Sarria Stephen, Wong Betty, García Martín Hector, Keasling Jay D, Peralta-Yahya Pamela
School of Chemistry and Biochemistry and ⊥School of Chemical and Biomolecular Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
ACS Synth Biol. 2014 Jul 18;3(7):466-75. doi: 10.1021/sb4001382. Epub 2014 Feb 27.
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.
当今生物燃料的体积热值过低，无法为能源密集型飞机、火箭和导弹提供动力。最近研究表明，蒎烯二聚体的体积热值与战术燃料JP - 10相似。为了提供可持续的蒎烯来源，我们对大肠杆菌进行了改造以生产蒎烯。我们组合表达了三种蒎烯合酶（PS）和三种香叶基二磷酸合酶（GPPS），最佳组合可实现约28 mg/L的蒎烯产量。我们推测蒎烯毒性限制了产量；然而，在当前滴度下毒性不应构成限制因素。由于GPPS会受到香叶基二磷酸（GPP）的抑制，为了增加该途径的通量，我们组合构建了GPPS - PS蛋白融合体。来自大冷杉的GPPS - PS融合体产生了32 mg/L的蒎烯，比之前在工程化大肠杆菌中报道的最高滴度提高了6倍。最后，我们研究了产蒎烯微生物的蒎烯异构体比例，发现异构体分布不仅取决于所使用的PS的种类，还取决于与PS配对的GPPS的种类。我们证明了可供PS环化的GPP浓度会改变蒎烯异构体比例。
