Identification and heterologous expression of enzymes degrading xyloglucan in biomass

1. IdentificationandheterologousexpressionofenzymesdegradingxyloglucaninbiomassIdentificationandheterologousexpressionofenzymesdegradingxyloglucaninbiomass State Research Institute of Genetics and Selections of Industrial Microorganisms (GosNIIgenetika) Project leader Dr. Sergei Yarotsky Technische Universität München, Dept. Microbiology Project leader Prof. Wolfgang Liebl Project is funded by the Ministry of education and Science of Russia and the German Federal Ministry of Education and Research Dr. Oksana Berezina GosNII Genetika

2. Xyloglucan is one of the major structural polysaccharides in primary plant cell walls Primaryplantcellwall, type I crosslinks of polygalacturonic acids pectin extensin cellulose microfibril xyloglucan Xyloglucan connects cellulose microfibrils by hydrogen bonding Dr. Oksana Berezina GosNII Genetika German Jurgens, 29 April 2011

3. In some plants xyloglucan is a storage polysaccharide Tamarindus indica is a tropical Asian evergreen tree «Köhler’s Medizinal-Pflanzen», 1887 Dr. Oksana Berezina GosNII Genetika German Jurgens, 29 April 2011

4. Xyloglucan structure Tamarind seed xyloglucan consist of cellotetraose units that have three branched backbone residues and a fourth unbranched backbone residue Xyl Xyl Xyl 11 1 α↓6βα↓6βα↓6ββ Glc → Glc → Glc → Glc → 1 4 1 4 1 4 1 4 Gal1 α↓2 Gal1 α↓2 Gal1 α↓2 Xyl Xyl Xyl 11 1 α↓6βα↓6βα↓6ββ Glc → Glc → Glc → Glc → 1 4 1 4 1 4 1 4 Xyl Xyl Xyl 11 1 α↓6βα↓6βα↓6ββ Glc → Glc → Glc → Glc → 1 4 1 4 1 4 1 4 Xylose may be derivatized with arabinose, and galactose with fucose depending on the plant type Dr. Oksana Berezina GosNII Genetika German Jurgens, 29 April 2011

5. Xyloglucan-degrading enzymes GH74, GH5, GH12, GH31, GH44, GH7, GH16 GH74: endo-glucanasesXEGs and exo-gluconasesOXG-RCBH Xyl Xyl Xyl 11 1 α↓6βα↓6βα↓6ββ Glc → Glc → Glc → Glc → 1 4 1 4 1 4 1 4 Gal1 α↓2 Xyl Xyl Xyl 11 1 α↓6βα↓6βα↓6ββ Glc → Glc → Glc → Glc → 1 4 1 4 1 4 1 4 Xyl Xyl Xyl 11 1 α↓6βα↓6βα↓6ββ Glc → Glc → Glc → Glc → 1 4 1 4 1 4 1 4 Gal1 α↓2 Gal1 α↓2 OXG-RCBHs release two glucose residues from reducing end of the main chain The first Glu must be unbranched. XEGs randomly hydrolyze glycosidic bonds of unbranched glucose residues Dr. Oksana Berezina GosNII Genetika German Jurgens, 29 April 2011

6. Project goals Identification, isolation and heterologous production in microbial hosts of new efficient enzymes degrading xyloglucan in plant biomass Xyloglucan utilization is part of the complete utilization of biomass, especially of byproducts from agriculture, food and feed production, and forestry, as part of a biorefinery process The novel enzymes may be also utilized: • for the partial digestion of xyloglucan to defined oligosaccharides; those may be assayed for their effect on human, animal or plant cells e.g. as nutraceuticals • for the definition of the structure of such oligosaccharides; concomitantly the mechanism of xyloglucan degradation will be elucidated Dr. Oksana Berezina GosNII Genetika 1

7. Project tasks • New genes encoding xyloglucan degrading enzymes will be isolated from bacteria and fungi • These genes will be expressed in appropriate hosts • Novel recombinant xyloglucanases will be purified and characterized • Best strains will be optimized the for enhancing xyloglucanase production • Structure and properties of the oligosaccharides produced from different types of xyloglucan by enzymatic hydrolysis will be investigated • Efficiency of xyloglucan hydrolysis with new recombinant enzymes in combination with commercial enzymes will be evaluated 1 Dr. Oksana Berezina GosNII Genetika

8. Tasks of the Russian side for the first stage of the project • Preparation of xyloglucanes of different structure (galacto-, arabino- and fuco-xyloglucan) from various plant sources • Screening VKPM collection for xyloglucan degrading fungi strains 1 Dr. Oksana Berezina GosNII Genetika

9. Preparation of xyloglucan from tamarind seeds Fruits and seeds of Tamarindus indica Tamarind seeds contain 70 % of xyloglucan Suspension of homogenized seeds Tamarind seed xyloglucan is used as a model substrate in study of enzymatic properties of xyloglucanases Dr. Oksana Berezina GosNII Genetika 1

10. Preparation of xyloglucan from tamarind seeds 1 2 3 1 2 3 4 5 • C (-) • C(+) XGMegazyme • Suspension of homogenized seeds • deproteinized and defatted XG • Purified tamarind seed XG • C (-) • Corn starch • Purified tamarind seed XG Dr. Oksana Berezina GosNII Genetika 2

11. VKPM - Russian National Collection of Industrial Microorganisms VKPM contains607fungi strains 25 strains utilizing plant biomass were selected 7of them produced thermostable enzymes hydrolyzing xyloglucan at 50-60°С Dr. Oksana Berezina GosNII Genetika 3

12. Effect of temperature on the activity of xyloglucan degrading enzymes Dr. Oksana Berezina GosNII Genetika 1

13. Thermostability of the fungi xyloglucan degrading enzymes Dr. Oksana Berezina GosNII Genetika

14. Conclusions • Xyloglucanes of different structure were isolated from tamarind and nasturtium seeds and apple fruits - Tamarind and nasturtium seeds contain storage galactoxyloglucane. • Apple fruits contain structural arabinofucogalactoxyloglucan • Fungi strains producing thermostable xyloglucan degrading enzymes were selected from VKPM collection Dr. Oksana Berezina GosNII Genetika 1

15. Future plans • New genes encoding fungi xyloglucan degrading enzymes will be isolated and expressed in Pichia pastoris • Novel recombinant xyloglucanases will be purified and characterized • Different types of xyloglucans will be used as a substrate for study of the enzyme properties Dr. Oksana Berezina GosNII Genetika 1