title: Enhancement of excretory production of an exoglucanase from Escherichia coli with phage shock protein A (PspA) overexpression.
abstract: Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Wang
authors.forename: Z B
authors.initials: ZB
authors.lastname: Fu
authors.forename: K L
authors.initials: KL
authors.lastname: Ng
authors.forename: C C
authors.initials: CC
authors.lastname: Lam
authors.forename: A K N
authors.initials: AK
authors.lastname: Chan
authors.forename: K F
authors.initials: KF
authors.lastname: Sze
authors.forename: W K R
authors.initials: WK
authors.lastname: Wong
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D040143:Cellulomonas / Q000201:enzymology / Q000235:genetics; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C069816:phage shock protein, Bacteria; D005959:Glucosidases