Background

Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15.

Result

After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in k_cat/K_m) and higher H₂O₂ tolerance (sevenfold improvement based on IC₅₀).

Conclusion

To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.

中文翻译：

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使用细菌细胞外蛋白分泌系统 (BENNY) 加速染料脱色过氧化物酶的定向进化。

背景

染料脱色过氧化物酶 (DyPs) 是一种含血红素的过氧化物酶，在工业生物催化和木质纤维素降解中显示出巨大的前景。通过使用大肠杆菌渗透诱导蛋白 Y (OsmY) 作为细菌细胞外蛋白分泌系统 (BENNY)，我们成功开发了一种简化的定向进化工作流程，以加速平菇菌株 PC15 中 DyP4 的蛋白质工程。

结果

经过 3 轮带有易错聚合酶链反应 (epPCR) 的随机诱变和 1 轮饱和诱变后，我们获得了 4D4 变体（I56V、K109R、N227S 和 N312S），它显示出多种理想的表型，包括更高的蛋白质产量和分泌，更高的比活性（ k _cat / K _m提高 2.7 倍）和更高的 H ₂ O ₂耐受性（基于 IC ₅₀提高七倍）。

结论

据我们所知，这是应用 OsmY 来简化定向进化工作流程和指导血红素蛋白（如 DyP4）的细胞外分泌的第一份报告。