Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E-coli

被引:70
作者
Jose, J [1 ]
Bernhardt, R [1 ]
Hannemann, F [1 ]
机构
[1] Univ Saarland, D-66041 Saarbrucken, Germany
关键词
surface display; autotransporter; Adx; (P450s) cytochrome P450; whole cell biocatalystl; dimers;
D O I
10.1016/S0168-1656(02)00030-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 h(-1) 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. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:257 / 268
页数:12
相关论文
共 31 条
[1]   DEGRADATION OF SECRETED PROTEINS IN ESCHERICHIA-COLI [J].
BANEYX, F ;
GEORGIOU, G .
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES-SERIES, 1992, 665 :301-308
[3]   Cytochrome P450: Structure, function, and generation of reactive oxygen species [J].
Bernhardt, R .
REVIEWS OF PHYSIOLOGY BIOCHEMISTRY AND PHARMACOLOGY, VOL 127, 1996, 127 :137-221
[4]   ASSEMBLY OF [FE2S2(SR)4]2-, [FE4S4(SR)4]2- IN AQUEOUS-MEDIA FROM IRON SALTS, THIOLS, AND SULFUR, SULFIDE, OR THIOSULFATE PLUS RHODANESE [J].
BONOMI, F ;
WERTH, MT ;
KURTZ, DM .
INORGANIC CHEMISTRY, 1985, 24 (25) :4331-4335
[5]  
CALI JJ, 1991, J BIOL CHEM, V266, P7774
[6]   ISOLATION AND EXPRESSION OF HUMAN 1,25-DIHYDROXYVITAMIN-D3 24-HYDROXYLASE CDNA [J].
CHEN, KS ;
PRAHL, JM ;
DELUCA, HF .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (10) :4543-4547
[7]   Directed evolution of microbial oxidative enzymes [J].
Cherry, JR .
CURRENT OPINION IN BIOTECHNOLOGY, 2000, 11 (03) :250-254
[8]   Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies [J].
Daugherty, PS ;
Chen, G ;
Iverson, BL ;
Georgiou, G .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (05) :2029-2034
[9]   Impact of the presequence of a mitochondrium-targeted precursor, preadrenodoxin, on folding, catalytic activity, and stability of the protein in vitro [J].
Goder, V ;
Beckert, V ;
Pfeil, W ;
Bernhardt, R .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1998, 359 (01) :31-41
[10]   OMPT ENCODES THE ESCHERICHIA-COLI OUTER-MEMBRANE PROTEASE THAT CLEAVES T7-RNA POLYMERASE DURING PURIFICATION [J].
GRODBERG, J ;
DUNN, JJ .
JOURNAL OF BACTERIOLOGY, 1988, 170 (03) :1245-1253