Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PPi-dependent 6-phosphofructokinase (PPi-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PPi-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PPi can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PPi-generating mechanism to drive glycolysis, as PPi-Pfk can simply use whatever PPi is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPPi-Pfk strain, unable to valorize PPi, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PPi. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/gx/h. These findings are both relevant for fundamental insight into dual ATP/PPi Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

中文翻译：

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热纤菌中的 6-磷酸果糖激酶反应既依赖于 ATP 又依赖于焦磷酸盐


热纤醋酸菌（以前称为热纤梭菌）是木质纤维素乙醇生产的潜在平台。由于其中心代谢的热力学驱动力低，其产物滴度低，阻碍了其工业应用。它同时具有功能性 ATP 和功能性 PPi 依赖性 6-磷酸果糖激酶 (PPi-Pfk)，其中只有后者负责低驱动力。在这里，我们表明，在用胞质焦磷酸酶和转醛醇酶取代 PPi-Pfk 后，天然 ATP-Pfk 能够携带全部糖酵解通量。有趣的是，体外几乎检测不到的 ATP-Pfk 活性仅是所需活性的一小部分，这表明它对糖酵解的贡献一直被低估。动力学模型表明，PPi 对 ATP-Pfk 的强烈抑制可以防止两种酶同时激活时出现的无效循环。因此，似乎不需要长期寻求的 PPi 生成机制来驱动糖酵解，因为 PPi-Pfk 可以简单地使用任何可用的 PPi，而 ATP-Pfk 补充了 PFK 通量的其余部分。 ΔPPi-Pfk 菌株的实验室进化无法评估 PPi，导致 GreA 转录延伸因子发生突变。这种突变可能导致 RNA 周转减少，暗示转录是合成代谢 PPi 的重要（且被低估）来源。与其他突变一起，产生了具有迄今为止最高的生物量特异性纤维二糖摄取率 2.2 g/gx/h 的热纤维素菌株。这些发现既有助于深入了解双 ATP/PPi Pfk 节点（这在其他微生物中并不罕见），也有助于进一步工程化 A. 用于综合生物加工的热纤维。