State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and Laboratory of Molecular Biochemical Engineering, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
Biotechnol Bioeng. 2019 Dec;116(12):3301-3311. doi: 10.1002/bit.27154. Epub 2019 Sep 4.
Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.
灵芝酸 3-羟基-羊毛甾-8,24-二烯-26-酸（GA-HLDOA）是一种来自中国传统药用高等真菌灵芝的抗肿瘤三萜类化合物，被认为是生物合成具有优异抗肿瘤活性的其他灵芝酸（GAs）的关键前体。我们之前的研究鉴定了灵芝中的 CYP5150L8 为羊毛甾醇氧化酶，并在酿酒酵母中实现了 GA-HLDOA 的异源生物合成。然而，灵芝或异源宿主中 GA-HLDOA 的产量较低，阻碍了其进一步的研究和应用。在本研究中，我们构建了一个双调控系统来平衡 CYP5150L8 和灵芝 P450 还原酶 iGLCPR 的表达，并对 CYP5150L8 表达、iGLCPR 表达和甘油利用进行了全面优化。然后，我们在优化条件下研究了最佳菌株在摇瓶中的发酵行为，获得了 154.45mg/L 的 GA-HLDOA 产量，比以前的报道高 10.7 倍。这项研究可能有助于 GA-HLDOA 的广泛应用和下游 GAs 生物合成中未知细胞色素 P450s 的发现。
