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. 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 are investigated. Iterated fed‐batch‐like microbioreactor cultivations are conducted under production conditions. The easy‐to‐produce green fluorescent protein (GFP) and a challenging antigen‐binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole‐genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. 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 remains stable in the production of GFP in long‐term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives have positive effects on long‐term stability. The 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.

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield

“…Construction of the reference strain BL21(DE3) expressing GFPmut3.1 from a single copy integrated into the genome is described elsewhere. 92 Since sfGFP exhibits fluorescence regardless of cellular localization, it was used as periplasmic reference protein. The co-translational dsbA SS was used to mitigate cytosolic fluorescence.…”

“…GFP was quantified using a Tecan analyzer infinite 200Pro (485/520 nm) and a calibration curve constructed with in-house-purified GFP as previously described. 92 …”

“…The reference genome of E. coli BL21(DE3) used for mapping of the RNA-seq reads had been previously determined in-house by whole genome sequencing. 92 The mapping was conducted with HISAT2 98 using the following adapted parameters: --score-min L,0.0,-0.2 --no-spliced-alignment --no-softclip. The remaining parameters of the program were left as default.…”

“…We used production systems with a single copy of the gene of interest integrated into the bacterial chromosome at the attTn7 site. Construction of the reference strain BL21­(DE3) expressing GFPmut3.1 from a single copy integrated into the genome is described elsewhere . Since sfGFP exhibits fluorescence regardless of cellular localization, it was used as periplasmic reference protein.…”

“…GFP was quantified using a Tecan analyzer infinite 200Pro (485/520 nm) and a calibration curve constructed with in-house-purified GFP as previously described. 92 Flow Cytometric Superoxide Measurement. Intracellular superoxide levels were measured flow cytometrically using CellROX Green reagent (Invitrogen, USA).…”