Investigating xylose metabolism in recombinant Saccharomyces cerevisiae via 13C metabolic flux analysis

Background: To engineer Saccharomyces cerevisiae for efficient xylose utilization, a fungal pathway consisting of xylose reductase, xylitol dehydrogenase, and xylulose kinase is often introduced to the host strain. Despite extensive in vitro studies on the xylose pathway, the intracellular metabolism rewiring in response to the heterologous xylose pathway remains largely unknown. In this study, we applied C-13 metabolic flux analysis and stoichiometric modeling to systemically investigate the flux distributions in a series of xylose utilizing S. cerevisiae strains. Results: As revealed by C-13 metabolic flux analysis, the oxidative pentose phosphate pathway was actively used for producing NADPH required by the fungal xylose pathway during xylose utilization of recombinant S. cerevisiae strains. The TCA cycle activity was found to be tightly correlated with the requirements of maintenance energy and biomass yield. Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production. The stoichiometric modeling also suggested that both the cofactor-imbalanced and cofactor-balanced pathways could lead to optimal ethanol production, by flexibly adjusting the metabolic fluxes in futile cycle. However, compared to the cofactor-imbalanced pathway, the cofactor-balanced xylose pathway can lead to optimal ethanol production in a wider range of fermentation conditions. Conclusions: By applying C-13-MFA and in silico flux balance analysis to a series of recombinant xylose-utilizing S. cerevisiae strains, this work brings new knowledge about xylose utilization in two aspects. First, the interplays between the fungal xylose pathway and the native host metabolism were uncovered. Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization. Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study. In addition, the impacts of cofactor balance issues on xylose utilization were systemically investigated. The futile pathways were identified as the key factor to adapt to different degrees of cofactor imbalances and suggested as the targets for further engineering to tackle cofactor-balance issues.