Functional Analysis of Deoxyhexose Sugar Utilization in Escherichia coli Reveals Fermentative Metabolism under Aerobic Conditions

Abstract

l-Rhamnose and l-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde, whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol, whereas under aerobic conditions, it should be oxidized into lactate and then channeled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on l-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes in E. coli K-12 MG1655 (a laboratory strain) and in E. coli Nissle 1917 (a human commensal strain) during anaerobic and aerobic growth on l-rhamnose and l-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol under anoxic conditions, allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on l-fucose, ¹³C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probiotic E. coli Nissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strain's prolonged laboratory use. IMPORTANCE E. coli's ability to survive in, grow in, and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolyzed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include l-fucose and l-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism in E. coli under anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by both E. coli strains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding of E. coli's metabolism and of its functioning in the bacterium's natural environment.

Keywords: anaerobic catabolic pathways; carbon metabolism; metabolism.

FIG 1

Metabolic network of 6-deoxyhexose catabolism in Escherichia coli. The genes encoding the various enzymatic steps are shown in italics. The genes and metabolites specific to fucose and rhamnose metabolism are shown in dark blue and medium blue, respectively. *, suspected reactions with no identified gene(s) (21). Methylgloxal metabolism is shown in gray. Pathway abbreviations: PP pathway, pentose-phosphate pathway; ED pathway, Entner-Doudoroff pathway; EMP pathway, Embden-Meyerhof-Parnas pathway (glycolysis); Mglyo pathway, methylglyoxal pathway; TCA cycle, tricarboxylic acid cycle. Metabolite abbreviations: l-fuc, l-fuculose; l-rha, l-rhamnulose; l-fucP, l-fuculose-1-phosphate; l-rhaP, l-rhamnulose-1-phosphate; S(R)-lactald, S(R)-lactaledyde; S(R)-lact, S(R)-lactate; S(R)-1,2-prop, S(R)-1,2-propanediol; Mglyo, methylglyoxal; g6p, glucose-6-phosphate; f6p, fructose-6-phosphate; fbp, fructose-1,6-biphosphate; dhap, dihydroxyacetone phosphate; gap, glyceraldehyde 3-phosphate; bpg, 1,3-biphosphoglycerate; 3pg, 3-phosphoglycerate; 2pg, 2-phosphoglycerate; pep, phosphoenolpyruvate; pyr, pyruvate; 6pgl, 6-phosphoglucono-δ-lactone; 6pg, 6-phosphogluconate; rb5P, ribulose-5-phosphate; r5p, ribose-5-phosphate; x5p, xylulose-5-phosphate; s7p, sedoheptulose-7-phosphate; e4p, erythrose-4-phosphate; acoa, acetyl coenzyme A; acp, acetyl phosphate; cit, citrate; icit, isocitrate; glx, glyoxylate; akg, α-ketoglutarate; succoa, succinyl-coenzyme A; suc, succinate; fum, fumarate; mal, malate; oaa, oxaloacetate.

FIG 2

(A to D) Growth profiles of E. coli K-12 MG1655 cultivated anaerobically on fucose (A) or rhamnose (B), and aerobically on fucose (C) or rhamnose (D). The cultures were grown in bioreactors on fucose or rhamnose as the sole carbon and energy source, under nitrogen atmosphere for anaerobic cultures and with a dissolved oxygen tension above 30% for aerobic cultures. The solid lines are the best fits obtained with PhysioFit (43).

FIG 3

Flux distribution in the central metabolism of Escherichia coli K-12 MG1655 growing anaerobically on fucose. Fluxes are given as a molar percentage of the specific fucose uptake rate, which was set to 1.

FIG 4

Flux distribution in the central metabolism of Escherichia coli K-12 MG1655 growing aerobically on fucose. Fluxes are given as a molar percentage of the specific fucose uptake rate, which was set to 1. The net extracellular fluxes measured (mmol · [g_CDW · h]⁻¹) are shown in blue. The growth rate was 0.43 ± 0.04 h⁻¹.

FIG 5

Quantitative analysis of redox and energy metabolism in Escherichia coli K-12 MG1655 grown aerobically on fucose. (A and B) The absolute fluxes (mmol · [g_CDW · h]⁻¹) of reactions linked to NADPH (A) and ATP (B) metabolism are shown, as calculated from carbon fluxes (Fig. 4). Abbreviations: idh, isocitrate dehydrogenase; pp, pentose-phosphate pathway; mae, malic enzyme; anab, anabolism; oxP, oxidative phosphorylation; subP, substrate-level phosphorylation; trans-nadh, transhydrogenases; fuc-upt, fucose uptake system; ngam, non-growth-associated maintenance.