Utilization of ATP-binding cassette exporter for hyperproduction of an exoprotein: construction of lipase-hyperproducing recombinant strains of Serratia marcescens.
The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.
Idei
A
A
Discovery Research Laboratory, Tanabe Seiyaku, Osaka, Japan.
Matsumae
H
H
Kawai
E
E
Yoshioka
R
R
Shibatani
T
T
Akatsuka
H
H
Omori
K
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D003470:Culture Media; D020689:Exopeptidases / Q000138:chemical synthesis / Q000378:metabolism; D015870:Gene Expression; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012706:Serratia marcescens / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003470:Culture Media; C079999:LipA protein, Bacteria; D011994:Recombinant Proteins; D008049:Lipase; D020689:Exopeptidases