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Main Title: Molecular genetic approaches to decrease the uncontrolled misincorporation of non-canonical branched chain amino acids into recombinant mini-proinsulin expressed in Escherichia coli
Author(s): García, Ángel Córcoles
Hauptmann, Peter
Neubauer, Peter
Type: Article
Abstract: 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.
Subject(s): non-canonical branched chain amino acids
genetic engineering
strain screening
Issue Date: 4-Mar-2022
Date Available: 25-May-2022
Language Code: en
DDC Class: 570 Biowissenschaften; Biologie
Sponsor/Funder: TU Berlin, Open-Access-Mittel – 2022
EC/H2020/643056/EU/Rapid Bioprocess Development/Biorapid
Journal Title: Microbial Cell Factories
Publisher: Springer Nature
Volume: 21
Article Number: 30
Publisher DOI: 10.1186/s12934-022-01756-x
EISSN: 1475-2859
TU Affiliation(s): Fak. 3 Prozesswissenschaften » Inst. Biotechnologie » FG Bioverfahrenstechnik
Appears in Collections:Technische Universität Berlin » Publications

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