酿酒酵母合成灵芝酸类似物的研究

Ganoderic acids (GAs), a kind of triterpenoids, which are unique secondary metabolites of the higher fungi Ganoderma, exhibit important biological activities including significant antitumor activities. As the precursors of GAs, GAs analogues are formed by the post-modification of lanosterol, the backbone of GAs. Owing to difficulties in genetic manipulation of Ganoderma, biosynthesis of GAs analogues in heterologous host using synthetic biology approaches and the identification of key enzymes of GAs biosynthetic pathway are extremely important. Identification of the CYP450s responsible for converting lanosterol into GAs analogues is a critical step for heterologous biosynthesis of GAs. As a platform in synthetic biology research, Saccharomyces cerevisiae is able to synthesize lanosterol, and is also a suitable host for expressing eukaryotic CYP450s. In this project, hypothetical CYP450s from Ganoderma will be expressed in S. cerevisiae to construct the biosynthetic pathway for heterologous production of GAs analogues. The results are expected to not only lay the foundation for elucidating the entire pathway of GAs biosynthesis, but also providing insights to synthetic biology studies on other bioactive terpenoids with the newly-identified enzymes, metabolic pathways and biocatalytic mechanisms.

灵芝酸是高等真菌灵芝特有的三萜类次级代谢产物，具有良好抗肿瘤等重要生物活性。灵芝酸类似物作为合成灵芝酸的前体，是灵芝酸骨架羊毛甾醇经后修饰得到的化合物。鉴于高等真菌灵芝的遗传操作困难，利用合成生物学手段在异源宿主中实现灵芝酸类似物的合成，进而发现灵芝酸生物合成途径关键元件，这些无疑是十分重要的。确定催化羊毛甾醇生成灵芝酸类似物的细胞色素P450酶， 是异源合成灵芝酸的关键一步。 作为合成生物学常用的宿主，酿酒酵母自身能合成羊毛甾醇且适合表达真核来源的细胞色素P450酶。本研究拟以酿酒酵母为宿主，表达灵芝来源的细胞色素P450酶的候选基因，构建酿酒酵母异源合成灵芝酸类似物的代谢通路。预期取得的结果将为灵芝酸生物合成途径的完整解析奠定基础，所发现的催化元件、代谢途径及有关催化机制还将为其它萜类化合物的合成生物学研究提供有益借鉴。

灵芝酸是具有良好抗肿瘤活性的三萜类化合物，是高等真菌灵芝特有的次级代谢产物。找到催化羊毛甾醇生成灵芝酸类似物的CYP450酶，是合成灵芝酸最关键的一步。本项目以遗传背景清楚、分子操作技术成熟、能天然合成灵芝酸前体——麦角固醇的酿酒酵母为宿主,从已报道的219个灵芝CYP基因中优先选择了72个可能参与灵芝酸生物合成的候选元件，发现CYP5150L8能催化羊毛甾醇第26位碳的三步连续氧化生成灵芝酸3-hydroxy-lanosta-8, 24-dien-26-oic acid (HLDOA)。最终，酿酒酵母工程菌发酵生产HLDOA产量为14.5 mg/L。此外，我们还发现在酿酒酵母中过表达CYP505D13能生产角鲨烯型三萜。通过对工程菌株的大规模发酵、产物分离纯化和核磁分析，我们鉴定了其中三个主要化合物的结构。它们分别是两个新发现的4,8-dihydroxy-22,23-oxidosqualene (ST-1), 8-hydroxy-2,3;22,23-squalene dioxide (ST-2)和一个已知的2,3; 22,23-squalene dioxide (ST-3)。本研究发现的关键催化元件，不仅有助于解析灵芝酸合成生物机制，而且对灵芝酸以及其它三萜类化合物的高效生物合成提供有益借鉴。