The 6-phosphofructokinase reaction in Acetivibrio thermocellus is both ATP- and pyrophosphate-dependent

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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Abstract

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.

Abstract Image

热肠乙酸弧菌中的 6-磷酸果糖激酶反应既依赖 ATP,也依赖焦磷酸。
热细胞醋酸梭菌(原热细胞梭菌)是木质纤维素乙醇生产的潜在平台。由于其中央代谢的热动力较低,产品滴度较低,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),其中只有后者对低驱动力负责。在这里,我们展示了在细胞质焦磷酸酶和反醛酸酶取代 PPi-Pfk 后,原生 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 的强烈抑制作用可以防止两种酶同时活跃时产生的徒劳循环。因此,似乎不需要人们长期寻求的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则补充PFK流的其余部分。ΔPPi-Pfk菌株无法利用PPi,其实验室进化导致GreA转录延伸因子发生突变。这种突变可能导致 RNA 翻转减少,暗示转录是合成 PPi 的一个重要来源(但被低估了)。这一突变与其他突变一起,使热菌菌株具有迄今为止最高的生物质特异性纤维生物糖吸收率(2.2 g/gx/h)。这些发现既有助于从根本上了解 ATP/PPi 双 Pfk 节点(这在其他微生物中并不罕见),也有助于进一步改造热菌,使其用于综合生物加工。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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