Method for reducing misincorporation of non-canonical branched-chain amino acids
The present invention relates to a method for producing a recombinant polypeptide of interest in a microbial host cell, comprising (a) introducing a polynucleotide encoding the polypeptide of interest into a microbial host cell which has been modified such that an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase activity (EC 4.3.1.19) is modulated in said microbial host cell as compared to the enzymatic activity in an unmodified microbial host cell, and (b) expressing said polypeptide of interest in said microbial host cell. Moreover, the present invention relates to a method for reducing misincorporation of at least one non-canonical branched-chain amino acid into a recombinant polypeptide of interest expressed in a microbial host cell.
1 . A method for producing a recombinant polypeptide of interest in a microbial host cell, the method comprising:
(a) selecting a microbial host cell with genomic DNA having a knock-out of a gene encoding an enzyme selected from the group consisting of acetohydroxyacid synthase (AHAS) isoform III, ketol-acid reductoisomerase (NADP(+)), aspartate kinase, and homoserine dehydrogenase,
(b) modifying said microbial host cell by introducing a first plasmid expressing a polynucleotide encoding said enzyme such that enzymatic activity of said enzyme is increased in said microbial host cell as compared to enzymatic activity of said enzyme in the microbial host cell with genomic DNA having a knock-out of a gene encoding said enzyme,
(c) introducing a second plasmid comprising a polynucleotide encoding the recombinant polypeptide of interest into said microbial host cell of (b),
(d) expressing said recombinant polypeptide of interest in said microbial host cell, and
wherein the recombinant polypeptide of interest expressed by said microbial host cell has lower misincorporation of non-canonical branched-chain amino acids selected from norvaline and norleucine as compared to a same recombinant polypeptide of interest expressed by an otherwise identical microbial host cell that has not been modified with said first plasmid.
2 . The method of claim 1 , wherein the method further comprises isolation of the recombinant polypeptide of interest from the microbial host cell, and purification of the recombinant polypeptide.
3 . The method of claim 2 , wherein the purification comprises enrichment of recombinant polypeptides of interest which do not comprise non-canonical branched-chain amino acids.
4 . The method of claim 1 , wherein the polynucleotide encoding the recombinant polypeptide of interest and/or the polynucleotide encoding the enzyme is operably linked to an inducible promoter.
5 . The method of claim 1 , wherein said microbial host cell is an Escherichia coli cell.
6 . The method of claim 1 , wherein the recombinant polypeptide of interest is an insulin or a proinsulin.
7 . The method of claim 6 , wherein the insulin is insulin lispro, insulin aspart, insulin glulisine, insulin detemir, insulin glargine, or a precursor thereof.