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CN-106119113-B - Organisms for the production of 1, 3-butanediol

CN106119113BCN 106119113 BCN106119113 BCN 106119113BCN-106119113-B

Abstract

A non-naturally occurring microorganism, the microorganism comprising a microorganism having a 1, 3-butanediol (1, 3-BDO) pathway, the 1, 3-butanediol (1, 3-BDO) pathway having at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme expressed in a sufficient amount to produce 1,3-BDO. A process for producing 1,3-BDO comprising culturing such a microorganism under a plurality of conditions for a time sufficient to produce 1,3-BDO.

Inventors

  • A.P. Bogard
  • M.J. Burke
  • OSTERHOUT ROBIN E.
  • Priti Fakia

Assignees

  • 基因组股份公司

Dates

Publication Date
20260512
Application Date
20100430
Priority Date
20090430

Claims (20)

  1. 1. A non-naturally occurring bacterium having a1, 3-butanediol (1, 3-BDO) pathway, wherein said bacterium comprises: (a) At least three exogenous nucleic acids encoding a1, 3-BDO pathway enzyme comprising (1) a 4-hydroxybutyryl-CoA dehydratase that converts 4-hydroxybutyryl-CoA to crotonyl-CoA, (2) a crotonase that converts crotonyl-CoA to 3-hydroxybutyryl-CoA, and (3) a 3-hydroxybutyryl-CoA reductase that converts 3-hydroxybutyryl-CoA to an alcohol of 1, 3-butanediol, or (B) At least four exogenous nucleic acids encoding a1, 3-BDO pathway enzyme comprising (1) a 4-hydroxybutyryl-CoA dehydratase that converts 4-hydroxybutyryl-CoA to crotonyl-CoA, (2) a crotonase that converts crotonyl-CoA to 3-hydroxybutyryl-CoA, (3) an aldehyde forming 3-hydroxybutyryl-CoA reductase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, and (4) a 3-hydroxybutyraldehyde reductase that converts 3-hydroxybutyraldehyde to 1, 3-butanediol, and Wherein the 1,3-BDO pathway enzyme is expressed in a sufficient amount to produce 1,3-BDO.
  2. 2. The non-naturally occurring bacterium of claim 1, wherein at least one of the exogenous nucleic acids is a heterologous nucleic acid.
  3. 3. The non-naturally occurring bacterium of claim 1, wherein the non-naturally occurring bacterium is in a medium containing less than 10% of dissolved oxygen in saturation.
  4. 4. The non-naturally occurring bacterium of claim 1, wherein the alcohol-forming 3-hydroxybutyryl-CoA reductase is encoded by one or more genes selected from the group consisting of adhE, adhE2, mcr, rcas_2929, nap1_02720, mgp2080_00535, and FAR.
  5. 5. The non-naturally occurring bacterium of claim 1, wherein said 3-hydroxybutyraldehyde reductase is encoded by one or more genes selected from the group consisting of alrA, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhI, P84067, mmsb, dhat, and 3hidh.
  6. 6. The non-naturally occurring bacterium of claim 1, wherein said aldehyde-forming 3-hydroxybutyryl-CoA reductase is encoded by one or more genes selected from acr1, sucD, bphG, bld, adhE, msed _0709, mcr, asd-2, saci_2370, ald, and eutE.
  7. 7. The non-naturally occurring bacterium of claim 1, wherein said 4-hydroxybutyryl-CoA dehydratase is encoded by one or more genes selected from ：fumA、fumB、fumC、fumH、fum1、MmcB、MmcC、hmd、BACCAP _02294、ANACOL_02527、NtherDRAFT_2368、dmdA、dmdB、crt、crt1、echpaaA、paaB、phaA、phaB、maoC、paaF、paaG、abfD、 Msed_1220、fadA、fadB、fadI、fadJ and fadR.
  8. 8. The non-naturally occurring bacterium of claim 1, wherein the crotonase is encoded by one or more genes selected from ：fumA、fumB、fumC、fumH、fum1、MmcB、MmcC、hmd、BACCAP _02294、ANACOL_02527、NtherDRAFT_2368、dmdA、dmdB、crt、crt1、echpaaA、paaB、phaA、phaB、maoC、paaF、paaG、abfD、Msed_1220、fadA、fadB、fadI、fadJ and fadR.
  9. 9. The non-naturally occurring bacterium of claim 8, wherein the bacterium is e.
  10. 10. A culture medium comprising the non-naturally occurring bacterium of claim 1.
  11. 11. A composition comprising the culture medium of claim 10 and biosynthesized 1,3-BDO.
  12. 12. A method for producing 1,3-BDO, the method comprising culturing a non-naturally occurring bacterium having a1, 3-butanediol (1, 3-BDO) pathway under a plurality of conditions for a time sufficient to produce 1,3-BDO, Wherein the bacteria comprise: (a) At least three exogenous nucleic acids encoding a1, 3-BDO pathway enzyme comprising (1) a 4-hydroxybutyryl-CoA dehydratase that converts 4-hydroxybutyryl-CoA to crotonyl-CoA, (2) a crotonase that converts crotonyl-CoA to 3-hydroxybutyryl-CoA, and (3) a 3-hydroxybutyryl-CoA reductase that converts 3-hydroxybutyryl-CoA to an alcohol of 1, 3-butanediol, or (B) At least four exogenous nucleic acids encoding a 1,3-BDO pathway enzyme comprising (1) a 4-hydroxybutyryl-CoA dehydratase that converts 4-hydroxybutyryl-CoA to crotonyl-CoA, (2) a crotonase that converts crotonyl-CoA to 3-hydroxybutyryl-CoA, (3) an aldehyde forming 3-hydroxybutyryl-CoA reductase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, and (4) a 3-hydroxybutyraldehyde reductase that converts 3-hydroxybutyraldehyde to 1, 3-butanediol, and Wherein the 1,3-BDO pathway enzyme is expressed in a sufficient amount to produce 1,3-BDO.
  13. 13. The method of claim 12, wherein the non-naturally occurring bacteria are in a medium containing less than 10% of dissolved oxygen in saturation.
  14. 14. The method of claim 12, wherein the alcohol forming 3-hydroxybutyryl-CoA reductase is encoded by one or more genes selected from the group consisting of adhE, adhE2, mcr, rcas_2929, NAP1_02720, MGP2080_00535, and FAR.
  15. 15. The method of claim 12, wherein the 3-hydroxybutyraldehyde reductase is encoded by one or more genes selected from the group consisting of alrA, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhI, P84067, mmsb, dhat, and 3hidh.
  16. 16. The method of claim 12, wherein the aldehyde-forming 3-hydroxybutyryl-CoA reductase is encoded by one or more genes selected from acr1, sucD, bphG, bld, adhE, msed _0709, mcr, asd-2, saci_2370, ald, and eutE.
  17. 17. The method of claim 12, wherein the 4-hydroxybutyryl-CoA dehydratase is encoded by one or more genes selected from the group consisting of ：fumA、fumB、fumC、fumH、fum1、MmcB、MmcC、hmd、BACCAP_02294、ANACOL_02527、NtherDRAFT_2368、dmdA、dmdB、crt、crt1、echpaaA、paaB、phaA、phaB、maoC、paaF、paaG、abfD、Msed_1220、fadA、fadB、fadI、fadJ and fadR.
  18. 18. The method of claim 12, wherein the crotonase is encoded by one or more genes selected from ：fumA、fumB、fumC、fumH、fum1、MmcB、MmcC、hmd、BACCAP_02294、ANACOL_02527、NtherDRAFT_2368、dmdA、dmdB、crt、crt1、echpaaA、paaB、phaA、phaB、maoC、paaF、paaG、abfD、Msed_1220、fadA、fadB、fadI、fadJ and fadR.
  19. 19. A process as set forth in claim 12 further comprising isolating 1,3-BDO from the culture.
  20. 20. The method of claim 19, wherein the separating comprises extraction, pervaporation, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, adsorption chromatography, and ultrafiltration.

Description

Organisms for the production of 1, 3-butanediol The application is a division of the application application with the application number of 201080029715.X, the application number of 30/2010 and the name of "organism for producing 1, 3-butanediol". Description of related application The present application claims priority from U.S. provisional application serial No. 61/174,473 filed on 4 months of 2009, 30, the entire contents of which are incorporated herein by reference. Background In general, the present invention relates to biosynthetic processes and organisms capable of producing organic compounds. More particularly, the present invention relates to non-naturally occurring organisms that can produce the chemical commodity 1, 3-butanediol. 1, 3-Butanediol (1, 3-BDO) is a four carbon diol traditionally produced from acetylene by the hydration of acetylene. The resulting acetaldehyde is then converted to 3-hydroxybutyraldehyde, which is then reduced to form 1,3-BDO. In recent years, acetylene has been replaced by cheaper ethylene as a source of acetaldehyde. 1,3-BDO is commonly used as an organic solvent for food flavoring. It is also used as a comonomer for polyurethane and polyester resins and is widely used as a hypoglycemic agent. Optically active 1,3-BDO is a useful starting material for the synthesis of bioactive compounds and liquid crystals. The basic commercial use of 1, 3-butanediol is subsequent dehydration to provide 1, 3-butadiene (Ichikawa et al, J.of Molecular CATALYSIS A-Chemical,256:106-112 (2006); ichikawa et al, J.of Molecular CATALYSIS A-Chemical,231:181-189 (2005)), a petrochemical product of 250 hundred million lb/year for use in the manufacture of synthetic rubber (e.g., tires), latex, and resins. The reliance on petroleum-based feedstocks for acetylene or ethylene necessitates the development of routes to 1, 3-butanediol and butadiene based on renewable feedstocks. Thus, there is a need to develop microorganisms and methods for producing 1,3-BDO using the same. The present invention meets this need and provides related advantages as well. Summary of The Invention In some embodiments, the invention is directed to a non-naturally occurring microorganism comprising a microorganism having a1, 3-butanediol (1, 3-BDO) pathway with at least one exogenous nucleic acid encoding a1, 3-BDO pathway enzyme expressed in a sufficient amount to produce 1,3-BDO. The 1,3-BDO pathway includes an enzyme selected from the group consisting of 2-amino-4-oxopentanoic Acid (AKP) thiolase, AKP dehydrogenase, 2-amino-4-hydroxypentanoate transaminase, 2-amino-4-hydroxypentanoate oxidoreductase (deamination), 2-oxo-4-hydroxypentanoate decarboxylase, 3-hydroxybutyraldehyde reductase, AKP transaminase, AKP oxidoreductase (deamination), 2, 4-dioxovalerate decarboxylase, 3-oxobutyraldehyde reductase (ketone reduction), 3-oxobutyraldehyde reductase (aldehyde reduction), 4-hydroxy-2-butanone reductase, AKP decarboxylase, 4-aminobutin-2-one transaminase, 4-aminobutin-2-one oxidoreductase (deamination), 4-aminobutin-2-one ammonia-lyase, butenone hydratase, AKP ammonia-lyase, acetoacetate decarboxylase, acetoacetyl-CoA reductase (CoA-dependent, aldehyde formation), acetyl-reductase (aldehyde formation), acetyl-alcohol-forming, acetyl-CoA-dependent CoA reductase (acetyl-3-hydroxy-CoA formation), butyryl-2-one reductase (butyryl-CoA formation), and butyryl-3-hydroxy-CoA reductase (butyryl-alcohol-forming). In some embodiments, the present invention is directed to a method for producing 1,3-BDO comprising culturing such non-naturally occurring microorganisms under conditions for a time sufficient to produce 1,3-BDO. Drawings FIG. 1 shows the pathway from alanine to 1, 3-BDO. The enzymes are A) AKP thiolase, B) AKP aminotransferase or AKP oxidoreductase (deamination), C) 2, 4-dioxovalerate decarboxylase, D) 3-oxobutyraldehyde reductase (aldehyde reduction), E) AKP decarboxylase, F) 4-aminobutin-2-one ammonia-lyase, G) butenone hydratase, H) 4-hydroxy, 2-butanone reductase, I) AKP ammonia-lyase, J) acetoacetate decarboxylase, K) 4-aminobutin-2-one aminotransferase or 4-aminobutin-2-one oxidoreductase (deamination), L) AKP dehydrogenase, M) 2-amino-4-hydroxypentanoate aminotransferase or 2-amino-4-hydroxypentanoate oxidoreductase (deamination), N) 2-oxo-4-hydroxypentanoate decarboxylase, O) 3-oxobutyrase (ketone reduction), and P) 3-hydroxybutyraldehyde reductase. Figure 2 shows the pathway from acetoacetyl-CoA to 1, 3-butanediol. The enzymes are A) acetoacetyl-CoA reductase (CoA-dependent, aldehyde-forming), B) 3-oxobutyraldehyde reductase (ketone-reducing), C) 3-hydroxybutyraldehyde reductase, D) acetoacetyl-CoA reductase (CoA-dependent, alcohol-forming), E) 3-oxobutyraldehyde reductase (aldehyde-reducing), F) 4-hydroxy, 2-butanone reductase, G) acetoacetyl-CoA reductase (ketone-reducing), H) 3-hydroxybutyryl-CoA reductase (aldehyde-forming) and I) 3-hydroxybutyryl-CoA reductase (alcohol-forming). Figure 3 shows the pathway