Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production.
Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production.
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DOI：
10.1111/tpj.12067
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发表时间：
2013-02
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通讯作者：
Chunyu Zhang;Rebecca E. Cahoon;Sarah C. Hunter;Ming Chen;Jixiang Han;E. Cahoon
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作者：
Chunyu Zhang;Rebecca E. Cahoon;Sarah C. Hunter;Ming Chen;Jixiang Han;E. Cahoon
单子叶植物中维生素E生育三烯酚的合成需要尿黑酸香叶基香叶基转移酶（HGGT），其催化尿黑酸和不饱和C20类异戊二烯香叶基香叶基二磷酸（GGDP）的缩合。相比之下，维生素E生育酚合成由尿黑酸植基转移酶（HPT）介导，其缩合尿黑酸和饱和的C20类异戊二烯植基二磷酸（PDP）。生育三烯酚合成的HGGT非依赖性途径也已显示通过尿黑酸合成的失调而发生。在本文中，这一途径的基础及其与HGGT结合时对维生素E生产的影响进行了探索。最初开发了拟南芥品系，其通过共表达拟南芥羟苯丙酮酸双加氧酶（HPPD）和大肠杆菌双功能分支酸脱氢酶/预苯酸脱氢酶（TyrA）来积累生育三烯酚和尿黑酸。当与vte 2 -1 HPT无效突变体杂交时，生育三烯酚的产生丧失，表明HPT通过非典型地使用GGDP作为底物来催化表达HPPD/TyrA的植物中的生育三烯酚合成。与此一致，重组拟南芥HPT仅在存在高摩尔比的GGDP：PDP时优先催化生育三烯酚前体香叶基香叶基苯醌的体外产生。此外，生育三烯酚水平在HPPD/TyrA系的早期生长阶段最高，但在生长后期PDP积累时相对于生育酚强烈下降。总的来说，这些结果表明，HPPD/TyrA诱导的生育三烯酚生产需要HPT，并在异戊烯基二磷酸池中相对于PDP富集GGDP时发生。最后，HPPD/TyrA和HGGT在拟南芥叶片和种子中的组合表达导致维生素E生产的大的加性增加，表明尿黑酸浓度限制HGGT催化的生育三烯酚合成。
Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT-independent pathway for tocotrienol synthesis has also been shown to occur by de-regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2-1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA-expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA-induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT-catalyzed tocotrienol synthesis.