Enhanced itaconic acid production in
Fungi, such as Aspergillus niger , having a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (Alg3) gene genetic inactivation, increased expression of a loss of aflR expression A (LaeA), or both, are described. In some examples, such mutants have several phenotypes, including an increased production of citric acid relative to the parental strain. Methods of using the disclosed fungi to make citric acid are also described, as are compositions and kits including the disclosed fungi. Further described are Aspergillus terreus fungi overexpressing the LaeA gene and the use of such fungi for the production of itaconic acid.
1. An isolated Aspergillus terreus fungus transformed with a heterologous nucleic acid construct comprising an Aspergillus species LaeA (loss of aflR expression A) gene, wherein the LaeA gene encodes a LaeA protein comprising an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 59 and the heterologous nucleic acid construct is inserted upstream of the coding region of the endogenous LaeA gene, and wherein expression of the Aspergillus species LaeA gene is increased in the transformed fungus compared to an A. terreus fungus that is not transformed with the heterologous nucleic acid construct.
2. The isolated A. terreus fungus of claim 1 , wherein the heterologous nucleic acid construct further comprises a heterologous promoter, a heterologous transcription terminator, a heterologous selective marker gene, or any combination thereof.
3. The isolated A. terreus fungus of claim 1 , wherein the Aspergillus species LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 41.
4. The isolated A. terreus fungus of claim 1 , wherein the Aspergillus species LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 59.
5. The isolated A. terreus fungus of claim 1 , wherein the heterologous nucleic acid construct comprises a promoter operably linked to the Aspergillus species LaeA gene, a transcription terminator and a selective marker gene.
6. The isolated A. terreus fungus of claim 5 , wherein:
the promoter comprises the A. nidulans gpdA promoter;
the Aspergillus species LaeA gene is an A. nidulans LaeA gene;
the transcription terminator comprises the A. nidulans TrpC transcription terminator; or
the selective marker gene comprises the A. oryzae pyrithiamine resistance (ptrA) gene.
7. The isolated A. terreus fungus of claim 6 , wherein the heterologous nucleic acid construct comprises the nucleotide sequence of SEQ ID NO: 109.
8. A method of making itaconic acid, comprising culturing the isolated A. terreus fungus of claim 1 under conditions that permit the fungus to make itaconic acid, thereby making itaconic acid.
9. The method of claim 8 , wherein the fungus is cultured in itaconic acid production media.
10. The method of claim 8 , wherein the heterologous nucleic acid construct further comprises a heterologous promoter, a heterologous transcription terminator, a heterologous selective maker gene, or any combination thereof.
11. The method of claim 10 , wherein the Aspergillus species LaeA gene in the heterologous nucleic acid construct is an A. nidulans or an A. niger LaeA gene.
12. The method of claim 9 , wherein the Aspergillus species LaeA gene encodes a protein having an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 59.
13. The method of claim 12 , wherein the Aspergillus species LaeA gene encodes a protein having the amino acid sequence of SEQ ID NO: 41 or the amino acid sequence of SEQ ID NO: 59.
14. The method of claim 8 , wherein the heterologous nucleic acid construct comprises a promoter operably linked to the Aspergillus species LaeA gene, a transcription terminator and a selective marker gene.
15. The method of claim 14 , wherein:
the promoter comprises the A. nidulans gpdA promoter;
the Aspergillus species LaeA gene is an A. nidulans or an A. niger LaeA gene;
the transcription terminator comprises the A. nidulans TrpC transcription terminator; or
the selective marker gene comprises the A. oryzae pyrithiamine resistance (ptrA) gene.
16. The method of claim 15 , wherein the heterologous nucleic acid construct comprises the nucleotide sequence of SEQ ID NO: 109.