title: Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines.
abstract: Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270μg/L, 61μg/L, and 3700μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.
authors: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA 94305, United States.
authors: Maureen
authors: M
authors: McKeague
authors: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA 94305, United States.
authors: Yen-Hsiang
authors: YH
authors: Wang
authors: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA 94305, United States.
authors: Aaron
authors: A
authors: Cravens
authors: Synthetic Genomics, La Jolla, CA 92037, United States.
authors: Maung Nyan
authors: MN
authors: Win
authors: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA 94305, United States. Electronic address: csmolke@stanford.edu.
authors: Christina D
authors: CD
authors: Smolke
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D053898:Biosynthetic Pathways / Q000235:genetics*; D002110:Caffeine / Q000096:biosynthesis* / Q000302:isolation & purification; D040503:Coffea / Q000502:physiology*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014970:Xanthines / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014970:Xanthines; C008514:methylxanthine; D002110:Caffeine
keywords: Methylxanthines; Plant natural products; Purine alkaloids; Synthetic biology; Yeast