Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production
ABSTRACTPseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production.P. fluorescenspossesses 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 inP. fluorescensare attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from theP. fluorescensgenome 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 ofP. fluorescensSIK W1 were deleted using the targeted gene knockout method. Deletion mutantP. fluorescensΔtliAΔprtAsecreted fusion proteins without TliA or protein degradation. Using wild-typeP. fluorescensas an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with theP. fluorescensΔtliAΔprtAdouble mutant irrespective of growth conditions. By homologous expression oftliAand the ABC transporter in a plasmid, TliA secreted fromP. fluorescensΔprtAandP. fluorescensΔtliAΔprtAcells was found to be intact, whereas that secreted from the wild-typeP. fluorescensandP. fluorescensΔtliAcells was found to be hydrolyzed. Our results demonstrate that theP. fluorescensΔtliAΔprtAdeletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.