The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System.

Achieving commercially significant yields of recombinant proteins in Bacillus subtilis requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the B. subtilis chromosome. Once a suitable promoter was selected, the expression levels of amyQ were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single amyQ gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting B. subtilis strains with industrial relevance.