CRISPR-Cas9 mediated metabolic engineering of a mucoid Bacillus licheniformis isolate for mass production of 2,3-butanediol

Bacillus licheniformis is a bacterial strain generally recognized as safe (GRAS) with a tremendous potential for 2,3-butanediol (BDO) synthesis. Naturally isolated B. licheniformis usually secretes mucoid exopolymers, which hinder the strain development and metabolite production. A non-mucoid strain was generated by deleting both the pgsBCAE operon encoding polyglutamate synthase and the sacB gene encoding levansucrase using the CRISPR-Cas9 system. In addition, lactate, glycerol and ethanol were identified as the major byproducts of B. licheniformis. The corresponding genes including ldhA encoding lactate dehydrogenase, dgp encoding D-alpha-glycerophosphatase, and adhE encoding alcohol dehydrogenase were deleted, resulting in the generation of a byproduct-free strain via flask culture. The fed-batch fermentation yielded more than 120 g/L of 2,3-BDO with negligible amounts of other byproducts using a corn steep liquor (CSL)-based medium. This study is the first of its kind to demonstrate the development of non-mucoid and byproduct-free strains for mass production of 2,3-BDO using a naturally isolated B. licheniformis strain.