SCALING UP E. COLI FROM THE LAB TO INDUSTRIAL CONDITIONS: LESSONS LEARNED TO ENGINEER ROBUST PROCESSES AND PRODUCTION HOSTS

摘要

For commercialization, strain and bioprocess developments need to be successfully transferred from the lab to industrial scale. Often, this step crucially decides about economic feasibility and survival of the approach. Accordingly, profound understanding of impact factors that hamper the successful scale-up is key, either to create novel microbial production platforms with enhanced robustness or to improve bioreactor design targeting minimized impact on cellular performance. Using an experimental scale-up simulator consisting of a stirred tank reactor (STR) and a plug flow reactor (PFR) Escherichia coli was exposed to typical large-scale mixing conditions in continuous experiments. Installing mixing times of about 110 seconds and simulating fluctuating availability of carbon and nitrogen sources, short-term responses revealed the repeated on/off switching of about 600 genes (Loeffler et al.,Metab Eng 2016; Simen et al. Microbial Biotechnol 2017). Dynamics of gene expression and protein formation were modelled using an agent-based approach and simulating large-scale conditions (Niess et al. Frontiers Microbiol., 2017). ATP balancing of gene expression and protein formation showed that maintenance demands increased by -50%. Thereof, strategies for genome reduction were deduced. Large-scale simulation revealed the dominating role of the alarmone ppGpp which triggers the on/off-switching of the stringent response. Accordingly, a novel chassis was engineered such that intracellular ppGpp levels were no more affected thereby disconnecting the extracellular stimulus from the intracellular response, even under nitrogen or carbon limitation. Experimental studies outline the energetic advantages of stringent response deficient production hosts. Additionally, changes were implemented in central metabolism finally yielding E. coli HGT (high glucose throughput, Michalowski et al., Metab Eng 2017). The patent-filed strain offers about 10 fold risen glucose uptake rates (relative to maintenance demands under glucose limitation) under resting conditions which is beneficial for large-scale production processes.