Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering

Monascus pigments have various food industry applications and are pharmacologically active. Genome sequencing-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been implemented to increase pigment production in Monascus. To increase pigment production in M. ruber KACC46666, the CRISPR/Cas9 system was used to introduce mutations in two negative regulator genes (MpigI and MpigI'), among other genes involved in the Monascus pigment biosynthesis pathway. Dual single-guide RNAs were constructed to inactivate MpigI and MpigI'. After CRISPR/Cas9 inactivation, yellow, orange, and red pigment expression in the resulting △MpigI16-7 strain (among several Cas9-mediated mutants studied) was 2.5-, 12.4-, and 18.5-fold, respectively, higher than that in the wild-type strain. This study provides valuable information regarding CRISPR-guided metabolic engineering for natural colorant production.