Oleaginous Yeasts for the Production of Sugar Alcohols Sujit S Jagtap1,2* ([email protected]), Ashwini A Bedekar2, Jing-Jing Liu1, Anshu Deewan1,2, and Christopher V Rao1,2 1DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois at Urbana-Champaign, Illinois. 2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Illinois. https://cabbi.bio/ Project Goal: The goal of this project is to investigate sugar alcohol production from plant- based sugars and glycerol in the oleaginous yeasts Rhodosporidium toruloides and Yarrowia lipolytica. We are also interested in understanding the mechanism of sugar alcohol production and the key genes involved in the polyol synthesis process. Sugar alcohols are commonly used as low-calorie, natural sweeteners. They have also been proposed by the Department of Energy as potential building blocks for bio-based chemicals. They can be used to produce polymers with applications in medicine and as precursors to anti-cancer drugs 1. Production of these sugar alcohols by yeast often results, from redox imbalances associated with growth on different sugars, accumulation of toxic intermediates, and as a cell response to the high osmotic pressure of the environment 2-3. The ability of yeast to naturally produce these sugar alcohols from simple sugars provides a potentially safer and more sustainable alternative to traditional chemical hydrogenation. In our study, we found that the oleaginous yeast R. toruloides IFO0880 produces D-arabitol during growth on xylose in nitrogen-rich medium 3. Efficient xylose utilization was a prerequisite for extracellular D-arabitol production. D-arabitol is an overflow metabolite associated with transient redox imbalances during growth on xylose. R. toruloides is also able to produce galactitol from galactose 2. In addition, R. toruloides was able to produce galactitol from galactose at reduced titers during growth in nitrogen-poor medium, which also induces lipid production. These results suggest that R. toruloides can potentially be used for the co-production of lipids and galactitol from galactose. We further characterized the mechanism for galactitol production, including identifying and biochemically characterizing the critical aldose reductase. Intracellular metabolite analysis was also performed to further understand galactose metabolism. We also explored sugar alcohol production in Y. lipolytica. It is known to produce erythritol during growth on glycerol. The heterologous overexpression of a sugar polyol phosphatase increases erythritol production two fold in Y. lipolytica. Intracellular metabolite analysis was also performed to further understand glycerol metabolism in wild type and engineered strains. We also overexpressed the key genes involved in glycerol assimilation pathways and the pentose phosphate pathway. R. toruloides and Y. lipolytica have traditionally been used for the production of lipids and lipid-based chemicals 4-5. Our work demonstrates that these non-model yeasts can also produce arabitol, galactitol, and erythritol. Collectively, our results further establishes that R. toruloides and Y. lipolytica can produce multiple value-added chemicals from a wide range of sugars and glycerol. References 1. Jagtap, S. S.; Rao, C. V., Microbial conversion of xylose into useful bioproducts. Applied Microbiology and Biotechnology 2018, 102 (21), 9015-9036. 2. Jagtap, S. S.; Bedekar, A. A.; Liu, J.-J.; Jin, Y.-S.; Rao, C. V., Production of galactitol from galactose by the oleaginous yeast Rhodosporidium toruloides IFO0880. Biotechnology for Biofuels 2019, 12 (1), 250. 3. Jagtap, S. S.; Rao, C. V., Production of d-arabitol from d-xylose by the oleaginous yeast Rhodosporidium toruloides IFO0880. Applied Microbiology and Biotechnology 2018, 102 (1), 143-151. 4. Zhang, S.; Ito, M.; Skerker, J. M.; Arkin, A. P.; Rao, C. V., Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation. Applied Microbiology and Biotechnology 2016, 100 (21), 9393-9405. 5. Zhang, S.; Jagtap, S. S.; Deewan, A.; Rao, C. V., pH selectively regulates citric acid and lipid production in Yarrowia lipolytica W29 during nitrogen-limited growth on glucose. Journal of Biotechnology 2019, 290, 10-15. The Center for Advanced Bioenergy and Bioproducts Innovation is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. .