Kardashliev Tsvetan, Weingartner Alexandra, Romero Elvira, Schwaneberg Ulrich, Fraaije Marco, Panke Sven, Held Martin
Department of Biosystems Science and Engineering ETH Zurich, Mattenstrasse 26 4058 Basel Switzerland
Institute of Biotechnology, RWTH Aachen University Worringerweg 3 52074 Aachen Germany.
Chem Sci. 2021 Oct 29;12(44):14766-14772. doi: 10.1039/d1sc02578c. eCollection 2021 Nov 17.
Biocatalysis is increasingly used for synthetic purposes in the chemical and especially the pharmaceutical industry. Enzyme discovery and optimization which is frequently needed to improve biocatalytic performance rely on high-throughput methods for activity determination. These methods should ideally be generic and applicable to entire enzyme families. Hydrogen peroxide (HO) is a product of several biocatalytic oxidations and its formation can serve as a proxy for oxidative activity. We designed a genetically encoded sensor for activity measurement of oxidative biocatalysts the amount of intracellularly-formed HO. A key component of the sensor is an HO-sensitive transcriptional regulator, OxyR, which is used to control the expression levels of fluorescent proteins. We employed the OxyR sensor to monitor the oxidation of glycerol to glyceraldehyde and of toluene to -cresol catalysed by recombinant expressing an alcohol oxidase and a P450 monooxygenase, respectively. In case of the P450 BM3-catalysed reaction, we additionally monitored -cresol formation a second genetically encoded sensor based on the phenol-sensitive transcriptional activator, DmpR, and an orthogonal fluorescent reporter protein. Single round screens of mutant libraries by flow cytometry or by visual inspection of colonies on agar plates yielded significantly improved oxidase and oxygenase variants thus exemplifying the suitability of the sensor system to accurately assess whole-cell oxidations in a high-throughput manner.
生物催化在化学工业尤其是制药工业中越来越多地用于合成目的。为提高生物催化性能而经常需要进行的酶发现和优化依赖于用于活性测定的高通量方法。理想情况下，这些方法应具有通用性，适用于整个酶家族。过氧化氢（HO）是几种生物催化氧化反应的产物，其形成可作为氧化活性的替代指标。我们设计了一种用于测量氧化生物催化剂活性（细胞内形成的HO量）的基因编码传感器。该传感器的一个关键组件是对HO敏感的转录调节因子OxyR，它用于控制荧光蛋白的表达水平。我们使用OxyR传感器监测了分别表达醇氧化酶和P450单加氧酶的重组体催化甘油氧化为甘油醛以及甲苯氧化为对甲酚的过程。在P450 BM3催化的反应中，我们还基于对苯酚敏感的转录激活因子DmpR和正交荧光报告蛋白，使用了第二个基因编码传感器监测对甲酚的形成。通过流式细胞术或通过目视检查琼脂平板上的菌落对突变文库进行单轮筛选，得到了显著改进的氧化酶和加氧酶变体，从而证明了该传感器系统适用于以高通量方式准确评估全细胞氧化反应。
