Molecular genetic approaches to decrease the uncontrolled misincorporation of non-canonical branched chain amino...

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.