a high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level高通量的方法来识别基因组变异的细菌代谢物生产商在单细胞水平.pdfVIP

Binder et al. Genome Biology 2012, 13:R40 /2012/13/5/R40 METHOD Open Access A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level Stephan Binder, Georg Schendzielorz, Norma Stäbler, Karin Krumbach, Kristina Hoffmann, Michael Bott and * Lothar Eggeling Abstract We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes. Background ‘random’ approaches, which are typically based on the Since the first demonstration of microbial product for- creation of mutant libraries containing nondirected mation more than a century ago [1], vitamins, antibiotics, changes in genotype with subsequent screening for phe- nucleotides, amino acids and organic acids have been notypes of interest. Both approaches have been successful produced in ever increasing quantities. For example, but the use of mutant libraries has proven to have dis- about three million tonnes of sodium glutamate are pro- tinct advantages. The reason is that the exact genomic duced each year as a small microbial molecule. Bacterial