DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, 1871, Denmark.
Plant Biochemistry Section, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.
Plant J. 2021 May;106(4):978-992. doi: 10.1111/tpj.15212. Epub 2021 Mar 22.
2-Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health-promoting functions, due to its hydrolysis product 2-phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.T. Aiton and demonstrated the feasibility of engineering 2PE production in Nicotiana benthamiana Domin. We used different combinations of genes from B. vulgaris and Arabidopsis thaliana (L.) Heynh. to demonstrate that: (i) BvBCAT4 performed more efficiently than AtBCAT4 in biosynthesis of both homophenylalanine and dihomomethionine; (ii) MAM1 enzymes were critical for the chain-elongated profile, while CYP79F enzymes accepted both chain-elongated methionine and homophenylalanine; (iii) aliphatic but not aromatic core structure pathway catalyzed the 2PE biosynthesis; (iv) a chimeric pathway containing BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in a two-fold increase in 2PE production compared with the B. vulgaris-specific chain elongation pathway; and (v) profiles of chain-elongated products and glucosinolates partially mirrored the profiles in the gene donor plant, but were wider in N. benthamiana than in the native plants. Our study provides a strategy to produce the important homophenylalanine and 2PE in a heterologous host. Furthermore, chimeric engineering of the complex 2PE biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution. The new-to-nature gene combinations have the potential for application in biotechnological and plant breeding.
2-苯乙基葡萄糖苷（2PE）来源于同型苯丙氨酸，是 Brassicales 目中植物的防御化合物。由于其水解产物 2-苯乙基异硫氰酸酯，它具有驱虫、抗菌和促进健康的功能，与多种生物学特性相关，因此 2PE 具有在农业和工业中应用的潜力。在这项研究中，我们从普通独行菜（Barbarea vulgaris W.T. Aiton）中鉴定出 2PE 生物合成的假定关键基因，并证明了在黄花烟草（Nicotiana benthamiana Domin.）中工程生产 2PE 的可行性。我们使用来自普通独行菜和拟南芥（Arabidopsis thaliana (L.) Heynh.）的不同基因组合来证明：（i）BvBCAT4 在同型苯丙氨酸和二氢甲硫氨酸的生物合成中比 AtBCAT4 更有效；（ii）MAM1 酶对链延长谱至关重要，而 CYP79F 酶接受链延长的甲硫氨酸和同型苯丙氨酸；（iii）脂肪族而非芳香族核心结构途径催化 2PE 的生物合成；（iv）含有 BvBCAT4、BvMAM1、AtIPMI 和 AtIPMDH1 的嵌合途径导致 2PE 产量比普通独行菜特异性的链延长途径增加了两倍；（v）链延长产物和硫代葡萄糖苷的图谱部分反映了供体植物的图谱，但在黄花烟草中的图谱比在天然植物中更宽。我们的研究为在异源宿主中生产重要的同型苯丙氨酸和 2PE 提供了一种策略。此外，对复杂的 2PE 生物合成途径进行嵌合工程，使我们能够详细了解单个酶的催化特性——这是理解生化进化的前提。新的天然基因组合有可能应用于生物技术和植物育种。
