Pathway and protein channel engineering of Bacillus subtilis for improved production of desthiobiotin and biotin

Biotin (vitamin B₇) is a crucial cofactor for various metabolic processes and has significant applications in pharmaceuticals, cosmetics, and animal feed. <i>Bacillus subtilis</i>, a well-studied Gram-positive bacterium, presents a promising host for biotin production due to its Generally Recognized as Safe (GRAS) status, robust genetic tractability, and capacity for metabolite secretion. This study focuses on the metabolic engineering of <i>B</i>. <i>subtilis</i> to enhance biotin biosynthesis. Initially, the desthiobiotin (DTB) and biotin synthesis ability of different <i>B</i>. <i>subtilis</i> strains were evaluated to screen for suitable chassis cells. Subsequently, the titers of DTB and biotin were increased to 21.6 mg/L and 2.7 mg/L, respectively, by relieving the feedback repression of biotin synthesis and deleting the biotin uptake protein YhfU. Finally, through engineering the access tunnel to the active site of biotin synthase (BioB) for reactants and modulating its expression, the biotin titer was increased to 11.2 mg/L, marking an 1130-fold improvement compared to the wild-type strain. These findings provide novel strategies for enhancing the production of DTB and improving the conversion efficiency of DTB to biotin.