Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science and Multidisciplinary Institut for Environment Studies "Ramon Margalef", University of Alicante, Alicante, Spain.
Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science and Multidisciplinary Institut for Environment Studies "Ramon Margalef", University of Alicante, Alicante, Spain; Biotechnology and Molecular Biology Group, Quevedo State Technical University, Quevedo, Ecuador.
N Biotechnol. 2018 May 25;42:62-70. doi: 10.1016/j.nbt.2018.02.011.
Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties.
二苯乙烯是天然稀缺的高附加值植物化合物，具有化学预防、药理和美容特性。生物生产策略包括工程化细菌、真菌和植物细胞系统的代谢。引人注目的是，最有效的策略之一是刺激野生葡萄细胞培养物，这导致细胞外培养基中大量白藜芦醇(resveratrol)积累。细胞培养物刺激和代谢工程策略的结合被证明对于羟基化衍生物白藜芦醇比二甲化衍生物紫檀芪更为有效，对于后者，分别使用了人羟化酶 HsCYP1B1-和葡萄 VvROMT 甲基转移酶转化的细胞培养物。玫瑰愈创木酚 O-甲基转移酶（OOMT）具有酶学特性，使其成为替代 VvROMT 的有吸引力的候选物，用于增强工程化葡萄细胞在刺激时对紫檀芪的生产水平。在这里，我们克隆了一个 Rosa hybrida OOMT 基因，并创建了一个适合农杆菌介导的植物转化的遗传构建体。首先使用农杆菌浸润的 N. benthamiana 叶片和转化的葡萄愈伤组织的蛋白提取物在体外评估了 OOMT 将白藜芦醇转化为紫檀芪的能力。用 RhOOMT 转化的葡萄细胞培养物产生了约 16mg/L 的紫檀芪培养物，在最佳条件下，细胞外分布高达总产量的 34%，这是迄今为止在植物系统中报道的最高产量。同时获得了约 1500-3000mg/L 的大量白藜芦醇的同时生产。我们的结果证明了一种可行的成功的代谢工程策略，可以生产紫檀芪，这是一种具有增强药理特性的白藜芦醇类似物。
