**4. Materials and Methods**

## *4.1. Bacterial Strains, Plasmids, and Reagents*

*Pseudomonas* sp. 7HK4 bacterial strain capable of using 7-hydroxycoumarin as the sole source of carbon and energy was isolated from soil by enrichment in mineral medium containing 0.05% of 7-hydroxycoumarin. For cloning purposes, *E. coli* DH5α bacteria (ϕ80 *lacZ*ΔM15 Δ(*lacZY-argF*)*U169 deoR recA1 endA1 hsdR17*(rK-mK+) *supE44 thi-1 gyrA96 relA1*) (Thermo Fischer Scientific, Lithuania) were used. *E. coli* BL21 (DE3) bacteria (F- *ompT gal dcm lon hsdS*B(rB-mB-) λ(DE3) [*lacI lacUV5-T7* gene 1, *ind1*, *sam7*, *nin5*]) (Novagen, Darmstadt, Germany) were used for gene expression studies.

All reagents used during this study are listed in Table S1 and plasmids are described in Table S2 in the Supplementary Material.

## *4.2. Bioconversions with Whole Cells*

*Pseudomonas* sp. 7HK4 bacteria were grown in mineral medium supplemented with 0.05% (*w*/*v*) of 7-hydroxycoumarin or glucose, as the sole carbon and energy source, at 30 ◦C with rotary aeration (180 rpm) for 48 h. *E. coli* BL21 (DE3) bacteria containing recombinant genes were grown in 30 mL of Brain Heart Infusion (BHI) medium at 30 ◦C and 180 rpm overnight. High density bacterial culture was centrifuged and resuspended in 30 mL of minimal C-750501 medium, in which the synthesis of proteins was induced with 1 mM of isopropyl-*β*-D-1-thiogalactopyranoside (IPTG) after 1.5 h of incubation at 20 ◦C and 180 rpm [65]. Incubation at 20 ◦C was continued for another 24 h. Both *Pseudomonas* sp. 7HK4 and *E. coli* cells were sedimented by centrifugation (3220× *g*, 15 min). The collected cells were washed twice with 15 mL of 0.9% NaCl solution. For whole-cell conversion experiments, cells from 20 mL of culture were resuspended in 1 mL of 50 mM potassium phosphate buffer (pH 7.2). All small-scale bioconversions with whole cells were made in 50 mM potassium phosphate buffer, pH 7.2, which contained 1–2 mM of the substrate. The reaction mixtures were kept in a thermoblock at

30 ◦C and 500 rpm. Bioconversion mixtures were centrifuged for 2 min at 10,000× *g*, and 100 μL of the supernatant were analyzed by UV-VIS spectroscopy (range 200–600 nm). Measurements were repeated to record the changes in the absorption intensity over time. All measurements were performed with PowerWave XS microplate reader.
