Fig. 6.

Production of norTAs in engineered yeast. (A) Simplified schematic of the TA biosynthetic pathway extended using ATLASx for de novo production of norTAs in yeast. Dashed arrows indicate abbreviated pathway steps. H6H, hyoscyamine 6β-hydroxylase/dioxygenase; HsCYP, human liver cytochrome P450; CPR, yeast NADPH-CYP450 reductase (Ncp1p); CYB5, human cytochrome b5. (B and C) Effect of human liver CYP expression on titers and the molar production ratio of (B) norhyoscyamine and (C) norscopolamine in engineered yeast. CYPs or a BFP control were coexpressed with Ncp1p fused to human cytochrome b5 (HsCYB5A) from low-copy plasmids in CSY1324, and metabolites in supernatant were quantified by LC–MS/MS after 96-h growth in selective media buffered to pH 5.8 with 0.1 M potassium phosphate (B) or unbuffered (C). Data represent the mean of n = 3 biologically independent samples (open circles), and error bars show the SD. Student’s two-tailed t test: *P < 0.05, **P < 0.01, ***P < 0.001. (D) LC–MS/MS multiple reaction monitoring chromatograms for norTAs produced de novo by CSY1324 expressing HsCYP2D6 and cultured as in B and C, or authentic chemical standards. Chromatograms are representative of n = 3 biologically independent samples.