Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

Abstract

Signal peptides and secretory carrier proteins are commonly used to secrete 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 previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis 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) compared to the wildtype. 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. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

Figure 1

The OsmY(WT)-Tfu0937 activity in the spent medium was measured with the pNPG assay using an absorbance at 405 nm. (a) The pNPG assay was performed in a 96-well microplate for OsmY(WT)-Tfu0937-expressing C41(DE3) cells cultivated in a 96-well deep well plates. The coefficient of variance was calculated using the absolute values measured at 405 nm. (b) The pNPG assay was conducted using 5–50 µL of spent medium. The activity value is linear to the volume of spent medium used with an R² = 0.992.

Figure 2

The OsmY-Tfu0937 activity in the spent medium of epPCR generated OsmY mutagenesis libraries was measured in a 96-well microplate. The activity was measured with the pNPG assay using an absorbance at 405 nm. The libraries were generated using (a) a low and (b) a high mutagenic rates. The activities of parental clones in column 6 were measured in the same 96-well microplate. The average of parental clones is shown as a solid red line and one standard deviation above and below the average are shown as dash red lines.

Figure 3

Characterization of improved OsmY variants in 50-mL flask cultures. The OsmY-Tfu0937 activity, measured with the pNPG assay using an absorbance at 405 nm (left axis), are plotted as bars. The cell optical densities monitored at 600 nm (right axis) are shown as symbols. Cultures were inoculated using 1:100 dilution. (a) OsmY(WT) and variants were expressed using C41(DE3) in 2 × TY auto-induction media and at 37 °C and 200 rpm. The bars and symbols of the variants are represented using different colours; OsmY(WT) (black), OsmY(V43A) (blue), OsmY(S19C) (orange) and OsmY(TOA4) (red). Experiments were performed in triplicate. (b) OsmY(WT)-Tfu0937 and OsmY(TOA4)-Tfu0937 were expressed using C41(DE3) in 2 × TY auto-induction media, at 30 °C or 25 °C, and 200 rpm. The bars and symbols of the sample are represented using different colours; OsmY(WT) at 30 °C (black), OsmY(TOA4) at 30 °C (red), OsmY(WT) at 25 °C (grey), OsmY(TOA4) at 25 °C (magenta).

Figure 4

The OsmY-Tfu0937 activity in the spent medium of single site-directed mutants was measured with the pNPG assay using an absorbance at 405 nm. Cells were cultivated in 20-mL of 2 × TY auto-induction media at 37 °C. Culture was inoculated using 1:100 dilution and 0.5 mL culture was sampled at various time points for activity assay. Experiments were performed in triplicate. The samples are represented using different colours; OsmY(WT)—black line, OsmY(L6P)—blue line, OsmY(S154R)—orange line and OsmY(V191E)—red line.

Figure 5

Subcellular localization of Tfu0937 and OsmY(M3)-Tfu0937. Proteins were expressed using pET24a-Tfu0937 and pET24a-OsmY(M3)-Tfu0937 in 2 × TY auto-induction medium at 37 °C for 15 h. The fractional Tfu0937 and OsmY(M3)-Tfu0937 activity in the extracellular, periplasmic and cytoplasmic fractions were measured with the pNPG assay using an absorbance at 405 nm. The enzyme activity in each fraction was calculated as a percentage of total activity measured in all three fractions. Experiments were performed in triplicate.

Figure 6

Fractional OsmY-Tfu0937 activity in the extracellular, periplasmic and cytoplasmic fractions of cells expressing Tfu0937 fused to either OsmY(WT) or OsmY(M3). Activity was measured with the pNPG assay using an absorbance at 405 nm. Experiments were performed in triplicate. Cells were cultivated in 2 × TY auto-induction medium at 37 °C for either 6 h or 24 h. The enzyme activity in each fraction was calculated as a percentage of total activity in all three fractions.

Figure 7

Protein secretion using OsmY(WT) and OsmY(M3) in (a) E. coli C41 (DE3) and (b) BL21(DE3). OsmY(WT)-Tfu0937 and OsmY(M3)-Tfu0937 were expressed using C41(DE3) and BL21(DE3) in 50-mL 2 × TY auto-induction media and at 37 °C and 250 rpm. Cultures were inoculated using 1:200 dilution. The cell optical densities monitored at 600 nm (right axis) are shown as lines. The activity of OsmY-Tfu0937 shown as bars was measured with the pNPG assay using an absorbance at 405 nm (left axis). The lines and bars of different variants are represented using different colours; OsmY(WT)—black, OsmY(TOA4)—blue and OsmY(M3)—red.

Figure 8

OsmY-Tf0937 secretion using C41(DE3) and BL21(DE3) in 400-mL 2 × TY auto-induction media, at 37 °C and 200 rpm. Cultures were inoculated using 1:100 dilution and samples were collected at 7 h and 24 h of cultivation. (a) SDS-PAGE of spent medium. Lane L: protein marker, Lane 1 and 5: OsmY(WT)-Tfu0937 in BL21(DE3), Lane 2 and 6: OsmY(M3)-Tfu0937 in BL21(DE3), Lane 3 and 7: OsmY(WT)-Tfu0937 in C41(DE3), Lane 4 and 8: OsmY(M3)-Tfu0937 in C41(DE3). (b) pNPG assay of the spent medium.

Figure 9

OsmY-RFP expression in E. coli BL21(DE3). Cells were cultivated in 20 mL of 2xTY auto-induction media at 37 °C using 1:100 dilution of inoculum. The supernatant and cells from 20 µL culture were used for fluorescence measurement using E_x = 584 nm and E_m = 607 nm. (a) Growth curve of the culture. (b) Fluorescence intensity of the spent medium. (c) Fluorescence intensity of the cells. The lines and bars of different variants are represented using different colours; OsmY(WT)-RFP—grey and OsmY(M3)-RFP—orange.

Figure 10

OsmY-Lipase 6B expressed in BL21(DE3) and C41(DE3). Cells were cultivated in 50 mL of 2xTY auto-induction media at 37 °C using 1:100 dilution of inoculum. The cell optical densities monitored at 600 nm are shown as lines. The activity of OsmY-Lipase6B in the spent medium measured with the pNPA assay using an absorbance at 405 nm are shown as bars. The lines and bars of different samples are represented using different colours; OsmY(WT)-Lipase 6B in C41(DE3)—black, OsmY(M3)-Lipase 6B in C41(DE3)—blue, OsmY(WT)-Lipase 6B in BL21(DE3)—grey and OsmY(M3)-Lipase 6B in BL21(DE3)—orange.