Improving the expression of secretory and membrane proteins in Escherichia coli through folding pathway engineering

Escherichia coli is one of the most widely used organisms for protein expression due to well-understood genetics, a rapid doubling time, and an ability to grow on inexpensive substrates. Despite the wide array of genetic tools available for protein expression in E. coli, the yield of medically and technologically relevant secretory proteins and membrane proteins (MPs) is typically low due to cellular toxicity and protein aggregation and/or degradation. The goal of this work is to facilitate the high-level production of these proteins by engineering the folding pathways responsible for protein export, membrane insertion and folding. In E. coli, MPs and certain secretory proteins are targeted to the inner membrane via the signal recognition particle (SRP) dependent pathway. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes both the highly hydrophobic signal sequences of SRP dependent secretory proteins and the hydrophobic transmembrane segments of MPs as they emerge from the ribosome. SRP then delivers ribosome nascent chain complexes to the FtsY receptor for cotranslational translocation of secretory proteins across the SecYEG pore or insertion and folding of MPs in the lipid bilayer in a process that may involve the molecular chaperone YidC. Here we show that the yields of secretory proteins and MPs can be improved by up to 3-fold by inactivating TF in the expression host, and that MP productivity can be further improved by increasing the cellular levels of YidC. These large gains in productivity are due to a reduction in toxicity and an improvement in protein export across or into the membrane on a per cell basis. We further describe the isolation of a regulatory mutant of the arabinose-inducible P BAD promoter that improves the growth and productivity of MP-producing cells twofold through a 65% decrease in transcription rates. This approach to streamline MP trafficking to the inner membrane could be combined with TF deficient expression hosts to achieve a more than 4-fold improvement in active MP expression and yields as high as 20 mg/L of culture.