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

Abstract

Summary Pollution from ethylene glycol, and plastics containing this monomer, represent a significant environmental problem. The investigation of its microbial metabolism therefore provides insights into the environmental fate of this pollutant and also enables its utilization as a carbon source for microbial biotechnology. Here, we reveal the genomic and metabolic basis of ethylene glycol metabolism in Pseudomonas putida KT2440. Although this strain cannot grow on ethylene glycol as sole carbon source, it can be used to generate growth‐enhancing reducing equivalents upon co‐feeding with acetate. Mutants that utilize ethylene glycol as sole carbon source were isolated through adaptive laboratory evolution. Genomic analysis of these mutants revealed a central role of the transcriptional regulator GclR, which represses the glyoxylate carboligase pathway as part of a larger metabolic context of purine and allantoin metabolism. Secondary mutations in a transcriptional regulator encoded by PP_2046 and a porin encoded by PP_2662 further improved growth on ethylene glycol in evolved strains, likely by balancing fluxes through the initial oxidations of ethylene glycol to glyoxylate. With this knowledge, we reverse engineered an ethylene glycol utilizing strain and thus revealed the metabolic and regulatory basis that are essential for efficient ethylene glycol metabolism in P. putida KT2440.

Bio-upcycling of polyethylene terephthalate

“…Pseudomonads are Gram-negative bacteria with a high potential for degrading synthetic plastics 5,36 , due to their versatile arsenal of catabolic enzymes 37 . Different Pseudomonas putida strains are known for their metabolism of a wide variety of substrates, including aromatics such as TA 38 , and aliphatics such as EG [39][40][41] . Indeed…”

Unraveling 1,4-Butanediol Metabolism in Pseudomonas putida KT2440

“…For measuring extracellular metabolites, samples taken from liquid cultivation were centrifuged for 3 min at 17,000 × g to obtain supernatant for High-Performance Liquid Chromatography (HLPC) analysis using a Beckman System Gold 126 Solvent Module equipped with a Smartline 2300 refractive index detector (Knauer, Berlin, Germany). 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 (Li et al, 2019).…”

“…To enhance its ability to grow on 1,4-butanediol, wildtype P. putida KT2440 was subjected to ALE. This method is known to enable the selection of mutated strains with enhanced properties toward specific environments, likely affecting transcriptional regulatory systems (Dragosits and Mattanovich, 2013;Lennen et al, 2019;Li et al, 2019). Cultures of P. putida KT2440 were serially re-inoculated to fresh media containing 20 mM 1,4butanediol ten times, as soon as growth was observed in form of optical densities above 0.8 (Figure 1).…”

“…In contrast to the wildtype they completely consume all carbon source, reaching maximum biomass concentrations of 1.05 ± 0.0 g cdw L −1 and 0.96 ± 0.02 g cdw L −1 after 33 h. This translates to an average biomass yield of 0.56 ± 0.025 g/g for the evolved strains. This relatively high yield is likely caused by the high degree of reduction of butanediol, providing considerable reducing equivalents especially in the initial oxidation reactions (Li et al, 2019). In contrast to the wildtype, the evolved strains only transiently accumulate low concentrations of 4-hydroxybutyrate, which are rapidly metabolized within 4 h (Figure 1).…”

“…In addition to the diamines, which are relatively valuable and can be extracted (Bednarz et al, 2017), typical PU monomers like adipic acid, 1,4-butanediol, and ethylene glycol are released during the process of depolymerization. Degradation pathways for ethylene glycol (Franden et al, 2018;Li et al, 2019) and adipic acid (Parke et al, 2001) are known. Yet, surprisingly little is known about the microbial catabolism of 1,4-butanediol.…”