Recombinant protein expression in proteome-reduced cells under aerobic and oxygen-limited regimes.

Industrial cultures are hindered by the physiological complexity of the host and the limited mass transfer capacity of conventional bioreactors. In this study, a minimal cell approach was combined with genetic devices to overcome such issues. A flavin mononucleotide-based fluorescent protein (FbFP) was expressed in a proteome-reduced Escherichia coli (PR). When FbFP was expressed from a constitutive protein generator (CPG), the PR strain produced 47% and 35% more FbFP than its wild type (WT), in aerobic or oxygen-limited regimes, respectively. Metabolic and expression models predicted more efficient biomass formation at higher fluxes to FbFP, in agreement with these results. A microaerobic protein generator (MPG) and a microaerobic transcriptional cascade (MTC) were designed to induce FbFP expression upon oxygen depletion. The FbFP fluorescence using the MTC in the PR strain was 9% higher than that of the WT bearing the CPG under oxygen limitation. To further improve the PR strain, the pyruvate dehydrogenase complex regulator gene was deleted, and the Vitreoscilla hemoglobin was expressed. Compared to oxygen-limited cultures of the WT, the engineered strains increased the FbFP expression more than 50% using the MTC. Therefore, the designed expression systems can be a valuable alternative for industrial cultivations.

Model-Based Characterization of E. coli Strains with Impaired Glucose Uptake.

The bacterium Escherichia coli is a widely used organism in biotechnology. For high space-time yields, glucose-limited fed-batch technology is the industry standard; this is because an overflow metabolism of acetate occurs at high glucose concentrations. As an interesting alternative, various strains with limited glucose uptake have been developed. However, these have not yet been characterized under process conditions. To demonstrate the efficiency of our previously developed high-throughput robotic platform, in the present work, we characterized three different exemplary E. coli knockout (KO) strains with limited glucose uptake capacities at three different scales (microtiter plates, 10 mL bioreactor system and 100 mL bioreactor system) under excess glucose conditions with different initial glucose concentrations. The extensive measurements of growth behavior, substrate consumption, respiration, and overflow metabolism were then used to determine the appropriate growth parameters using a mechanistic mathematical model, which allowed for a comprehensive comparative analysis of the strains. The analysis was performed coherently with these different reactor configurations and the results could be successfully transferred from one platform to another. Single and double KO mutants showed reduced specific rates for substrate uptake qSmax and acetate production qApmax; meanwhile, higher glucose concentrations had adverse effects on the biomass yield coefficient YXSem. Additional parameters compared to previous studies for the oxygen uptake rate and carbon dioxide production rate indicated differences in the specific oxygen uptake rate qOmax. This study is an example of how automated robotic equipment, together with mathematical model-based approaches, can be successfully used to characterize strains and obtain comprehensive information more quickly, with a trade-off between throughput and analytical capacity.