Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using beta-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Automated summary

In this study, eight enzymes involved in the biosynthesis of crocins were identified in two Verbascum species. Four of them were similar to enzymes found in Buddleja and were identified to be involved in crocin biosynthesis. These enzymes were able to efficiently produce crocins using beta-carotene and zeaxanthin as substrates in bacterial and plant systems. The discovery of these enzymes, which have a broad substrate spectrum, provides new opportunities for biotechnological production of apocarotenoids.