Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing
Autor: | Gerald Striedner, Martin Wagenknecht, Matteo Schiavinato, Monika Cserjan-Puschmann, Reingard Grabherr, Christoph Köppl, Heinz Himmelbauer, Artur Schuller, Juliane C. Dohm |
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Rok vydání: | 2020 |
Předmět: |
0106 biological sciences
Mutant Green Fluorescent Proteins Biology Lac repressor medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology law.invention chemistry.chemical_compound law 010608 biotechnology RNA polymerase Gene expression medicine Escherichia coli T7 RNA polymerase Promoter Regions Genetic Gene 010401 analytical chemistry General Medicine Recombinant Proteins 0104 chemical sciences chemistry Biochemistry Recombinant DNA Molecular Medicine medicine.drug |
Zdroj: | Biotechnology journal. 16(3) |
ISSN: | 1860-7314 |
Popis: | The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. We investigated the stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations. We conducted iterated fed-batch-like microbioreactor cultivations under production conditions. We used the easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole-genome sequencing analyses, we identified mutations that allowed cells to grow unhindered despite recombinant protein production. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21Q expression system remained stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives had positive effects on long-term stability. Our results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial relevant production conditions is an efficient strain development tool for production hosts. This article is protected by copyright. All rights reserved. |
Databáze: | OpenAIRE |
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