Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis.
Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis.
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DOI：
10.1007/s00253-015-6496-2
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发表时间：
2015-09
影响因子：
5
通讯作者：
Peters RJ
中科院分区：
文献类型：
--
作者：
Kitaoka N;Wu Y;Xu M;Peters RJ
细胞色素P450（CYP）催化的氧化反应在植物天然产物的生物合成中起着重要作用。与此同时，CYP是最具挑战性的酶之一，以功能上的特点，由于重组表达这些膜相关的单加氧酶的困难。在研究水稻二萜类化合物生物合成的过程中，我们开发了一种在大肠杆菌中功能表达相关CYP的合成生物学方法。在某些情况下，仅观察到两种密切相关的旁系同源物之一的活性，尽管似乎清楚的是，相关反应是生产已知二萜类所必需的。在这里，我们报告的重组表达系统的优化，使表征不仅这些以前的cycitrant CYP，但也发现了额外的活动相关的水稻二萜类化合物的生物合成。特别令人感兴趣的是，发现CYP 701 A8催化顺式海松二烯的3β-羟基化，这可能与momilactone生物合成有关，而发现CYP 71 Z6和7催化多个反应，其中CYP 71 Z6催化经由2α-羟基-对映-异贝壳杉烯产生2α，3 α-二羟基-对映-异贝壳杉烯，CYP 71 Z7催化2α-羟基-对映-木薯二烯和对映-木薯二烯-2-酮生成3α-羟基-对映-木薯二烯-2-酮，可能分别与谷草酮和植物卡桑的生物合成有关。
The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3β-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy- ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2- one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.