Ethanol production by recombinant hosts

• US 5,487,989 A
• Filed: 09/17/1992
• Issued: 01/30/1996
• Est. Priority Date: 08/31/1988
• Status: Expired due to Term

• Current Assignee: BP Corporation North America Incorporated (BP PLC)
• Original Assignee: Bioenergy International LLC (PTTGC Innovation America Corporation)
• Inventors: Fowler, David E., Horton, Philip G., Ben-Bassat, Arie
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): GRIMES, ERIC BURTON
• Application Number:
  US07/946,290
• Time in Patent Office: 1,230 Days
• Field of Search: 435/165, 435/852, 435/252.3
• US Class Current: 435/165
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/065 with microorganisms other than yeasts
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01037 Xylan 1,4-beta-xylosidase (3.2.1.37)
• CPC Class Codes: G06T 1/00 General purpose image data processing
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y10S 435/852 Klebsiella

Abstract

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Claims

1. A method for producing ethanol from cellulose-containing biomass, comprising the steps of:

A. contacting, in a first reaction vessel, said biomass with a polysaccharase such that cellulose in said biomass is broken down into simpler oligosaccharides and/or monosaccharides,

wherein said contacting is carried out at a temperature of from about 40°

C. to about 60°

C. and a pH of from about 4.5 to about 5.0;

B. producing from said first reaction vessel a sugar solution comprising said simpler oligosaccharides and/or monosaccharides;

C. introducing said sugar solution into a fermentor which comprises gram-negative enteric recombinant host microorganisms capable of fermenting said simpler oligosaccharides and/or monosaccharides into ethanol; and

D. fermenting said simpler oligosaccharides and/or monosaccharides into ethanol at a temperature of from about 30°

C. to about 35°

C. and a pH of about 6.0,

wherein said microorganism is capable of fermenting monosaccharides into ethanol, and comprises a recombinant host, other than Escherichia coli, comprising a first heterologous DNA coding for alcohol dehydrogenase and pyruvate decarboxylase, wherein said heterologous DNA is from Zymomonas mobilis and wherein said host expresses said heterologous DNA at a sufficient functional level so as to facilitate the production of ethanol as the primary fermentation product by said host,

wherein said host also produces a polysaccharase, and said host further comprises a second heterologous DNA segment, the expression product of which is said polysaccharase.

2. A method according to claim 1, wherein a first stream from said fermentor is withdrawn and used to cool the sugar solution produced in Step B.

3. A method according to claim 2, wherein said first stream is introduced into said first vessel after cooling said sugar solution produced in Step B.

4. A method according to claim 1, wherein the sugar solution produced in Step B is passed through an ultrafiltration unit having an upper molecular weight cut-off ultrafiltration membrane to obtain an ultrafiltration product solution and a second solution,

said ultrafiltration product solution comprising predominantly molecules having a molecular weight below the molecular weight cut-off of said ultrafiltration membrane, said product solution comprising at least some of said oligosaccharides and/or monosaccharides obtained from Step B, and

said second solution comprising predominantly molecules having a molecular weight above the molecular weight cut-off of said ultrafiltration membrane.

5. A method according to claim 4, wherein said product solution predominantly comprises molecules having a molecular weight of less than about 25,000.

6. A method according to claim 4, wherein the ultrafiltration product solution is subjected to reverse osmosis to obtain a first stream comprising predominantly water and a second stream comprising at least some of the oligosaccharides and/or monosaccharides.

7. A method according to claim 6, wherein said stream comprising predominantly water is recycled to the first reaction vessel.

8. A method according to claim 1, wherein the sugar solution produced in Step B is subjected to reverse osmosis to obtain a first stream comprising predominantly water and a second stream comprising at least some of the oligosaccharides and/or monosaccharides.

9. A method according to claim 8, wherein said stream comprising predominantly water is recycled to the first reaction vessel.

10. A method according to claim 1, wherein the contacting in said first reaction vessel is carried out at a temperature of from about 50°

C. to about 60°

C.

11. A method according to claim 1, wherein the contacting in said first reaction vessel is carried out at a temperature of from about 50°

C. to about 55°

C.

12. A method according to claim 1, wherein enzymes which break down cellulose into simpler oligosaccharides and/or monosaccharides are added to said fermentor.

13. The method of claim 1, wherein said host is selected from the group consisting of Erwinia, Klebsiella and Xanthomonas.

14. The method of claim 13, wherein said host is selected from the group consisting of Erwinia and Klebsiella.

15. The method of claim 14, wherein said host is Klebsiella oxytoca M5A1(pLOI555), ATCC 68564, deposited Mar. 14, 1991.

16. The method of claim 1, wherein said host has been transformed with a plasmid comprising genes coding for alcohol dehydrogenase and pyruvate decarboxylase, wherein said host expresses said genes to produce alcohol dehydrogenase and pyruvate decarboxylase at a sufficient functional level to facilitate the production of ethanol as the primary fermentation product by said host.

17. The method of claim 16, wherein said plasmid comprises Zymomonas mobilis genes coding for alcohol dehydrogenase and pyruvate decarboxylase.

18. The method of claim 16, wherein said plasmid further comprises a lac promoter which directs the expression of said genes coding for alcohol dehydrogenase and pyruvate decarboxylase.

19. The method of claim 16, wherein said plasmid has been designated pLOI555.

20. The method of claim 1, wherein said polysaccharase is a cellulolytic enzyme.

21. The method of claim 20, wherein said polysaccharase is selected from the group consisting of an endoglucanase, cellobiohydrolase, β

-glucosidase, and β

-glucanase.

22. The method of claim 21, wherein polysaccharase is an expression product of a celD gene.

23. The method of claim 22, wherein said celD gene is derived from Clostridium thermocellum.

24. The method of claim 21, wherein said polysaccharase is at least partially secreted by said host.

25. The method of claim 21, wherein said polysaccharase is accumulated in said host.

26. A method for producing ethanol from cellulose-containing biomass, comprising the steps of:

A. contacting, in a first reaction vessel, said biomass with a polysaccharase such that oligosaccharide in said biomass is broken down into simpler oligosaccharides and/or monosaccharides,

wherein said contacting is carried out at a temperature of from about 40°

C. to about 60°

C. and a pH of from about 4.5 to about 5.0;

B. producing from said first reaction vessel a sugar solution comprising said simpler oligosaccharides and/or monosacchrides;

C. introducing said sugar solution into a fermentor which comprises gram-negative enteric recombinant host microorganisms capable of fermenting said simpler oligosaccharides and/or monosaccharides into ethanol; and

D. fermenting said simpler oligosaccharides and/or monosaccharides into ethanol at a temperature of from about 30°

C. to about 35°

C. and a pH of about 6.0,

wherein said microorganism is capable of fermenting both monosaccharides and oligosaccharides into ethanol, and said microorganism further comprises a recombinant host comprising first heterologous DNA from Zymomonas mobilis coding for alcohol dehydrogenase and pyruvate decarboxylase, respectively, wherein said host

(A) further comprises genes coding for proteins which enable said host to transport and metabolize an oligosaccharide, and

(B) expresses said genes and said heterologous DNA at a level such that ethanol is produced as the primary fermentation product by said host from the metabolism of said oligosaccharide, and

wherein said recombinant host also produces a polysaccharase, and said host further comprises a second heterologous DNA segment, the expression product of which is said polysaccharase.

27. The method of claim 26, wherein said recombinant host is selected from the group consisting of Erwinia and Klebsiella.

28. The method of claim 27, wherein said recombinant host is Klebsiella oxytoca M5A1(pLOI555), ATCC 68564, deposited Mar. 14, 1991.

29. The method of claim 26, wherein said oligosaccharide is selected from the group consisting of dimers and trimers.

30. The method of claim 26, wherein said polysaccharase is a cellulolytic enzyme.

31. The method of claim 30, wherein said polysaccharase comprises the expression product of a cellulase gene of Cellulomonas fimi, and said host secretes at least some of said polysaccharase.

32. The method of claim 26, wherein said polysaccharase is selected from the group consisting of an endoglucanase, cellobiohydrolase, β

-glucosidase, β

-glucanase and hemicellulase, arabinosidase.

33. The method of claim 32, wherein said polysaccharase is an expression product of a celD gene.

34. The method of claim 33, wherein said celD gene is derived from Clostridium thermocellum.

35. The method of claim 26, wherein said host further comprises an additional heterologous DNA segment, the expression product of which is a protein involved in the transport of mono- and/or oligosaccharides into the recombinant host.

36. The method of claim 26, wherein said polysaccharase is at least partially secreted by said host.

37. The method of claim 26, wherein said polysaccharase is accumulated in said host.

38. The method of claim 37, wherein said host further comprises an additional heterologous DNA segment, the expression product of which is an additional polysaccharase that is at least partially secreted by said host.

39. The method of claim 38, wherein said additional polysaccharase comprises the expression product of a cellulase gene of Cellulomonas fimi.