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The uncontrolled incorporation of non-canonical branched chain amino acids (ncBCAAs) such as norleucine, norvaline and β-methylnorleucine into recombinant proteins in  E. coli  production processes is a crucial problem in the pharmaceutical industry, since it can lead to the production of altered proteins with non-optimal characteristics. Despite various solutions, to date there are no engineered strains that exhibit a reduced accumulation of these ncBAAs. In this study, novel  E. coli  K-12 BW25113 strains with exogenous tunable expression of target genes of the BCAA biosynthetic pathway were developed. For this purpose, single gene knock-outs for  thrA ,  ilvA ,  leuA ,  ilvIH ,  ilvBN ,  ilvGM  and  ilvC  were complemented with plasmids containing the respective genes under control of an arabinose promoter. These clones were screened in a mL-bioreactor system in fed-batch mode under both standard cultivation conditions and with pyruvate pulses, and induction of a min-proinsulin. Screening was performed by comparing the impurity profile of the recombinant mini-proinsulin expressed of each clone with the  E. coli  BW25113 WT strain, and the most promising clones were cultivated in a 15L Screening showed that up-regulation of  ilvC ,  ilvIH  and  ilvGM,  and downregulation of  leuA  and  ilvBN  trigger a reduction of norvaline and norleucine accumulation and misincorporation into mini-proinsulin. The stirred tank bioreactor cultivations confirmed that up-regulation of  ilvIH  and  ilvGM  were most effective to reduce the ncBCAA misincorporation. This novel approach for a reduced ncBCAA misincorporation may be solution to this old challenging problem in the large-scale production of human therapeutics.

microbial cell factories

Molecular genetic approaches to decrease the uncontrolled misincorporation of non-canonical branched chain amino acids into recombinant mini-proinsulin expressed in Escherichia coli