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 would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and in Saccharomyces cerevisiae by employing the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from Anisodus acutangulus were first characterized. Then, we produced N-methylpyrrolinium in vitro from l-ornithine via a combination of the three cascade enzymes, ornithine decarboxylase from Erythroxylum coca, putrescine N-methyltransferase from Anisodus tanguticus, and DAOs from A. acutangulus. Construction of the plant biosynthetic pathway in E. coli and S. cerevisiae resulted in de novo bioproduction of N-methylpyrrolinium with titers of 3.02 and 2.07 mg/L, respectively. Metabolic engineering of the yeast strain to produce N-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 N-methylpyrrolinium but also lays the foundation for heterologous biosynthesis of N-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.
Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.
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