*2.2. Bioconversion Experiments by Using Whole Cells of Pseudomonas sp. 7HK4*

Time course experiments using whole cells of *Pseudomonas* sp. 7HK4 pre-grown in the presence of 7-hydroxycoumarin showed that the cells converted coumarin, 6-hydroxycoumarin, and 6,7-dihydroxycoumarin in addition to 7-hydroxycoumarin, according to the changes in the UV-VIS spectra, as shown in Figure S2 in the Supplementary Material. UV absorption maxima were dropping down over time, although the biotransformation rates of 6-hydroxycoumarin, 6,7-dihydroxycoumarin, and coumarin were approximately 5-fold to 10-fold lower, respectively. After the completion of bioconversions, there were no visible spectra observed for the residual aromatic compound in the reaction mixtures with 7-hydroxycoumarin, except for the reactions with 6-hydroxycoumarin, 6,7-dihydroxycoumarin, and coumarin, which had non-disappearing UV absorption maxima at 260–270 nm wavelengths. These spectra are similar to that of 3-phenylpropionic acid (data not shown), suggesting that 7-hydroxycoumarin-induced 7HK4 strain can only catalyze the hydrolysis and reduction of lactone moiety of coumarin, 6-hydroxycoumarin, and 6,7-dihydroxycoumarin producing 3-(2-hydroxyphenyl)-propionic, 3-(2,5-dihydroxyphenyl)-propionic, and 3-(2,4,5-trihydroxyphenyl)-propionic acids, respectively, comparable to similar biotransformations in other microorganisms [9–16]. In addition, uninduced *Pseudomonas* sp. 7HK4 cells grown in the presence of glucose showed delayed and slower conversion of 7-hydroxycoumarin. The bioconversion process only started 0.5 h after the addition of substrate, suggesting that 7-hydroxycoumarin induced its own metabolism. In addition, uninduced cells did not catalyze any conversion or showed delayed and much slower biotransformations for coumarin, 6,7-dihydroxycoumarin, and 6-hydroxycoumarin, respectively. This demonstrates that in *Pseudomonas* sp. 7HK4 bacteria, the metabolism of 7-hydroxycoumarin is an inducible process.

It has been shown previously that 3-(2-hydroxyphenyl)-2-propenoic and 3-(2-hydroxyphenyl)-propionic acids, as shown in Figure 1B, are the intermediates in a known coumarin metabolic pathway in several microorganisms [9–11,14,16]. By analogy, it was suggested that 3-(2,4-dihydroxyphenyl)-propionic acid may be an intermediate metabolite in 7-hydroxycoumarin catabolism, as shown in Figure 1B. The cells of 7HK4 strain pre-cultivated in the presence of 7-hydroxycoumarin catalyzed no conversion of 3-(2-hydroxyphenyl)-2-propenoic or 3-(2-hydroxyphenyl)-propionic acid, however 3-(2,4-dihydroxyphenyl)-propionic acid was straightforwardly consumed. The HPLC-MS analysis of the bioconversion mixtures showed that *Pseudomonas* sp. 7HK4 cells cultivated in the presence of 7-hydroxycoumarin produce 3-(2,4-dihydroxyphenyl)-propionic acid as an intermediate metabolite, as shown in Figure 2. In 7HK4 bacteria grown on glucose, the aforementioned compound was not observed suggesting that 3-(2,4-dihydroxyphenyl)-propionic acid is an intermediate metabolite during 7-hydroxycoumarin degradation.

**Figure 2.** *Pseudomonas* sp. 7HK4 bacteria were grown in the presence of 7-hydroxycoumarin, and produced metabolites were analyzed by HPLC-MS. UV 254 nm trace of metabolites (**A**). UV and MS spectra of peaks with retention times 5.082 min (**B**,**D**) and 5.759 min (**C**,**E**). The negative ions [M−H]<sup>−</sup> generated are at m/z 181.00 (3-(2,4-dihydroxyphenyl)-propionic acid) and 161.05 (7-hydroxycoumarin).

#### *2.3. Identification of 7-Hydroxycoumarin-Inducible Proteins*

To elucidate which enzymes are involved in 7-hydroxycoumarin metabolism, *Pseudomonas* sp. 7HK4 cells were cultivated in a minimal medium supplemented with 7-hydroxycoumarin (0.3 mM) or glucose (0.3 mM) as the sole carbon and energy source. Several 7-hydroxycoumarin-inducible proteins of different molecular mass were observed by using SDS-PAGE analysis of cell-free extracts from *Pseudomonas* sp. 7HK4, as shown in Figure 3A.

Three bands corresponding to the inducible proteins of 23, 32, and 50 kDa, as shown in Figure 3A, were excised from the SDS-PAGE gel and analyzed by MS-MS sequencing. The genome sequence of strain 7HK4 was obtained via Illumina sequencing as described in Materials and Methods. For the identification of corresponding genes of inducible proteins, the sequences of the identified peptides were searched against the partial 7HK4 genome sequence. Thus, the genome fragment was discovered, encoding a 31.2 kDa protein containing 16 aa-long sequence, as shown in the bolded sequence in Figure 3A, identical to that found in 7-hydroxycoumarin-inducible ~32 kDa protein.

**Figure 3.** (**A**) SDS-PAGE analysis of cell-free extracts of *Pseudomonas* sp. 7HK4. Bacteria were cultivated in the presence of glucose (lane 1) or 7-hydroxycoumarin (lane 2). M—molecular mass ladder (kDa). The arrows indicate 7-hydroxycoumarin-inducible 23, 32, and 50 kDa proteins. The peptide sequences determined by MS-MS are given on the right. The sequence in bold was identified after the peptide sequences obtained by MS-MS were compared with the partially sequenced genome of *Pseudomonas* sp. 7HK4. (**B**) Organization of *hcd* genes in *Pseudomonas* sp. 7HK4 bacteria. The arrows indicate open reading frames (ORFs) encoding HcdA, HcdB, and HcdC. The gene for the 7-hydroxycoumarin-inducible protein of 31.2 kDa is marked by an asterisk.
