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

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%

Cellulose Rigid, crystalline polysaccharide Cellulose microfibrils are resistant to simple enzymatic degradation

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

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

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

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

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

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

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

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

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

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)

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

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

Bacterial Cell CSBM ScaG ScaC ScaJ ScaF ScaD Dockerin-bearing enzyme ScaB Artzi et al. 2014

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?....

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

iBAQ Values LFQ Values Gene nameProtein compositionCB ICB IIMCC IMCC IISG ISG II CB ICB IIMCC IMCC IISG ISG II Clocl_3306ScaA Clocl_3305ScaB Clocl_2745ScaF Clocl_1799ScaE Clocl_1978ScaG Clocl_3334ScaJ Clocl_4212ScaM(b) Clocl_3303ScaD Clocl_3304ScaC Clocl_3395ScaH/L Clocl_4158ScaM Scaffoldins ScaA CBM3 ScaB ScaE ScaD ScaC LC-MS/MS Analysis – High-Molecular-Weight Fractions

iBAQ Values LFQ Values Gene nameProtein compositionCB ICB IIMCC IMCC IISG ISG II CB ICB IIMCC IMCC IISG ISG II Clocl_3306ScaA Clocl_3305ScaB Clocl_2745ScaF Clocl_1799ScaE Clocl_1978ScaG Clocl_3334ScaJ Clocl_4212ScaM(b) Clocl_3303ScaD Clocl_3304ScaC Clocl_3395ScaH/L Clocl_4158ScaM Scaffoldins Artzi et al ScaG CSBM ScaM(b) CBM2 ScaF LC-MS/MS Analysis – Low-Molecular-Weight Fractions

LC-MS/MS Analysis Artzi et al. 2015

Enzymatic activity assays Artzi et al. 2015

Enzymatic activity assays Artzi et al (submitted)

Enzymatic activity assays Artzi et al (submitted)

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 Cellulolytic Hemicellulolytic

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

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

CSBM pEC 50 [nM] DocGH48XDocAXDocH/LDocB CBM

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

The findings may suggest an evolutionary progress of horizontal gene transfer in order to improve the efficiency of plant cell wall degradation

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

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

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

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

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