Establishment of a Visible Reporter System in Zymomonas mobilis through Random Mutagenesis and Rational Design of a Chromoprotein eforRed.

IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
ACS Synthetic Biology Pub Date : 2025-07-18 Epub Date: 2025-07-01 DOI:10.1021/acssynbio.4c00872
Xinyu Yang, Ning Yang, Siqi Wu, Shuche He, Wei Jiang, WeiXuan Zhong, Jun Du, Guimin Zhang, Xia Wang, Shihui Yang
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Abstract

The lack of reporter-gene systems that can measure the activities of biological parts quantitatively and qualitatively impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. Chromoproteins are homologues of fluorescent proteins, which are good reporter-gene candidates for their special absorption of natural light, making them visible to the naked eye without additional instrumentation. In this study, a fluorescent chromoprotein eforRed was selected to establish the visible reporter system in Zymomonas mobilis, a non-model ethanologenic Gram-negative bacterium with many excellent industrial properties to be developed as a biorefinery chassis for lignocellulosic biochemical production. Coupled with random error-prone PCR and protein rational design, the spectral characteristics of the eforRed chromoprotein were enhanced, particularly the fluorescence intensity. Mechanistic studies revealed that substitutions at the amino acid residues K201 and T24 situated on the surface region of eforRed, especially the double-mutant K201H-T24V, could optimize the protein optical characteristics by concurrently increasing intrinsic fluorescence and elevating protein expression levels. Finally, a visible reporter-gene system was successfully established in Z. mobilis by expressing the mutant eforRedK201H-T24V under a strong promoter like Pgap-4S or PT7 that gave colonies a visible red color for phenotypic screening and retained high fluorescence intensity for quantitative analysis. This study thus not only constructed a robust visible reporter-gene system in the non-model bacterium Z. mobilis but also helped expand the applications of chromoproteins in biotechnology and synthetic biology, especially in non-model microorganisms.

通过随机诱变建立运动单胞菌可见报告系统及合理设计一种色素蛋白。
缺乏能够定量和定性地测量生物部分活动的报告基因系统阻碍了强大细胞工厂的发展,以满足快速增长的生物制造的需求。色蛋白是荧光蛋白的同源物,荧光蛋白是很好的报告基因候选者,因为它们对自然光的特殊吸收,使它们在没有额外仪器的情况下肉眼可见。本研究选择荧光蛋白eforRed在运动酵母单胞菌(一种非模式产乙醇革兰氏阴性菌,具有许多优异的工业性能,可作为木质纤维素生化生产的生物炼制基础)中建立可见报告系统。结合随机易出错PCR和蛋白合理设计,增强了eforRed蛋白的光谱特征,特别是荧光强度。机制研究表明,在eforRed表面的氨基酸残基K201和T24上,尤其是双突变体K201H-T24V的取代可以通过增加内在荧光和提高蛋白质表达水平来优化蛋白质的光学特性。最后,通过在pgp - 4s或PT7等强启动子下表达突变体eforRedK201H-T24V,成功地在Z. mobilis中建立了可见报告基因体系,使菌落呈现可见红色,便于表型筛选,并保持高荧光强度,便于定量分析。因此,本研究不仅在非模式细菌Z. mobilis中构建了一个强大的可见报告基因系统,而且有助于扩大色素蛋白在生物技术和合成生物学中的应用,特别是在非模式微生物中的应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
8.00
自引率
10.60%
发文量
380
审稿时长
6-12 weeks
期刊介绍: The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.
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