Deleting mrdA and mrcB to significantly improve extracellular recombinant protein production in Escherichia coli

Abstract

In this work, two key genes (mrdA and mrcB) involved in cell wall biosynthesis were deleted (singly and doubly) in Escherichia coli, and the effects on extracellular recombinant protein production were investigated. The mrdA and mrcB genes encode penicillin-binding protein (PBP) 2 and PBP1B, respectively. The growth of deletion mutants was not significantly inhibited compared with control cells, but cell morphology was altered. The concentration of intracellular soluble peptidoglycan was increased in deletion mutants, especially the double deletion mutant. Extracellular protein production was significantly improved in deletion mutants compared with controls, in the order double deletion > single deletion of mrcB > single deletion of mrdA. Extracellular green fluorescent protein production by the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-gfp was most significantly enhanced compared with control cells (2.6-fold). Extracellular production of recombinant fibroblast growth factor receptor 2 and collagen E4 was also improved in deletion mutants compared with control cells. Extracellular amylase activity of the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-amyk was increased 5.2-fold relative to control cells, but activity in BL21 ΔmrdA::pET28a-amyk and BL21 ΔmrcB::pET28a-amyk single deletion mutants was only increased 1.7- and 4.3-fold, respectively. Extracellular α-galactosidase activity of deletion mutants was also improved, especially the double deletion mutant (2.6-fold). Membrane permeability of deletion mutants was improved compared with control cells, which might increase extracellular recombinant protein production.