CoER : Biofuels Department of Microbiology • Faculty of Natural Sciences

UNIVERSITEIT • STELLENBOSCH • UNIVERSITY

jou kennisvennoot • your knowledge partner

Construction of cellulolytic Saccharomyces cerevisiae strains for

consolidated bioprocessing

WH van Zyl1, R Den Haan1, SH Rose1, DC la Grange1, R van Rooyen1, JE McBride2 and LR Lynd2

1Department of Microbiology, University of Stellenbosch,

Stellenbosch, South Africa2Chemical and Biochemical Engineering Program, Thayer School of

Engineering, Dartmouth College, Hanover, NH 03755, USA

2

Content

4. Functional expression of cellobiohydrolases in yeast

3. Conversion of amorphous cellulose to yeast biomass

6. Acknowledgments

1. Next generation technologies for cellulose conversion

2. What is Consolidated Bioprocessing?

5. Recent advances towards realizing CBP

7. SANERI Senior Chair of Energy Research : Biofuels

3

Next generation technologies for cellulose conversion

4

Lignocellulose composition

Sugarcane bagasseLignin28%

Arabinan2%

Xylan25%

Cellulose46%

Hexoses(fermentable) Pentoses

(fermentable)

Non-fermentablesugars

high energy aromatics

Technologies for Cellulose Conversion

5

Enzymatic hydrolysis of biomass

Ligninases(laccases, lignin peroxidases,Mn-peroxidases)

Cellulases(endoglucanases, cellobiohydrolases,-glucosidases)

Hemicellulases(xylanases, -xylosidases

-arabinofuranosidases-glucuronidases)

Esterases(feruloyl esterases,coumaroyl esterases)

Technologies for Cellulose Conversion

6

Biomass Processes for EtOH productionBiologically-Mediated

Event

Enzyme Hydrolysis Processing Strategy1

(Each box represents a bioreactory - not to scale)

CellulaseProduction

LignocelluloseHydrolysis

HexoseFermentation

PentoseFermentation

SHF: Separate Hydrolysis & Fermentation

SHF

O2

SSF: Simultaneous Saccharification & Fermentation

SSF

O2

SSCF: Simultaneous Saccharification & Co-Fermenation

SSCF

O2

CBP: Consolidated Bioprocessing

CBP

Technologies for Cellulose Conversion

7

Consolidated BioProcessing (CBP)

8

Microbiology and Molecular Biology Reviews 66: 506-577 (2002)

Fundamentals of Microbial Cellulose Utilization

Consolidated BioProcessing (CBP)

9

Current Opinion in Biotechnology 16:577–583 (2005)

Consolidated BioProcessing (CBP)Consolidated bioprocessing :

update

10

Advances in Biochemical Engineering / Biotechnology (2007)

Consolidated bioprocessing : update (2)

Consolidated BioProcessing (CBP)

11

Applied Microbiology & Biotechnology (2010) [corrected proofs]

Consolidated BioProcessing (CBP)Consolidated bioprocessing :

update (3)

12Applied Microbiology & Biotechnology (2010) [corrected proofs]

Consolidated BioProcessing (CBP)Consolidated bioprocessing: update (3)

Good activity

Low activity

Very low activity

No activity

WT GE WT WT GE WT WT GE WT GE WT GE WT GE WT GE WT GE WT GE

NA

Utilize cellobiose

Utilize xylobiose NA NA

E E E E E E E E E E E E B B L L E

E E E E E E E E E E B B L L ENA NA NA NA NA NA

NA NA NA NA

NA NA

High temperature

K.m

arx

ian

us

T. sa

ccha

roly

ticu

m

Grow on xylose

Ferment glucose to ethanol

Ferment xylose to ethanol

Resistant to hydrolysate inhibitors

GRAS status

Low pH

Lac

tic

acid

bac

teri

a

Breakdown crystalline cellulose

Breakdown amorphous cellulose

Breakdown hemicellulose

C. a

ce

tob

uty

lic

um

Grow on glucose

H. p

oly

mo

rph

a

S.c

ere

vis

iae

E.c

oli

Z.m

ob

ilis

K.o

xy

toc

a

P.s

tip

itis

13

Ethanol production from sugar

Spent

yeast

Crashing

Sugar

extraction

Sugarcane\

Sugarbeet

Sweet

sorghum

Fuel

blending

Alcoholrecovery

Distillation & dehydration

Storage

tank

Yeast

Fermentation

Technologies for Ethanol Production

Sugar

Storage

tank

14

Ethanol production from cellulosics

Spent

material

Pre-treatment

Cooling &

conditioning

Chipping

Grinding

Agric Res

Woody

Material

Grasses

Water

mixing

tank

Steam explosion

~200ºC

Cellulases YeastAlcoholrecovery

Fuel

blending

Saccharification Fermentation Distillation & dehydration

Storage

tank

Technologies for Ethanol Production

Largest Component of Recalcitrance Barrier:Cost of Cellulase

a) Hinman et al. 1991. Appl. Biotechnol. Bioeng. 34/35:639-657.

b) Hettenhaus & Glassner, 1997 (http://www.ceassist.com/assessment.htm).

c) NREL, 1998. Bioethanol from the corn industry. DOE/GO-1009-577.

d) Schell, 2004. ASM Natl Meeting; McMillan, 2004. DOE/NASULGS Biomass & Solar Energy Workshops.

e) Genencor & Novozyme, 2004. Press releases (e.g. http://www.genencor.com/wt/groc/pr 109831360).

f) Petiot, Novozymes, Platts Cellulosic Ethanol & Second Generation Biofuels, 2007.

g) Sheridan (Novozymes) Nature Biotech, 2008.

0.01

0.10

1.00

10.00

1990 1995 2000 2005 2010

Es

tim

ate

d C

ellu

las

e C

os

t

($/g

al E

tOH

)

Year

2008

g

2007

f

e

2004

d

2000

c

1997

b

a

1991

15

16

Consolidated BioProcessing (CBP)

OO

O

O

O

Glu Man Gal

Xyl Ara

Ethanol + CO2

P TYFG

Glycosyl

Hydrolases

17

Ethanol production from cellulosics

Spent

material

Pre-treatment

Cooling &

conditioning

Chipping

Grinding

Agric Res

Woody

Material

Grasses

Water

mixing

tank

Steam explosion

~200ºC

Cellulases

Fuel

blending

Saccharification

Alcoholrecovery

Distillation & dehydration

Storage

tank

Yeast

Fermentation

Technologies for Ethanol Production

18Spent

material

Pre-treatment

Cooling &

conditioning

Chipping

Grinding

Agric Res

Woody

Material

Grasses

Water

mixing

tank

Steam explosion

~200ºC

Fuel

blending

Alcoholrecovery

Distillation & dehydration

Storage

tank

Cellulolytic Yeast

Saccharification & Fermentation

Technologies for Ethanol Production

Ethanol production from cellulosics

19

Conversion of amorphous celluloseto yeast biomass

20

∑ host - glucose

Cellobiose utilization by S. cerevisiae

SC media;5g/L cellobiose; 30°C / pH adjusted to 6.0 (KOH)

∑ySF1 - cellobiose

∑ host - cellobiose

∑ySF1 - glucose

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 10 20 30 40 50

OD

60

0

Time (hrs)

max = 0.20

max = 0.21

max = 0.21

Van Rooyen, R., B. Hahn-Hägerdal, D.C. La Grange, W.H. Van Zyl. 2005. Construction

and characterization of cellobiose-growing and fermenting Saccharomyces cerevisiae

strains. J. Biotechnol 20: 284 – 295.

Cellobiose utilization in yeast

ΣySF1 = S. cerevisiae expressing S.fibuligeraβ-glucosidase (BGL1) gene from 2μ plasmid.

21

Co-expression of endoglucanase & β-glucosidase in S. cerevisiae

[REF] = Y294 [yEP352]::fur1; [EG1] = Y294 [EG1]::fur1[SFI] = Y294 [BGL1]::fur1; [CEL5] = Y294 [EG1+BGL1]::fur1

1: 2:

3: 4:

SC Glucose SC cellobiose

SC CMC YPD PASC

22

[REF] [EG1]

[SFI] [CEL5]

Den Haan, R., S.H. Rose, L.R. Lynd, and W.H. Van Zyl. 2007. Hydrolysis and fermentation of amorphous

cellulose by recombinant Saccharomyces cerevisiae. Met. Eng. 9: 87–94.

Growth on amorphous cellulose (PASC)

Conversion of amorphous cellulose

23

Functional expression of cellobiohydrolases in yeast

24

CBH expression in S. cerevisiae

ENO1p ENO1tA. niger CBHBENO1P ENO1TA. niger CBHBpDLG82 =

ENO1 ENO1tT. reesei CBH2ENO1P ENO1TT. reesei CBH2pAZ21 =

ENO1p ENO1tT.reesei CBH1 ENO1P ENO1TT.reesei CBH1 pAZ22 =

= XYNSEC secretion signal

P. chrysosporium CBH1-4ENO1p ENO1tENO1P ENO1TpDLG100 =

P. chrysosporium CBH1-4ENO1p ENO1tENO1P ENO1TpCBH1-4 =

= HisTag

Functional CBH expression: a long-standing but elusive goal!

Expression of cellobiohydrolases in yeast

25

Y294[CBH1] ~20X conc.

Y294[CBH1] ~150X conc.

Y294[CBH1] ~150X conc. treated with

Pngase

+ control CBH1 ~250 ng,

treated with Pngase

206,675115,75898,003

54,604

37,390

29,559

20,366

7,036

MW (Da)

CBH1 cellobiohydrolase production by yeast

Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl. 2007. Functional

expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of

cellulose to ethanol. Enzyme Microb. Technol. 40: 1291–1299.

Expression of cellobiohydrolases in yeast

26

CBH expressed in yeast

mU

nits

/g d

ry c

ell w

eigh

t

1

10

100

1000

10000

CBH1 CBH1-4 CBHB CBH2 CBH1Aerobic

CBH1Anaerobic

•CBH1 requirements calculated based on ratio of CBH1 to other cellulase components in T. reesei cellulase mixtures to allow growth rate of 0.02 hr-1

2.6% of t.c.p.

Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl. 2007. Functional expression of

cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme

Microb. Technol. 40: 1291–1299.

Cellobiohydrolase production by yeast

Expression of cellobiohydrolases in yeast

27

Recent advances towards realizing CBP

28

Mascoma CorporationTechnical facilities, Lebanon, NH, USA

(www.mascoma.com)

Leading Investment, Unprecedented Focus on CBP

Technical Focus: Overcoming the biomass recalcitrance barrier and enablingthe emergence of a cellulosic biofuels industry via pioneering CBP technology integrated with advanced pretreatment

Partners in Mascoma’s CBP Organism Development Effort

• Dartmouth College

• University of Stellenbosch

• VTT • BioEnergy Science Center

Three Platforms

1. T. saccharolyticum, thermophilic bacterium able to use non-glucose sugars2. C. thermocellum, thermophilic cellulolytic bacterium3. Yeast engineered to utilize cellulose and ferment glucose and xylose

• Department of Energy

Multiple chances to succeed near-term & long-term29

Subsequent expression of cellobiohydrolases!

12% SDS-PAGE, silver staining

170 KDa130 KDa

95 KDa

72 KDa

55 KDa

43 KDa

34 KDa

26 KDa

30

Mascoma Cellulolytic Yeast

Mar, 2007 to Dec, 2008: >3,000-fold improvement in expression levels

Cellulase expression in Mascoma Yeast(robust C5/C6 fermenting) vs Time

T. reesei cellulase

in yeast

(Reinikainen

et al., 1992)

T. reesei cellulase

in Mascoma Yeast

(March '07)

Proprietary

cellulase in

Mascoma Yeast

(March '08)

Cellulase

expression

in Mascoma yeast

(October '08)

Cellulase expression Time-line

Cell

ula

se (

mg

/g D

CW

)

0.62 0.03 2.413

100

20

40

60

80

100

120Total cellulase (mg/g)

0Cellulase

expression

in Mascoma yeast

(December '08)

31

Enzyme Reduction on Hardwood

Equivalent performance with 2.5-fold less added enzyme

Further reduction likely

Mascoma CBP Strain (robust C5/C6 fermenting yeast) + 22% w/w unwashed Pretreated Hardwood + Commercial cellulase

0

5

10

15

20

25

30

35

40

45

0 20 40 60 80 100 120 140 160

Fermentation Time (hours)

Eth

an

ol (g

/L)

Mascoma non-cellulolytic yeast + baseline

commercial cellulase

Mascoma cellulolytic yeast + only 40% of

baseline commercial cellulase

32

Conversion of Paper Sludge to Ethanol: Proof of CBP Concept

85% cellulose conversion with production of recoverable ethanol with no added cellulase!

Mascoma CBP Yeast, no added cellulase

Mascoma non-CBP Yeast, w/10 FPU loadingcellulases

Non-CBP Yeast

Mascoma CBP technology on 18% w/w paper sludge (1 mg/g TS b-glucosidase and 1 mg/g TS xylanase added)

0

10

20

30

40

50

0 20 40 60 80 100 120 140

Fermentation Time (hours)

Eth

an

ol Y

ield

(g

/L)

33

Rome, NY Pilot & Demonstration Plant

January 2008

November 200834

35

Shaunita Rose

Riaan den Haan

Acknowledgements

Danie la Grange

Stellenbosch University, South Africa

Dartmouth College, USA

Ronél van Rooyen

John McBride Lee Lynd

VTT, Finland

Marja Ilmen Merja Penttilä

36

Microbiology

Chem EngProc Eng

Chair of Energy Research : Biofuels (members)

37

Cellulosics biofuels production value chain:

1. The CoER : Biofuels positions itself in the conversion technologies, but acknowledges the importance of establishing the whole value chain.

2. These includes both biochemical and thermochemical processes and integration of the processes if applicable

Technologies for Cellulose Conversion

Primary

Biomass

Production

Biomass

Transport

Biomass

Primary

Processing

Biomass

Conversion

to Biofuels

and By-

products

Product

Distribution

& Marketing

38

BiologicalProcessing• Pretreatment• Fermentation• Separation

Non-BiologicalProcessing

• Gasification• Fast pyrolysis• Power generation• Synthesis & separation

Biomass Biorefinery Concept

Steam

Process Power

Ethanol

CellulosicBiomass

Biologically-derivedChemicals (potentially several)

Thermochemically-derivedChemicals(potentially several)

Animal feed

Exported Power/BiofuelsResidues

Technologies for Cellulose Conversion

Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third

Chapter? South African Journal of Science 99: 499 – 507.

39

Biomass potential of Africa at large

Ratio of the energy content of the biomass on abandoned agriculture lands relative to the current primary energy demand at the country level. The energy content of biomass is assumed to be 20 kJ g−1. Source: Campbell et al. (2008)

40

South Africa’s potential:Renewable biomass available

1. ResiduesAgriculturalMaize stover 6.7 Mt/a (118 PJ/a)Sugar cane bagasse 3.3 Mt/a (58 PJ/a)Wheat straw 1.6 Mt/a (28 PJ/a)Sunflower stalks 0.6 Mt/a (11 PJ/a)Agricultural subtotal 12.3 Mt/a (214 PJ/a)Forest industryLeft in forest 4.0 Mt/a (69 PJ/a)Saw mill residue 0.9 Mt/a (16 PJ/a)Paper & board mill sludge 0.1 Mt/a (2 PJ/a)Forest industry subtotal 5.0 Mt/a (87 PJ/a)

2. Energy crops From 10% of available land 67 Mt/a (1 171 PJ/a)

(Marrison and Larson, 1996)3. Invasive plant species 8.7 Mt (151 PJ)

Total, annual basis 93 Mt/a (1 622 PJ/a)Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third

Chapter? South African Journal of Science 99: 499 – 507.

Tot

al

Ene

rgy

crop

s

Bur

ned

gras

ses

41

South Africa’s potential:Biofuels production

Maize to Ethanol = 430 L/ton Biomass to ethanol = 280 L/ton

Biomass to liquid (BtL) = 570 L/tonBiomass to upgraded bio-oils = 310 L/ton

BtL (50%)

0

5000

10000

15000

20000

25000

30000

Diesel

Ethanol

Petrol

Volum

es

in M

L

Mai

ze

For

est

bio

was

te

Inva

sive

plan

ts

Sub

tota

l

Cur

r fu

el

Str

ategy

targ

et

Agr

icul

tbio

was

te

Upgraded bio-oil (70% of residue)

(70% of residue)

42

Construction of cellulolytic S. cerevisiae

Thank you!