1. Lignocellulosic biomass characteristics Tuan-hua David Ho, Ph.D ( 賀端華 ) Institute of Plant & Microbial Biology Academia Sinica 02-27891709 tho@sinica.edu.tw

2. Carbohydrate and cell walls Outline Basic sugar chemistry Sucrose metabolism Starch metabolism Cell walls--structure and function Cell wall degrading enzymes

3. Basic sugar biochemistry

4. Polysaccharides can get complicated such as mixed polymers with branches.

6. Glucose-P ADPG Amylose Amylopection Starch biosynthesis ADPG pyrophosphorylase Branching enzymes Soluble starch synthase Bound starch synthase Chloroplast Other plastids

7. Winkled pea mutant deficient in a starch branching enzyme Waxy mutant in maize: amylopectin replaces amylose due to deficient in granule bound starch synthase

8. Wild type potato Transgenic potato with a bacterial ADPG pyrophosphorylase not subject to feedback inhibition Iodine stained potato slices

9. Shrunken maize mutant: deficient in sucrose synthase Sucrose Glucose + Fructose Invertase Sucrose synthase Fructose + UDPG UTP Synthesis of starch or cellulose Sucrose-P synthase

10. Starch degradation during cereal grain germination

11. Cell walls determine the shape and size of a cell. Protoplasts are always spherical, but walled cells are different in shape and size.

12. Involvement of cell walls in cell differentiation Shape and size of cells Aging and senescence Vascular system formation Fiber formation Abscission formation Fruit ripening Defense against pathogens and predators Sensing changes in the environment

13. Layers of walls: middle lamella, primary walls and secondary walls A new wall begins in the “phragomosome”/”phragmoplast” in a dividing cell.

14. From sugar to polysaccharides: Glucose (sugar) -------> glucan (such as  (1-->4)glucan, cellulose) Mannose -------> mannan Glactose -------> galactan Xylose -------> xylan Xylose and glucose -------> xyloglucan (hemicellulose) Glactose ----> oxidized to glacturonic acid ------> galacturonan Techniques used to determine the structure of complexpolysaccharides: 1. Gas-liquid chromatography (GLC)--composition 2. Mass spectrometry--structure 3. Nuclear magnetic resonance---interacting groups 4. Electrone microscopy---”seeing is believing”

15. Cellulose fibrils in the primary cell wall are oriented perpendicular to the main axis of the cell. H-bondings between the cellulose fibrils provide the strength of the wall.

16. Cell wall proteins

17. Dicots and some monocots Most of the monocots

18. Cell wall biosynthesis requires ER, Golgi, and plasma membrane. Components remain soluble until they can be cross-linked at the cell surface.

19. From starch and other sources Polysaccharides

20. Cellulose microfibrils are assembled at the surface of plasms memberane. Cellulose synthase “rosettes” in action!

21. Cellulose synthase First isolated from certain bacteria (Acetobactor xylinum and Agrobacterium tumefaciens) mutants--CesA genes Apparent homologs of CesA have been isolated from cotton, Arabidopsis, etc.

22. Thickness of walls remains the same when a cell expands

23. Cell Expands

25. XET (xyloglucan endotransglycosylase) cuts and rejoins the tethering hemicellulose molecules, “Expansins” loosen the interactions between cellulose fibrils and the tethering hemicellulose molecules

26. Lignin is an important component in cell walls

31. Pre-treatment Cellulose hydrolysis Sugars Fermentation Ethanol Plant Materials (rice straws, wood chips, etc) Search for enzymes/microbes for lignocellulosic bio-ethanol formation Hemi-cellulases Ligninases Cutinases Proteases (?) Cellulases X 3 Cell Walls Liganins Cuticle

32. Biological sources of cell wall degrading enzymes Rice straw composts Guts of herbivorous animals Guts of wood/grass consuming insects Guts of grass consuming fishes and aquatic organisms

33. Integrative Processes Gene Cloning Microbes Isolation Composts Microbes from ATCC etc. Data Bases Recombinant Protein Characterization Transformation into energy crops Gene Selection Protein Production Environments Metagenomic Libraries Proteomics Protein Engineering Protein isolation, purification Genomic libraries Enzymatic treatments by adding to feedstock In planta deconstruction & bioconversion

34. SDS-PAGE zymogram of endoglucanse activities from Geobaccillus spp. Substrate: 0.1% CMC Cellulases: endoglucanases, exoglucanases (such as CBH),  -glucosidases

35. 2-D gel zymogram detection for endoglucanse activities Substrate: 0.4% CMC

36. Cloning of endoglucanase genes via activity staining Geobaccillus spp. Geobaccillus thermodentrificans

37. Endoglucanase activities in gut tissues of Taiwanese grasshopper

38. SDS-PAGE zymogram of xylanase from Geobaccillus spp. Substrate: Birch wood xylan

39. Xalanase activities in gut tissues of Taiwanese grasshopper

40. Laccase activities in Basdiomyctous fungi 1,4 benzenediol + O 2 ------> quinone + H 2 O (Laccase is a ligninase)

41. Detection of laccase activity on SDS-PAGE Source: Pycnoporus cinnabarinus

42. Take home messages Lignocellulosic materials are virtually the cell walls of plants Plant cell walls are complicated interlocking polymers of cellulose, hemicellulose, lignins, pectins and proteins A combination of enzymes are needed to “deconstruct” the cell wall complex Much effort is needed to search for novel cell wall degrading enzymes/microbes