Characterization of Catechol-1,2-Dioxygenase (Acdo1p) From and Investigation of Its Role in the Catabolism of Aromatic Compounds.

Gallic acid, protocatechuic acid, catechol, and pyrogallol are only a few examples of industrially relevant aromatics. Today much attention is paid to the development of new microbial factories for the environmentally friendly biosynthesis of industrially relevant chemicals with renewable resources or organic pollutants as the starting material. The non-conventional yeast, , possesses attractive properties for industrial bio-production processes such as thermo- and osmotolerance. An additional advantage is its broad substrate spectrum, with tannins at the forefront. The present study is dedicated to the characterization of catechol-1,2-dioxygenase (Acdo1p) and the analysis of its function in tannic acid catabolism. Acdo1p is a dimeric protein with higher affinity for catechol ( = 0.004 ± 0.001 mM, = 15.6 ± 0.4 s) than to pyrogallol ( = 0.1 ± 0.02 mM, = 10.6 ± 0.4 s). It is an intradiol dioxygenase and its reaction product with catechol as the substrate is -muconic acid. G1212/YIC102-AYNI1-ACDO1-6H, which expresses the gene under the control of the strong nitrate-inducible promoter, achieved a maximum catechol-1,2-dioxygenase activity of 280.6 U/L and 26.9 U/g of dry cell weight in yeast grown in minimal medium with nitrate as the nitrogen source and 1.5% glucose as the carbon source. In the same medium with glucose as the carbon source, catechol-1,2-dioxygenase activity was not detected for the control strain G1212/YIC102 with expression under the regulation of its respective endogenous promoter. Gene expression analysis showed that is induced by gallic acid and protocatechuic acid. In contrast to the wild-type strain, the strain with a deletion of the gene was unable to grow on medium supplemented with gallic acid or protocatechuic acid as the sole carbon source. In summary, we propose that due to its substrate specificity, its thermal stability, and its ability to undergo long-term storage without significant loss of activity, catechol-1,2-dioxygenase (Acdo1p) is a promising enzyme candidate for industrial applications.