For the production of biopharmaceuticals in E. coli, genome integrated expression systems offer significant advantages. They are released from plasmid-mediated metabolic load and there is no variation in gene dosage of the gene of interest (GOI) during the production process. However, in T7-based genome integrated expression systems, long-term cultivation under production conditions leads to mutation and inactivation of the T7 RNA polymerase (RNAP). This phenomenon can be excluded in systems based on host RNAP specific promoters, as full functionality of the host RNAP is required for cell growth. Aim of this work was to investigate the suitability of two host RNAP dependent promoters in genome integrated expression systems. We applied a promoter engineering strategy that allowed expression control of the model protein GFP by introducing lac operators (lacO) to the constitutive T5N25 and T7A1 promoter sequences. Seven promoter/operator combinations with the model proteins GFP and an antibody Fab’ fragment were investigated regarding expression strength, tunability, basal expression and cell growth in plasmid-based and plasmid-free BL21 expression systems. The resulting set of production clones was cultivated and compared under fed-batch like conditions in micro-titer fermentations and for detailed characterization lab-scale fed-batch cultivations were conducted. We showed that in genome-integrated expression systems the number of lacO sequences must be well balanced. Three and two lacOs exhibit low basal expression but result in a complete stop in recombinant protein production in partially induced cultures. Whereas expression systems regulated by a sole lacO and lacIQ on the chromosome exhibit very low basal expression, are highly efficient in recombinant protein production and are tunable on a cellular level. We hypothesize that this phenomenon is associated with the autoregulation of the lac repressor (LacI). The affinity of LacI to the lacOs of the GOI must be lower than the affinity of LacI to the lacOs of the endogenous lac operon. Otherwise, LacI autoregulation cannot take place, which leads to disturbance of lac repressor-mediated regulation of transcription.

Bioprocess control: Deeper insights into LacI autoregulation and how to exploit it

2019 Spring National Meeting

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