Expression in Escherichia coli of native and chimeric phenolic acid decarboxylases with modified enzymatic activities and method for screening recombinant E. coli strains expressing these enzymes.
Four bacterial phenolic acid decarboxylases (PAD) from Lactobacillus plantarum, Pediococcus pentosaceus, Bacillus subtilis, and Bacillus pumilus were expressed in Escherichia coli, and their activities on p-coumaric, ferulic, and caffeic acids were compared. Although these four enzymes displayed 61% amino acid sequence identity, they exhibit different activities for ferulic and caffeic acid metabolism. To elucidate the domain(s) that determines these differences, chimeric PAD proteins were constructed and expressed in E. coli by exchanging their individual carboxy-terminal portions. Analysis of the chimeric enzyme activities suggests that the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity. In order to test phenolic acid toxicity, the levels of growth of recombinant E. coli displaying and not displaying PAD activity were compared on medium supplemented with different concentrations of phenolic acids and with differing pHs. Though these acids already have a slight inhibitory effect on E. coli, vinyl phenol derivatives, created during decarboxylation of phenolic acids, were much more inhibitory to the E. coli control strain. To take advantage of this property, a solid medium with the appropriate pH and phenolic acid concentration was developed; in this medium the recombinant E. coli strains expressing PAD activity form colonies approximately five times smaller than those formed by strains devoid of PAD activity.
Barthelmebs
L
L
Laboratoire de Microbiologie UMR-INRA, ENSBANA, Université de Bourgogne, 21000 Dijon, France.
Diviès
C
C
Cavin
J F
JF
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002109:Caffeic Acids / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D002109:Caffeic Acids; D003373:Coumaric Acids; D003470:Culture Media; D011993:Recombinant Fusion Proteins; D002262:Carboxy-Lyases; C417714:phenolic acid decarboxylase; C040048:caffeic acid