In vivo implementation of a synthetic metabolic pathway for the carbon-conserving conversion of glycolaldehyde to acetyl-CoA

Abstract

Ethylene glycol (EG) derived from plastic waste or CO2 can serve as a substrate for microbial production of value-added chemicals. Assimilation of EG proceeds though the characteristic intermediate glycolaldehyde (GA). However, natural metabolic pathways for GA assimilation have low carbon efficiency when producing the metabolic precursor acetyl-CoA. In alternative, the reaction sequence catalyzed by EG dehydrogenase, d-arabinose 5-phosphate aldolase, d-arabinose 5-phosphate isomerase, d-ribulose 5-phosphate 3-epimerase (Rpe), d-xylulose 5-phosphate phosphoketolase, and phosphate acetyltransferase may enable the conversion of EG into acetyl-CoA without carbon loss. We investigated the metabolic requirements for in vivo function of this pathway in Escherichia coli by (over)expressing constituting enzymes in different combinations. Using 13C-tracer experiments, we first examined the conversion of EG to acetate via the synthetic reaction sequence and showed that, in addition to heterologous phosphoketolase, overexpression of all native enzymes except Rpe was required for the pathway to function. Since acetyl-CoA could not be reliably quantified by our LC/MS-method, the distribution of isotopologues in mevalonate, a stable metabolite that is exclusively derived from this intermediate, was used to probe the contribution of the synthetic pathway to biosynthesis of acetyl-CoA. We detected strong incorporation of 13C carbon derived from labeled GA in all intermediates of the synthetic pathway. In presence of unlabeled co-substrate glycerol, 12.4% of the mevalonate (and therefore acetyl-CoA) was derived from GA. The contribution of the synthetic pathway to acetyl-CoA production was further increased to 16.1% by the additional expression of the native phosphate acyltransferase enzyme. Finally, we demonstrated that conversion of EG to mevalonate was feasible albeit at currently extremely small yields.

From trash to cash: current strategies for bio-upcycling of recaptured monomeric building blocks from poly(ethylene terephthalate) (PET) waste

“…This last method has attracted attention due to its safety, using natural agents and non-toxic chemicals, and is considered an emerging method for MVA production with a promising future. 134 Wagner et al 135 investigated the in vivo functioning of the alternative synthetic pathway, described as the D-arabinose 5-phosphate (Ara5P)-dependent glycolaldehyde assimilation (GAA) pathway, using a strain of E. coli as the host organism to examine the enzymatic requirements for converting EG into the AcCoA-derived product MVA. 135 The reaction sequence catalyzed by six enzymes: EG dehydrogenase, D-arabinose 5-phosphate aldolase, D-arabinose 5-phosphate isomerase, D-ribulose 5-phosphate 3-epimerase, D-xylulose 5-phosphate phosphoketolase and phosphate acetyltransferase can allow the conversion of EG into AcCoA without carbon loss.…”

“…134 Wagner et al 135 investigated the in vivo functioning of the alternative synthetic pathway, described as the D-arabinose 5-phosphate (Ara5P)-dependent glycolaldehyde assimilation (GAA) pathway, using a strain of E. coli as the host organism to examine the enzymatic requirements for converting EG into the AcCoA-derived product MVA. 135 The reaction sequence catalyzed by six enzymes: EG dehydrogenase, D-arabinose 5-phosphate aldolase, D-arabinose 5-phosphate isomerase, D-ribulose 5-phosphate 3-epimerase, D-xylulose 5-phosphate phosphoketolase and phosphate acetyltransferase can allow the conversion of EG into AcCoA without carbon loss. 135 The engineered E. coli was capable to produce only a total concentration of 10.80 mM MVA was produced for engineered E. coli after 46 h of cultivation in the presence of 500 mM EG and 250 mM glycerol.…”

“…135 The engineered E. coli was capable to produce only a total concentration of 10.80 mM MVA was produced for engineered E. coli after 46 h of cultivation in the presence of 500 mM EG and 250 mM glycerol. 135 This result indicate that the Ara5P-dependent GAA pathway is functional albeit with very low yields. Furthermore, the acetate secretion was three times higher in the presence of EG (500 mM EG supplemented with 55 mM glycerol) than in the absence of the compound.…”

Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol

“…Antibiotics were added when required at standard concentrations (chloramphenicol, 35 µg mL -1 ; kanamycin, 50µg mL -1 ; ampicillin, 100 µg mL -1 ; spectinomycin, 100 µg mL -1 ). One litre of M9 mineral medium contained: 20 g glucose, 18…”

“…The assimilation of EG typically proceeds via the intermediate glycolaldehyde. This versatile compound can be assimilated through various pathways, such as the natural tartronic semialdehyde pathway 13,15 , or non-natural acetyl-CoA-yielding routes [16][17][18] .…”