title: A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates.
abstract: The high yields required for the economical production of chemicals and fuels using microbes can be difficult to achieve due to the complexities of cellular metabolism. An alternative to performing biochemical transformations in microbes is to build biochemical pathways in vitro, an approach we call synthetic biochemistry. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chemical isoprene. We construct an in vitro synthetic biochemical pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, University of California, Los Angeles, California.
authors.forename: Tyler P
authors.initials: TP
authors.lastname: Korman
authors.forename: Bobby
authors.initials: B
authors.lastname: Sahachartsiri
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Vinokur
authors.forename: David
authors.initials: D
authors.lastname: Eisenberg
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D053898:Biosynthetic Pathways; D002070:Butadienes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D006019:Glycolysis; D055772:Green Chemistry Technology; D045782:Hemiterpenes / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D008798:Mevalonic Acid / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010420:Pentanes / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D002070:Butadienes; D045782:Hemiterpenes; D010420:Pentanes; C005059:isoprene; D009249:NADP; D000105:Acetyl Coenzyme A; D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D008798:Mevalonic Acid
keywords: biofuel; commodity chemicals; green chemistry; in vitro synthesis; isoprenoids; metabolic engineering