Premium
Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses
Author(s) -
Lee Pat S.,
Lee Kelvin H.
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20342
Subject(s) - mutant , escherichia coli , biology , secretion , gene , phenotype , proteomics , dna microarray , protein biosynthesis , messenger rna , translation (biology) , recombinant dna , microarray , mutagenesis , plasmid , gene expression , microbiology and biotechnology , biochemistry
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 α‐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. © 2004 Wiley Periodicals, Inc.