Methods and compositions for improving the production of products in microorganisms

• US 7,943,363 B2
• Filed: 07/28/2009
• Issued: 05/17/2011
• Est. Priority Date: 07/28/2008
• Status: Expired due to Fees

• Current Assignee: Qteros, Inc., University Of Massachusetts
• Original Assignee: Qteros, Inc., University Of Massachusetts
• Inventors: Schmalisch, Matthias, Blanchard, Jeffrey, Leschine, Susan, Fabel, John, Petit, Elsa
• Primary Examiner(s): PROUTY, REBECCA E
• Application Number:
  US12/510,994
• Publication Number:
  US 20100035320A1
• Time in Patent Office: 658 Days
• Field of Search: None
• US Class Current: 435/252.3
• 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: 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
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel
• CPC Class Codes: Y10S 435/842 Clostridium

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:

a. a nucleic acid sequence encoding at least one transcriptional regulator from C. phytofermentans; and

b. a nucleic acid sequence encoding

i. at least one hydrolase from C. phytofermentans, wherein said nucleic acid is selected from Table 6;

or

ii. at least one ABC-transporter from C. phytofermentans, wherein said nucleic acid is selected from ABC-transporter genes in Table 7.

2. The cassette of claim 1, wherein said at least one hydrolase is selected from the group consisting of Cphy407, Cphy408, Cphy0430, Cphy3854, Cphy0857, Cphy0694, and Cphy1929.

3. The cassette of claim 1, wherein said at least one ABC-transporter is selected from the group consisting of Cphy3854, Cphy3855, Cphy3857, Cphy3858, Cphy3859, Cphy3860, Cphy3861, and Cphy3862.

4. The cassette of claim 1, wherein said at least one transcriptional regulator from C. phytofermentans is selected from Table 8.

5. The cassette of claim 1, further comprising a regulatory sequence.

6. The cassette of claim 5, wherein said regulatory sequence is operably linked to said at least one hydrolase, said at least one ABC-transporter, or said at least one transcriptional regulator.

7. The cassette of claim 1, wherein said isolated polynucleotide cassette is inserted into an expression vector.

8. The vector of claim 7, wherein at least one of said nucleic acid sequences is methylated.

9. The vector of claim 8, wherein said methylation prevents restriction of said said nucleic acid sequences by a Clostridium endonuclease.

10. The isolated polynucleotide cassette of claim 1, wherein said at least one transcriptional regulator is Cphy1187.

11. The isolated polynucleotide cassette of claim 1, wherein said at least one hydrolase is Cphy3207.

12. The isolated polynucleotide cassette of claim 1, wherein said at least one transcriptional regulator is Cphy1187 and the hydrolase is Cphy3207.

13. A recombinant microorganism comprising:

a. an isolated nucleic acid sequence encoding at least one transcriptional regulator from C. phytofermentans; and

b. an isolated nucleic acid sequence encoding

i. at least one hydrolase from C. phytofermentans, wherein said isolated nucleic acid is selected from Table 6;

or

ii. at least one ABC-transporter from C. phytofermentans, wherein said isolated nucleic acid is selected from ABC-transporter genes in Table 7,

wherein at least one of said isolated nucleic acid sequence encoding at least one transcription regulator and said isolated nucleic acid sequence encoding at least one hydrolase or at least one ABC-transporter from C. phytofermentans is heterologous to the microorganism or is an additional copy of an endogenous nucleic acid sequence.

14. The recombinant microorganism of claim 13, 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.

15. The recombinant microorganism of claim 13, wherein said at least one hydrolase is selected from the group consisting of Cphy407, Cphy408, Cphy0430, Cphy3854, Cphy0857, Cphy0694, and Cphy1929.

16. The recombinant microorganism of claim 13, wherein said at least one ABC-transporter is selected from the group consisting of Cphy3854, Cphy3855, Cphy3857, Cphy3858, Cphy3859, Cphy3860, Cphy3861, and Cphy3862.

17. The recombinant microorganism of claim 13, wherein said hydrolase is selected from the group consisting of Cphy407, Cphy408, Cphy0430, Cphy3854, Cphy0857, Cphy0694, and Cphy1929;

and wherein said ABC-transporter is selected from the group consisting of Cphy3854, Cphy3855, Cphy3857, Cphy3858, Cphy3859, Cphy3860, Cphy3861, and Cphy3862.

18. The recombinant microorganism of claim 13, wherein said hydrolase is Cphy3207.

19. The recombinant microorganism of claim 13, wherein said transcriptional regulator is Cphy1187 and said hydrolase is Cphy3207.

20. The recombinant microorganism of claim 13, wherein said Cphy1187 modulates the expression of Cphy3207.

21. The recombinant microorganism of claim 13, wherein said recombinant microorganism is a Clostridium microorganism.

22. The recombinant microorganism of claim 13, wherein said recombinant microorganism is a Clostridium phytofermentans microorganism.

23. The recombinant microorganism of claim 13, wherein said recombinant microorganism is a Clostridium thermocellum microorganism.

24. The recombinant microorganism of claim 13, wherein at least one of said isolated nucleic acid sequence encoding at least one transcription regulator and said isolated nucleic acid sequence encoding at least one hydrolase or at least one ABC-transporter from C. phytofermentans is heterologous to the microorganism.

25. The recombinant microorganism of claim 13, wherein at least one of said isolated nucleic acid sequence encoding at least one transcription regulator and said isolated nucleic acid sequence encoding at least one hydrolase or at least one ABC-transporter from C. phytofermentans is an additional copy of an endogenous nucleic acid sequence.