Evaluation of cell factory performance through determination of intracellular metabolites using LC-MS/MS

A major objective in biotechnology is the improvement of the efficiency of host microorganisms used as cell factories. Engineering a strain capable of producing high amounts of a desired biochemical is a multi-step process consisting of design, construction, and analysis of the constructed cell factory. In order to address the function or disfunction of the engineered cells,systems biology tools are employed by using the multi “omics” approach (genomics, transcriptomics, proteomics, metabolomics and fluxomics). Metabolomics is a tool aimed at a quantitative understanding of metabolism. By quantification of intracellular metabolite changes over time, under given genetic and environmental perturbations, key regulatory nodes may be identified in the cellular metabolic network. In target metabolome analysis, the quantification is applied to only one or a small group of metabolites and is very useful for studying the effect of a genetic modification. The approach of targeted metabolomics was applied for the analysis of metabolite extracts from Saccharomyces cerevisiae and Lacctococus lactis. The analytical technique employed was ion-pair liquid chromatography tandem mass pectrometry (LC-MS/MS). The unique combination of sensitivity and specificity of this technique allowed qualitative and quantitative analysis of low metabolite concentrations. The mains steps involved were: (i) metabolite sample preparation by quenching, extraction, sample purification using dispersive Solid Phase Extraction; (ii) quantitative analysis, (iii) data analysis and interpretation. The established analytical method covers analysis of sixty metabolites from glycolysis,cofactors, coenzymes and nucleotides. Implementation of this method provides a powerful new tool in future cell factory design and characterization.