Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113

Abstract

cis-Dihydrodiendiols are valuable compounds, finding multiple application as chiral synthons in organic chemistry. The biotechnological route for the generation of cis-dihydrodiendiols involves the dihydroxylation of aromatic compounds, catalyzed by Rieske non-heme iron dioxygenases. To date, numerous examples of recombinant E. coli, harboring such dioxygenases, can be found in the literature. Nevertheless, there is only a minor number of publications, addressing the E. coli catalyzed degradation of cis-dihydrodiendiols into catechols via dehydrogenases. Identification and elimination of such dehydrogenase catalyzed degradation is key for the establishment of enhanced recombinant E. coli platforms pursuing the production of cis-dihydrodiendiols. Here, we provide a fast and easy strategy for the identification of promiscuous alcohol dehydrogenases in E. coli BW25113, catalyzing the degradation of cis-dihydrodiendiols into catechols. This approach is based on the screening of dehydrogenase deficient KEIO strains, regarding their incapability of degrading a cis-dihydrodiendiol of choice. • Novel screening strategy for E. coli BW25113 dehydrogenase knock-outs, incapable of degrading cis-dihydrodiendiols was validated for cis-1,2-dihydrocatechol as substrate • Corresponding knock-outs can be used for recombinant production of cis-dihydrodiendiols • Simple analysis based on liquid chromatography with diode array detector (HPLC-DAD)

Keywords

• Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113