Enhancing functional production of a chaperone-dependent lipase in <it>Escherichia coli </it>using the dual expression cassette plasmid

• Main Authors: Quyen Thi Dinh, Vu Chi Hai, Thu Le Giang Thi
• Format: Article
• Language: English
• Published: BMC 2012-03-01
• Series: Microbial Cell Factories
• Subjects: <it>Ralstonia </it>sp. M1; Lipase; Chaperone; Functional expression; Secretion
• Online Access: http://www.microbialcellfactories.com/content/11/1/29

<p>Abstracts</p> <p>Background</p> <p>The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from <it>Ralstonia </it>sp. M1 were overexpressed in <it>E. coli </it>and the lipase was successfully refolded <it>in vitro</it>. The purpose of this study was to enhance the production of the active lipase LipA from <it>Ralstonia </it>sp. M1 in the heterologous host <it>E. coli </it>without <it>in vitro </it>refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.</p> <p>Results</p> <p>To produce more active lipase from <it>Ralstonia </it>sp. M1 in <it>E. coli </it>without <it>in vitro </it>refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (<it>E. coli </it>BL21/pELipAB^a+ pELipB1^kand BL21/pELipAB^a+ pELipB3^k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1^a, BL21/pELipAB-LipB3^a, BL21/pELipA-LipB1^a, and BL21/pELipA-LipB3^a) were constructed. The two-plasmid co-expression systems (<it>E. coli </it>BL21/pELipAB^a+ pELipB1^kand BL21/pELipAB^a+ pELipB3^k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system <it>E. coli </it>BL21/pELipAB^adid. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1^aand BL21/pELipAB-LipB3^ayielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system <it>E. coli </it>BL21/pELipAB^a, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded <it>in vivo </it>into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems <it>E. coli </it>BL21/pELipAB-LipB1^aand <it>E. coli</it>/pELipAB-LipB3^asecreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system <it>E. coli </it>pELipAB^adid, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1^aand BL21/pELipA-LipB3^a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in <it>E. coli </it>BL21/pELipAB^a+ pELipB1^k, BL21/pELipAB^a+ pELipB3^k, BL21/pELipAB-LipB1^a, and BL21/pELipAB-LipB3^a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.</p> <p>Conclusions</p> <p>Our findings demonstrated that a dual expression cassette plasmid system <it>E. coli </it>could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) <it>in vivo </it>and an improved dual expression cassette plasmid system <it>E. coli </it>could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.</p>