Salinosporamide Biosynthesis and Engineering

描述（由申请人提供）：Salinosporamide A是一种有效的抗癌药物，在从专性海洋细菌Salinispora tropica中发现仅三年后，于2006年5月进入治疗多发性骨髓瘤的一期人体临床试验。这种新型的海洋天然产物具有密集功能化的γ -内酰胺- β -内酯药效团，负责与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的药物靶点。尽管它具有临床前景和新颖的化学结构，但没有关于这种发酵药物如何自然产生的报道。阐明盐孢酰胺的生物合成途径将为长期影响盐孢酰胺a的商业化生产提供许多机会，并通过合理的代谢工程为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工艺提供了许多难得的机会，这些工艺可能作为生物催化剂具有应用价值。我们已经对热带葡萄球菌的5.2 Mbp基因组进行了测序，并通过基因组挖掘、诱变和蛋白质表达确定了盐孢酰胺生物合成基因簇，在此基础上，我们提出了新的资助申请：1)阐明两种新型生物合成构建块氯乙基丙二酰辅酶a和β -羟基环己烯丙氨酸的生物合成；2)表征盐孢酰胺合成酶；3)对新的盐孢酰胺类似物进行基因工程和生物学评价；4)对盐孢酰胺耐药20S蛋白酶体p单元进行表征，为未来第二代药物的开发提供蛋白酶体耐药模型。

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.