title: Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid.
abstract: BACKGROUND
  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 Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.
  
  
  RESULTS
  To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro 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 (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, 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 in vivo 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 E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, 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-LipB1a and BL21/pELipA-LipB3a) 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 E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.
  
  
  CONCLUSIONS
  Our findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr, Caugiay 10600, Hanoi, Vietnam. quyen@ibt.ac.vn
authors.forename: Thi Dinh
authors.initials: TD
authors.lastname: Quyen
authors.forename: Chi Hai
authors.initials: CH
authors.lastname: Vu
authors.forename: Giang Thi Thu
authors.initials: GT
authors.lastname: Le
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D043367:Ralstonia / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D008049:Lipase