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1期人体临床试验，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γ-内酰胺-β-内酯药效团，该药效团负责其与20 S蛋白酶体的不可逆结合，20 S蛋白酶体是一种在癌症生物学中验证的新药靶点。尽管其临床前景和新颖的化学结构，但没有关于这种发酵药物是如何自然产生的报道。阐明盐孢菌酰胺的生物合成途径将为影响盐孢菌酰胺A的长期商业生产提供许多机会，并通过合理的代谢工程为SAR研究提供随时获得新的基于发酵的化学变体。此外，其独特的化学结构提供了许多难得的机会来发现新的生物合成过程，这些过程可能具有作为生物催化剂的应用价值。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。tropica中，并通过基因组挖掘、诱变和蛋白质表达鉴定了盐孢菌酰胺生物合成基因簇，我们在该新的授权申请中提出1）阐明两种新的生物合成结构单元氯乙基丙二酰-CoA和β-羟基环己烯基丙氨酸的生物合成，2）表征盐孢菌酰胺合成酶，一种前所未有的用于γ-内酰胺-β-内酯组装的杂合聚酮脱氢酶-肽合成酶，3）基因工程和生物学评价新的盐孢菌酰胺类似物，和4）表征盐孢菌酰胺抗性20 S蛋白酶体p-单元作为蛋白酶体抗性的模型，用于第二代药物的未来开发。

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.