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Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

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 α-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.

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Acknowledgments

This study was supported in part by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canada Research Chair (CRC) program. We greatly appreciate the E. coli strain and plasmids provided by Benita Westerlund-Wilkstrom (p5′UTR, pMCS3′UTR, and MKS12) and Luis Angel (pEHLYA2-SD).

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  1. Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Niju Narayanan, Manal Khan & C. Perry Chou

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  1. Niju Narayanan
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  2. Manal Khan
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  3. C. Perry Chou
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Correspondence to C. Perry Chou.

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Narayanan, N., Khan, M. & Chou, C.P. Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion. J Ind Microbiol Biotechnol 37, 349–361 (2010). https://doi.org/10.1007/s10295-009-0680-2

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  • DOI: https://doi.org/10.1007/s10295-009-0680-2

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