Horticultural Sciences Department, University of Florida, Gainesville, FL, U.S.A.
Biochem Soc Trans. 2023 Oct 31;51(5):1957-1966. doi: 10.1042/BST20230532.
Synthetic biology creates new metabolic processes and improves existing ones using engineered or natural enzymes. These enzymes are often sourced from cells that differ from those in the target plant organ with respect to, e.g. redox potential, effector levels, or proteostasis machinery. Non-native enzymes may thus need to be adapted to work well in their new plant context ('plantized') even if their specificity and kinetics in vitro are adequate. Hence there are two distinct ways in which an enzyme destined for use in plants can require improvement: In catalytic properties such as substrate and product specificity, kcat, and KM; and in general compatibility with the milieu of cells that express the enzyme. Continuous directed evolution systems can deliver both types of improvement and are so far the most broadly effective way to deliver the second type. Accordingly, in this review we provide a short account of continuous evolution methods, emphasizing the yeast OrthoRep system because of its suitability for plant applications. We then cover the down-to-earth and increasingly urgent issues of which enzymes and enzyme properties can - or cannot - be improved in theory, and which in practice are the best to target for crop improvement, i.e. those that are realistically improvable and important enough to warrant deploying continuous directed evolution. We take horticultural crops as examples because of the opportunities they present and to sharpen the focus.
合成生物学利用工程化或天然酶来创建新的代谢途径并改进现有的代谢途径。这些酶通常来源于与目标植物器官不同的细胞，例如氧化还原电位、效应物水平或蛋白质稳定机制。因此，即使非天然酶在体外具有足够的特异性和动力学，它们也可能需要适应新的植物环境（“植物化”）才能很好地发挥作用。因此，一种注定要在植物中使用的酶有两种截然不同的改进方式：在催化特性（如底物和产物特异性、kcat 和 KM）方面，以及在与表达酶的细胞环境的一般兼容性方面。连续定向进化系统可以实现这两种类型的改进，迄今为止，这是实现第二种类型改进的最广泛有效的方法。因此，在这篇综述中，我们简要介绍了连续进化方法，重点介绍了酵母 OrthoRep 系统，因为它适用于植物应用。然后，我们讨论了哪些酶和酶特性在理论上可以或不能得到改进，以及在实践中哪些酶最适合作为作物改良的目标，即那些在实际上可以改进且重要到足以值得部署连续定向进化的酶。我们以园艺作物为例，因为它们提供了机会，并且可以更聚焦。
