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Title: An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme      
dateReleased:
07-16-2014
description:
Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80Δ KlCAR1 reference strain) and IMS0367 (Klcar1Δ Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-56060
refinement:
raw
alternateIdentifiers:
56060
keywords:
functional genomics
dateModified:
09-05-2014
availability:
available
types:
gene expression
name:
Kluyveromyces lactis
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-56060/E-GEOD-56060.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-56060/E-GEOD-56060.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56060
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress

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