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 (MP... More
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.
