*2.8. Expression of the HcdC Protein*

HcdC was expressed from the plasmid p2K4PH in *E. coli* BL21. The addition of *E. coli* extracts containing the HcdC protein did not cause decolorization of the *meta*-cleavage products of 3-(2,3-dihydroxyphenyl)-propionic acid, pyrocatechol, 3-methylcatechol, or 4-methylcatechol formed in the presence of the HcdB extradiol dioxygenase. Also, the addition of NAD(P)+ to these reaction mixtures did not induce decolorization of the *meta*-cleavage products [50]. To confirm the function of HcdC, *E. coli* BL21 cells were transformed with p4pmPmo and pCDF-BC plasmids. The expression of *hcdA*, *hcdB,* and *hcdC* genes in *E. coli* cells was confirmed by SDS-PAGE, the enzymes migrated as 62, 31, and 20 kDa bands, respectively, as shown in Figure S15 in the Supplementary Material.

The bioconversion of 3-(2,4-dihydroxyphenyl)-propionic acid was conducted in *E. coli* cells containing all three recombinant proteins. Later, the reaction mixture was incubated with NH4Cl, and the reaction products were analyzed by HPLC-MS. The ions [M−H]<sup>−</sup> and [M+H]+ with masses of 210.00 and 212.00, respectively, were not detected, compared to the bioconversion mixture with *E. coli* cells containing *hcdAB* genes only. This showed a complete conversion of *meta*-cleavage product of the catechol derivative, therefore no picolinic acid derivative could be obtained. No reaction products of (*2E*,*4E*)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid hydrolysis by the HcdC protein were identified. We suggest that the later compound was converted to succinic acid (**17**), which entered the Krebs cycle, and (*E*)-2-hydroxy-4-oxopent-2-enoic acid (**16**), which could be further converted by *E. coli* cells, thus complicating the extraction of these reaction products. Nevertheless, it may be concluded that all three enzymes encoded by the *hcdABC* operon are responsible for the catabolism of 3-(2,4-dihydroxyphenyl)-propionic acid in *Pseudomonas* sp. 7HK4 bacteria.
