Gonzalez Jacqueline E, Long Christopher P, Antoniewicz Maciek R
Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
Metab Eng. 2017 Jan;39:9-18. doi: 10.1016/j.ymben.2016.11.003. Epub 2016 Nov 11.
Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in C metabolic flux analysis (C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated C-MFA using the optimal tracers [1,2-C]glucose, [1,6-C]glucose, [1,2-C]xylose and [5-C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-C]glucose and [U-C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.
葡萄糖和木糖是木质纤维素生物质分解产生的两种最丰富的糖类。虽然大肠杆菌中需氧葡萄糖代谢已得到较好理解，但到目前为止，仅有少数研究关注厌氧葡萄糖代谢，且尚无关于木糖代谢的碳通量研究。在缺乏经实验验证的通量图的情况下，诸如MOMA和RELATCH等基于约束的方法无法用于指导新的代谢工程设计。在这项工作中，我们通过使用碳代谢通量分析（C-MFA）的最新技术，对需氧和厌氧条件下的葡萄糖和木糖代谢进行全面表征，解决了当前理解中的这一关键差距。具体而言，我们通过进行平行标记实验并使用最佳示踪剂[1,2-C]葡萄糖、[1,6-C]葡萄糖、[1,2-C]木糖和[5-C]木糖，通过综合C-MFA分析数据，对每种条件下的精确代谢通量进行了量化。我们还通过应用[U-C]葡萄糖和[U-C]木糖示踪剂，量化了生物质组成的变化并确认了大分子的周转。我们证明，在厌氧生长条件下，脂质有显著周转，且相当一部分CO源自生物质周转。使用基因敲除菌株，我们还证明β-氧化对于木糖厌氧生长至关重要。对不同生长条件下的辅因子平衡（NADH/FADH、NADPH和ATP）进行定量分析，为能量和氧化还原代谢的相互作用以及对大肠杆菌细胞生理学的影响提供了新的见解。
