Withers Sydnor T, Gottlieb Shayin S, Lieu Bonny, Newman Jack D, Keasling Jay D
Department of Chemical Engineering, University of California, Berkeley, California, USA.
Appl Environ Microbiol. 2007 Oct;73(19):6277-83. doi: 10.1128/AEM.00861-07. Epub 2007 Aug 10.
We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6,051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.
我们开发了一种克隆萜烯合酶基因的新方法。该方法依赖于异戊二烯基二磷酸前体对萜烯的固有毒性，这导致了生长受限的表型。当这些前体被萜烯合酶消耗时，生长恢复正常。我们已经证明，这种方法能够从表达倍半萜烯生产酶——紫穗槐二烯合酶的克隆群体中富集工程化大肠杆菌。此外，我们在工程化大肠杆菌中富集了来自产异戊二烯的枯草芽孢杆菌菌株6051的基因组DNA文库，该大肠杆菌能产生高水平的异戊烯基二磷酸和二甲基烯丙基二磷酸。通过筛选发现了两个基因（yhfR和nudF），其蛋白质产物直接作用于异戊二烯基二磷酸前体并产生异戊烯醇。在基于甲羟戊酸的异戊烯基焦磷酸生物合成途径工程化的大肠杆菌中表达nudF会产生异戊烯醇。
