CcCHIL, a type IV chalcone isomerase that can improve (2S)-naringenin production in Saccharomyces cerevisiae

(2S)-Naringenin, a crucial precursor in plant flavonoid synthesis, holds significance in diverse biological processes and potential therapeutic applications for human diseases. The economically valuable Citrus reticulata 'Chachi' citrus cultivar, renowned for its flavonoid-rich peel, was subjected to integrated transcriptomic and metabolomic analysis in this study to reveal patterns of flavonoid accumulation. The analysis revealed a strong correlation between the expression pattern of the gene encoding type IV chalcone isomerase (chalcone isomerase like, CHIL), CcCHIL, and the accumulation of flavonoids in C. reticulata 'Chachi' peel. Previous studies have demonstrated that type IV chalcone isomerase can enhance flavonoid accumulation in citrus and improve the catalytic efficiency of chalcone synthase in vitro, thereby increasing the titer of (2S)-naringenin. In this study, we constructed a genetically engineered yeast strain capable of de novo synthesis of (2S)-naringenin. We found that CcCHIL can increase (2S)-naringenin production in engineered yeast by 13.60 %. Subsequently, we conducted experiments using CHILs from various species and found that GmCHIL from Glycine max can increase (2S)-naringenin production in engineered yeast by 45.35 %. Sequence alignment, molecular docking predictions and site-directed mutagenesis showed that Asn151 is one of the key sites for CHIL improving (2S)-naringenin production. Our study unveiled CcCHIL's crucial role in flavonoid biosynthesis in C. reticulata 'Chachi' peel and paving the way for future synthesis of intricate and economically valuable flavonoids.