An enhanced toluene dioxygenase platform for the production of cis-1,2-dihydrocatechol in Escherichia coli BW25113 lacking glycerol dehydrogenase activity.

• A highly efficient biotechnological platform for cis -1,2-dihydrocatechol production was established. • A strongly regulated toluene dioxygenase plasmid for the dearomatizing cis -1,2-dihydroxylation of benzene was developed. • An unforeseen downstream reaction consuming cis -1,2-dihydrocatechol, yielding catechol, was detected. • The native glycerol dehydrogenase in E. coli was identified as the main responsible enzyme for catechol generation. • A yield of 1.4 g L−1 cis -1,2-dihydrocatechol was achieved at semi-preparative scale. The compound cis -1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 Δ gldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41 g L−1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis. [ABSTRACT FROM AUTHOR]