Tailoring Escherichia coli for the l-Rhamnose P_BAD Promoter-Based Production of Membrane and Secretory Proteins

Abstract

Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli l-rhamnose P promoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an l-rhamnose concentration-dependent manner. By monitoring protein production in real-time in E. coli wild-type and an l-rhamnose catabolism deficient mutant, we demonstrate that the l-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to l-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated l-rhamnose transport and l-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an l-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an l-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.

Synthetic negative feedback circuits using engineered small RNAs

“…Mean fluorescence is reduced with increasing concentrations of l -rhamnose, with a 92% reduction in mean output reached by saturation at 0.001 mg/ml l -rhamnose (Figure 3B ). This concentration corresponds with saturating levels of the RhaS-P rhaBAD induction system and agrees with data from strains incapable of l -rhamnose metabolism or when a non-metabolisable inducer of l -rhamnose is used ( 100 , 103 ). This result confirmed the hypothesis that varying the concentration of sRNA in the cell would allow the output of the autorepressor circuit to be varied independently of aTc.…”

Synthetic negative feedback circuits using engineered small RNAs

“…Mean fluorescence is reduced with increasing concentrations of L-rhamnose, with saturation observed at 0.001 mg/ml L-rhamnose ( Figure 3B). This concentration corresponds with saturating levels of the RhaS-P rhaBAD induction system and agrees with data from strains incapable of L-rhamnose metabolism or when a non-metabolisable inducer of L-rhamnose is used (89,92) . This result confirmed the hypothesis that varying the concentration of sRNA in the cell would allow the output of the autorepressor circuit to be varied independently of aTc.…”

Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region

“…In the previous series of experiments a mild induction protocol was used (0.05 mM IPTG for 2 h at 30 °C), so that differences in protein synthesis could be assessed in the absence of a metabolic load on the cell. The concern about metabolic load largely related to the Sec translocon, which is believed to be a bottleneck in the production of periplasmic proteins [31,32]. When production levels of periplasmic proteins are too high, the translocon can become saturated and the recombinant protein may be retained in the cytoplasm.…”