NSERC-DFG SUSTAIN: SysDevOx - Systematic development of new oxidative biocatalysts for the sustainable production of pharmaceutical compounds

虽然许多药物仍然来自天然产物，但药物制造过程通常需要合成修饰，称为半合成，以将自然界中发现的代谢物转化为临床相关药物。生物系统在温和条件下可持续地产生代谢物，而半合成通常使用有毒化学品、昂贵的催化剂和/或苛刻的不可持续条件。酶可以为复杂天然产物的选择性修饰提供可持续的替代方案。氧化酶如细胞色素P450单加氧酶在这方面是特别相关的，因为它们可以活化否则不反应的C-H键。微生物细胞色素P450单加氧酶已经用于化学酶促合成。然而，迄今为止，植物细胞色素P450单加氧酶的生物技术潜力被严重忽视。这与细胞色素P450单加氧酶在植物代谢中起关键作用的事实形成对比，因此通过进化来氧化植物代谢物。通过这个项目，我们希望缩小这一差距，并开发植物细胞色素P450单加氧酶作为氧化生物催化剂，用于特权生物碱药效团的可持续功能化。具体来说，我们将进行有针对性的筛选活动，以确定植物细胞色素P450单加氧酶，这些酶可以氧化已经用作药物或具有高药物开发潜力的植物生物碱。使用一种新的序列挖掘方法，结合序列相似性网络和正交群推理分析，我们将合理地选择一个CA库。200个细胞色素P450单加氧酶，将针对一组30种市售植物生物碱进行筛选。通过在植物宿主本氏烟草中同时瞬时共表达多达25个细胞色素P450单加氧酶基因，然后去复制来实现所需的通量。将在面包酵母中验证阳性命中。10-100 mg规模的大规模生物转化将证明新发现的生物催化剂与生物技术应用相关，并能够对氧化产物进行全面的结构表征。德国（Franke，Leibniz大学汉诺威）和加拿大（Dang，不列颠哥伦比亚省大学）之间的跨学科合作项目建立在互补的专业知识（Franke：序列相似性网络，结构解析; Dang：生物碱生物合成，氧化酶，正群推理分析）上。总之，本项目开发的新型生物催化剂将促进过渡到可持续生产过程中的药用相关生物碱衍生物。

Although many drugs are still derived from natural products, drug manufacturing processes commonly require synthetic modifications, called semisynthesis, to convert metabolites found in nature into clinically relevant drugs. Whereas biological systems produce metabolites sustainably under mild conditions, semisynthesis often employs toxic chemicals, expensive catalysts and/or harsh non-sustainable conditions. Enzymes could provide a sustainable alternative for selective modifications of complex natural products. Oxidative enzymes such as cytochrome P450 monooxygenases are particularly relevant in this regard, as they can activate otherwise unreactive C-H bonds. Microbial cytochrome P450 monooxygenases have already been employed for chemoenzymatic syntheses. The biotechnological potential of cytochrome P450 monooxygenases from plants, however, has been drastically neglected so far. This stands in contrast to the fact that cytochrome P450 monooxygenases play key roles in plant metabolism and are therefore primed by evolution to oxidize plant metabolites. With this project, we want to close this gap and develop plant cytochrome P450 monooxygenases as oxidative biocatalysts for the sustainable functionalization of privileged alkaloid pharmacophores. Specifically, we will perform a targeted screening campaign to identify plant cytochrome P450 monooxygenases that oxidize plant alkaloids which are either already used as drugs or show high potential for drug development. Using a new sequence mining approach with sequence similarity networks and orthogroup inference analyses, we will rationally select a library of ca. 200 cytochrome P450 monooxygenases that will be screened against a panel of 30 commercially available plant alkaloids. The required throughput is achieved by transient co-expression of up to 25 cytochrome P450 monooxygenase genes simultaneously in the plant host Nicotiana benthamiana followed by dereplication. Positive hits will be verified in baker’s yeast. Large scale biotransformations in the 10-100 mg scale will demonstrate that the newly found biocatalysts are relevant for biotechnological applications and enable full structural characterization of oxidized products. This interdisciplinary collaborative project between a German (Franke, Leibniz University Hannover) and a Canadian group (Dang, University of British Columbia) is built on complementary expertise (Franke: sequence similarity networks, structure elucidation; Dang: alkaloid biosynthesis, oxidative enzymes, orthogroup inference analyses). Taken together, the novel biocatalysts developed in this project will facilitate the transition to sustainable production processes for medicinally relevant alkaloid derivatives.