Elucidating the xylose metabolising
properties of Scheffersomyces
stipitis using a genome scale
metabolic model
Kevin Correia, Goutham Vemuri, Radhakrishnan Mahadevan
Pathway Tools Conference, Menlo Park, CA
March 6th, 2013
• Challenges with lignocellulose fermentation
• Xylose metabolism in yeast
• Nature’s lignocellulose metabolizer: Scheffersomyces
• S. stipitis genome-scale metabolic models
• Exploring xylitol production mechanisms
• Conclusions and Future Work
Lignocellulose fermentation
• Lignocellulose feedstocks offer a sustainable
source of biomass for biofuels and
• They contain a variety of fermentable sugars:
glucose, xylose, arabinose
• Xylose content can range from 10-30% of the
biomass in softwood, hardwood, and
herbaceous agriculture residue
Pentose fermentation in S. cerevisiae
• Wild-type Saccharomyces cerevisiae cannot efficiently
ferment pentose sugars: xylose and arabinose
• S. cerevisiae has been genetically engineered to
metabolise xylose via:
– Yeast oxidoreductive pathway (Scheffersomyces stipitis)
– Bacterial isomerase pathway (Thermus thermophilus)
• Xylitol accumulates as a by-product with the yeast pathway
due to a cofactor imbalance
Pentose fermentation in yeasts
• Two enzymes exists in yeast for xylose reductase:
– NADPH-dependent xylose reductase (Candida utilus)
– NADPH/NADH-dependent xylose reductase (Scheffersomyces stipitis,
Pachysolen tannophilus)
• C. utilus has excessive xylitol production
• P. tannophilus produces 13-30% xylitol under oxygen limiting conditions
• This suggests other redox balancing mechanisms exist in S. stipitis
Scheffersomyces stipitis
• Found in the gut of wood digesting
• Can ferment all major components
of lignocellulos biomass: glucose,
mannose, xylose, arabinose,
rhamnose, cellobiose
• 48% ethanol yield from xylose
• Little to no xylitol production
Scheffersomyces stipitis:
genome scale metabolic models
• iBB814: Balaji Balagurunathan et al. Reconstruction and
analysis of a genome-scale metabolic model
for Scheffersomyces stipitis. Microbial Cell
Factories 2012, 11:27
• iSS884: Caspeta et al. Genome-scale metabolic
reconstructions of Pichia stipitis and Pichia pastoris and in
silico evaluation of their potentials. BMC Systems
Biology 2012, 6:24
• iTL885: Liu et al. A constraint-based model
of Scheffersomyces stipitis for improved ethanol
production. Biotechnology for Biofuels 2012, 5:7
iBB814: Xylose reductase study
iBB814: Xylose reductase study
In silico production of xylitol
• Balaguruthan, Caspeta and Liu show that xylitol is not a fermentation byproduct in their models, but fail to explore metabolic mechanisms
• Simulations in our study show that arabinitol is a byproduct during ethanol
fermentation, and xylitol if FVA is used
Jeppsson et al. Appl Environ Microbiol.
1995 July; 61(7): 2596–2600.
Study objectives
• Develop a comprehensive S. stipitis model by reviewing
the published models and literature
• Run batch and chemostat experiments to fine-tune model
parameters; analyse gene expression
• Evaluate metabolic mechanisms that lead to xylitol
• Overlay chemostat and gene expression data over the
metabolic model to gain insight into regulation in S. stipitis
Proposed mechanisms leading to reduced
xylitol production
Xylose reductase cofactor specificity
Crabtree effect and robustness analysis
Alternative oxidase
Suboptimal growth
Xylitol yield sensitivity to
aeration and XR cofactor specificity
Xylitol yield and the Crabtree effect
• Crabtree positive yeasts have
high uptake rates of substrate
and low uptake of oxygen
• Crabtree negative yeasts
have lower substrate uptake
rates and metabolism is
sensitive to oxygen uptake
Redox balancing with alternative
Joseph-Horne et al.
Biochim Biophys Acta. 2001 Apr 2;1504(2-3):17995.
Xylitol yield sensitivity to AOX
Ethanol yield sensitivity to AOX
Xylitol yield and suboptimal growth
• An alternative mechanism to account for low xylitol yields in
S. stipitis is suboptimal growth
• S. stiptis often has lower growth rate in microaerobic
conditions when grown on xylose, relative to glucose
Succinate bypass
• Jeffries (2009) proposed a succinate bypass that allows S.
stipitis to convert NADH to NADPH
NADPH production envelope
• Simulations show that the bypass leads to suboptimal
growth compared to NADH kinase
• We compiled a comprehensive S. stipitis genome
scale model from published and unpublished
• We evaluated metabolic mechanisms leading to
xylitol production
– Cofactor specificity, suboptimal growth and oxygen
sensitive metabolism have a greater sensitivity to
xylitol yield than alternative oxidase
Next steps
• Integrate chemostat results, metabolic model,
and gene expression
• Perform additional experiments in different
conditions to explore xylitol production in S.
• Dr. Radhakrishnan Mahadeven, University of Toronto
• Peter Y. Li, University of Toronto
• Dr. Goutham Vemuri, BioAmber
• Xin Wen, University of Guelph
• Dr. Hung Lee, University of Guelph
• Bioconversion Network