title: Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses.
abstract: Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification. However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression. The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, New York 14853-5201, USA.
authors.forename: Pat S
authors.initials: PS
authors.lastname: Lee
authors.forename: Kelvin H
authors.initials: KH
authors.lastname: Lee
mesh_terms: D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D011994:Recombinant Proteins