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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

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Department of Chemical and Biological Engineering

Reaction Pathway Analysis of the (Bio)conversion of (Bio)macromolecules

Linda J. Broadbelt

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Department of Chemical and Biological Engineering Northwestern University

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Multiscale modeling of chemical reactivity

Atomic Scale Transition states Quantum effects Elementary reaction steps Mesoscale Reaction dynamics Molecular dynamics Continuum scale Reactor design Mechanism validation

> 10-10 m >100 m 10-10 s 104 s Length Time

Department of Chemical and Biological Engineering

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Atomic Scale. Transition states. Quantum effects. Elementary reaction steps Mesoscale. Reaction dynamics. Molecular dynamics Continuum scale. Reactor design. Mechanism validation

> 10-10 m >100 m 10-10 s 104 s Length Time

Department of Chemical and Biological Engineering

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Department of Chemical and Biological Engineering

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Thermochemical conversion Catalysis Biocatalysis

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Department of Chemical and Biological Engineering

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Thermochemical conversion Catalysis Biocatalysis

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Boiler

Bridgwater, A. V. Therm. Sci., 2004, 8, 21.

Chemicals Transport Fuels, etc. Electricity Heat Charcoal Applications Extraction Upgrading Boiler Co-firing Engine Turbine Gasification Biomass

(switchgrass, stover, etc.)

Solid Char Yield ~12% Gas Yield ~13% Liquid Bio-Oil Yield ~75%

Fast Pyrolysis

Pyrolysis Heat Process Heat

How can we use non-food biomass to replace fossil fuels?

Chemicals Transport Fuels, etc. Electricity Heat Extraction Upgrading Co-firing Engine Turbine Gasification Liquid Bio-Oil Yield ~75%

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How can we model fast pyrolysis?

Char + Gases Cellulose k1 Active Cellulose Volatiles k2 k3

Shafizadeh, F. J. Anal. Appl. Pyrolysis 1982, 3, 283.

Empirical

Levoglucosan Cellulose k1 k2 6Char + 5H2O Active Cellulose 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 k3 k4

Calonaci, M.; Grana, R.; Barker Hemings, E.; Bozzano, G.; Dente, M.; Ranzi, E. Energy Fuels 2010, 24, 5727.

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Cellulose

C, CO, CO2, H2O, H2

  • Exp. yields at 500°C:

Levoglucosan 59 wt% Glycolaldehyde 6.7 wt% 5-HMF 2.8 wt% 2-Furaldehyde 1.3 wt% Formic acid 6.4 wt%

1,3-Dehydration, subsequent elimination 1,2-Dehydration and hydrolysis + dehydration Cyclic Grob fragmentation, hydrolysis, dehydration Glycosidic bond cleavage Retro Diels-Alder reactions Condensation of small fragments

Postulate mechanisms based on known products

Vinu, R.; Broadbelt, L. J. Energy

  • Environ. Sci. 2012, 5, 9808.

Patwardhan, P.; Satrio, J. A.; Brown,

  • R. C.; Shanks, B. H. J. Anal. Appl.

Pyrolysis 2009, 86, 323.

…

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Kinetic parameters needed for every reaction

?

Levoglucosan 59 wt% heterolytic homolytic

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Glycosidic bond cleavage

Mayes, H. B.; Broadbelt, L. J. J. Phys. Chem. A 2012, 116, 7098.

(multiple steps) (multiple steps)

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O OH HO OH O OH HO O OH

+

O O OH HO O OH O OH HO OH O O O OH HO OH O HO O OH HO OH O O O OH HO O OH O OH HO O O O OH HO O OH O OH HO HO O

+

O OH HO O

+

O OH HO OH O HO

New picture of cellulose unraveling

Initiation Depropagation

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Mayes, H. B.; Broadbelt, L. J. J. Phys. Chem. A 2012, 116, 7098.

O OH HO O OH O OH HO OH OH O H3C

  • Quantum mechanics (Gaussian 09 rev B)

– DFT (M06-2X/6-311+G(3df,2p)//M06-2X/6-31+G(2df,p)) – Implicit solvent to model pyrolysis electrostatic environment

  • Transition-state-theory
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Validation

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.

  • Kinetic parameters used in neat cellulose pyrolysis

microkinetic model

  • Predicted levoglucosan yield compared to experiment

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Microkinetic model provides detailed product speciation

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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.

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Microkinetic model further tracks species time evolution

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.

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for cellulose pyrolysis at 500°C at 1 atm

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Department of Chemical and Biological Engineering

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Thermochemical conversion Catalysis Biocatalysis

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?

heterolytic homolytic

Cellulose Hemicellulose Lignin Inorganic salts

Extending the microkinetic model

Patwardhan, P. R.; Satrio, J. A; Brown, R. C.; Shanks, B. H. Bioresour. Technol. 2010, 101, 4646.

wt % Yield

10 20 30 40 50 60 5-HMF Anhydro xylopyranose Levoglucosan - furanose Formic acid Glycolaldehyde Levoglucosan - pyranose Cellulose, Neat Cellulose + 0.006 mmol NaCl / g cellulose

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Experimental Results

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Determine effect of Na+ on select pyrolysis reactions

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Mayes, H. B.; Nolte, M. W.; Beckham, G. T.; Shanks, B. H.; Broadbelt, L.J. ACS Catal., 2015, 5, 192.

HO O OH OH OH OH

3

HO O OH OH OH HO O OH OH OH

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HO O OH OH

18

OH ‒H2O ‒H2O

12

‒H2O

B.

‒H2O ‒H2O ‒H2O ‒H2O

23 27 15

OH O OH HO HO OH

1

O OH HO HO OH OH O HO HO OH OH O OH HO OH OH O OH HO OH OH O OH HO OH OH O OH HO OH OH O HO HO OH

24 25 26 28

‒H2O ‒H2O ‒H2O

C.

OH O HO HO HO OH ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O OH O HO HO OH OH O HO HO OH OH O HO HO OH OH O HO HO OH OH O HO HO OH

29 2 16 30 31 32

D.

O OH HO OH OH HO O O HO OH HO O O HO HO OH O OH HO HO OH O O HO O HO OH HO OH OH O OH HO OH HO HO O OH OH OH OH HO OH OH OH O OH HO O OH OH OH HO O OH OH O O O HO HO OH OH O OH HO OH HO O OH OH OH HO O OH OH ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O ‒H2O

1 3 4 5 6 7 8 9 17 15 12 10 11 13 14

‒H2O ‒H2O ‒H2O ‒H2O ‒H2O

16

OH O HO HO OH

2

OH O HO HO HO OH

A.

E.

‒H2O O O HO OH HO O OH HO OH OH O OH HO OH HO O OH HO OH OH O OH HO OH HO O OH HO OH OH HO

7 17 33

‒H2O ‒H2O ‒H2O ‒H2O ‒H2O O OH HO OH HO

6 36 35 34

G.

O OH HO HO OH O OH HO HO O

41 40

‒H2O ‒H2O OH O OH HO HO OH OH O O H HO HO OH

1 2

F.

O O HO OH HO

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‒H2O ‒H2O ‒H2O ‒H2O

37 38 39

O O HO HO

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O O HO HO O O HO OH O O HO OH

H.

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HO O OH OH OH OH

3

‒H2O O OH HO OH HO OH O OH HO OH HO OH O HO OH HO OH O OH HO O HO ‒H2O

44 43 45

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.

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Incorporation into kinetic model

Key products wt% yields from pyrolysis with 0.00 to 0.34 mmol NaCl / g cellobiose

levoglucosan 5-HMF

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CO2

Zhou, X.; Nolte, M. W.; Mayes, H. B.; Shanks, B. H.; Broadbelt, L. J. AIChE Journal, 2016, 62(3), 766-777 and 778-791 .

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Insight: Na+ favoring competing dehydration reactions

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Department of Chemical and Biological Engineering

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Thermochemical conversion Catalysis Biocatalysis

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Metabolic Models

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Modeling as a key component of metabolic engineering toolbox

R1: A  2C R2: A  B R3: B  D + E …. Reaction N C M F G H I J L K A B D E N O R1 R2 R3 R1 R2 R3 RN A B C D E … ….

  • 1

2

  • 1

1

  • 1

…. …. …. … … … 1 1 …. …. Reactions Metabolites S matrix

Maximize vproduct Subject to N · v = 0 ai ≤ vi ≤ bi

https://www.e-education.psu.edu/files/worldofweather/image/Section5/Katrina_track_gfs_ensemble_18Z_August27%20(Medium).png

Contador, et al. Metabolic Engineering (2009)

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A B C D F E A B C D F E A B C D F E P

Media Changes Reaction Knockouts Heterologous Expression

Metabolic Models

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What can we model?

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Reaction Network (Mechanism) as Foundation of Metabolic Models

  • Reactants, intermediates

and products

  • Reactions
  • Thermodynamic parameters

DG1 DG3 DG4 DG5 DG7 DG6 DG8 DG9 DG10 DG11 DG13 DG12 DG14 DG15 DG16 DG17 DG18

  • Kinetic parameters

k1 k2 k4 k6 k7 k9 k3 k8 k5 k10 k12 k13 k15 k16 k14 k17 k11 k18

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Computer-Generated Reaction Networks to Fill Gaps or Identify Novel Reactions

  • Graph Theory
  • Reaction Matrix

Operations

  • Connectivity

Scan

  • Uniqueness

Determination

  • Property

Calculation

  • Termination

Criteria Reactants Reaction Types Reaction Rules

k1 k2 k4 k6 k7 k9 k3 k8 k5 k10 k12 k13 k15 k16 k14 k17 k11 k18 DG1 DG3 DG4 DG5 DG7 DG6 DG8 DG9 DG10 DG11 DG13 DG12 DG14 DG15 DG16 DG17 DG18

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Bond-Electron Representation Allows Implementation of Chemical Reaction

ij entries denote the bond order between atoms i and j ii entries designate the number of nonbonded electrons associated with atom i

methane methyl radical ethylene

C 0 1 1 1 1 H 1 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 0 C 0 2 1 0 0 1 C 2 0 0 1 1 0 H 1 0 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H 1 0 0 0 0 0 C 1 1 1 1 H 1 0 0 0 H 1 0 0 0 C 1 1 1 1 H 1 0 0 0 H 1 0 0 0 H 1 0 0 0

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Reaction Operation H 0 1 0 C 1 0 0 H• 0 0 1 H 0 0 1 C• 0 1 0 H 1 0 0 + 0 -1 1

  • 1 1 0

1 0 -1

Reactant Matrices Reactant Matrix Reordered Reactant Matrix Product Matrix

C 0 1 1 1 1 H 1 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 0 H• 1 C 0 1 1 1 1 0 H 1 0 0 0 0 0 H 1 0 0 0 0 0 H 1 0 0 0 0 0 H 1 0 0 0 0 0 H• 0 0 0 0 0 1 H 0 1 0 0 0 0 C 1 0 0 1 1 1 H• 0 0 1 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H 0 0 1 0 0 0 C• 0 1 0 1 1 1 H 1 0 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H • + CH4

  • CH3 + H2

Chemical Reaction as a Matrix Addition Operation

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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

Formulation of Reaction Operators Using EC System

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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

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Formulation of Reaction Operators Using EC System

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  • 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 Generalized Enzyme Function Examined at the i.j.k Level

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Example of a Generalized Enzyme Reaction

  • EC 4.2.1.2 (fumarate hydratase)
  • EC 4.2.1.3 ( aconitate hydratase)

H-C-C-O-H C=C + H2O Generalized enzyme reaction (EC 4.2.1) H-C-C-O-H C=C + H2O

  • - - -

+

HO2C CO2H OH HO2C CO

2H

H O H

+

HO2C CO2H CO2H H O H HO2C CO2H HO CO2H

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A + B + A + B

C C D C

I.J.K L.M.N Q.R.S

D

+ A + B

E Generation 1 Generation 2 Generation 3

A + B C D E

I.J.K L.M.N Q.R.S

I.J.K L.M.N Q.R.S I.J.K L.M.N Q.R.S

Generation

Discovery of Novel Biosynthetic Routes

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P

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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

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Application to Biobased Chemical Production

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P

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Analysis of Longer Pyruvate Pathways

4 Reactions 5 Reactions

Reactants of the final step # of pathways F 1259 Y 36 Z 32 C 24 Total Pathways 1410

Numerous Novel Candidate Pathways

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P

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Experimental Confirmation

Enzyme 1 Enzyme 2 Enzyme 3

Experimental Demonstration of Novel Reaction

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Stine, A.; Zhang, M.; Roo, S.; Tyo, K.E.J.; Broadbelt, L. J. Manuscript accepted.

P

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Using Reaction Rule to Identify Enzymes

Reaction of Interest Reaction Operator 82 rxns out > 9,000

Experimental Confirmation Finding Putative Enzymes based on Generalized Operators

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Selecting Similar Reactions

Reaction of Interest Reaction Operator

Experimental Confirmation Are there natural subgroups?

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Measuring Similarity

Reaction of Interest Reaction Operator

Dissimilarity Score = 1 Dissimilarity Score = 2 Dissimilarity Score = 55

Experimental Confirmation Define Reaction Similarity

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Measuring Similarity Experimental Confirmation Reactions as graph with weighted edges

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Dividing Reactions Into Groups Experimental Confirmation Reducing similarity

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Dividing Reactions Into Groups

All edges Edges with Cost < 50 Edges with Cost < 30 Edges with Cost < 20 Edges with Cost < 15 Edges with Cost < 14 Edges with Cost < 12 Edges with Cost < 10 Edges with Cost < 8 Edges with Cost < 6 Edges with Cost < 5 Edges with Cost < 4 Edges with Cost < 3 Edges with Cost < 2 Edges with Cost < 1

Experimental Confirmation

Identifying candidate enzymes through reaction similarity

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Reaction of Interest Reaction Rule

Dissimilarity Score = 1 Dissimilarity Score = 55

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Experimental Confirmation

Enzyme 1 Enzyme 2 Enzyme 3

Concentration of P (uM) Time (s) 50 mM F

Experimental Demonstration of Novel Reaction

41

Stine, A.; Zhang, M.; Roo, S.; Tyo, K.E.J.; Broadbelt, L. J. Manuscript accepted.

P

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Design of Reaction Operators

Most Specific (Known Reactions) Promiscuity

  • f an Enzyme

Potential Reactions Across All Enzymes Biomimetic Catalysis Most General (2329 Operators)

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Inverting Retaining

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 Koshland, D. E., Jr. Biol. Rev. 1953, 28, 413.

Fungal cellulases

How do we further constrain kinetic parameters?

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Consistent feature of carbohydrate-active enzymes

  • T. reesei Cel6A

Rouvinen, J., et al. Science, 1990, 249, 380.

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Many puckering options

Mayes, H. B.; Broadbelt, L. J.; Beckham, G. T. J. Amer. Chem. Soc. 2014, 136, 1008.

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Simulation allows us to test hypotheses and determine contributions to the reaction coordinate

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O O H O H O O H O O R H O H

D221 D175

Mayes, H. B.; Knott, B. C.; Broadbelt, L. J.; Ståhlberg, J.; Beckham, G. T. Chem. Sci. 2016, DOI: 10.1039/c6sc00571c.

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Department of Chemical and Biological Engineering

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Thermochemical conversion Catalysis Biocatalysis

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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

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