Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing adipate, 6-aminocaproate, hexamethylenediamine or caprolactam related thereto

Abstract

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as adipate, 6-aminocaproate, hexamethylenediamine or caprolactam. Also provided herein are methods for using such an organism to produce adipate, 6-aminocaproate, hexamethylenediamine or caprolactam.

1 . A non-naturally occurring microbial organism (NNOMO) comprising:
 (A) a methanol metabolic pathway (MMP), wherein said organism comprises at least one exogenous nucleic acid encoding a MMP enzyme (MMPE) expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises:
 a methanol methyltransferase (EM1) and a methylenetetrahydrofolate reductase (EM2); and 
   (B) (i) an adipate pathway (AdiP),
 (ii) a 6-aminocaproate (6-ACA) pathway (6-ACAP), 
 (iii) a hexamethylenediamine (HMDA) pathway (HNMAP), or 
 (iv) a caprolactam pathway (CapP). 
   
     
     
         2 . The organism of  claim 1 , wherein
 (a) said organism comprises an AdiP, and wherein
 (1) said organism comprises at least one exogenous nucleic acid encoding an AdiP enzyme (AdiPE) expressed in a sufficient amount to produce adipate, wherein said AdiP comprises (i) a 3-oxoadipyl-CoA thiolase (EA1); (ii) a 3-oxoadipyl-CoA reductase (EA2); (iii) a 3-hydroxyadipyl-CoA dehydratase (EA3); (iv) a 5-carboxy-2-pentenoyl-CoA reductase (EA4); and (v) an adipyl-CoA hydrolase (EA11A), an adipyl-CoA ligase (EA11B), an adipyl-CoA transferase (EA11C) or a phosphotransadipylase/adipate kinase (EA11D), 
 (2) the organism comprises two, three, four or five exogenous nucleic acids, each encoding an AdiPE; and/or 
 (3) said at least one exogenous nucleic acid encoding an AdiPE is a heterologous nucleic acid; 
   (b) said organism comprises a 6-ACAP, and wherein
 (1) said organism comprises at least one exogenous nucleic acid encoding a 6-ACAP enzyme (6-ACAPE) expressed in a sufficient amount to produce 6-ACA, wherein said 6-ACAP comprises (i) an EA1; (ii) an EA2; (iii) an EA3; (iv) an EA4; (v) adipyl-CoA reductase (aldehyde forming) (EA5); and (vi) a 6-ACA transaminase (EA6A) or a 6-ACA dehydrogenase (EA6B); 
 (2) the organism comprises two, three, four, five or six exogenous nucleic acids, each encoding a 6-ACAPE; and/or 
 (3) said at least one exogenous nucleic acid encoding a 6-ACAPE is a heterologous nucleic acid; 
   (c) said organism comprises a HMDAP, and wherein
 (1) said organism comprises at least one exogenous nucleic acid encoding a HMDA pathway enzyme (HMDAPE) expressed in a sufficient amount to produce HMDA, wherein said HMDAP comprises (i) an EA1; (ii) an EA2; (iii) an EA3; (iv) an EA4; (v) an EA5; (vi) an EA6A or an EA6B; (vii) a 6-aminocaproyl-CoA/acyl-CoA transferase (EA7A) or 6-aminocaproyl-CoA synthase (EA7B); (viii) a 6-aminocaproyl-CoA reductase (aldehyde forming) (EA9); and (ix) a HMDA transaminase (EA10A) or a HMDA dehydrogenase (EA10B); 
 (2) the organism comprises two, three, four, five, six, seven, eight or nine exogenous nucleic acids, each encoding a HMDAPE; and/or 
 (3) said at least one exogenous nucleic acid encoding a HMDAPE is a heterologous nucleic acid; or 
   (d) said organism comprise a CapP, and wherein
 (1) said organism comprises at least one exogenous nucleic acid encoding a CapP enzyme (CapPE) expressed in a sufficient amount to produce caprolactam, wherein said CapP comprises
 (a) (i) an EA1; (ii) an EA2; (iii) an EA3; (iv) an EA4; (v) an EA5; (vi) an EA6A or an EA6B; and (vii) EA7A or EA7B; or 
 (b) (i) an EA1; (ii) an EA2; (iii) an EA3; (iv) an EA4; (v) an EA5; (vi) an EA6A or an EA6B; and (vii) an amidohydrolase (EA8); 
 wherein said CapP optionally further comprises a spontaneous cyclization, which converts a 6-aminocaproyl-CoA to caprolactam; 
 
 (2) the organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a CapPE; and/or 
 (3) said at least one exogenous nucleic acid encoding a CapPE is a heterologous nucleic acid. 
   
     
     
         3 . The organism of  claim 1 , wherein the MMP comprises:
 (i) an EM1, an EM2, a methylenetetrahydrofolate dehydrogenase (EM3), a methenyltetrahydrofolate cyclohydrolase (EM4), and a formyltetrahydrofolate deformylase (EM5); or   (ii) an EM1, an EM2, an EM3, an EM4 and a formyltetrahydrofolate synthetase (EM6);   wherein the MMP optionally further comprises (i) a formate dehydrogenase (EM8); (ii) a formate hydrogen lyase (EM15); or (iii) an EM15 and an EM16.   
     
     
         4 . The organism of  claim 1 , wherein:
 (a) said organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE;   (b) said at least one exogenous nucleic acid encoding a MMPE is a heterologous nucleic acid;   (c) said organism comprises one or more gene disruptions, wherein said one or more gene disruptions occur in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 , and/or amino acids, by said microbial organism, and wherein said one or more gene disruptions confers increased production of adipate, 6-ACA, HMDA or caprolactam in said microbial organism;   (d) one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, lactate, formate, CO 2  and/or amino acids by said microbial organism, has attenuated enzyme activity or expression levels;   (e) said at least one exogenous nucleic acid is a heterologous nucleic acid;   (f) said organism is in a substantially anaerobic culture medium; and/or   (g) said organism is a species of bacteria, yeast, or fungus.   
     
     
         5 . The organism of  claim 1 , further comprising a formaldehyde assimilation pathway (FAP), wherein said organism comprises at least one exogenous nucleic acid encoding a FAP enzyme (FAPE) expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass, and wherein
 (A) (i) said FAP comprises a hexulose-6-phosphate (H6P) synthase (EF1) and a 6-phospho-3-hexuloisomerase (EF2);
 (ii) said FAP comprises a dihydroxyacetone (DHA) synthase (EF3), and optionally further comprises a DHA kinase (EF4); or 
 (iii) said FAP comprises an EF1, an EF2, an EF3, and further an EF4; and/or 
   (B) the intermediate is (i) a H6P, a fructose-6-phosphate (F6P), or a combination thereof, or (ii) a DHA, a DHAP, or a combination thereof, and/or   (C) the organism comprises two exogenous nucleic acids, each encoding a FAPE.   
     
     
         6 . (canceled) 
     
     
         7 . A method for producing adipate, 6-ACA, HMDA or caprolactam, comprising culturing the organism of  claim 1  under conditions and for a sufficient period of time to produce adipate, 6-ACA, HMDA or caprolactam;
 (i) wherein said method optionally further comprises separating the adipate, 6-ACA, HMDA or caprolactam from other components in the culture,
 wherein the separation optionally comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, or ultrafiltration; and/or 
 
 (ii) wherein the organism is optionally a Crabtree positive, eukaryotic organism, and wherein the organism is cultured in a culture medium comprising glucose. 
 
     
     
         8 . A bioderived adipate, 6-ACA, HMDA or caprolactam, or an intermediate thereof, produced according to the method of  claim 7 ; wherein
 said bioderived adipate, 6-ACA, HMDA or caprolactam has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source; and/or   said bioderived adipate, 6-ACA, HMDA or caprolactam has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.   
     
     
         9 . A culture medium comprising the bioderived adipate, 6-ACA, HMDA or caprolactam of  claim 8 , wherein
 said bioderived adipate, 6-ACA, HMDA or caprolactam optionally has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source;   said bioderived adipate, 6-ACA, HMDA or caprolactam has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%; and/or   said culture medium is optionally separated from the NNOMO having the AdiP, 6-ACAP, HMDAP or CapP.   
     
     
         10 . A composition comprising said bioderived adipate, 6-ACA, HMDA or caprolactam of  claim 8 , and a compound other than said bioderived adipate, 6-ACA, HMDA or caprolactam;
 wherein said compound other than said bioderived adipate, 6-ACA, HMDA or caprolactam optionally is a trace amount of a cellular portion of a NNOMO having an AdiP, 6-ACAP, HMDAP or CapP.   
     
     
         11 . A biobased product or a composition comprising said bioderived adipate, 6-ACA, HMDA or caprolactam, or an intermediate thereof of  claim 8 , wherein the composition is a cell lysate or culture supernatant thereof. 
     
     
         12 . The biobased product of  claim 11 , wherein said bioderived or biobased product is wherein said bioderived or biobased product is selected from the group consisting of a polymer, plastic, epoxy resin, nylon, nylon-6, nylon 6-6, textile, polyurethane, plasticizer, unsaturated polyester, fiber, clothing, polyester polyol, polyurethane, lubricant component, PVC, food additive, food ingredient, flavorant, gelling aid, food, oral or other medicinal coating, and an oral or other medicinal product, or any combination thereof. 
     
     
         13 . The biobased product of  claim 11 , wherein said biobased product comprises
 (a) at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived adipate, 6-ACA, HMDA or caprolactam; and/or   (b) a portion of said bioderived adipate, 6-ACA, HMDA or caprolactam as a repeating unit.   
     
     
         14 . A molded product obtained by molding the biobased product of  claim 11 . 
     
     
         15 . A process for producing the biobased product of  claim 11 , comprising chemically reacting said bioderived adipate, 6-ACA, HMDA or caprolactam with itself or another compound in a reaction that produces said biobased product. 
     
     
         16 . (canceled) 
     
     
         17 . A method for producing a polymer, comprising chemically of enzymatically converting the bioderived adipate, 6-ACA, HMDA or caprolactam of  claim 8  to the polymer. 
     
     
         18 . (canceled) 
     
     
         19 . A method of producing formaldehyde, comprising culturing the organism of  claim 1  under conditions and for a sufficient period of time to produce formaldehyde; and wherein the formaldehyde is consumed to provide a reducing equivalent or to incorporate into bioderived adipate, 6-ACA, HMDA or caprolactam or target product. 
     
     
         20 . A method of producing an intermediate of glycolysis and/or an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing the organism of  claim 5  under conditions and for a sufficient period of time to produce the intermediate, and wherein the intermediate is consumed to provide a reducing equivalent or to incorporate into bioderived adipate, 6-ACA, HMDA or caprolactam or target product. 
     
     
         21 . The method of  claim 19 , wherein the organism is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source. 
     
     
         22 . The organism of  claim 1 ,
 wherein said AdiP, 6-ACAP, HMDAP or CapP further comprises (i) a PEP carboxylase (EFR16A) or PEP carboxykinase (EFR16B); (ii) a pyruvate carboxylase (EFR17); (iii) a malate dehydrogenase (EFR18); (iv) a malic enzyme (EFR19); and/or (v) a fumarase (EFR20A), fumarate reductase (EFR20B), succinyl-CoA synthetase (EFR20C), succinyl-CoA ligase (EFR20D), or succinyl-CoA transferase (EFR20E);   wherein optionally said AdiP, 6-ACAP, HMDAP or CapP comprises (1) (i) EFR16A or EF16B, (ii) EFR18, and (iii) EFR20A, EFR20B, EFR20C, EFR20D, or EFR20E; (2) (i) EFR17, (ii) EFR18 and (iii) EFR20A, EFR20B, EFR20C, EFR20D, or EFR20E; or (3) (i) EFR19 and (ii) EFR20A, EFR20B, EFR20C, EFR20D, or EFR20E.   
     
     
         23 . The organism of  claim 1 , further comprising a formate reutilization pathway (FRP), and wherein:
 (i) said organism comprises at least one exogenous nucleic acid encoding a FRP enzyme (FRPE) expressed in a sufficient amount to produce formaldehyde, pyruvate or acetyl-CoA, wherein said FRP comprises: (1) a formate reductase (EFR1); (2) (i) a formate ligase (EFR2A), a formate transferase (EFR2B), or a formate synthetase (EFR2C), and (ii) a formyl-CoA reductase (EFR3); (3) (i) a formyltetrahydrofolate synthetase (EFR4), (ii) a methenyltetrahydrofolate cyclohydrolase (EFR5), (iii) a methylenetetrahydrofolate dehydrogenase (EFR6) and (iv) a formaldehyde-forming enzyme (EFR7) or spontaneous; (6) (i) an EFR4, (ii) an EFR5, (iii) an EFR6, (iv) a glycine cleavage system (EFR8), (v) a serine hydroxymethyltransferase (EFR9), and (vi) a serine deaminase (EFR10); (7) (i) an EFR1, (ii) an EFR4, (iii) an EFR5, (iv) an EFR6, (v) an EFR8, (vi) an EFR9, and (vii) an EFR10; (8) (i) an EFR2A, an EFR2B or an EFR2C, (ii) an EFR3, (iii) an EFR4, (iv) an EFR5, (v) an EFR6, (vi) an EFR8, (vii) an EFR9, and (viii) an EFR10; (9) (i) an EFR7 or spontaneous, (ii) an EFR4, (iii) an EFR5, (iv) an EFR6, (v) an EFR8, (vi) an EFR9, and (vii) an EFR10; and (10) (i) an EFR4, (ii) an EFR5, (iii) an EFR6, (iv) a methylenetetrahydrofolate reductase (EFR11), and (v) an acetyl-CoA synthase (EFR12);   (ii) the organism comprises two, three, four, five, six, seven or eight exogenous nucleic acids, each encoding a FRPE; and/or   (iii) said at least one exogenous nucleic acid encoding a FRPE is a heterologous nucleic acid;   wherein the FRP further comprises (i) a pyruvate formate lyase (EFR13); (ii) a pyruvate dehydrogenase (EFR14A), a pyruvate ferredoxin oxidoreductase (EFR14B), or a pyruvate:NADP+ oxidoreductase (EFR14C); (iii) a formate dehydrogenase (EFR15); or (iv) an EFR14A, EFR14B, or EFR14C; and an EFR15.