Salinosporamide Biosynthesis and Engineering

盐孢酰胺A是一种有效的抗癌剂，于2006年5月进入第一阶段人体临床试验，用于 从专性海洋细菌中发现多发性骨髓瘤仅三年后的治疗 热带盐生孢属。这种新型海洋天然产物具有浓密的官能化Y-内酰胺-对内酯 负责其与新药靶标20S蛋白酶体的不可逆结合的药效团 在癌症生物学方面得到了验证。尽管它在临床上很有希望，而且它的化学结构也很新颖，但还没有报道 关于这种发酵药物是如何自然产生的。生物合成途径的阐明 盐孢酰胺类药物将提供许多机会来影响盐孢子酰胺A的商业化 长期生产，并提供容易获得的新的发酵为基础的化学变种用于SAR 通过合理的代谢工程进行研究。此外，其独特的化学结构提供了许多 发现新的生物合成过程的难得机会，这些过程可能作为生物催化剂具有应用价值。 建立在坚实的初步数据基础上，我们已经对S。 Tropica，并通过基因组挖掘、诱变等方法鉴定了盐孢酰胺生物合成基因簇。 和蛋白质的表达，我们建议在这项新的拨款申请中：1)阐明两者的生物合成 新型生物合成基团氯乙基丙二酸辅酶A和对羟基环己烯丙氨酸，2) 一种史无前例的混合型聚酮合成酶--盐孢子胺合成酶的特性 Y-内酰胺-对内酯组装的合成酶，3)基因工程和生物学评价 盐孢子胺类似物，以及4)将抗盐孢子胺的20S蛋白酶体p-单位表征为 第二代药物未来发展的蛋白酶体耐药模型。

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 y-lactam-p-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 p-hydroxycyclohexenylalanine, 2) characterize the salinosporamide synthetase, an unprecedented hybrid polyketide synthase-peptide synthetase for Y-lactam-p-lactoneassembly, 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-generationdrugs.