Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme.
Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme.
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DOI：
10.1186/s13036-015-0022-z
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发表时间：
2015
影响因子：
5.6
通讯作者：
Jensen PE
中科院分区：
文献类型：
--
作者：
Gnanasekaran T;Vavitsas K;Andersen-Ranberg J;Nielsen AZ;Olsen CE;Hamberger B;Jensen PE
植物萜类化合物以其多样性、立体化学复杂性以及作为药物、食品添加剂和化妆品的商业利益而闻名。开发在异源宿主中生产这些化合物的生物技术方法可以增加其市场可用性，降低其成本，并提供可持续的生产平台。在这种情况下，我们的目的是生产抗菌二萜异海松酸从锡特卡云杉。异海松酸是使用香叶基香叶基二磷酸作为前体分子合成的，其被叶绿体中的二萜合酶环化，随后被细胞色素P450，CYP 720 B4氧化。我们瞬时表达的异海松酸途径在本氏烟草叶和提高其生产力的表达的两个限速步骤的途径（提供一般的前体二萜）。这种共表达导致使用GC-MS和LC-MS方法检测到的异海松二烯和异海松酸的积累增加3倍。我们还表明，修饰或删除CYP720 B4的跨膜螺旋不会改变酶活性，并导致异海松酸在渗透叶中的成功积累。此外，我们证明，修饰的膜锚是一个功能性的CYP720 B4酶时，叶绿体靶向肽的先决条件。我们报道了在用修饰的酶渗透后四天异海松酸的45 - 55 μ g/g植物干重的积累。云杉的二萜类化合物代谢途径可能完全定位于N. benthamiana和CYP720B4的N-末端序列的一些修饰可以促进植物P450在质体中的表达。萜类化合物的生物合成与光合作用的耦合为光驱动的萜类化合物的生物合成奠定了基础。
Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45–55 μg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes. It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.