Salinosporamide Biosynthesis and Engineering

描述(申请人提供)：盐孢酰胺A是一种有效的抗癌药物，在从热带盐孢杆菌专属海洋细菌中发现仅三年后，于2006年5月进入第一阶段人类临床试验，用于治疗多发性骨髓瘤。这种新型海洋天然产物具有密集功能化的伽马-内酰胺-β-内酯药效团，负责其与20S蛋白酶体的不可逆结合，20S蛋白酶体是癌症生物学中验证的新药靶点。尽管它的临床前景和它新颖的化学结构，但还没有关于这种发酵药物是如何自然产生的报道。盐孢子酰胺生物合成途径的阐明将为影响盐孢子胺A的长期商业生产提供大量机会，并为通过合理的代谢工程进行SAR研究提供新的基于发酵的化学变体。此外，其独特的化学结构为发现新的生物合成过程提供了许多难得的机会，这些过程可能作为生物催化剂具有应用价值。在坚实的初步数据基础上，我们已经对热带葡萄球菌5.2Mbp的基因组进行了测序，并通过基因组挖掘、突变和蛋白质表达鉴定了盐孢酰胺生物合成基因簇，在这项新的拨款申请中，我们建议1)阐明两个新的生物合成构件氯乙基丙二酰辅酶A和β-羟基环己基丙氨酸的生物合成，2)表征盐孢酰胺合成酶，这是一种前所未有的用于伽马-内酰胺-β-内酯组装的混合型聚酮合成酶-肽合成酶，3)对新的盐孢子胺类似物进行基因工程和生物评价，以及4)表征耐盐孢子胺的20S蛋白体蛋白酶体单位，作为未来第二代药物开发的蛋白质组耐药模型。

DESCRIPTION (provided by applicant): Salinosporamide A is a potent anticancer agent that entered phase 1 human clinical trials in May 2006 for the treatment of multiple myeloma only three years after its discovery from the obligate marine bacterium Salinispora tropica. This novel marine natural product possesses a densely functionalized gamma-lactam-beta-lactone pharmacophore that is responsible for its irreversible binding to the 20S proteasome, a new drug target validated in cancer biology. Despite its clinical promise and its novel chemical structure, there are no reports on how this fermented drug is naturally created. The elucidation of the biosynthetic pathway to the salinosporamides will provide a number of opportunities to impact how salinosporamide A is commercially produced in the long-term and to afford ready access to new fermentation-based chemical variants for SAR studies through rational metabolic engineering. In addition, its unique chemical structure provides a number of rare opportunities to discover new biosynthetic processes that may have applied value as biocatalysts. Building upon a solid preliminary data foundation in which we have sequenced the 5.2 Mbp genome of S. tropica and identified the salinosporamide biosynthetic gene cluster through genome mining, mutagenesis, and protein expression, we propose in this new grant application to 1) elucidate the biosynthesis of the two novel biosynthetic building blocks chloroethylmalonyl-CoA and beta-hydroxycyclohexenylalanine, 2) characterize the salinosporamide synthetase, an unprecedented hybrid polyketide synthase-peptide synthetase for gamma-lactam-beta-lactone assembly, 3) genetically engineer and biologically evaluate new salinosporamide analogs, and 4) characterize the salinosporamide resistant 20S proteasome p-unit as a model for proteasome resistance for the future development of second-generation drugs.