Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii
Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii
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DOI：
10.1073/pnas.1106222108
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发表时间：
2011-07-26
影响因子：
11.1
通讯作者：
Chappell, Joe
中科院分区：
文献类型：
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作者：
Niehaus, Tom D.;Okada, Shigeru;Chappell, Joe
葡萄球菌的生物合成被认为类似于角鲨烯的生物合成，角鲨烯是所有真核生物中甾醇代谢所必需的代谢物。角鲨烯是由两个法呢基二磷酸（FPP）分子的初始缩合形成前角鲨烯二磷酸（PSPP），然后经过还原重排形成角鲨烯。原则上，botryococcene可能是由前角鲨烯中间体的另一种重排产生的。由于这些提出的相似性，我们预测葡萄球菌合酶将类似于角鲨烯合酶，因此从布朗葡萄球菌品种B中分离出角鲨烯脱氢酶样基因。而B. braunii确实具有至少一个典型的角鲨烯合成酶，其他三个角鲨烯合成酶样（SSL）基因都不直接编码葡萄球菌的生物合成。SSL-1催化PSPP的生物合成，SSL-2催化双法呢基醚的生物合成，而SSL-3似乎不能直接利用FPP作为底物。然而，当在体内和体外将类葡萄球菌酶的组合混合在一起时，观察到稳健的葡萄球菌（SSL-1+SSL-3）或角鲨烯生物合成（SSL 1 +SSL-2）。这些发现是出乎意料的，因为角鲨烯合酶，一种古老的和可能的其他葡萄球菌三萜脱氢酶的祖先，催化单个酶单位内的两步反应，而没有中间释放，但在B中。braunii，这些活动似乎已经分离，并相互依赖地发展为专门的三萜油生产大于500 MYA。SSL-1和SSL-3基因以不同的构型共表达，作为独立的基因，作为基因融合体，或靶向细胞内膜，也证明了工程化甚至更高效率的葡萄球菌生物合成的潜力。
Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.