Time-resolved analysis of Bacillus subtilis DB104 Spo0A-mutant transcriptome profile and enhancement of recombinant protein release

Abstract

Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruptionon sporulation have been extensively studied, a comprehensive understanding of thegenomic response throughout growth phases remain elusive. Here, we examined the transcriptomicchanges in Spo0A mutant strain, R211E, and wild-type across a time-course RNAseqto identify impacted biological processes and pathways. The R211E strain, which exhibitssporulation deficiency, was constructed using the clustered regularly interspaced short palindromicrepeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the criticalrole of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressedgenes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism,and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducingbiofilm formation and enhancing flagellar gene expression. Up-regulated metabolicpathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbersduring vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression,resulting in reduced cell viability in the stationary phase. We also introduce thenovel potential of R211E in a recombinant protein expression system that facilitated proteinrelease into the supernatant, providing valuable insight for future research in metabolic engineeringand efficient production systems in B. subtilis.