Alkaloids synthesis via enzymatic cascades

四氢异喹啉生物碱（Tetrahydroisoquinoline alkaloids，THIA）是一类结构和功能多样的植物代谢产物，具有重要的治疗活性，如抗菌的小檗碱、镇痛的吗啡和抗精神病的阿朴啡。一些THIA可以从植物中提取，但收率低，化学全合成存在多步反应和低选择性。THIA的新的立体选择性途径受到高度追捧，因为它们是特权药效团。我们最近开创了一种无细胞酶级联方法，我们知道每种酶的确切性质，因此有一个通用的合成系统，使已知的和新的THIA。开发用于级联反应的关键酶包括将L-酪氨酸转化为多巴胺的酪氨酸酶和脱羧酶、可以合成醛的转氨酶和Pictet-Spenglerase去甲乌药碱合酶（NCS）。此外，我们还有其他酶可用于合成THIA前体或修饰产物，如甲基转移酶。我们在这个博士学位的目标是开发NCS酶用于更广泛的苯乙胺和使用定制酶后THIA形成小分子生成，通过可扩展的过程。研究将首先集中在开发NCS反应与一系列苯乙胺探测接受的底物以及NCS诱变，基于我们目前的机制理解。计算机模拟将是指导底物设计，合成和测试与NCS突变体至关重要。我们还将为THIA多样化开发植物P450，以获得生物碱和非天然类似物。然后将使用选定的酶来构建酶途径，以建立有效的可持续级联到新的/天然的单一异构体生物碱。酶系统将在体外的可扩展性进行优化，我们将研究两种或三种酶在同一质粒和固定化酶支持物上的共表达。总的来说，最终目标是设计酶和途径，以更广泛地使用，包括可扩展的过程，以这个非常重要的生物活性生物碱家族。

Tetrahydroisoquinoline alkaloids (THIAs) are a structurally and functionally diverse group of plant metabolites with significant therapeutic activities, such as the anti-bacterial berberine, analgesic morphine and anti-psychotic aporphine. Some THIAs can be extracted from plants which is low yielding and chemical total synthesis suffers from multistep procedures and low reaction selectivity. New stereoselective routes to THIAs are highly sought after as they are privileged pharmacophores. We have recently pioneered a cell free enzyme cascade approach where we know the exact properties of each enzyme and thus have a versatile synthetic system to make known and novel THIAs. Key enzymes developed for use in the cascades include tyrosinases and decarboxylases that convert L-tyrosine into dopamine, transaminases that can synthesise aldehydes and the Pictet-Spenglerase norcoclaurine synthase (NCS). In addition, we have other enzymes that can be used in the synthesis of THIA precursors or modify products such as methyl transferases. Our aim in this PhD is to develop the NCS enzymes for use with a wider range of phenethylamines and the use of tailoring enzymes after THIA formation for small molecule generation, via scalable processes.Studies will initially focus on developing the NCS reaction with a range of phenethylamines to probe the substrates accepted as well as NCS mutagenesis, based on our current mechanistic understanding. In silico modelling will be essential for guiding substrate design, synthesis and testing with NCS mutants. We will also develop plant P450s for THIA diversification to access alkaloids and un-natural analogues. Selected enzymes will then be used to construct enzyme pathways to establish efficient sustainable cascades to novel/natural single isomer alkaloids. Enzyme systems will be optimised for scalability in vitro, and we will investigate the co-expression of two or three enzymes on the same plasmid and immobilised enzyme supports. Overall, the end goal is to engineer enzymes and pathways for wider use including scalable processes to this very important family of bioactive alkaloids.