Metabolic role of pyrophosphate-linked phosphofructokinasepfkfor C1 assimilation inMethylotuvimicrobium alcaliphilum20Z

Abstract

Background 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. Results In this study, we performed gene knockout and RNA-seq experiments inMethylotuvimicrobium alcaliphilum20Z 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 inM. alcaliphilum20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture inpfkknockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation topfkknockout. A number of mutations, as well as gene expression profiles, were identified inpfkALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain. Conclusions 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.