265511 Metabolic Network Modeling of Redox Balancing and Ethanol Production in Scheffersomyces Stipitis

Tuesday, October 30, 2012: 12:31 PM
Westmoreland Central (Westin )
Meng Liang1, Min Hea Kim1, Q. Peter He2, Thomas Jeffries3 and Jin Wang4, (1)Department of Chemical Engineering, Auburn University, Auburn, AL, (2)Chemical Engineering, Tuskegee University, Tuskegee, AL, (3)Bacteriology, University of Wisconsin-Madison, Madison, WI, (4)Chemical Engineering, Auburn University, Auburn, AL

Keywords: Scheffersomyces stipitis; Pichia stipitis; stoichiometric model; xylose fermentation; ethanol production; xylitol production; redox balance; xylose reductase; xylitol dehydrogenase

In view of rising concerns over energy sustainability, global warming and feed stock availability, lignocellulosic ethanol has been identified as one of the most promising long-term renewable energy sources 1. However, many barriers exist for industrializing lignocellulosic ethanol processes. The effective conversion of xylose, as the second abundant mono-saccharide and representative pentose, to ethanol is one of the major barriers of commercial application of lignocellulosic ethanol processes.

Scheffersomyces stipitis (previous named Pichia stipitis), as the most promising native strain for xylose fermentation 2, has shown good overall performance on hydrolysate 3.  Understanding its metabolism, especially the central carbon metabolism, is very important to improve the strain, or to use it as a gene provider for other strains, or to provide metabolism adjustment for other strains.  Redox couples NAD+/NADH and NADP+/NADPH have been reported to play an important role in energy metabolism and product formation yeasts 4.  Specifically for S. stipitis, it has been reported that its product distribution is very sensitive to oxygen transfer rate due to redox imbalance.  Therefore, understanding the mechanism of redox balance and tuning up the redox conditions have significant impacts on ethanol production for S. stipitis, particularly for xylose fermentation.

In spite of the abundant experimental evidence regarding the role of redox balance in xylose utilization 5, there is a lack of understanding on the mechanism and cellular details of how redox imbalance affects the distribution of different metabolic products, especially considering that the cofactor specificities of the enzymes change according to culture conditions 6.  In this work, we reconstructed the central carbon metabolic network model of S. stipitis, and used the model to gain better understanding of its metabolic capabilities, as well as studied the influence of redox balance to products distribution.

First, the topology of central carbon metabolism of S. stipitis was identified and the metabolic network was reconstructed by integrating genomic (Pichia stipitis v2.0, KEGG), biochemical (ChEBI, KEGG) and physiological information available for this microorganism and other related yeast. The stoichiometry of the metabolic reactions was used in combination with biosynthetic requirements for growth and pseudo-steady state mass balances over intracellular metabolites for the quantification of metabolic fluxes using flux balance analysis (FBA). The model was employed to perform in silico characterization of the phenotypic behavior of S. stipitis grown on different carbon sources (glucose and xylose) with various oxygen supplies. The model predictions are in general agreement with published and our own experimental results. The carbon flux through pentose phosphate pathway was consistent with published data7 when glucose was used as carbon source and aerobic culture was applied. The effects on growth of single reaction deletions were assessed and essential biochemical reactions were identified for different carbon sources. Phenotype phase plane analysis was applied to the model to study the interactive influence of sugar (glucose or xylose) and oxygen. Several phase planes have been identified, all of which are both sugar and oxygen limited except one is solely oxygen-limited.

Following the development of the stoichiometric model, the influence of redox imbalance on products distribution was studied stoichiometrically. Specifically for xylose metabolism, the cofactor specificity of XR and XDH play very important roles for redox balance. The influence of alternating the cofactor specificities of the two enzymes has recently been studied by protein-engineering the two enzymes to reversing or adding or enhancing the affinity to the other cofactor.  It has been shown that alternating the cofactor specificities of the enzymes could not always improve the strain performance as desired 8.  With the model properly constrained, the influences of changing cofactor specificities of XR and XDH have been investigated. The results provide important insight on how the alternations of the specificities of the enzymes change the redox balance and product distribution.  The increase of affinity of XR to NADH will promote the ethanol production and depress the xylitol production while the increase of affinity of XDH to NADPH does not always improve the ethanol production, especially if XR already has a high affinity to NADH. A comparison of flux distribution involved in redox balance with different carbon sources and oxygenation conditions also provides insights on the role of redox balance in the metabolism of glucose/xylose and by-products such as xylitol, glycerol and acetic acid. By performing multivariate statistical analysis tools, we were able to illustrate how the cofactor specificities of the enzymes influenced the redox balance and carbon flux distribution.


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