DegP-coexpression minimizes inclusion-body formation upon overproduction of recombinant penicillin acylase in Escherichia coli.
We demonstrated the enhancement of recombinant penicillin acylase (PAC) production in Escherichia coli by increasing the intracellular concentration of the periplasmic protease DegP. Using appropriate host/vector systems (e.g., HB101 harboring pTrcKnPAC2902 or MDDeltaP7 harboring pTrcKnPAC2902) in which the expression of the pac gene was regulated by the strong trc promoter, the overproduction of PAC was often limited by periplasmic processing and inclusion bodies composed of protein aggregates of PAC precursors were formed in the periplasm. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The specific PAC activity reached an extremely high level of 674 U/L/OD(600) for MDDeltaP7 harboring pTrcKnPAC2902 and pKS12 under optimum culture conditions. However, such improvement in the production of PAC was not observed for the expression systems (e.g., MDDeltaP7 harboring pCLL2902) in which the periplasmic processing was not the step limiting the production of PAC. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme is shown to be not absolutely required for the formation of active PAC in E. coli. In addition, the steps limiting the production of PAC are identified and the reasons for the formation of PAC inclusion bodies are discussed here.
Lin
W J
WJ
Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan, Republic of China.
Huang
S W
SW
Chou
C P
CP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins; D002479:Inclusion Bodies / Q000378:metabolism*; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism; D033903:Periplasmic Proteins; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; C061682:DegP protease; D012697:Serine Endopeptidases; D010399:Penicillin Amidase