Transcriptome analysis reveals novel enzymes for apo-carotenoid biosynthesis in saffron and allows construction of a pathway for crocetin synthesis in yeast

: Crocus sativus is generally considered the source of saffron spice which is rich in apo-carotenoid compounds such as crocins, crocetin, picrocrocin, and safranal, which possess effective pharmacological activities. However, little is known about the exact genes involved in apo-carotenoid biosynthesis in saffron and the potential mechanism of specific accumulation in the stigma. In this study, we integrated stigmas at different developmental stages to perform in-depth transcriptome and dynamic metabolomic analyses to discover the potential key catalytic steps involved in apo-carotenoid biosynthesis in saffron. A total of 61 202 unigenes were obtained, and 28 regulators and 32 putative carotenogenic genes were captured after the co-expression network analysis. Moreover, 15 candidate genes were predicted to be closely related to safranal and crocin production, in which one aldehyde dehydrogenase (CsALDH3) was validated to oxidize crocetin dialdehyde into crocetin and a crocetin-producing yeast strain was created. In addition, a new branch pathway that catalyses the conversion of geranyl-geranyl pyrophosphate to copalol and ent-kaurene by the class II diterpene synthase CsCPS1 and three class I diterpene synthases CsEKL1/2/3 were investigated for the first time. Such gene to apo-carotenoid landscapes illuminate the synthetic charactersistics and regulators of apo-carotenoid biosynthesis, laying the foundation for a deep understanding of the biosynthesis mechanism and metabolic engineering of apo-carotenoids in plants or microbes.