Stereoinversion via alcohol dehydrogenases enables complete catabolism of β-1-type lignin-derived aromatic isomers

Abstract

ABSTRACTSphingobiumsp. SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened β-1-type dimer. Recently, LdpA-mediated catabolism oferythro-DGPD was reported in SYK-6, but the catabolic pathway forthreo-DGPD was heretofore unknown. Here we elucidated the catabolism ofthreo-DGPD, which proceeds through conversion toerythro-DGPD. Whenthreo-DGPD was incubated with SYK-6, the Cα alcohol groups ofthreo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of β-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced toerythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase andldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated thatldpCandldpBare predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four β-1-type stereoisomers through successive oxidation and reduction reactions of the Cα alcohol group ofthreo-DGPD with a net stereoinversion using multiple dehydrogenases.IMPORTANCEIn many catalytic depolymerization processes of lignin polymers, aryl–ether bonds are selectively cleaved, leaving carbon–carbon bonds between aromatic units intact, including dimers and oligomers with β-1 linkages. Therefore, elucidating the catabolic system of β-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. In this work, we found thatthreo-DGPD was converted by successive stereoselective oxidation and reduction at the Cα-position by multiple alcohol dehydrogenases toerythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native β-1-type dimer catabolic system.