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. A method of expressing a heterologous protein, comprising:
culturing an isolated fungus in the genus Aspergillus under conditions that permit the fungus to express a heterologous protein; thereby expressing the heterologous protein,
wherein:
the isolated fungus comprises a polynucleotide comprising a glucoamylase promoter operably linked to a heterologous protein coding sequence, and
the isolated fungus comprises a genetic inactivation of an mstC gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2.
2. The method of claim 1 , wherein the isolated fungus in the genus Aspergillus is Aspergillus niger ( A. niger ).
3. The method of claim 1 , wherein the mstC gene is genetically inactivated by nonsynonymous mutation or by insertional mutation.
4. The method of claim 1 , wherein:
the mstC gene comprises a nucleic acid molecule comprising a nucleotide sequence having at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 3, or nucleotides 82 to 1764 of SEQ ID NO: 1.
5. The method of claim 1 , wherein:
the mstC gene encodes a protein comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.
6. The method of claim 1 , wherein the heterologous protein is a heterologous enzyme, or a chimeric protein.
7. The method of claim 6 , wherein expression of the heterologous enzyme is increased by at least 2-fold as compared to expression of the heterologous enzyme in a fungus comprising a native mstC gene.
8. A method of degrading a biomass comprising cellulose, comprising:
incubating the biomass with an isolated fungus in the genus Aspergillus under conditions that permit the fungus to express a heterologous cellulolytic enzyme, thereby degrading the biomass, wherein:
the isolated fungus comprises a polynucleotide comprising a glucoamylase promoter operably linked to a heterologous cellulolytic enzyme coding sequence, and
the isolated fungus comprises a genetic inactivation of an mstC gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2.
9. The method of claim 8 , further comprising incubating the biomass with ionic liquids.
10. A method of expressing a heterologous protein, comprising:
culturing an isolated fungus in the genus Neurospora under conditions that permit the fungus to express a heterologous protein; thereby expressing the heterologous protein,
wherein:
the isolated fungus comprises a polynucleotide comprising a glucoamylase promoter operably linked to a heterologous protein coding sequence, and
the isolated fungus comprises a genetic inactivation of an NCU01633 gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11.
11. The method of claim 10 , wherein the isolated fungus in the genus Neurospora is Neurospora crassa ( N. crassa ).
12. The method of claim 10 , wherein the NCU01633 gene is genetically inactivated by nonsynonymous mutation or by insertional mutation.
13. The method of claim 10 , wherein the NCU01633 gene comprises at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, 10, or nucleotides 652 to 2250 of SEQ ID NO: 10.
14. The method of claim 10 , wherein the NCU01633 gene encodes a NCU01633 protein comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 11.
15. The method of claim 10 , wherein the heterologous protein is a heterologous enzyme, or a chimeric protein.
16. The method of claim 15 , wherein expression of the heterologous enzyme is increased by at least 2-fold as compared to expression of the heterologous enzyme in a fungus comprising a native NCU01633 gene.
17. A method of degrading a biomass comprising cellulose, comprising:
incubating the biomass with an isolated fungus in the genus Neurospora under conditions that permit the fungus to express a heterologous cellulolytic enzyme, thereby degrading the biomass, wherein:
the isolated fungus comprises a polynucleotide comprising a glucoamylase promoter operably linked to a heterologous cellulolytic enzyme coding sequence, and
the isolated fungus comprises a genetic inactivation of an NCU01633 gene encoding a polypeptide comprising monosaccharide transporter activity and an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11.
18. The method of claim 17 , further comprising incubating the biomass with ionic liquids.