Claims
- 1. A recombinant host cell comprising:
a first heterologous polynucleotide segment comprising a sequence encoding a polysaccharase polypeptide under the transcriptional control of a surrogate promoter, said promoter capable of causing increased expression of said polysaccharase polypeptide; and a second heterologous polynucleotide segment comprising a sequence encoding a secretory polypeptide, wherein expression of said first and second polynucleotide segments results in the increased production of a polysaccharase by the recombinant host cell.
- 2. The recombinant host cell of claim 1 wherein production is selected from the group consisting of activity, amount, and a combination thereof.
- 3. The recombinant host cell of claim 2 wherein said polysaccharase polypeptide is secreted.
- 4. The recombinant host cell of claim 2 wherein said host cell is a bacterial cell.
- 5. The recombinant host cell of claim 4 wherein said host cell is a Gram-negative bacterial cell.
- 6. The recombinant host cell of claim 5 wherein said host cell is a facultatively anaerobic bacterial cell.
- 7. The recombinant host cell of claim 6 wherein said host cell is selected from the family Enterobacteriaceae.
- 8. The recombinant host cell of claim 7 wherein said host is selected from the group consisting of Escherichia and Klebsiella.
- 9. The recombinant host cell of claim 8 wherein said Escherichia is selected from the group consisting of E. coli B, E. coli DH5α, E coli KO4 (ATCC 55123), E. coli KO11 (ATCC 55124), E. coli KO12 (ATCC 55125) and E. coli LY01, K. oxytoca M5A1, and K. oxyloca P2 (ATCC 55307).
- 10. The recombinant host cell of claim 2 wherein said polysaccharase is selected from the group consisting of glucanase, endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, endo-1,4-β-xylanase, α-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, or a combination thereof.
- 11. The recombinant host cell of claim 10 wherein said polysaccharase is glucanase.
- 12. The recombinant host cell according to claim 10, wherein said polysaccharase is an expression product of a celZ gene.
- 13. The recombinant host cell of claim 12 wherein said celZ gene is derived from Erwinia chrysanthemi.
- 14. The recombinant host cell of claim 2 wherein said second heterologous polynucleotide segment comprises at least one pul gene or out gene.
- 15. The recombinant host cell of claim 2 wherein said second heterologous polynucleotide segment is derived from a bacterial cell selected from the family Enterobacteriaceae.
- 16. The recombinant host cell of claim 15 wherein said bacterial cell is selected from the group consisting of K. oxytoca, E. carotovora, E. carotovora subspecies carotovora, E. carotovora subspecies atroseptica, and E. chrysanthemi.
- 17. The recombinant host cell of claim 2 wherein said surrogate promoter comprises a polynucleotide fragment derived from Zymomonas mobilis.
- 18. The recombinant host cell of claim 17 wherein said surrogate promoter comprises a nucleic acid having the sequence provided in SEQ ID NO: 1, or a fragment thereof.
- 19. The recombinant host cell of any one of claims 1-18 wherein said host cell is ethanologenic.
- 20. A recombinant ethanologenic host cell comprising a heterologous polynucleotide segment encoding a polysaccharase under the transcriptional control of an exogenous surrogate promoter.
- 21. The recombinant host cell of claim 20 wherein said host cell is a bacterial cell.
- 22. The recombinant host cell of claim 21 wherein said host cell is a Gram-negative bacterial cell.
- 23. The recombinant host cell of claim 22 wherein said host cell is a facultatively anaerobic bacterial cell.
- 24. The recombinant host cell of claim 23 wherein said host cell is selected from the family Enterobacteriaceae.
- 25. The recombinant host cell of claim 24 wherein said host is selected from the group consisting of Escherichia and Klebsiella.
- 26. The recombinant host cell of claim 25 wherein said Escherichia and Klebsiella are selected from the group consisting of E. coli B, E. coli DH5α, E. coli KO4 (ATCC 55123), E. coli KO11 (ATCC 55124), E. coli KO12 (ATCC 55125), E. coli LY01, K. oxytoca M5A1 and K. oxytoca P2 (ATCC 55307).
- 27. The recombinant host cell of claim 20 wherein said polysaccharase is selected from the group consisting of glucanase, endoglucanase, exoglucanase, cellobiohydrolase, α-glucosidase, endo-1,4-α-xylanase, β-xylosidase, β-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, or a combination thereof.
- 28. The recombinant host cell of claim 27 wherein said polysaccharase is glucanase.
- 29. The recombinant host cell according to claim 28 wherein said polysaccharase is an expression product of a celZ gene.
- 30. The recombinant host cell of claim 29 wherein said celZ gene is derived from Erwinia chrysanthemi.
- 31. The recombinant host cell of claim 20 wherein said surrogate promoter comprises a polynucleotide fragment derived from Zymomonas mobilis.
- 32. The recombinant host cell of claim 31 wherein said surrogate promoter comprises a polynucleotide segment having the sequence provided in SEQ ID NO: 1, or a fragment thereof.
- 33. A recombinant ethanologenic Gram-negative bacterial host cell comprising:
a first heterologous polynucleotide segment comprising a sequence encoding a first polypeptide; and a second heterologous polynucleotide segment comprising a sequence encoding a secretory polypeptide, wherein production of the first polypeptide by the host cell is increased.
- 34. The recombinant host cell of claim 33 wherein said first polypeptide is secreted.
- 35. The recombinant host cell of claim 33 wherein said host cell is a facultatively anaerobic bacterial cell.
- 36. The recombinant host cell of claim 35 wherein said host cell is selected from the family Enterobacteriaceae.
- 37. The recombinant bacterial host cell of claim 36 wherein said host cell is selected from the group consisting of Escherichia and Klebsiella.
- 38. The recombinant bacterial host cell of claim 37 wherein said Escherichia and Klebsiella are selected from the group consisting of E. coli B, E coli DH5α, E. coli KO4 (ATCC 55123), E. coli KO11 (ATCC 55124), E. coli KO12 (ATCC 55125) E. coli LY01, K. oxytoca M5A1, and K. oxytoca P2 (ATCC 55307).
- 39. The recombinant bacterial host cell of claim 33 wherein said first polypeptide is a polysaccharase.
- 40. The recombinant bacterial host cell of claim 39 wherein said polysaccharase is of increased activity.
- 41. The recombinant host cell of claim 39 wherein said polysaccharase is selected from the group consisting of glucanase, endoglucanase, exoglucanase, cellobiohydrolase, α-glucosidase, endo-1,4-α-xylanase, β-xylosidase, β-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase, α-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, or a combination thereof.
- 42. The recombinant host cell of claim 41 wherein said polysaccharase is glucanase.
- 43. The recombinant host cell according to claim 42 wherein said glucanase is an expression product of a celZ gene.
- 44. The recombinant host cell of claim 43 wherein said celZ gene is derived from Erwinia chrysanthemi.
- 45. The recombinant host cell of claim 33 wherein said second heterologous polynucleotide segment comprises at least one pul gene or out gene.
- 46. The recombinant host cell of claim 45 wherein said second heterologous polynucleotide segment is derived from a bacterial cell selected from the family Enterobacteriaceae.
- 47. The recombinant host cell of claim 46 wherein said bacterial cell is selected from the group consisting of K. oxytoca, E. carotovora, E. carotovora subspecies carotovora, E. carotovora subspecies atroseptica, and E. chrysanthemi.
- 48. A method for enzymatically degrading an oligosaccharide comprising the steps of:
providing a oligosaccharide; and contacting said oligosaccharide with a host cell comprising a first heterologous polynucleotide segment comprising a sequence encoding a polysaccharase under the transcriptional control of a surrogate promoter, said promoter capable of causing increased expression of said polysaccharase; and a second heterologous polynucleotide segment comprising a sequence encoding a secretory polypeptide, wherein expression of said first and second polynucleotide segments results in the increased production of polysaccharase activity by the recombinant host cell such that the oligosaccharide is enzymatically degraded.
- 49. The method of claim 48 wherein said polysaccharase is secreted.
- 50. The method of claim 48 wherein said host cell is ethanologenic.
- 51. The method of claim 48 wherein said method is conducted in an aqueous solution.
- 52. The method of claim 48 wherein said method is used for simultaneous saccharification and fermentation.
- 53. The method of claim 48 wherein said oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin, and any combination thereof.
- 54. A method of identifying a surrogate promoter capable of increasing the expression of a gene-of-interest in a host cell, said method comprising:
fragmenting a genomic polynucleotide from an organism into one or more fragments; placing said gene-of-interest under the transcriptional control of at least one fragment; introducing said fragment and gene-of-interest into a host cell; and identifying a host cell having increased expression of said gene-of-interest whereby said increased expression indicates that the fragment is a surrogate promoter.
- 55. A method of making a recombinant host cell for use in simultaneous saccharification and fermentation comprising:
introducing into said host cell a first heterologous polynucleotide segment comprising a sequence encoding a polysaccharase under the transcriptional control of a surrogate promoter, said promoter capable of causing increased expression of said polysaccharase; and introducing into said host cell a second heterologous polynucleotide segment comprising a sequence encoding a secretory polypeptide, wherein expression of said first and second polynucleotide segments results in the increased production of a polysaccharase by the recombinant host cell.
- 56. The recombinant host cell of claim 55 wherein production is selected from the group consisting of activity, amount, and a combination thereof.
- 57. The recombinant host cell of claim 55 or 56 wherein said polysaccharase polypeptide is secreted.
- 58. The method of claim 55, 56, or 57 wherein said host cell is ethanologenic.
- 59. A vector comprising the polynucleotide sequence of pLOI2306 (SEQ ID NO: 12).
- 60. A host cell having a vector comprising the polynucleotide sequence of pLOI2306 (SEQ ID NO: 12).
- 61. A method of making a recombinant host cell integrant comprising:
introducing into said host cell a vector comprising the polynucleotide sequence of pLOI2306 (SEQ ID NO: 12); and identifying a host cell having said vector stably integrated.
- 62. A method for expressing a polysaccharase in a host cell comprising:
introducing into said host cell a vector comprising the polynucleotide sequence of pLOI2306 (SEQ ID NO: 12); and identifying a host cell expressing said polysaccharase.
- 63. The method of any one of claims 60-62 wherein said host cell is ethanologenic.
- 64. A method for producing ethanol from an oligosaccharide source comprising,
contacting said oligosaccharide source with a ethanologenic host cell comprising a first heterologous polynucleotide segment comprising a sequence encoding a polysaccharase under the transcriptional control of a surrogate promoter, said promoter capable of causing increased expression of said polysaccharase; and a second heterologous polynucleotide segment comprising a sequence encoding a secretory polypeptide, wherein expression of said first and second polynucleotide segments results in the increased production of polysaccharase activity by the ethanologenic cell such that the oligosaccharide source is enzymatically degraded and fermented into ethanol.
- 65. The host cell of claim 64 wherein said polysaccharase is selected from the group consisting of glucanase, endoglucanase, exoglucanase, cellobiohydrolase, α-glucosidase, endo-1,4-α-xylanase, β-xylosidase, β-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, or a combination thereof.
- 66. The host cell of claim 65 wherein said polysaccharase is glucanase.
- 67. The host cell according to claim 66 wherein said glucanase is an expression product of a celZ gene.
- 68. The host cell of claim 67 wherein said celZ gene is derived from Erwinia chrysanthemi.
- 69. The host cell of claim 64 wherein said second heterologous polynucleotide segment comprises at least one pul gene or out gene.
- 70. The host cell of claim 64 wherein said host cell is selected from the family Enterobacteriaceae.
- 71. The host cell of claim 64 wherein said host cell is selected from the group consisting of Escherichia and Klebsiella.
- 72. The host cell of claim 64, wherein said host cell is selected from the group consisting of E. coli KO4 (ATCC 55123), E. coli KO11 (ATCC 55124), E. coli KO12 (ATCC 55125), K. oxytoca M5A1, and K. oxytoca P2 (ATCC 55307).
- 73. The host cell of claim 64, wherein said polysaccharase is of increased activity.
- 74. The method of claim 64, wherein said method is conducted in an aqueous solution.
- 75. The method of claim 64, wherein said oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin, and any combination thereof.
- 76. The method according to claim 64, wherein said first heterologous polynucleotide segment is, or derived from, pLOI2306 (SEQ ID NO: 12).
RELATED INFORMATION
[0001] This application claims priority to U.S. provisional Application No. 60/136,376, entitled “RECOMBINANT HOSTS SUITABLE FOR SIMULTANEOUS SACCHARIFICATION AND FERMENTATION,” filed on May 26, 1999, incorporated herein in its entirety by this reference. The contents of all patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.
GOVERNMENT SPONSORED RESEARCH
[0002] This work was supported, in part, by grants from the United States Department of Agriculture, National Research Initiative (95-37308-1843; 98-35504-6177), and United States Department of Energy (DE-FG02-96ER20222)
Provisional Applications (1)
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Number |
Date |
Country |
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60136376 |
May 1999 |
US |
Continuations (1)
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Number |
Date |
Country |
Parent |
09579395 |
May 2000 |
US |
Child |
10377406 |
Feb 2003 |
US |