Clostridium XII
Engineering Improved Cellulosomes
Michael E. Himmel
National Renewable Energy Laboratory
BioEnergy Science Center - 2012
Focus area 2: Biomass Deconstruction and Conversion
Conversion of Biomass to Fuels
Recalcitrance and multi-scale complexity
• transport phenomena - tissue/cellular scale
• microfibril/matrix interaction -
switchgrass
Meters
cellular/macromolecular
• cellulose morphology - molecular scale
CLSM
Stereo
Nanometers
TEM
cell debris
AFM
milled biomass
vascular bundle
secondary
cell wall
xylem
cellulose microfibrils
SEM
Acid (hot water) pretreatment
• Delaminates cell
walls/increases porosity
after hot water
pretreatment
• Solubilizes hemicellulose
• CAFI3 switchgrass
samples (Purdue)
before
middle
lamella
plant cell
debris
cell wall
cell wall
1 µm
1 µm
cell lumen
Innovation for Our Energy Future
Alkaline pretreatment
• Erodes wall surfaces
• Solubilizes lignins
• CAFI3 switchgrass
after lime pretreatment
samples (Texas A&M)
before
exposed cellulose microfibrils
cell wall surface
5 µm
Innovation for Our Energy Future
enzymes
free
confined
localized secretion
complexed
large complexed
isolated free
small complexed
cell tethered
matrix confined
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
Free vs. complexed enzymes
Trichoderma reesei
C. cellulolyticum
Our strategy is information based
Molecular Structure
(experimental parameters)
X-ray crystallography
Structure diversity (genomics)
Homology modeling
Physical Biochemistry
Numerical Models
(experimental parameters)
(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)
F*
Hydrolysis
Hydrolysis
{…}
Hydrolysis +
Processivity
Surface
binding
Recognition
Processivity
Initial
processivity/decrystallization by
cellobiose
Processivity
RC
We take a reductionist approach
Example: T. reesei Cel7A
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
Example: Improving Cel7A through enhanced understanding
• Our approach to enhanced cellulose conversion: use experiments
and modeling as complementary tools
Carbohydratebinding module
Linker
Catalytic
Domain
Cellulose
1
0
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
The C. thermocellum Cellulosome
1 primary scaffoldin
4 anchoring scaffoldins
91 enzymes
Fontes et. al. (2010)
Illustration of Enzymatic Mechanisms
Bryon Donohoe & Mike Resch, NREL
What advantage from highly articulated GHs ?
Mike Crowley, NREL
C. thermocellum
cellulosome
GH
GH
GH
GH
1
2
3
4
5
GH
GH
GH
GH
6
7
8
9
CBM3
1 primary scaffoldin
4 different anchoring scaffoldins
72
various
dockerins
GH
proteins
with
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
SdbA
CBM3
At least 92 potential places for
cell-wall-bound enzymes?
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
Orf2p
CBM3
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
GH
GH
GH
GH
GH
GH
GH
GH
GH
1
2
3
4
5
6
7
8
9
CBM3
CBM3
CBM3
CBM3
CipA
1
OlpC
(Cthe_0452)
cell
2
3
OlpB
4
GH
CBM3
CBM3
CBM3
5
6
7
Cthe_0736
OlpA
??
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 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.
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
*in collaboration with D. Wilson
C. bescii CelA
GH48
B. pumilus GH48
H. Chejuensis
GH48
T. fusca GH48
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
Computational scheme to characterize product expulsion
Initial
Final
Chen and Brady,
Cornell University
Reaction coordinate
Understanding T. thermocellum CbhA
Dockerin
PDB
CBM4
NREL 2009
Ig-GH9
PDB
Vlad Lunin & Markus Alahuta, NREL
Fn31- Fn32
NREL 2009
CBM3b
Bayer et al 2009
Clostridium thermocellum CbhA X-ray structures
SP
•
CBM
4
IG
GH
9
FN
3
CBM3
b
DO
C
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.

•
FN
3
The CBM4 binding pocket with bound cellobiose
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.
Molecular dynamics simulation snapshot of
CbhA CBM4 with cellohexaose
Domain swapping leads to an enhanced cellulase
Domain-swapping doubles activity of CbhA!
Wild type CbhA
New cellulase
Wild-type CbhA
24
“Coated” C. cellulolyticum morphology
Enzymatic domain
Dockerin domain
CBM3
Cohesin domain
CipC (Scaffoldin)
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)