Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (‘plantize’) enzymes from prokaryotes – especially exotic prokaryotes – to function well in mild, plant-like conditions.

中文翻译：

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使用连续定向进化来改进植物应用中的酶

微生物宿主中酶和其他蛋白质的连续定向进化能够通过在体内同时大规模执行超突变和选择，以最少的手动输入来超越经典定向进化。如果目标酶的活性可以与宿主细胞的生长相结合，则可以通过选择生长来简单地提高活性。像所有定向进化一样，连续版本不需要目标的先验机械知识。因此，连续定向进化是修饰植物或非植物酶以用于植物代谢研究和工程的一种强大的新方法。在这里，我们首先描述酿酒酵母的基本特征OrthoRep 系统用于连续定向进化，并将其与其他系统进行简要比较。然后，我们以 THI4 噻唑合酶为例，逐步说明可以部署 OrthoRep 以进化主要代谢酶的三种方式，并说明获得的突变结果。我们最后概述了 OrthoRep 的应用，满足不断增长的需求 (i) 改变植物酶的特性，这些酶注定返回植物，以及 (ii) 适应（“种植”）来自原核生物——尤其是外来原核生物的酶——在温和的，植物般的条件。