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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Metab Eng. 2015 May 14;30:96–104. doi: 10.1016/j.ymben.2015.05.001

Figure 4.

Figure 4

Microbial production of cis-N-methylstylopine, protopine, and sanguinarine in optimized culture conditions. (A) Production of cis-N-methylstylopine, protopine, and sanguinarine yeast strains under optimized growth conditions. CSY968 contains PsTNMT integrated and CSY969 contains both PsTNMT and PsMSH integrated. P6H variants were expressed from a low-copy plasmid in CSY969. Metabolite production is determined by LC-MS analysis of culture media after indicated strains were grown for 96 h (CSY968) or 10 d (CSY969, CSY969 with P6H). Data is reported as the mean ± s.d. of at least 3 independent experiments. (B) LC-MS analysis of growth media of CSY969 with EcP6H. Traces are shown for a no EcP6H enzyme control strain (left) and an engineered strain expressing EcP6H (right). EICs for compounds corresponding to protopine (354 m/z), dihydrosanguinarine (334 m/z), and sanguinarine (332 m/z) are shown. A sanguinarine standard is included for comparison. (C) MS2 spectra for the sanguinarine standard (left) and the 332 EIC peak produced from the engineered yeast strain (right). LC-MS traces and fragmentation patterns are representative of at least 3 independent experiments.