The role of GlobalYeast in the world-wide transition to a sustainable bio-based economy

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1 The role of GlobalYeast in the world-wide transition to a sustainable bio-based economy

2 CEO: Marcelo do Amaral CSO: Johan Thevelein Leuven Rio de Janeiro Business Development & Service to Industry Research & Development Brazilian Belgian Start-up company July 2015

3 Mission Developing and delivering superior industrial yeast strains for the biobased economy Industrial production of * First- and second-generation bioethanol * Bio-based chemicals

4 One of the greatest challenges of our times Transition from petroleum-based economy to bio-based economy

5 Sugar Biofuels Cell factory micro-organisms YEAST Biomass Bio-based chemicals

6 The yeast S. cerevisiae is the most popular micro-organism in industrial biotechnology High fermentative activity under aerobic and anaerobic conditions Very robust under industrial process conditions Most used and widest experience in industrial fermentations Best understood organism at the molecular level Impressive accessibility and versatility for metabolic engineering Extensive array of tools and genomic collections

7 Bioethanol production 1 st generation feedstock Food crops sucrose starch PRO Well-established mature commercial markets CON Food versus fuel debate Sustainability issues with some substrates Existing commercial yeast strains Established industry Economic viability well established Incremental improvements Still disruptive improvements possible. Glucoamylasesecreting yeast Wild-yeast contamination in Brazil

8 1G feedstocks Corn Corn & Wheat Sugar beets Molasses Sugar cane juice + molasses + other feedstocks

9 Bioethanol production 2 nd generation feedstock Waste streams Energy crops PRO Cheap and abundant High sustainability CON S. cerevisiae cannot naturally utilize pentose sugars GMO Large amounts of inhibitors New yeast strains needed with disruptive improvements New industrial process needed Economic viability not yet clearly established

10 2G feedstocks Waste streams Forest residues Recycled paper Corn cob & stover Wheat straw Empty fruit bunches Bagasse + many other potential feedstocks

11 2G feedstocks Bioenergy crops Elephant grass (Miscanthus) Giant reed (Arundo) Poplar (Populus)

12 Requirements for yeast in 2G : Ghent talk Requirements for 2G yeast Inability of S. cerevisiae to utilize C5 sugars: xylose and arabinose Xylose: up to 35% of all sugars in lignocellulosic biomass Expression of bacterial xylose isomerase GlobalYeast has several proprietary bacterial xylose isomerases with high activity in yeast Inability of S. cerevisiae to tolerate the very high levels of inhibitors present in lignocellulose hydrolysates (acetic acid, furfural, HMF, etc.) (2011 ) Mutagenesis, genome shuffling, evolutionary adaptation with alternation selection conditions, rational engineering, etc. GlobalYeast: 2G industrial yeast strain with excellent performance in concentrated, undetoxified lignocellulose hydrolysates

13 Lignocellulose hydrolysate

14 Concentration Efficient cofermentation of glucose and xylose in undetoxified, concentrated bagasse hydrolysates glucose T18 HAA1* 1gDW/L ethanol Glucose g% Xylose g% 4 Glycerol g% 3 2 xylose Acetic Acid g% 1 Ethanol %v/v Time (h)

15 Steady improvement of 2G strain performance glucose ethanol xylose

16 Major approach for strain improvement Polygenic analysis of complex traits We improve the performance of existing industrial strains by the exchange of the strain s natural alleles by proprietary superior alleles Proprietary superior alleles Existing industrial strain Not protected Superior industrial strain Patented and traceable The major advantages of our approach as compared to classical breeding, mutagenesis or selection is the predictability of the improvement and the minimization of the risk of side effects

17 Major approach for strain improvement Polygenic analysis of complex traits We take advantage of S. cerevisiae s very large biodiversity 3500 S. cerevisiae natural and industrial strains in our collection Large variation in industrially-important properties Virtually all properties of industrial interest are polygenic High-throughput + small-scale fermentations with relevant industrial conditions

18 Major approach for strain improvement Polygenic analysis of complex traits Pooled-segregant wholegenome sequence analysis Pooled Extraction of genomic DNA ± segr. 2 parent strains Whole-genome sequence analysis QTL mapping (Illumina) SNP var. freq.

19 Major approach for strain improvement Polygenic analysis of complex traits Polygenic analysis of high acetic acid tolerance I II III IV Fermentation performance Glucose + 2% acetic acid (ph 5.2) V VI VII VIII GSE16-T18-HAA1 * IX X XI XII haa1* GSE16-T18 XIII XIV XV XVI

20 Reduced glycerol production Acetic acid tolerance Thermotolerance Osmotic stress tolerance Ethanol tolerance Ethanol accumulation capacity Xylose fermentation capacity Heavy metal tolerance Inhibitor tolerance Collection of proprietary superior alleles CRISPR-Cas9 Existing industrial strain Not protected Superior industrial strain Patented and traceable

21 Growth drivers of the bioethanol industry Increasing awareness of climate change Growing political support for sustainable economy Economic growth in developing countries Huge increase in import of fossil fuels for transport Huge increase in import invoice for transport fuels Example of Ethiopia uses 80% of its export revenue to buy refined petrol products huge financial burden for the country whatever percentage replacement by locally produced bioethanol huge financial savings for the country

22 Ethanol mandates around the world

23 Ethanol mandates around the world 64 countries From 5% (many) to 27.5% (Brazil) ethanol required in gasoline Steady increase in percentage Increase in 2016: Queensland, Finland, Norway, Vietnam, India, Indonesia, Argentina and Brazil Belgium 5% 10% on 1 January 2017 Limiting factor: national production capacity for bioethanol India: since 2014, 5% mandate, finally reached only in 2016; next goal: 10% Angola 10%; Ethiopia 5-10%; Mozambique 10%; Sudan 5%; Zimbabwe 10-15% (540 million $ fuel import per year)

24 Goal Play a significant supporting role in the transition to the bio-based economy Industrial production of * First- and second-generation bioethanol * Bio-based chemicals

25 Acknowledgments Team in Brazil Marcelo do Amaral Sergio Oki Carlos Marcondes Paula Naomi Isabela Sá Team in Belgium Steve Swinnen Maria Foulquié Mekonnen Demeke Georg Hubmann Saju Varghese Daniela Arruda Costa Stephen Castermans Dannele Echemendia

26 Thank you for your attention

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