Method for reducing misincorporation of non-canonical branched-chain amino acids

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.