| Autor: |
Heinz Himmelbauer, Juliane C. Dohm, Monika Cserjan-Puschmann, Gerald Striedner, Martin Wagenknecht, Reingard Grabherr, Christoph Köppl, Matteo Schiavinato, Artur Schuller |
| Rok vydání: |
2020 |
| Předmět: |
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| DOI: |
10.22541/au.159769219.92858284 |
| 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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