贯叶金丝桃素中间体的大肠杆菌异源合成

Hyperforin is a polyprenylated acylphloroglucinol derivative from Hypericum perforatum (St. John's wort). It exhibits antidepressant activity by a novel mechanism of action, antibiotic activity against gram-positive bacteria, and antitumoral activity in vivo. This compound has attracted great interests worldwide as a plant natural medicine. Presently, hyperforin is mainly produced by extraction from H. perforutum. However, the concentration is low and the extraction is not facile enough to meet the growing demand. Total synthesis poses many challenges for chemists, and the tedious steps and poor yields make it practically valueless in therapeutics. Biosynthesis of plant natural products from recombinant microorganisms has arisen as a competitive alternative to traditional chemistry-based routes. Nevertheless, the synthesis pathway of hyperforin is not completely clear and there is no successful case of biosynthesis of acylphloroglucinol derivatives in microorganisms yet. To ascertain the metabolic pathway of hyperforin in H. perforutum, and express this heterologous biosynthetic pathway in engineered Escherichia coli for the production of hyperforin, is innovative and economically valuable. In this study, three modules: α-ketoisovalerate module, isobutyryl-CoA module and malonyl-CoA module will be constructed in E.coli, and combinatorial optimization of transcriptional and translational levels of these three modules will lead to high production of isobutyryl-CoA and malonyl-CoA, the two substrates of hyperforin skeleton. We synthesized the E. coli codon-optimized isovalerophenone synthase (VPS) gene. On the other hand, using Digital Gene Expression Profiling, analysis of differentially expressed genes and 3',5'-RACE，we try to clone the key enzyme, isobutyrophenone synthase (BUS) from H. perforutum. Then introduce VPS or BUS into the engineered E.coli, in order to achieve effective heterologous production of hyperforin.

贯叶金丝桃素是贯叶连翘的重要活性成分，具有抗抑郁、抗肿瘤等多种药理活性，是目前国际上研究的热点天然药物。但贯叶金丝桃素在贯叶连翘中的含量很低，提取困难；化学合成步骤繁琐，产率低；因此从基因的水平上解析、组合及优化植物中的合成途径，构建微生物细胞工厂，实现贯叶金丝桃素的异源合成，具有很好的应用前景和经济价值。目前还没有此类藤黄酚类天然产物异源合成的报道。本项目拟通过构建α酮异戊酸、异丁酰辅酶A及丙二酰辅酶A三个功能模块，利用密码子优化、启动子优选、RBS调控等分子生物学手段对各模块进行单独及协同调控，实现高产异丁酰辅酶A及丙二酰辅酶A的大肠杆菌底盘细胞组建。人工合成异戊酰苯合酶；同时利用数字化基因表达谱、差异基因表达分析、3'和5'-RACE等技术进一步从贯叶连翘中克隆关键基因异丁酰苯合酶；组合入大肠杆菌底盘细胞，构建细胞工厂实现贯叶金丝桃高素中间体的高效异源合成。

贯叶金丝桃素是贯叶连翘的重要活性成分，具有抗抑郁、抗肿瘤等多种药理活性，是目前国际上研究的热点天然药物。但贯叶金丝桃素在贯叶连翘中的含量很低，提取困难；化学合成步骤繁琐，产率低；因此构建微生物细胞工厂，实现贯叶金丝桃素的异源合成，具有很好的应用前景和经济价值。目前还没有此类藤黄酚类天然产物异源合成的报道。本项目首先构建了利用支链氨基酸脱氢酶复合体生产异丁酰辅酶A和异戊酰辅酶A的大肠细胞底盘细胞，以及经由羟甲基戊二酰辅酶A（HMG-CoA）合成异戊酰辅酶A的大肠杆菌底盘细胞，并对其进行了优化。然后，从贯叶连翘中克隆挖掘到一个III型PKS合酶HpCHS，并将HpCHS和啤酒花来源的异戊酰苯合酶HlVPS以及草莓来源的FvCHS基因引入支链辅酶A底盘细胞，首次实现了贯叶金丝桃素中间体异丁酰间苯三酚及其衍生物异戊酰间苯三酚的生物合成，为贯叶金丝桃素的工业化生产奠定物质基础，同时也为此类结构相似的天然产物，如啤酒花中的重要成份苦味酸等的生物合成提供了理论和案例参考。本项目还发现HlVPS，FvCHS以及HpCHS除了合成支链间苯三酚外，还能合成支链吡喃酮副产物，支链吡喃酮具备用作平台化合物的潜力。通过蛋白结构解析、理性设计、定向进化等提高VPS等的产物专一性及活性，还有待深入研究。