Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
Department of Drug Discovery, Moffitt Cancer Center & Research Institute, Stabile Research Building, 12902 Magnolia Dr, Tampa, FL, 33612, USA.
Sci Rep. 2021 Mar 5;11(1):5290. doi: 10.1038/s41598-021-84859-6.
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.
信号肽和分泌载体蛋白通常用于在革兰氏阴性菌中分泌异源重组蛋白。大肠杆菌渗透压诱导蛋白 Y（OsmY）是一种载体蛋白，可将靶蛋白分泌到细胞外，我们之前已将其应用于细菌胞外蛋白分泌系统（BENNY）来加速定向进化。在这项研究中，我们首次在载体蛋白上应用随机和组合诱变来提高总分泌目标蛋白的产量。经过一轮随机诱变并结合发现的突变后，确定 OsmY（M3）（L6P、V43A、S154R、V191E）为最佳载体蛋白。与野生型相比，OsmY（M3）在大肠杆菌 BL21（DE3）和 C41（DE3）菌株中分别使 Tfu0937β-葡萄糖苷酶的分泌量增加了 3.1±0.3 倍和 2.9±0.8 倍。与野生型相比，OsmY（M3）在不同培养温度（37°C、30°C 和 25°C）下也产生了更多的 Tfu0937 分泌物。表达蛋白的亚细胞分级分离证实了 OsmY 在蛋白分泌中的重要作用。培养 15 小时后，总可溶性蛋白中有 80.8±12.2%被分泌。当与红色荧光蛋白或枯草芽孢杆菌脂肪酶融合时，与野生型相比，OsmY（M3）也产生了更多的分泌蛋白。在这项研究中，与野生型相比，OsmY（M3）变体提高了三种来源不同且具有不同性质的蛋白质的细胞外产量，这清楚地表明了其广泛的应用。本工作中在载体蛋白上进行的随机和组合诱变的使用也可以进一步扩展到用于在大肠杆菌中生产分泌蛋白的其他信号肽或载体蛋白。
