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

Abstract

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