BioEnergy Science Center: An Integrated Strategy to Understand Biomass Recalcitrance

Brian H. Davison,

Oak Ridge National Laboratory

BioEnergy Science Center

www.bioenergycenter.org

2 Presentation_name

Fig. II.2. Biosynthesis of primary and secondary walls: from genes to polymers. A.

Fig. II.2. Biosynthesis of primary and secondary walls: from genes to polymers. A.

The challenges: Lignocellulosic

biomass is complex and heterogeneous

Figure II.3Figure II.3

3

Switchgrass – Atomic force microscopy (AFM)

NREL, Ding, et al, unpublished results

Crystalline cellulose microfibrils

4

• Overcoming this recalcitrance barrier will cut processing costs significantly and be used in most conversion processes.

• This requires an integrated,

multi-disciplinary approach. • BESC believes

biotechnology-intensive solutions offer greatest potential.

Removal of the Recalcitrance Barrier

Rural Employment

Expanded Markets

Human Resource

Development

Energy Security &

Sustainability

Other Fuels, Chemicals

Cellulosic Ethanol

Plants with Improved Sugar

Release

Biomass Deconstruction •Enzyme - Microbe - Substrate Interface

Cell Walls •Biosynthesis •Structure •Recalcitrance Pathways

More Effective Microbes

Enabling Technologies •Systems Biology •Biomass Characterization •Pretreatment

More Effective Pretreatment

Und

erst

andi

ng

Inno

vatio

n

Soci

etal

Ben

efits

Enabling Intellectual

Legacy

Versatile, New Manufacturing

Platform

More Effective Combinations

Access to the sugars in lignocellulosic biomass is the current critical barrier for cellulosic biofuels

5

BioEnergy Science Center (BESC) A multi-institutional, DOE-funded center performing basic and applied science dedicated to understanding biomass recalcitrance and improving yields of biofuels from cellulosic biomass

University of Georgia University of Tennessee

Cornell University Dartmouth College

West Virginia University Georgia Institute of Technology

University of California--Riverside North Carolina State University

University of California—Los Angeles

Oak Ridge National Laboratory National Renewable Energy Laboratory Samuel Roberts Noble Foundation ArborGen, LLD Ceres, Incorporated Mascoma Corporation DuPont GreenWood Resources

300+ People in 17 Institutions

www.bioenergycenter.org

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BESC is organized into three focus areas to understand biomass recalcitrance

Better Plants Better Microbes

Better Tools and Combinations

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Systems Biology: • Philosophical approach to consider

biology as integrated complex microbial and metazoan systems including:

– Molecular complexes & interactions – Molecular networks including cell signaling and

gene regulation

• Experimental approach to generate and analyze HTP data

Recombinant engineering is the intentional manipulation of organisms via altered genes or gene expression. Synthetic biology accelerates the process by manipulating entire pathways not just single of several genes.

A

D

B

C

F

E

I

H

G

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ORNL (and others) has “omic” capabilities for Systems Biology Single component • Gene • Transcript (mRNA) • Protein • Metabolite

“All” components • Genomics • Transcriptomics • Proteomics • Metabolomics

Together these data can provide a deeper picture of how an organism is functioning. This can help identify where improvements need to be made

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Do I need a why feedstock – switchgrass and poplar slide

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feruloyl CoA O

CoAS

OH

OCH3

O

H

OH

4-coumaraldehyde

HOH2C OH

4-coumaroyl alcohol

O

R- O

OH

OH

caffeoyl shikimic acid or quinic acid

4-coumaroyl shikimic acid or quinic acid

O OH

R- O

Phenylalanine

H lignin

O

CoAS

OH

4-coumaroyl CoA

O

CoAS

OH

OH

caffeoyl CoA

HCT

C3H

HCT

PAL HOOC

cinnamate

CCoAOMT

HOOC OH

4-coumaric acid

C4H

4CL

coniferaldehyde O

H

OH

OCH3

OCH3

coniferyl alcohol

OH HOH2C

G lignin

CAD

5-hydroxyconiferaldehyde

O

H

OH

OCH3

OH sinapaldehyde

O

H

OH

OCH3

CH3O

5-hydroxyconiferyl alcohol

OCH3

OH

OH

HOH2C

F5H

F5H

CAD

CCR

CCR

Lignin pathway

Agrobacterium- mediated

transformation of switchgrass

X. Fu and Z. Wang (Noble), J. Mielenz (ORNL), support from USDA/DOE

The Samuel Roberts

NOBLE Foundation

COMT

sinapyl alcohol

OCH3

OH

OCH3

HOH2C S lignin CAD

COMT

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Genetic Block in Lignin Biosynthesis in Switchgrass Increases Ethanol Yields

Wild-type (L) and 3 transgenic switchgrass plants (R)

Ethanol yield

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

Wild-type switchgrass

Etha

nol Y

ield

per

Wei

ght

of B

iom

ass

(g/g

)

Noble Foundation transgenic

switchgrass

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Top performing transgenic greenhouse plants must be evaluated in the field

• Greenhouse plants have minimal stresses • The stresses in a field may result in plants responding

differently • First year field-grown data is qualitatively consistent and

second-year field grown data is better

Baxter, et al., “Two-year field analysis of reduced recalcitrance transgenic switchgrass,” Plant Biotech J, 2014.

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Cell wall biosynthesis database

Sugar release assay

High-throughput screening pipeline

Collected ~1300 samples for Populus association and activation-tag study

Mining variation to identify key genes in biomass composition and sugar release

• Create genetic marker map to identify allelic variation

• Identify marker trait association

Establish common gardens for association and activation-tag populations with thousands of plants 100 mi

200 km

Skagit (Sedro Woolley)

Skykomish (Monroe)

Puyallup (Orting) Columbia (Longview)

Existing collections (N = 500; 1–2 trees/site) New collections (N = 580; 140–160 trees/site)

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Start with Nature’s Best: Feedstocks

Some feedstocks are less recalcitrant than others

Sequential hydrolysis & fermentation, fungal cellulase (15 mg Ctec2/g solids), yeast

0

20

40

60

80

100

120

140

160

180

200

Eth

anol

(mg/

g gl

ucan

)

Low Lignin Comparator

High Lignin Comparator

Populus trichocarpa No pretreatment other than autoclaving

Even in the same species!

Variation in diameter from different 3-year-old individuals from a common garden

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• Combines common gardens, phenotyping, GWAS and sequencing of ~1000 lines by JGI

Sugar release analysis shows up to 1.4x lower lignin, 2.7x increase in sugar release, and up to 2.4x higher ethanol yield compared to wild-type genotypes.

We have identified natural variants from the genome-wide association population with: increased levels of sugar release, high productivity in field trials across

diverse sites, and causal alleles.

• These genotypes can be clonally replicated and planted on a large scale in near-term bioenergy plantations.

Discovering and utilizing natural variants: Populus association genetics study

QTN - Lignin

False Discovery Threshold

-log 1

0 (p)

0

1

0

20

30

40

Chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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Transcriptional regulation of flavonoid and phenylpropanoid pathways in Populus

Novel isoform

EPSP ancestral isoform

Isoform has DNA-binding motif

Isoform is expressed in xylem instead of chloroplasts

EPSP variants have >2.5X more sugar release than controls

Selecting TOP Lines Recalcitrance Mechanisms

Q3: Scientific achievements.

Association mapping study of 1,100 Populus sequenced natural variants

Identified natural variants with low

lignin

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Forage Genetics plans to commercialize BESC invention in lignin regulation • The invention provides a genetic mechanism for the reduction of lignin biosynthesis while increasing

concentration of desirable flavonoids. • Reduced lignin content increases digestibility and nutritional value of animal feedstocks such as

alfalfa, corn and sorghum. • Forage Genetics plans to evaluate commercial viability of this technology in alfalfa, corn and

sorghum forage crops as animal feedstocks.

Forage Genetics International is the union of industry-leading forage companies whose history of alfalfa innovations dates back to the 1950s. Brought together in 1991, we've leveraged our collective strength to advance the forage industry and meet the needs of a diverse and growing world. Our breeding expertise combined with our proprietary germplasm base and global reach allows us to develop unique seed varieties for diverse growing conditions, making us the world leader in value-added genetics. We're proud to provide not only the seed in the bag, but the expertise, research and technology that help growers succeed. foragegenetics.com

US 2015/0353948

BESC co-inventors Sara Jawdy and Lee Gunter evaluating growth performance of rice plants carrying the lignin reducing-flavonoid enhancing mechanism .

The novel protein motif (represented in green) is responsible for regulating lignin biosynthesis and has shown decreases in lignin content of up 25% in Medicago hairy roots (a model system for alfalfa).

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Biomass Fermentation Options: Reduction of Process Steps by Using CBP

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Snapshot of the metabolic pathways involved in central glycolysis and fermentation from BMBP pathway map http://ca.expasy.org/cgi-bin/show_thumbnails.pl

GLUCOSE ETHANOL

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GLUCOSE ETHANOL

CELLUBIOSE

CELLULOSE

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Access biodiversity for new microbes • State-of-the-art cultivation techniques to

isolate novel high-temperature microbes with powerful lignocellulolytic enzymes – Collect samples from thermal biotopes – Establish primary enrichment cultures at

relevant temperatures and conditions

Sampling at Yellowstone National Park, October 2007 and July 2008

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C. thermocellum is effective at CBP

• Clostridium thermocellum, a thermophilic lignocellulose degrading anaerobe, is the most effective cellulolytic microbe.

• C. thermocellum uses multiple glucosyl hydrolase mechanisms: – Cellulosomes – Free cellulases – Free cellulolytic enzyme complexes

• High solubilization with minimal pretreatment is possible using C. thermocellum and other bacterial systems compared to industry standard SSF using fungal cellulases.

• Microbial solubilization of biomass selectively targets the carbohydrates

Paye, M.D., et al., Biotechnol. for Biofuels. (2016); Xu et al., Science Advances (2016).

Solubilization of washed mid-season switchgrass by various biocatalysts. Xylan (white) and glucan (black) solubilization from washed mid-season switchgrass by various bacteria or SSF with yeast and fungal cellulase after 5 days. (Paye et al.)

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NAD(P)H NAD(P)

+

Pyruvate

Acetyl-CoA

Acetyl-P Acetaldehyde

2 H+

NAD+

NAD+

NADH

NADH Pi

CoA ADP

ATP

L-Lactic Acid

Acetic Acid Ethanol

Cellobiose

NADH

NAD+

NADH NAD+

H2

2 H+

Fdoxidized

Fdreduced formate

H2

Biswas et al., "Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum" Biotechnol. Biofuels, 2015

C. thermocellum ethanol yield is poor due to side reactions

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NAD(P)H NAD(P)

+

Pyruvate

Acetyl-CoA

Acetyl-P Acetaldehyde

2 H+

NAD+

NAD+

NADH

NADH Pi

CoA ADP

ATP

L-Lactic Acid

Acetic Acid Ethanol

Cellobiose

NADH

NAD+

NADH NAD+

H2

2 H+

Fdoxidized

Fdreduced formate

H2

Ethanol yield 81% of theoretical

However strain grows very slowly – 10 days to consume 5 g/L cellobiose (lean medium) - secretes pyruvate, valine, alanine

Biswas et al., "Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum" Biotechnol. Biofuels, 2015

Highest C. thermocellum ethanol yield to date: “quad mutant” (pfl- hydG- ldh- pta-ack-)

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Single microbial gene linked to increased ethanol tolerance

Arg734 NAD Leu704

Fe

40 g/L ethanol

• A mutated alcohol dehydrogenase (AdhE) with altered co-factor specificity was shown to enhance ethanol tolerance in Clostridium thermocellum, a candidate consolidated bioprocessing microbe.

• The simplicity of the genetic basis for this ethanol-tolerant phenotype informs rational engineering of mutant microbial strains for cellulosic ethanol production.

• Illustrates systems biology approach including molecular modeling, ‘omics, physiological measurements and leadership class computing facilities.

Brown, et al., PNAS, 2011

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Resequencing via 454

Resequencing an ethanol tolerant C. thermocellum mutant

Williams et al. Appl Microbiol Biotechnol (2007)

gDNA resequenced via microarray

Brown S. D., et al. submitted. U.S. 61/346,660.

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Specific Activitya (Std dev) NADH NADPH

WT 2.7 (0.18) 0.025 (0.005)

EA <0.005b 0.052 (0.007)

adhE*(EA) <0.005 0.12 (0.03) a µg NAD(P)H oxidized.mg crude extract protein-1.min-1 b Below assay detection limit

0

0.5

1

1.5

2

2.5

Ethanol Acetate Formate Lactate

WT adhE*

g/L

Mutant ADH co-factor specificity changes to NADPH dependence

Carbon flow also effected in C. thermocellum containing

mutant ADH Mutation in NADH binding domain of ADH

Studies underway to further optimize carbon and electron flow for

productivity advances

Brown S. D., et al. submitted. U.S. 61/346,660.

Carbon and electron flow partition differently in AdhE mutant strain

34 Yee et al., Biotechnology for Biofuels 7:75, 2014.

Conversion (mg/g glucan loaded) for C. thermocellum mutant M1570 and wild-type DSM 1313 strains on both transgenic (T1-3-TG) and wild-type (T1-3-WT) switchgrass, which were pretreated with dilute acid. The standard deviation is from the average of triplicate buffered serum bottle fermentations.

0

50

100

150

200

250

DSM 1313(wild-type)

M1570 DSM 1313(wild-type)

M1570

Con

vers

ion

(mg/

g gl

ucan

load

ed)

C. thermocellum strain

GlucoseAcetic AcidLactic AcidEthanol

Transgenic Switchgrass

Wild-type Switchgrass

Significance • First report of use of a microbe engineered to

produce increased amounts of a biofuel on a bioenergy feedstock modified for the same purpose. Results demonstrate the potential additive advantages from combining a modified feedstock with an engineered consolidated bioprocessing microorganism.

Outcome • Fermentation of the modified COMT switchgrass

by C. thermocellum mutant M1570 had superior conversion relative to the wild-type control switchgrass line with an increase in conversion of approximately 20%.

• Ethanol was the primary product, accounting for 90% of the total metabolites with conversion of 0.19 g ethanol/g glucan loaded and 0.27 g liberated.

Combining modified switchgrass with engineered C. thermocellum improves yield

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Farming for Fuels lessons reach thousands of students through hands-on science activities • BESC in collaboration with the Creative Discovery Museum (CDM) in Chattanooga, Tennessee,

developed hands-on lesson plans for students in 4th, 5th and 6th grades. • Farming for Fuel lessons educate students about the carbon cycle, lignocellulosic biomass as substrate

for the production of biofuels and the technical and economic obstacles to a bio-based fuel economy.

“Hub and Spoke” model allows economical outreach national outreach using partnering with regional science centers and museums. Over six years, the outreach program has steadily expanded from Chattanooga across Tennessee to currently active hubs in Georgia, Texas, Michigan, Illinois, Florida, Oklahoma, Idaho, Montana, Washington, Oregon and Utah.

Community outreach in bioenergy science education is becoming self-sustaining

Science Night events reach thousands of families • In the last 2 years, >100 Science Nights were presented nation-wide reaching more than 25,000

students, parents and teachers.

A marker of self-sustaining success is that now 75% of the support for the hands-on activities now come from the schools, hubs, and other sources.

This approach has allowed BESC to steadily increase hands-on science contacts to over 25,000 in the last year and over 145,000 students, parents, and teachers in the past six years.

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Recalcitrance of biomass can be overcome

Better Plants Better Microbes

Better Tools and Combinations

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Acknowledgements

BESC Collaborators: ORNL: Kelsey Yee (now Genomatica), Jonathan Mielenz (ret.), Alex Dumitrache, Olivia Thompson (now at UGA), Miguel Rodriguez, Tim Tschaplinski, Steve Brown, Adam Guss, Udaya Kalluri Mascoma: Erin Wiswall, David Hogsett Dartmouth: Lee Lynd, Dan Olson, Julie Paye NREL: Rob Sykes, Mark Davis, Erica Gjersing Noble/UNTexas: Rick Dixon, C. Fu UTK: Neal Stewart, H Baxter, M. Mitra

Funding by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research for BioEnergy Science Center and for the Biofuels SFA.