PlantSynBio: Chassis design for sustainable production of high value terpenoids in the crop species tomato

植物产生一系列对人类有用的化学物质，如药品、香料、食品添加剂和农用化学品。一类化合物是萜类化合物，可以用作农用化学品，因为它们对许多昆虫都有活性，但对哺乳动物无毒，可生物降解，并且与可持续农业实践中使用的方法兼容。然而，由于产量低以及种植植物和提取化合物所需的时间、空间和成本，直接从植物中生产这些化合物可能具有挑战性。通过生物技术的进步，现在有可能在一种易于种植的作物品种的植物中设计生产高价值化学品，从而为生产植物衍生的农用化学品提供了一种可持续的方法。在这个项目中，我们将使用生物技术方法对番茄进行工程改造，以在水果中生产高价值的萜类化合物，并证明将化学与生物技术和农业相结合，为自然资源有限的化学品的可持续生产提供了一个平台。我们预计，获得可持续的萜类化合物生产生物工程平台将广泛应用于学术界和工业界。在这个项目中，我们将培训对21世纪劳动力至关重要的技术人员。通过在佐治亚州州立植物园、密歇根州立大学和明尼苏达大学举办的年度活动和模块，我们将向公众宣传植物系统在化学品可持续生产方面的潜力。目前已知有5万多种植物萜类化合物，它们是由通用的C5构建块通过萜烯合成酶、细胞色素p450和糖基转移酶的活性合成的，具有植物防御功能，并作为授粉者和共生体的引诱剂。然而，萜类生物合成基因在植物中的异源表达存在问题，因为激活产物倾向于通过内源性细胞色素p450和糖基转移酶转化为偶联物或其他衍生物。利用尖端的基因编辑和转化技术，该项目将通过产生两个具有最小萜类生物合成能力的新型番茄底盘，为植物合成生物学开发新的实验平台和范例，从而实现番茄果实中高价值萜类分子的工程设计。我们的科学目标是：(i)利用新生分生组织产生的基因编辑系快速确认番茄果实中参与三萜和四萜生物合成的细胞色素P450和糖基转移酶；（ii）使用最先进的基因编辑和转化方法，在番茄中开发两种底盘，以消耗水果中的三萜和四萜（类胡萝卜素）；（iii）构建合成两种高价值萜类化合物的生物工程番茄系，作为概念验证。在这个项目中，我们将培养最先进的合成生物学科学家，以促进植物合成生物学的新范式。通过与三家机构的年度活动和模块，我们将让公众参与到可持续化学品生产的工厂系统的力量中来。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Plants produce a diverse set of chemicals that are of use to humans as pharmaceuticals, fragrance, food additives, and agrichemicals. One class of compounds are terpenoids which can be used as agrichemicals as they are active against many insects but non-toxic to mammals, biodegradable, and compatible with methods used in sustainable agriculture practices. However, production of these compounds directly from plants can be challenging due to low yield as well as the time, space, and costs required to grow the plants and extract the compounds. Through advances in biotechnology, it is now possible to engineer production of high value chemicals in plants in a crop species which is simple to grow thereby providing a sustainable approach to production of plant-derived agrichemicals. In this project, we will use biotechnology approaches to engineer tomato to produce high value terpenoids in fruit and demonstrate that merging chemistry with biotechnology and agriculture provides a platform for sustainable production of chemicals with limited availability from natural sources. We anticipate that access to a sustainable bio-engineered platform for terpenoid production will be of broad use to both the academic and industry sectors. In this project, we will train personnel in technologies critical to a 21st century workforce. Through annual events and modules at the State Botanic Garden of Georgia, Michigan State University, and University of Minnesota, we will inform the public of the potential of plant systems for sustainable production of chemicals.Over 50,000 plant terpenoids are known which are synthesized from universal C5 building blocks via the activity of terpene synthases, cytochrome P450s, and glycosyl transferases and function in plant defense, and as attractants to pollinators and symbionts. However, heterologous expression of terpenoid biosynthetic genes in plants is problematic due to the tendency for conversion of the activated products into conjugates or other derivatives via endogenous cytochrome P450s and glycosyl transferases. Using cutting edge gene editing and transformation technologies, this project will develop new experimental platforms and a paradigm for plant synthetic biology by generating two novel tomato chassis with minimal terpenoid biosynthetic capacity that will enable engineering of high value terpenoid molecules in tomato fruit. Our scientific aims are to (i) Rapidly confirm cytochromes P450 and glycosyl transferases involved in tri- and tetra-terpene biosynthesis in tomato fruit using de novo meristem generated gene-edited lines; (ii) Develop two chassis in tomato that are depleted of tri- and tetraterpenes (carotenoids) in fruit using state-of-the-art gene editing and transformation methods; and (iii) Construct bioengineered tomato lines that synthesize two high value terpenoids as a proof-of-concept. Within this project we will train scientists in state-of-the-art synthetic biology to facilitate new paradigms in plant synthetic biology. Through annual events and modules with three institutions, we will engage the lay public in the power of plant systems for sustainable chemical production.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.