Use FACS Sorting in Metabolic Engineering of Escherichia coli for Increased Peptide Production

Abstract

Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced as recombinant forms by microbial fermentation. Targeted metabolic engineering of the production strains has usually been the approach taken to increase protein production, and this approach requires sufficient knowledge about cell metabolism and regulation. Random screening is an alternative approach that could circumvent the knowledge requirement, but is hampered by lack of suitable high-throughput screening methods. We developed a novel fluorescence-activated cell sorting (FACS) method to screen for cells with increased peptide production. Using a model peptide rich in certain amino acids, we showed that increased fluorescence clones sorted from a plasmid expression library contained genes encoding rate-limiting enzymes for amino acid synthesis. These expression clones showed increased peptide production. This demonstrated that FACS could be used as a very powerful tool for metabolic engineering. It can be generally applied to other products or processes if the desired phenotype could be correlated with a fluorescence or light scattering parameter on the FACS.