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阐明阻碍嗜热栖热梭菌乙醇生产的中心代谢氧化还原障碍。

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum.

作者信息

Thompson R Adam, Layton Donovan S, Guss Adam M, Olson Daniel G, Lynd Lee R, Trinh Cong T

机构信息

Bredesen Center for Interdisciplinary Research and Graduate Education, The University of Tennessee, Knoxville and Oak Ridge National Laboratory, Oak Ridge, TN, USA; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, TN, USA.

出版信息

Metab Eng. 2015 Nov;32:207-219. doi: 10.1016/j.ymben.2015.10.004. Epub 2015 Oct 21.

Abstract

Clostridium thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of C. thermocellum, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for C. thermocellum which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model's capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. In silico predictions of a redox-imbalanced genotype incapable of growth were confirmed in vivo, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of C. thermocellum and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain C. thermocellum to achieve high ethanol yields while requiring minimal genetic manipulations. The model can be applied to design C. thermocellum as a platform microbe for consolidated bioprocessing to produce ethanol and other reduced metabolites.

摘要

嗜热栖热菌是一种厌氧、革兰氏阳性的嗜热细菌,因其能够将木质纤维素生物质发酵为乙醇而备受关注。然而,由于嗜热栖热菌复杂且尚未完全了解的分支代谢,以及在某些情况下的溢流代谢,乙醇产量较低。在这项工作中,我们开发了一种嗜热栖热菌的预测性化学计量代谢模型,该模型纳入了当前的认识状态,特别关注非典型糖酵解酶中的辅因子特异性以及复杂的能量、氧化还原和发酵途径,旨在辅助代谢工程研究。我们验证了该模型涵盖亲本菌株和为显著扰动氧化还原和能量途径而设计的衍生突变体的实验观察表型的能力。代谢通量分布显示,工程菌株中关键代谢分支点(如磷酸烯醇丙酮酸、丙酮酸、乙酰辅酶A和辅因子节点)发生了显著变化,以引导电子和碳通量以提高乙醇合成,表现最佳的菌株与亲本菌株相比,乙醇产量和滴度提高了一倍。在体内证实了对无法生长的氧化还原失衡基因型的计算机预测,并且使用突变菌株作为平台来探究阻碍高效乙醇生产的中心代谢中的氧化还原瓶颈。结果突出了嗜热栖热菌氧化还原代谢的稳健性以及将还原铁氧还蛋白的电子通量简化为NAD(P)H以实现高乙醇产量的必要性。该模型进一步用于设计一种代谢工程策略,以在表型上限制嗜热栖热菌,从而在需要最少基因操作的情况下实现高乙醇产量。该模型可应用于将嗜热栖热菌设计为用于联合生物加工以生产乙醇和其他还原代谢物的平台微生物。

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