Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli.
Bovine adrenodoxin (Adx) was expressed on the surface of Escherichia coli as a monomeric fusion protein with the translocation unit of the AIDA-I autotransporter. The fusion protein remained anchored in the outer membrane by the beta-barrel of the autotransporter. Dimeric Adx molecules were formed spontaneously on the bacterial surface with high efficiencies. Adx dimers could be activated to biological function by chemical incorporation of the [2Fe-2S] cluster. By adding purified adrenodoxin reductase and P450 CYP11A1, a whole cell biocatalyst system was obtained, which effectively synthesized pregnenolone from cholesterol. Addition of artificial membrane constituents or detergents, which was indispensable before to get functional steroidal P450 enzymes, was not necessary. The whole cell activity (0.21 nmol x h(-1) x nmol(-1) CYP11A1) was in the same range as obtained earlier for reconstitution assays. The whole cell system developed here is an easy to handle, stable tool for the expression of membrane-associated P450 enzymes without the need of microsome preparation or reconstitution of artificial membrane vesicles. Moreover, it is the first report on functional dimer formation of a protein anchored on the surface of E. coli after being transported as a monomer. This seems to be a special feature of the autotransporter translocation unit, containing a beta-barrel, motile in the outer membrane and opens a new dimension for the surface display of multimeric proteins.
Jose
Joachim
J
Pharmazeutische und Medizinische Chemie, Universität des Saarlandes, Postfach 151150, 66041, Saarbrücken, Germany. j.jose@mx.uni-saarland.de
Bernhardt
Rita
R
Hannemann
Frank
F
D018830:Adhesins, Escherichia coli / Q000096:biosynthesis / Q000235:genetics; D000325:Adrenodoxin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme