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Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY).
Bioresources and Bioprocessing ( IF 4.3 ) Pub Date : 2019-05-31 , DOI: 10.1186/s40643-019-0255-7
Abdulrahman H A Alessa 1 , Kang Lan Tee 1 , David Gonzalez-Perez 1 , Hossam E M Omar Ali 1 , Caroline A Evans 1 , Alex Trevaskis 1 , Jian-He Xu 2 , Tuck Seng Wong 1
Affiliation  

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 kcat/Km) 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:

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。
更新日期:2019-05-31
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