Directed evolution is often limited by the throughput of accurate screening methods. Here we demonstrate the feasibility of utilizing a singular transcription factor (TF)-system that can be refactored in two ways (both as an activator and repressor). Specifically, we showcase the use of previously evolved 5-halo- or 6-halo-tryptophan-specific TF biosensors suitable for the detection of a halogenated tryptophan molecule in vivo. We subsequently validate the biosensor’s utility for two halogenase-specific halo-tryptophan accumulation screens. First, we isolated 5-tryptophan-halogenase, XsHal, from a mixed pool of halogenases with 100% efficiency. Thereafter, we generated a targeted library of the catalytic residue of 6-tryptophan halogenase, Th-Hal, and isolated functioning halogenases with 100% efficiency. Lastly, we refactor the TF circuit to respond to the depletion of halogenated tryptophan and prototype a high-throughput biosensor-directed evolution scheme to screen for downstream enzyme variants capable of promiscuously converting halogenated tryptophan. Altogether, this work takes a significant step toward the rapid and higher throughput screening of halogenases and halo-tryptophan converting enzymes to further reinforce efforts to enable high-level bioproduction of halogenated chemicals.

中文翻译：

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基于荧光的卤代色氨酸工程酶筛选

定向进化通常受到精确筛选方法的通量的限制。在这里，我们展示了利用单一转录因子（TF）系统的可行性，该系统可以通过两种方式重构（既作为激活子又作为抑制子）。具体来说，我们展示了先前进化的 5-卤-或 6-卤-色氨酸特异性 TF 生物传感器的使用，适用于体内卤化色氨酸分子的检测。随后，我们验证了生物传感器在两种卤化酶特异性卤代色氨酸积累筛选中的实用性。首先，我们从卤化酶混合池中以 100% 的效率分离出 5-色氨酸卤化酶 XsHal。此后，我们生成了 6-色氨酸卤化酶、Th-Hal 催化残基的靶向文库，并以 100% 的效率分离出功能性卤化酶。最后，我们重构了 TF 电路以响应卤化色氨酸的消耗，并构建了高通量生物传感器引导的进化方案原型，以筛选能够混杂转化卤化色氨酸的下游酶变体。总而言之，这项工作朝着卤化酶和卤代色氨酸转化酶的快速和更高通量筛选迈出了重要一步，以进一步加强卤化化学品高水平生物生产的努力。