Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis

Area of Science:

• Microbiology
• Metabolic Engineering
• Synthetic Biology

Background:

• Bacillus licheniformis is a valuable microbial factory for biochemicals and enzymes.
• Limited genetic tools hinder metabolic engineering and gene function studies in B. licheniformis.

Purpose of the Study:

• To develop and validate an integrated CRISPR interference (CRISPRi) system for gene expression repression in B. licheniformis.
• To demonstrate the utility of the CRISPRi system for multiplex metabolic engineering to enhance L-valine production.

Main Methods:

• Construction of an integrated CRISPRi system in B. licheniformis.
• Testing CRISPRi efficiency on endogenous genes (yvmC, cypX, alsD, pta, ldh, rpsC) with repression efficiencies from 45.02% to 94.00%.
• Simultaneous repression of multiple genes, including those in by-product synthesis and L-valine degradation pathways (alsD, bcd).

Main Results:

• The CRISPRi system achieved high repression efficiencies for individual and multiple target genes.
• Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) increased L-valine titer by 90.48% and 80.09%, respectively.
• Combined gene repression in strain DW9i△leuA/pHYi-alsD-bcd resulted in 1.27-fold and 2.89-fold increases in L-valine titers in flask and bioreactor cultures, respectively.

Conclusions:

• The developed CRISPRi system is a feasible and effective tool for multiplex metabolic engineering in B. licheniformis.
• This system facilitates functional genome studies and optimization of biochemical production in B. licheniformis.