title: Lipase and protease double-deletion mutant of Pseudomonas fluorescens suitable for extracellular protein production.
abstract: 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 ΔtliA ΔprtA 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 ΔtliA ΔprtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens ΔprtA and P. fluorescens ΔtliA ΔprtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens ΔtliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens ΔtliA ΔprtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.
authors.affiliation: Korea Science Academy of KAIST, Busan, South Korea.
authors.forename: Myunghan
authors.initials: M
authors.lastname: Son
authors.forename: Yuseok
authors.initials: Y
authors.lastname: Moon
authors.forename: Mi Jin
authors.initials: MJ
authors.lastname: Oh
authors.forename: Sang Bin
authors.initials: SB
authors.lastname: Han
authors.forename: Ki Hyun
authors.initials: KH
authors.lastname: Park
authors.forename: Jung-Gon
authors.initials: JG
authors.lastname: Kim
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Ahn
mesh_terms: D003470:Culture Media / Q000737:chemistry; D055786:Gene Knockout Techniques; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D008049:Lipase; D010447:Peptide Hydrolases