Exploring and Engineering Novel Strong Promoters for High-Level Protein Expression in Bacillus subtilis DB104 through Transcriptome Analysis.

Bacillus subtilis is widely employed for recombinant protein expression. B. subtilis DB104 offers a distinct advantage as a protein expression host because it is an extracellular protease-deficient derivative of B. subtilis 168. We have conducted a time-course transcriptome analysis of B. subtilis DB104 in a prior study. In the present study, we identified 10 genes that exhibited strong expression at each time point or all, based on transcriptome data. Subsequently, we assessed the strength of 12 promoters that transcribe these genes using enhanced green fluorescent protein (eGFP) as a reporter. Among these promoters, Psdp and PskfA had the highest expression levels. At 24 h, these two promoters exhibited 34.5- and 38.8-fold higher strength, respectively, than the strength of P43, the control promoter. Consequently, these two promoters were selected for further development. We enhanced these promoters by optimizing spacer length, promoter sequence, Shine-Dalgarno sequence, regulator binding sites, and terminator sequences. As a result, we successfully engineered the most potent protein expression cassette, Psdp-4, which exhibited a 3.84-fold increase in strength compared to the original Psdp promoter. Furthermore, we constructed an expression cassette for a human epidermal growth factor (hEGF) using Psdp-4 to evaluate its general application. The expression level of His tagged hEGF, quantified using ImageJ analysis and applied to SDS-PAGE, reached the highest yield of 103.9 µg/mL under the control of Psdp-4 at 24 h. The expressed hEGF protein was purified, and its bioactivity was confirmed through a cell proliferation assay using HT-29 cells. Our work demonstrates the construction of a highly efficient expression system for B. subtilis DB104 based on transcriptome data and promoter engineering. This system enables rapid, inducer-free protein expression within 24 h. It can be used as a valuable tool for various industrial applications.

Time-Course Transcriptome Analysis of Bacillus subtilis DB104 during Growth.

Bacillus subtilis DB104, an extracellular protease-deficient derivative of B. subtilis 168, is widely used for recombinant protein expression. An understanding of the changes in gene expression during growth is essential for the commercial use of bacterial strains. Transcriptome and proteome analyses are ideal methods to study the genomic response of microorganisms. In this study, transcriptome analysis was performed to monitor changes in the gene expression level of B. subtilis DB104 while growing on a complete medium. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, K-mean cluster analysis, gene ontology (GO) enrichment analysis, and the function of sigma factors were used to divide 2122 differentially expressed genes (DEGs) into 10 clusters and identified gene functions according to expression patterns. The results of KEGG pathway analysis indicated that ABC transporter is down-regulated during exponential growth and metabolic changes occur at the transition point where sporulation starts. At this point, several stress response genes were also turned on. The genes involved in the lipid catabolic process were up-regulated briefly at 15 h as an outcome of the programmed cell death that postpones sporulation. The results suggest that changes in the gene expression of B. subtilis DB104 were dependent on the initiation of sporulation. However, the expression timing of the spore coat gene was only affected by the relevant sigma factor. This study can help to understand gene expression and regulatory mechanisms in B. subtilis species by providing an overall view of transcriptional changes during the growth of B. subtilis DB104.

Transcriptome Analysis Reveals Immunomodulatory Effect of Spore-Displayed p75 on Human Intestinal Epithelial Caco-2 Cells.

Lacticaseibacillus rhamnosus GG (LGG) can promote intestinal health by modulating the immune responses of the gastrointestinal tract. However, knowledge about the immunomodulatory action of LGG-derived soluble factors is limited. In our previous study, we have displayed LGG-derived p75 protein on the spore surface of Bacillus subtilis. The objective of this study was to determine the effect of spore-displayed p75 (CotG-p75) on immune system by investigating transcriptional response of Caco-2 cells stimulated by CotG-p75 through RNA-sequencing (RNA-seq). RNA-seq results showed that CotG-p75 mainly stimulated genes involved in biological processes, such as response to stimulus, immune regulation, and chemotaxis. KEGG pathway analysis suggested that many genes activated by CotG-p75 were involved in NF-ĸB signaling and chemokine signaling pathways. CotG-p75 increased cytokines and chemokines such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, CCL20, CCL22, and IL1B essential for the immune system. In particular, CotG-p75 increased the expression levels of NF-ĸB-related genes such as NFKBIA, TNFAIP3, BIRC3, NFKB2, and RELB involved in immune and inflammatory responses. This study provides genes and pathways involved in immune responses influenced by CotG-p75. These comprehensive transcriptome profiling could be used to elucidate the immunomodulatory action of CotG-p75.

Surface display of p75, a Lactobacillus rhamnosus GG derived protein, on Bacillus subtilis spores and its antibacterial activity against Listeria monocytogenes.

Lactobacillus rhamnosus p75 protein with peptidoglycan hydrolase (PGH) activity is one of the key molecules exhibiting anti-apoptotic and cell-protective activity for human intestinal epithelial cells. In this study, with the goal of developing new probiotics, the p75 protein was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchoring motif. The PGH activity, stability, and the antibacterial activity of the spore-displayed p75 (CotG-p75) protein were also investigated. The PGH activity of the CotG-p75 against peptidoglycan extracted from B. subtilis was confirmed by the ninhydrin test. Under various harsh conditions, compared to the control groups, the PGH activities of CotG-p75 were very stable in the range of pH 3-7 and maintained at 70% at 50 °C. In addition, the antibacterial activity of CotG-p75 against Listeria monocytogenes was evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells of L. monocytogenes by up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall of L. monocytogenes was partially damaged by the treatment with CotG-p75. Our preliminary results show that CotG-p75 could be a good candidate for further research to develop new genetically engineered probiotics.