Engineering of quorum-sensing systems for improved production of alkaline protease by Bacillus subtilis

Abstract

Aim: Engineering of Rap-Phr quorum-sensing systems of Bacillus subtilis and subsequent evaluation of the transcription of the aprE gene, encoding a major extracellular alkaline protease. Methods and Results: Addition of synthetic Phr pentapeptides to the growth medium, or overproduction of prePhr peptides, slightly improved the transcription of the aprE gene in B. subtilis. Disruption of certain rap genes similarly improved the transcription of the aprE gene. The production of extracellular proteolytic enzymes was increased when the rapA mutation was combined with a degU32 (Hy) mutation for hyper-secretion. Conclusions: Certain Rap-Phr systems of B. subtilis seem to suppress extracellular AprE production. Although this may be an important feature under natural conditions, repression of AprE production by these systems is not desirable under fermentation conditions. Significance and Impact of the Study: Although the levels of aprE transcriptional increase in this study are moderate, engineering of Rap-Phr systems may be used to improve the yield of Bacillus strains that are used for the production of the extracellular protease AprE, or Bacillus strains that use of the aprE promoter for the production of a heterologous protein.

A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

“…Finally, B. subtilis is commonly used commercially to produce secreted enzymes such as the protease AprE, and Rap-Phr systems have been the targets of studies designed to improve AprE expression, which is under AbrB control (56). In light of the fact that RapP functions to inhibit the phosphorelay, we note that the presence of rapP or the 80-kb rapP-containing plasmid could be counterproductive to the overexpression of AprE or other proteins whose expression is similarly regulated by the phosphorelay.…”

Forty Years in the Making: Understanding the Molecular Mechanism of Peptide Regulation in Bacterial Development

“…Thus, the myriad peptides present in the bacterial natural environment (theoretically 20 5 for a peptide of length 5), particularly in the mammalian host of a pathogen, could interfere with the physiology of microorganisms capable of importing them. Comparison of interaction kinetics could be used to define the determinants of specificity between Rap proteins and Phr peptides and may be exploited for the design of molecules that could modulate bacterial physiology for medical or industrial applications [32]. Conversely, given the distribution of TPR-containing proteins in eukaryotic cells, bacterial peptides like the Phr of pathogenic bacilli could exert signaling functions in the mammalian host [33],[34].…”

High copy number and highly stable Escherichia coli–Bacillus subtilis shuttle plasmids based on pWB980

“…The highest activity was 2738 U/mL with pUC980-2-pelN, which was nearly 3 times than that of pWB980. It also was the highest expression level in Bacillus [27,28]. It is worth noting that the plasmids pUC980-3, pUC980-4, pUC980-6 produced activities lower than 200 U/mL.…”

“…The alkaline protease activity was measured using the modified method described by Tjalsma et al [28]. One standard enzyme unit was defined as the as the amount of enzyme that hydrolyzes 1 μg casein minute under the situation of pH 9.0, 50 °C.…”