BACKGROUND Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. RESULTS To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. CONCLUSION Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

中文翻译：

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将途径阐明与酵母工程相结合，生产抗肥胖剂雷公藤红素的前体聚普诺酸。


背景雷公藤红素是一种有前景的抗肥胖剂，可作为蛋白质激素瘦素的敏化剂。尽管其活性很强，但用于进一步药理学研究的雷公藤红素和雷公藤红素衍生物的可持续来源仍然缺乏。结果 为了阐明雷公藤红素生物合成途径并在酿酒酵母中重建它，我们挖掘了雷公藤红素的根转录组，并鉴定了四种氧化角鲨烯环化酶和 49 种细胞色素 P450 作为参与雷公藤红素生物合成早期步骤的候选酶。通过对本塞姆氏烟草中的候选基因进行功能筛选，TwOSC4 被鉴定为一种新型氧化角鲨烯环化酶，可产生木栓苷（推测为雷公藤红醇的三萜类主链）。此外，还发现作用于 TwOSC4 下游的三个 P450（CYP712K1、CYP712K2 和 CYP712K3）可有效氧化木栓素并形成可能的雷公藤红醇生物合成中间体 29-羟基-木栓醇和 polpunonic 酸。为了促进木栓素的生产，通过删除 UBC7 构建了酵母菌株 AM254，这使得木栓素滴度增加了五倍。该平台通过 CYP712K1 进一步扩展，可生产 polpunonic 酸，并提供了一种从酵母培养基中轻松提取产物的方法，使 polpunonic 酸滴度达到 1.4 mg/L。结论我们的研究阐明了雷公藤红醇生物合成的早期步骤，并为未来在工程酵母菌株中生物技术生产这种具有药理学前景的化合物铺平了道路。