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Abstract：
Terpenoids are the most diverse natural products with many industrial applications and are all synthesized from simple precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized by two distinct metabolic pathways - cytosolic mevalonate (MVA) pathway for C15 sesquiterpene and C30 triterpene, and plastidic methylerythritol phosphate (MEP) pathway for C10 monoterpene and C20 diterpene. A number of studies have altered the metabolic gene expressions in either the MVA or MEP pathway to increase terpene production; however, it remains unknown if the alteration of the acetyl-CoA pool in plastid fatty acid biosynthesis can influence terpenoid flux. Here, we focused on the fact that acetyl-CoA is the precursor for both fatty acid biosynthesis in plastid and terpene biosynthesis in cytosol, and the metabolic impact of increased plastidic acetyl-CoA level on the cytosolic terpene biosynthesis was investigated. In tobacco leaf infiltration studies, the acetyl-CoA carboxylase complex (the enzyme supplying malonyl-CoA in plastid) was partially inhibited by overexpressing the inactive form of biotin carboxyl carrier protein (BCCP) by a negative dominant effect. Overexpression of BCCP showed 1.4-2.4-fold increase of sesquiterpenes in cytosol; however, surprisingly overexpression of BCCP linked to truncated HMG-CoA reductase (tHMGR) by a cleavable peptide 2A showed 20-40-fold increases of C15 sesquiterpenes (α-bisabolol, amorphadiene, and valerenadiene) and a 6-fold increase of C30 β-amyrin. α-Bisabolol and β-amyrin production reached 28.8 mg g-1 and 9.8 mg g-1 dry weight, respectively. Detailed analyses showed that a large increase in flux was achieved by the additive effect of BCCP- and tHMGR-overexpression, and an enhanced tHMGR activity by 2A peptide tag. Kinetic analyses showed that tHMGR-2A has a three-fold higher kcat value than tHMGR. The tHMGR-2A-BCCP1 co-expression strategy in this work provides a new insight into metabolic cross-talks and can be a generally applicable approach to over-produce sesqui- and tri-terpene in plants.
摘要：
萜类化合物是具有许多工业应用的最多样化的天然产物，并且都是从简单的前体合成的。异戊烯基二磷酸(IPP)及其异构体二甲基烯丙基二磷酸(DMAPP)。在植物中,IPP通过两种不同的代谢途径合成-C15倍半萜和C30三萜的胞质甲羟戊酸（MVA）途径，C10单萜和C20二萜的质体甲基赤藓糖醇磷酸酯（MEP）途径。许多研究已经改变了MVA或MEP途径中的代谢基因表达，以增加萜烯的产量；然而，尚不清楚质体脂肪酸生物合成中乙酰辅酶A池的改变是否会影响萜类化合物的通量。这里,我们专注于乙酰辅酶A是质体中脂肪酸生物合成和细胞质中萜生物合成的前体，并研究了质体乙酰辅酶A水平增加对胞质萜烯生物合成的代谢影响。在烟叶渗透研究中，乙酰辅酶A羧化酶复合物（在质体中提供丙二酰辅酶A的酶）通过负显性作用过度表达生物素羧基载体蛋白（BCCP）的非活性形式而被部分抑制。BCCP的过表达显示细胞质中倍半萜的1.4-2.4倍增加；然而，令人惊讶的是，通过可切割的肽2A与截短的HMG-CoA还原酶（tHMGR）连接的BCCP过表达显示C15倍半萜（α-没药醇，amorphadiene,和戊二烯）和C30β-淀粉苷增加6倍。α-没药醇和β-淀粉素的产量达到28.8mgg-1和9.8mgg-1干重，分别。详细的分析表明，通过BCCP和tHMGR过表达的加性效应实现了通量的大幅增加，和通过2A肽标签增强的tHMGR活性。动力学分析表明，tHMGR-2A的kcat值比tHMGR高三倍。这项工作中的tHMGR-2A-BCCP1共表达策略提供了对代谢交叉对话的新见解，并且可以成为在植物中过度生产倍半萜和三萜的普遍适用方法。
