Opera Medica et Physiologica

CRISPR/Cas9-Redacted Bacillus Subtilis Strain as the Producer of Extracellular Metalloproteinase of B. Pumilus

Published ahead of print September 22, 2022; Printed September 23, 2022; OM&P 2022 Volume 9 Issue 3, pages 121-127; doi:10.24412/2500-2295-2022-3-121-127
Abstract: 

B. pumilus metalloproteinase was firstly isolated and characterized by Kazan Federal University scientists. Primary structure analysis showed that the novel enzyme has no analogs among prokaryotic enzymes and occupies an intermediate position between two large families of the metzinkin clan metalloproteinases – adamalysins and astacins. These families are mainly represented by eukaryotic enzymes, which play an important role in human life and health. A more detailed study of the structure and functions of novel metalloproteinase requires an efficient expression system. B. pumilus metalloproteinase gene (mprBp) was cloned into the pGP382 expression vector under a strong constitutive promoter of the degQ36 gene (PdegQ36). The resulting construct was used to transform B. subtilis Δ6 strain. This strain was constructed by CRISPR/Cas9 genome editing technology with deletion of some prophage genes of B. subtilis 168. The functional role of prophage genes is poorly understood. It is possible that prophage deletion will increase the expression of secreted enzymes. For the transformed strain we determined the dynamics of growth and accumulation of proteolytic activity by hydrolysis of azocasein. The dynamics of proteolytic activity accumulation by this strain has a different character in contrast to the protease-deficient strains carrying the gene of the investigated enzyme. The result of this work was to obtain an effective producer strain of adamalizin-like metalloproteinase of B. pumilus, which can be used in the production of the enzyme for subsequent studies.