**1. Introduction**

The compound 7-hydroxycoumarin (**5**), also known as umbelliferone, is one of the most abundant plant-derived secondary metabolites. It is a parent compound of other, more complex natural furanocoumarins and pyranocoumarins in higher plants [1–3]. In the case of plant damage, plants produce a high diversity of natural coumarins as a defense mechanism against insect herbivores as well as fungal and microbial pathogens [4]. For example, simple hydroxycoumarins have antibacterial activity against *Ralstonia solanacearum*, *Escherichia coli*, *Klebsiella pneumoniae*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* [4–8]. Despite the toxic effects of coumarins, it has been shown that microorganisms evolve to gain the ability to metabolize such compounds.

It has been shown previously that a number of soil microorganisms, such as *Pseudomonas*, *Arthrobacter*, *Aspergillus*, *Penicillium*, and *Fusarium* spp. can grow on coumarin (**1**) as a sole source of carbon [9–16]. The key intermediate during coumarin catabolism in bacteria is 3-(2-hydroxyphenyl)-propionic acid (**4**) [10,11,16]. The bioconversion of coumarin to 3-(2-hydroxyphenyl)-propionic acid can be achieved in two different metabolic pathways, as shown in Figure 1A. Bacteria belonging to the *Arthrobacter* genus enzymatically hydrolyze the lactone moiety to give 3-(2-hydroxyphenyl)-2-propenoic acid (**2**), and then

reduce a double bond by using a NADH-dependent enzyme to yield 3-(2-hydroxyphenyl)-propionic acid [11]. In the case of *Pseudomonas* sp. 30-1 and *Aspergillus niger* ATCC 11394 cells, coumarin is initially reduced to dihydrocoumarin (**3**) by a NADH-dependent oxidoreductase and only then enzymatically hydrolyzed [9,10,14]. *Arthrobacter* and *Pseudomonas* species are capable of oxidizing 3-(2-hydroxyphenyl)-propionic acid to 3-(2,3-dihydroxyphenyl)-propionic acid by using specific flavin-binding aromatic hydroxylases [16]. However, no data are available on further conversions of 3-(2,3-dihydroxyphenyl)-propionic acid in these bacteria. Also, no microorganisms or enzymes implicated in the metabolism of any hydroxycoumarin have been identified to date.

**Figure 1.** (**A**) Coumarin metabolic routes in *Arthrobacter* spp. (a, b), as well as *Pseudomonas* and *Aspergillus* spp. (c, d). (**B**) Proposed metabolic pathway of 7-hydroxycoumarin in *Pseudomonas* sp. 7HK4 bacteria. **1**—coumarin; **2**—3-(2-hydroxyphenyl)-2-propenoic acid; **3**—dihydrocoumarin; **4**—3-(2-hydroxyphenyl)-propionic acid; **5**—7-hydroxycoumarin; **6**—3-(2,4-dihydroxyphenyl)-propionic acid; **a**—putative coumarin hydrolase; **b**—NADH:*o*-coumarate oxidoreductase; **c**—dihydrocoumarin:NAD[NADP] oxidoreductase; **d**—putative dihydrocoumarin hydrolase.

Here we describe a *Pseudomonas* sp. 7HK4 strain capable of utilizing 7-hydroxycoumarin as the sole carbon and energy source, and a catabolic pathway of 7-hydroxycoumarin in these bacteria. Analysis of 7-hydroxycoumarin-inducible proteins led to the identification of the genome locus encoding 7-hydroxycoumarin catabolic proteins. The corresponding genes were cloned and heterologously expressed in *E. coli* system. The functions of the recombinant proteins were determined and enzyme activities towards various substrates were evaluated. We show that *Pseudomonas* sp. 7HK4 encodes a distinct 7-hydroxycoumarin metabolic route, which utilizes a flavin monooxygenase responsible for *ipso*-hydroxylation of 3-(2,4-dihydroxyphenyl)-propionic acid (**6**).

#### **2. Results**

#### *2.1. Screening and Identification of 7-Hydroxycoumarin-Degrading Microorganisms*

By the means of enrichment culture using various coumarin derivatives, an aerobic strain 7HK4 degrading 7-hydroxycoumarin was isolated from the garden soil in Lithuania (DSMZ accession number DSM 107615). This bacterium was tested for its ability to grow on several coumarin derivatives, such as coumarin, 3-hydroxycoumarin, 4-hydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin, 6,7-dihydroxycoumarin, and 7-methylcoumarin, as the sole carbon and energy source in a minimal salt medium. However, of all the aforementioned compounds, the strain 7HK4 was able to utilize 7-hydroxycoumarin only. The strain utilized glucose, which was used as a control substrate in whole-cell reactions. Data on biochemical analysis of this strain are given in the Supplementary Material. The nucleotide sequence of 16S rRNA gene was determined by sequencing of the cloned DNA fragment, which was obtained by PCR amplification. The strain 7HK4 showed the highest

16S rDNA sequence similarity to that from *Pseudomonas* genus and was similar to 16S rDNA from *Pseudomonas mandelii* species according to the phylogenetic analysis, as shown in Figure S1 in the Supplementary Material.
