BACKGROUND Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. RESULTS We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and isopentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a flux control coefficient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway flux) in the isoprene-producing strain and therefore exerted significant control over the flux though the MEP pathway. At higher dxs expression levels, the intracellular concentration of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the flux towards isoprene. The higher intracellular concentrations of MEcPP led to the efflux of this intermediate into the growth medium. DISCUSSION These findings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineering of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids.

中文翻译：

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通过代谢控制分析研究甲基赤藓糖醇4-磷酸途径用于生产微生物萜类化合物的方法。

背景技术萜类化合物作为化学构件和药物备受关注。在微生物中，可以通过磷酸甲基赤藓糖醇（MEP）或甲羟戊酸（MVA）途径合成萜类化合物。尽管MEP途径具有较高的理论产率，但是由于对途径的调控了解甚少，因此代谢工程几乎没有成功。结果我们对表达异源异戊二烯合酶基因（ispS）的大肠杆菌中的MEP途径进行了代谢控制分析。ispS的表达导致异戊烯基焦磷酸酯（IPP）/二甲基烯丙基焦磷酸酯（DMAPP）的积累并严重损害了细菌的生长，但是ispS和异戊烯基二磷酸异构酶（idi）的共表达恢复了正常的生长和野生型IPP / DMAPP的水平。靶向蛋白质组学和代谢组学分析提供了该途径的定量描述，该途径被随机化编码1-脱氧木酮糖5-磷酸合酶（Dxs）的基因中的核糖体结合位点所扰动。Dxs在产生异戊二烯的菌株中的通量控制系数为0.35（即，Dxs活性增加1％导致途径通量增加0.35％），因此对通过MEP途径的通量进行了重要控制。在较高的dxs表达水平下，2-C-甲基-D-赤藓糖醇-2,4-环焦磷酸盐（MEcPP）的细胞内浓度与其他MEP途径中间体相反，后者的浓度线性依赖于Dxs的丰度。这表明消耗MEcPP的4-羟基-3-甲基丁-2-烯-1-基二磷酸合酶（IspG）变得饱和，因此限制了通向异戊二烯的通量。较高的细胞内MEcPP浓度导致该中间体流出到生长培养基中。讨论这些发现表明，Dxs，Idi和IspG以及代谢产物的出口对于MEP途径的代谢工程非常重要，并将促进微生物生产有价值的类异戊二烯的进一步方法。