The catabolism of ethylene glycol by RHA1 and its dependence on mycofactocin.

Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the locus, predicted to encode ycofactocin-dependent lcohol egradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the and genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither Δ nor Δ RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the Δ mutant depleted glycolaldehyde but not EG from culture media. These results indicate that encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, , predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in , a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks.