BioEnergy Science Center - 2012 Focus area 2: Bio mass Deconstruction and Conversion

1. BioEnergy Science Center - 2012Focus area 2: Biomass Deconstruction and Conversion Clostridium XIIEngineering Improved CellulosomesMichael E. HimmelNational Renewable Energy Laboratory

2. Conversion of Biomass to Fuels

3. Recalcitrance and multi-scale complexity switchgrass Meters • transport phenomena - tissue/cellular scale • microfibril/matrix interaction - cellular/macromolecular • cellulose morphology - molecular scale CLSM Stereo Nanometers TEM cell debris milled biomass AFM vascular bundle secondary cell wall xylem SEM cellulose microfibrils

4. Acid (hot water) pretreatment after hot water pretreatment • Delaminates cell walls/increases porosity • Solubilizes hemicellulose • CAFI3 switchgrass samples (Purdue) before middle lamella plant cell debris cell wall cell wall cell lumen 1 µm 1 µm Innovation for Our Energy Future

5. Alkaline pretreatment after lime pretreatment • Erodes wall surfaces • Solubilizes lignins • CAFI3 switchgrass samples (Texas A&M) before exposed cellulose microfibrils cell wall surface 5 µm Innovation for Our Energy Future

6. enzymes matrix confined localized secretion large complexed cell tethered isolated free small complexed free confined complexed diffuse localized concentrated digestion T. reesei enzymes C. cellulolyticum bacteria α-Cel7a::15 nm Au digested scalloped surface 200 nm cell wall 200 nm cell wall

7. Free vs. complexed enzymes Trichoderma reesei C. cellulolyticum CL

8. Our strategy is information based F* Hydrolysis Hydrolysis { … } Hydrolysis + Processivity Surface binding Processivity Processivity Initial processivity/decrystallization by cellobiose RC Recognition Physical Biochemistry (experimental parameters) Numerical Models (subsets to entire system) Molecular dynamics QM/MM Multi-scale modeling Code development Force fields Supercomputers Protein purification Physical chemical analyses MS and spectro. analyses Special and HTP activity testing Mechanistic Model (kinetic and thermodynamic) Molecular Structure (experimental parameters) X-ray crystallography Structure diversity (genomics) Homology modeling

9. We take a reductionist approach Example: T. reeseiCel7A Linker peptide • Define function and functionality • Spring action • Interactions with substrate and water Binding Domain • Adsorption, binding energy • Mobility on cellulose surface • Interaction with broken strands 800,000 atoms Cellulose Substrate • Define most likely form of cell wall cellulose • How does pretreatment change it? • Are other isomorphs better substrates? Catalytic Domain • Free energy of motion of cellodextrin in tunnel • Exiting of cellobiose • QM/MM of reaction and structural changes

10. Example: Improving Cel7A through enhanced understanding • Our approach to enhanced cellulose conversion: use experiments and modeling as complementary tools Catalytic Domain Carbohydrate-binding module Linker Cellulose

11. 1 nm 1 nm CBM1 translates along cellulose, pausing every 1 nm Four residues form strategic hydrogen bonds: Y5, Q7, N29, Y32 Homology at these sites is conserved across many cellulases and species: Beckham et al., JPCB 2010

12. The C. thermocellumCellulosome 1 primary scaffoldin 4 anchoring scaffoldins 91 enzymes Fontes et. al. (2010)

13. Illustration of Enzymatic Mechanisms Bryon Donohoe & Mike Resch, NREL

14. What advantage from highly articulated GHs ? Mike Crowley, NREL

15. GH 1 4 2 8 9 5 6 7 3 1 CBM3 4 1 3 5 7 8 9 2 6 CBM3 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 CBM3 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH OlpA ? Cthe_0735 ? C. thermocellum cellulosome 1 primary scaffoldin 4 different anchoring scaffoldins 72 various proteins with dockerins At least 92 potential places for cell-wall-bound enzymes? SdbA GH GH GH GH GH GH GH GH 4 9 2 3 8 5 6 7 CBM3 Orf2p CipA OlpC (Cthe_0452) cell 1 2 3 OlpB 4 Cthe_0736 5 ? 6 7

16. Purifiedcellulosome performance • Cellulosomes perform better on the substrates they were grown on. • Cellulosomes grown on cellobiose perform poorly on Avicel and PTSG.

17. Understanding cellulosomes: the critical enzymes GH48 and GH9 (CbhA)

18. Family 48 cellulases are essential components of CBP organisms • Family 48 cellulases are essential components in several biomass-degrading bacteria. • Deletion of CelS reduces the activity of C. thermocellum by more than 40%. • Product inhibition is a major problem. • Understanding and improving these cellulases will lead to better microbes.

19. Four new structures of GH family 48 from NREL • We have solved the structures of C. bescii, B. pumillus, H. chejuensis and T. fusca GH48 enzymes in addition to the two already known unique structures • B. pumillus GH48 stands out from the others enzymes due to its enlarged loops near the active site tunnel C. bescii CelA GH48 B. pumilus GH48 H. Chejuensis GH48 T. fusca GH48 *in collaboration with D. Wilson

20. Comparison of family 48 cellulases CelA, CelS, CelF (Blue) Cel48(Red) Cel48 Tm ~ 45°C CelF Tm ~ 55°C CelS Tm ~ 65°C CelA Tm ~ 85°C

21. Computational scheme to characterize product expulsion Initial Final Chen and Brady, Cornell University Reaction coordinate

22. Ig-GH9 PDB Understanding T. thermocellumCbhA IG GH9 CBM4 FN31 FN32 CBM3b D Dockerin PDB CBM4 CBM3b Fn31- Fn32 Bayer et al 2009 NREL 2009 NREL 2009 Vlad Lunin & Markus Alahuta, NREL

23. SP Clostridium thermocellumCbhA X-ray structures CBM4 IG GH9 FN3 FN3 DOC CBM3b The CBM4 binding pocket with bound cellobiose Molecular dynamics simulation snapshot of CbhA CBM4 with cellohexaose • The structures of three new modules of CbhA have been solved • A family 4 carbohydrate binding module (CBM4) and two fibronectin(III)-like modules. • CBM4 binds to cellobiose, where the aromatic side chains of tyrosine 110 and tryptophan 68 constitute the main interactions with one glucose unit of cellobiose. • Tryptophan 118 is a unique feature of CbhA CBM4 and other clostridial CBM4s. • Our structural and computational studies indicate a possible role in binding for Trp118 • Treatment of dilute acid pretreated corn stover with Fn(III)-like domains showed no significant improvement in digestion relative to Spezyme CP alone. • The role of the fibronectin domains in CbhA might not be related to digestion.

24. Wild-type CbhA Domain swapping leads to an enhanced cellulase Domain-swapping doubles activity of CbhA! Wild type CbhA New cellulase 24

25. Enzymatic domain Dockerin domain CBM3 Cohesin domain CipC (Scaffoldin) “Coated” C. cellulolyticum morphology

26. Using CHARMM (MD) to begin to visualize these systems Mike Crowley, NREL

27. Acknowledgements • Steve Decker • Roman Brunecky • Shi-You Ding • Bryon Donohoe • John Baker • Yannick Bomble • Qi Xu • Peter Ciesielski • Deanne Sammond • Mike Resch • John Yarbrough • Michael Crowley • Marcus Alahuhta • Vladimir Lunin • Ed Bayer (Weizmann) • David Wilson (Cornell) • Maxim Kostylev (Cornell) • Adam Guss (ORNL) • Bob Hettich (ORNL) • Rich Giannone (ORNL) • Lee Lynd (Dartmouth) • Dan Olsen (Dartmouth) • Mo Chen (Cornell) • John Brady (Cornell) • Igor Zhulin (UT-Knoxville)