Biocatalysis for Carbohydrate Conversion

Andreas S. BommariusGeorgia Institute of Technology

ChBE, also CHEMDeveloping and Advancing Opportunities in the Bioeconomy

Atlanta, GA; March 10-11, 2015

Non-Ideal Biocatalyst

Reaction Constraints

Compromised Process

Conventional Development

Reaction Constraints

Create the Ideal Biocatalyst

“Ideal” Process

Ideal Development

Paradigm shift in biotechnology process development

S.G. Burton, D.A. Cowan, and J.M. Woodley, "The Search for the Ideal Biocatalyst", Nature Biotechnol. 2002, 20, 37-45

PerspectivePathway to developing a biocatalyst useful in synthesis has evolved

A.S. Bommarius, J.K. Blum, and M.J. Abrahamson, Curr. Opin. Chem. Biol. 2011, 15, 194-200

Contents

• Overview• Deconstruction of cellulose• Synthetic reactions with carbohydrates

– Biomonomers• 1,3-propanediol (PDO)• Furandicarboxylic acid (FDCA)

– Bulk Chemicals• Fructose from Glucose• Ascorbic acid

Summary and Perspective

• Process opportunities exist for cellulose, hemicellulose, and lignin

• To succeed, products from renewables have to feature superior properties w.r.t. products from non-renewables, not just feature “Greenness”

• Request: set the goal to RBI (i.e. the faculty) to develop process routes (incl. catalysts, solvents) – from defined raw materials to defined products, or– to products with defined properties.

O

OO

OH

OH

OH

HOHO

OHO

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OH

OH

HOHO

OHO

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HOHO

OHO

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OCH3

OCH3

O

O

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OH

OCH3

OCH3

H3CO

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HO

H3CO

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OCH3

OCH3

OHO

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H3CO

OH

OCH3

OCH3O

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OH

OCH3

OCH3

OCH3

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Lignin: 15-25% Complex network of aromatic compounds High energy content Treasure trove of novel chemistry

Hemicellulose: 23-32% A collection of 5- and 6-carbon sugars linked together in long, substituted chains- branched Xylose, arabinose, glucose, mannose and galactose

Cellulose: 38-50% Long chains of beta-linked glucose Semicrystalline structure

Major ComponentsIn LignocellulosicBiomass

J.D. McMillan, NREL

MI Pretreatment on Lignocelluloses

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120Pe

rcen

tage

lign

in d

isso

lved

Solid loading (g/L)

SBL SEWSSEB SELP

MI Ctrl

Temp: 25°CTime: 5 min

MI: 100%

Mechanical mixture of Avicel and lignin (1:1 w/w)

• MI – efficient delignifier• Extract lignin without dissolving, degrading or altering cellulose crystal structure

Bagasse (SEB) and Wheat straw (SEWS) provided by Dr. G. Zacchi

1-methylimidazole m.p. b.p.

MI - 6oC 198oC

Y. Kang et al., Biotechnol. Progr. 2015, 31, 25-34

New PSE fellowship recipient: Thomas Kwok

Cellobiohydrolase is a molecular machine

U.S. Department of Energy Genome Programs (http://genomics.energy.gov)

Cellobiohydrolase (CBH, exo-glucanase, E.C. 3.2.1.x) consists of several domains that help to pre-organize the cellulose chain; some of the major issues are: - kinetics on a heterogeneous surface, and - processivity: number of glucose/cellobiose units cleaved during a binding event

Cellulose-binding domain (CBD)

Catalytic domain

• Heterogeneous biocatalysis

Cellulase attack on cellulose

Find chain end

Cellobiohydrolase I acting on cellulose

Source: NREL (www.nrel.gov)

1. Adsorption

2. Find chain end 3. E:S complex (thread into tunnel)

4. Hydrolysis(bond cleavage, expulsion, de-crystallization)

Beckham GT et al., J. Phys. Chem. B, 2011, 115, 4118–4127 Bansal P et al., Biotechnology Advances, 2009, 27, 833-848

Rate slowdown along conversion• Rate slow-down is not simply due to substrate depletion• Importance of rate-order

dX/dt = [Enzymes cleaving the β-glycosidic bond]*kdX/dt = k*[Eadsorbed,active]*SdX/dt = k*[Eadsorbed,active]*So(1-X)If no rate hindrances, dX/dt ~(1-X)

• Crystallinity influences the order of the reaction

Apparent first order reaction with amorphous cellulose

No apparent rate order with crystalline cellulose (Avicel®)

-12

-10

-8

-6

-4

-2

0-3 -2.5 -2 -1.5 -1 -0.5 0

ln(1-X)

ln(d

X/dt

)

10

Experimental results

• Restart rates account for most of the rate reduction, higher restart rates – the remainder is attributed to clogging

• Hydrolysability (α), Kad decrease with conversion, [E]ads,max does not vary strongly, reactivity (k) no noticeable trend

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80

Nor

mal

ized

par

amet

er v

alue

Conversion (%)

HydrolysabilityKad[E]ads,maxReactivity (k)

0

0.5

1

1.5

2

2.5

3

3.5

0 20 40 60 80 100

Rat

e (g

luco

se in

10

min

., m

g/m

L)

Conversion (%)

Restart

Uninterrupted

Due to clogging

11P. Bansal, et al., Bioresour Technol., 2012, 107, 243-250

Kinetic studies: summary

Total cellulose Accessible Hydrolysable

Accessible/total ≡ [E]ads,maxHydrolysable/Accessible = αRate/([E]ads,active) = k[E]ads,active/[E]ads = f

12

P. Bansal, et al., Bioresour Technol., 2012, 107, 243-250

Factors involved in determining the raterate = k*[Eads,active]*So(1-X) (So – initial substrate conc.)rate = k*[Eads]*f*So(1-X)

f = 1 or α/y, where y = [E]ads/[E]ads,max

Ratio of accessible to total cellulose decreases (adsorption studies) Productive adsorption - fraction of hydrolyzable cellulose

Unproductive adsorption: obstacles, improper orientation of cellulose chain, exhaustion of reactive sites, etc. Cause cannot be explicitly measured or determined but productive adsorption related to hydrolyzability

Intrinsic reactivity decreases with conversion

Top Value Added Chemicals from Biomass (2004 DOE report )

Bio Process for Soronatm: an example of both Metabolic Engineering and Biocatalysis

OOH

OHHOHO

OH

HO OH

HO OHOH

HO OPO3=O

O

O O

O

n 3G

3GT

HO OPO3=OH

HO OGene 3

Gene 4

Gene 1

Gene 2Glucose

“T”

Competition between chemical and biological route on novel polymer

component: 1,3-propanediol

1,3-propanediol

acrolein ethylene oxideH2O, [H2]

Degussa/Dupontprocess

CO, H2Shell process

glucose

E. coli Dupont/Genencor process

Propanediol (PDO) from Cornstarch

Insert Genesin Microbe

Grow “Bugs”Process microbes,separate product,purify, polymerize

and form into end-use

Yeast Bacterium

Glucose Glycerol PDO

1,3 Propanediol3G

TerephthalateT

OO--0-CH2-CH2-CH2-0-C-- ---C--( )

DuPont Soronatm

credit: Ray Miller, Dupont

2,5-FDCA (2,5-furandicarboxylic acid): building block for biopolymers

2,5-FDCA is a building block for polyethylene furanoate (PEF) or polypropylene furanoate (PPF) polyesters from renewables

PET Plastic• Petroleum-based• Food packaging (e.g., soda

and water bottles)• Textiles (e.g., polyester)• 19.1 Megatons by 2017

Smithers Pira organization. 2012.

PEF Plastic• Bio-based

• From Hydroxymethylfurfural(HMF)

• Avantium (YXY), Bird Engineering (Netherlands)

• Material properties superior to those of PET

Poly(ethylene terephthalate) vs. poly(ethylene furanoate)

O

OO

O

n

OO

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O

n

Burgess, et al. Macromolecules 2014. dx.doi.org/10.1021/ma5000199

Survey of routes to FDCAGlucose ↔

Harrison B. Rose Georgia Institute of Technology

Hydroxymethyl-furfural oxidase

(HMFO)

HFMO is a very new enzyme- Flavoprotein- O2-dependent

W.P. Dijkman, ACS Catalysis 2015, 5, 1833-39

Food: glucose isomerase (GI), the commercially most important biocatalyst (!?)

E.C. 5.3.1.5.

Tetramer, composed of two dimers

Subunit: 43 kDa

Mg catalytically essential, also requirement for Co; Mg/Ca-ratio critical for proper activity

Found originally as a xylose isomerase

Starch

Liquefaction

Saccharification

Isomerization

Slurry(40% solid)

pH 3.5-4.2

Requires:pH 6.0-6.2, Ca++

Thermo-tolerant:105°C Short Step95 °C 1-2 hr

α-amylasebreaks starch into 10-13 sugar units

NaOH

Requires:pH 4.2-4.5

GlucoamylaseBreaks into glucosemonomers

HCl

Glucose IsomeraseConverts glucose to fructose

NaOH

42% Fructose

Requires:pH 7.8

Chrom

.

Enrichm

ent$$$$

Final Product:55% Fructose

90% Fructose

Example of a Sub-optimal Process

Crabb and Shetty. (1999). Curr Opinion Micro 2:252-256

A More Efficient Process

Starch

Liquefaction

Saccharification

Isomerization

Slurry(40% solid)

pH 3.5-4.2

Requires:pH 6.0-6.2, Ca++

Thermo-tolerant:105°C Short Step95 °C 1-2 hr

α-amylasebreaks starch into 10-13 sugar units

NaOH

Requires:pH 4.2-4.5

GlucoamylaseBreaks into glucosemonomers

HCl

Glucose IsomeraseConverts glucose to fructose

NaOH

42% Fructose

Requires:pH 7.8

Chrom

.

Enrichm

ent$$$$

Final Product:55% Fructose

90% Fructose

X

X

X

X______________________________

Fine chemicals, vitamins: Process routes to ascorbic acid

Reichstein-Grüssner Sorbitol Fermentation Glucose Fermentationsynthesis

Hydrogenation

Fermentation

Oxidation/Hydrolysis

Acetonization

Esterification

Lactonization

D-Glucose

Diacetone-L-sorbose

D-Sorbitol

2-keto-L-gulonic Acid

L-Sorbose

Methyl 2-keto-L-gulonic Acid

Ascorbic Acid

Fermentation

L-Sorbose

Fermentation

Fermentation

One step Process

ChemicalProcessingTechnology

Ascorbic AcidChotani, G. et al. , Biochimica et Biophysica Acta 1543 (2000) 434-455

Process routes to ascorbic acidTable 20.3: Features of the steps in the Reichstein-Grüssner synthesis Step Yield

(%) cycle time (h); T,p, cat, solvent

Work-up steps [S] (g/L)

Biggest challenges

Hydrogenation 95 2; 140°C, 80-125 bar; Ra-Ni, H2O/MeOH

hot filtration, ion exchange, filtration

Sorbitol oxidation

90 24; 30°C, 2 atm pH 5-6 → 2; H2O

centrifugation, deionization, crystallization

200 Sterility & 2 atm O2 requ.; Ni tank material toxic

Acetonization 85 24; 30°C,135°C /3 Torr; acetone /H2SO4, ether

2x distillation, filtration, vac. Distillation

50

Oxidation 90 6; 50°C; pH H2O, acid, acetone; Pd/C

Precipitation, filtration, drying

Hydrolysis/ rearrangement

85 2; 100°C; pH 2: HCl/MeOH, ClHC, EtOH

distillation, re-crystallization, evaporation

N2/CO2-atm required

ClHC: chlorinated hydrocarbon

Current state of affairs : One-step biological production of 2-keto-L-gulonic acid (2-KLG)

Gluconic Acid 2-keto-D-Gulonic Acid

2,5-diketo-D-Gulonic Acid

D-Glucose

Ascorbic Acid

2-KLG Recovery via Crystallization

Esterification/LactonizationRecovery

E1 E2

E4

E3

E1- glucose dehydrogenaseE2- gluconic acid dehydrogenaseE3- 2-keto-D-gluconic acid dehydrogenaseE4- 2,5-diketo-D-gluconic acid reductase

Chotani, G., Biochimica et Biophysica Acta 1543 (2000) 434-455

2-keto-L-Gulonic Acid (2-KLG)

Messages From This Presentation[Andreas Bommarius]

• Possible applications of the insights/techniques/ findings/opportunities in this presentation – Lowering cost of clean raw materials (glucose, xylose)– Combine use of chemo/bio/catalysis to innovative products

• Barriers and challenges to success– Cellulose hydrolysis to glucose still too expensive– Catalyses compartmentalized, unoptimized for $2-10/kg

products• Additional research opportunities

– (ligno)cellulose structure, cellulase kinetics – Catalyses in cascades and in benign, mostly aqueous solvents