Development of a simplified and generalized CRISPR-Cas9 methodology for the optimization of the production of enzymes of industrial interest by bacteria of the genus Bacillus

Abstract

The subtilisin serine protease alkaline (AprE) is an important exoprotease enzyme produced by Bacillus subtilis that has important biotechnological applications. In this sense, this project intends to establish a generalized methodology based on CRISPR for genome editing of Bacillus subtilis guided by gRNA using the Case system to carry out targeted mutation in the aprE promoter in order to prevent repressor proteins from identifying their region of binding in the promoter, and with that does not occur the repression of the expression of this protease. For this, we will use a commercial Cas9, with the ability of cleavage without increased specificity, that will be complexed to the chosen sequences (gRNA transcribed in vitro). The introduction of the Cas9 complex and gRNA created in vitro into the B. subtilis cell will be via electroporation. This is one of the differentials of this project: to establish the conditions for using the in vitro ribonucleoprotein complex to genetically modify the Bacillus subtilis strain in order to repress expression of the aprE gene. This system differs from that based on plasmids or viruses with respect to how quickly the nuclease can act since the components do not need to be expressed. To confirm the generality of the methodology, it will be tested in Bacillus amylloliquefasciens FZB42. After obtaining the appropriate mutants, statistical methodologies of process optimization will be performed to direct the physiology of the strains in order to further increase the production of the protein of interest.