Production of 1,3-propanediol via condensation of glycolaldehyde and c1-derived formyl-coa

Abstract

This invention provides a non-naturally occurring genetically modified microbial organism that has been engineered to produce glyceraldehyde, glycerol, 3-hydroxypropionaldehyde or 1,3-propanediol using a pathway which enables the condensation of formyl-CoA with glycolaldehyde. This invention also provides a method for the production of glyceraldehyde, glycerol, 3-hydroxypropionaldehyde or 1,3-propanediol using a pathway which enables the condensation of formyl-CoA with 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 1,3-PROPANEDIOL VIACONDENSATION OF GLYCOLALDEHYDE ANDCl-DERIVED FORMYL-COATECHNICAL FIELD

[0001] The present disclosure generally relates to a method for producing 1,3 -propanediol (1,3- PDO), in particular, this disclosure describes three methods for the production of 1, -propanediol. Firstly, the present disclosure provides a method for the production of 1,3 -propanediol from glycerol via coupling with oxidation of Cl compounds such as methane, methanol, formaldehyde or formate to CO2 Secondly, the present disclosure provides a method for producing 1,3- propanediol via condensation of glycolaldehyde and Cl -derived formyl-CoA, with the glycolaldehyde being derived from oxidation of ethylene glycol. Thirdly, the present disclosure provides a method for producing 1,3 -propanediol via condensation of glycolaldehyde and Cl- derived formyl-CoA, with the glycolaldehyde being itself produced via condensation of formaldehyde and Cl-denved formyl-CoA. Additionally, this disclosure describes a 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 1,3-propanediol.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 1,3-propanediol from glycerol via coupling with oxidation of Cl compounds such as methane, methanol, formaldehyde or formate to CO2.

[0004] Embodiment 1. A method for producing 1,3-propanediol from glycerol comprising one or more of the following: a) oxidation of C 1 compounds such as methane, methanol, formaldehyde or formate toform COz, for example by the method described in any one of Embodiments 6-10; 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 any one of Embodiments 11 , 25 and 26; c) production of glycerol from Cl compounds such as methane, methanol, formaldehyde or formate through iterative condensation comprising condensation of formyl-CoA with formaldehyde (for example by the method described in any one of Embodiments 30-32) followed by condensation of formyl-CoA with glycolaldehyde (for example by the method described in any one of Embodiments 12-15).

[0005] Embodiment 2. The method of Embodiment 1, comprising generating 3- hydroxypropionaldehyde (i e , 3-hydroxypropional) 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 1,3 -propanediol comprising:(a) providing 3-hydroxypropionaldehyde by the method of Embodiment 2 or 3; and(b) converting 3-hydroxypropionaldehyde to 1,3 -propanediol by contacting 3- hydroxypropionaldehyde and an NADH cofactor with an alcohol dehydrogenase.

[0008] Embodiment 5. The method of Embodiment 4, wherein the alcohol dehydrogenase is selected from:10009| Embodiment 6. The method of Embodiment 4 or 5, where the NADH cofactor is provided by the oxidation of Cl compounds such as methane, methanol, formaldehyde or formate to carbon dioxide.

[0010] Embodiment 7. The method of Embodiment 6, wherein the oxidation comprises generating methanol and NAD+by contacting methane and NADH with a methane monooxygenase, wherein the methane monooxygenase is selected from:

[0011] Embodiment 8. The method of Embodiment 7, wherein the oxidation further comprises generating formaldehyde and NADH by contacting the methanol and NAD” with a methanol dehydrogenase or methanol oxidase, wherein the methanol dehydrogenase or methanol oxidase is selected from:

[0012] Embodiment 9. The method of Embodiment 8, wherein the oxidation further comprises generating formate and NADH by contacting the formaldehyde and NAD+with an enzyme 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 formyltransferase / hydrolase complex; and e) a methylene-tetrahydrofolate dehydrogenase, a methenyl-tetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase.

[0013] Embodiment 10. The method of Embodiment 9, wherein the oxidation further comprises generating CO2 and NADH by contacting the formate and NAD+with a formate dehydrogenase.

[0014] In another aspect, provided herein is a method for producing 1,3 -propanediol via condensation of glycolaldehyde and Cl -derived formyl-CoA.

[0015] Embodiment 11. A method for producing glycerol from glycolaldehyde comprising condensation of glycolaldehyde with formyl-CoA, comprising forming glyceryl-CoA from glycolaldehyde, forming glyceraldehyde from glyceryl-CoA and forming glycerol from glyceraldehyde.

[0016] Embodiment 12. The method of Embodiment 11 , 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.

[0017] Embodiment 13 The method of Embodiment 12, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0018] Embodiment 14. The method of Embodiment 13, wherein the TPP-dependent enzyme is selected from:

[0019] Embodiment 15. The method of any one of Embodiments 12-14, further comprising forming formyl-CoA from one-carbon (Cl) compound.

[0020] Embodiment 16. The method of Embodiment 15, wherein forming formyl-CoA comprises contacting the one-carbon (Cl) compound selected from formaldehyde or formate with an enzyme.

[0021] Embodiment 17. The method of Embodiment 16, wherein the Cl compound is formaldehyde, and the enzyme is an acyl-CoA reductase.

[0022] Embodiment 18. The method of Embodiment 17, wherein the acyl-CoA reductase is selected from:

[0023] Embodiment 19. The method of Embodiment 16, 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.

[0024] Embodiment 20. The method of Embodiment 19, wherein the acyl-CoA transferase is selected from:

[0025] Embodiment 21. The method of Embodiment 19, wherein the acyl-CoA synthase is selected from:

[0026] Embodiment 22. The method of Embodiment 19, wherein the formate kinase and the phosphate formyltransferase pair is selected from:

[0027] Embodiment 23. The method of any one of Embodiments 11-22, wherein forming glyceraldehyde comprising contacting the glyceryl-CoA with an acyl-CoA reductase catalyzing the conversion of glyceryl-CoAto glyceraldehyde.

[0028] Embodiment 24. The method of Embodiment 23, wherein the acyl-CoA reductase is selected from:

[0029] Embodiment 25. The method of any one of Embodiments 11-22, wherein forming glycerol comprises contacting the glyceraldehyde with an alcohol dehydrogenase.

[0030] Embodiment 26. The method of Embodiment 25, wherein the alcohol dehydrogenase is selected from:

[0031] Embodiment 27. The method of any one of Embodiments 11-26, further comprising forming glycolaldehyde via oxidation of ethylene glycol.

[0032] Embodiment 28. The method of Embodiment 27, wherein forming glycolaldehyde comprises contacting the ethylene glycol with an alcohol dehydrogenase selected from:

[0033] Also provided herein is a method for producing 1,3 -propanediol 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.

[0034] Embodiment 29. A method of producing glycolaldehyde from formaldehyde, comprising forming glycolyl-CoA from formyl-CoA and forming glycolaldehyde from glycolyl-CoA.

[0035] Embodiment 30. The method of Embodiment 29, wherein forming glycolyl-CoA comprises contacting formaldehyde and formyl-CoA (generated by the method described in Embodiments 15-22) with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxyacyl-CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase.

[0036] Embodiment 31 The method of Embodiment 30, wherein the TPP-dependent enzyme is 2-hydroxyacyl-CoA synthase or 2-hydroxyacyl-CoA lyase.

[0037] Embodiment 32. The method of Embodiment 30, wherein the TPP-dependent enzyme is selected from:

[0038] Embodiment 33. The method of any one of Embodiments 29-32, wherein forming glycolaldehyde comprises contacting the glycolyl-CoA with an acyl-CoA reductase catalyzing the conversion of glycolyl-CoA to glycolaldehyde.

[0039] Embodiment 34. The method of Embodiment 33, wherein the acyl-CoA reductase is selected from:

[0040] Embodiment 35. A method for producing 1,3 -propanediol from Cl compounds such as methane or methanol, comprising forming formaldehyde (for example, by the method described in Embodiments 7 and 8), forming formyl-CoA (for example, by the method described in Embodiments 16-22), forming glycolaldehyde from formaldehyde (for example, by the method described in Embodiments 29-34), forming glycerol from glycolaldehyde (for example, by the method described in Embodiments 11-28), and the conversion of glycerol to 1,3 -propanediol (for example, by the method described in Embodiments 1-10).

[0041] Embodiment 36. The method of any one of Embodiments 1 to 35, wherein the enzyme used in the method is isolated from a microorganism.10042| Embodiment 37. The method of any one of Embodiments 1 to 35, wherein the enzyme used in the method is contained in a microorganism.

[0043] Embodiment 38. A genetically modified microorganism providing 1, -propanediol by the method of Embodiment 1.

[0044] Embodiment 39. A genetically modified microorganism providing glycerol by the method of Embodiment 11.

[0045] Embodiment 40. A genetically modified microorganism providing glycolaldehyde by the method of Embodiment 29.

[0046] Embodiment 41. A genetically modified microorganism providing 1,3 -propanediol by the method of Embodiment 35.

[0047] Embodiment 42. The microorganism of any one of Embodiments 38 to 41, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

[0048] Embodiment 43. The microorganism of any one of Embodiments 38 to 42, wherein the microorganism is bacteria, yeast or fungi, including but not limited to Escherichia sp., Bacillussp., 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.

[0049] Also provided herein is an enzyme comprising or consisting of an amino acid sequence selected from any amino acid sequence as set forth herein 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.

[0050] Also provided herein is a genetically modified microorganism comprising any enzyme disclosed herein.

[0051] Also provided herein is an enzyme or a genetically modified microorganism for use in any method disclosed herein.

[0052] In some embodiments, the genetically modified microorganism is FucO and / or frmR deactivated.

[0053] 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.

[0054] The present disclosure is illustrated by the following non-limiting examples

[0055] 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

[0056] 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.

[0057] 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 FhMPT-dependent oxidation of formaldehyde to formate.

[0058] 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; MID A: methylene-tetrahydromethanopterin dehydrogenases; FCH: methenyl-H4F cyclohydrolase; FTFL: formate-H4F ligase; FAE: formaldehyde activating; MCH: methenyl-FUMPT cyclohydrolase enzyme; MCH: methenyl-H4MPT cyclohydrolase.

[0059] Figure 2 is a scheme illustrating the generation of formyl-CoA and condensation of formyl- CoA with formaldehyde to form glycolyl-CoA. a) Pathway for production of glycolic acid from formate via ATP utilizing ACS for activation to generate formyl-CoA, HACS for condensation of formyl-CoA with formaldehyde to generate glycolyl-CoA, a key intermediate in the 1,3-PDO production pathway, and lastly termination by endogenous thioesterases to form glycolic acid, b) Testing several homologues of ACS for activity in activating formate to formyl-CoA using an in- vitro assay. Glycolate production demonstrates the activity of both ACS and HACS enzymes in the pathway, c) Testing the same ACS homologues in-vivo, using the same HACS enzymes. Production of glycolate demonstrates activity of both the ACS and HACS.

[0060] 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.

[0061] Figure 3 is a scheme illustrating an alternative method for the generation of formyl-CoA, and condensation of formyl-CoA with formaldehyde to form glycolyl-CoA. a) Pathway for production of glycolic acid from formate via ATP utilizing FOK and PTA for activation to generate formyl-CoA, HACS for condensation of formyl-CoA with formaldehyde to generate glycolyl- CoA, a key intermediate in the 1 ,3-PDO production pathway, and lastly termination by endogenous thioesterases to form glycolic acid, b) Testing FOK and PTA for activity in activating formate to formyl-CoA using an in-vitro assay. Glycolate production demonstrates the activity of FOK, PTA, and HACS enzymes in the pathway.

[0062] FOK-PTA: formate kinase-phosphate formyltransferase derived from Coriobactenia bacterium and DtbHACS: 2-hydroxyacyl-CoA synthase derived from Deltaproteobacteria bacterium.

[0063] Figure 4 is a scheme illustrating the production of glycerol from ethylene glycol and formaldehyde, a) Pathway for producing 1,3 -propanediol from ethylene glycol coupled to generating formyl-CoA and reducing equivalents from Cl feedstocks, b) Testing several ACR, HACS, and ADH combinations for glycerol production via whole-cell bioconversion. Glycerol production demonstrates the activity of ACR, HACS, and ADH enzymes in the pathway.

[0064] 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; FucO: lactaldehyde reductase; GDHt: glycerol dehydratase.

[0065] Figure 5 is a scheme illustrating the production of 1,3 -propanediol from formaldehyde, a) Pathway for the production of 1,3 -propanediol from Cl compounds coupled to the generation of formyl-CoA and reducing equivalents from Cl feedstocks, b) Testing ACR, HACS, and ADH combinations for glycerol production in different chassis via whole-cell bioconversion. Glycerol production demonstrates the activity of ACR, HACS, and ADH enzymes in the pathway.

[0066] 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; FucO: lactaldehyde reductase; GDHt: glycerol dehydratase.

[0067] Figure 6 is a scheme illustrating the production of 1,3 -propanediol from glycerol coupled to oxidation of Cl feedstocks to CO2. a) Pathway for the production of 1,3 -propanediol from glycerol requires NADH for the ADH step, b) NADH can be provided by the oxidation of Cl compounds such as methane, methanol, formaldehyde, or formate to CO2. c) Demonstration of 1,3-PDO production from glycerol with different concentrations in whole-cell bioconversion.

[0068] 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; FucO: lactaldehyde reductase; GDHt: glycerol dehydratase.

[0069] Figure 7 is demonstrating of the NAD(P)H generation from formaldehyde.

[0070] Figure 8 is demonstrating of the NAD(P)H generation from methanol by monitoring the formate accumulation.

[0071] Figure 9 is the screening of formate dehydrogenase for NAD(P)H generation from formate.

[0072] Figure 10 is the NAD(P)H generation by combination of formaldehyde oxidation and formate oxidation.

[0073] Figure 11 is the 1,3 -propanediol production from glycerol using formate to provide NAD(P)H.DETAILED DESCRIPTION OF THE INVENTION

[0074] 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

[0075] 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.

[0076] EXAMPLES

[0077] EXAMPLE 1: INTERCONVERSION OF ONE-CARBON COMPOUNDS TO GENERATE FORMYL-COA

[0078] 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. coll 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 stearothermophilus (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).

[0079] 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. Hydroxym ethyl -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 m ethenyl -H4MPT by methylene-tetrahydromethanopterin dehydrogenases MtdA or MtdB. Methenyl-FUMPT was converted to formyl-FUMPT 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.

[0080] 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- Co A 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 by a set of phosphotransacylase-formate kinase (PTA-FOK, e.g., set forth in Table 6 below) pair catalyzereversible phosphorylation of formyl-CoAto 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.

[0081] 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), lO mM ofMgCh, 150 pMTPP, lO mM of CoA, 50 mM of formate, 10 mM of formaldehyde, 5 uM 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 analysed 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 2b, 2c below).

[0082] Furthermore, we have tested the formyl-CoA generation from formate via FOK-PTA route. A 1 mL of cell-free bioconversion reaction contains 100 mM of potassium phosphate buffer (pH6.9), 10 mMofMgC12, 150 pMTPP, 10 mMof CoA, 50 mM of formate, 20 mM of formaldehyde, 2 pM of HACS, 2 |iM of ACK (FOK) and 2 LIM 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 3b below).

[0083] EXAMPLE 2: PRODUCTION OF GLYCEROL FROM ETHYLENE GLYCOL AND ONE-CARBON COMPOUNDS

[0084] This example demonstrates the glycerol production from different feed stocks, including but not limited to ethylene glycol (C2) and methanol (Cl). When glycerol is produced, it can be further converted to 1 ,3-propanediol as presented in Figure 4.

[0085] When ethylene glycol is utilized as the feedstock for generating glycerol as an intermediate in the production of 1 ,3-propanediol, it can be firstly converted to glycolaldehyde by overexpression of Escherichia coli (EcfucO, Genbank accession: P0A9S1) or other ADH variants drive (e.g., ADH set forth in Table 3 below) (Figure 4 below).

[0086] Glycolaldehyde can be directly condensed with formyl-CoA to generate glyceryl-CoA (Figure 4a below). The condensation of glycolaldehyde and formyl-CoA 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).

[0087] 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 (EcFucO, Genbank accession: P0A9S1) or other ADH variants drive (e.g., ADH set forth in Table 3 below) the reaction that converts glyceraldehyde to glycerol.

[0088] 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 hours with a shaking speed of 1000 rpm. Subsequently, the cells were transferred into NBS medium with 2% glycerol at 30 °C for 16 h with a shaking speed of 1000 rpm. The cells were then transferred into NBS medium with 1.5% glucose for 4 h, subsequently, IPTG with the final concentration of 0.05 mM was added, and the culture was further incubated at 30 °C for 24 hours to induce protein expression. The cells were centrifuged at 4,000 rpm for 8 minutes, and the supernatant was discarded. 1 mL M9 wash buffer was added to the 2 mL deep well plate, and the mixture was thoroughly mixed. This washing step was repeated twice. The cells were resuspended in the M9 wash buffer. 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 4b below).

[0089] EXAMPLE 3: PRODUCTION OF GLYCEROL THROUGH ONE CARBON COMPOUNDS

[0090] This example demonstrates the glycerol production from different one-carbon feedstocks, including but not limited to methanol. When glycerol is produced, it can be further converted to 1,3-propanediol as presented in Figure 5.

[0091] When Cl compounds are utilized as the feedstock for generating glycerol as an intermediate in the production of 1,3-propanediol, the Cl compounds are first converted to formaldehyde and formyl-CoA through the interconversion module (Figures 1-3). Formaldehyde and formyl-CoA condensation leads to the 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). Glycolyl-CoA is further converted to glycolaldehyde via theactivation of acyl-CoA reductase (ACR) from Salmonella lyphimurium (StEutE, Genbank accession: P41793) or other ACR variants (e.g., ACR set forth in Table 2 below). Glycolaldehyde can be condensed with formyl-CoA to generate glyceryl-CoA 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).

[0092] 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 (EcDkgB, Genbank accession: NC 000913) or other ADH variants drive (e.g., ADH set forth in Table 3 below) the reaction that converts glyceraldehyde to glycerol (Figure 5a below)

[0093] The whole-cell bioconversion of glycerol production from formaldehyde was carried out to demonstrate the feasibility of this pathway (Figure 5 below). This was tested in E. coli strain MG1655(DE3) del (fucO, gldA, glpK, tesA, tesB,fadM, ybgC, ydil, yciA, aldA, aldB, puuC, pall), adhP, yjgB, yahK, yqhD, adhE, mgsA, lldD, did, IdhA, frmA, fdhF, fdnG, fdoG, glcD) expressing several functional enzymes, such as acyl-CoA reductase from Listeria monocytogenes (LmACR), and 2-hydroxyacyl-CoA synthase from Chloroflexi bacterium (CfhHACS) The cell culture and bioconversion process were done as above description. The glycerol was identified and quantified by HPLC, indicating the pathway for glycerol from formaldehyde is reliable Through the in vivo bioconversion, the strain can produce 40 uM glycerol in one hour and 60 pM glycerol in three hours.

[0094] EXAMPLE 4: PRODUCTION OF 1,3-PROPANEDIOL FROM GLYCEROL|0095| This example demonstrates the implementation of 1,3-PDO production from glycerol which is produced in Example 2, 3 or fed externally. Glycerol is converted to 3- hydroxypropionaldehyde by glycerol dehydratase (GDHt), such as dhaB123 from Klebsiella pneumonia or other microorganisms (e g., GDHt set forth in Table 3 below). 3- hydroxypropionaldehyde is then converted to 1,3-PDO by alcohol dehydrogenase (Fig. 6 below; enzymes set forth in Table 3 below), requiring the addition of NADH cofactor. The NADH cofactor can be derived from the oxidation of Cl compounds as mentioned in Example 1.

[0096] The whole-cell bioconversion of glycerol to 1,3-propanediol via glycerol dehydratase and alcohol dehydrogenase and alcohol dehydrogenase was carried out to demonstrate the feasibility of this pathway (Figure 6a). This was tested in E. coli strain BW25113 (DE3) del (fdhF, fdnG, fdoG, sthA) expressing GDHt from Klebsiella pneumoniae, activator protein GdrAB, and Yqhd from E. coli. The E. coli cells were cultured in LB medium with 2% glycerol at 37 °C for 4 hours with a shaking speed of 1000 rpm. Subsequently, the cells were transferred into NBS medium with 2% glycerol at 30 °C for 16 h with a shaking speed of 1000 rpm. The cells were then transferred into NBS medium with 1.5% glucose for 4 h, subsequently, IPTG and Coenzyme B12 with the final concentration of 0.5 mM and 0.015 mM were added, respectively, and the culture was further incubated at 37 °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 at OD 30 in the M9 buffer with 15 pM of Coenzyme B12. Glycerol was initially added to the reaction as the substrate. The mixture was incubated at 37 °C for 24 hours with a shaking speed of 1000 rpm. The sample of the reaction mixture was collected for HPLC analysis. The 1,3-PDO was identified and quantified by HPLC, indicating the pathway for 1,3-PDO from glycerol is reliable. Through the in vivo bioconversion, the strain can produce 25 g / L glycerol in 24 hours (Figure 6b below).

[0097] EXAMPLE 5: GENERATION OF NAD(P)H FROM FORMALDEHYDE BY NATIVE DETOXIFICATION SYSTEM

[0098] This example is to demonstration of NADH generation from formaldehyde by its native detoxification system. In E. coli, formaldehyde can be detoxified by oxidation of formaldehyde to formate with FrmA and FrmB and generate one NADH as example 1 described. While normally the expression of FrmA and FrmB is repressed by enzyme FrmR.

[0099] In this invention, a formate dehydrogenase knockout strain BWA3 (BW25113 delta fdhF, fdoG, fdnG) was constructed, which is able to accumulate formate, and used as a host. frmR was further deleted in BWA3 to release the protein expression repression, alternatively, FrmAB were overexpressed in a plasmid pCDFduet under the control of T7 promoter. The bioconversion was carried out as described above with a final OD of 0.25 (low cell loading) by adding 10 mM formaldehyde as the substrate, and formate accumulation was monitored by HPLC. The BWA3shows the lowest activity due to the repression of FrmR, knockout frmR slightly improved the activity, while the flux is still less than 1 mM / OD / h. With overexpression of FrmAB in the plasmid, formaldehyde was consumed at a flux of 5 mM / OD / h (Figure 7), which was further confirmed by monitoring the NADH generation using crude enzyme assay.FOIOO] EXAMPLE 6: GENERATION OF NADH BY METHANOL DEHYDROGENASEFOlOl] This example is to demonstrate the NADH generation from methanol by using methanol dehydrogenase. Oxidation of methanol to formaldehyde can be catalyzed by NAD+-dependent methanol dehydrogenases (MDH), MDH from Bacillus methanolicus MGA3 (BmMDH), Bacillus stearothermophilus (BsMDH) (Metab. Eng. 39:49-59, 2017) and Cupriavidus necator (Appl. Microbiol. Biotechnol. 100:4969-4983, 2016), and other MDHs. Formaldehyde can be further oxidized to formate, which may generate another NADH as described above.

[0102] To check the feasibility of this reaction, MDH was overexpressed in the plasmid pCDFduet under the control of T7 promoter, and a bioconversion was performed same as example 5 described with 500 mM methanol. Among the tested MDHs, CnMDH shows the best performance, which gives a flux of 1.2 mM / OD / h (Figure 8).

[0103] EXAMPLE 7: GENERATION OF NAD(P)H FROM FORMATE BY FORMATE DEHYDROGENASE.

[0104] This example is to demonstrate the NADH generation from formate by using formate dehydrogenase (FDH), including MvaFDH from Mycolicihacterium vaccae (gene accession number: Q93GV1 ), BsFDH from Bacillus sp. (gene accession number: D6CJK0), CboFDH from Candida, boidinii (gene accession number: 013437) or other microorganisms (e.g., FDH set forth in Table 4 below). To use the formate consumption as an indicator of NADH generation was confirmed using crude enzyme assays using both NAD+or NADP+as a cofactor, and found the FDH prefers to use NAD+as the cofactor. After the confirmation, different FDH was evaluated in a bioconversion format which using formate consumption as an indicator. MvaFDH showed the best performance with 1 .25 mM / OD / h when 10 mM formate was used, the formate consumption increased to 1.75 mM / OD / h when 20 mM formate was used (Figure 9). BsFDH has similar performance to MvaFDH and better than other tested FDHs.

[0105] This was further combined with the oxidation of formaldehyde to formate and then formate to COz. The bioconversion was performed the same as described above with 1 mM formaldehyde. In BWA3, only a little amount of formaldehyde was consumed which is the same as before, and almost same amount of formate was accumulated due to the knockout of native formate dehydrogenase. When BsFDH was overexpressed in BWA3, still the same that only about 0.1 mM formaldehyde was consumed, but the formate was further consumed by BsFDH. When FrmAB was further overexpressed in BWA3 or BWA3 / pCDFduet-BsFDH, it consumed all the formaldehyde (1 mM), with more than 0.9 mM formate accumulation in BWA3-frmAB, and 0.65 mM formate was left with extra BsFDH overexpression (Figure 10). There is a significant amount of formate accumulation which indicates the formate dehydrogenase activity is not high enough, further engineering or bioprospecting is needed to build a more balanced NADH generation module.

[0106] EXAMPLE 8: PRODUCTION OF 1,3-PROPANEDIOL FROM GLYCEROL AND ONE CARBON COMPOUND

[0107] This example is to demonstrate the 1,3 -propanediol production from glycerol with Cl compounds to provide NAD(P)H. One NAD(P)H is needed to produce one 1,3-propanediol from glycerol (Figure 6a), the NAD(P)H can be from the oxidation of glycerol which lower the final carbon yield, or it can be used cheaper one carbon feedstock to provide NADH for 1 ,3-propanediol production. As is shown in figure 6b one methanol can provide up to 3 NAD(P)H, one formaldehyde can provide two NAD(P)H, and formate can provide one NAD(P)H.To demonstrate the feasibility of using Cl to provide NAD(P)H for 1,3-propanediol, we used formate to provide NAD(P)H as an example. This was carried out the same as example 4 described above. BsFDH was co-overexpressed with GDHt, YqhD and PntAB for 1,3-propanediol production, 40 g / L glycerol and different amount of formate was added as the feedstock. 23 mM 1,3-PDO was produced without formate addition and 28 mM 1,3-PDO was produced when 50 mM formate was added (Figure 11).

[0108] EXAMPLE 9: OVERVIEW OF METHODS USED FOR GENETIC MANIPULATION

[0109] 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 intovectors, 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.

[0110] 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 A- red recombinase. The resulting strain is grown under 30°C with L-arabinose for induction of A- 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.

[0111] All molecular biology techniques are performed with standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).) or bymanufacturer 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)

[0112] Expression of selected enzyme variants was achieved using plasmid-based gene expression by cloning the desired gene(s) into pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes and by utilizing In-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, CA). Linear DNA fragments for insertion were created via PCR of the open reading frame of interest (for genes native to E. coli) or by gene synthesis of the codon optimized gene. Genes were synthesized by GeneArt (Life Technologies, Carlsbad, CA). Resulting In-Fusion reaction products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, CA), and clones identified by PCR screening were further confirmed by DNA sequencing. As a host for these vectors, an engineered strain of E. coli based on MG1655(DE3), BW25113 or ATCC8739 with knockouts for formaldehyde (A / hwA), formate ( \fdhE EfdnG 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.

[0113] EXAMPLE 10: BIOCONVERSTON CONDITIONS USED FOR IN VIVO PRODUCTION OF GLYCEROL AND 1,3-PROPANEDIOL

[0114] NBS minimal medium with 25.72 mM KH2PO4, 28.71 mM K2HPO4, 26.5 mM (NH4)2HPO4and lmM MgS04, 5.92 pMFeCh, 0.84 pM CoCh, 0.59 pM CuCh, 1.47 pMZnCh, 0.83 pM Na2MoO4and 0.81 pM FFBOi, supplemented with 20 g / L glycerol or 15 g / L glucose as carbon source was used in cell culture. Antibiotics (100 pg / mL carbenicillin, 100 pg / mL spectinomycin and 50 pg / mL kanamycin) are included when appropriate. M9 medium with All chemicals are obtained from Fisher Scientific Co. (Pittsburg, PA) and Sigma-Aldrich Co. (St. Louis, MO).

[0115] In vivo product synthesis (whole-cell bioconversion) was conducted using M9 minimal media (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4C1, 0.5 g / L NaCl, 2 mM MgSO4, 100 pM CaCl2, and 15 pM thiamine-HCl) unless otherwise stated. Cells were initially grown in 96-deep well plates (USA Scientific, Ocala, FL) containing 0.5 mL of the above NBS medium. A single colony of the desired strain was cultivated 4-6 hours in LB medium with 2% glycerol andappropriate antibiotics and used as the inoculum (1%). Cells were then transferred into NBS medium with 2% glycerol and incubated at 30°C and 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) overnight for 14-16 hours. Subsequently, the cells were transferred into NBS medium with 1.5% glucose and incubated until an OD600 of ~0.4 was reached, at which point appropriate amounts of inducer(s) (isopropyl [3-D-l -thiogalactopyranoside and cumate) and Coenzyme B12 were added. Plates were incubated for a total of 24 hours post-inoculation.

[0116] Cells from the above pre-cultures were then centrifuged (4000 rpm, 22°C), washed with the above M9 minimal media without any carbon source, and resuspended with 1 mL of above minimal media containing indicated amounts of carbon source. Appropriate substrates 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, the cells were pelleted by centrifugation and the sample of the reaction mixture was collected for the HPLC analysis.

[0117] EXAMPLE 11: HPLC ANALYSIS OF GLYCEROL, 1,3-PROPANEDIOL AND OTHER COMPOUNDS

[0118] The concentrations of glycerol, 1,3-PDO, and other compounds were determined via ionexclusion HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a refractive index detector and an HPX-87H organic acid column (Bio-Rad, Hercules, CA) with operating conditions to optimize peak separation. The mobile phase is 30 mM H2SO4 with the flow rate of 0.3 mL / min and the column temperature of 55 °C.

[0119] Table 1. List of 2-hydroxyacyl-CoA synthase (HACS) used for the condensation of 3- hydroxyaldehyde and formyl-CoA

[0120] Table 2. List of acyl-CoA reductase (ACR)

[0121] Table 3. List of enzymes used for the conversion of intermediate to 1 ,3- propanediolNote: GDHt: glycerol dehydratase; ADH: alcohol dehydrogenase.

[0122] Table 4. List of enzymes used for the interconversion of one-carbon compounds to generate formyl-CoA

[0123] Note: sMMO: soluble methane monooxygenase; pMMO: Particulate methane monooxygenase; MDH: methanol dehydrogenase; AOD: alcohol oxidase; FDH: formate dehydrogenase; ACS: acyl-CoA synthase.

[0124] Table 5. List of acyl-CoA transferases (ACT) variants used for the interconversion of one-carbon compounds to generate formyl-CoA

[0125] 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

[0126] Various embodiments have been described herein with reference to the accompanying drawings. It will, however, be evident that various modifications and changes may be made thereto, and additional embodiments may be implemented, without departing from the broader scope of the invention as set forth in the claims that follow Further, other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of one or more embodiments of the invention disclosed herein. It is intended, therefore, that this application and the examples herein be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following listing of exemplary claims.

Claims

Claims1. A genetically modified microorganism performing a glycerol-to- 1 ,3 -propanediol pathway in combination with at least one of the following: a. oxidation of Cl compounds such as methane, methanol, formaldehyde or formate to CO2; b. conversion of ethylene glycol to glycerol via condensation of glycolaldehyde with formyl-CoA derived from Cl compounds such as methane, methanol, formaldehyde or formate; and c. conversion of Cl compounds such as methane, methanol, formaldehyde or formate to glycerol 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- hy droxypropionaldehy de .

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

4. The genetically modified microorganism of any one of the claims 1-3, further comprising an overexpressed methane monooxygenase catalyzing the conversion of methane to methanol.

5. The genetically modified microorganism of any one of the claims 1-4, further comprising an overexpressed enzyme catalyzing the conversion of methanol to formaldehyde, wherein the enzyme is selected from methanol dehydrogenase or alcohol oxidase.

6. The genetically modified microorganism of any one of the claims 1-5, further comprising an ovcrcxprcsscd 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-dcpcndcnt formaldehyde dehydrogenase and a hydrolase d. a formaldehyde activating enzyme (FAE), a methylene -tetrahydromethanopterin dehydrogenase, a methenyl-tetrahydromethanopterin cyclohydrolase and a formyltransferase / hydrolase complex; and e. a methylene-tetrahydrofolate dehydrogenase, a methenyl-tetrahydrofolate cyclohydrolasc and a formyltctrahydrofolatc dcformylasc.

7. The genetically modified microorganism of any one of the claims 1-6, further comprising an ovcrcxprcsscd formate dehydrogenase enzyme catalyzing the conversion of formate to CO2.

8. The genetically modified microorganism of any one of claims 1-7, 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; and c. an acyl-CoA transferase converting formate to formyl-CoA; and an acyl-CoA synthase converting formate to formyl-CoA.

9. The genetically modified microorganism of any one of claims 1-8, 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.

10. The genetically modified microorganism of claim 9, 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.

11. The genetically modified microorganism of claim 10, the one or more enzymes further include an overexpressed acyl-CoA reductase enzyme catalyzing the conversion of glyccryl-CoA to glyceraldehyde.

12. The genetically modified microorganism of claim 11, the one or more enzymes further include an overexpressed alcohol dehydrogenase catalyzing the conversion of glyceraldehyde to glycerol.

13. The genetically modified microorganism of claim 12, the one or more enzyme further include an ovcrcxprcsscd TPP-dcpcndcnt 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.

14. The genetically modified microorganism of claim 13, the one or more enzyme further include an overexpressed acyl-CoA reductase catalyzing the conversion of glycolyl- CoA to glycolaldehyde.

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

16. 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.

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

18. 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.

19. 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).

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

21. 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.

22. An enzyme, wherein the enzyme is an alcohol dehydrogenase (ADH) selected from Table3.

23. 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.

24. An enzyme, wherein the enzyme is an alcohol dehydrogenase derived from microorganisms including but not limited to EcDkgB (GcnBank accession: NC_OOO913), EcYahK (GenBank accession: P75691), EcFucO (GenBank accession: P0A9S1), and EcYqhD (GenBank accession: Q46856) derived from Escherichia coli.

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

26. 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.

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

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

29. An enzyme, wherein the enzyme is a formate dehydrogenase (FDH) selected from Table4.

30. An enzyme, wherein the enzyme is an FDH 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. An 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 Table5.

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 kinases (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 phosphoacyltransfcrasc (PTA) selected from Table6.

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 1-14, comprising the enzyme of any one of claims 15-38.

40. The genetically modified microorganisms of any one of claims 1-14 and 39, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

41. The genetically modified microorganisms of any one of claims 1-14 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., Melhylorubrum sp., Candida sp., Kluyveromyces sp., Aspergillus sp., Pennicilium sp., Rhi .opus 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; andc. contacting formate with an acyl-CoA transferase or an acyi-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 17-19.

44. Amethod for the production of glycolaldehyde comprising contacting glycolyl-CoA with an acyl-CoA reductase, optionally the acyl-CoA reductase is the enzyme of claim 20 or 21.45 A method for the production of glyceryl-CoA comprising contacting formyl-CoA and glycolaldehyde with a TPP-dependent enzyme selected from 2-hydroxyacyl-CoA synthase, 2-hydroxy acyl -CoA lyase, oxalyl-CoA decarboxylase or benzaldehyde lyase, optionally the TPP-dependent enzyme is the enzyme of any one of claims 17-19.

46. Amethod for the production of glyceraldehyde comprising:(a) providing glyceryl-CoA obtained by the method of claim 45; and(b) converting glyceryl-CoAto 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 20 or 21.47 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 22-24.48 Amethod 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 15 or 16.

49. Amethod for the production of 1,3 -propanediol:(a) providing 3-hydroxypropionaldehyde obtained by the method of claim 48; and(b) converting 3-hydroxypropionaldehyde to 1,3 -propanediol by contacting of 3- hydroxypropionaldehyde with an alcohol dehydrogenase, optionally the alcohol dehydrogenase is the enzyme of any one of claims 22-24.50 An enzyme of any one of claims 15-38, for use in the method of any one of claims 42-49.

51. A genetically modified microorganism comprising the enzyme of any one of claims 15- 38, for use in the method of any one of claims 42-49.

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

53. The genetically modified microorganisms for use 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 claims 1-14 and 39-41, for use in the method of any one of claims 42-49.

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