Improving the activity of surface displayed cytochrome P450 enzymes by optimizing the outer membrane linker.
Anchorage of recombinant proteins onto the outer membrane of gram-negative bacteria is an attractive solution for protein library screening and whole cell biocatalysis if a membrane environment is required or mass transfer into the cell is limiting. Autotransporters have been successfully applied for surface display of various heterologous proteins. Still, many underlying parameters for achieving active enzymes are not known. Here, we systematically tested different linkers between passenger and the membrane embedded β-barrel of the autotransporter. The linker can have influence on aspects such as steric orientation of the passenger, distance to the outer membrane and accessibility of active sites. Six linker variants for display of the cytochrome P450 reductase were tested. Cytochrome c reduction by the cytochrome P450 reductase varied fivefold and was highest by introduction of a flexible glycine-serine region. When these variants were co-expressed with surface displayed CYP1A2, product concentration for paracetamol differed between 0.22 μM and 2.5 μM and for resorufin between 0.23 μM to 1 μM. The best glycine/serine containing sequence, that turned out to be best for CPR display, was then introduced into the linker for displaying CYP1A2. In comparison, up to 7.9 μM paracetamol and up to 1.69 μM resorufin were obtained with this new variant. The differences were not caused by changes in the number of displayed enzymes. To our knowledge, this is the first systematic study on engineering the linker for surface display of recombinant enzymes.
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany. Electronic address: paul.quehl@uni-muenster.de.
Paul
P
Quehl
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany. Electronic address: jan.schueuermann@uni-muenster.de.
Jan
J
Schüürmann
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany; Autodisplay Biotech GmbH, Merowingerplatz 1a, D-40225 Düsseldorf, Germany. Electronic address: mail.hollender@gmail.com.
Joel
J
Hollender
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany. Electronic address: joachim.jose@uni-muenster.de.
Joachim
J
Jose
D000082:Acetaminophen / Q000737:chemistry* / Q000378:metabolism; D018830:Adhesins, Escherichia coli / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D020134:Catalytic Domain; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D007700:Kinetics; D008958:Models, Molecular; D010078:Oxazines / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D018830:Adhesins, Escherichia coli; D010078:Oxazines; D011993:Recombinant Fusion Proteins; D000082:Acetaminophen; C014180:resorufin; D045304:Cytochromes c; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2
Autodisplay; Autotransporter, outer membrane linker; Cytochrome P450 1A2; Cytochrome P450 reductase