Ethanol production in gram-positive microbes

• US 5,916,787 A
• Filed: 06/07/1995
• Issued: 06/29/1999
• Est. Priority Date: 08/31/1988
• Status: Expired due to Term

• Current Assignee: University of Florida (State University System of Florida)
• Original Assignee: University of Florida (State University System of Florida)
• Inventors: Ingram, Lonnie O'Neal, Barbosa-Alleyne, Maria D. F.
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Saidha, Tekchand
• Application Number:
  US08/475,925
• Time in Patent Office: 1,483 Days
• Field of Search: 435/161, 435/163, 435/252.31, 435/252.3, 435/320.1, 435/165, 435/162
• US Class Current: 435/161
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• 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 101/01001 Alcohol dehydrogenase (1.1.1.1)
• CPC Class Codes: C12Y 102/04001 Pyruvate dehydrogenase (acetyl-transferring) (1.2.4.1)
• CPC Class Codes: G06T 1/00 General purpose image data processing
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

Claims

1. A Gram-positive bacterium which has been transformed with heterologous genes encoding alcohol dehydrogenase and pyruvate decarboxylase wherein said genes are expressed at sufficient levels to confer upon said Gram-positive bacterium transformant the ability to produce ethanol as a fermentation product.

2. The Gram-positive bacterium, according to claim 1, wherein said host is selected from the group consisting of Bacillus, Lactobacillus, Streptococcus, Fibribacter, Ruminococcus, Pediococcus, Cytophaga, Cellulomonas, Bacteroides, and Clostridium.

3. The Gram-positive bacterium according to claim 2, wherein said host is a Bacillus sp.

4. The Gram-positive bacterium, according to claim 3, wherein said Bacillus sp. is selected from the group consisting of B. subtilis and B. polymyxa.

5. The Gram-positive bacterium, according to claim 1, which has been transformed with Z. mobilis genes encoding alcohol dehydrogenase and pyruvate decarboxylase.

6. The Gram-positive bacterium according to claim 1, wherein said bacterium is further transformed with a gene encoding an enzyme which degrades oligosaccharides.

7. The Gram-positive bacterium, according to claim 6, wherein said enzyme which degrades oligosaccharides is a polysaccharase.

8. The Gram-positive bacterium according to claim 7, wherein said polysaccharase is selected from the group consisting of cellulolytic, xylanolytic, and starch-degrading enzymes.

9. The Gram-positive bacterium, according to claim 1, wherein said heterologous genes are incorporated onto the chromosome of said bacterium.

10. A method for the production of ethanol, said method comprising transforming a Gram-positive bacterial host with heterologous genes encoding pyruvate decarboxylase and alcohol dehydrogenase wherein said genes are expressed at sufficient levels to result in the production of ethanol as a fermentation product.

11. The method, according to claim 10, wherein said host is selected from the group consisting of Bacillus, Lactobacillus, Streptococcus, Fibribacter, Ruminococcus, Pediococcus, Cytophaga, Cellulomonas, Bacteroides, and Clostridium.

12. The method, according to claim 11, wherein said host is a Bacillus sp.

13. The method, according to claim 12, wherein said Bacillus sp. is selected from the group consisting of B. subtilis and B. polymyxa.

14. The method, according to claim 10, wherein said Gram-positive bacterium has been transformed with Z. mobilis genes encoding alcohol dehydrogenase and pyruvate decarboxylase.

15. The method, according to claim 10, wherein said bacterium is further transformed with a gene encoding an enzyme which degrades oligosaccharides.

16. The method, according to claim 15, wherein said enzyme which degrades oligosaccharides is a polysaccharase.

17. A method for reducing the accumulation of acidic metabolic products in the growth medium of Gram-positive bacteria, said method comprising transforming said bacteria with heterologous genes which express alcohol dehydrogenase and pyruvate decarboxylase at sufficient levels to result in the production of ethanol as a fermentation product.

18. A plasmid designated pLOI1500.