Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

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Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens Delta tliA Delta prtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens Delta tliA Delta prtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens Delta prtA and P. fluorescens Delta tliA Delta prtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens Delta tliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens Delta tliA Delta prtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.
Publisher
AMER SOC MICROBIOLOGY
Issue Date
2012-12
Language
English
Article Type
Article
Keywords

GRAM-NEGATIVE BACTERIA; ESCHERICHIA-COLI; SECRETION; EXPRESSION; CLONING; PLASMIDS; IDENTIFICATION; CHROMOSOME; AERUGINOSA; SUBSTRATE

Citation

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, v.78, no.23, pp.8454 - 8462

ISSN
0099-2240
DOI
10.1128/AEM.02476-12
URI
http://hdl.handle.net/10203/97165
Appears in Collection
RIMS Journal Papers
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