Methanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product. In this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain. This study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

中文翻译：

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焦磷酸盐连接的磷酸果糖激酶pfk在C1同化中的代谢作用，在甲基杯状微藻20Z中。

甲烷营养生物是生物技术应用中有前途的生物催化剂，它们具有利用单一碳（C1）原料生产高价值化合物的能力。了解甲烷营养细菌在不同参数下的生物学网络行为对于系统生物学和代谢工程至关重要。有趣的是，甲烷氧化细菌具有焦磷酸依赖性的6-磷酸果糖激酶（PPi-PFK），而不是ATP依赖性的6-磷酸果糖激酶，这表明它们有潜力用作研究无机焦磷酸盐（PPi）和PPi依赖性糖酵解作用的有前途的模型在细菌中。基因敲除实验以及整体组学方法可用于研究基因功能以及建立依赖基因产物的调控网络。在这个研究中，我们在甲基杯状微微生物20Z中进行了基因敲除和RNA-seq实验，以研究PPi-PFK在细胞在甲烷和甲醇上生长时在C1代谢中的功能作用，从而突出了其在M.alcaliphilum 20Z中对C1同化的代谢重要性。我们进一步进行了自适应实验室进化（ALE），以研究pfk基因敲除菌株的调控结构。进行了全基因组重测序和RNA测序方法来表征适应pfk基因敲除的遗传和代谢反应。在pfk ALE菌株中鉴定了许多突变以及基因表达谱，以克服不足的C1同化途径，这限制了未进化菌株的生长。