Development of biosensors highly responsive to <i>N</i>-acetylneuraminic acid in <i>Bacillus subtilis</i>.

Jiaqi Sun; Yanting Cao; Xueqin LÜ; Jianghua LI; Long Liu; Guocheng DU; Jian Chen; Yanfeng Liu

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Development of biosensors highly responsive to N-acetylneuraminic acid in Bacillus subtilis.

Author: Jiaqi SUN ¹ ; Yanting CAO ¹ ; Xueqin LÜ ¹ ; Jianghua LI ¹ ; Long LIU ¹ ; Guocheng DU ¹ ; Jian CHEN ¹ ; Yanfeng LIU ¹
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1. Key Laboratory of Sugar Chemistry and Biotechnology, Ministry of Education, College of Bioengineering, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: Bacillus subtilis; N-acetylneuraminic acid; biosensor; transcription factor
MeSH: N-Acetylneuraminic Acid/metabolism*; Bacillus subtilis/metabolism*; Promoter Regions, Genetic/genetics*; Binding Sites; Biosensing Techniques
From: Chinese Journal of Biotechnology 2023;39(5):2502-2516
CountryChina
Language:Chinese
Abstract: Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.