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. 2023 Feb 9:11:1125544.
doi: 10.3389/fbioe.2023.1125544. eCollection 2023.

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

Affiliations

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

Nils Wagner et al. Front Bioeng Biotechnol. .

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.

Keywords: Ara5P-dependent GAA pathway; Escherichia coli; acetyl-CoA; arabinose 5-phosphate; ethylene glycol; glycolaldehyde; synthetic metabolic pathway.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Synthetic Ara5P-dependent GAA pathway (black arrows) for the carbon-conserving conversion of the C2 compound ethylene glycol (EG) into acetyl-CoA (AcCoA). Enzymes required for operation of the new route are colored in red. The natural route for glycolaldehyde (GA) assimilation in E. coli (grey arrows) was disrupted by deletion of GA dehydrogenase, AldA (red cross). Dashed grey arrows indicate multiple reaction steps between shown intermediates. Arrows indicate the biosynthetic sense of the reactions. Metabolites: AcP, acetyl phosphate; Ara5P, d-arabinose 5-phosphate; GA3P, glyceraldehyde 3-phosphate; Glyc, glycolate; Glyox, glyoxylate; Ribu5P, d-ribulose 5-phosphate; TarSA, tatronate semialdehyde; Xylu5P, d-xylulose 5-phosphate. Enzymes: FucOMut, I6L L7V mutant of native L-1,2-propanediol oxidoreductase; FsaA, arabinose 5-phosphate aldolase; KdsD, d-arabinose 5-phosphate isomerase; Rpe, D-ribulose 5-phosphate 3-epimerase; Pkt, heterologous phosphoketolase; Pta, phosphate acetyltransferase.
FIGURE 2
FIGURE 2
In vitro demonstration of the synthetic Ara5P-dependent GAA pathway. The reaction mix contained the his-tag purified enzymes FsaA (60 μg L−1), KdsD (60 μg L−1), Rpe (50 μg L−1) and Ca-Pkt (80 μg L−1). Reactions were performed at 37°C in a 1.5 mL reaction tube rotary shaken at 220 rpm. In control reactions either GA3P, Ca-Pkt or FsaA were omitted. Error bars indicate standard error of the mean (n = 2).
FIGURE 3
FIGURE 3
Production of acetate from 13C2-EG via the synthetic Ara5P-dependent GAA pathway. (A) Carbon transition from EG into acetate using the synthetic pathway. Green circles represent EG derived carbon, whereas carbon from glycerol co-substrate is shown in blue circles. Arrows indicate the biosynthetic sense of the reactions. (B) Maximum detected M+0 and M+2 acetate concentrations during the cultivation of engineered E. coli strains overexpressing different enzyme combinations of the synthetic Ara5P-dependent GAA pathway in the presence of unlabeled glycerol (55 mM) and labelled 13C2 EG (500 mM). Overexpressed enzymes are indicated below the axis (plus sign). Error bars indicate standard error of the mean (n = 2). Abbreviations not previously introduced: Pyr, pyruvate; AckA, native acetate kinase; PoxB, native pyruvate oxidase.
FIGURE 4
FIGURE 4
Growth and product kinetics of strain EG4 (E. coli ΔyqhD ΔaldA lacI q + pZA23_fucOMut_fsaA_Ca-pkt + pZS13_kdsD) for the conversion of acetate from 13C2-EG. (A) Time course of optical density (OD600), glycerol concentration and total acetate concentration during cultivation of EG4 expressing all enzymes of the Ara5P-dependent GAA pathway with exception of Rpe in the presence (+EG) and absence (-EG) of EG. (B) Time course of labelled acetate fraction M+2 during cultivation of EG4. All data refer to experiments performed in minimal medium (M9) supplemented with 55 mM glycerol and 500 mM 13C2 EG. Error bars indicate standard error of the mean (n = 2).
FIGURE 5
FIGURE 5
In vivo conversion of the C2-compound GA into MVA by strain GA1 (E. coli ΔyqhD ΔaldA lacI q + pZA23_fsaA_Ca-pkt + pZS33_kdsD_rpe + pMEV-7). Cells were incubated in minimal M9 medium supplemented with 1 mM IPTG, 10 mM glycerol and 10 mM 13C2-GA at 30°C. Error bars indicate standard error of the mean (n = 2). (A) Carbon transition during GA assimilation via synthetic pathway in cellular context. Dashed lines imply more than one involved reaction between shown intermediates. Blue or green filled circles indicate that the carbon atom may be derived from glycerol or GA, respectively. Black arrows indicate the proposed reaction sequence from GA into MVA in the biosynthetic direction. Grey lines refer to side reactions by enzymes involved in the central carbon metabolism. (B) Substrate consumption, MVA production and biomass formation during incubation time. (C) Relative abundance of isotopologues of Ara5P-dependent GAA pathway intermediates and metabolites of the central carbon metabolism after 1 h of incubation. Relative abundance was measured from extracts of intracellular metabolites with the exception of MVA, which was measured in the supernatant (extracellular). P5P sugars (which are Ara5P, Ribu5P, Xylu5P and D-ribose 5-phosphate) and GA3P/DHAP are shown as pools since they could not be separated by LC/MS. Abbreviations not previously introduced: 2-OG, 2-oxoglutarate; AA-CoA, acetoacetyl-CoA; AMP, adenosine monophosphate; Asp, aspartate; CMP, cytosine monophosphate; DHAP, dihydroxyacetonphosphate; ED, Entner-Doudoroff; Ery4P, d-erythrose 4-phosphate; F6P, d-fructose 6-phosphate; G6P, D-glucose 6-phosphate; Glu, glutamate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; Ici, iso-citrate; Leu, leucine; Mal, malate; MVA*, extracellular mevalonate; PEP, phosphoenolpyruvate, Ribo5P, ribose 5-phosphate, Trp, tryptophan.
FIGURE 6
FIGURE 6
Investigation of various metabolic optimization approaches for the conversion of 13C2-GA into MVA. Cells were incubated in M9 medium supplemented with 1 mM IPTG, 10 mM glycerol and 10 mM 13C2 GA at 30°C. Strain GA1 (E. coli ΔyqhD ΔaldA lacI q + pZA23_fsaA_Ca-pkt + pZS33_kdsD_rpe + pMEV-7) was used as reference strain. Using strain EC0 (E. coli ΔyqhD ΔaldA lacIq) expressing the MVA pathway from the pMEV-7 plasmid as the starting point, different enzymes of the synthetic Ara5P-dependent GAA pathway were overexpressed either from plasmids or by replacing the native promoter of the native genes in the chromosome by the strong constitutive proD promoter. Genetic modifications (shown in table) of each strain are marked with a plus sign. Relative enrichment of 13C-carbon found in the product MVA after 24 h of incubation was calculated by dividing amount of labelled C-atoms by total amount of C-atoms summed up for each detected isotopologue fraction of MVA. Error bars indicate standard error of the mean (n ≥ 2).

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