Abstract
Betalains are a class of natural pigments found in plants of the Caryophyllales order. Betanin is the dominant betalain on the food colour market, even though over 80 other variants are known. Recombinant production of betanin has recently gained interest as a cost-efficient and sustainable alternative to traditional plant extraction, but the production of other betalain variants remains largely unexplored. We selected three glucuronosyltransferases from Amaranthus hypochondriacus, Chenopodium quinoa and Celosia argentea var. cristata and screened the enzymes in vivo in betanin-producing Saccharomyces cerevisiae. Instead of producing amaranthin, two of the enzymes led to the formation of the betalain bougainvillein-rI (betanidin 5-O-β-sophoroside). When expressed together with a UDP-dehydrogenase that allowed the synthesis of UDP-glucuronic acid, each enzyme converted betanin to amaranthin. Integration of the glucuronosyltransferases in a Yarrowia lipolytica betanin producer strain directly resulted in amaranthin production. In fed-batch fermentation, 2.97 g/L ± 29.3 mg/L of amaranthin was produced. Co-expression of the glucuronosyltransferases with a malonyltransferase from Hylocereus polyrhizus led to the formation of 6'-O-malonyl-amaranthin in S. cerevisiae and Y. lipolytica. This study expands the portfolio of natural food colourants that can efficiently be produced through microbial fermentation and contributes to elucidating the biosynthesis pathway of betalains.