Restructuring of the periplasmic space for the efficient production of recombinant proteins in a reduced-genome Escherichia coli

Abstract

Escherichia coli periplasm has a suitable environment for the production of proteins with essential disulfide bonds. The periplasm, however, has not been widely employed for the production of recombinant proteins because of its limited space and secretion capacity, degradation of recombinant proteins by proteases, and aggregation of proteins overexpressed in the periplasm. To overcome these problems, we have restructured E. coli periplasm by removing unnecessary genes encoding periplasmic proteins that interfere the expression of heterologous proteins in the periplasm. Forty six out of 170 periplasmic genes (27% of total periplasmic genes) were deleted from the E. coli MG1655 genome by a rapid markerless deletion system. With the periplasm-restructured E. coli, expression of alkaline phosphatase and human leptin was examined. In the restructured periplasm, a twofold increase in the production of the alkaline phosphatase was obtained and 2.5 times more leptin was produced in a soluble form than that obtained with E. coli MG1655. Comparative proteomics of the periplasm-restructured strain (PRS46) and MG1655 confirmed the targeted deletions, and a total of 10 different periplasmic proteins were identified whose abundance changed significantly between the PRS46 and MG1655: some genes (aphA, glpQ) were overexpressed by more than 6-fold in the PRS46 and others (xylF, araF) were repressed 2-fold. Interestingly, several periplasmic proteins (Dcp, TolB, PstS, CysP, GltI, LptA) were newly induced. These identified periplasmic proteins were involved in nutrient transports, central intermediary metabolisms, and cell envelope formation. This periplasm-restructured E. coli will be beneficial for the production of a wide range of recombinant proteins through increasing the protein productivity and solubility.