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

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 plasmidless

super-secreting B. subtilis strains with industrial relevance.