Gene targets for improved enzyme production in fungi
Fungi that are genetically inactivated for the mstC gene (or a homolog thereof) are provided, which can also be genetically modified to increase production of heterologous proteins from a glucoamylase promoter. Methods of using these fungi, for example to degrade a biomass, are also provided.
1. An isolated Aspergillus fungus, comprising:
a genetic inactivation of a mstC gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, and
a polynucleotide comprising a glucoamylase promoter operably linked to a heterologous protein coding sequence encoding a heterologous protein,
wherein said heterologous protein coding sequence is heterologous to the isolated Aspergillus fungus, and wherein said heterologous protein coding sequence is heterologous or homologous to the glucoamylase promoter.
2. The isolated Aspergillus fungus of claim 1 , wherein the isolated fungus is Aspergillus niger ( A. niger ).
3. The isolated Aspergillus fungus of claim 1 , wherein the mstC gene is genetically inactivated by nonsynonymous mutation or by insertional mutation.
4. The isolated Aspergillus fungus of claim 1 , wherein the mstC gene prior to genetic inactivation comprises a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or nucleotides 82 to 1764 of SEQ ID NO: 1.
5. The isolated Aspergillus fungus of claim 1 , wherein the mstC gene prior to genetic inactivation encodes a protein comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence SEQ ID NO: 2.
6. The isolated Aspergillus fungus of claim 1 , wherein the heterologous protein is a heterologous enzyme, or a chimeric protein.
7. The isolated Aspergillus fungus of claim 6 , wherein the heterologous enzyme is a beta-glucosidase (BG), a glycoside hydrolase, a carbohydrate esterase, a protease, a lipase, a liginase, a cellulase, or a hemicellulase.
8. A method of expressing a heterologous protein, comprising:
culturing the isolated Aspergillus fungus of claim 1 under conditions that permit the fungus to express the heterologous protein; thereby expressing the heterologous protein.
9. The method of claim 8 , wherein the isolated Aspergillus fungus is Aspergillus niger ( A. niger ).
10. The method of claim 8 , wherein the mstC gene is genetically inactivated by nonsynonymous mutation or by insertional mutation.
11. The method of claim 8 , wherein the mstC gene prior to genetic inactivation comprises a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or nucleotides 82 to 1764 of SEQ ID NO: 1.
12. The method of claim 8 , wherein the mstC gene prior to genetic inactivation encodes a protein comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence SEQ ID NO: 2.
13. The method of claim 8 , wherein the heterologous protein is a heterologous enzyme, or a chimeric protein.
14. The method of claim 13 , wherein expression of the heterologous enzyme is increased by at least 2-fold as compared to expression of the heterologous enzyme in a corresponding Aspergillus fungus comprising a native mstC gene that has not been genetically inactivated.
15. A method of degrading a biomass comprising cellulose, comprising:
incubating the biomass with the isolated Aspergillus fungus of claim 1 under conditions that permit the Aspergillus fungus to express the heterologous protein from the glucoamylase promoter, thereby degrading the biomass,
wherein the heterologous protein is a cellulolytic enzyme.
16. The method of claim 15 , further comprising incubating the biomass with ionic liquids.
17. A method of increasing glucoamylase production in an Aspergillus fungus, comprising:
culturing the isolated Aspergillus fungus of claim 1 under conditions that permit the Aspergillus fungus to express native glucoamylase; thereby increasing production of glucoamylase relative to a corresponding Aspergillus fungus comprising a native mstC gene that has not been genetically inactivated.
18. The isolated Aspergillus fungus of claim 4 , wherein the mstC gene prior to genetic inactivation comprises a nucleic acid molecule comprising a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or nucleotides 82 to 1764 of SEQ ID NO: 1.
19. The isolated Aspergillus fungus of claim 4 , wherein the mstC gene prior to genetic inactivation comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or nucleotides 82 to 1764 of SEQ ID NO: 1.
20. The isolated Aspergillus fungus of claim 5 , wherein the mstC gene prior to genetic inactivation encodes a protein comprising an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.
21. The isolated Aspergillus fungus of claim 5 , wherein the mstC gene prior to genetic inactivation encodes a protein comprising the amino acid sequence of SEQ ID NO: 2.
22. An isolated Neurospora fungus, comprising:
a genetic inactivation of a NCU01633 gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11, and
a polynucleotide comprising a glucoamylase promoter operably linked to a heterologous protein coding sequence encoding a heterologous protein,
wherein said heterologous protein coding sequence is heterologous to the isolated Neurospora fungus, and wherein said heterologous protein coding sequence is heterologous to or homologous to the glucoamylase promoter.
23. The isolated Neurospora fungus of claim 22 , wherein the Neurospora fungus is Neurospora crassa ( N. crassa ).
24. The isolated Neurospora fungus of claim 22 , wherein the NCU01633 gene is genetically inactivated by nonsynonymous mutation or by insertional mutation.
25. The isolated Neurospora fungus of claim 22 , wherein the NCU01633 gene prior to genetic inactivation comprises a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, or nucleotides 652 to 2250 of SEQ ID NO: 10.
26. The isolated Neurospora fungus of claim 22 , wherein the NCU01633 gene prior to genetic inactivation comprises a nucleic acid molecule comprising the nucleotide sequence of a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10, or nucleotides 652 to 2250 of SEQ ID NO: 10.
27. The isolated Neurospora fungus of claim 22 , wherein the NCU01633 gene prior to genetic inactivation encodes a protein comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 11.
28. The isolated Neurospora fungus of claim 22 , wherein the NCU01633 gene prior to genetic inactivation encodes a protein comprising the amino acid sequence of SEQ ID NO: 11.
29. The isolated Neurospora fungus of claim 22 , wherein the heterologous protein is a beta-glucosidase (BG), a glycoside hydrolase, a carbohydrate esterase, a protease, a lipase, a liginase, a cellulase, or a hemicellulase.
30. A method of expressing a heterologous protein, comprising:
culturing the isolated Neurospora fungus of claim 22 under conditions that permit the fungus to express the heterologous protein; thereby expressing the heterologous protein.
31. A method of degrading a biomass comprising cellulose, comprising:
incubating the biomass with the isolated Neurospora fungus of claim 22 under conditions that permit the Neurospora fungus to express the heterologous protein from the glucoamylase promoter, thereby degrading the biomass,
wherein the heterologous protein is a cellulolytic enzyme.
32. A method of increasing glucoamylase production in an Neurospora fungus, comprising:
culturing the isolated Neurospora fungus of claim 22 under conditions that permit the Neurospora fungus to express native glucoamylase; thereby increasing production of glucoamylase relative to an Neurospora fungus comprising a native NCU01633 gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11.