State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and Laboratory of Molecular Biochemical Engineering & Advanced Fermentation Technology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Biotechnol Bioeng. 2018 Jul;115(7):1842-1854. doi: 10.1002/bit.26583. Epub 2018 Mar 8.
Ganoderic acid (GA), a triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, possesses antitumor and other significant pharmacological activities. Owing to the notorious difficulty and immaturity in genetic manipulation of higher fungi as well as their slow growth, biosynthesis of GAs in a heterologous host is an attractive alternative for their efficient bioproduction. In this study, using Saccharomyces cerevisiae as a host, we did a systematic screening of cytochrome P450 monooxygenase (CYP450) gene candidates from G. lucidum, which may be responsible for the GA biosynthesis from lanosterol but have not been functionally characterized. As a result, overexpression of a CYP450 gene cyp5150l8 was firstly found to produce an antitumor GA, 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) in S. cerevisiae, as confirmed by HPLC, LC-MS and NMR. A final titer of 14.5 mg/L of HLDOA was obtained at 120 hr of the yeast fermentation. Furthermore, our in vitro enzymatic experiments indicate that CYP5150L8 catalyzes a three-step biotransformation of lanosterol at C-26 to synthesize HLDOA. To our knowledge, this is the first report on the heterologous biosynthesis of GAs. The results will be helpful to the GA biosynthetic pathway elucidation and to future optimization of heterologous cell factories for GA production.
灵芝酸（GA）是一种来自传统中药灵芝的三萜类化合物，具有抗肿瘤和其他重要的药理活性。由于高等真菌遗传操作的难度和不成熟以及生长缓慢，在异源宿主中生物合成 GA 是其高效生物生产的一种有吸引力的替代方法。在这项研究中，我们使用酿酒酵母作为宿主，对灵芝中可能负责从羊毛甾醇生物合成 GA 但尚未进行功能表征的细胞色素 P450 单加氧酶（CYP450）基因候选物进行了系统筛选。结果，首次发现过表达 CYP450 基因 cyp5150l8 可在酿酒酵母中产生抗肿瘤 GA，3-羟基-羊毛甾烷-8,24-二烯-26 酸（HLDOA），这通过 HPLC、LC-MS 和 NMR 得到证实。在酵母发酵 120 小时后，HLDOA 的终产量达到 14.5mg/L。此外，我们的体外酶实验表明 CYP5150L8 催化羊毛甾醇在 C-26 位的三步生物转化，合成 HLDOA。据我们所知，这是首次报道 GA 的异源生物合成。研究结果将有助于 GA 生物合成途径的阐明以及未来优化用于 GA 生产的异源细胞工厂。
