Department of Energy, Environmental, and Chemical Engineering, Washington University, St. Louis, MO, USA.
Department of Chemical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA; College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
Metab Eng. 2017 Jan;39:247-256. doi: 10.1016/j.ymben.2016.12.008. Epub 2016 Dec 23.
Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this study, C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37°C in stage 1, and then the temperature was transitioned to 20°C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (late production phase) in the high producing strain, reaching a final concentration of 1.5mmol/L. On the contrary, ~0.02mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).
微生物发酵条件是动态的，由于转录诱导、营养消耗或孵育条件的变化。在这项研究中，我们使用 C 代谢通量分析来描述两种具有截然不同生产力的紫色素产生大肠杆菌菌株，并分析它们在发酵过程中受到外部干扰时的代谢调整。这两种菌株首先在 37°C 下在第 1 阶段生长，然后在第 2 阶段将温度转换为 20°C，以优化紫色素合成途径的表达。诱导后，高生产力菌株在第 3 阶段紫色素产量最低，但在第 4 阶段（早期生产阶段）和第 5 阶段（晚期生产阶段）加速生产，最终浓度达到 1.5mmol/L。相比之下，低生产力菌株仅获得约 0.02mmol/L 的紫色素。为了了解每个阶段的时间代谢变化，我们通过快速周转游离代谢物的同位素标记分析进行了 C-MFA。结果表明，在早期生长阶段，中心代谢中通量比保持惊人的稳定。然而，在后期，高产菌株显著调整了其通量分布，其特征是戊糖磷酸途径和 TCA 循环上调，来自乙酸利用的回流，几乎没有合成代谢通量，以及升高的维持损耗，以补偿由于紫色素过度产生而导致的营养物质消耗和一些构建块的流失。具有更强启动子的低生产力菌株通过增强 TCA 循环和乙酸溢出的通量，在第 5 阶段调整了其相对通量，同时表现出较低的生物量生长和最小的紫色素合成通量。有趣的是，在培养基中添加紫色素前体（色氨酸）抑制了高产菌株但增强了低生产力菌株的生产力，这导致了对未知途径调节（例如代谢物通道化）的假设。
