PT - JOURNAL ARTICLE AU - Lombe, Blaise Kimbadi AU - Zhou, Tingan AU - Caputi, Lorenzo AU - Ploss, Kerstin AU - O’Connor, Sarah E. TI - Biosynthetic Origin of the Methoxy Group in Quinine and Related Cinchona Alkaloids AID - 10.1101/2024.09.24.614729 DP - 2024 Jan 01 TA - bioRxiv PG - 2024.09.24.614729 4099 - http://biorxiv.org/content/early/2024/09/26/2024.09.24.614729.short 4100 - http://biorxiv.org/content/early/2024/09/26/2024.09.24.614729.full AB - Quinine is a historically important natural product containing a methoxy group that is assumed to be incorporated at a late pathway stage. Here we show that the methoxy group in quinine and related Cinchona alkaloids is introduced onto the starting substrate tryptamine. Feeding studies with Cinchona plantlets definitively show that 5-methoxytryptamine is utilized as a quinine biosynthetic intermediate in planta. We discover the biosynthetic genes that encode the responsible oxidase and methyltransferase, and we use these genes to reconstitute the early steps of the Cinchona alkaloid biosynthetic pathway in Nicotiana benthamiana to produce a mixture of methoxylated and desmethoxylated Cinchona alkaloid intermediates. Importantly, we show that the co-occurrence of both tryptamine and 5-methoxytryptamine substrates, along with the substrate promiscuity of downstream pathway enzymes, enable parallel formation of both methoxylated and desmethoxylated alkaloids in Cinchona pubescens.Competing Interest StatementThe authors have declared no competing interest.