Callaghan Melanie M, Thusoo Eashant, Sharma Bishal D, Getahun Fitsum, Stevenson David M, Maranas Costas, Olson Daniel G, Lynd Lee R, Amador-Noguez Daniel
Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.
Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA; Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA.
Metab Eng. 2023 Nov;80:254-266. doi: 10.1016/j.ymben.2023.10.006. Epub 2023 Nov 1.
Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. C and H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water (HO) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply HO labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli-harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that HO labeling recapitulates previous reversibility and thermodynamic findings obtained with established C and H labeled nutrient substrates. Furthermore, we exemplify the utility of this HO labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly H-labeled nutrient substrates.
稳定同位素示踪剂是微生物代谢定量分析的有力工具，可用于阐明代谢途径、定量代谢通量以及评估反应和途径的热力学。碳和氢代谢通量分析通常依赖于同位素标记的碳底物，如葡萄糖。然而，与碳示踪剂相比，氢标记的营养底物由于成本高且可用性有限，其应用面临局限性。此外，对于代谢纤维素、半纤维素或木质纤维素生物质等复杂底物的工业相关细菌进行同位素示踪研究具有挑战性，因为难以获得这些作为同位素标记的底物。在本研究中，我们研究了重水（H₂¹⁸O）作为一种经济实惠、底物中性的同位素示踪剂用于研究中心碳代谢的潜力。我们应用H₂¹⁸O标记来研究三种工业相关细菌——嗜热栖热菌、运动发酵单胞菌和大肠杆菌——中糖酵解反应的可逆性，这些细菌具有独特热力学的不同糖酵解途径。我们证明H₂¹⁸O标记概括了先前使用已确立的碳和氢标记营养底物获得的可逆性和热力学研究结果。此外，我们通过将这种H₂¹⁸O标记方法应用于高底物浓度的嗜热栖热菌发酵（在这种情况下使用传统示踪剂不切实际）来举例说明其效用，从而确定糖酵解酶磷酸果糖激酶是高底物浓度发酵过程中的主要瓶颈，并揭示了将指导未来工程努力以提高这种纤维素分解生物中乙醇产量的关键见解。这项研究证明了重水作为一种与底物无关的同位素示踪剂用于检查中心碳代谢反应的通量和可逆性的效用，其产生的生物学见解与使用昂贵的氢标记营养底物获得的见解相当。
