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

Abstract

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.

FIG. 1

Comparison of the amino acid sequences of LPPAD (accession no. U63827), PPPAD (accession no. AJ276891), BPPAD (accession no. AJ278683), and BSPAD (accession no. AF017117). The sequences were aligned using the Clustal program. Identical residues are shaded. The cluster of 18 amino acids which corresponds to the variable region is boxed. The conserved regions are indicated (1 to 10). The numbers on the right correspond to the amino acid position in the protein sequence.

FIG. 2

(a) Structure of the PAD proteins expressed in E. coli under the control of their own promoters. The positions of the ClaI sites of pdc and padA and the SpeI site of pdc are indicated. (b) Structure of the four chimeric proteins. The promoter which controls chimeric gene expression in E. coli is indicated. Restriction sites are indicated as follows: H, HindIII; P, PstI; Sa, SacI; K, KpnI; S, SmaI. Names of genes are on the right.

FIG. 3

SDS-PAGE of crude cell extracts from recombinant clones of E. coli TG1 expressing native and chimeric PADs. Lanes: 1, molecular mass standards (SDS-PAGE standards; Bio-Rad); 2, TG1(pJPAD14); 3, TG1(pJPLP113PP); 4, TG1(pJPADP6); 5, TG1(pJPP113LP); 6, TG1(pJDC9). Molecular size markers are indicated on the left.

FIG. 4

Effect of p-coumaric acid addition in LB medium at different pHs on growth of E. coli TG1(pJDC9) (A) and TG1(pJPAD14) (B). Cultures were inoculated to an initial density of 0.1 OD₆₀₀ unit and incubated for 20 h at 37°C with shaking. A growth of 100% refers to the control cultures lacking p-coumaric acid in which final biomass levels at pH 5.2, 6.2, and 7.2 resulted in 1.8, 2, and 2.1 OD₆₀₀ units, respectively.

FIG. 5

Growth of E. coli TG1(pJDC9) (■), E. coli TG1(pJBS121LP) (●), and E. coli TG1(pJPAD14) (▴) supplemented (filled symbols) or not (open symbols) with p-coumaric acid (3 mM) at pH 6.2. Residual p-coumaric acid concentrations in cultures of E. coli TG1(pJDC9) (–□–), E. coli TG1(pJBS121LP) (–○–), and E. coli TG1(pJPAD14) (–▵–) were measured by UV spectrophotometry.