(528b) Combined 2H and 13C Metabolic Flux Analysis Enables Novel Discoveries in Zymomonas Mobilis Metabolism Used for Renewable Biofuel Production

Zymomonas mobilis is an alpha-proteo bacterium ideal for metabolic engineering of biofuels such as isobutanol from renewable feedstocks owing to its unique properties; namely, strong resilience to lignotoxins present in lignocellulose-derived feedstocks, a simple metabolic network, fast and efficient glucose utilization, and low biomass yield. In this study, metabolic flux analysis (MFA) of wild-type Z. mobilis was performed using ²H- and ¹³C-labeled glucose tracers and subsequent measurement of downstream intracellular metabolites by high-resolution LC-MS. With successful, simultaneous fitting of data from seven different isotopic tracers (1-¹³C, 3-¹³C, 6-¹³C, 1,2-¹³C, 2-²H, 4-²H, and 5-²H glucose) into a single, statistically acceptable flux map, we were able to i) identify succinate dehydrogenase (SDH)-like activity and inactivity of a tricarboxylic acid (TCA) cycle shunt, ii) elucidate pathway thermodynamics, iii) determine select enzyme co-factor specificity and their substrate stereo-specificity and, iv) confirm a metabolic imbalance preventing efficient xylose catabolism in Z. mobilis.

Firstly, ²H and ¹³C MFA has identified SDH-like activity in Z. mobilis. Specifically, before this study, it was not clear whether Z. mobilis has SDH activity because only one out of four SDH subunits was annotated in the Z. mobilis genome. Upon addition of an SDH reaction to the model, non-zero flux and correct succinate labeling patterns were observed. Unexpectedly, although two Z. mobilis enzymes have strong sequence similarity to known cyanobacterial enzymes responsible for a TCA cycle shunt—driven by α-ketoglutarate decarboxylase and succinic semialdehyde dehydrogenase—MFA confirms that this shunt is inactive in Z. mobilis and succinate is not derived from it under the conditions tested.

We then inferred the thermodynamic favorability of many Z. mobilis reactions after calculating Gibbs free energies (∆G) from forward and reverse fluxes as determined accurately by MFA. Specifically, we demonstrated the large in vivo thermodynamic favorability of the Entner-Doudoroff (ED) pathway that is supported by measured, high intracellular concentrations of ED pathway intermediates—before now, this was only a computational hypothesis.

Cofactor specificity of select Z. mobilis enzymes and their unknown substrate stereo-specificity were identified—possible only from ²H labeling experiments. MFA reveals that i) glucose-6-phosphate dehydrogenase is only NADP⁺-dependent, ii) glyceraldehyde-3-phosphate (GAP) dehydrogenase is only NAD⁺-dependent with, iii) the C1 hydrogen from GAP being transferred exclusively to the si face of NAD⁺, and iv) transhydrogenase-like activity is present in Z. mobilis and operates by transferring a hydrogen from NADPH exclusively to the si face of NAD⁺.

Lastly, MFA confirms there is a metabolic imbalance in Z. mobilis pentose phosphate pathway due to an absent transaldolase preventing it from metabolizing xylose as a sole carbon source and causing an overflow of erythrose-4-phosphate, excretion of shikimate, and an intracellular shikimate concentration 1000-fold higher than in E. coli. Future MFA experiments to investigate this imbalance would enable rational engineering of Z. mobilis strains to obtain higher product yields from glucose- and xylose-rich renewable feedstocks.