Elucidating redox balance shift in Scheffersomyces stipitis' fermentative metabolism using a modified genome-scale metabolic model

Abstract

Background: Scheffersomyces stipitis is an important yeast species in the field of biorenewables due to its desired capacity for xylose utilization. It has been recognized that redox balance plays a critical role in S. stipitis due to the different cofactor preferences in xylose assimilation pathway. However, there has not been any systems level understanding on how the shift in redox balance contributes to the overall metabolic shift in S. stipitis to cope with reduced oxygen uptake. Genome-scale metabolic network models (GEMs) offer the opportunity to gain such systems level understanding; however, currently the two published GEMs for S. stipitis cannot be used for this purpose, as neither of them is able to capture the strain's fermentative metabolism reasonably well due to their poor prediction of xylitol production, a key by-product under oxygen limited conditions.

Results: A system identification-based (SID-based) framework that we previously developed for GEM validation is expanded and applied to refine a published GEM for S. stipitis, iBB814. After the modified GEM, named iDH814, was validated using literature data, it is used to obtain genome-scale understanding on how redox cofactor shifts when cells respond to reduced oxygen supply. The SID-based framework for GEM analysis was applied to examine how the environmental perturbation (i.e., reduced oxygen supply) propagates through the metabolic network, and key reactions that contribute to the shifts of redox and metabolic state were identified. Finally, the findings obtained through GEM analysis were validated using transcriptomic data.

Conclusions: iDH814, the modified model, was shown to offer significantly improved performance in terms of matching available experimental results and better capturing available knowledge on the organism. More importantly, our analysis based on iDH814 provides the first genome-scale understanding on how redox balance in S. stipitis was shifted as a result of reduced oxygen supply. The systems level analysis identified the key contributors to the overall metabolic state shift, which were validated using transcriptomic data. The analysis confirmed that S. stipitis uses a concerted approach to cope with the stress associated with reduced oxygen supply, and the shift of reducing power from NADPH to NADH seems to be the center theme that directs the overall shift in metabolic states.

Keywords: Genome-scale metabolic network model (GEM); Phenotype phase-plane analysis; Redox balance; Scheffersomyces stipitis; System identification.

Fig. 1

Demonstration of the SID based framework for GEM refinement

Fig. 2

3D phenotype phase planes for the two GEMs. a Growth PhPP for iBB814, b growth PhPP for iDH814, c CO₂ production PhPP for iBB814, d CO₂ production PhPP for iDH814, e ethanol production PhPP for iBB814, f ethanol production PhPP for iDH814, g xylitol production PhPP for iBB814, h xylitol production PhPP for iDH814. Uptake and flux values given in mmol/gCDW/h. Growth rate given in h⁻¹

Fig. 3

2D phenotype phase plots with LO indicated by red line for a iBB814 and b iDH814. Uptake values given in mmol/gCDW/h

Fig. 4

PCA loading values visualized on central carbon metabolic network maps for a iBB814 and b iDH814 along the LO

Fig. 5

Experimental ethanol production and NADPH stoichiometric coefficient in XR vs OUR. As can be seen, the linear fits for XRratio and ethanol production indicate that the three points are in the same phenotypic phase

Fig. 6

Visualization of the metabolic network response induced by the reduced oxygen uptake