Building Microbial Hosts for Heterologous Production of N-methylpyrrolinium.

N-methylpyrrolinium-derived alkaloids like tropane alkaloids, nicotine, and calystegines are valuable plant source specialized metabolites bearing pharmaceutical or biological activity. Microbial synthesis of the critical common intermediate N-methylpyrrolinium (NMP) would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and Saccharomyces cerevisiae by employment of the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from Anisodus acutangulus were first characterized. Then, we presented the production of N-methylpyrrolinium in vitro from L-ornithine (ORN) via combination of the three cascade enzymes, ornithine decarboxylase (ODC) from Erythroxylum coca, putrescine N-methyltransferase (PMT) from Anisodus tanguticus, and diamine oxidases (DAOs) from A. acutangulus. Construction of the plant biosynthetic pathway into E. coli and S. cerevisiae resulted in de novo bio-production of N-methylpyrrolinium with titers of 3.02 mg/L and 2.07 mg/L, respectively. Metabolically engineering of the yeast strain to produce N-methylpyrrolinium via decreasing the flux to the product catabolism pathway and improving cofactor supply resulted in a final titer of 17.82 mg/L. This study not only presents the first microbial synthesis of N-methylpyrrolinium but also lays the foundation of heterologous biosynthesis of N-methylpyrrolinium-derived alkaloids. More importantly, the strains constructed can serve as important alternative tools to identify undiscovered pathway enzymes with a synthetic biology strategy.