Biochemical and molecular investigations of benzoic acid metabolism in Hypericum calycinum cell cultures

Xanthones are polyketide scaffolds with astounding pharmacological potency. Elicitor-treated cell cultures of Hypericum calycinum (Hc) accumulate prenylated xanthones. Benzoyl-CoA is required for the catalysis of the first committed step of their biosynthesis. In Hypericum, the formation of benzoyl-CoA proceeds by the CoA-dependent non-s-oxidative route. This route was biochemically established but only a peroxisomal cinnamate-CoA ligase (CNL) was defined at the molecular level. Molecular information of the other genes in the pathway remained elusive. The aim of the current work was cDNA cloning, functional characterization and subcellular localization of benzaldehyde dehydrogenase (BD) and benzoate-CoA ligase (BZL) from xanthone-accumulating yeast extract-treated Hc cells. BD catalyzes the conversion of benzaldehyde to benzoic acid, which is metabolized to benzoyl-CoA by BZL. Based on publicly available transcriptomic resources and homology-based cloning strategies, the first plant full-length HcBZL cDNA was isolated. The 1647 bp coding sequence encoded a ~60 kDa soluble protein with pI 6.6. The HcBD cDNA had a 1506 bp coding sequence and it encoded a soluble protein with 54.2 kDa subunit mass and pI 6.01. Substrate screening with 10 potential substrates revealed that HcBD primarily catalyzed the conversion of trans-cinnamaldehyde and benzaldehyde to their corresponding acids. Out of 24 potential substrates offered to HcBZL in a luciferase-based substrate specificity assay, benzoic acid was the only aromatic acid to be activated. Additionally, short-chain fatty acids (C3-C7) were converted to their CoA thioesters. Expression analyses indicated the upregulation of both HcBD and HcBZL after yeast extract treatment of cultured cells, which preceded xanthone accumulation. The maximum xanthone content was 4.7 mg/g dry weight. The results suggested the in vivo participation of the two genes in the CoA-dependent non-s-oxidative route, which directs carbon flux from the benzenoid biosynthetic pathway towards the xanthone biosynthetic route. Subcellular localization of HcBD and HcBZL primarily to the cytoplasm of the plant cell helped postulate the trafficking of reaction intermediates of the CoA-dependent non-s-oxidative route from the peroxisomes to the cytoplasm of the plant cell.