Scalable Biosynthesis

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Summary: Researchers in Prof. Christina Smolke’s laboratory have engineered yeast strains for high-yield, scalable, environmentally-friendly production of methylxanthines from simple carbon and nitrogen sources. Methylxanthines are valuable, plant-derived xanthine-based purine products such as caffeine, theobromine, and theophylline (an asthma medication). Traditionally, they are produced by either extraction or chemical synthesis. Extraction has low yield due to low accumulation of metabolites and it requires intensive time and resources to grow the plant source. Chemical synthesis has low yield and purity due to the multiple reactions and it requires the use of harsh chemicals. Instead, these methylxanthines can be efficiently fermented in yeast that have been reprogrammed to direct their metabolic flux away from endogenous purine biosynthesis while maintaining their ability to grow normally. In addition, the biosynthetic pathways of the engineered yeast provide a foundation for low-cost production of a broad range of end products - natural purine alkaloids and novel, non-naturally occurring derivatives. This technology could enable a high-yield, reliable industrial scale process to biomanufacture a range of complex bioactive molecules for therapeutics, drug discovery, or food chemistry.   Stage of Research: The inventors have engineered S. cerevisiae strains for production of all monomethylated (1-, 3-, or 7- methylxanthine), dimethylated (theophylline, paraxanthine, and theobromine), and trimethylated (caffeine) xanthine products. In bench scale batch fermentation, they demonstrated de novoproduction of caffeine (270 ug/L), theophylline (61 ug/L) and 3-methylxanthine (3700 ug/L).   Applications: Biosynthesis of therapeutic agents - sustainable industrial scale production of known therapeutic molecules such as theophylline Drug discovery - low cost supply to exploit pharmaceutical applications of natural and non-natural xanthine-based alkaloids for screening to identify new therapeutic agents Food chemistry - efficient, secure source of precursor chemicals and end products for plant-derived compounds such as caffeine and theobromine Bioremediation - platform to develop strategies to remove or degrade environmental waste   Advantages: Efficient, scalable, controlled production: microbial strains can be engineered for industrial scale fermentation potential for higher yield than natural plant sources which have low accumulation of secondary metabolites higher purity than chemical synthesis which requires multiple reactions with undesired products secure, sustainable supply with shorter production time and lower resource requirements for fermentation compared with growing plant hosts simple, low cost carbon and nitrogen sources (e.g., sugar and ammonia) Environmentally friendly synthesis - microbial fermentation does not use the undesirable chemicals, solvents and harsh reaction conditions needed for multi-step chemical synthesis Flexible pathways with broad range of end products: microbial pathways can be engineered to produce methylxanthines that have never been synthesized, including non-natural compounds could expand engineered strains across many industrial microbial platforms with same fermentation infrastructure enables inexpensive supply for drug discovery and testing of non-natural compounds or compounds based on semisynthetic derivatives