Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN 37996, USA.
Appl Microbiol Biotechnol. 2012 Aug;95(4):1083-94. doi: 10.1007/s00253-012-4197-7. Epub 2012 Jun 9.
Elementary mode (EM) analysis based on the constraint-based metabolic network modeling was applied to elucidate and compare complex fermentative metabolisms of Escherichia coli for obligate anaerobic production of n-butanol and isobutanol. The result shows that the n-butanol fermentative metabolism was NADH-deficient, while the isobutanol fermentative metabolism was NADH redundant. E. coli could grow and produce n-butanol anaerobically as the sole fermentative product but not achieve the maximum theoretical n-butanol yield. In contrast, for the isobutanol fermentative metabolism, E. coli was required to couple with either ethanol- or succinate-producing pathway to recycle NADH. To overcome these "defective" metabolisms, EM analysis was implemented to reprogram the native fermentative metabolism of E. coli for optimized anaerobic production of n-butanol and isobutanol through multiple gene deletion (8-9 genes), addition (6-7 genes), up- and downexpression (~6-7 genes), and cofactor engineering (e.g., NADH, NADPH). The designed strains were forced to couple both growth and anaerobic production of n-butanol and isobutanol, which is a useful characteristic to enhance biofuel production and tolerance through metabolic pathway evolution. Even though the n-butanol and isobutanol fermentative metabolisms were quite different, the designed strains could be engineered to have identical metabolic flux distribution in "core" metabolic pathways mainly supporting cell growth and maintenance. Finally, the model prediction in elucidating and reprogramming the native fermentative metabolism of E. coli for obligate anaerobic production of n-butanol and isobutanol was validated with published experimental data.
基于基于约束的代谢网络建模的基本模式 (EM) 分析被应用于阐明和比较大肠杆菌的复杂发酵代谢，以强制厌氧生产正丁醇和异丁醇。结果表明，正丁醇发酵代谢 NADH 不足，而异丁醇发酵代谢 NADH 过剩。大肠杆菌可以作为唯一的发酵产物在厌氧条件下生长并生产正丁醇，但不能达到最大的理论正丁醇产量。相比之下，对于异丁醇发酵代谢，大肠杆菌需要与乙醇或琥珀酸生产途径相耦合以回收 NADH。为了克服这些“缺陷”代谢，进行了 EM 分析以重新编程大肠杆菌的天然发酵代谢，通过多个基因缺失 (8-9 个基因)、添加 (6-7 个基因)、上调和下调表达 (~6-7 个基因)以及辅助因子工程（例如 NADH、NADPH）来优化正丁醇和异丁醇的厌氧生产。设计的菌株被迫将正丁醇和异丁醇的生长和厌氧生产耦合在一起，这是通过代谢途径进化来提高生物燃料生产和耐受性的有用特性。尽管正丁醇和异丁醇发酵代谢有很大的不同，但可以对设计的菌株进行工程改造，使其在主要支持细胞生长和维持的“核心”代谢途径中具有相同的代谢通量分布。最后，通过使用已发表的实验数据验证了模型在阐明和重新编程大肠杆菌的天然发酵代谢以强制厌氧生产正丁醇和异丁醇方面的预测。
