Abstract
Gallic acid, gallic aldehyde, and gallic alcohol are polyphenolic compounds with promising antioxidant and therapeutic properties. Despite their biological significance, a complete microbial biosynthetic route for their production from simple carbon sources has not been established. We engineered Corynebacterium glutamicum to produce gallic acid and its two reduced derivatives via a synthetic pathway extended from the shikimate pathway. Introduction of a mutant 4-hydroxybenzoate hydroxylase conferred protocatechuate hydroxylation activity in C. glutamicum. Among tested mutants, the Y385F/L200V mutant exhibited the highest gallic acid production, reaching 4.03 g/l with a yield of 5.95% in flask cultures. To enable gallic aldehyde biosynthesis, carboxylic acid reductases (CARs) from various microbial sources were screened. Of these, MpCAR exhibited the highest catalytic activity toward gallic acid, producing 0.66 g/l of gallic aldehyde in an NCgl0324-deleted strain. Further reduction of gallic aldehyde to gallic alcohol was achieved using the endogenous aromatic aldehyde reductase encoded by NCgl0324 in C. glutamicum, as confirmed by Q-TOF mass analysis. Overexpression of qsuB encoding 3-dehydroshikimate dehydratase improved carbon flux from 3-dehydroshikimate toward PCA and significantly enhanced the gallic compound production. In 5-l fed-batch fermentation, engineered strains produced up to 12.0 g/l gallic acid, 1.14 g/l gallic aldehyde, and 172.4 AU*s gallic alcohol, respectively, representing 82-86% increases compared to flask cultures. This study reports the first complete microbial biosynthetic route for gallic acid, gallic aldehyde, and gallic alcohol from D-glucose. Our work highlights C. glutamicum as a robust microbial platform for sustainable production of value-added gallic polyphenols through pathway design and metabolic engineering.