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

Clostridium XIIEngineering Improved CellulosomesMichael E. HimmelNational Renewable Energy Laboratory

Conversion of Biomass to Fuels

•transport phenomena - tissue/cellular scale

•microfibril/matrix interaction - cellular/macromolecular

•cellulose morphology - molecular scale

Recalcitrance and multi-scale complexityswitchgrass

SEM

AFM

Nanometers

Meters

cellulose microfibrils

vascular bundle

secondary cell wall

CLSM

TEM

Stereo

cell debris

xylem

milled biomass

Innovation for Our Energy Future

•Delaminates cell walls/increases porosity

•Solubilizes hemicellulose

•CAFI3 switchgrass samples (Purdue)

Acid (hot water) pretreatment

before

cell lumen

middle lamella

cell wall

cell wall

after hot water pretreatment

plant cell debris

1 µm 1 µm

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•Erodes wall surfaces

•Solubilizes lignins•CAFI3 switchgrass samples (Texas A&M)

before

after lime pretreatment

cell wall surface5 µm

exposed cellulose microfibrils

Alkaline pretreatment

matrix confined

digestion

free confined complexed

diffuse localized concentrated

enzymes

isolated free cell tetheredsmall complexedlarge complexedlocalized secretion

cell wall

digested

cell wall

bacteria

scalloped surface

α-Cel7a::15 nm Au

T. reesei enzymes C. cellulolyticum

200 nm200 nm

CL

Trichoderma reeseiFree vs. complexed enzymes

C. cellulolyticum

Molecular Structure (experimental parameters)

Our strategy is information based

X-ray crystallographyStructure diversity (genomics)Homology modeling

Physical Biochemistry(experimental parameters)

Protein purificationPhysical chemical analysesMS and spectro. analysesSpecial and HTP activity testing

Numerical Models(subsets to entire system)

Molecular dynamics QM/MMMulti-scale modelingCode developmentForce fieldsSupercomputers

Mechanistic Model (kinetic and thermodynamic)

F*

RCSurface binding

Recognition Initial processivity/decrystallization by

cellobiose

Hydrolysis

Processivity

{ … }

Hydrolysis + Processivity

Hydrolysis

Processivity

Catalytic DomainCellulose Substrate

Linker peptide

800,000 atoms

We take a reductionist approach

Binding Domain• Adsorption, binding energy

• Mobility on cellulose surface• Interaction with broken strands

• Define function and functionality• Spring action• Interactions with substrate and water

• Free energy of motion of cellodextrin in tunnel• Exiting of cellobiose• QM/MM of reaction and structural changes

• Define most likely form of cell wall cellulose• How does pretreatment change it?• Are other isomorphs better substrates?

Example: T. reesei Cel7A

10

Example: Improving Cel7A through enhanced understanding

• Our approach to enhanced cellulose conversion: use experiments and modeling as complementary tools

Catalytic DomainCarbohydrate-

binding module Linker

Cellulose

CBM1 translates along cellulose, pausing every 1 nm

Four residues form strategic hydrogen bonds: Y5, Q7, N29, Y32

1 nm 1 nm

Homology at these sites is conserved across many cellulases and species:

Beckham et al., JPCB 2010

Fontes et. al. (2010)

1 primary scaffoldin

4 anchoring scaffoldins

91 enzymes

The C. thermocellum Cellulosome

Illustration of Enzymatic Mechanisms

Bryon Donohoe & Mike Resch, NREL

What advantage from highly articulated GHs ?

Mike Crowley, NREL

cell1

2

3

4

5

6

7

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

2 3 4 5 76 8 9CBM3

1

GH GH GH GH GH GH GH GH GH

GH

OlpA

OlpB

2 3 4 5 76 8 9CBM3

GH

1

GH GHGH GH GH GH GH GH

Orf2p

2 3 4 5 76 8 9

CBM3

1

GHGH GHGH GH GH GH GH GH

SdbA

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?

CipA

2 3 4 5 76 8 9CBM3

1

GHGH GHGH GH GH GH GH GH

?

OlpC (Cthe_0452)

Cthe_0736

?

Cthe_0735 ?

Purified cellulosome performance

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

Understanding cellulosomes: the critical enzymes

GH48 and GH9 (CbhA)

• 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.

Family 48 cellulases are essential components of CBP organisms

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

CelA, CelS, CelF (Blue) Cel48(Red)

CelF Tm ~ 55°C CelS Tm ~ 65°C CelA Tm ~ 85°CCel48 Tm ~ 45°C

Comparison of family 48 cellulases

Initial Final

Reaction coordinateChen and Brady, Cornell University

Computational scheme to characterize product expulsion

Understanding T. thermocellum CbhA

Dockerin

CBM3bBayer et al 2009

CBM4NREL 2009 Fn31- Fn32

NREL 2009

PDB

Ig-GH9PDB

CBM4 IG

GH9 FN31 FN32 CBM3b

D

Vlad Lunin & Markus Alahuta, NREL

Clostridium thermocellum CbhA X-ray structures

• 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.

SP CBM4 IG GH9

FN3

FN3

CBM3b

DOC

Molecular dynamics simulation snapshot of CbhA CBM4 with cellohexaose

The CBM4 binding pocket with bound cellobiose

Domain swapping leads to an enhanced cellulase

Wild type CbhA

New cellulase

Domain-swapping doubles activity of CbhA!

24

Wild-type CbhA

“Coated” C. cellulolyticum morphology

CBM3CipC

(Scaffoldin)

Enzymatic domainDockerin domain

Cohesin domain

Using CHARMM (MD) to begin to visualize these systems

Mike Crowley, NREL

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)