Serine-glyoxylate aminotranferases from methanotrophs using different C1-assimilation pathways

But, Sergey Y.; Егорова, С. В.; Khmelenina, V. N.; Trotsenko, Yu. A.

doi:10.1007/s10482-018-1208-4

Cited by 10 publications

(5 citation statements)

References 34 publications

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“…The comparable growth of the Δ pyc and Δ gcv knockout strains to that of the wild type indicated that these enzymes are not essential for CO 2 -dependent growth of this organism. Interestingly, we observed a growth defect by the Δ sga knockout strain, a similar phenotype to that recently observed in M. alcaliphilum 20Z R , which does not require exogenously supplied CO 2 for growth ( 24 ). Collectively, the Δ sga and Δ pyc knockout strain phenotypes suggest that the serine-glyoxylate aminotransferase is involved in M. capsulatus Bath oxaloacetate and/or glyoxylate conversion, but that a canonical serine cycle that would be dependent on a phosphoenolpyruvate or pyruvate carboxylase is not essential for M. capsulatus Bath CO 2 -dependent growth.…”

Section: Resultssupporting

confidence: 87%

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath

Henard

Xiong

et al. 2021

Appl Environ Microbiol

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The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we identified that CO 2 was requisite for RubisCO-encoding Methylococcus capsulatus Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO 2 assimilation during cultivation with both CH 4 and CO 2 as carbon sources. Marker-exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO 2 -assimilating metabolic pathways indicated that a complete serine cycle is not required while RubisCO is essential for growth of this bacterium. 13 CO 2 tracer analysis showed that CH 4 and CO 2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO 2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13 C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO 2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO 2 via RubisCO, which may play a more pivotal role in the Earth’s biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO 2 -assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH 4 and CO 2 . Importance The importance of RubisCO and CO 2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO 2 and RubisCO are essential for M. capsulatus Bath growth. 13 CO 2 tracing experiments supported that RubisCO mediates CO 2 fixation and a non-canonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dual CH 4 /CO 2 -utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO 2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases CH 4 and CO 2 .

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Section: Resultssupporting

confidence: 87%

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath

Henard

Xiong

et al. 2021

Appl Environ Microbiol

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“…It has been found that the expression of the serine cycle was more activated on methanol and deletion of PPi-PFK showed more defects on this pathway, suggesting the operation of a functional serine cycle. In agreement with this observation, when shifting the substrate from methane to methanol, our current studies showed the activation of serine cycle on methanol-grown for energy regulation [10,16,18], while the serine cycle only carries negligible flux for growth on methane [8]. In summary, those results demonstrated the impact of pfk knockouts on the metabolic phenotype is more expressive and also showed to have a different influence on the metabolism of grown on methane and methanol.…”

Section: Discussionsupporting

confidence: 90%

“…Compared to methane, the growth rate of M. alcaliphilum 20Z on methanol is much higher [16]. Moreover, it was reported that Methylotuvimicrobium species could accumulate a large amount of glycogen up to 30% dry cell weight and formate when cells are grown on methanol [10,17].…”

Section: Different Transcriptional Response Of Ppi-pfk Knockout On Mementioning

confidence: 99%

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

et al. 2020

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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.

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“…A high relative abundance of reads associated with one-carbon metabolism such as YgfZ, a folate-binding regulatory protein 79 and sequences related to the serine-glyoxylate cycle (Table 3 ) were identified. Serine-glyoxylate cycle is a carbon assimilation pathway found in aerobic methanotrophs belonging to the classes Alpha-, Gammaproteobacteria, and the phylum Verrucomicrobia 80 . Sequences annotated as crucial enzymes for methanotrophic metabolism such as methane monooxygenase, methanol dehydrogenase or hydroxypyruvate reductase 81 , 82 were present in the two replicates of As Burgas metagenome.…”

Section: Resultsmentioning

confidence: 99%

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

DeCastro

Doane

Dinsdale

et al. 2021

Sci Rep

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In the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-d-galactopyranoside (ONPG) and p-Nitrophenyl-β-d-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.

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Serine-glyoxylate aminotranferases from methanotrophs using different C1-assimilation pathways

Cited by 10 publications

References 34 publications

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath

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

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

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