蜜环菌来源的倍半萜醇芳香酸酯类活性天然产物的生物合成研究

Melleolide A and derivatives constitute a remarkable group of sesquiterpene aryl esters derived from basidiomycetous tree pathogen Armillaria mellea (honey mushroom). These natural products feature an intriguing molecular architecture composed of an orsellinic acid (2, 4-dihydroxy-6-methylbenzenecarboxylic acid) moiety esterified to a tricyclic sesquiterpene (protoilludane) alcohol. The unique structure of melleolides revealed an unusual biosynthetic mechanism and potential as pharmaceuticals exhibiting two distinct structure-activity relationships for their cytotoxic and antifungal bioactivities. The melleolides biosynthetic gene cluster (arm) from Armillaria mellea has been characterized by bioinformatic analysis. Herein, we try to investigate the genetics and biochemical mechanism of melleolides biosynthesis. We aim to construct biosynthetic pathway of melleolides in heterologous expression system in which Aspergillus nidulans (An) as host by stepwise strategy. Elucidation of on-pathway intermediates isolated from An transformants, heterologous expression of key enzymes encoded by biosynthetic gene cluster arm in Saccharomyces cerevisiae and Escherichia coli system, and in vitro biochemical reactions will be performed to investigate the biosynthetic pathway of melleolides and biochemical mechanism at the gene-protein level. We focused on mechanism for double bond rearrangement (∆2, 4 and ∆2, 3) and hydroxylation of 4-protoilludene to 2-protoilluden-4-ol, as well as redox reactions which generate complexity of melleolides. The study on the biosynthesis of melleolides is expected to discover some distinguished enzymes participating in double bond rearrangement and formation of molecular scaffold with intriguing functions and novel catalytic mechanisms. This will shed light on following studies especially on mechanism of key enzymes and generation of novel chemical derivatives with better bioactivities.

Melleolide A等是从真菌Armillaria mellea分离得到的倍半萜醇芳香酸酯类活性天然产物。这类化合物特征性的原伊鲁烷型倍半萜和苔色酸基团的存在表明其独特的生物合成机制。本课题通过对A. mellea的全基因组生物信息学分析，确定了melleolides的生物合成基因簇arm。拟利用构巢曲霉（An）途径重建系统构建melleolides类化合物的生物合成途径，同时结合An转化子中间体化合物分离鉴定、酿酒酵母表达系统等表达关键酶，并结合体外生化酶学实验，从基因-蛋白水平揭示melleolide A等的生物合成途径和酶学基础。重点研究原伊鲁烷型倍半萜醇单元双键重排、C-4羟基化和多个氧化酶的催化机制。本研究以期发现melleolides生物合成中参与双键重排和氧化还原修饰的特殊功能酶和新的催化机制，并有望发现生物学活性优于已报道melleolides类化合物的活性分子。

Melleolides是从蜜环菌中分离得到的倍半萜醇芳香酸酯类天然产物。本课题利用OSMAC策略中蜜环菌CPCC 401429分离鉴定14个melleolides类化合物，其中包括7个新化合物，转录组测序揭示杂萜melleolides化合物生物合成机制。利用农杆菌介导遗传转化策略构建melleolides类化合物高产基因工程菌Am-OE::armR。结合比较基因组学、酿酒酵母系统和GC-MS分析平台，对蜜环菌DSM 3731和CPCC 401429中倍半萜合酶异源表达。共成功表达12个倍半萜合酶，其中多个STSs属于新的亚家族Clade IV，该类群倍半萜合酶功能复合新颖，均能合成多个（种）倍半萜分子。利用代谢组和蛋白质组学技术阐明探究melleolides生物合成调控机制，深入挖掘melleolides类分子，并对melleolides类化合物继续深入分离鉴定和构效关系评价。运用酵母同源重组技术构建了多个表达载体，利用构巢曲霉异源表达系统研究melleolides类化合物生物合成机制，同时构建CRISPR-Cas9敲除系统针对arm基因簇中关键基因敲除构建敲除突变株。课题对melleolides类化合物生物合成关键酶包括在E. coli中实现卤代酶和糖基转移酶，在酿酒酵母系统中实现聚酮合酶和P450氧化酶等的功能表达。课题利用利用共培养策略挖掘蜜环菌和巴西青霉发酵次级代谢产物，共挖掘化合物15个，其中新化合物6个，拓宽了蜜环菌的应用潜力。研究首次通过OSMAC和RNA-seq技术对蜜环菌代谢产物挖掘，结合比较基因组学和酿酒酵母系统对蜜环菌CPCC 401429分类地位和倍半萜合酶功能鉴定。课题还获得melleolides类化合物高产基因工程菌，并结合蛋白组学技术等对该类杂萜化合物的生物合成和调控机制探讨，为后续继续开展melleolides类化合物的生物合成奠定基础。