Methods and Compositions for Improving The production Of Products In Microorganisms

• US 20100028966A1
• Filed: 07/28/2009
• Published: 02/04/2010
• Est. Priority Date: 07/28/2008
• Status: Abandoned Application

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Blanchard, Jeffrey, Leschine, Susan, Fabel, John, Petit, Elsa
• Application Number:
  US12/510,952
• Publication Number:
  US 20100028966A1
• Time in Patent Office: Days
• US Class Current: 435/165
• CPC Class Codes: C07K 14/33 from Clostridium (G)
• CPC Class Codes: C12N 1/22 Processes using, or culture media containing, cellulose or hydrolysates thereof
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12P 7/065 with microorganisms other than yeasts
• CPC Class Codes: C12P 7/08 produced as by-product or from waste or cellulosic material substrate
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Methods and compositions are provided for improving the production of products, such as fuel products like ethanol, in microorganisms. In particular, methods and compositions are described for improving ethanol production utilizing genes identified in Clostridium phytofermentans.

Claims

1. An isolated polynucleotide cassette comprising two or more of:

(i) a sequence encoding a Clostridium phytofermentans hydrolase;

(ii) a sequence encoding a C. phytofermentans ATP-binding cassette (ABC) transporter; and

(iii) a sequence encoding a C. phytofermentans transcriptional regulator.

2. The cassette of claim 1, wherein the hydrolase is selected from the group consisting of Cphy3368, Cphy3367, Cphy1799, Cphy1800, Cphy2105, Cphy1071, Cphy0430, Cphy1163, Cphy3854, Cphy1929, Cphy2108, Cphy3158, Cphy3207, Cphy3009, Cphy3010, Cphy2632, Cphy3586, Cphy0218, Cphy0220, Cphy1720, Cphy3160, Cphy2276, Cphy1714, Cphy0694, Cphy3202, Cphy3862, Cphy0858, Cphy1510, Cphy2128, Cphy1169, Cphy1888, Cphy2919, and Cphy1612.

3. The cassette of claim 1, wherein the ABC transporter is selected from the group consisting of Cphy1529, Cphy1530, Cphy1531, Cphy3858, Cphy3859, Cphy3860, Cphy2569, Cphy2570, Cphy2571, Cphy2654, Cphy2655, Cphy2656, Cphy3588, Cphy3589, Cphy3590, Cphy3210, Cphy3209, Cphy3208, Cphy2274, Cphy2273, Cphy2272, Cphy2268, Cphy2267, Cphy2266, Cphy2265, Cphy2012, Cphy2011, Cphy2010, Cphy2009, Cphy1717, Cphy1716, Cphy1715 Cphy1451, Cphy1450, Cphy1449, Cphy1448, Cphy1134, Cphy1133, and Cphy1132.

4. The cassette of claim 1, wherein the cassette comprises a sequence encoding a Clostridium phytofermentans hydrolase;

and a sequence encoding a C. phytofermentans ATP-binding cassette (ABC) transporter.

5. The cassette of claim 1, wherein the cassette comprises a sequence encoding a Clostridium phytofermentans hydrolase;

and a sequence encoding a C. phytofermentans transcriptional regulator.

6. The cassette of claim 1, wherein the cassette comprises a sequence encoding a Clostridium phytofermentans ATP-binding cassette (ABC) transporter;

and a sequence encoding a C. phytofermentans transcriptional regulator.

7. The cassette of claim 1, comprising a sequence encoding a C. phytofermentans transcriptional regulator and a nucleic acid sequence to which the transcriptional regulator binds.

8. The cassette of claim 7, wherein the nucleic acid sequence is operably linked to a promoter.

9. A recombinant microorganism comprising one or more of:

(i) an exogenous nucleic acid encoding a Clostridium phytofermentans hydrolase;

(ii) an exogenous nucleic acid encoding a C. phytofermentans ATP-binding cassette (ABC) transporter; and

(iii) an exogenous nucleic acid encoding a C. phytofermentans transcriptional regulator.

10. The microorganism of claim 9, wherein the hydrolase is selected from the group consisting of Cphy3368, Cphy3367, Cphy1799, Cphy1800, Cphy2105, Cphy1071, Cphy0430, Cphy1163, Cphy3854, Cphy1929, Cphy2108, Cphy3158, Cphy3207, Cphy3009, Cphy3010, Cphy2632, Cphy3586, Cphy0218, Cphy0220, Cphy1720, Cphy3160, Cphy2276, Cphy1714, Cphy0694, Cphy3202, Cphy3862, Cphy0858, Cphy1510, Cphy2128, Cphy1169, Cphy1888, Cphy2919, and Cphy1612.

11. The microorganism of claim 9, wherein the ABC transporter is selected from the group consisting of Cphy1529, Cphy1530, Cphy1531, Cphy3858, Cphy3859, Cphy3860, Cphy2569, Cphy2570, Cphy2571, Cphy2654, Cphy2655, Cphy2656, Cphy3588, Cphy3589, Cphy3590, Cphy3210, Cphy3209, Cphy3208, Cphy2274, Cphy2273, Cphy2272, Cphy2268, Cphy2267, Cphy2266, Cphy2265, Cphy2012, Cphy2011, Cphy2010, Cphy2009, Cphy1717, Cphy1716, Cphy1715 Cphy1451, Cphy1450, Cphy1449, Cphy1448, Cphy1134, Cphy1133, and Cphy1132.

12. The microorganism of claim 9, wherein said microorganism is selected from the group consisting of Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum.

13. The microorganism of claim 9, wherein said microorganism comprises a sequence encoding a Clostridium phytofermentans hydrolase;

and a sequence encoding a C phytofermentans ATP-binding cassette (ABC) transporter.

14. The microorganism of claim 9, wherein said microorganism comprises a sequence encoding a Clostridium phytofermentans hydrolase;

and a sequence encoding a C. phytofermentans transcriptional regulator.

15. The microorganism of claim 9, wherein said microorganism comprises a sequence encoding a Clostridium phytofermentans ATP-binding cassette (ABC) transporter;

and a sequence encoding a C. phytofermentans transcriptional regulator.

16. A method of producing ethanol comprising culturing at least one recombinant microorganism of claim 9.

17. The method of claim 16, wherein the substrate is selected from the group consisting of saw dust, wood flour, wood pulp, paper pulp, paper pulp waste steams, one or more grasses, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, corn grind, distillers grains, and pectin.

18. A method of processing a substrate of a hydrolase, the method comprising:

providing a recombinant microorganism that exogenously expresses a Clostridium phytofermentans hydrolase; and

supplying the substrate of the hydrolase to the microorganism, such that the substrate is processed to form a product.

19. The method of claim 18, wherein the microorganism exogenously expresses a Clostridium phytofermentans ATP-binding cassette (ABC) transporter that transports the product.

20. A product for production of a biofuel comprising:

a carbonaceous biomass, a microorganism that is capable of direct hydrolysis and fermentation of said biomass, wherein said microorganism is modified to provide enhanced activity of one or more cellulases.

21. The product of claim 20, wherein the carbonaceous biomass is a lignocellulosic biomass.

22. A process for producing a biofuel, the process comprising:

(a) contacting a carbonaceous biomass with;

a microorganism that is capable of direct hydrolysis and fermentation of said biomass, wherein the microorganism is modified to enhance activity of one or more cellulase enzymes; and

(b) allowing sufficient time for said hydrolysis and fermentation to produce a biofuel.