Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. 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 solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once-enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. 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 workflow from putative UPO gene to preparative scale enantioselective biotransformations.

中文翻译：

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模块化的两种酵母菌种分泌系统，用于真菌过氧合酶的生产和制备

真菌非特异性过氧化酶（UPOs）是令人关注的生物催化剂。使用模块化分泌系统提供对新型UPO的访问是这项工作的主要目标。UPO代表一类酶，可在广泛的底物范围内催化多种氧基官能化反应。它们以分泌的糖基化蛋白的形式出现，带有糖化血红素硫醚活性位点，仅依靠过氧化氢作为氧源。真菌过氧化酶广泛分布在整个真菌界，因此可以使用多种UPO基因序列。但是，在一次快速生长的，适合高通量筛选的生物体中，UPO的异源生产只有通过密集的定向进化运动才能成功实现。在这里，我们开发并应用了基于模块化金门技术的分泌系统，允许首次酵母生产四个活性UPO，它们的一步纯化和制备规模的对映选择性转化应用。Golden Gate装置设计为可广泛应用，由三种模块类型组成：i）指导分泌的信号肽面板，ii）UPO基因，以及iii）用于纯化和GFP分离检测的蛋白质标签。我们显示可以通过组合测试每个UPO基因的17个信号肽来选择最佳的信号肽，以成功的UPO分泌。模块化附加型系统适用于酿酒酵母，并已转移至巴斯德毕赤酵母的附加型和染色体整合表达盒中。在巴斯德毕赤酵母中摇瓶产量最高可分泌24 mg / L的UPO酶，它用于苯乙胺衍生物的制备规模转化达到ee的98.6％。我们的结果证明了从假定的UPO基因到制备规模的对映选择性生物转化的快速工作流程。