Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain.
Commun Biol. 2021 May 12;4(1):562. doi: 10.1038/s42003-021-02076-3.
Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once-enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.
真菌非特异性过氧化物酶（UPO）是一类能够在广泛的底物范围内催化多功能氧化反应的酶。它们作为分泌的、糖基化的蛋白质存在，具有血红素硫醇酯活性位点，并依赖过氧化氢作为氧源。然而，它们在适合高通量筛选的快速生长的生物体中的异源生产仅成功过一次——这得益于密集的定向进化运动。我们开发并应用了一种基于模块化 Golden Gate 的分泌系统，首次在酵母中生产了四种活性 UPO，并对其进行了一步纯化，并在制备规模上进行了对映选择性转化。Golden Gate 设计为通用型，由三种模块类型组成：i）用于分泌的信号肽，ii）UPO 基因，和 iii）用于纯化和 GFP 分裂检测的蛋白质标签。模块化的附加型系统适用于酿酒酵母，并已转移到巴斯德毕赤酵母的附加型和染色体整合表达载体中。巴斯德毕赤酵母的摇瓶生产可产生高达 24mg/L 的分泌 UPO 酶，该酶可用于制备规模转化苯乙胺衍生物，达到 98.6%ee。我们的结果表明，UPO 的快速、模块化酵母分泌工作流程可产生制备规模的对映选择性生物转化。
