title: Microbial synthesis of pinene.
abstract: The volumetric heating values of today's biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ~28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.
authors.affiliation: School of Chemistry and Biochemistry and ⊥School of Chemical and Biomolecular Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Sarria
authors.forename: Betty
authors.initials: B
authors.lastname: Wong
authors.forename: Hector
authors.initials: H
authors.lastname: García Martín
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.forename: Pamela
authors.initials: P
authors.lastname: Peralta-Yahya
mesh_terms: D028202:Abies / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000081008:Bicyclic Monoterpenes; D056804:Biofuels; D001643:Bridged Bicyclo Compounds; D004926:Escherichia coli / Q000378:metabolism; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D039821:Monoterpenes / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000081008:Bicyclic Monoterpenes; D056804:Biofuels; D001643:Bridged Bicyclo Compounds; D039821:Monoterpenes; D011993:Recombinant Fusion Proteins; C010789:beta-pinene; D007535:Isomerases; C040245:pinene cyclase II; D019753:Intramolecular Lyases; C044526:geranyl-diphosphate cyclase; C005451:alpha-pinene