Metabolic engineering of Bacillus subtilis toward the efficient and stable production of C₃₀-carotenoids

Commercial carotenoid production is dominated by chemical synthesis and plant extraction, both of which are unsustainable and can be detrimental to the environment. A promising alternative for the mass production of carotenoids from both an ecological and commercial perspective is microbial synthesis. To date, C₃₀ carotenoid production in Bacillus subtilis has been achieved using plasmid systems for the overexpression of biosynthetic enzymes. In the present study, we employed a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system to develop an efficient, safe, and stable C₃₀ carotenoid-producing B. subtilis strain, devoid of plasmids and antibiotic selection markers. To this end, the expression levels of crtM (dehydrosqualene synthase) and crtN (dehydrosqualene desaturase) genes from Staphylococcus aureus were upregulated by the insertion of three gene copies into the chromosome of B. subtilis. Subsequently, the supply of the C₃₀ carotenoid precursor farnesyl diphosphate (FPP), which is the substrate for CrtMN enzymes, was enhanced by expressing chromosomally integrated Bacillus megaterium-derived farnesyl diphosphate synthase (FPPS), a key enzyme in the FPP pathway, and abolishing the expression of farnesyl diphosphate phosphatase (YisP), an enzyme responsible for the undesired conversion of FPP to farnesol. The consecutive combination of these features resulted in a stepwise increased production of C₃₀ carotenoids. For the first time, a B. subtilis strain that can endogenously produce C₃₀ carotenoids has been constructed, which we anticipate will serve as a chassis for further metabolic engineering and fermentation optimization aimed at developing a commercial scale bioproduction process.