title: Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion.
abstract: Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli has been technically problematic due to protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis. To overcome these problems, an alternative approach was explored in this study by extracellular secretion of PalB via two Sec-independent secretion systems, i.e., the alpha-hemolysin (type I) and the modified flagellar (type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Both shaker flask and bioreactor cultivations were performed to characterize the developed PalB expression/secretion systems. Bioactive PalB was expressed and secreted extracellularly either as a HlyA fusion (i.e., PalB-HlyA via type I system) or an intact protein (via type III system). However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. Also importantly, the secretion strategy appeared to have a minimum impact on cell physiology. PalB secretion via the type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via the type III system was slow with lower specific PalB activities but effective cell growth.
authors.affiliation: Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
authors.forename: Niju
authors.initials: N
authors.lastname: Narayanan
authors.forename: Manal
authors.initials: M
authors.lastname: Khan
authors.forename: C Perry
authors.initials: CP
authors.lastname: Chou
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000096:biosynthesis* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D006460:Hemolysin Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica