Engineering of Riboflavin Transporter and Enzymes in Bacillus subtilis Facilitates High-Efficiency FAD Synthesis from RF

IF 4.5 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-05-11 DOI:10.1021/acssynbio.6c00153

Yunlong Wu, Yuhao Sun, Huan Miao, Xiaole Xia, Ling Gao

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引用次数: 0

Abstract

The biosynthesis of flavin adenine dinucleotide (FAD), an essential redox coenzyme, is limited by inefficient substrate utilization and low catalytic activity. To address this, an “Uptake-Catalysis” system was constructed in Bacillus subtilis for FAD synthesis from riboflavin (RF). First, the Streptomyces davawensis transporter (RibM) was coexpressed with native FAD synthase (BsFADs) at a 2:1 ratio, which increased RF consumption to 51%. Subsequently, the isothermal compressibility perturbation engineering (ICPE) strategy was adopted, generating the optimum mutant BsFADs^P61W+R202Y, which increased catalytic efficiency (k_cat/K_m) toward RF and FMN by 83% and 73%, respectively. Mechanistic analysis revealed that this improvement was due to a 39% shortening of RF substrate channel. Finally, FAD titer peaked at 563 mg/L with a conversion rate (cr-FAD) of 92% in shaking-flask fermentation optimization. This work achieved a 225-fold increase in FAD production, validating the effectiveness of the uptake-catalysis synergistic strategy, thereby establishing an efficient and scalable synthesis paradigm.

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枯草芽孢杆菌核黄素转运体及酶工程促进RF高效合成FAD。

黄素腺嘌呤二核苷酸（FAD）是一种重要的氧化还原辅酶，其生物合成受到底物利用率低和催化活性低的限制。为解决这一问题，在枯草芽孢杆菌中构建了一个以核黄素（RF）为原料合成FAD的“摄取催化”体系。首先，将链霉菌（Streptomyces davawensis）转运体（RibM）与天然FAD合成酶（BsFADs）以2:1的比例共表达，使RF消耗增加到51%。随后，采用等温压缩扰动工程（ICPE）策略，生成最优突变体BsFADsP61W+R202Y，对RF和FMN的催化效率（kcat/Km）分别提高了83%和73%。机理分析表明，这种改进是由于射频衬底通道缩短了39%。在摇瓶发酵优化中，FAD效价最高达到563 mg/L，转化率（cr-FAD）为92%。这项工作使FAD的产量增加了225倍，证实了吸收催化协同策略的有效性，从而建立了一种高效和可扩展的合成范例。

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来源期刊

ACS Synthetic Biology 生物-

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.