Directed evolution for improved total secretory protein production in Escherichia coli

Production of secretory protein in Gram-negative bacteria simplifies downstream processing in recombinant protein production, accelerates protein engineering, and advances synthetic biology. Signal peptides and secretory carrier proteins are commonly used to effect the secretion of heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have successfully applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate the directed evolution workflow. In this study, we applied directed evolution on OsmY to enhance its total secretory protein production. After just one round of directed evolution followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C). Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis , OsmY(M3) also produced more secretory protein compared to the wildtype. This is the first report of applying directed evolution on a carrier protein to enhance total secretory protein production. The methodology can be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli and other bacteria. In this study, OsmY(M3) improved the production of three proteins, originating from diverse organisms and with diverse properties, in secreted form, clearly demonstrating its wide-ranging applications. ### Competing Interest Statement The authors have declared no competing interest. * dATP : deoxyadenosine triphosphate dCTP : deoxycytidine triphosphate dGTP : deoxyguanosine triphosphate dNTP : deoxynucleotide dTTP : deoxythymidine triphosphate DyP4 : dye-decolorizing peroxidase 4 from Pleurotus ostreatus strain PC15 Em : emission wavelength E. coli : Escherichia coli Ex : excitation wavelength epPCR : error-prone polymerase chain reaction HTS : high-throughput screening Mw : molecular weight pNPA : p -nitrophenyl acetate pNPG : p -nitrophenyl-β-D-glucopyranoside OD600 : optical density at 600 nm OsmY : osmotically-inducible protein Y PCR : polymerase chain reaction RFP : red fluorescent protein WT : wildtype