Genetically modified clostridium thermocellum engineered to ferment xylose
One aspect of the invention relates to industrial bioconversion of the xylose portion of biomass materials into fuels and chemicals. Another aspect of the invention relates to industrial bioconversion of the xylan portion of biomass materials into fuels and chemicals. In one embodiment, the invention is directed to the bacterium Clostridium thermocellum , a highly cellulolytic organism that has much potential as a biocatalyst in a consolidated bioprocess configuration. In some embodiments, the invention is a genetic modification that confers the ability to ferment xylose to C. thermocellum and the strains created with this modification. In some embodiments, the genetic modification is composed of two genes contained in an operon from T. saccharolyticum . The genes express proteins with xylose isomerase (XI) and xylulokinase activites (XK). In other embodiments, the invention relates to a recombinant Clostridium thermocellum host cell capable of fermenting xylan.
1. A recombinant Clostridium thermocellum host cell capable of fermenting xylan, wherein the host cell comprises one or more heterologous polynucleotides encoding one or more xylanase enzymes, wherein the polynucleotide comprises a nucleotide sequence from any one of the nucleotide sequences of SEQ ID NOs: 5-21,
wherein the host cell further comprises one or more additional heterologous polynucleotides encoding a xylose isomerase and/or a xylulokinase, and
wherein the heterologous polynucleotide that encodes the xylose isomerase comprises the nucleotide sequence of SEQ ID NO: 1 and the heterologous polynucleotide that encodes the xylulokinase comprises the nucleotide sequence of SEQ ID NO: 2.
2. The recombinant Clostridium thermocellum host cell of claim 1 , wherein one or more heterologous polynucleotides are integrated at a specific site in the genome of the host cell.
3. The recombinant Clostridium thermocellum host cell of claim 2 , wherein the integration of at least one of the heterologous polynucleotides occurs at the site of a specific gene in the genome of the host cell.
4. The recombinant Clostridium thermocellum host cell of claim 3 , wherein the specific gene is disrupted as a result of integration of the heterologous polynucleotide.
5. The recombinant Clostridium thermocellum host cell of claim 3 , wherein the specific gene is deleted as a result of integration of the heterologous polynucleotide.
6. The recombinant Clostridium thermocellum host cell of claim 4 , wherein the host cell cannot make the protein product of the disrupted gene.
7. The recombinant Clostridium thermocellum host cell of claim 5 , wherein the host cell cannot make the protein product of the deleted gene.
8. The recombinant Clostridium thermocellum host cell of claim 1 , wherein one or more heterologous polynucleotides are present in the host cell in an extrachromosomal plasmid.
9. The recombinant Clostridium thermocellum host cell of claim 1 , wherein one or more heterologous polynucleotides are randomly integrated into the genome of the host cell.
10. The recombinant Clostridium thermocellum host cell of claim 1 , wherein the host cell ferments xylan to ethanol.
11. A co-culture comprising the recombinant Clostridium thermocellum host cell of claim 1 and at least one other host cell.
12. The co-culture of claim 11 , wherein the host cells express the same xylan metabolizing enzymes.
13. The co-culture of claim 11 , wherein the host cells express different xylan metabolizing enzymes.
14. The co-culture of claim 11 , wherein the host cells express at least one common xylan metabolizing enzyme.
15. The recombinant Clostridium thermocellum host cell of claim 1 , wherein the one or more heterologous polynucleotides that encode a xylanase enzyme encode a polypeptide comprising an amino acid sequence from any one of the amino acid sequences of SEQ ID NOs: 22-38.
16. The recombinant Clostridium thermocellum host cell of claim 1 , further comprising one or more heterologous polynucleotides encoding one or more xylose transporters, wherein the polynucleotide comprises a nucleotide sequence from any one of the nucleotide sequences of SEQ ID NOs: 39-50.
17. The recombinant Clostridium thermocellum of claim 16 , wherein the one or more heterologous polynucleotides that encode a xylose transporter encode a polypeptide comprising an amino acid sequence from any one of the amino acid sequences of SEQ ID NOs: 51-62.
18. The recombinant Clostridium thermocellum host cell of claim 1 , wherein the host cell further comprises a lactate dehydrogenase promoter and terminator.
19. The recombinant Clostridium thermocellum host cell of claim 1 , wherein one or more heterologous polynucleotides encoding one or more xylanse enzymes are expressed.
20. The recombinant Clostridium thermocellum host cell of claim 19 , wherein expression of one or more heterologous polynucleotides confers to the recombinant host cell the ability to ferment xylan.
21. The recombinant Clostridium thermocellum host cell of claim 1 , further comprising a deletion of one or more genes endogenous to the host cell.
22. The recombinant Clostridium thermocellum host cell of claim 21 , wherein the deletion of one or more genes endogenous to the host cell comprises the hypoxanthine phosphoribosyl transferase (HPT) gene.
23. The recombinant Clostridium thermocellum host cell of claim 21 , wherein the deletion of one or more genes endogenous to the host cell comprises the phosphotransacetylase (PTA) gene.
24. The recombinant Clostridium thermocellum of claim 1 , wherein the heterologous polynucleotide that encodes a xylose isomerase encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
25. The recombinant Clostridium thermocellum host cell of claim 1 , wherein expression of the heterologous polynucleotide encoding the xylose isomerase confers to the recombinant host cell the ability to ferment xylose.
26. The recombinant Clostridium thermocellum of claim 1 , wherein the heterologous polynucleotide that encodes a xylulokinase encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.