Ethylene Glycol Metabolism by Pseudomonas putida

Abstract

dIn this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism byPseudomonas putidaKT2440

“…D) suggests that the rate of ethylene glycol uptake or glycolate export from the periplasm is not affected, thus pointing to a possible transport effect on the aldehyde intermediates. That said, pedE and pedI are very highly expressed in both strains, and it is known that they are involved in the oxidation of a variety of alcohols and aldehydes including ethylene glycol (Arias et al ., ; Mückschel et al ., ). The essentiality of the Ped cluster was confirmed by the deletion of the pedE‐pedI cluster (PP_2673‐PP_2780) in the E6 strains, which eliminated their ability to grow on ethylene glycol (Supporting Information Fig.…”

“…Analytes were eluted using a 300 × 8 mm organic acid resin column together with a 40 × 8 mm organic acid resin precolumn (both from CS Chromatographie, Langerwehe, Germany) with 5 mM H 2 SO 4 as mobile phase at a flow rate of 0.7 ml min −1 at 70°C. (Mückschel et al ., ) A list with retention times and detection limits is shown in the Supporting Information Table S4.…”

“…Initially, the diol is converted into glyoxylate in a series of oxidation reactions catalysed by a set of redundant dehydrogenases, with PP_0545, PedI, PedE and PedH as predominant enzymes (Fig. 1A) (Mückschel et al, 2012;Wehrmann et al, 2017). Further oxidation to oxalate also occurs in wholecell biotransformations, but generally only relatively small traces of this dead end-product are observed (Mückschel et al, 2012).…”

Ethylene Glycol Metabolism in the Acetogen Acetobacterium woodii

“…However, Asiatic clams as well as the fungus Tricholoma matsutake were reported to produce ethylene glycol (30,31), and it is also known to be a product of ethylene metabolism in a number of higher plants (32). Whereas many aerobic bacteria utilize ethylene glycol by oxidation to glycolaldehyde and glycolate followed by an oxidase reaction to glyoxylate (33)(34)(35)(36), anaerobic bacteria use the alternative route via the acetaldehyde resulting from ethylene glycol dehydration catalyzed by the very oxygen-sensitive diol dehydratase (13,28,29). As recently shown, growth on 1,2-PD leads to the formation of bacterial microcompartments in the cytoplasm of Acetonema longum and A. woodii cells (15,37).…”

Coexistence of two d‐lactate‐utilizing systems in Pseudomonas putida KT2440

“…It has been reported that this enzyme is associated with the glycolate utilization trait in E. coli (Pellicer et al ., ). However, P. putida KT2440 could not use glycolate as the sole carbon source for growth (Mückschel et al ., ), leaving the function of this enzyme uncertain. Growth on and utilization of d ‐lactate in minimal medium was fully abolished only when both Fe‐S d ‐iLDH and glycolate oxidase were absent.…”