A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates

• DOI: 10.1002/pro.2436
• Publication Date: 2014-02-06T09:39:49Z
• Authors: Tyler P. Korman; Bobby Sahachartsiri; Dan Li; Jeffrey M. Vinokur; David Eisenberg; James U. Bowie
• Keywords: 0301 basic medicine; Biophysics; Mevalonic Acid; in vitro synthesis; Phosphoenolpyruvate; 03 medical and health sciences; Adenosine Triphosphate; Hemiterpenes; Acetyl Coenzyme A; Pentanes; Pyruvic Acid; Butadienes; Escherichia coli; Other Information and Computing Sciences; isoprenoids; green chemistry; Computation Theory and Mathematics; Green Chemistry Technology; Biosynthetic Pathways; Metabolic Engineering; biofuel; Biochemistry and Cell Biology; metabolic engineering; commodity chemicals; Glycolysis; NADP

AbstractThe 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.