Fig. 3

Optimization of N-methylpyrrolinium production in yeast. a Engineered biosynthetic pathway for production of N-methylpyrrolinium (NMPy) from putrescine and stepwise substrate and product tracking. The proposed mechanism for formation of the 4MAB acid side product via activity of an endogenous yeast enzyme (ALD) is shown. Extracted ion chromatogram MRM traces are shown for each metabolite along the pathway and for authentic standards using the highest precursor ion/product ion transition for each metabolite (Supplementary Table 3). Control represents strain CSY1235 expressing SPE1, AsADC, and speB on a low-copy plasmid (pCS4239). Chromatogram traces are representative of three biological replicates. Enzyme symbols: PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; ALD, aldehyde dehydrogenase. b Characterization of the subcellular localization of putrescine N-methyltransferase and N-methylputrescine oxidase by epifluorescence microscopy. Microscopy was performed on wild-type yeast expressing N- or C-terminal GFP-tagged AbPMT1 or NtMPO1 from low-copy plasmids. Scale bar, 10 μm. c Effect of N- and C-terminal truncations to methylputrescine oxidase on NMPy production in engineered yeast. Wild-type (WT) enzymes and indicated truncations were expressed from low-copy plasmids (pCS4233-4238) in CSY1235 together with SPE1, AsADC, and speB (pCS4239) and AbPMT1 (pCS4193). d Effect of aldehyde dehydrogenase gene disruptions on production of the 4MAB acid side product and NMPy in engineered yeast. Plus and minus symbols indicate presence or absence of functional enzyme, respectively. For a, c, d, strains were cultured in selective (YNB-DO) media with 2% dextrose at 30 °C for 48 h before LC-MS/MS analysis. All data represent the mean of n = 3 biologically independent samples (open circles in c, d) and error bars show standard deviation. Student’s two-tailed t-test: *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated, statistical significance is shown relative to the corresponding control (CSY1235). Source data of Fig. 3c and d are provided as a source data file