1. From Biomass to Biofuels: Exploiting The Cellulosome of Clostridium clariflavum for Plant Cell Wall Degradation Lior Artzi Prof. Ed Bayer Weizmann Institute of Science August 25 th -27 th, Valencia, Spain

2. Plant Cell Wall The most abundant renewable source of carbon on earth Composed of a variety of polysaccharides Cellulose ~40% Hemicellulose ~30% Lignin (noncarbohydrate) ~20%

3. Cellulose Rigid, crystalline polysaccharide Cellulose microfibrils are resistant to simple enzymatic degradation https://public.ornl.gov

4. Degradation of cellulose to glucose monomers can be beneficial to the world population: Recycling of cellulosic industrial and agricultural waste by conversion of glucose to bioethanol Harvesting the energy encapsulated in biomass can help liberate society from complete dependence on unsustainable fuel sources

5. Production of Cellulosic Ethanol Cellulosic feedstock Pretreatment with chemicals or heat Hydrolysis of cellulose by enzymes FermentationDistillation Ethanol

6. Cellulose-degrading microorganisms Many microorganisms are capable of plant cell wall degradation Fungi Aerobic and anaerobic bacteria Degradation is carried out by cellulases and hemicellulases that are secreted from the cell Anaerobic cellulolytic bacteria possess a special type of complex in which these enzymes are organized together The cellulosome

7. High-molecular-weight, multi-enzyme complex Secreted from the bacterial cell Anchored to the cell surface Released into the extracellular medium Composed of catalytic and non-catalytic structural subunits First discovered in the anaerobic, thermophilic bacterium Clostridium thermocellum

8. Clostridium thermocellum cellulosome Cell Cellulose Enzymatic subunits II II Anchoring protein CBM Scaffoldin subunit Type I Cohesin and Dockerin Type II Cohesin and Dockerin Proximity and targeting effect

9. Clostridium clariflavum – who are you? Gram positive, anaerobic, thermophilic bacterium First isolated from a thermophilic methanogenic bioreactor Utilizes cellulose and cellobiose as sole carbon sources Shiratori et al., 2006

10. 16S rRNA gene phylogenetic tree Izquierdo et al., 2012

11. Clostridium clariflavum – who are you? C. clariflavum’s genome was sequenced recently Putative enzymes and scaffoldins were revealed Glycoside hydrolases C. thermocellum Scaffoldins A. cellulolyticus (Izquierdo et al., 2012) Resemble

12. Clostridium clariflavum – who are you? Gram positive, anaerobic, thermophilic bacterium First isolated from thermophilic methanogenic bioreactor Utilizes cellulose and cellobiose as sole carbon sources C. clariflavum’s genome was sequenced recently Putative enzymes and scaffoldins were revealed Glycoside hydrolases C. thermocellum Scaffoldins A. cellulolyticus Shiratori et al., 2006

13. Bioinformatics analysis 49 cohesin sequences, 13 different scaffoldins A. cellulolyticus – 16 scaffoldins C. thermocellum – 8 scaffoldins 79 dockerin-containing protein sequences 75 type-I dockerins 4 type-II X-dockerins Annotation of dockerin-containing enzymes by using Carbohydrate Active Enzymes database (CAZY) 41 glycoside hydrolases (GHs) 14 carbohydrate esterases (CEs) 2 polysaccharide lyases (PLs)

14. Type I Cohesins Type II Cohesin Dockerin X-Dockerin SLH Domain CBM ScaDScaM CBM2 ScaO BIL CARDB DUF11FN3 ScaM(a) CBM2 ScaE ScaJScaC ScaM(b) CBM2 ScaF ScaG CSBM ScaA CBM3 ScaBScaH/L GH48 Scaffoldin system of C. clariflavum Artzi et al. 2014

15. Results The largest cellulosome complex is constructed from ScaA, ScaB and ScaC This complex can contain up to 160 (!) enzymatic subunits ScaC ScaA CBM3 X5 ScaB X4 8X5X4=160 C. clariflavum 8X4X3=96 9X7=63 A. cellulolyticus C. thermocellum

16. Bacterial Cell CSBM ScaG ScaC 1 2 3 4 ScaJ ScaF ScaD 1 2 3 Dockerin-bearing enzyme ScaB 12345 Artzi et al. 2014

17. The interactions between the recombinant modules create an ideal architectural model… But is the prediction actually fulfilled in-vivo? And which are the most important catalytic units in the complexes?....

18. Cultivation of C. clariflavum Three sources of carbon were used: Cellobiose (CB) Microcrystalline cellulose (MCC) Acid-pretreated switchgrass (SG) Isolation of the high-molecular-weight complexes High Mw Low Mw

21. iBAQ Values LFQ Values Gene nameProtein compositionCB ICB IIMCC IMCC IISG ISG II CB ICB IIMCC IMCC IISG ISG II Clocl_3306ScaA1.00 1.0000.2381.5280.3270.7390.191 Clocl_3305ScaB0.230.020.180.060.100.03 0.1450.0070.2030.0140.0520.006 Clocl_2745ScaF0.070.200.020.120.050.09 0.0320.0350.0120.0200.0190.012 Clocl_1799ScaE0.070.000.080.020.090.03 0.0530.0010.1030.0060.0520.005 Clocl_1978ScaG0.041.680.000.730.012.91 0.0100.1600.0010.0610.0030.196 Clocl_3334ScaJ0.040.000.010.000.020.00 0.0090.0000.0050.0000.0030.000 Clocl_4212ScaM(b)0.020.510.020.620.050.41 0.0110.0640.0140.0890.0180.034 Clocl_3303ScaD0.01 0.030.050.04 0.0050.0030.007 0.0200.008 Clocl_3304ScaC0.010.000.010.00 0.0050.0000.0090.0000.0010.000 Clocl_3395ScaH/L0.00 0.01 0.0020.0010.0020.0010.0020.001 Clocl_4158ScaM0.000.010.000.020.000.02 0.001 0.0030.0020.001 Scaffoldins ScaA CBM3 ScaB ScaE ScaD ScaC LC-MS/MS Analysis – High-Molecular-Weight Fractions

22. iBAQ Values LFQ Values Gene nameProtein compositionCB ICB IIMCC IMCC IISG ISG II CB ICB IIMCC IMCC IISG ISG II Clocl_3306ScaA1.00 1.0000.2381.5280.3270.7390.191 Clocl_3305ScaB0.230.020.180.060.100.03 0.1450.0070.2030.0140.0520.006 Clocl_2745ScaF0.070.200.020.120.050.09 0.0320.0350.0120.0200.0190.012 Clocl_1799ScaE0.070.000.080.020.090.03 0.0530.0010.1030.0060.0520.005 Clocl_1978ScaG0.041.680.000.730.012.91 0.0100.1600.0010.0610.0030.196 Clocl_3334ScaJ0.040.000.010.000.020.00 0.0090.0000.0050.0000.0030.000 Clocl_4212ScaM(b)0.020.510.020.620.050.41 0.0110.0640.0140.0890.0180.034 Clocl_3303ScaD0.01 0.030.050.04 0.0050.0030.007 0.0200.008 Clocl_3304ScaC0.010.000.010.00 0.0050.0000.0090.0000.0010.000 Clocl_3395ScaH/L0.00 0.01 0.0020.0010.0020.0010.0020.001 Clocl_4158ScaM0.000.010.000.020.000.02 0.001 0.0030.0020.001 Scaffoldins Artzi et al. 2015 ScaG CSBM ScaM(b) CBM2 ScaF LC-MS/MS Analysis – Low-Molecular-Weight Fractions

23. LC-MS/MS Analysis Artzi et al. 2015

24. Enzymatic activity assays Artzi et al. 2015

25. Enzymatic activity assays Artzi et al. 2015 (submitted)

26. Enzymatic activity assays Artzi et al. 2015 (submitted)

27. Fraction I cellulosomes Fraction II cellulosomes CBM3 ScaA CSBM ScaG CBM3 ScaA x7 ScaB CBM3 ScaA x5 (i) Complex 1 ScaE (ii) Complex 2 ScaF (i) Complex 1 ScaM(b) CBM2 (ii) Complex 2 (iii) Complex 3 Artzi et al. 2015 Cellulolytic Hemicellulolytic

28. Conclusions The variety of expressed cellulosomes reveal two complementary mechanisms of action: Cell-bound cellulosomes Cell-free cellulosomes Higher presence of hemicellulases in the second fractions The MCC cellulosome had the strongest catalytic activity on all substrates The highest ScaA quantity Biomass degradation activity which approaches the degradation capabilities of the C. thermocellum cellulosome A potential candidate for industrial applications of biomass degradation

29. The Bayer lab: Prof. Edward A. Bayer Dr. Yoav Barak Dr. Ely Morag Dr. Sarah Moraïs Dr. Yael Vazana Dr. Lital Davidi Johanna Stern Melina Shamshoum Yonit Ben David Olga Zhivin Vered Israeli Amarantha Kahn Lizi Hazan Eva Lamed Acknowledgements

31. CSBM pEC 50 [nM] DocGH48XDocAXDocH/LDocB CBM

32. Discussion The genome sequence of C. clariflavum is rich in cellulosomal genes that create an intricate cellulosomal system with a great number of possible cellulosome assemblies Its scaffoldins show high homology to the A. cellulolyticus scaffoldins: Similar architecture High sequence homology of cohesin modules In addition, there is high sequence similarity of dockerin modules from both species The enzyme sequences have the highest homology to C. thermocellum enzymes

33. The findings may suggest an evolutionary progress of horizontal gene transfer in order to improve the efficiency of plant cell wall degradation http://2011.igem.org/Team:Imperial_College_London/Project_Gene_Overview

34. Cohesin-dockerin interactions The detected cohesin-dockerin interactions suggest a large number of cellulosome architectures

35. Most of the complexes are cell-associated, except for one complex, assembled on ScaE, that is a cell-free complex, containing up to 56 enzymes Although ScaE is a cell free cellulosome, the CBM modules that belong to ScaA target the entire complex to the substrate ScaA CBM3 ScaE

36. All examined type-II cohesins interacted with both X- dockerin A and X-dockerin H/L The combined interaction with the two scaffoldins allows a large number of possible cellulosome assemblies CBM3

37. Goals Exploring putative cellulosome proteins present in the genome Discover the modular arrangement of cellulosomal structures

38. Type I Cohesins Type II Cohesin Dockerin X-Dockerin SLH Domain CBM ScaDScaM CBM2 ScaO BIL CARDB DUF11FN3 ScaM(a) CBM2 ScaE ScaJScaC ScaM(b) CBM2 ScaF ScaG CSBM ScaA CBM3 ScaBScaH/L