Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
Department of Genome Sciences, University of Washington, Seattle, Washington 98195, United States.
ACS Synth Biol. 2020 Dec 18;9(12):3408-3415. doi: 10.1021/acssynbio.0c00348. Epub 2020 Nov 12.
Genetic modifications of living organisms and proteins are made possible by a catalogue of molecular and synthetic biology tools, yet proper screening assays for genetic variants of interest continue to lag behind. Synthetic growth-coupling (GC) of enzyme activities offers a simple, inexpensive way to track such improvements. In this follow-up study we present the optimization of a recently established GC design for screening of heterologous methyltransferases (MTases) and related pathways in the yeast . Specifically, upon testing different media compositions and genetic backgrounds, improved GC of different heterologous MTase activities is obtained. Furthermore, we demonstrate the strength of the system by screening a library of catechol O-MTase variants converting protocatechuic acid into vanillic acid. We demonstrated high correlation ( = 0.775) between vanillic acid and cell density as a proxy for MTase activity. We envision that the improved MTase GC can aid evolution-guided optimization of biobased production processes for methylated compounds with yeast in the future.
通过一系列分子和合成生物学工具，可以实现对生物体和蛋白质的遗传修饰，但对于感兴趣的遗传变异体的适当筛选检测仍然滞后。酶活性的合成生长偶联（GC）为跟踪此类改进提供了一种简单、廉价的方法。在这项后续研究中，我们对最近建立的用于筛选酵母中异源甲基转移酶（MTase）和相关途径的 GC 设计进行了优化。具体来说，在测试不同的培养基组成和遗传背景后，获得了不同异源 MTase 活性的改进 GC。此外，我们通过筛选将原儿茶酸转化为香草酸的儿茶酚 O-甲基转移酶变体文库来证明该系统的优势。我们证明了香草酸与细胞密度（作为 MTase 活性的替代物）之间具有高度相关性（ = 0.775）。我们设想，改进的 MTase GC 可以在未来帮助基于酵母的甲基化化合物生物基生产过程的进化引导优化。
