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

# metadata.dc.description.abstract[].value
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 plasmidless

super-secreting B. subtilis strains with industrial relevance.

# metadata.dc.relation.ispartof[].value
International Journal of Molecular Sciences, 2024, vol. 13, p. 6957

# metadata.dc.source[].value
Articles publicats en revistes (Biologia, Sanitat i Medi Ambient)

# metadata.dc.title[].value
The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System