Multi-omics reveal the molecular basis of LrAN2 overexpression in black goji callus promoting the excessive accumulation of petanin, a promising food blue colorant

Lycium ruthenicum fruit (LRF) is a potential source of natural colorant in a wide pH range due to richness in monoacylated anthocyanin petanin. However, the yield of LRF largely lags behind the market demand in food industry. In this study, overexpressing LrAN2 (LrAN2OE), an anthocyanin activator in LRF, produced excessive acylated anthocyanins including petanin in L. ruthenicum callus (LRC). Physiological analysis indicates that LrAN2OE extensively orchestrate the redox homeostasis, including antioxidant enzymes system and antioxidants with low molecular weight. KEGG enrichment analysis indicates that LrAN2 hierarchically orchestrate the acylated anthocyanin biosynthesis in LRC at multi-omics level, such as small RNAome, transcriptome, and metabolome. The anthocyanin yield of LrAN2OE callus cultured by 5% sucrose or glucose is significantly enhanced. Furthermore, the anthocyanin yield approach to 96.23 mg/g dry weight, approximately 3.5 folds of that in LRF, when LrAN2OE suspension cells are cultured in liquid MS medium supplement with 5% sucrose and 100 mM NaCl. Notably, anthocyanins extracted from LrAN2OE callus are stable in pH 1.0–2.0, pH 7.0, and pH 9.0, showing red or blue color within 30 days at 4°C. Our findings suggest that LrAN2 is perfect gene resource for metabolic engineering of acylated anthocyanins in LRC, a promising chassis producing acylated anthocyanins. Taken together, LRC metabolically engineered by LrAN2 is a potential source of natural food color, which facilitates to make up the shortfall of LRF as source of natural colorant in future food industry.