Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Appl Microbiol Biotechnol. 2024 May 6;108(1):320. doi: 10.1007/s00253-024-13166-7.
The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.
糙皮侧耳（Cyclocybe aegerita）中的非特异性过氧化物酶（UPO）（AaeUPO）可以在没有辅助因子的情况下使用过氧化氢作为氧供体选择性地氧化 C-H 键，这引起了工业界的极大关注。许多研究都致力于通过采用酶定向进化、利用合适的启动子和筛选分泌肽等策略来提高在毕赤酵母中表达的 AaeUPO 的整体活性。在这些先前研究的基础上，本研究的目的是通过增加基因拷贝数、共表达分子伴侣和优化培养条件，进一步提高具有改进活性的突变体 AaeUPO（PaDa-I）的表达。我们的结果表明，携带大约三个表达盒拷贝数的菌株，并共表达蛋白质二硫键异构酶，使用 2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）作为底物时，酶活体积增加了约 10.7 倍。在优化培养条件后，该菌株的酶活体积进一步增加了约 48.7%，达到 117.3 U/mL。此外，PaDa-I 的催化结构域对 R-2-苯氧丙酸具有区域选择性羟化作用。本研究的结果可能有助于 UPO 的工业应用。 关键点： • 通过增加基因拷贝数和共表达蛋白质二硫键异构酶，可以显著提高 PaDa-I 的催化结构域的分泌量。 • 优化培养条件后，使用 2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）作为底物时，酶活体积可达 117.3 U/mL。 • PaDa-I 的催化结构域可以催化 R-2-苯氧丙酸发生特异性羟化反应，生成 R-2-（4-羟苯氧）丙酸。
