Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp.
The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30 degrees C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml. OD (optical density) (28 microg/ml. OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.
Ahn
J H
JH
R&D Center, Creagene Inc., Seo-gu, Taejon 302-858, Korea.
Pan
J G
JG
Rhee
J S
JS
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D010957:Plasmids; D011549:Pseudomonas / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011995:Recombination, Genetic
D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; D008049:Lipase