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Characterization of a Protease Hyper-Productive Mutant of Bacillus pumilus by Comparative Genomic and Transcriptomic Analysis

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Abstract

Bacillus pumilus BA06 has great potential for the production of alkaline proteases. To improve the protease yield, classical mutagenesis to combine the physical and chemical mutagens was performed to obtain a protease hyper-productive mutant SCU11. The full genome sequences of BA06 and SCU11 strains were assembled through DNA sequencing using the PacBio sequencing platform. By comparative genomics analysis, 147 SNPs and 15 InDels were found between these two genomes, which lead to alternation of coding sequence in 15 genes. Noticeable, the gene (kinA) encoding sporulation kinase A is interrupted by introducing a stop codon in its coding region in BA06. Interestedly, this gene is reversely corrected in SCU11. Furthermore, comparative transcriptome analysis revealed that kinA and two positive regulatory genes (DegU and Spo0A) were upregulated in transcription in SCU11. In terms of the transcriptional data, upregulation of a phosphorylation cascade starting with KinA may enhance Spo0A phosphorylation, and thus activate expression of the gene aprE (encoding major extracellular protease) through repression of AbrB (a repressor of aprE) and activation of SinI, an antagonist of SinR (a repressor of aprE). In addition, the other genes involved in various metabolic pathways, especially of membrane transport and sporulation, were altered in transcription between these two strains. Conclusively, our transcriptome data suggested that upregulation degU and spo0A, as well as kinA, may at least partially contribute to the high production of alkaline protease in SCU11.

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Data Availability

The PacBio SMRT sequencing dataset of B. pumilus BA06 was deposited at NCBI SRA database (Accession no: PRJNA515162). The SMRT and Illumina sequencing datasets of B. pumilus SCU11 have been deposited at NCBI SRA database with accession numbers of PRJNA606253 and PRJNA515152, respectively. The RNA-seq reads of B. pumilus BA06 and SCU11 are available in NCBI SRA database with accession numbers of PRJNA475378 and PRJNA480562.

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Funding

This work was financially supported by a grant (2016NZ2016) from the Department of Sciences and Technology of Sichuan province. The funding sponsor had no role in the design of the study, in the collections, analysis, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.

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  1. College of Life Sciences, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, Sichuan University, Chengdu, 610064, Sichuan, People’s Republic of China

    Yong-Cheng Liu, Lin-Li Han, Tian-Yu Chen, Yan-Bing Lu & Hong Feng

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  1. Yong-Cheng Liu
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  2. Lin-Li Han
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HF and YCL conceived and designed the experiments; YCL, LLH, TYC, and YBL performed the experiments; YCL, YBL, and HF analyzed the data; HF and YCL wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Hong Feng.

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Liu, YC., Han, LL., Chen, TY. et al. Characterization of a Protease Hyper-Productive Mutant of Bacillus pumilus by Comparative Genomic and Transcriptomic Analysis. Curr Microbiol 77, 3612–3622 (2020). https://doi.org/10.1007/s00284-020-02154-5

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