Department of Chemical and Biomolecular Engineering, North Carolina State University, EB-1, 911 Partners Way, Raleigh, NC, 27695-7905, USA.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.
Extremophiles. 2020 Jul;24(4):511-518. doi: 10.1007/s00792-020-01172-2. Epub 2020 May 15.
The key difference in the modified Embden-Meyerhof glycolytic pathway in hyperthermophilic Archaea, such as Pyrococcus furiosus, occurs at the conversion from glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) where the typical intermediate 1,3-bisphosphoglycerate (1,3-BPG) is not present. The absence of the ATP-yielding step catalyzed by phosphoglycerate kinase (PGK) alters energy yield, redox energetics, and kinetics of carbohydrate metabolism. Either of the two enzymes, ferredoxin-dependent glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) or NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), responsible for this "bypass" reaction, could be deleted individually without impacting viability, albeit with differences in native fermentation product profiles. Furthermore, P. furiosus was viable in the gluconeogenic direction (growth on pyruvate or peptides plus elemental sulfur) in a ΔgapnΔgapor strain. Ethanol was utilized as a proxy for potential heterologous products (e.g., isopropanol, butanol, fatty acids) that require reducing equivalents (e.g., NAD(P)H, reduced ferredoxin) generated from glycolysis. Insertion of a single gene encoding the thermostable NADPH-dependent primary alcohol dehydrogenase (adhA) (Tte_0696) from Caldanaerobacter subterraneus, resulted in a strain producing ethanol via the previously established aldehyde oxidoreductase (AOR) pathway. This strain demonstrated a high ratio of ethanol over acetate (> 8:1) at 80 °C and enabled ethanol production up to 85 °C, the highest temperature for bio-ethanol production reported to date.
在嗜热古菌（如 Pyrococcus furiosus）中，经修正的 Embden-Meyerhof 糖酵解途径的关键区别在于从甘油醛-3-磷酸 (GAP) 转化为 3-磷酸甘油酸 (3-PG) 时，典型的中间产物 1,3-二磷酸甘油酸 (1,3-BPG) 不存在。磷酸甘油酸激酶 (PGK) 催化的产生 ATP 的步骤的缺失改变了能量产生、氧化还原能学和碳水化合物代谢的动力学。负责此“旁路”反应的两种酶中的任何一种，即依赖铁氧还蛋白的甘油醛-3-磷酸铁氧还蛋白氧化还原酶 (GAPOR) 或依赖 NADP 的非磷酸化甘油醛-3-磷酸脱氢酶 (GAPN)，单独缺失不会影响生存能力，尽管在天然发酵产物谱上存在差异。此外，P. furiosus 在ΔgapnΔgapor 菌株中能够在生糖方向（在丙酮酸或肽加元素硫上生长）生存。乙醇被用作需要从糖酵解产生的还原当量（例如 NAD(P)H、还原型铁氧还蛋白）的潜在异源产物（例如异丙醇、丁醇、脂肪酸）的替代物。插入一个来自 Caldanaerobacter subterraneus 的单基因，编码耐热 NADPH 依赖的伯醇脱氢酶（adhA）（Tte_0696），导致通过先前建立的醛氧化还原酶 (AOR) 途径产生乙醇的菌株。该菌株在 80°C 时表现出乙醇与乙酸盐的高比例（>8:1），并且能够在高达 85°C 的温度下生产乙醇，这是迄今为止报道的生物乙醇生产的最高温度。
