Dataset Information

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z.

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 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. 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.

SUBMITTER: Nguyen AD

PROVIDER: S-EPMC7298851 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z.

Nguyen Anh Duc AD Nam Gayoung G Kim Donghyuk D Lee Eun Yeol EY

Microbial cell factories 20200616 1

<h4>Background</h4>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, ...[more]

PMID: 32546161

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Project description:BackgroundMethane is the primary component of natural gas and biogas. The huge abundance of methane makes it a promising alternative carbon source for industrial biotechnology. Herein, we report diamine compound, putrescine, production from methane by an industrially promising methanotroph Methylomicrobium alcaliphilum 20Z.ResultsWe conducted adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z because putrescine highly inhibits the cell growth. The evolved strain 20ZE was able to grow in the presence of 400 mM of putrescine dihydrochloride. The expression of linear pathway ornithine decarboxylase genes from Escherichia coli and Methylosinus trichosporium OB3b allowed the engineered strain to produce putrescine. A higher putrescine titer of 12.44 mg/L was obtained in the strain 20ZE-pACO with ornithine decarboxylase from M. trichosporium OB3b. For elimination of the putrescine utilization pathway, spermidine synthase (MEALZ_3408) was knocked out, resulting in no spermidine formation in the strain 20ZES1-pACO with a putrescine titer of 18.43 mg/L. Next, a genome-scale metabolic model was applied to identify gene knockout strategies. Acetate kinase (MEALZ_2853) and subsequently lactate dehydrogenase (MEALZ_0534) were selected as knockout targets, and the deletion of these genes resulted in an improvement of the putrescine titer to 26.69 mg/L. Furthermore, the putrescine titer was improved to 39.04 mg/L by overexpression of key genes in the ornithine biosynthesis pathway under control of the pTac promoter. Finally, suitable nitrogen sources for growth of M. alcaliphilum 20Z and putrescine production were optimized with the supplement of 2 mM ammonium chloride to nitrate mineral salt medium, and this led to the production of 98.08 mg/L putrescine, almost eightfold higher than that from the initial strain. Transcriptome analysis of the engineered strains showed upregulation of most genes involved in methane assimilation, citric acid cycle, and ammonia assimilation in ammonia nitrate mineral salt medium, compared to nitrate mineral salt medium.ConclusionsThe engineered M. alcaliphilum 20ZE4-pACO strain was able to produce putrescine up to 98.08 mg/L, almost eightfold higher than the initial strain. This study represents the bioconversion of methane to putrescine-a high value-added diamine compound.

Project description:Background: Rare Earth Elements (REEs) control methanol utilization in both methane- and methanol-utilizing microbes. It has been established that the addition of REEs leads to the transcriptional repression of MxaFI-MeDH [a two-subunit methanol dehydrogenase (MeDH), calcium-dependent] and the activation of XoxF-MeDH (a one-subunit MeDH, lanthanum-dependent). Both enzymes are pyrroquinoline quinone-dependent alcohol dehydrogenases and show significant homology; however, they display different kinetic properties and substrate specificities. This study investigates the impact of the MxaFI to XoxF switch on the behavior of metabolic networks at a global scale. Results: In this study we investigated the steady-state growth of Methylomicrobium alcaliphilum 20ZR in media containing calcium (Ca) or lanthanum (La, a REE element). We found that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca. Three complementary global-omics approaches-RNA-seq transcriptomics, proteomics, and metabolomics-were applied to investigate the mechanisms of improved growth vs. carbon conversion. Cells grown with La showed the transcriptional activation of the xoxF gene, a homolog of the formaldehyde-activating enzyme (fae2), a putative transporter, genes for hemin-transport proteins, and nitrate reductase. In contrast, genes for mxaFI and associated cytochrome (mxaG) expression were downregulated. Proteomic profiling suggested additional adjustments of the metabolic network at the protein level, including carbon assimilation pathways, electron transport systems, and the tricarboxylic acid (TCA) cycle. Discord between gene expression and protein abundance changes points toward the possibility of post-transcriptional control of the related systems including key enzymes of the TCA cycle and a set of electron-transport carriers. Metabolomic data followed proteomics and showed the reduction of the ribulose-monophosphate (RuMP) pathway intermediates and the increase of the TCA cycle metabolites. Conclusion: Cells exposed to REEs display higher rates of growth but have lower carbon conversion efficiency compared to cells supplemented with Ca. The most plausible explanation for these physiological changes is an increased conversion of methanol into formate by XoxF-MeDH, which further stimulates methane oxidation but limits both the supply of reducing power and flux of formaldehyde into the RuMP pathway.

Dataset Information

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z.

Publications

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z.

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