Fig. 3: Betalain biosynthesis-based reporters.

a Three betalain-based transcriptional reporter systems were constructed based on three different biosynthetic pathways. In all the reporters, DOPA dioxygenase (MjDOD) is the reporter gene that is controlled by the biosensor and other genes are constitutively expressed. The betanin reporter is based on the strain KM204, where BvCYP76AD1 hydroxylates tyrosine into L-DOPA that is subsequently oxidized to betalamic acid. This is then further oxidized by BvCYP6AD1 into cyclo-DOPA and glucosylated by DOPA-5-glucosyltransferase to cyclo-DOPA-glucoside. Finally, betalamic acid and cyclo-DOPA-glucoside spontaneously condense to make betanin. The betaxanthin reporter is based on strain KM205, where CYP76AD5 is used to hydroxylate tyrosine into L-DOPA, which is then further oxidized into betalamic acid. Betalamic acid will spontaneously react with amino acids available in the cells to make betaxanthins. The O-dianisidine-betacyanin reporter is also based on strain KM205. In this case, however, L-DOPA is directly fed to the cells and oxidized by MjDOD to betalamic acid, which subsequently reacts strongly with O-dianisidine to produce O-da-betacyanin. b The addition of supplements to strain KM205 modulates the color output. The addition of O-da results in the appearance of a red color. Color intensity is strongly increased upon supplementation with both O-da and L-DOPA. c The color produced by the O-da-betacyanin reporter can be easily detected by eye. Inducing the strain KM205 with a dilution series ranging from 10 pM to 1 μM CP55940 for 16 h and supplementing it with L-DOPA and O-da results in different intensities of red, where the effect of 100 pM CP55940 could still be detected by eye. d–f The output of the O-da-betacyanin reporter can be quantified by measuring the absorbance of the pigment at 520 nm. d A dose–response curve of the betacyanin reporter strain induced with CP55940 shows an apparent EC₅₀ of 7 nM. e For THC, the dose–response curve reveals an apparent EC₅₀ of 2 µM. f Dose response for JWH-018 shows an apparent EC₅₀ of 221 nM. For (d–f), data presented as mean + /− standard deviation. n = 3 biologically independent samples. Source data are provided in the Source Data file.