Department of Chemical and Biological Engineering; Science and Technology Center, Tufts University, Medford, MA 02155, USA.
Appl Microbiol Biotechnol. 2012 Feb;93(4):1651-61. doi: 10.1007/s00253-011-3528-4. Epub 2011 Aug 18.
Taxa-4(5),11(12)-diene is the first dedicated intermediate in the metabolic pathway responsible for synthesizing the anticancer compound Taxol. In this study, the heterologous production of taxadiene was established in and analyzed between K- and B-derived Escherichia coli strains. First, recombinant parameters associated with precursor metabolism (the upstream methylerythritol phosphate (MEP) pathway) and taxadiene biosynthesis (the downstream pathway) were varied to probe the effect different promoters and cellular backgrounds have on taxadiene production. Specifically, upstream MEP pathway genes responsible for the taxadiene precursors, dimethylallyl diphosphate and isopentenyl diphosphate, were tested with an inducible T7 promoter system within K and B E. coli strains. Whereas, inducible T7, Trc, and T5 promoters were tested with the plasmid-borne geranylgeranyl diphosphate synthase and taxadiene synthase genes responsible for the downstream pathway. The K-derivative produced taxadiene roughly 2.5-fold higher than the B-derivative. A transcriptomics study revealed significant differences in pyruvate metabolism between the K and B strains, providing insight into the differences observed in taxadiene biosynthesis and targets for future metabolic engineering efforts. Next, the effect of temperature on cell growth and taxadiene production was analyzed in these two strains, revealing similar phenotypes between the two with 22°C as the optimal production temperature. Lastly, the effect of indole on cell growth was investigated between the two strains, showing that the K-derivative demonstrated greater growth inhibition compared to the B-derivative.
Taxa-4(5),11(12)-二烯是负责合成抗癌化合物紫杉醇的代谢途径中的第一个专用中间产物。在这项研究中，在 K 和 B 衍生的大肠杆菌菌株中建立了 taxadiene 的异源生产并进行了分析。首先，改变了与前体代谢（上游甲基赤藓醇磷酸 (MEP) 途径）和 taxadiene 生物合成（下游途径）相关的重组参数，以探究不同启动子和细胞背景对 taxadiene 生产的影响。具体来说，使用 K 和 B 大肠杆菌菌株中的诱导型 T7 启动子系统测试了负责 taxadiene 前体二甲基烯丙基二磷酸和异戊烯二磷酸的上游 MEP 途径基因。而，使用带有质粒的香叶基香叶基二磷酸合酶和负责下游途径的 taxadiene 合酶基因测试了诱导型 T7、Trc 和 T5 启动子。K 衍生物产生的 taxadiene 比 B 衍生物高约 2.5 倍。转录组学研究揭示了 K 和 B 菌株之间丙酮酸代谢的显着差异，为观察到的 taxadiene 生物合成差异和未来代谢工程努力的目标提供了深入了解。接下来，分析了这两种菌株中温度对细胞生长和 taxadiene 生产的影响，发现两种菌株的表型相似，22°C 是最佳生产温度。最后，研究了两种菌株之间吲哚对细胞生长的影响，结果表明 K 衍生物比 B 衍生物表现出更大的生长抑制。
