Dataset Information

Rare Earth Elements Alter Redox Balance in Methylomicrobium alcaliphilum 20ZR.

ABSTRACT: 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.

SUBMITTER: Akberdin IR

PROVIDER: S-EPMC6277846 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Rare Earth Elements Alter Redox Balance in Methylomicrobium alcaliphilum 20ZR.

Akberdin Ilya R IR Collins David A DA Hamilton Richard R Oshchepkov Dmitry Y DY Shukla Anil K AK Nicora Carrie D CD Nakayasu Ernesto S ES Adkins Joshua N JN Kalyuzhnaya Marina G MG

Frontiers in microbiology 20181127

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 diffe ...[more]

PMID: 30542328

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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.

Dataset Information

Rare Earth Elements Alter Redox Balance in Methylomicrobium alcaliphilum 20ZR.

Publications

Rare Earth Elements Alter Redox Balance in <i>Methylomicrobium alcaliphilum</i> 20Z<sup>R</sup>.

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