Comprehensive Analysis of Glucose and Xylose Metabolism in Escherichia coli Under Aerobic and Anaerobic Conditions By 13c-Metabolic Flux Analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. In this work, we have applied parallel labeling experiments and integrated ¹³C metabolic flux analysis (¹³C-MFA) to elucidate metabolism of glucose and xylose in E. coli under aerobic and anaerobic conditions. While aerobic glucose metabolism is well understood, until now, there have been only a handful of studies focusing on anaerobic glucose metabolism in E. coli, and no ¹³C-MFA studies on xylose metabolism have been published. As such, this work fills a gap in current understanding regarding metabolism of these two important sugars. Specifically, in this study we performed parallel labeling experiments with [1,2-¹³C]glucose, [1,6-¹³C]glucose, [1,2-¹³C]xylose and [5-¹³C]xylose, the four isotopic tracers that were determined to be optimal for ¹³C-MFA. These same tracers were used to perform ¹³C-MFA of an E. coli strain that can co-utilize xylose and glucose, creating a complete picture of the metabolism of these sugars. Additionally, isotopic labeling experiments were performed with [U-¹³C]glucose and [U-¹³C]xylose to measure, for the first time, turnover of macromolecules including proteins, RNA and lipids. A common assumption in ¹³C-MFA is that turnover of macromolecules can be neglected. Here, we demonstrate that this assumption is largely valid for E. coli grown under aerobic conditions. However, under anaerobic growth conditions, significant turnover of lipids was detected for both glucose and xylose. Additionally, there was significant turnover of RNA when cells were grown anaerobically on xylose. Taken together, this study provides valuable new information on metabolic conversions of two important sugars by E. coli that can be used for future metabolic engineering efforts.