Use of an enzyme-coupled biosensor to engineer a BIA fermentation pathway from glucose in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) represent a large family of natural products rich in potential bioactivities including analgesics, antitumor candidates, antitussives, and antibiotic.  There have been ample recent advances in engineering S. cerevisiae strains that can convert fed intermediate reticuline into multiple products of interest, including morphine, codeine, noscapine, and dihydrosanguinarine in S. cerevisiae.  These pathways contain numerous P450 enzymes, a class of enzyme that frequently demonstrate superior expression in eukaryotic hosts.  However, direct fermentation of reticuline from central metabolism in yeast requires an elusive tyrosine hydroxylase activity to produce L-DOPA.  To identify an enzyme capable of catalyzing this activity, we constructed an enzyme-coupled biosensor: production of a colored, fluorescent metabolite in the presence of L-DOPA.  Further, we used this screenable phenotype to isolate a mutant that preferentially performs the desired hydroxylation by lowering the undesired further oxidation of L-DOPA to the melanin biosynthetic pathway.  Replacing the enzyme for biosensor metabolite production with the reticuline biosynthetic enzymes enabled the production of this major BIA branchpoint intermediate.