초록
<P><I>N</I>-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 <I>N</I>-methylpyrrolinium would allow for sustainable production of <I>N</I>-methylpyrrolinium-derived alkaloids. Here, we achieve the production of <I>N</I>-methylpyrrolinium both in <I>Escherichia coli</I> and in <I>Saccharomyces cerevisiae</I> by employing the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from <I>Anisodus acutangulus</I> were first characterized. Then, we produced <I>N</I>-methylpyrrolinium <I>in vitro</I> from <SMALL>L</SMALL>-ornithine via a combination of the three cascade enzymes, ornithine decarboxylase from <I>Erythroxylum coca</I>, putrescine <I>N</I>-methyltransferase from <I>Anisodus tanguticus</I>, and DAOs from <I>A. acutangulus</I>. Construction of the plant biosynthetic pathway in <I>E. coli</I> and <I>S. cerevisiae</I> resulted in <I>de novo</I> bioproduction of <I>N</I>-methylpyrrolinium with titers of 3.02 and 2.07 mg/L, respectively. Metabolic engineering of the yeast strain to produce <I>N</I>-methylpyrrolinium via decreasing the flux to the product catabolism pathway and improving the cofactor supply resulted in a final titer of 17.82 mg/L. This study not only presents the first microbial synthesis of <I>N</I>-methylpyrrolinium but also lays the foundation for heterologous biosynthesis of <I>N</I>-methylpyrrolinium-derived alkaloids. More importantly, the strains constructed herein can serve as important alternative tools for identifying undiscovered pathway enzymes with a synthetic biology strategy.</P><BR>[FIG OMISSION]</BR>