Effect of heat‐shock proteins for relieving physiological stress and enhancing the production of penicillin acylase in Escherichia coli

Abstract

High-level expression of recombinant penicillin acylase (PAC) using the strong trc promoter system in Escherichia coli is frequently limited by the processing and folding of PAC precursors (proPAC) in the periplasm, resulting in physiological stress and inclusion body formation in this compartment. Periplasmic heat-shock proteins with protease or chaperone activity potentially offer a promise for overcoming this technical hurdle. In this study, the effect of the two genes encoding periplasmic heat-shock proteins, that is degP and fkpA, on pac overexpression was investigated and manipulation of the two genes to enhance the production of recombinant PAC was demonstrated. Both DeltadegP and DeltafkpA mutants showed defective culture performance primarily due to growth arrest. However, pac expression level was not seriously affected by the mutations, indicating that the two proteins were not directly involved in the pathway for periplasmic processing of proPAC. The growth defect caused by the two mutations (i.e., DeltadegP and DeltafkpA) was complemented by either one of the wild-type proteins, implying that the function of the two proteins could partially overlap in cells overexpressing pac. The possible role that the two heat-shock proteins played for suppression of physiological stress caused by pac overexpression is discussed.

Enhancing Functional Expression of Heterologous Burkholderia Lipase in Escherichia coli

“…C20 Isolated from the food waste of a food processing plant [23] Plasmid pAR3GRO P araB ::groEL/ES, Cm R Lab stock [52] pAR3KJ P araB ::dnaK/J::grpE, Cm R Lab stock [52] pARDegP P araB ::degP, Cm R Lab stock [53] pARDsbA P araB ::dsbA, Cm R Lab stock [54] pARDsbC P araB ::dsbC, Cm R Lab stock [54] pARFkpA P araB ::fkpA, Cm R Lab stock [55] pARllDsbA P araB ::lldsbA, Cm R Lab stock [8] pARllDsbC P araB ::lldsbC, Cm R Lab stock [8] pARSkp P araB ::skp, Cm R Lab stock [42] pARSurA P araB ::surA, Cm R Lab stock pDest-555 P tac ::69his::T7PK, Ap R Chatterjee and Esposito [34] pDest-556 P tac ::69his::skp, Ap R Chatterjee and Esposito [34] pHisperiMBP P tac ::69his::mbp, Ap R Waugh and co-workers [56] pDsbALipll P T7 ::dsbA:: E. coli has several folding modulators to assist the folding of nascent polypeptides, particularly during stressful conditions. These folding modulators, including trigger factor, DnaK/J-GrpE, and GroEL/ES in the cytoplasm and Dsb-family chaperones, DegP, FkpA, Skp, and SurA in the periplasm, can be potentially useful for developing their bioactive conformation upon the high-level production of heterologous proteins [17].…”

Use of folding modulators to improve heterologous protein production in Escherichia coli

“…Meanwhile FkpA, which like trigger factor possesses both chaperone and PPIase activity, enhanced production of a wide range of scFv fragments by up to 10-fold when overproduced [ 173 ], while its fusion to various scAb fragments also led to increased solubility and higher functional yields [ 177 ]. FkpA co-production also led to increased hydrolysis of ampicillin by a catalytic scFv [ 178 ] and enhanced the production of penicillin acylase [ 179 ].…”

Application of proteomics in biotechnology – Microbial proteomics

“…Additionally, the final product has to be tested and a proof for quantitative removal of host proteins is necessary [93]. Of particular importance are influence of temperature [97,98] and global physiological changes in E. coli during high cell density cultivation [99][100][101]. Overexpression of recombinant proteins, a high cell density cultivation, and cultivation under extreme conditions (starving, elevated temperature, etc.)…”