Fig. 5.

De novo production of the non-canonical tropane alkaloid cinnamoyltropine in yeast. a Tandem MS/MS spectra of extracellular medium of CSY1251 (gray), CSY1282 (red), or a genuine cinnamoyltropine standard (blue) for a parent mass of m/z⁺ = 272. Blue diamond indicates parent compound peak. b–i Validation of EcCS acyltransferase activity on cinnamic acid and α-tropine via substrate feeding. Strains were transformed with combinations of plasmids expressing AtPAL1 (low-copy plasmid pCS4252) and/or At4CL5 and EcCS (high-copy plasmid pCS4207), and then cultured in media with different supplemented substrates, as follows: b CEN.PK2 + At4CL5 + EcCS + 0.1 mM trans-cinnamic acid; c CEN.PK2 + At4CL5 + EcCS + 0.5 mM α-tropine; d CEN.PK2 + AtPAL1 + At4CL5 + EcCS; e CEN.PK2 + AtPAL1 + At4CL5 + EcCS + 0.5 mM α-tropine; f CSY1251 + At4CL5 + EcCS; g CSY1251 + At4CL5 + EcCS + 0.2 mM trans-cinnamic acid; h CSY1251 + AtPAL1 + At4CL5 + EcCS; i 25 nM cinnamoyltropine standard. For a–h, yeast strains were cultured in selective media (YNB-DO + 2% dextrose + 5% glycerol) at 25 °C for 72 h prior to LC-MS/MS analysis. All traces are representative of n = 3 biologically independent samples