Abstract
Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known functions.
Highlights
The technological progress in genomic research led to a dramatic increase of knowledge of whole genome sequences, opening the demand of understanding the complex and dynamic processes of gene expression, proteomics and metabolic pathways
Due to the convoluted state of cell metabolism, where many metabolites are involved in different pathways, it is difficult and sometimes even impossible to establish a direct link between genes and metabolites [6]
High-throughput Insertion Point Determination An insertion point determination was established using the TAIL-PCR method [20] for our in-house C. glutamicum transposon mutant library. This transposon mutant library was created by the aid of the vector pAT6100 [13] and the restriction deficient wild type derivative C. glutamicum Res167
Summary
The technological progress in genomic research led to a dramatic increase of knowledge of whole genome sequences, opening the demand of understanding the complex and dynamic processes of gene expression, proteomics and metabolic pathways. While a rapid development in multiparallel analytical methods for transcriptomics and proteomics has already taken place [1] the field of metabolomics still lacks appropriate techniques. Compared to the other Omics-platforms, metabolomics closely reflects cell activity at the functional level and is often directly correlated with the cellular phenotype. Observed changes in transcriptome and proteome level do not always correspond to phenotypic alterations [6]. Due to the convoluted state of cell metabolism, where many metabolites are involved in different pathways, it is difficult and sometimes even impossible to establish a direct link between genes and metabolites [6]
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