Project description:Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and is also generated in the degradation of plastics such as PET. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode mycofactocin-dependent alcohol degradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither ΔmadA nor ΔmftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the ΔmadA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad 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, aldA2, predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 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 Rhodococcus for the upgrading of plastic waste streams.
Project description:Transcriptome studies confirm the nitrogen limited physiological state of both the wild-type and mutant cells. In addition, multiple differentially expressed genes involved in the synthesis and consumption of pools of acetyl-CoA, acetoacetyl-CoA and 3-hydroxybutyryl-CoA, key metabolites for PHA and TAG synthesis, were identified. An enrichment analysis of differentially expressed genes in the nitrogen starved wild-type versus the isogenic RHA1_ro02104 mutant strain identified genes in the mutant involved in fatty acid and lipid as well as genes involved in acyl-CoA hydrolysis and triacylglycerol degradation. An 8 x 15K array study using total RNA recovered from triplicate cultures of Rhodococcus jostii RHA1 under nitrogen rich and nitrogen starved conditions and triplicate cultures of Rhodococcus jostii RHA1 TadA-homolog deletion mutants (2104) under nitrogen rich and nitrogen starved conditions.
