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

尽管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，将自然界中发现的代谢物转化为临床相关的药物。虽然生物系统在温和的条件下可持续地产生代谢物，但半合成通常使用有毒化学品、昂贵的催化剂和/或严酷的不可持续条件。酶可以为复杂天然产物的选择性修饰提供一种可持续的替代方案。细胞色素P450单加氧酶等氧化酶在这方面特别相关，因为它们可以激活原本不活跃的C-H键。微生物细胞色素P450单加氧酶已被用于化学酶的合成。然而，迄今为止，植物细胞色素P450单加氧酶的生物技术潜力一直被严重忽视。这与细胞色素P450单加氧酶在植物新陈代谢中发挥关键作用的事实相反，因此细胞色素P450单加氧酶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。具体地说，我们将进行有针对性的筛选活动，以确定氧化植物生物碱的植物细胞色素P450单加氧酶，这些植物生物碱要么已经用作药物，要么显示出很高的药物开发潜力。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约200个细胞色素P450单加氧酶的文库，该文库将与一组30种商业可用植物生物碱进行筛选。所需的吞吐量是通过瞬时共表达多达25个细胞色素P450单加氧酶基因在植物宿主本氏烟草中同时表达，然后去复制来实现的。阳性结果将在面包师酵母中得到验证。在10-100毫克范围内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(法兰克，汉诺威莱布尼茨大学)和加拿大(DANG，不列颠哥伦比亚省大学)之间的这一跨学科合作项目建立在互补专业知识的基础上(法兰克：序列相似性网络，结构说明；DANG：生物碱生物合成，氧化酶，正交群推理分析)。综上所述，该项目中开发的新型生物催化剂将促进向医学相关生物碱衍生物的可持续生产工艺的过渡。

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.