In a recently published study, we developed a simple methodology to monitor Escherichia coli cell integrity and lysis during bioreactor cultivations, where we intentionally triggered leakiness. In this follow-up study, we used this methodology, comprising the measurement of extracellular alkaline phosphatase to monitor leakiness and flow cytometry to follow viability, to investigate the effect of process parameters on a recombinant E. coli strain producing the highly valuable vascular endothelial growth factor A165 (VEGF-A165) in the periplasm. Since the amount of soluble product was very little (<500 μg/g dry cell weight), we directly linked the effect of the three process parameters temperature, specific uptake rate of the inducer arabinose and specific growth rate (μ) to cell integrity and viability. We found that a low temperature and a high μ were beneficial for cell integrity and that an elevated temperature resulted in reduced viability. We concluded that the recombinant E. coli cells producing VEGF-A165 in the periplasm should be cultivated at low temperature and high μ to reduce leakiness and guarantee high viability. Summarizing, in this follow-up study we demonstrate the usefulness of our simple methodology to monitor leakiness and viability of recombinant E. coli cells during bioreactor cultivations.
Keywords: Alkaline phosphatase; Flow cytometry; Monitoring; Outer membrane integrity; VEGF‐A165.
© 2016 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.