Adaptive evolution in producing microtiter cultivations generates genetically stable Escherichia coli production hosts for continuous bioprocessing

Abstract

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 BL21 Q 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 BL21 Q expression system remained stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21 Q 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.

Online estimation of changing metabolic capacities in continuous Corynebacterium glutamicum cultivations growing on a complex sugar mixture

“…Thereby, process intensification in comparison with regular batch or fed‐batch operations is feasible. Applications of continuous microbial bioprocesses are versatile, ranging from large scale ethanol production (Sahm & Bringer‐Meyer, 1987 ) to recent developments for recombinant protein production (Kittler et al, 2020 ; Schuller et al, 2020 ). Despite technological advantages such as reduced culture volumes and potential for continuous downstream integration (Gagnon et al, 2018 ), physiological instabilities still hamper the implementation of robust continuous processes (Kopp et al, 2019 ).…”

“…During continuous processes, selection pressure on the culture introduces an additional element of complexity. Besides of process instabilities caused by the metabolic burden of recombinant gene expression (Hoffmann & Rinas, 2004 ; Neubauer et al, 2003 ; Schuller et al, 2020 ), the selection pressure in continuous operation can also lead to favorable biological adaptions (Wortel et al, 2016 ). Phenotype changes are caused by genetic (Arensdorf et al, 2002 ; Jansen et al, 2005 ; Wenger et al, 2011 ) and nongenetic (Herwig & von Stockar, 2002 ; Wright et al, 2020 ) adaptions and their regulatory implications have to be considered (Sato et al, 2016 ).…”