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：

---

Acetivibrio thermocellus 中的 6-磷酸果糖激酶反应是 ATP 和焦磷酸盐依赖性的


Acetivibrio thermocellus（以前称为 Clostridium thermocellum）是木质纤维素乙醇生产的潜在平台。由于其中央代谢的热力学驱动力低，其工业应用受到低产物滴度的阻碍。它具有功能性 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，在被胞质焦磷酸酶和转醛缩酶取代 PPi-Pfk 后，天然 ATP-Pfk 能够携带完整的糖酵解通量。有趣的是，几乎无法检测到的体外 ATP-Pfk 活性仅为所需活性的一小部分，这表明其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活跃时出现的徒劳循环。因此，似乎不需要一个长期寻求的 PPi 产生机制来驱动糖酵解，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使 PPi 增值，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（但被低估）来源。与其他突变一起，这导致 A. thermocellus 菌株具有迄今为止最高的生物量特异性纤维二糖摄取率，为 2.2 g/gx/h。这些发现都与对双 ATP/PPi Pfk 节点的基本认识有关，这在其他微生物中并不少见，以及 A 的进一步工程化。 用于整合生物工艺的 Thermocellus。