Boosting extracellular protein secretion of Escherichia coli via perturbing cell wall

ABSTRACT

Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Herein, extracellular protein secretion was enhanced in E. coli by deleting two D,D-carboxypeptidase genes (dacA and dacB, single and double deletion) to perturb the cell wall peptidoglycan network. Deletion of dacA and dacB enhanced the accumulation of intracellular soluble peptidoglycan in E. coli and affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion of dacA and dacB appears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased >2.0-fold compared with control cells, and the double deletion mutant was increased 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with control cells. Additionally, the extracellular recombinant amylase activity of single deletion mutants BL21-ΔdacA-pETDuet-amyk and BL21-ΔdacB-pETDuet-amyk was increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased >2.0-fold. Deletion of dacA and dacB increased the outer membrane permeability, which could explain the enhanced extracellular protein secretion.

IMPORTANCE Cell surface structure stabilization is important for extracellular secretion of proteins in E. coli. As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Herein, we perturbed the peptidoglycan network by deleting dacA and dacB genes encoding D,D-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity of E. coli as a microbial cell factory for extracellular secretion of proteins and chemicals.