Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.

/pdf/biosynthesis-of-medicinal-tropane-alkaloids-in-yeast-1dxm94dxr2.pdf

Biosynthesis of medicinal tropane alkaloids in yeast.

https://pubs.rsc.org/en/content/articlepdf/2021/np/d0np00031k

The scaffold-forming steps of plant alkaloid biosynthesis.

Advanced Strategies for Production of Natural Products in Yeast.

Biosynthesis and Regulation of Salicylic Acid and N-Hydroxypipecolic Acid in Plant Immunity

Natural nonproteinogenic amino acids vastly outnumber the well-known 22 proteinogenic amino acids. Such amino acids are generated in specialized metabolic pathways. In these pathways, diverse biosynthetic transformations, ranging from isomerizations to the stereospecific functionalization of C-H bonds, are employed to generate structural diversity. The resulting nonproteinogenic amino acids can be integrated into more complex natural products. Here we review recently discovered biosynthetic routes to freestanding nonproteinogenic α-amino acids, with an emphasis on work reported between 2013 and mid-2019.

Biosynthetic Pathways to Nonproteinogenic α-Amino Acids.

Tropine and pseudotropine with opposite stereospecific configurations as platform compounds are central building blocks in both biosynthesis and chemical synthesis of pharmacologically important tropane and nortropane alkaloids. The supply of plant-derived tropine and pseudotropine still heavily depends on either plant extraction or chemical synthesis. Advances in synthetic biology prompt the microbial synthesis of various valuable chemicals. With the biosynthetic pathway elucidation of tropine and pseudotropine in several Solanaceae plants, the key genes were sequentially identified. Here, the enzymes responsible for converting N-methylpyrrolinium into tropine and pseudotropine from Anisodus acutangulus were characterized. Reconstruction of the six-step biosynthetic pathways into Saccharomyces cerevisiae provides cell chassis producing tropine and pseudotropine with 0.13 and 0.08 mg/L titers from simple feedstocks in a shake flask, respectively. The strains described not only offer alternative sources of these central intermediates and their derived alkaloids but also provide platforms for pathway enzyme discovery.

De Novo Production of the Plant-Derived Tropine and Pseudotropine in Yeast.

Insights into Pipecolic Acid Biosynthesis in Huperzia serrata

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.

Building Microbial Hosts for Heterologous Production of N-Methylpyrrolinium.

The invention provides a method for preparing N-methyl pyrroline through combined catalysis of three enzymes. The method comprises the following steps: taking L-ornithine as a substrate, and carryingout combined catalysis by using ornithine decarboxylase EcODC, putrescine N-methyltransferase AtPMT and amine oxidase AaDAO3 to obtain the N-methyl pyrroline. The method can also be realized by fermentation of a genetically engineered bacterium expressing the three enzymes. The method is environmentally friendly, has mild reaction conditions, and has an industrial development prospect.

Method for preparing N-methyl pyrroline

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Papers

De Novo Production of the Plant-Derived Tropine and Pseudotropine in Yeast.

Insights into Pipecolic Acid Biosynthesis in Huperzia serrata

Building Microbial Hosts for Heterologous Production of N-Methylpyrrolinium.

Method for preparing N-methyl pyrroline