Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Appl Environ Microbiol. 2010 Dec;76(24):8004-10. doi: 10.1128/AEM.01917-10. Epub 2010 Oct 22.
Isobutene is an important commercial chemical used for the synthesis of butyl rubber, terephthalic acid, specialty chemicals, and a gasoline performance additive known as alkylate. Currently, isobutene is produced from petroleum and hence is nonrenewable. Here, we report that the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (ScMDD) can convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene. Whole cells of Escherichia coli producing ScMDD with an N-terminal 6×His tag (His(6)-ScMDD) formed isobutene from 3-HMB at a rate of 154 pmol h(-1) g cells(-1). In contrast, no isobutene was detected from control cells lacking ScMDD. His(6)-ScMDD was purified by nickel affinity chromatography and shown to produce isobutene from 3-HMB at a rate of 1.33 pmol min(-1) mg(-1) protein. Controls showed that both His(6)-ScMDD and 3-HMB were required for detectable isobutene formation. Isobutene was identified by gas chromatography (GC) with flame ionization detection as well as by GC-mass spectrometry (MS). ScMDD was subjected to error-prone PCR, and two improved variants were characterized, ScMDD1 (I145F) and ScMDD2 (R74H). Whole cells of E. coli producing ScMDD1 and ScMDD2 produced isobutene from 3-HMB at rates of 3,000 and 5,888 pmol h(-1) g cells(-1), which are 19- and 38-fold increases compared to rates for cells producing His(6)-ScMDD. This showed that genetic modifications can be used to increase the rate at which ScMDD converts 3-HMB to isobutene. Because 3-HMB can be produced from l-leucine, ScMDD has a potential application for the production of renewable isobutene. Moreover, isobutene is a gas, which might simplify its purification from a fermentation medium, substantially reducing production costs.
异丁烯是一种重要的商业化学品，用于合成丁基橡胶、对苯二甲酸、特种化学品和一种称为烷基化的汽油性能添加剂。目前，异丁烯是从石油中生产的，因此是非可再生的。在这里，我们报告称酿酒酵母甲羟戊酸二磷酸脱羧酶（ScMDD）可以将 3-羟基-3-甲基丁酸（3-HMB）转化为异丁烯。带有 N 端 6×His 标签（His(6)-ScMDD）的产 ScMDD 的大肠杆菌全细胞以 154 pmol h(-1) g 细胞(-1)的速率从 3-HMB 形成异丁烯。相比之下，缺乏 ScMDD 的对照细胞未检测到异丁烯。His(6)-ScMDD 通过镍亲和层析纯化，并显示以 1.33 pmol min(-1) mg(-1)蛋白的速率从 3-HMB 产生异丁烯。对照表明，His(6)-ScMDD 和 3-HMB 都是可检测到异丁烯形成所必需的。通过气相色谱（GC）和火焰电离检测以及 GC-质谱（MS）鉴定异丁烯。对易错 PCR 进行 ScMDD 处理，并对两种改进的变体进行了表征，即 ScMDD1（I145F）和 ScMDD2（R74H）。产生 ScMDD1 和 ScMDD2 的大肠杆菌全细胞以 3000 和 5888 pmol h(-1) g 细胞(-1)的速率从 3-HMB 产生异丁烯，与产生 His(6)-ScMDD 的细胞的速率相比，分别增加了 19 倍和 38 倍。这表明遗传修饰可用于提高 ScMDD 将 3-HMB 转化为异丁烯的速率。由于 3-HMB 可以从 L-亮氨酸生产，因此 ScMDD 有可能用于生产可再生异丁烯。此外，异丁烯是一种气体，这可能使其从发酵培养基中纯化变得简单，大大降低生产成本。
