Salinosporamide Biosynthesis and Engineering

Salinosporamide A是一种有效的抗癌药物，于2006年5月进入1期人体临床试验， 在从专性海洋细菌中发现多发性骨髓瘤仅三年后， 热带盐孢菌这种新的海洋天然产物具有密集官能化的γ-内酰胺-β-内酯 药效团负责其与20 S蛋白酶体的不可逆结合，20 S蛋白酶体是一种新的药物靶点 在癌症生物学中得到验证。尽管它的临床前景和其新颖的化学结构， 这种发酵药物是如何自然产生的阐明了生物合成途径， Salinosporamide将提供许多机会来影响Salinosporamide A在商业上的应用。 长期生产，并随时获得用于SAR的新的发酵化学变体 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一些 发现可能具有生物催化剂应用价值的新生物合成过程的难得机会。 建立在坚实的初步数据基础上，我们已经测序了5.2 Mbp的S。 并通过基因组挖掘、诱变、 和蛋白质表达，我们建议在这个新的赠款申请，1）阐明生物合成的两个 新的生物合成结构单元氯乙基丙二酰-CoA和对羟基环己烯基丙氨酸，2） 表征盐孢菌酰胺合成酶，一种前所未有的杂合聚酮酶-肽 用于Y-内酰胺-β-内酯组装合成酶，3）新的基因工程和生物学评价 salinosporamide类似物，和4）将salinosporamide抗性20 S蛋白酶体p-单元表征为 蛋白酶体耐药模型，用于未来第二代药物的开发。

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.