Salinosporamide Biosynthesis and Engineering

描述（由申请人提供）：Salinosporamide A是一种有效的不可逆蛋白酶体抑制剂，目前处于治疗多发性骨髓瘤和其他癌症的Ib期人体临床试验中。这种海洋细菌天然产物具有独特的作用机制，基于其-内酰胺-？-内酯药效团，不同于fda唯一批准的蛋白酶体抑制剂，肽硼酸硼替佐米。在最后的支持期间，我们建立了盐孢酰胺组装的生物合成基础，并在卤化，预苯生物化学和聚酮前体供应中发现了一些新的酶促反应。这些基础知识的翻译使我们能够通过基因工程合理设计新的盐孢酰胺类似物进行生物学评价。这项工作有助于确定盐孢酰胺家族抗癌药物的结构-活性关系。尽管迄今为止我们取得了重大进展，但由于其前所未有的新型分子构建块组装，我们对盐孢酰胺的生物合成方式仍然只有粗略的了解。尽管这个正在进行的研究项目的发现带来了新的机遇，但仍有许多问题有待解决。酶发现、合成生物学、化学酶合成、基因组挖掘和蛋白酶体生物化学等领域的机会都非常适合这个天然产物生物合成项目。为了实现本申请中概述的广泛目标，我们提出了一个涉及五个具体目标的多学科项目。首先，我们计划对SalC进行功能和结构表征

DESCRIPTION (provided by applicant): Salinosporamide A is a potent irreversible proteasome inhibitor presently in phase Ib human clinical trials for the treatment of multiple myeloma and other cancers. This marine bacterial natural product has a distinctive mechanism of action based on its ?-lactam-?-lactone pharmacophore that differs from the only FDA-approved proteasome inhibitor, the peptide boronate bortezomib. During the last period of support, we established the biosynthetic foundation of salinosporamide assembly and discovered a number of novel enzymatic reactions in halogenation, prephenate biochemistry, and polyketide precursor supply. Translation of this basic knowledge allowed us to rationally design through genetic engineering new salinosporamide analogues for biological evaluation. This work helped determine the structure-activity relationships within the salinosporamide family of anticancer agents. Despite our significant progress to date, we still only have a cursory understanding of how salinosporamide is biosynthesized due to its unprecedented assembly from novel molecular building blocks. Numerous questions remain, while new opportunities have surfaced in response to discoveries made in this ongoing research program. Opportunities in enzyme discovery, synthetic biology, chemoenzymatic synthesis, genome mining, and proteasome biochemistry are uniquely suited for this natural product biosynthetic program. To accomplish the broad goals outlined in this application, we propose a multidisciplinary project involving five specific aims. First, we plan to functionally and structurally characterize the SalC ketosynthase and its key biosynthetic role in the formation of the ?-lactam-?-lactone core of salinosporamide. Second, we will apply the function of SalC to develop a streamlined chemoenzymatic synthesis of salinosporamide derivatives based on a focused library of ?-lactam-?-lactones from synthetic acylamino acid thioesters with recombinant salinosporamide biosynthetic enzymes. Third, we aim to functionally characterize the biosynthetic enzymes responsible for the synthesis of salinosporamide's novel amino acid residue, cyclohexenylalanine, which is paramount to its potent proteasome binding affinity. Fourth, we will functionally characterize the dedicated proteasome ?-subunit SalI and its hypothesized role in S. tropica self-resistance against salinosporamide. And fifth, we plan to develop new crotonyl-CoA reductase-based expression systems for the engineered biosynthesis of new polyketide synthase extender units with halogenated (fluorine and chlorine) and branched side chains for the design of new polyketide molecules.