Influence of expression vector design on the simultaneous heterologous production of cytochromes and rusticyanin from Acidithiobacillus ferrooxidans in Escherichia coli and Vibrio natriegens

Heterologous protein production is often employed to accelerate protein characterization and interaction studies. This can be especially relevant for proteins from acidophiles since these microorganisms only produce low biomass. However, the production of proteins from acidophiles in neutrophilic hosts, e.g. Escherichia coli, often poses a great challenge. Correct protein folding and co-factor integration need to be achieved despite the difference in extracellular and periplasmic pH. The Gram-negative acidophile Acidithiobacillus ferrooxidans possesses the remarkable ability to transfer electrons through its outer membrane to exocellular iron. This requires an electron transfer chain that spans both membranes and the periplasm and consists of three c-type cytochromes and the copper redox protein rusticyanin. We aim to investigate the functionality of this exocellular electron transfer chain and reconstructed it in several E. coli expression strains and Vibrio natriegens Vmax X2. We previously reported that E. coli and V. natriegens were able to express the genes from a pET16b-based vector construct and translocated all proteins to the correct cell compartment. Recent experiments focused on the impact of the expression vector design on the production of our four proteins of interest. The genes were encoded on a pRSF-1b vector, significantly reducing plasmid size compared to the pET16b-based construct. Additionally, we investigated a pCOLADuet-1-based construct where the inner membrane c-type cytochrome Cyc2 is under the control of a separate T7 promoter. The use of the pCOLADuet-1-based construct improved the production of Cyc2 in V. natriegens, while the viability of the E. coli hosts was reduced. This indicates that V. natriegens might be an interesting chassis for the production of integral membrane proteins. Additionally, V. natriegens possess a distinct advantage over E. coli for the production of c-type cytochromes. It does not require the co-expression of the cytochrome c maturation genes from an additional plasmid under aerobic conditions.