Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; Ammon-Pinizzotto Biopharmaceutical Innovation Center, University of Delaware, Newark, DE, USA.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA.
Metab Eng. 2022 Jul;72:161-170. doi: 10.1016/j.ymben.2022.03.011. Epub 2022 Mar 18.
Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO fixation in the biosphere and in the development of biological processes - alone or in cocultures, under both autotrophic and mixotrophic conditions - for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using C-metabolic flux analysis (C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. C-MFA during mixotrophic growth using the parallel tracers [1-C]fructose, [1,2-C]fructose, [1,2,3-C]fructose, and [U-C]asparagine found that externally supplied CO contributed the majority of carbon consumed. All internally-produced CO from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.
利用 Wood-Ljungdahl 途径（WLP）进行二氧化碳固定的产乙酸菌（acetogens）在生物圈中固定 CO 以及开发单独或共培养的生物过程中发挥着重要作用，这些过程可以在自养和混合营养条件下进行，用于生产化学品和燃料。迄今为止，在实验验证和量化其核心代谢途径的反应通量方面，相关工作的报道十分有限。在这里，我们使用 C 代谢通量分析（C-MFA）来研究产乙酸菌 Clostridium ljungdahlii 的核心代谢模型，这需要开发一种新的定义培养基。通过向定义的培养基中添加 1mM 蛋氨酸来替代酵母提取物，可以实现自养、异养和混合营养生长。我们的 C-MFA 发现不完全的三羧酸循环和不活跃的核心途径/反应，特别是氧化戊糖磷酸途径、Entner-Doudoroff 途径和苹果酸脱氢酶的途径/反应。在使用平行示踪剂 [1-C]果糖、[1,2-C]果糖、[1,2,3-C]果糖和 [U-C]天冬酰胺进行混合营养生长期间的 C-MFA 发现，外部供应的 CO 贡献了大部分消耗的碳。来自天冬酰胺和果糖分解代谢的所有内部产生的 CO 都被 WLP 消耗。虽然果糖的糖酵解是活跃的，但它不是产生 ATP、NADH 和乙酰辅酶 A 的主要贡献者。尽管存在有机碳，但糖异生反应仍然活跃。天冬酰胺通过转化为苏氨酸和随后裂解生成乙醛酸和甘氨酸，以及通过脱氨生成延胡索酸和随后的苹果酸的补料作用转化为丙酮酸而被同等地分解。天冬酰胺分解代谢的两条途径都产生乙酰辅酶 A，要么直接通过丙酮酸，要么间接通过 WLP。基于我们的数据，辅因子化学计量预测通过依赖于铁氧还蛋白的转氢酶（Nfn）反应的通量基本为零。相反，几乎所有由氢化酶反应产生的 NADPH 都被 WLP 消耗。氢化酶反应和糖酵解产生的还原型铁氧还蛋白主要用于通过 RNF/ATP 酶系统产生 ATP，其余的被 WLP 消耗。RNF/ATP 酶产生的 NADH 完全通过 WLP 消耗。
