Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109.
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824.
Plant Physiol. 2018 Jan;176(1):524-537. doi: 10.1104/pp.17.01330. Epub 2017 Nov 9.
Flowers of produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencing-based approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while encodes an enzyme that oxidizes trans-chrysanthemal into trans-chrysanthemic acid. Transient coexpression of and together with in leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host.
植物产生一组被称为除虫菊酯的化合物，这些化合物是具有商业重要性的杀虫剂，对飞行昆虫具有很强的毒性，但对大多数脊椎动物没有毒性。除虫菊酯分子是一种酯，由反式菊酸或其修饰形式除虫菊酸和三种醇之一组成，这三种醇似乎是由茉莉酸衍生而来的，分别为茉莉酮、茉莉醇和毕那醇。菊花酰二磷酸合酶（CDS）是参与反式菊酸合成的第一个酶，其特性以前已被描述，并分离出其基因。植物除了产生反式菊酰二磷酸外，还会产生游离的反式菊醇，但负责将反式菊醇转化为相应醛，然后再转化为酸的酶尚未报道。我们使用基于 RNA 测序的方法和共表达相关性分析来鉴定几个候选基因，这些基因编码可能的反式菊醇和反式菊醛脱氢酶。我们使用体外生化测定和异源表达在植物体内相结合的方法，对这些基因编码的蛋白质进行了功能表征，证明了 编码一种将反式菊醇氧化为反式菊醛的酶，而 编码一种将反式菊醛氧化为反式菊酸的酶。在 叶片中转瞬共表达 和 以及 会导致反式菊酸以及其他几种副产物的产生。大多数（58%）的反式菊酸被糖基化或其他修饰。总的来说，这些数据确定了除虫菊酯生物合成中的关键步骤，并证明了在异源宿主中生产这些防御化合物成分的代谢工程的可行性。
