Data from: Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

甾体生物碱（steroid alkaloids）已被证实可展现涵盖抗肿瘤与抗真菌活性在内的多种药理效应。阐明这类分子的生物合成通路，对于通过生物工程实现其可持续生产至关重要。本研究针对环巴胺（cyclopamine）的生物合成通路展开探究，环巴胺是一种具备良好抗肿瘤活性的甾体生物碱。当前环巴胺的唯一来源为野生采集的加州藜芦（Veratrum californicum），其供给量十分有限。为阐明环巴胺生物合成通路的早期阶段，本研究以环巴胺的积累特征作为基因表达的预设模型，借助模式匹配算法Haystack对加州藜芦的RNA测序（RNA-seq）数据集进行了分析。通过在Sf9昆虫细胞中重构候选基因，本研究发现了4种可催化甾体生物碱生物合成前六个步骤的酶，这些酶能够以胆固醇为底物合成维拉嗪（verazine）——一种被预测为环巴胺的前体物质。其中3种酶为细胞色素P450（cytochromes P450），剩余1种为γ-氨基丁酸转氨酶（γ-aminobutyrate transaminase），这四种酶协同可将胆固醇转化为维拉嗪。