Article
Extended Data Fig. 10 | Time courses of de novo TA and precursor
production in pseudo-fed-batch cultures of CSY1297 and CSY1298. a–e,
TA-producing strains CSY1297 and CSY1298 (with pCS4213) were respectively
cultured in non-selective or selective (leucine dropout) media with 50 mM
2-OG and 15 mg l−1 Fe2+ under pseudo-fed-batch conditions at 25 °C for 120 h.
Culture supernatants were sampled for PLA (a), tropine (b), hyoscyamine (c)
and scopolamine (d) titres by LC–MS/MS analysis and optical density at 600 nm
(OD 600 ) (e) every 24 h. Cultures were supplemented with additional dextrose,
glycerol and amino acids to final concentrations of 2%, 2% and 1× at 72 h
(vertical dotted line). Data indicate the mean of n = 3 biologically independent
samples and error bars denote s.d. Note that no littorine accumulation was
observed in any samples. f–i, LC–MS/MS verification of de novo medicinal TA
production in CSY1297 and CSY1298. Panels show LC–MS/MS chromatograms
of hyoscyamine (f, g) and scopolamine (h, i) detected in CSY1297 and CSY1298
cultures grown in non-selective medium or selective medium, respectively
(samples from experiment in c and d, 120 h), and of authentic 100 nM chemical
standards. Primary MRM transitions used for detection and quantification are
shown in f and h; additional characteristic transitions are shown in g and i
(‘**’ indicates any detected fragment mass in product ion scan). Traces are
representative of n = 3 biologically independent samples.