Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose
Host cells, comprising Kluveryomyces expressing heterologous cellulases produce ethanol from cellulose In addition, multiple host cells expressing different heterlogous cellulases can be co-cultured together and used to produce ethanol from cellulose The recombinant yeast strains and co-cultures of the yeast strains can be used to produce ethanol on their own, or can also be used in combination with externally added cellulases to increase the efficiency of sacchanfication and fermentation processes.
1. A thermotolerant yeast host cell comprising
1. at least one heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase;
2. at least one heterologous polynucleotide comprising a nucleic acid which encodes a β-glucosidase;
3. at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase I; and
4. at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase II;
wherein the thermotolerant yeast host cell can grow at a temperature of at least 35° C. or above,
wherein the endoglucanase, β-glucosidase, cellobiohydrolase I, and cellobiohydrolase II are secreted; and
wherein the host cell is capable of producing ethanol from Avicel.
2. The thermotolerant yeast host cell of claim 1 , wherein the β-glucosidase is a Saccharomycopsis fibuligera β-glucosidase comprising the amino acids of SEQ ID NO-40.
3. The thermotolerant yeast host cell of claim 1 , wherein the cellobiohydrolase I is a Talaromyces emersonii cellobiohydrolase I comprising the amino acids of SEQ ID NO:23.
4. The thermotolerant yeast host cell of claim 3 , wherein the Talaromyces emersonii cellobiohydrolase I is fused to a cellulose binding module (CBM).
5. The thermotolerant yeast host cell of claim 4 , wherein the CBM is the CBM of Trichoderma reesei Cbh1 comprising amino acids 503 to 535 of SEQ ID NO:27 or Humicola grisea Cbh1 comprising amino acids 492 to 525 of SEQ ID NO: 21.
6. The thermotolerant yeast host cell of claim 1 , wherein the cellobiohydrolase 11 is a Chrysoporium lucknowense cellobiohydrolase 11 comprising the amino acids of SEQ ID NO:25.
7. The thermotolerant yeast host cell of claim 1 , wherein the endoglucanase is an Humicola grisea, Thermoascus aurantiacus, Taalaromyces emersonii, Trichoderma reesei, Coptotermes lacteus, Coptotermes formosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis, Mastotermes darwinensis, Nasutitermes walkeri, Saccharomycopsis fibuligera, Chrysosporium lucknowense, Reticulitermes speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Chaetomium thermophilum, Aspergillus terreus, Neurospora Crassa, Reticulitermes flavipes , or Arabidopsis thaliana endoglucanase.
8. The thermotolerant yeast host cell of claim 1 , further comprising a heterologous polynucleotide comprising a nucleic acid which encodes a second endoglucanase.
9. The thermotolerant yeast host cell of claim 1 , wherein the β-glucosidase is a Saccharomycopsis fibuligera β-glucosidase comprising the amino acids of SEQ ID NO:40, the cellobiohydrolase I is a Taalaromyces emersonii cellobiohydrolase I comprising the amino acids of SEQ ID NO:23, and the cellobiohydrolase II is a Chrysosporium lucknowense cellobiohydrolase II comprising the amino acids of SEQ ID NO 25.
10. The thermotolerant yeast host cell of claim 9 , further comprising a second heterologous polynucleotide comprising a nucleic acid which encodes a second endoglucanase, and wherein the endoglucanase and the second endoglucanase are selected from the group consisting of Humicola grisea, Thermoascus aurantiacus, Taalaromyces emersonii, Trichoderma reesei, Coptotermes lacteus, Coptotermes formosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis, Mastotermes darwinensis, Nasutitermes walkeri, Saccharomycopsis fibuligera, Chrysosporium lucknowense, Reticulitermes speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Neosartorya fischeri, Chaetomium thermophilum, Aspergillus terreus, Neurospora Crassa, Reticulitermes flavipes , or Arabidopsis thaliana endoglucanases.
11. The thermotolerant yeast host cell of claim 1 , wherein the host cell is a Kluveromyces host cell.
12. The thermotolerant yeast host cell of claim 1 , wherein the host cell is a Saccharomyces cerevisiae host cell.
13. The thermotolerant yeast host cell of claim 1 , wherein the host cell is capable of fermenting pentose sugars.
14. The thermotolerant yeast host cell of claim 13 , wherein the host cell is capable of fermenting xylose.
15. The thermotolerant yeast host cell of claim 14 , wherein the host cell expresses xylose isomerase, xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase, transaldolase or a combination thereof.
16. The thermotolerant yeast host cell of claim 15 , wherein the host cell expresses xylose isomerase, xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase.
17. The thermotolerant yeast host cell of claim 13 , wherein the host cell does not express the GRE3 gene encoding aldose reductase.
18. A method of fermenting cellulose comprising culturing the host cell of claim 1 in medium that contains cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose.
19. A culture comprising the host cell of claim 1 and cellulose.
20. The thermotolerant yeast host cell of claim 1 , wherein the endoglucanase is an Aspergillus kawachii, Heterodera schachtii, Hypocrea jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Chaetomium globosum , or Aspergillus fumigatus endoglucanase.
21. The thermotolerant yeast host cell of claim 9 , further comprising a second heterologous polynucleotide comprising a nucleic acid which encodes a second endoglucanase, and wherein the endoglucanase and the second endoglucanase are selected from the group consisting of Aspergillus kawachii, Heterodera schachtii, Hypocrea jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Chaetomium globosum , and Aspergillus fumigatus endoglucanase.
22. The thermotolerant yeast host cell of claim 1 , wherein the endoglucanase is a Neosartorya fischeri endoglucanase comprising the amino acids of SEQ ID NO:37.
23. The thermotolerant yeast host cell of claim 1 , wherein the thermotolerant yeast host cell can grow at temperatures of at least 38° C. or above.
24. The thermotolerant yeast host cell of claim 1 , wherein the thermotolerant yeast host cell can grow at temperatures of at least 40° C. or above.
25. The thermotolerant yeast host cell of claim 1 , wherein the thermotolerant yeast host cell can grow at temperature from 40° C. to 50° C.
26. The thermotolerant yeast host cell of claim 9 , wherein the Talaromyces emersonii cellobiohydrolase I is fused to a cellulose binding module (CBM).
27. A thermotolerant yeast host cell comprising
(a) at least one heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase;
(b) at least one heterologous polynucleotide comprising a nucleic acid which encodes a β-glucosidase;
(c) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase I; and
(d) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase II;
wherein the thermotolerant yeast host cell can crow at a temperature of at least 35° C. or above,
wherein the cellobiohydrolase I, the cellobiohydrolase I, or both is fused to a cellulose binding module (CBM); and
wherein the host cell is capable of producing ethanol from Avicel.