodiments 1-14).

[0049] Embodiment 44. The method of any one of Embodiments 1 to 43, wherein the enzyme used in the method is isolated from a microorganism.

[0050] Embodiment 45. The method of any one of Embodiments 1 to 43, wherein the enzyme used in the method is contained in a microorganism.

[0051] Embodiment 46. A genetically modified microorganism providing 1,4-butanediol from glycerol by the method of any one of Embodiments 2 to 14.

[0052] Embodiment 47. A genetically modified microorganism further providing glycerol by the method of any one for Embodiment 15 to 36.

[0053] Embodiment 48. A genetically modified microorganism further providing glycerol from Cl compounds by the method of any one for Embodiment 31 to 36.

[0054] Embodiment 49. The microorganism of any one of Embodiments 46 to 48, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

[0055] Embodiment 50. The microorganism of any one of Embodiments 46 to 49, 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. and Trichoderma sp.

[0056] Also provided herein is an enzyme comprising or consisting of an amino acid sequence selected from any amino acid sequence as set forth herein (such as those in Tables 1 -6) or an amino acid sequence having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5%) sequence identity thereto.

[0057] Also provided herein is a genetically modified microorganism comprising any enzyme disclosed herein.

[0058] Also provided herein is an enzyme or a genetically modified microorganism for use in any method disclosed herein.

[0059] In some embodiments, the genetically modified microorganism is aldB knocked-out, aldB+aldA knocked-out, and / or FucO and / or ackA-Pta deactivated.

[0060] 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.

[0061] The present disclosure is illustrated by the following non-limiting examples.

[0062] 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

[0063] 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.

[0064] Figure 1 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 1 CMPT-dependent oxidation of formaldehyde to formate.

[0065] 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-I I4MPT cyclohydrolase enzyme; MCH: methenyl-H4MPT cyclohydrolase.

[0066] Figures 2A-2B illustrate the generation of formyl-CoA from formate and the generation of glycolate from the condensation of formaldehyde and formyl-CoA. Figure 2A) shows the reaction driven by ACS and HACS; and Figure 2B) shows the reaction driven by a set of FOK-PTA and HACS.

[0067] ACS: acyl-CoA synthase (BsACS: Bacillus subtilis, EcACS: Escherichia coli, MhACS: Marinithermus hydrothermalis, StACSstab: Salmonella typhimurium), ApbHCAS: 2- hydroxyacyl-CoA synthase derived from Alphaproteobacteria bacterium, FOK-PTA: formate kinase-phosphate formyl transferase derived from Coriobacteriia bacterium and DtbHACS: 2- hydroxyacyl-CoA synthase derived from Deltaproteobacteria bacterium.

[0068] Figure 3 is a scheme illustrating the production of 1,4-butanediol from different feedstock through the condensation of 3-hydroxypropinoaldehyde and formyl-CoA.

[0069] 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; ADH: alcohol dehydrogenase; DDR: diol dehydratase; GDHt: glycerol dehydratase.

[0070] Figure 4 is the demonstration of 2,4-dihydroxybutyric acid production from 3- hydroxypropionaldehyde in vitro using purified enzymes.

[0071] Figure 5 is the demonstration of 2,4-dihydroxybutyric acid production from 3- hydroxy propion aldehyde in vivo.

[0072] Figure 6 is the production of 2,4-dihydroxybutyric acid from 3 -hydroxy propion aldehyde in vivo.

[0073] Figure 7 is the effect of aldehyde / alcohol dehydrogenase on 3-hydroxypropinoaldehyde consumption.

[0074] Figure 8 is the effect of double aldehyde dehydrogenase knockout on 3- hydroxy propino aldehyde consumption.

[0075] Figure 9 shows 2,4-dihydroxybutyric acid production from glycerol and formate.

[0076] Figure 10 is the demonstration of 1 ,4-butanediol production from glycerol and formate.

[0077] Figure 11 is a scheme illustrating the production of 3-hydroxypropionaldehyde from glycerol, ethylene glycol and Cl compounds through formyl-CoA elongation (FORCE) pathway.

[0078] Figure 12 shows the production of 3-hydroxypropinoaldehyde from glycerol.

[0079] Figure 13 A) is a scheme illustrating the production of 1,4-butanediol from ethylene glycol coupled to generation of formyl-CoA and reducing equivalents from Cl compounds; B) shows glycerol production from glycolaldehyde where ethylene glycol is a feedstock; C) shows glycerol production from glyceraldehyde where different ADH variants are tested.

[0080] 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; ADH: alcohol dehydrogenase; DDR: diol dehydratase; GDHt: glycerol dehydratase; FucO: lactaldehyde reductase.

[0081] Figure 14 A) is a scheme illustrating the production of glycerol from Cl compounds coupled to generation of formyl-CoA and reducing equivalents from Cl compounds; B) shows glycerate production from Cl compounds through the condensation with formyl-CoA; C) shows glycerol production from C1 compounds through the condensation with formyl-CoA.

[0082] 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; ADH: alcohol dehydrogenase; DDR: diol dehydratase; GDHt: glycerol dehydratase.

[0083] DETAILED DESCRIPTION OF THE INVENTION

[0084] 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.

[0085] 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.

[0086] EXAMPLE 1: INTERCONVERSION OF ONE-CARBON COMPOUNDS TO GENERATE FORMYL-COA

[0087] Formyl-CoA can be provided through the interconversion of Cl compounds including methane, methanol, formaldehyde, and formate (Figure 1 below) with suitable enzymes (e.g., enzymes set forth in Table 4 below). Methane is readily available Cl source from natural gas, landfills, and agriculture. Biological oxidation of methane to methanol is catalyzed by methane monooxygenases. Functional expression of soluble methane monooxygenase (sMMO) fromMethylococcus capsulatus is demonstrated in E. coli as a host (bioRxiv 2021. 08.05.455234). Subsequent oxidation of methanol to formaldehyde can be catalyzed by NAD+-dependent methanol dehydrogenases (MDH), MDH from Bacillus methanolicus MGA3 (BmMDH), Bacillus steamthermophilus (BsMDH) (Metab. Eng. 39:49-59, 2017) and Cupriavidus necator (Appl. Microbiol. Biotechnol. 100:4969-4983, 2016), PQQ (pyrroloquinoline quinone)-dependent MDH (MxaFI, or XoxF), or oxygen-dependent alcohol oxidase (AOD). When AOD is used, a catalase (CTA) is needed for de-toxicity of hydrogen peroxide (e.g., enzymes set forth in Table 4 below).

[0088] Formaldehyde can be directly oxidized to formic acid catalyzed by four cofactor-dependent formaldehyde oxidation pathways, involving glutathione (GSH), tetrahydrofolate (H4F, THF), tetrahydromethanopterin (H4MPT), or mycothiol (MSH). Formaldehyde spontaneously reacts with glutathione (GSH) to form S-hydroxymethyl-GS. Hydroxymethyl -GS is then converted to formyl- GS by hydroxymethyl-GS dehydrogenase (FrmA). Formyl-GS is then converted to formate by formyl-GS hydrolase (FrmB). Formaldehyde also spontaneously reacts with mycothiol to form an S-hydroxymethylmycothiol same as GSH-dependent route, and then it was converted to S- formylmycothiol by MD-FalDH, which is further hydrolyzed to formate and MSH via a hydrolase. When H4MPT was used as the cofactor, formaldehyde condensed with H4MPT to generate methylene-H4MPT spontaneously or by formaldehyde activating enzyme (FAE), methylene - H4MPT is then converted to metheny1-H4MPT by methylene-tetrahydromethanopterin dehydrogenases MtdA or MtdB. Methenyl-H iMPT was converted to formyl -H 1 MPT by methenyl- H4MPT cyclohydrolase (MCH), and then converted to formate with the help of formyltransferase / hydrolase FHC. Formaldehyde spontaneously reacts with H4F to form methylene-H4F, and then contacting with MtdA it was converted to methenyl -H4F which is then converted to formyl-H4F by methenyl-H4F cyclohydrolase. Formyl-H4F can be further converted to formate by formate-H4F ligase FTFL.

[0089] Alternatively, formaldehyde oxidation to formyl-CoA can be catalyzed by various acylating aldehyde dehydrogenase candidates (Nat. Chem. Biol. 15:900-906, 2019). Formyl-CoA can either be fed to the Cl elongation platform or further converted to CO2 to generate energy. Formyl-CoA hydrolysis to formate can be catalyzed by two different families of enzymes. Acyl- CoA transferases (ACT, e.g., set forth in Table 5 below) can catalyze reversible CoA transfer from various CoA donor, such as 2,4-dihydroxybutyryl-CoA or acetyl-CoA to formate. A set of phosphotransacylase-formate kinase (PTA-FOK, e.g., set forth in Table 6 below) pair can alsocatalyze a reversible phosphorylation of formyl-CoA to formyl-phosphate, followed by dephosphorylation to formate generating 1 ATP. While these two reactions are considered fully reversible, AMP-forming acyl-CoA synthetases are favored toward formate activation to formyl- CoA (ACS, e.g., set forth in Table 4 below).F0090] We have demonstrated various pathways for formyl-CoA generation, with glycolate formed through the condensation of formaldehyde and formyl-CoA serving as a representative indicator. To generate formyl-CoA from formate, 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, 50 mM of formate, 10 m of formaldehyde, 5 pM of HACS and 1 pM of ACS. The reaction mixture was incubated at room temperature for overnight, and the reaction was terminated by adding 2.5 pL of 10 M NaOH then incubated at room temperature for 30 minutes. A 2.5 pL of 10 M HO 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 glycolate from the reaction indicates that formyl-CoA could be generated from formate via the activation of ACS (acyl-CoA synthase) (Figure 2A below). In addition to the cell-free bioconversion system, we investigated formyl-CoA generation in vivo using Escherichia coli MG1655 expressing ACS and HACS. The cells were cultured in TB medium at 37 °C for 2.5 hours with a shaking speed of 200 rpm. Subsequently, 50 pL of 0.1 M IPTG was added, and the culture was further incubated at 30 °C for 24 hours to induce protein expression. The cells were then centrifuged at 5,000 rpm, 4 °C for 5 minutes, and the supernatant was discarded. A 10 mL M9 wash buffer was added to the tube, and the mixture was thoroughly mixed. This washing step was repeated twice. The cells were resuspended in M9 wash buffer to achieve a cell density of OD600 = 50; the final cell density in the reaction was OD600 = 10. Formaldehyde and formate were initially added to the reaction at final concentrations of 10 mM and 20 mM, respectively. The mixture was incubated at 30 °C for 3.5 hours with a shaking speed of 200 rpm. A 1 mL sample of the reaction mixture was collected for HPLC analysis. Additional formaldehyde and formate, both at final concentrations of 10 mM and 20 mM, were added to the reaction. At 6 hours, formate at a final concentration of 20 mM was reintroduced. The reaction was prolonged to 20 hours and analyzed by HPLC. Glycolate was observed in the reaction, indicating the generation of formyl-CoA from formate in vivo by selected ACS (Figure 2A below).

[0091] Furthermore, we have tested the formyl-CoA generation from formate via FOK-PTA route. A 1 mL of cell-free bioconversion reaction contains 100 M of potassium phosphate buffer (pH6.9), 10 mM of MgC12, 150 |_iM TPP, 10 mM of Co A, 50 mM of formate, 20 mM of formaldehyde,2 M of HACS, 2 pM of ACK (FOK) and 2 pM of PTA. The reaction mixture was incubated, terminated, and analyzed in the same manner as mentioned above. The HPLC result showed that 1 .45 mM of glycolate was produced from the reaction, confirming the generation of formyl -CoA from formate, catalyzed by a set of FOK-PTA (formyl-CoA kinase and phosphotransacylase) (Figure 2B below).EXAMPLE 2: OVERVIEW OF 1,4-BUTANEDIOL PRODUCTION THROUGH CONDENSATION OF 3-HYDROXYPROPINO ALDEHYDES AND FORMYL-COA

[0092] This example demonstrates the implementation of 1,4-butanediol production through condensation of 3-hydroxypropinoaldehydes and formyl-CoA followed by derivation to 1,4- butanediol (Figure 3 below). Feedstocks of varying carbon lengths can be incorporated into the 1,4-butanediol production pathway through different aldehyde intermediates, such as 3- hydroxypropionaldehyde, glycolaldehyde and formaldehyde. The condensation can be catalyzed by HACS (e.g., HACS set forth in Table 1 below), including but not limited to beach sand metagenome BsmHACS (GenBank accession: HAK63664.1), DhcHACS from Dehalococcoidia bacterium (GenBank accession: PWB41796.1), CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), to generate 2,4-dihydroxybutyryl-CoA. 2,4-dihydroxybutyryl-CoA is further converted to 2,4-dihydroxybutyric acid (also referred to as 2,4-dihydroxybutanoic acid) through the action of a thioesterase (such as TesB from E. coli or Pseudomonas putida (McMahon, M.D. and Prather, K.L.J. Appl. Environ. Microbiol. 2014, 80: 1042-1050)), CoA transferase (ACT, e.g., set forth in Table 5 below), or phosphotransacylase-acid kinase (PTA-ACK, e.g., set forth in Table 6 below). 2,4-dihydroxybutyryl-CoA is reduced to 2, 4-dihydroxy butyraldehyde (also referred to as 2,4-dihydroxybutanal) by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Alcohol dehydrogenase (ADH) such as enzyme from Escherichia coli (EcYqhD, GenBank accession: Q46856) and other ADH variants (e.g., ADH set forth in Table 3 below) drives the reaction that converts 2,4-dihydroxybutanal to 1 ,2,4-butanetriol. 1 ,2,4-butanetriol is then converted to 4-hybroxybutanal by diol dehydratase (DDR) from Klebsiella oxytoca (KoPddABC, GenBank accession: Q59470, Q59471, Q59472) and other DDR variants (e.g., DDR set forth in Table 3 below). The final step is a conversion of 4-hydroxybutanal to 1 ,4-butanediol by alcohol dehydrogenase (Figure 3 below; enzymes set forth in Table3 below).

[0093] EXAMPLE 3. PRODUCTION OF 2,4-DIHYDROXYBUTYRYL-COA BY CONDENSATION OF 3-HYDROXYPROPINALDEHYDE AND FORMYL-COA USING PURIFIED ENZYMES.

[0094] This example demonstrates the production of 2,4-dihydroxybutyryl-CoA by condensation of 3-hydroxypropinoaldehyde (3-HPA) and formyl-CoA using purified enzymes using 2,4- dihydroxybutyric acid as a benchmark. In this example, ACK-PTA route was employed to provide formyl-CoA for condensation.

[0095] Expression of selected enzyme variants was achieved using plasmid-based gene expression by cloning the desired gene(s) into pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes and by utilizing In-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, CA). Linear DNA fragments for insertion were created via PCR of the open reading frame of interest (for genes native to E. coli) or by gene synthesis of the codon optimized gene. Genes were synthesized by GeneArt (Life Technologies, Carlsbad, CA). Resulting In-Fusion reaction products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, CA), and clones identified by PCR screening were further confirmed by DNA sequencing.

[0096] Overnight cultures of the expression strains were grown in LB, which was used to inoculate 25 mL TB medium in a 250 mL baffled flask at 1 %. The culture was grown at 30°C and 250 rpm in an orbital shaker until ODeoo reached 0.4-0.6, at which point expression was induced with 0.1 mM IPTG. 24 hours post inoculation, cells were harvested by centrifugation. The cell pellets were washed once with cold 9 g / L NaCl solution and stored at -80°C until needed. Antibiotics were included where appropriate at the following concentrations: ampicillin (100 pg / mL), carbenicillin (50 pg / mL), and spectinomycin (50 pg / mL).

[0097] For protein purification, E. coli cell pellets expressing the desired his-tagged enzymes were prepared as described above. The frozen cell pellets were resuspended in cold lysis buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 10 mM imidazole) to an approximate OD600 of 40, to which 1 mg / mL of lysozyme and 250 U of Benzonase nuclease was added. The mixture was further treated by sonication on ice using a Branson Sonifier 250 (5 minutes with a 25% duty cycle and output control set at 3), and centrifuged at 7500xg for 15 minutes at 4°C. The supernatant was applied to a chromatography column containing 1 mL TALON metal affinity resin (Clontech Laboratories, Inc., Mountain View, CA), which had been pre-equilibrated with the lysis buffer. The column wasthen washed first with 10 mL of the lysis buffer and then twice with 20 mL of wash buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 20 mM imidazole). The his-tagged protein of interest was eluted with 1-2 applications of 4 mL elution buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 250 mM imidazole). The eluate was collected and applied to a 10,000 MWCO Amicon ultrafiltration centrifugal device (Millipore, Billerica, MA), and the concentrate (-100 pL) was washed twice with 4 mL of 50 mM KPi pH 7.4 for desalting. Protein concentrations were estimated by the Bradford method. Purified protein was saved in 20 pL aliquots at -80°C until needed.

[0098] SDS-PAGE was performed using NuPAGE 12% Bis-Tris Protein Gels with SDS running buffer and stained with SimplyBlue SafeStain according to manufacturer protocols (ThermoFisher Scientific, Waltham, MA).

[0099] In vitro purified enzyme reaction was done in a reaction containing 50 mM HEPES pH 7.8, 5 mM ATP, 10 mM MgCh, 5 mM CoA, 0.2 mM TPP, 2 pM CaoHACS or CfhHACS, 1 pM PTA, 1 pM ACK, 30 mM sodium formate and 10 mM 3-hydroxypropinoaldehyde. Reactions were incubated at 30°C for 18 hours unless otherwise specified. 1 / 20 of the reaction volume of 10 M NaOH solution was added to terminate the reactions. After 30 min hydrolysis, 1 / 20 of the reaction volume of 10 N H2SO4 was added to neutralize the pH. Samples were centrifuged at 20817xg for 15 minutes and the supernatant analyzed by HPLC as described below.

[0100] Quantification of product and substrate concentrations was performed via HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a DAD and an Supelco aphera-NH2 column (5pm*4.6mm*250mm) with operating conditions to optimize peak separation (1 ml / min flowrate, 0.1% HiPG4:CAN=70:30 as the mobile phase, column temperature 40°C).

[0101] The results indicate that both CaoHACS and CfhHACS were able to produce 2,4- dihydroxybutyric acid through the condensation of 3-hydroxypropinoaldehyde and formyl-CoA (Figure 4)

[0102] EXAMPLE 4: SCREENING OF DIFFERENT HACS FOR 2,4- DIHYDROXYBUTYRIC ACID PRODUCTION

[0103] The purpose of this example is to screen better HACS for 2,4-dihydroxybutyric acid production by condensation of 3-hydroxypropinoaldehyde and formyl-CoA. Eight HACS candidates from previous study were tested here, including ApbHACS (JGI15), DhcHACS(JGI20), CabHACS (JGI23), CdcHACS (JGI24), MeOXC4, CaoHACS (JGIH48), CfhHACS (JG1H65), BbHACS (JG119). Acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT) was used for formyl-CoA generation.

[0104] To compare HACS for 2,4-dihydroxybutyric acid production in vivo, vectors were engineered to independently control the expression of various HACS candidates and the acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), with HACS under control of the IPTG- inducible T7 promoter in pCDFDuet-1 and CaAbfT under control of a cumate-inducible T5 promoter in pETDuet-1. As a host for these vectors, an engineered strain of E. coli based on MG1655 (DE3) with knockouts for formaldehyde (AfrmA) and formate (AfdhF AfdnG AfdoG) oxidation as well as for glycolate utilization (AglcD) was used.

[0105] In vivo product synthesis was conducted using M9 minimal media (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4CI, 0.5 g / L NaCl, 2 mM MgSO4, 100 pM CaCh, and 15 jiM thiamine- HC1) unless otherwise stated. Cells were initially grown in 96-deep well plates containing 0.5 mL of the above media further supplemented with 20 g / L glycerol, 10 g / L tryptone, and 5 g / L yeast extract. A single colony of the desired strain was cultivated overnight (14-16 hours) in LB medium with appropriate antibiotics and used as the inoculum (1%). Antibiotics (100 pg / mL carbenicillin, 100 pg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30°C and 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until an GD600 of ~0.4 was reached, at which point appropriate amounts of inducer(s) (isopropyl p-D- 1 -thiogalactopyranoside and cumate) were added. Plates were incubated for a total of 24 hours post-inoculation.

[0106] Cells from the above pre-cultures were then centrifuged (4000 rpm, 22°C), washed with the above minimal media without any carbon source, and resuspended with 1 mL of above minimal media containing indicated amounts of carbon source. 10 mM 3-hydroxypropinoaldehyde and 30 mM formate were added at 0 hr and were incubated at 30 °C and 1000 rpm in Digital Microplate Shaker (Fisher Scientific). After incubation at 30°C for 2 hour, the cells were pelleted by centrifugation and the media was analyzed using HPLC.

[0107] The results indicate that 6 out of 8 tested enzymes have condensation activity and DhcHACS has higher activity than other tested enzymes, it produced 35 mg / L 2,4- dihydroxybutyric acid (Figure 5).

[0108] The HACS performance on 2,4-dihydroxybutyric acid production was further evaluated in an engineered strain MG1655(DE3) del (glcD, frmA, fdhF, fdoG, fdnG, yqhD, aldB) that has alower 3-hydroxypropinoaldehyde background consumption. By adding 10 mM 3- hydroxy propino aldehyde and 20 mM formate to a final OD of 20 for bioconversion at 30 °C for 21 hours, DhcHACS and CaoHACS have the highest 2,4-dihydroxybutyric acid production (154 mg / L and 153 mg / L respectively), CfhHACS and ApbHACS has good performance too (Figure 6)

[0109] EXAMPLE 5. ENGINEERING OF THE HOST STRAIN TO HAVE A LOWER 3- HYDROXYPROPINOALDEHYDE BACKGROUND CONSUMPTION

[0110] The purpose of this example is to decrease the 3-hydroxypropinoaldehyde background consumption in the host strain by knockout the genes responsible for 3HPA background consumption. The aldehyde dehydrogenases and alcohol dehydrogenases were deactivated individually in BW251 13 del (frmA, fdhF, fdoG, FdnG, glcD) (also referred to as BWA5R).

[0111] A bioconversion with the newly constructed strains was performed as described above. Cells were cultured in TB medium for 24 horns, centrifuged at 4000 x g for 10 min, discarded the broth and washed the cells with M9 once. And then the cells were resuspended in 1 mL M9 with 2 mM 3-hydroxypropinoaldehyde, shaken at 30 °C for 2 hours. The residual 3- hydroxy propino aldehyde in the supernatant was measured using 3-methyl-2-benzothiazolone hydrazone (MBTH) method as reported before.

[0112] The results indicate that knockout aldB has the highest impact on 3- hydroxypropinoaldehyde consumption, and then yiaY, yahK, etc. (Figure 7).

[0113] Seven aldehyde dehydrogenases were further deactivated in BW25113 del (frmA, fdhF, fdoG, FdnG, glcD, aldB), and the 3-HPA background consumption of newly constructed was evaluated using the same method. The results showed that 3-HPA consumption was further decreased and aldB+aldA knockout has the lowest consumption (Figure 8).

[0114] EXAMPLE 6. PRODUCTION OF 2,4-DIHYDROXYBUTYRIC ACID FROM GLYCEROL AND FORMATE

[0115] This example demonstrates the 2,4-dihydroxybutyric acid production from glycerol and formate. Glycerol was converted to 3-hydroxypropinoaldehyde by glycerol dehydratase and formate was converted to formyl-CoA by CaAbfT, and then they were condensed to produce 2,4- dihydroxybutyryl-CoA and converted to 2,4-dihydroxybutyric acid (Figure 9A).

[0116] This was tested in BW25113(DE3) del (frmA, fdhF, fdnG, fdoG, glcD). Glycerol dehydratase from Klebsiella peneumoniae, DhcHACS and CaAbfT were co-overexpressed for thispurpose. The bioconversion was performed the same as described above. Cells were cultured in NBS and induced with appropriate concentration of 1PTG and cumate. Bioconversion was done in a final OD of 20 for 3 hours. The result indicates that the cells can produce 2,4-dihydroxybutyric acid from glycerol and formate (Figure 9B).

[0117] EXAMPLE 7: PRODUCTION OF 1,4-BUTANEDIOL FROM 1,2,4- BUTANETRIOL

[0118] This example demonstrates the 1,4-butanediol production from 1,2,4-butanetriol. 1,2,4- butanetriol can be first converted to 4-hydroxybutanal by 1,2-diol dehydratase, including dehydratase from Klebsiella pneumoniae (Genbank accession: Q59476, 008505, Q59475) and other dehydratases (e.g., DDR set forth in Table 3 below). 4-hydroxybutanal is further converted to 1 ,4-butanediol by 1 ,4-butanediol dehydrogenase, including YqhD from E. coli and other alcohol dehydrogenases (e.g., alcohol dehydrogenase set forth in Table 3 below).

[0119] Whole cell bioconversion of 1,2,4-butanetriol to 1,4-butanediol via glycerol dehydratase was carried out to demonstrate feasibility of this pathway (Figure 10A). Escherichia coli MJMG0237(MG1655(DE3)AadhEAmgsAAlldDAdldAldhAAglcDAfrmAAfdhFAfdoGAfdnGAad hPAyjgBAyahKAyqhDAaldAAaldBApuuCApatDAgshA) expressing GDHt from Klebsiella pneumoniae (gene bank accession: Q59476, 008505, Q59475), activator protein GdrAB, and alcohol dehydrogenase YqhD was inoculated into LB medium and incubated at 37 degrees Celsius until OD reached 0.6-0.8. Cells were induced by adding 50 pM IPTG and 15 pM coenzyme B12 after 2.5 hours, and incubated for another 21.5h (30 degrees Celsius). Cells were then collected by centrifugation at 6000rpm for 5 minutes and washed once with IX M9-N (M9 medium without ammonia) before being resuspended with IX M9-N to an OD600 of 60. The total volume of 1 mL of reaction mixture contains cells at OD600 30, M9-N medium, 30 mM of 4-hydroxybutanal (or1.2.4-butanetriol), 5 mM of KC1, 5 mM of MgCh, and 15 pM of Coenzyme Bl 2. The mixture was incubated at 30 C for 5 h. The reaction was terminated by centrifugation at 12000 rpm for 10 min, and then the amount of 1,4-butanediol was detected by HPLC as described above. When 30 mM 4-hydroxybutanal was added to the system, 0.27 g / L 1,4-butanediol was produced, and 0.1 g / L1.4-butanediol was produced from 30 mM 1,2,4-butanetriol (Figure 10C).

[0120] EXAMPLE 8: PRODUCTION OF 1,4-BUTANEDIOL FROM GLYCEROL

[0121] This example demonstrates the 1,4-butanediol production from glycerol through 1,2,4- butanetriol (Figure 10A). Glycerol is firstly converted to 3 -hydroxypropion aldehyde by glyceroldehydratase, followed by a condensation with formyl-CoA to generate 2,4-dihydroxybutyryl-CoA as mentioned above. 2,4-dihydroxybutyryl-CoA is subsequently converted to 2,4-dihydroxybutanal via the activation of acyl-CoA reductase (ACR) (e.g., ACR set forth in Table 2 below). Alcohol dehydrogenase (ADH) such as enzyme from Escherichia coli (EcYqhD, GenBank accession: Q46856) and other ADH variants (e.g., ADH set forth in Table 3 below) drives the reaction that converts 2,4-dihydroxybutanal to1.2.4-bulanetriol. 1,2,4-butanetriol is then converted to 4-hybroxybutanal by diol dehydratase (DDR) from Klebsiella oxytoca (KoPddABC, GenBank accession: Q59470, Q59471, Q59472) and other DDR variants (e.g., DDR set forth in Table 3 below). The final step is a conversion of 4-hydroxybutanal to 1 ,4-butanediol by alcohol dehydrogenase (Figure 3 below; enzymes set forth in Table 3 below).

[0122] To produce 1 ,4-butanediol from glycerol, the required enzymes were overexpressed in MJMG0237 same as described in Example 7. And an in vitro assay was carried out to demonstrate the feasibility of 1,4-butanediol production from glycerol. After washing with M9-N, cells were resuspended to an OD of 40, and lysed using B-Per cell lysis buffer. Cell-free bioconversion was carried out in a reaction contains 50 mM MOPS-K buffer, pH 8.0, 15 uM TPP, 10 mM MgC12, 2 mM CoA, 2.5 mM NAD+, 2.5 mM NADH, 10 uM vitamin B12, 10 uL of enzymes. 30 mM 3- hydroxy propion aldehyde or 40 g / L glycerol and 30 mM formate was added to start the reaction, the reaction was incubated at 30 °C for 4 hours and the products was analyzed by HPLC. 76.7 pM1.4-butanediol was produced from 30 mM 3-hydroxypropionaldehyde, and 90 pM of 1,4- butanediol was accumulated when 40 g / L glycerol was used as the substrate (Figure 10B) .EXAMPLE 9: PRODUCTION OF 3-HYDROXYPROPIONALDEHYDE FROM GLYCEROL, ETHYLENE GLYCOL AND ONE-CARBON COMPOUNDS

[0123] This example demonstrates the 3-hydroxypropionaldehyde production from different feedstocks, including but not limited to glycerol (C3), ethylene glycol (C2) and methanol (Cl). When 3-hydroxypropionaldehyde is produced, it can be further condensed with formyl-CoA generated from Cl compounds to form 2,4-dihydroxybutyryl-CoA and finally converted to 1,4- butanediol as presented in Figure 3.

[0124] When 3-hydroxypropionaldehyde is produced from glycerol. Glycerol dehydratase (GDHt) needs to be overexpressed for 3-hydroxypropionaldehyde production from glycerol (Figure 11).

[0125] When 3-hydroxypropionaldehyde is produced from ethylene glycol, two iterative condensations are needed for 1,4-butanediol production (Figure 11). Ethylene glycol is firstly converted to glycolaldehyde by E. coli FucO. Glycolaldehyde can be condensed with formyl-CoA to generate glyceryl-CoA, a reaction catalyzed by HACS, including CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), RhbHACS from Rhodocyclaceae bacterium (GenBank accession: MBL8483477.1), PdsHACS from Pseudonocardia sediminis (GenBank accession: WP_130292058.1) or other HACS variants (e.g., HACS set forth in Table 1 below). Glyceryl-CoA can be reduced to glyceraldehyde by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Glyceraldehyde was further converted to glycerol by glyceraldehyde reductase (ADH), such as yqhD, yahK from E. coli, GLD1 from Trichoderma reesei (e.g., enzymes set forth in Table 3 below). Glycerol can be converted to 3-hydroxypropionaldehyde as mentioned above by glycerol dehydratase GDHt, dhaB123 from Klebsiella pneumonia or other microorganisms (e.g., GDHt set forth in Table 3 below), and then further convert to 1,4-butanediol through the route described in Example 1.

[0126] When 3-hydroxypropionaldehyde is produced from methanol or other Cl compounds, one more iterative condensation is needed to generate glycolaldehyde for 1,4-butanediol production compare to using ethylene glycol as co-substrate (Figure 11). Methanol or other Cl compounds were firstly converted to formaldehyde and formyl-CoA through the interconversion module (Figure 1). Formaldehyde and formyl-CoA condensed together to generate glycolyl-CoA by HACS , including CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), RhbHACS from Rhodocyclaceae bacterium (GenBank accession: MBL8483477.1), PdsHACS from Pseudonocardia sediminis (GenBank accession: WP_130292058.1) or other HACS variants (e.g., HACS set forth in Table 1 below), and further converted to glycolaldehyde with ACR variants from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). And glycolaldehyde can be used as a substrate for next round condensation with formyl-CoA to generate glyceryl-CoA that catalyzed by above mentioned HACS variants (e.g., HACS set forth in Table 1 below). Glyceryl-CoA can be reduced to glyceraldehyde by acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Glyceraldehyde was further converted to glycerol by glyceraldehyde reductase (ADH), such as yqhD, yahK fromE. coli, GLD1 from Trichoderma reesei (e.g., enzyme set forth in Table 3 below). Glycerol can be converted to 3-hydroxypropionaldehyde as mentioned above by glycerol dehydratase (GDHt), and then further convert to 1 ,4-butanediol through the route described in Example 2.

[0127] Whole cell bioconversion was carried out to convert glycerol to 3-hydroxypropionaldehyde through the activation of glycerol dehydratase (GDHt). Escherichia coli BL21 expressing GDHt derived from Klebsiella pneumoniae with the activator protein, GdrA / B was grown in LB medium. The total volume of 1 mL of reaction mixture contains a cell suspension with an OD600 of 30, M9 medium, 50 mM of glycerol, 5 mM of KC1, 5 mM of MgCh, and 15 pM of Coenzyme B12. The mixture was incubated at 30°C for 5 hours. The reaction mixture was centrifuged at 12,000 rpm for 10 mins and subjected to a colorimetric assay to measure the concentration of 3- hydroxy propion aldehyde. A 100 pL of the supernatant was mixed with 75 L of 0.01 M DL- tryptophan solution (Sigma-Aldrich) in 0.05 M HC1 and 300 pL of 37% v / v HC1. The reaction mixture was incubated at 37°C for 20 mins and measured using a spectrophotometer at an absorbance of 560 nm. 3-hydroxypropionaldehyde from Aikon Chem was used as a standard. The colorimetric assay results demonstrate the conversion of glycerol to 3-hydroxypropionaldehyde, catalyzed by the selected GDHt (Figure 12 below).

[0128] EXAMPLE 10: PRODUCTION OF GLYCEROL FROM C2 COMPOUNDS

[0129] This example demonstrates the implementation of glycerol production through C 1+B io™ platform from C2 feed stocks, including but not limited to ethylene glycol. C2 feedstock was first converted to glycolaldehyde, glycolaldehyde can be further condensed with formyl-CoA that is generated from Cl compounds to form glyceryl-CoA which will be converted to glycerol, and finally converted to 1,4-butanediol as presented in Figure 13.

[0130] When ethylene glycol is utilized as the feedstock for glycerol production, it is firstly converted to glycolaldehyde by E. coli FucO or other ADH variants (e.g., ADH set forth in Table 3). Glycolaldehyde can be condensed with formyl-CoA to generate glyceryl-CoA that catalyzed by HACS, including CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), RhbHACS from Rhodocyclaceae bacterium (GenBank accession: MBL8483477.1), PdsHACS from Pseudonocardia sediminis (GenBank accession: WP_130292058.1) or other HACS variants (e.g., HACS set forth in Table 1 below).

[0131] Glyceryl-CoA is subsequently converted to glyceraldehyde via the activation of acyl-CoA reductase (ACR) from Salmonella typhimurium (StEutE, Genbank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Alcohol dehydrogenases (ADH) such as enzyme from Escherichia coli (EcYqhD, Genbank accession: Q46856) (e.g., ADH set forth in Table 3 below) drives the reaction that converts glyceraldehyde to glycerol. FucO shows better activity that drives the reaction from glyceraldehyde to glycerol (Figure 13C).

[0132] The whole-cell bioconversion of glycerol production from ethylene glycol and formaldehyde was carried out to demonstrate the feasibility of this pathway (Figure 4 below). This was tested in E. coli strain MG1655(DE3) del (eutE, gldA, glpK, tesA, tesB,fadM, ybgC, ydil, yciA, aldA, aldB, puuC, patD, adhP, yjgB, yahK, yqhD, adhE, mgsA, UdD, did, IdhA, frmA, fdhF, fdnG, fdoG, glcD) expressing several functional enzymes, such as acyl-CoA reductase from Listeria monocytogenes (LmACR), 2-hydroxyacyl-CoA synthase from Chloroflexi bacterium (CfhHACS) and alcohol dehydrogenase from E. coli (EcfucO). The E. coli cells were cultured in LB medium with 2% glycerol at 37 °C for 4 horns 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, 1PTG 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. Ethylene Glycol and formaldehyde were initially added to the reaction at final concentrations of 50 mM and 5 mM, respectively. The mixture was incubated at 30 °C for 1 hour with a shaking speed of 1000 rpm. The sample of the reaction mixture was collected for HPLC analysis. The glycerol was identified and quantified by HPLC, indicating the pathway for glycerol from ethylene glycol and formaldehyde is reliable Through the in vivo bioconversion, the strain can produce 200 pM glycerol in one hour (Figure 13B below).

[0133] EXAMPLE 11: PRODUCTION OF GLYCEROL FROM ONE-CARBONCOMPOUNDS

[0134] This example demonstrates the glycerol production from one-carbon feed stocks, including but not limited to formaldehyde (Cl) and methanol (Cl).

[0135] When glycerol is produced from methanol or other Cl compounds, the Cl compounds are firstly converted to formaldehyde and formyl-CoA through the interconversion module. Formaldehyde and formyl-CoA condensation lead to generation of glycolyl-CoA by HACS activation, including CfhHACS from Chloroflexi bacterium (GenBank accession: PKN81274.1), RhbHACS from Rhodocyclaceae bacterium (GenBank accession: MBL8483477.1), PdsHACS from Pseudonocardia sediminis (GenBank accession: WP_ 130292058.1) or other HACS variants (e.g., HACS set forth in Table 1 below), and further convert to glycolaldehyde with ACR variants from Salmonella typhimurium (StEutE, GenBank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Glycolaldehyde is then condensed with formyl-CoA to form glyceryl-CoA, converted to glyceraldehyde and finally converted to glycerol as described above.

[0136] This was tested in two strains MJI0301 and MJI0308 which are MG1655(DE3) derivatives, MJI0301 has thioesterases (yciA, tesA, tesB, ybgC, ydil and fadM) knockout in MG1655(DE3) del (frmA, fdhF, fdnG, fdoG, glcD). FucO and ackA-Pta were further deactivated in MJI0301 to give MJI0308. MJI0301 and MJI0308 harboring pCDFduet-CfhHACS and pET-PCT5-LmACR were cultured in NBS medium and induced with 75 u M 1PTG and 50 u M cumate. After 24 hours of cultivation, cells were harvested and washed with M9 medium, Cells were then resuspended in M9 with 5 mM formaldehyde to a final OD of 20 for bioconversion at 30 °C for 1 hour and 3 hours.

[0137] After 3 hours, 686 pM glycerate was accumulated in MJI0301, 766 pM glycerate was produced in MJI0308 (Figure 14B). Moreover, 20 pM and 61 pM of glycerol were produced in MJI0301 and MJI0308 respectively (Figure 14C).

[0138] EXAMPLE 12: OVERVIEW OF METHODS USDED FOR GENETIC MANIPULATION

[0139] 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 geneinserts 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.

[0140] When inserted into chromosome, CRISPR is used and genetic sites of tesB and adhE are suitable loci, although others could be used. The CRISPR method is based on the method developed by Jiang et al. (Jiang, Y., et al. Appl. Environ. Microbiol. 81:2506-2514 (2015)). First, the host strain is transformed with plasmid pCas, the vector for expression of Cas9 and X-red recombinase. The resulting strain is grown under 30°C with L-arabinose for induction of X-red recombinase expression, and when OD reaches -0.6, competent cells are prepared and transformed with pTargetF (AddGene 62226) expressing sgRNA and N20 spacer targeting the locus and template of insertion of target gene. The template is the inserted gene plus Ml -93 promoter with -500 bp sequences homologous with upstream and downstream of the insertion locus, constructed through overlap PCR with usage of Phusion polymerase or synthesized by GenScript (Piscataway, NJ) or GeneArt® (Life Technologies, Carlsbad, CA). The way to switch N20 spacer of pTargetF plasmid is inverse PCR with the modified N20 sequence hanging at the 5' end of primers with usage of Phusion polymerase and followed by self- ligation with usage of T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Transformants that grow under 30°C on solid media (LB+Agar) supplemented with spectinomycin and kanamycin (or other suitable antibiotic) are isolated and screened for the chromosomal gene insert by PCR. The sequence of the gene insert, which is amplified from genomic DNA through PCR using Phusion polymerase, is further confirmed by DNA sequencing. The pTargetF can then be cured through IPTG induction, and pCas can be cured through growth under higher temperature like 37-42°C.

[0141] 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).

[0142] 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 (AfrmA), formate ( \fdhr AfdnG AfdoG) oxidation and aldehyde consumption (including adhP, yjgB, yqhD, adhE, etc) can be used, which we expected could compete or interfere with the analysis of our pathway.

[0143] EXAMPLE 13: FERMENTATION CONDITIONS USED FOR IN VIVO PRODUCTION OF 1,4-BUTANEDIOL

[0144] MOPS minimal medium (Neidhardt et al. J. Bacteriol.119736-47 (1974)) with 125 mM MOPS and Na2HPO4 in place of K2HPO4 (2.8 mM), supplemented with 20 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract, 100 pM FeSO4, 5 mM (NH4)2SO4, and 30 mM NH4CI is used for fermentations. If required, 55 g / L of CaCOa 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).

[0145] 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 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 ODsoo as -0.05. After inoculation, flasks are incubated in a NBS 124 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, N.I) 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 (1PTG) (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. If required, lactic acid (20 m ) is added at 0, 24, and 48 hours.

[0146] 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.

[0147] EXAMPLE 14: HPLC ANALYSIS OF 1,4-BUTANEDIOL

[0148] A 375 pL of 0.1 M KH2PO4 buffer at pH 8.0 and 500 p L of ethyl acetate containing 4 mM of n-decane were added to 125 pL of a cell-free reaction mixture saturated with NaCl. The mixture was thoroughly mixed using a vortex and centrifuged at 13,680g for 1 min. The organic phase was dried over anhydrous Na2SO4 and then directly used for GC analysis. GC analysis was conducted on a Shimadzu SPL-2014 system equipped with FID-2014 detector and an Agilent DB-5 GC column (30 mm*0.25 mm, 0.25 pm). Injector temperature was maintained at 250 °C with the flow rate of 1 mL / min. Temperature program was set as 80°C / hold 5 min; 20°C per min to 250 °C, and hold 5 min.

[0149] Table 1. List of 2-hydroxyacyl-CoA synthase (HACS) used for the condensation of 3- hydroxypropionaldehyde and formyl-CoA

[0150] Table 2. List of acyl-CoA reductase (ACR)

[0151] Table 3. List of enzymes used for the conversion of intermediate to 1 ,4-butanediol

[0152] Note: DDR: diol dehydratase; GDHt: glycerol dehydratase; ADH: alcohol dehydrogenase.

[0153] Table 4. List of enzymes used for the interconversion of one-carbon compounds to generate formyl-CoA

[0154] Note: sMMO: soluble methane monooxygenase; pMMO: Particulate methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; ACS: acyl-CoA synthase.

[0155] Table 5. List of acyl-CoA transferases (ACT) variants used for the interconversion of one-carbon compounds to generate formyl-CoA

[0156] Table 6. List of pairs of acyl-CoA kinases (ACK) and phosphoacyltransferases (PTA) used for the interconversion of one-carbon compounds to generate formyl-CoA

[0157] 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 ormore 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 comprising the pathway for 1,4-butanediol production in combination with at least one of the following:(a) production of 1,4-butanediol from glycerol via the condensation of 3- hydroxypropionaldehyde with formyl-CoA derived from Cl compounds such as methane, methanol, formaldehyde or formate;(b) production of glycerol from ethylene glycol via the condensation of glycolaldehyde with formyl-CoA derived from Cl compounds such as methane, methanol, formaldehyde or formate; and(c) production of glycerol from Cl compounds such as methane, methanol, formaldehyde or formate through iterative condensation of formyl-CoA with formaldehyde followed by glycolaldehyde.

2. The genetically modified microorganism of claim 1, further comprising an overexpressed glycerol dehydratase catalyzing the conversion of glycerol to 3 -hydroxypropionaldehyde.

3. The genetically modified microorganism of claim 2, 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 for converting 3- hydroxypropi on aldehyde and formyl-CoA to 2,4-dihydroxybutyryl-CoA.

4. The genetically modified microorganism of claim 3, further comprising an overexpressed acyl-CoA reductase catalyzing the conversion of 2,4-dihydroxybutyryl-CoA to 2,4- dihydroxybutanal.

5. The genetically modified microorganism of claim 4, further comprising an overexpressed alcohol dehydrogenase catalyzing the conversion of 2,4-dihydroxybutanal to 1,2,4-butanetriol.

6. The genetically modified microorganism of claim 5, further comprising an overexpressed diol dehydratase catalyzing the conversion of 1 ,2,4-butanetriol to 4-hydroxybutanal.

7. The genetically modified microorganism of claim 6, further comprising an overexpressed alcohol dehydrogenase catalyzing the conversion of 4-hydroxybutanal to 1,4-butanediol.

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

9. The genetically modified microorganism of any one of claims 1 -8, further comprising an overexpressed enzyme catalyzing the conversion of formaldehyde to formate, 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 -tetrahydrofol ate cyclohydrolase and a formyltetrahydrofolate deformylase.

10. The genetically modified microorganism of any one of claims 1-9, further comprising an overexpressed enzyme 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.1 1 . The genetically modified microorganism of any one of claims 1 -10, further comprising at least one of the following:(a) an overexpressed alcohol dehydrogenase catalyzing the conversion of ethylene glycol to glycolaldehyde; and(b) one or more enzymes used in pathway for the production of glycolaldehyde from Cl compounds such as methane, methanol, formaldehyde or formate.

12. The genetically modified microorganism of claim 11, the one or more enzymes include 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 glycolaldehyde and formyl-CoA to glyceryl-CoA.

13. The genetically modified microorganism of claim 12, further comprising an overexpressed acyl-CoA reductase enzyme catalyzing the conversion of glyceryl-CoA to glyceraldehyde.

14. The genetically modified microorganism of claim 13, further comprising an overexpressed alcohol dehydrogenase catalyzing the conversion of glyceraldehyde to glycerol.

15. The genetically modified microorganism of claim 14, the one or more enzymes further include 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 formaldehyde and formyl-CoA to glycolyl-CoA.

16. The genetically modified microorganism of claim 15, further comprising an overexpressed acyl-CoA reductase catalyzing the conversion of glycolyl-CoA to glycolaldehyde.

17. An enzyme, wherein the enzyme is a glycerol dehydratase (GDHt) selected from Table 3.

18. An enzyme, wherein the enzyme is a GDHt 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.

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

20. 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.

21. 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).

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

23. 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.

24. An enzyme, wherein the enzyme is an alcohol dehydrogenase (ADH) selected from Table 3.

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

26. An enzyme, wherein the enzyme is an alcohol dehydrogenase 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.

27. An enzyme, wherein the enzyme is a diol dehydratase (DDR) selected from Table 3.

28. An enzyme, wherein the enzyme is a DDR 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 a methanol dehydrogenase (MDH) or alcohol oxidase (AOD) selected from Table 4.

30. 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 4 or an amino acid sequence having at least 90% sequence identity thereto.

31. An enzyme, wherein the enzyme is an acyl-CoA synthase (ACS) selected from Table 4.

32. enzyme, wherein the enzyme is an ACS 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.

33. An enzyme, wherein the enzyme is an acyl-CoA transferase (ACT) selected from Table 5.

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

35. An enzyme, wherein the enzyme is an acyl-CoA kinase (ACK) selected from Table 6.

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

37. An enzyme, wherein the enzyme is a phosphoacyltransferase (PT A) selected from Table 6.

38. An enzyme, wherein the enzyme is a PTA comprising an amino acid sequence selected from any amino acid sequence as set forth in Table 6, or an amino acid sequence having at least 90% sequence identity thereto.

39. The genetically modified microorganism of any one of claims 1-16, comprising the enzyme of any one of claims 17-38.

40. The genetically modified microorganism of any one of claims 1 -16 and 39, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

41. The genetically modified microorganism of any one of claims 1-16 and 39, wherein the microorganism 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., Pichia sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. and Trichoderma sp..

42. A method for the production of formyl-CoA comprising one of the following:(a) contacting formaldehyde with an acylating formaldehyde dehydrogenase;(b) contacting formate with a formate kinase forming formyl-phosphate and contacting formyl-phosphate with a phosphate formyltransferase, optionally the formate kinase is the enzyme of claim 35 or 36, and / or the phosphate formyltransferase is the enzyme of claim 37 or 38; and(c) contacting formate with an acyl-CoA transferase or an acyl-CoA synthase, optionally the acyl-CoA transferase is the enzyme of claim 33 or 34, and / or the acyl-CoA synthase is the enzyme of claim 31 or 32.

43. A method for the production of glycolyl-CoA comprising contacting formyl-CoA and formaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2- hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase, optionally the TPP- dependent enzyme is the enzyme of any one of claims 19-21.

44. A method for the production of glycolaldehyde comprising contacting glycolyl-CoA with an acyl-CoA reductase, optionally the acyl-CoA reductase is the enzyme of claim 22 or 23.

45. A method for the production of glycolyl-CoA comprising contacting formyl-CoA and glycolaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2- hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase, optionally the TPP- dependent enzyme is the enzyme of any one of claims 19-21.

46. A method for the production of glyceraldehyde comprising:(a) providing glyceryl-CoA obtained by the method of claim 45 ; and(b) converting glyceryl-CoA to glyceraldehyde by contacting glyceryl-CoA with an acyl- CoA reductase catalyzing the conversion of glyceryl-CoA to glyceraldehyde, optionally the acyl-CoA reductase is the enzyme of claim 22 or 23.

47. A method for the production of glycerol comprising:(a) providing glyceraldehyde obtained by the method of claim 46; and(b) converting glyceraldehyde to glycerol by contacting glyceraldehyde with an alcohol dehydrogenase, optionally the alcohol dehydrogenase is the enzyme of any one of claims 24-26.

48. A method for the production of 3-hydroxypropionaldehyde comprising:(a) providing glycerol obtained by the method of claim 47; and(b) converting glycerol to 3-hydroxypropionaldehyde by contacting glycerol with a glycerol dehydratase, optionally the glycerol dehydratase is the enzyme of claim 17 or 18.

49. A method for the production of 1,4-butanediol comprising:(a) providing 3-hydroxypropionaldehyde obtained by the method of claims 48;(b) converting 3-hydroxypropionaldehyde to 2,4-dihydroxybytyryl-CoA by contacting formyl-CoA and 3-hydroxypropionaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase, optionally the TPP-dependent enzyme is the enzyme of any one of claims 19-21;(c) converting 2,4- dihydroxybutyryl-CoA to 2,4-dihydroxybutanal by contacting dihydroxybutyryl-CoA with acyl-CoA reductase, optionally the acyl-CoA reductase is the enzyme of claim 22 or 23;(d) converting 2,4-dihydroxybutanal to 1,2,4-butanetriol by contacting 2,4- dihydroxybutanal with alcohol dehydrogenase, optionally the alcohol dehydrogenase is the enzyme of any one of claims 24-26;(e) converting 1,2,4-butanetriol to 4-hydroxybutanal by contacting 1,2,4-butanetriol with diol dehydratase, optionally the diol dehydratase is the enzyme of claim 27 or 28; and(f) converting 4-hydroxybutanal to 1 ,4-butanediol by contacting 4-hydroxybutanal with alcohol dehydrogenase, optionally the alcohol dehydrogenase is the enzyme of any one of claims 24-26.

50. An enzyme of any one of claims 17-38, for use in the method of any one of claims 43-49.

51. A genetically modified microorganism comprising the enzyme of any one of claims 17-38, for use in the method of any one of claims 43-49.

52. The genetically modified microorganism of claim 51 , wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

53. The genetically modified microorganism of claim 51, wherein the microorganism 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., Pichia sp., Yarrowia sp., Methylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhizopus sp. and Trichoderma sp..

54. A genetically modified microorganism of any one of claims 1-16 and 39-41, for use in the method of any one of claims 43-49.

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