Enhanced citric acid production in aspergillus with inactivated asparagine-linked glycosylation protein 3 (ALG3), and/or increased laeA expression
Provided herein are 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 (Lae), or both. 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 provided, as are compositions and kits including the disclosed fungi.
1. A method of making citric acid, comprising culturing an isolated Aspergillus species fungus transformed with at least one Aspergillus LaeA (a loss of aflR expression A) gene under conditions that permit the fungus to make citric acid, thereby making citric acid, wherein said LaeA gene encodes a protein having at least 80% sequence identity to the polypeptide of SEQ ID NO: 41 or SEQ ID NO: 59.
2. The method of claim 1 , wherein the Aspergillus LaeA gene encodes a protein comprising SEQ ID NO: 41 or SEQ ID NO: 59.
3. The method of claim 1 , wherein the fungus has been transformed with at least one Aspergillus LaeA gene, wherein said LaeA gene has at least 80% sequence identity to nucleotides 1-236 and 367-1252 of SEQ ID NO: 40, or to nucleotides 1-230 and 373-1267 of SEQ ID NO: 58.
4. The method of claim 3 , wherein the Aspergillus LaeA gene comprises nucleotides 1-236 and 367-1252 of SEQ ID NO: 40, or nucleotides 1-230 and 373-1267 of SEQ ID NO: 58.
5. The method of claim 1 , wherein the fungus further comprises a genetic inactivation of an endogenous dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (Alg3) gene, wherein the Alg3 gene is genetically inactivated by complete or partial deletion mutation or by an insertional mutation, wherein the endogenous Alg3 gene prior to the genetic inactivation encodes a protein having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
6. The method of claim 5 , wherein prior to genetic inactivation, the Alg3 gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
7. The method of claim 1 , wherein the fungus further comprises a genetic inactivation of an endogenous Alg3 gene, wherein the Alg3 gene is genetically inactivated by complete or partial deletion mutation or by insertional mutation, wherein the endogenous Alg3 gene prior to the genetic inactivation has at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
8. The method of claim 7 , wherein prior to genetic inactivation, the Alg3 gene comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
9. The method of claim 1 , wherein the Aspergillus species is Aspergillus niger ( A. niger ).
10. The method of claim 9 , wherein the A. niger is A. niger strain American Type Culture Collection (ATCC) 11414 or A. niger strain 11414KusA.
11. The method of claim 1 , wherein the fungus is cultured in media comprising at least 50 g/1 sugar and 8 to 15 ppb manganese.
12. The method of claim 1 , further comprising isolating the citric acid made by culturing the fungus.