Next Generation Sequencing in Predicting Gene Function in Podophyllotoxin Biosynthesis
Next Generation Sequencing in Predicting Gene Function in Podophyllotoxin Biosynthesis
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DOI：
10.1074/jbc.m112.400689
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发表时间：
2013-01-04
影响因子：
4.8
通讯作者：
Lewis, Norman G.
中科院分区：
文献类型：
--
作者：
Marques, Joaquim V.;Kim, Kye-Won;Lewis, Norman G.
鬼臼属物种是 (-)-鬼臼毒素的来源，鬼臼毒素是一种芳基四氢化萘木酚素，用于半合成各种强效且广泛使用的癌症治疗药物。然而，其生物合成途径仍然很大程度上未知，最后一个明确证明的中间体是 (-)-matairesinol。在此，对鬼臼和鬼臼转录组进行了大规模并行测序，并对相应的组装进行了随后的生物信息学分析。组装过程的验证首先通过用先前确定的参与鬼臼毒素生物合成的各种基因以及其他候选生物合成途径基因的组装序列进行确认来实现。该贡献描述了后者中的两个的特征，即细胞色素 P450、来自 P. hexandrum 的 CYP719A23 和来自 P. peltatum 的 CYP719A24。两种酶都能够通过催化亚甲二氧基桥的形成将(-)-matairesinol转化为(-)-pluviatolide，并且不作用于测试的其他可能的底物。有趣的是，本文描述的酶与生物碱生物合成中的亚甲二氧基桥形成酶高度相似，而与木脂素生物合成酶更相似的候选酶对所使用的底物没有催化活性。因此，这一总体策略使得能够方便地进一步鉴定假定参与(-)-鬼臼毒素生物合成的酶，并强调了下一代测序和生物信息学探测和推断药用植物生物合成途径的演绎能力。
Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.