An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose

The biosynthesis of benzylisoquinoline alkaloids such as morphine requires tyrosine oxidases, which are prone to overoxidation. A colorimetric readout that co-opts betaxanthin enzymes now enables discovery of an improved oxidase that, with other enzymes, makes reticuline in yeast. Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate ( S )-reticuline from glucose in Saccharomyces cerevisiae . To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L -3,4-dihydroxyphenylalanine ( L -DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L -DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L -tyrosine to ( S )-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae , will enable low-cost production of many high-value BIAs.