Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today's agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell's growth rate to the target gene's function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

中文翻译：

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将连续定向进化应用于植物酶的潜力

在当今的农业环境和植物生物技术应用中，植物进化产生的酶可能不是使产量和质量最大化的最佳酶。通过改善酶的性能，应该有可能减轻动力学特性或酶不稳定性目前对产量和质量的限制。通过应用定向进化，可以比自然地更快地优化酶，这需要在体外​​突变靶基因并筛选或选择具有所需特征的突变基因产物。连续定向进化是一种更有效和可扩展的版本，它通过易错靶基因的复制以及宿主细胞的生长速率与靶基因功能的耦合，在体内同时完成了诱变和选择步骤。然而，已发布的连续系统需要自定义质粒装配，并且没有便利的多功能平台。我们讨论了两种适用于酶的连续定向进化的系统，即酿酒酵母中的OrthoRep和大肠杆菌中的EvolvR，以及我们为使每种系统适应高通量植物酶工程应用而进行的试点工作。为了测试我们的改良系统，我们使用了硫胺素合成酶THI4，该酶先前被确定为主要的改良候选物。我们改编的OrthoRep系统显示出高效植物酶工程的前景。以及我们为使每个系统适应高通量植物酶工程应用而进行的试点工作。为了测试我们的改良系统，我们使用了硫胺素合成酶THI4，该酶先前被确定为主要的改良候选物。我们改编的OrthoRep系统显示出高效植物酶工程的前景。以及我们为使每个系统适应高通量植物酶工程应用而进行的试点工作。为了测试我们的改良系统，我们使用了硫胺素合成酶THI4，该酶先前被确定为主要的改良候选物。我们改编的OrthoRep系统显示出高效植物酶工程的前景。