Fig. 4

Optimization of de novo tropine biosynthesis in yeast. a Engineered pathway for production of tropine from N-methylpyrrolinium and proposed spontaneous side reactions producing hygrine. Putative major and minor side reactions in yeast are indicated by bold and dotted arrows, respectively. b Production of tropine and related intermediates with expression of AbPYKS, AbCYP82M3, and DsTR1 in engineered yeast. Indicated genes were expressed from low-copy plasmids in CSY1246; plus and minus symbols indicate presence or absence of enzyme. c Effect of growth temperature on spontaneous hygrine production in CSY1248. d Summary of genetic optimization to increase tropine production in engineered yeast. Minus symbol indicates absence of gene; ‘p’ and ‘i’ indicate gene expression from low-copy plasmid or genomic integration, respectively. Strains were cultured in selective (YNB-DO) media (b, d: CSY1247) or non-selective (YNB-SC) media (c, d: CSY1246, 1248, 1249, 1251) with 2% dextrose at 30 °C or 25 °C for 48 h before LC-MS/MS analysis. e Optimization of media conditions to improve tropine production in CSY1251. Minus symbols indicate absence of media component. Strains were cultured for 72 h prior to LC-MS/MS analysis. YNB yeast nitrogen base, YP yeast extract plus peptone, SC synthetic complete amino acid mixture, Dex dextrose, Gal galactose, Raf raffinose, Suc sucrose, Tre trehalose, Ara arabinose, Sor sorbitol, Gly glycerol. For b–e, data represent the mean of n = 3 biologically independent samples (open circles) and error bars show standard deviation. Student’s two-tailed t-test: *P < 0.05, **P < 0.01, ***P < 0.001. f Cell growth and tropine production in shake-flask cultures of CSY1251 over 6 days. Low-density (LDC) and high-density (HDC) cultures were grown in non-selective media (YNB-G; YNB + 1 × amino acids + 2% dextrose + 5% glycerol) at 25 °C and 300 rpm. At t = 72 h (dotted vertical line), cultures were supplemented with additional amino acids, dextrose, and glycerol to final concentrations of 1×, 2%, and 2%, respectively. Metabolite titers were quantified by LC-MS/MS. Data indicate the mean of n = 3 biologically independent samples and error bars show standard deviation. Source data of Fig. 4b–f are provided as a source data file