The bacteria Escherichia coli (E. coli), can utilize glycerol as sole carbon source under aerobic and anaerobic conditions (Clomburg et al., 2022, Gonzalez et al., 2008). In the later context, the first step of glycerol metabolization involves the NAD+ dependent glycerol dehydrogenase (GldA; EC 1.1.1.6), which catalyzes the reversible oxidation of glycerol to dihydroxyacetone (Piattoni et al., 2013, Subedi et al., 2008). This enzyme belongs to the family III polyol dehydrogenases and harbors a catalytically active Zn2+ ion in the active site (Ruzheinikov et al., 2001, Zhang et al., 2019). In addition to the substrate glycerol, GldA from E. coli K-12 exhibits promiscuous affinity towards the C2-C4 alcohols 1,2-ethanediol, 1-amino-2-propanol, 1,2-propanediol, 3-amino-1,2-propanediol, 3-mercapto-1,2-propanediol, 3-chloro-1,2-propanediol, 1,2-butanediol, 1,3-butanediol and 2,3-butanediol (Kelley and Dekker, 1984, Zhang et al., 2010). It is worth noting that prior to 1985, some publications referred to GldA from E. coli K-12 as D-1-amino-2-propanol:NAD+ oxidoreductase (Kelley and Dekker, 1985). Thus, it is striking that an enzyme involved in the central metabolism as GldA, actually exhibits affinity towards a wide substrate panel for several short-chain diols. To the best of our knowledge, there are no reports regarding the GldA mediated conversion of larger compounds with more than four C-atoms.

E. coli strains are frequently utilized for the recombinant expression of Rieske non-heme iron dioxygenase (ROs) genes, such as the toluene dioxygenase (TDO) from Pseudomonas putida F1, employing different genetic backgrounds like JM109 (Vila et al., 2013), BL21(DE3) (Osifalujo et al., 2022), or BW25113 (Wissner et al., 2021). Such TDO platforms were designed to catalyze the cis-dihydroxylation of aromatic compounds to the corresponding valuable cis-dihydrocatechols, which are employed as chiral synthons in the synthesis of various natural products (Borra et al., 2019, Hudlicky, 2018). However, we recently described the unforeseen degradation of the valuable chiral cyclic C6 compound cis-dihydrocatechol 1 to catechol 1a by E. coli BW25113 as well as by E. coli JM109(DE3) (Wissner et al., 2020a). Thus, the utilization of the whole cell-based platform E. coli BW25113 pAD18-TDO, customized for the production of the cis-diols 1, surprisingly resulted in 71% 1a as main product. By using the knock-out strain E. coli BW25113 ΔgldA we showed that the undesired generation of 1a could be abolished nearly completely, strongly pointing out that GldA was the main responsible enzyme for the dehydrogenation of 1 (Wissner et al., 2020b). However, a detailed study of GldA specifically expressing it for the conversion of cis-dihydrocatechol derivatives has not been reported to date.

Therefore, in this work we focused on confirming such observations by cloning the gldA encoding gene in a highly tunable plasmid and employing a clean genetic background lacking GldA for expression. Thus, we overexpressed GldA using the pBAD18 plasmid into E. coli BW25113 ΔgldA and explored its substrate scope towards C6-C10cis-diols, including cis-dihydrocatechol 1 and its enantiomerically pure derivatives 2-5 (Fig. 1). This substrate panel allowed us to correlate substrate acceptance regarding steric demand.