Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis

Abstract

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no ¹³C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated ¹³C-MFA using the optimal tracers [1,2-¹³C]glucose, [1,6-¹³C]glucose, [1,2-¹³C]xylose and [5-¹³C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-¹³C]glucose and [U-¹³C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO₂ originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH₂, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.

Keywords: Co-factor balances; Flux estimation; Metabolism; Model validation; Parallel labeling experiments.

Figure 1

Biomass composition analysis of wild-type E. coli grown aerobically and anaerobically on glucose and xylose.

Figure 2

Expected (black bars) and measured (red bars) mass isotopomer distributions for five metabolites (valine, serine, phenylalanine, aspartate, and palmitate) from tracer experiments with [U-¹³C]glucose and [U-¹³C]xylose. Presence of incompletely labeled mass isotopomers, especially under anaerobic conditions, indicates significant biomass turnover.

Figure 3

Mass isotopomer distributions for the glycolytic intermediates 3-phosphoglycerate (3PG), phosphoenolpyruvate (PEP), and pyruvate (Pyr) from tracer experiments with [U-¹³C]glucose and [U-¹³C]xylose. Presence of unlabeled mass isotopomers (M+0), especially during anaerobic growth on [U-¹³C]xylose, indicates that significant biomass turnover occurs.

Figure 4

(A) β-oxidation pathway with genes encoding each reaction. (B) Growth rates of wild-type E. coli and knockout strains ΔfadD, ΔfadK, ΔfadDΔfadK grown aerobically and anaerobically on glucose and xylose as substrates. The double-knockout strain ΔfadDΔfadK did not grow on xylose under anaerobic conditions.

Figure 5

Validation of metabolic network models for ¹³C-MFA. Sum of squared residual (SSR) values are shown for models containing various dilution reactions. For the aerobic cultures, inclusion of CO₂ dilution was necessary to obtain an acceptable SSR value (below the red dotted line). For the anaerobic cultures, CO₂ and lipid dilution were necessary to achieve an acceptable SSR value.

Figure 6

Metabolic flux maps for E. coli grown in batch culture at four growth conditions: aerobic and anaerobic growth on glucose and xylose, respectively. Fluxes were determined using integrated ¹³C-MFA by simultaneously fitting labeling data from two tracers for each substrate. For glucose, [1,2-¹³C]glucose and [1,6-¹³C]glucose tracers were used. For xylose, [1,2-¹³C]xylose and [5-¹³C]xylose tracers were used. Complete flux results are provided is Supplemental Materials.

Figure 7

Production and consumption rates of key co-factors in metabolism NADH//FADH₂, NADPH, and ATP, during aerobic and anaerobic growth on glucose (Gluc) or xylose (Xyl). “Other” in NADPH panel represents the contribution of malic enzyme to NADPH production. “Other” in ATP panel represents the estimated ATP maintenance cost (here, assuming P/O ratio = 2.0).