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Ib期人体临床试验中，用于治疗多发性骨髓瘤和其他癌症。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，不同于唯一的FDA批准的蛋白酶体抑制剂，肽硼酸硼替佐米。在支持的最后一段时间，我们建立了salinosporamide组装的生物合成基础，并发现了一些新的酶促反应的卤化，预苯酸生物化学，和聚酮前体供应。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抗癌药物salinosporamide家族内的结构-活性关系。尽管我们迄今为止取得了重大进展，但由于salinosporamide是由新型分子构建块前所未有地组装而成的，因此我们对salinosporamide是如何生物合成的仍然只有粗略的了解。许多问题仍然存在，而新的机会已经浮出水面，以回应在这个正在进行的研究计划中所取得的发现。在酶的发现，合成生物学，化学酶合成，基因组挖掘和蛋白酶体生物化学的机会是唯一适合这个天然产物生物合成计划。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，我们计划在功能和结构上表征SalC 酮合酶和它的关键生物合成作用的形成？内酰胺-？- Salinosporamide内酯核心。第二，我们将应用SalC的功能，开发一种基于？-内酰胺-？-用重组盐孢菌酰胺生物合成酶从合成的酰基氨基酸硫酯合成内酯。第三，我们的目标是功能特性的生物合成酶负责合成salinosporamide的新的氨基酸残基，环己烯基丙氨酸，这是至关重要的，其有效的蛋白酶体结合亲和力。第四，我们将功能特性的专用蛋白酶体？- SalI亚基及其在S.热带植物对Salinosporamide的自身抗性。第五，我们计划开发新的基于巴豆酰辅酶A还原酶的表达系统，用于设计新的聚酮化合物分子，设计具有卤代（氟和氯）和支链侧链的新聚酮化合物合酶扩展单元的工程生物合成。

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.