Augustin Megan M, Ruzicka Dan R, Shukla Ashutosh K, Augustin Jörg M, Starks Courtney M, O'Neil-Johnson Mark, McKain Michael R, Evans Bradley S, Barrett Matt D, Smithson Ann, Wong Gane Ka-Shu, Deyholos Michael K, Edger Patrick P, Pires J Chris, Leebens-Mack James H, Mann David A, Kutchan Toni M
Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO, 63132, USA.
Monsanto Company, 700 Chesterfield Parkway West, St Louis, MO, 63017, USA.
Plant J. 2015 Jun;82(6):991-1003. doi: 10.1111/tpj.12871.
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.
甾体生物碱已被证明具有广泛的药理作用，包括抗癌和抗真菌活性。了解这些分子的生物合成对于可持续生产的生物工程至关重要。在此，我们研究了环杷明的生物合成途径，环杷明是一种具有前景的抗肿瘤甾体生物碱。环杷明的供应有限，因为目前的来源完全是从野生植物加州藜芦中采集的。为了阐明环杷明合成途径的早期阶段，我们使用环杷明积累谱作为基因表达的预定义模型，通过模式匹配算法Haystack对加州藜芦RNA测序数据集进行分析。在Sf9昆虫细胞中对候选基因进行重构，发现了四种酶，它们催化甾体生物碱生物合成的前六个步骤，生成环杷明的预测前体维拉嗪。其中三种酶是细胞色素P450，第四种是γ-氨基丁酸转氨酶；它们共同作用从胆固醇生成维拉嗪。
