[Screening and engineering of a protocatechuic acid decarboxylase for efficient biosynthesis of catechol]

Catechol (CA) is an important chemical and pharmaceutical intermediate with wide applications. At present, CA is produced by phenol hydroxylation with non-renewable petrochemical resources, which causes serious environmental pollution. Hence, the biosynthesis of CA attracts much attention recently. However, due to the low activities of protocatechuic acid (PCA) decarboxylases, the production efficiency of biosynthetic catechol is too low to meet the requirements of large-scale industrial production. To improve the yield of CA, we screened 21 PCA decarboxylases from different species. RbAroY originated from Rikenellaceae had the best catalytic performance. The whole-cell biocatalyst ER11 with RbAroY was able to produce CA at a titer of 13.54 g/L. Then, the online tool HotSpot Wizard was employed to measure the enzyme stability, which revealed 10 potential mutation sites causing significant decreases in Gibbs free energy. The whole-cell biocatalyst ERT01 with the mutated RbAroY^G99A could produce CA at a titer of 15.16 g/L, which increased by 12% compared with that of the wild-type whole-cell biocatalyst. After optimization of the biocatalytic conditions, the whole-cell biocatalyst ERT01 was able to produce CA at a titer of 25.70 g/L with PCA as the substrate. Finally, with the fermentation broth of 3-dehydroshikimate as the substrate, the whole-cell biocatalyst DER03 expressing both 3-dehydroshikimate dehydratase and PCA decarboxylase realized the production CA at a titer of 29.55 g/L, which is currently the highest biosynthetic titer reported. This study provides a reference for the industrial production of CA by biosynthesis.