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Biosynthesis pathway of n-methylpyrrolidine and construction

Toluidine alkaloids such as anisodamine and cyperine have important biological activities and clinical medicinal value.These alkaloids are mainly obtained by traditional extraction methods of the original plants, and the microbial heterologous synthesis technology based on synthetic biology provides a new strategy for the preparation of medicinal natural products.In the biosynthesis of n-methylpyrrolidine, l-ornithine was used as the precursor, which was catalyzed by ornithine.





In order to build heterologous cells and synthesis of N - methyl pyrroline chassis XiaoYouLi team flat plume and xiao-dong li zhou research group, the researchers conducted the following research: 1) the first parsing the source of medicinal plant three points AaDAO2 amine oxidase and AaDAO3 biochemical function, source and coupling league coca ornithine decarboxylase EcODC and putrescine sources of anisodus tanguticus - N - methyl transferase to a pot in vitro enzyme catalysis synthesis and screening of EcODC, AtPMT and AaDAO3 in vitro synthesis of N - methyl pyrroline optimal catalytic module;2) the three structural genes in this module were further introduced into escherichia coli and saccharomyces cerevisiae to construct the corresponding chassis cells and conduct the shaking fermentation test. The target compound n-methylpyrrolium was successfully obtained with the yield of 3.02 and 2.07mg/L respectively.3) on the basis of this, in order to further increase production, the research also knock in addition to the yeast cells chassis in N - methyl pyrroline competition metabolic pathways related enzymes ALD4, ALD5 and HFD1, at the same time express SAM2 strengthened through a synthetic key enzyme AtPMT catalytic synthesis of cofactors required for SAM, eventually yeast cells chassis in N - methyl pyrroline yield reached 17.82 mg/L, compared with the original strain increased 8.6 times.
This study was the first to construct a microbial chassis cell for the heterologous synthesis of n-methylpyrrolidine, which laid an important foundation for the analysis and heterologous synthesis of medicinal alkaloids precursors of n-methylpyrrolidine.
The research was also supported by zhejiang university of traditional Chinese medicine professor zhang chuanyin and funded by the Chinese Academy of Sciences.The characterization of metabolite profiles and small molecule NMR was supported by the metabolomics and protein interaction technology platform of the center for public technical services of the center for molecular plant excellence


           

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