Abstract
This work reports the use of metabolic profiling and PCR techniques as a means of studying the growth of Streptomyces tenjimariensis . Metabolic profiles were created from gas chromatography–mass spectrometry analysis of extracts prepared through extraction of the intracellular contents followed by fractionation of the polar metabolites and trimethylsilyl derivatization. All chromatograms yielded profiles of between 100 and 200 metabolites.The major metabolite groups including organic acids, amino acids, sugars, sugar alcohols, and phosphatidyl sugar alcohols were identified and their profiles correlated to growth stage and antimicrobial activity. Statistical analysis including analysis of variance, hierarchical cluster analysis, and principal component analysis suggested that differences between the major growth phases could be distinguished, and may be used to establish patterns amongst key metabolites. The expression of four enzyme genes involving substrate utilization and istamycin biosynthesis was detected with reverse transcriptase-PCR across four time points during the growth of S. tenjimariensis .
Highlights
Analysis of select compounds and metabolites has been used extensively to elucidate specific metabolic pathways, it is more widely being applied to cataloguing all the biochemicals associated with an organism’s metabolism [1]
As with established gene expression technologies, it is assumed that when metabolite profiles are compared across stages of growth,variations in metabolites produced in response to specific environmental cues and stresses can be identified.Such an approach has numerous industrial applications including narrowing a list of target compounds, identifying disease biomarkers, optimizing growth conditions, or verifying product quality
This work reports the use of metabolic profiling as a means of distinguishing between the growth phases of S. tenjimariensis and gene expression of the enzymes AMT, deoxy-scyllo-inosose synthase (DOI) and myo-inositol dehydrogenase (MYO) involved in the biosynthesis of aminoglycoside antibiotics
Summary
Analysis of select compounds and metabolites has been used extensively to elucidate specific metabolic pathways, it is more widely being applied to cataloguing all (or most of) the biochemicals associated with an organism’s metabolism (or metabolome) [1]. Differential gene expression identifies the genes expressed in response to specific environmental cues or stresses, while metabolic profiling measures the metabolites. When combined, these techniques promise a powerful means of assigning functions to previously uncharacterized genes, as in the field of functional genomics [6,7], or toward understanding the coordinated processes of whole organisms as is the goal of systems biology [8,9]. This work reports the use of metabolic profiling as a means of distinguishing between the growth phases of S. tenjimariensis and gene expression of the enzymes AMT, DOI and MYO involved in the biosynthesis of aminoglycoside antibiotics. Gene expression of AMY was detected with PCR
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