Mining, identification, and application of nitrogen-responsive promoters in Bacillus licheniformis

Abstract

Promoters are crucial expression elements in synthetic biology, and Bacillus licheniformis serves as an excellent chassis cell for industrial production. However, the diversity and quantity of available promoter elements remain particularly limited. Existing promoters are categorized into constitutive and carbon source-inducible types, both exhibiting deficiencies in transcriptional strength and diversity of environmental signal responsiveness. As essential nutrients for microbial growth comparable to carbon sources, nitrogen sources hold significance. The development of nitrogen source-responsive promoters is vital for enriching synthetic biology toolkits and enhancing chassis cell performance. This study initially predicted nitrogen-responsive promoter elements through genomic analysis. Using enhanced green fluorescent protein (egfp) as a reporter gene, transcriptional initiation characteristics were evaluated. Results demonstrated that PglnR and Pgcv promoters could initiate transcription in response to sodium glutamate, with transcriptional intensities 200% and 100% higher than the control group at 36 h. Subsequently, these screened promoters (PglnR and Pgcv) were employed to mediate the expression of transglutaminase gene from Streptomyces mobaraensis. Under optimized conditions (37 °C, 10 g/L soluble starch, and 10 g/L glutamine), recombinant strains exhibited enhanced secretory expression. The maximum extracellular enzyme activity reached 2.61 U/mL. In fed-batch fermentation using a 5-L glass fermentor, the BL-TG3 recombinant strain achieved peak enzyme activity of 14.7 U/mL. The discovery, characterization, and application of novel nitrogen-responsive promoters establish a foundation for optimizing B. licheniformis expression systems.

Keywords

Promoter / Bacillus licheniformis / Transglutaminase / Nitrogen source / Expression system

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Funding

Key Technologies Research and Development Program(2020YFA0907700)

the Basic Research Program of Jiangsu and supported by the Jiangsu Basic Research Center for Synthetic Biology(BK20233003)

Wuxi Industrial Innovation Research Institute Pilot Technology Pre-research Project(XD24024)

the National Natural Foundation of China(32172174)

Program for Jiangsu Provincial Excellent Scientific and Technological Innovation Team(2024ZB371)

RIGHTS & PERMISSIONS

Jiangnan University

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