In
Pseudomonas aeruginosa and
Pseudomonas putida, EG is converted into glyoxylic acid under the action of dehydrogenase and finally enters the TCA cycle through different routes
[4][5][6][7]. At present, the metabolic pathway of EG in
P. putida KT2440 is the most widely studied. The metabolism pathways in utilizing EG have been well demonstrated in
P. putida KT2440, in comparison to other bacteria, and related enzymes have been identified. In
P. putida KT2440, two functionally redundant periplasmic quinoproteins, PedE and PedH, catalyze EG into glycolaldehyde
[8]. PedE and PedH are both pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs), and their expression depend on Ca
2+ and lanthanide metal ions, respectively
[8]. Once glycolaldehyde is produced, the two cytoplasmic aldehyde dehydrogenases, PP_0545 and PedI, catalyze it into glycolic acid, and glyoxylic acid is further generated via the membrane anchored oxidase GlcDEF. The glyoxylic acid is converted into acetyl-CoA and enters the TCA cycle to be catalyzed by a series of enzymes
[9]. Additionally, there are another two alternative pathways to convert glyoxylic acid, one of which is catalyzed by isocitrate lyase (AceA) and glyoxylic acid can condense with succinic acid to form isocitrate. The other is catalyzed by malate synthase (GlcB) and glyoxylic acid condenses with acetyl-CoA to form malic acid. However, due to the removal of CO
2 and the restriction of the amount of acetyl-CoA,
P. putida KT2440 cannot use EG as the sole carbon source for growth
[9]. Researchers engineered
P. putida KT2440 by overexpressing glycolate oxidase to remove the glycolate metabolic bottleneck and produced an engineered strain that can efficiently metabolize EG
[9]. After that, mutants of
P. putida KT2440 that utilize EG as the sole carbon source were isolated through adaptive laboratory evolution, and the metabolism and regulation mechanism of EG in
P. putida KT2440 was further clarified
[10].
P. putida JM37 was reported to be able to use EG as the sole carbon source for growth because there is another pathway to use glyoxylic acid compared to
P. putida KT2440. Glyoxylic acid is converted into tartrate semialdehyde under the catalysis of glyoxylate carboxylase (Gcl) and then tartrate semialdehyde is converted into glycerate acid, catalyzed by hydroxypyruvate isomerase (Hyi) and tartrate semialdehyde reductase (GlxR). Glycerate acid can be further converted into 2-phosphoglycerate and enter the TCA cycle
[11].
Wild-type
E. coli cannot use EG as the sole carbon source for growth
[12]. In 1983, researchers first reported an
E. coli strain capable of using EG as the sole carbon source from the propylene glycol using mutants. They identified the increased activities of propanediol oxidoreductase, glycolaldehyde dehydrogenase, and glycolate oxidase in the mutants
[12]. Based on this discovery, researchers began to design and construct engineered
E. coli that could use EG to convert PET monomers into high value chemicals.
EG is assimilated and oxidized into glycolaldehyde and, subsequently, into glycolic acid under the catalysis of 1,2-propanediol oxidoreductase mutant (fucO) and glycolaldehyde dehydrogenase (aldA), respectively. Glycolic acid can be metabolized into glyoxylic acid by glycolate dehydrogenase (GlcDEF)
[13]. Similar to
P. putida, glyoxylic acid is further condensed into acetyl-CoA by the linear glycerate pathway or converted into isocitrate and malate catalyzed by AceA and GlcB, respectively. An engineered
E. coli can take EG as the sole carbon source to produce glycolate by expressing fucO mutant (I7L/L8V) and aldA. Experiments identified that oxygen concentration was as an important metabolic valve, and flux to 2-phosphoglycerate was the primary route in the assimilation of EG as a substrate combining modeling
[7][14]. Additionally, EG can be efficiently utilized in
E. coli by optimizing the gene expression (fucO and aldA) and adding a growth medium with a low concentration of glycerol or a mixture of amino acids
[13]. Although
E. coli MG1655 contains the endogenous glyoxylic acid metabolism pathway, the EG-utilizing ability of the engineered
E. coli still needs to be improved
[14]. Introducing a heterologous pathway or unblocking the rate-limiting steps of the EG metabolic pathway in
E. coli may further enhance the assimilation of EG.
E. coli has a clear genetic background and simple genetic operations compared to other bacteria, so it is easier to engineer it to transform EG into high value chemicals.