Overview

1. Kevin Correia, Goutham Vemuri, Radhakrishnan MahadevanPathway Tools Conference, Menlo Park, CAMarch 6th, 2013 • Elucidating the xylose metabolising properties of Scheffersomyces stipitis using a genome scale metabolic model

2. Overview • Challenges with lignocellulose fermentation • Xylose metabolism in yeast • Nature’s lignocellulose metabolizer: Scheffersomyces stipitis • S. stipitis genome-scale metabolic models • Exploring xylitol production mechanisms • Conclusions and Future Work

3. Lignocellulose fermentation • Lignocellulose feedstocks offer a sustainable source of biomass for biofuels and biochemicals • 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

4. 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

5. 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

6. Scheffersomyces stipitis • Found in the gut of wood digesting beetles • Can ferment all major components of lignocellulos biomass: glucose, mannose, xylose, arabinose, rhamnose, cellobiose • 48% ethanol yield from xylose • Little to no xylitol production

7. 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

8. iBB814: Xylose reductase study

9. iBB814: Xylose reductase study

10. Jeppsson et al. Appl Environ Microbiol. 1995 July; 61(7): 2596–2600. In silico production of xylitol • Balaguruthan, Caspeta and Liu show that xylitol is not a fermentation by-product 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

11. 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 production • Overlay chemostat and gene expression data over the metabolic model to gain insight into regulation in S. stipitis metabolism

12. Proposed mechanisms leading to reduced xylitol production • Xylose reductase cofactor specificity • Crabtree effect and robustness analysis • Alternative oxidase • Suboptimal growth

13. Xylitol yield sensitivity to aeration and XR cofactor specificity

14. 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

15. Redox balancing with alternative oxidase Joseph-Horne et al. Biochim Biophys Acta. 2001 Apr 2;1504(2-3):179-95.

16. Xylitol yield sensitivity to AOX

17. Ethanol yield sensitivity to AOX

18. 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

19. Succinate bypass • Jeffries (2009) proposed a succinate bypass that allows S. stipitis to convert NADH to NADPH

20. NADPH production envelope • Simulations show that the bypass leads to suboptimal growth compared to NADH kinase

21. Conclusions • We compiled a comprehensive S. stipitis genome scale model from published and unpublished models • 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

22. Next steps • Integrate chemostat results, metabolic model, and gene expression • Perform additional experiments in different conditions to explore xylitol production in S. stipitis

23. Acknowledgments • 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