A novel P450 enzyme assay utilizing an NADP⁺-based biosensor.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2025-07-16 DOI:10.1007/s10529-025-03599-z

Sifan Shangguan, Taichang Wang, Di Zhao, Guobin Zhang, Yisang Zhang, Ruiming Wang, Junqing Wang, Jing Su

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引用次数: 0

Abstract

Purpose: High-throughput screening methods for cytochrome P450 enzymes (P450s), such as colorimetric, mass spectrometric, and fluorescence-based assays, often face limitations in throughput, real-time monitoring, and versatility.

Methods: To address these challenges, we developed a novel biosensor leveraging glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation for real-time monitoring of intracellular NADP⁺ levels, enabling P450 activity detection. The sensor was applied to monitor P450 activity by tracking intracellular NADP⁺ dynamics, as P450s catalyze diverse substrate reactions and convert NADPH to NADP⁺ via their electron transport system. To enhance detection precision, intracellular NADP⁺ synthesis was reduced by knocking down NADPH-dependent aldehyde reductase (YqhD), minimizing background fluorescence interference.

Results: The sensor exhibited a linear NADP⁺ detection range of 1 μM to 10 mM, suitable for P450 assays. The sensor's performance was validated by comparing P450 activities in engineered strains with traditional gas chromatography.

Conclusion: The developed biosensor demonstrates its potential as a robust, real-time screening tool for P450 enzyme studies.

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利用NADP+为基础的生物传感器的新型P450酶测定。

目的：细胞色素P450酶（P450）的高通量筛选方法，如比色法、质谱法和荧光法，通常面临通量、实时监测和通用性的限制。为了解决这些挑战，我们开发了一种新型的生物传感器，利用葡萄糖-6-磷酸脱氢酶和双分子荧光互补来实时监测细胞内NADP+水平，从而实现P450活性检测。该传感器通过跟踪细胞内NADP+动态来监测P450的活性，因为P450催化多种底物反应并通过其电子传递系统将NADPH转化为NADP+。为了提高检测精度，通过敲除nadph依赖性醛还原酶（YqhD）来减少细胞内NADP+的合成，最大限度地减少背景荧光干扰。结果：该传感器具有1 μM ~ 10 mM的线性NADP+检测范围，适用于P450的检测。通过与传统气相色谱法比较工程菌株的P450活性，验证了该传感器的性能。结论：所开发的生物传感器显示了其作为P450酶研究的强大实时筛选工具的潜力。

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

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.