Engineering a cpGFP-based biosensor for enhanced quantification of glycolate production in Escherichia coli.

IF 5.6 1区化学 Q1 CHEMISTRY, ANALYTICAL Talanta Pub Date : 2025-05-01 Epub Date: 2025-01-13 DOI:10.1016/j.talanta.2025.127529

Qiwei Wang, Zhifen Huang, Sen Ma, Mingxue Ma, Sheng Ye, Si Liu

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Abstract

The growing demand for glycolate, fueled by economic development, requires the advancement of production methods. Escherichia coli (E. coli), a preferred host for glycolate production, has undergone extensive metabolic engineering to improve yield. Developing rapid and precise methods for quantifying glycolate concentration is essential for screening high-yielding strains. Here, we present the engineering of a novel circularly permuted green fluorescent protein (cpGFP)-based glycolate sensor, termed GLYCO. GLYCO exhibits high specificity (minimal interference from other metabolites), stability (no decrease in performance after 15 days at -80 °C), and ease of detection via fluorescence measurement, enabling effective in vitro glycolate quantification. GLYCO spans a quantification range from 10 μM to 1 mM, facilitating effective monitoring of glycolate production in metabolically engineered E. coli strains. This biosensor represents a significant advancement in the metabolic engineering toolkit, facilitating efficient detection and optimization of glycolate production in E. coli, with potential applications in industrial biotechnology.

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设计一种基于cpgfp的生物传感器，用于增强大肠杆菌中乙醇酸生产的定量。

在经济发展的推动下，对乙醇酸的需求不断增长，要求生产方法的进步。大肠杆菌（E. coli）是乙醇酸生产的首选宿主，它经过了广泛的代谢工程来提高产量。发展快速、精确的乙醇酸浓度定量方法是筛选高产菌株的必要条件。在这里，我们提出了一种新的循环排列绿色荧光蛋白（cpGFP）为基础的乙醇酸传感器的工程，称为GLYCO。GLYCO具有高特异性（来自其他代谢物的干扰最小），稳定性（在-80°C下15天后性能不下降），易于通过荧光测量检测，能够有效地进行体外乙醇酸定量。GLYCO的定量范围从10 μM到1 mM，有助于有效监测代谢工程大肠杆菌菌株的乙醇酸生产。这种生物传感器代表了代谢工程工具包的重大进步，促进了大肠杆菌中乙醇酸生产的有效检测和优化，具有潜在的工业生物技术应用前景。

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

Talanta 化学-分析化学

CiteScore

12.30

自引率

4.90%

发文量

861

审稿时长

29 days

期刊介绍： Talanta provides a forum for the publication of original research papers, short communications, and critical reviews in all branches of pure and applied analytical chemistry. Papers are evaluated based on established guidelines, including the fundamental nature of the study, scientific novelty, substantial improvement or advantage over existing technology or methods, and demonstrated analytical applicability. Original research papers on fundamental studies, and on novel sensor and instrumentation developments, are encouraged. Novel or improved applications in areas such as clinical and biological chemistry, environmental analysis, geochemistry, materials science and engineering, and analytical platforms for omics development are welcome. Analytical performance of methods should be determined, including interference and matrix effects, and methods should be validated by comparison with a standard method, or analysis of a certified reference material. Simple spiking recoveries may not be sufficient. The developed method should especially comprise information on selectivity, sensitivity, detection limits, accuracy, and reliability. However, applying official validation or robustness studies to a routine method or technique does not necessarily constitute novelty. Proper statistical treatment of the data should be provided. Relevant literature should be cited, including related publications by the authors, and authors should discuss how their proposed methodology compares with previously reported methods.