Natural CCD2 Variants and RNA Interference for Boosting Crocin Biosynthesis in Tomato

Crocin biosynthesis involves a complex network of enzymes with biosynthetic and modifier enzymes, and the manipulation of these pathways holds promise for improving human health through the broad exploitation of these bioactive metabolites. Crocins play a significant role in human nutrition and health, as they exhibit antioxidant and anti-inflammatory activity. Plants that naturally accumulate high levels of crocins are scarce, and the production of crocins is highly limited by the characteristics of the crops and their yield. The CCD2 enzyme, initially identified in saffron, is responsible for converting zeaxanthin into crocetin, which is further modified to crocins by aldehyde dehydrogenases and glucosyltransferase enzymes. Crops like tomato fruits, which naturally contain high levels of carotenoids, offer valuable genetic resources for expanding synthetic biology tools. In an effort to explore CCD2 enzymes with improved activity, two CCD2 alleles from saffron and Crocosmia were introduced into tomato, together with a UGT gene. Furthermore, in order to increase the zeaxanthin pool in the fruit, an RNA interference construct was introduced to limit the conversion of zeaxanthin to violaxanthin. The expression of saffron CCD2, CsCCDD2L, led to the creation of transgenic tomatoes with significantly high crocins levels, reaching concentrations of 4.7 mg/g dry weight. The Crocosmia allele, CroCCD2, also resulted in high crocins levels, reaching a concentration of 2.1 mg/g dry weight. These findings underscore the importance of enzyme variants in synthetic biology, as they enable the development of crops rich in beneficial apocarotenoids.