Department of Chemical and Biological Engineering
1
Reaction Pathway Analysis of the (Bio)conversion of
(Bio)macromolecules
Linda J. Broadbelt
Department of Chemical and Biological Engineering
Northwestern University
Department of Chemical and Biological Engineering
2
Multiscale modeling of chemical reactivity
104 s
Time
Continuum scale
Reactor design
Mechanism validation
10-10 s
Mesoscale
Reaction dynamics
Molecular dynamics
Atomic Scale
Transition states
Quantum effects
Elementary reaction
steps
> 10-10 m
Length
>100 m
Department of Chemical and Biological Engineering
3
104 s
Time
Continuum scale.
Reactor design.
Mechanism validation
10-10 s
Mesoscale.
Reaction dynamics.
Molecular dynamics
Atomic Scale.
Transition states.
Quantum effects.
Elementary reaction
steps
> 10-10 m
Length
>100 m
Department of Chemical and Biological Engineering
Thermochemical
conversion
Catalysis
4
Biocatalysis
Department of Chemical and Biological Engineering
Thermochemical
conversion
Catalysis
5
Biocatalysis
6
How can we use non-food biomass
to replace fossil fuels?
Extraction
Process
Heat
Gas
Yield ~13%
Upgrading
Gasification
Biomass
(switchgrass,
stover, etc.)
Fast
Pyrolysis
Liquid
Bio-Oil
Yield ~75%
Chemicals
Transport
Fuels, etc.
Turbine
Electricity
Engine
Solid
Char
Yield ~12%
Pyrolysis
Heat
Co-firing
Heat
Boiler
Bridgwater, A. V. Therm. Sci., 2004, 8, 21.
Charcoal
Applications
7
How can we model fast pyrolysis?
Empirical
k1
Cellulose
k2
Volatiles
k3
Char + Gases
Active
Cellulose
Shafizadeh, F. J. Anal. Appl. Pyrolysis 1982, 3, 283.
k3
k1
Active
Cellulose
k4
Cellulose
k2
0.95 Hydroxy-acethaldeyde + 0.25 Glyoxal +
0.20 CH3CHO + 0.20 C3H6O + 0.25 5-HMF +
0.16 CO2 + 0.23 CO + 0.1 CH4 + 0.9 H2O + 0.61 Char
Levoglucosan
6Char + 5H2O
Calonaci, M.; Grana, R.; Barker Hemings, E.; Bozzano, G.; Dente, M.;
Ranzi, E. Energy Fuels 2010, 24, 5727.
8
Postulate mechanisms based on known products
Cellulose
Exp. yields at 500°C:
Glycosidic bond
cleavage
Retro Diels-Alder
reactions
1,2-Dehydration and
hydrolysis + dehydration
Cyclic Grob fragmentation,
hydrolysis, dehydration
1,3-Dehydration,
subsequent elimination
Condensation of
small fragments
Vinu, R.; Broadbelt, L. J. Energy
Environ. Sci. 2012, 5, 9808.
Levoglucosan
59 wt%
Glycolaldehyde
6.7 wt%
5-HMF
2.8 wt%
2-Furaldehyde
1.3 wt%
Formic acid
6.4 wt%
C, CO, CO2, H2O, H2
…
Patwardhan, P.; Satrio, J. A.; Brown,
R. C.; Shanks, B. H. J. Anal. Appl.
Pyrolysis 2009, 86, 323.
9
Kinetic parameters needed for every reaction
Levoglucosan
59 wt%
Glycosidic bond
cleavage
homolytic
(multiple steps)
?
(multiple steps)
heterolytic
Mayes, H. B.; Broadbelt, L. J. J. Phys. Chem. A 2012, 116, 7098.
10
New picture of cellulose unraveling
OH
• Quantum mechanics (Gaussian 09 rev B)
H3C
– DFT (M06-2X/6-311+G(3df,2p)//M06-2X/6-31+G(2df,p))
O
O
HO
OH
– Implicit solvent to model pyrolysis electrostatic environment
• Transition-state-theory
OH
O
O
HO
HO
O
OH
OH
OH
O
O
HO
O
OH
HO
O
O
O
OH
OH
OH
Initiation
OH
O
O
HO
OH
HO
O
OH
O
O
HO
O
O
+
OH
HO
HO
O
O
OH
OH
OH
Depropagation
OH
O
HO
O
OH
OH
HO
O
O
O
+
HO
HO
O
OH
OH
O
+
HO
HO
O
O
OH
Mayes, H. B.; Broadbelt, L. J. J. Phys. Chem. A 2012, 116, 7098.
OH
HO
O
OH
O
OH
11
Validation
• Kinetic parameters used in neat cellulose pyrolysis
microkinetic model
• Predicted levoglucosan yield compared to experiment
Vinu R; Broadbelt LJ. Energy Environ. Sci. 2012, 5, 9808-9826; Zhou X et al. Ind. Eng. Chem. Res. 2014, 53,
13274–13289; Zhou X et al. Ind. Eng. Chem. Res. 2014, 53, 13290–13301.
Patwardhan, P. Satrio, J. A. Brown, R. C.; Shanks, B. H. J. Anal. Appl. Pyrolysis 2009, 86, 323.
12
Microkinetic model provides
detailed product speciation
Vinu R; Broadbelt LJ. Energy Environ. Sci. 2012, 5, 9808-9826; Zhou X et al. Ind. Eng. Chem. Res. 2014, 53,
13274–13289; Zhou X et al. Ind. Eng. Chem. Res. 2014, 53, 13290–13301.
13
Microkinetic model further tracks
species time evolution
for cellulose pyrolysis at 500°C at 1 atm
Vinu R; Broadbelt LJ. Energy Environ. Sci. 2012, 5, 9808-9826; Zhou X et al. Ind. Eng. Chem. Res. 2014, 53,
13274–13289; Zhou X et al. Ind. Eng. Chem. Res. 2014, 53, 13290–13301.
Department of Chemical and Biological Engineering
Thermochemical
conversion
Catalysis
14
Biocatalysis
15
Extending the microkinetic model
Experimental Results
homolytic
Levoglucosan - pyranose
?
heterolytic
Glycolaldehyde
Formic acid
Levoglucosan - furanose
Cellulose, Neat
Cellulose + 0.006 mmol
NaCl / g cellulose
Anhydro xylopyranose
Cellulose
Hemicellulose
Lignin
Inorganic salts
5-HMF
0
10
20
30
40
50
60
wt % Yield
Patwardhan, P. R.; Satrio, J. A; Brown, R. C.;
Shanks, B. H. Bioresour. Technol. 2010, 101, 4646.
16
Determine effect of Na+ on select pyrolysis reactions
A.
17
O
HO
2 OH
HO
O
HO
HO
OH
5
OH
HO
OH
OH
‒H2O
HO
OH
O
HO
OH
OH
‒H2O
HO
OH
HO
OH
OH
O
HO
OH
1
OH
O
OH
HO
16
OH
HO
HO
OH
O
HO
23
HO
HO
OH
3
OH
OH
O
HO
OH
OH
OH
OH
O
10
‒H2O
‒H2O
1 OH
OH
28 OH
O
O
HO
OH OH
18
OH
O
HO
HO
OH
OH
HO
OH
O
OH
‒H2O
OH
OH
O
OH
O
31
OH
26
OH
HO
HO
‒H2O
HO
OH 16
O
HO
OH
O
HO
HO
O
37
HO
O O
OH
40
2
OH
‒H2O
O
O
HO
HO
H O OH
OH
41
H.
OH
OH
O
HO
OH
3
‒H2O
HO
OH
OH
HO
HO
OH
OH
OH
HO
OH
‒H2O
O
HO
OH
30
OH
O
HO OH
42
OH
HO
‒H2O
O
HO
OH
O
43
OH
OH
O
HO
OH
HO
2
HO
HO
OH
15
‒H2O
‒H2O
OH
27
HO
OH
‒H2O
HO OH
OH
O
O
HO
29
‒H2O
32 OH
‒H2O
OH
O
HO
OH
O
OH
‒H2O
O
38
G.
HO
7
O
HO
14
25
‒H2O
OH
‒H2O
HO
OH
O
HO
HO
O
OH
‒H2O
‒H2O
OH
HO
HO
O
‒H2O
D.
HO
OH
‒H2O
OH
O
‒H2O
O
HO
‒H2O
O
8
HO
‒H2O
9
1
12
OH
8
OH
24
OH
O
O
OH
HO
13
HO
HO
HO
HO
C.
B.
O
HO
HO
OH
O
HO
O
O
OH
OH
15
‒H2O
OH
O
O
HO
O
39
O
33
O
HO
‒H2O
OH
HO
F.
HO
HO
OH
34
HO
O
OH
O
HO
OH
‒H2O
OH
11
O
OH
O
HO
‒H2O
‒H2O
12
OH
OH
‒H2O
‒H2O
‒H2O
HO
‒H2O
O
HO
OH
O
OH
OH
O
O
HO
OH
HO
O
HO
OH
OH
HO
OH
HO
O
OH
OH
‒H2O
OH
HO
‒H2O
OH
O
HO
HO
10
‒H2O
‒H2O
O
‒H2O
7
3
OH
7
OH
HO
OH
35
OH
17
‒H2O
O
HO
OH
OH
‒H2O
OH
O
OH
HO
OH
6
O
HO
OH
‒H2O
‒H2O
6
O
HO
‒H2O
OH
4 OH O
HO
OH
36
E.
OH
OH
OH
OH
O
HO
HO
44
OH
OH
OH
‒H2O
45
O
HO
HO
Mayes, H. B.; Nolte, M. W.; Beckham, G. T.; Shanks, B. H.; Broadbelt, L.J. ACS Catal., 2015, 5, 192.
Mayes, H. B.; Tian, J.; Nolte, M. W.; Shanks, B. H.; Beckham, G. T.; Gnanakaran, S.;
Broadbelt, L. J. J. Phys. Chem. B, 2014, 118, 1990.
OH
17
Incorporation into kinetic model
Key products wt% yields from pyrolysis with 0.00 to 0.34 mmol NaCl / g cellobiose
levoglucosan
CO2
5-HMF
Zhou, X.; Nolte, M. W.; Mayes, H. B.; Shanks, B. H.; Broadbelt, L. J. AIChE Journal, 2016, 62(3), 766-777
and 778-791 .
18
Insight: Na+ favoring competing dehydration reactions
18
Department of Chemical and Biological Engineering
Thermochemical
conversion
Catalysis
19
Biocatalysis
20
Metabolic Models
Modeling as a key component of metabolic
engineering toolbox
R2
K
Reactions
A
B
R3
R2
A
-1
-1
0
….
B
0
1
-1
….
2
0
0
….
0
0
1
….
0
0
1
R1
D
E
R3
….
R1
RN
N
L
J
M
O
Metabolites
C
C
D
H
R1: A  2C
R2: A  B
R3: B  D + E
….
Reaction N
….
…
…
F
…
I
…
E
S matrix
G
Maximize vproduct
Subject to
N·v=0
ai ≤ vi ≤ bi
Contador, et al. Metabolic Engineering (2009)
https://www.e-education.psu.edu/files/worldofweather/image/Section5/Katrina_track_gfs_ensemble_18Z_August27%20(Medium).png
21
Metabolic Models
What can we model?
Reaction
Knockouts
B
A
Media Changes
E
B
D
A
Heterologous
Expression
E
B
D
A
E
D
P
C
F
C
F
C
F
22
Reaction Network (Mechanism) as
Foundation of Metabolic Models
•Reactants, intermediates
and products
DG1
k1
•Thermodynamic parameters
DG9
DG6
k6
k5
DG4
k4
•Kinetic parameters
k2
DG5
•Reactions
DG3
k3
DG7 DG8
k7 k8
DG11
k11
DG10
k10
k9
DG16
k16
DG13
DG14
k14
DG15
k15
k13
DG12
k12
DG18
k18
DG17
k17
23
Computer-Generated Reaction Networks to
Fill Gaps or Identify Novel Reactions
• Graph Theory
• Reaction Matrix
Operations
• Connectivity
Reactants
Scan
Reaction • Uniqueness
Types
Determination
Reaction • Property
Rules
Calculation
• Termination
Criteria
DG1
k1
k2
DG5
k5
DG4
k4
DG9
DG3
k3
DG6
k6
DG7 DG8
k7 k8
DG11
k11
DG10
k10
k9
DG16
k16
DG13
DG14 k13
k14
DG15
k15
DG12
k12
DG18
k
DG17 18
k17
24
Bond-Electron Representation Allows
Implementation of Chemical Reaction
C
H
H
H
H
01111
10000
10000
10000
10000
methane
C 1111
H 1000
H 1000
H 1000
methyl radical
C
C
H
H
H
H
021001
200110
100000
010000
010000
100000
ethylene
ij entries denote the bond order between atoms i and j
ii entries designate the number of nonbonded electrons
associated with atom i
25
Chemical Reaction as a Matrix Addition
Operation
H • + CH4
•CH3 + H2
Reactant
Matrices
C
H
H
H
H
01111
10000
10000
10000
10000
H• 1
C
H
H
H
H
H•
Reaction Operation
H 001
0 -1 1
H 010
C• 0 1 0
C 1 0 0 + -1 1 0
H 100
1 0 -1
H• 0 0 1
Reactant
Matrix
Reordered
Reactant Matrix
011110
100000
100000
100000
100000
000001
H
C
H•
H
H
H
010
100
001
010
010
010
000
111
000
000
000
000
Product
Matrix
H
C•
H
H
H
H
001
010
100
010
010
010
000
111
000
000
000
000
26
Formulation of Reaction Operators Using
EC System
Enzyme commission (EC) code number provides
systematic names for enzymes
EC i.j.k.l
unique enzyme
i
the main class
j
the specific functional groups
k
cofactors
l
specific to the substrates
27
Formulation of Reaction Operators Using
EC System
Enzyme commission (EC) code number provides
systematic names for enzymes
EC i.j.k.l
unique enzyme
i
the main class
j
the specific functional groups
k
cofactors
l
specific to the substrates
28
Generalized Enzyme Function Examined
at the i.j.k Level
•More than 9,000 specific enzymatic reactions
(i.j.k.l)
•Fewer than 300 generalized enzyme
functions cover 55% of reactions
•Novel enzyme functions should be expected
through genomic sequencing, proteomics and
protein engineering
29
Example of a Generalized Enzyme Reaction
Generalized
enzyme reaction
(EC 4.2.1)
• EC 4.2.1.2 (fumarate hydratase)
OH
CO2H
HO2C
HO2C
CO2H
+
O
H
H
H-C-C-O-H
- - - -
• EC 4.2.1.3 ( aconitate hydratase)
HO
HO2C
CO2H
CO2H
HO2C
CO2H
CO2H
+
O
H
H
C=C + H2O
30
Discovery of Novel Biosynthetic Routes
I.J.K
A+B
C
I.J.K
L.M.N
Q.R.S
A+B
L.M.N
I.J.K
L.M.N
Q.R.S
C
D
I.J.K
L.M.N
Q.R.S
C
+A+B
Q.R.S
E
C
+A+B
D
D
P
Generation Generation
0
1
Generation
2
E
Generation
3
31
Implications for Novel Pathway Development
Given a novel reaction (reactant/product),
can we identify enzymes (catalysts) that
could be engineered (evolved) to carry this
novel biotransformation ?
If A gives B under 4.2.1 action,
then target enzymes within the 4.2.1 class
32
Application to Biobased Chemical Production
P
33
Analysis of Longer Pyruvate Pathways
Numerous Novel Candidate Pathways
P
4 Reactions
5 Reactions
Reactants of the final step
F
Y
Z
C
Total Pathways
# of pathways
1259
36
32
24
1410
34
Experimental Confirmation
Experimental Demonstration of Novel Reaction
P
Enzyme 1
Enzyme 2
Enzyme 3
Stine, A.; Zhang, M.; Roo, S.; Tyo, K.E.J.; Broadbelt, L. J. Manuscript accepted.
35
Using Reaction
Experimental
Confirmation
Rule to Identify Enzymes
Finding Putative Enzymes based on Generalized
Operators
Reaction Operator
Reaction of Interest
82 rxns out > 9,000
36
Experimental
Selecting
Similar
Confirmation
Reactions
Are there natural subgroups?
Reaction of Interest
Reaction Operator
37
Experimental
Measuring
Similarity
Confirmation
Define Reaction Similarity
Reaction of Interest
Reaction Operator
Dissimilarity Score = 1
Dissimilarity Score = 55
Dissimilarity Score = 2
38
Measuring Similarity
Experimental
Confirmation
Reactions as graph with weighted edges
39
Experimental
Dividing
Reactions
Confirmation
Into Groups
Reducing similarity
40
Dividing Reactions
Experimental
Confirmation
Into Groups
Identifying candidate enzymes through reaction similarity
Reaction of Interest
Reaction Rule
Dissimilarity Score = 1
Dissimilarity Score = 55
EdgesAll
with
edges
Cost < 1
8
6
5
3
4
2
50
30
20
15
14
12
10
41
Experimental Confirmation
P
Enzyme 1
Enzyme 2
Enzyme 3
Concentration of P (uM)
Experimental Demonstration of Novel Reaction
50 mM F
Time (s)
Stine, A.; Zhang, M.; Roo, S.; Tyo, K.E.J.; Broadbelt, L. J. Manuscript accepted.
Design of Reaction Operators
Most Specific
(Known Reactions)
Promiscuity
of an Enzyme
Most General
(2329 Operators)
Potential
Reactions Across
All Enzymes
Biomimetic Catalysis
43
How do we further constrain kinetic parameters?
Inverting
Fungal cellulases
Retaining
Koshland, D. E., Jr. Biol. Rev. 1953, 28, 413.
Payne, C. M.*; Knott, B. C.*; Mayes, H. B.*; Hansson, H.; Himmel, M. E.; Sandgren, M.;
Ståhlberg, J.; Beckham, G. T. Chem. Rev. 2015, 115, 1308. *equal contributors
44
Consistent feature of carbohydrate-active enzymes
T. reesei Cel6A
Rouvinen, J., et al. Science, 1990, 249, 380.
45
Many puckering options
Mayes, H. B.; Broadbelt, L. J.; Beckham, G. T. J. Amer. Chem. Soc. 2014, 136, 1008.
46
Simulation allows us to test hypotheses and
determine contributions to the reaction coordinate
D221
O
O
H
O
O R
H
O
H
H
OO
O
H
O
O
D175
Mayes, H. B.; Knott, B. C.; Broadbelt, L. J.; Ståhlberg, J.; Beckham, G. T. Chem. Sci. 2016, DOI: 10.1039/c6sc00571c.
Department of Chemical and Biological Engineering
Thermochemical
conversion
Catalysis
47
Biocatalysis
48
Acknowledgements
•
•
•
•
•
•
•
•
•
•
Dr. Gregg T. Beckham
Dr. Xiaowei Zhou
Dr. Vinu Ravikrishnan
Dr. Brent H. Shanks
Dr. Keith Tyo
Dr. Heather B. Mayes
Andrew P. Stine
Jennifer L. Greene
Michael W. Nolte
Dr. Brandon Knott