Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,4-butanediol related thereto

1 . A non-naturally occurring microbial organism (NNOMO) having a methanol metabolic pathway (MMP), wherein said organism comprises at least two exogenous nucleic acids encoding MMP enzymes (MMPE) expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises:

(i) a methanol dehydrogenase (EM9) and a formaldehyde activating enzyme (EM10), wherein EM9 converts methanol to formaldehyde and wherein EM10 converts formaldehyde to methylene-THF; and

(ii) a methanol methyltransferase (EM1) and a methylenetetrahydrofolate reductase (EM2), wherein EM1 converts methanol to methyl-THF and EM2 converts methyl-THF to methylene-THF.

2 . The microbial organism of claim 1 , wherein:

the MMP further comprises:

(i) a methylenetetrahydrofolate dehydrogenase (EM3), a methenyltetrahydrofolate cyclohydrolase (EM4) and a formyltetrahydrofolate deformylase (EM5); or

(ii) an EM3, an EM4 and a formyltetrahydrofolate synthetase (EM6).

3 . The microbial organism of claim 1 , further comprising a 1,4-butanediol (BDO) pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a BDO pathway (BDOP) enzyme expressed in a sufficient amount to produce BDO, and wherein the BDOP comprises:

(i) a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), and a 1,4-butanediol dehydrogenase (EB8);

(ii) an EB3, an EB4, a 4-hydroxybutyryl-CoA transferase (EB11) or a 4-hydroxybutyryl-CoA synthetase (EB12), an EB7, and an EB8;

(iii) an EB3, an EB4, an EB11 or a EB12, and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15);

(iv) an EB3, an EB4, an EB5, an EB6, and an EB15;

(v) an EB3, an EB4, a 4-HB reductase (EB13), and an EB8;

(vi) an EB3, an EB4, an EB5, a 4-hydroxybutyryl-phosphate reductase (EB14), and an EB8;

(vii) a succinyl-CoA reductase (alcohol forming) (EB10), an EB5, an EB6, an EB7, and an EB8;

(viii) an EB10, an EB5, an EB6, and an EB15;

(ix) an EB10, an EB11 or an EB12, an EB7, and an EB8;

(x) an EB10, an EB11 or an EB12, and an EB15;

(xi) an EB10, an EB13, and an EB8;

(xii) an EB10, an EB5, an EB14 and an EB8;

(xiii) a succinate reductase (EB9), an EB4, an EB5, an EB6, an EB7, and an EB8;

(xiv) an EB9, an EB4, an EB11 or an EB12, an EB7, and an EB8;

(xv) an EB9, an EB4, an EB11 or an EB12, and an EB15;

(xvi) an EB9, an EB4, an EB5, an EB6, and an EB15;

(xvii) an EB9, an EB4, an EB13, and an EB8; and or

(xviii) an EB9, an EB4, an EB5, an EB14, and an EB8.

4 . The microbial organism of claim 1 , wherein

(a) said microbial organism further 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 BDO in said microbial organism relative to BDO production in a microbial organism without said one or more gene disruptions; and/or

(b) wherein 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.

5 . The microbial organism of claim 1 , further comprising a formaldehyde assimilation pathway (FAP), wherein said microbial 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.

6 . The microbial organism of claim 1 , wherein

(a) said at least one exogenous nucleic acid is a heterologous nucleic acid;

(b) said microbial organism is in a substantially anaerobic culture medium; and/or

(c) said microbial organism is a species of bacteria, yeast, or fungus.

7 . A method for producing BDO, comprising culturing the microbial organism of claim 3 under conditions and for a sufficient period of time to produce BDO.

8 . A bioderived BDO produced according to the method of claim 7 ; wherein:

(a) said bioderived BDO has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source; and/or

(b) said bioderived BDO 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 BDO of claim 8 ; wherein:

(a) said bioderived BDO has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source; and/or

(b) said culture medium is separated from the NNOMO having the BDOP.

10 . The microbial organism of claim 1 , wherein the MMP further comprises a formate dehydrogenase (EM8), a formate hydrogen lyase (EM15) or a hydrogenase (EM16).

11 . The microbial organism of claim 1 , wherein the organism further comprises three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE.

12 . The microbial organism of claim 3 , wherein the BDOP further comprises a succinyl-CoA transferase (EB1) or a succinyl-CoA synthetase (EB2A).

13 . The microbial organism of claim 3 , further comprises four, five, six or seven exogenous nucleic acids, each encoding a BDOP enzyme.

14 . The microbial organism of claim 5 , wherein said FAP further comprises a hexulose-6-phosphate synthase (EF1) or a 6-phospho-3-hexuloisomerase (EF2).

15 . The microbial organism of claim 5 , wherein said FAP further comprises a dihydroxyacetone synthase (EF3) or a dihydroxyacetone kinase (EF4).

16 . The microbial organism of claim 5 , wherein the intermediate is (i) a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof; or (ii) a dihydroxyacetone, a dihydroxyacetone phosphate, or a combination thereof.

17 . The microbial of claim 5 , wherein the microbial organism comprises two exogenous nucleic acids, each encoding a FAPE.

18 . The method of claim 7 , wherein said method further comprises separating the BDO from other components in the culture.

19 . The method of claim 18 , wherein the separation 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.