engineering yeasts for next generation ethanol production

27
Engineering yeasts for next generation ethanol production Riaan den Haan 1 , D.C. la Grange 1 , M. Mert 1 , H. Kroukamp 1 , M. Saayman 1 , M. Viktor 1 , J.E. McBride 3 , L.R. Lynd 3 , M. Ilmen 4 , M. Penttilä 4 , J.F. Görgens 2 , M. Bloom 1 , W.H. van Zyl 1 (1) Depts. of Microbiology, and (2) Process Engineering Stellenbosch University, South Africa (3) Mascoma Corporation, Lebanon, NH (4) VTT Technical Research Centre of Finland

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Engineering yeasts for next generation ethanol production. Riaan den Haan 1 , D . C. la Grange 1 , M. Mert 1 , H . Kroukamp 1 , M. Saayman 1 , M. Viktor 1 , J.E. McBride 3 , L.R. Lynd 3 , M. Ilmen 4 , M. Penttilä 4 , J.F. Görgens 2 , M. Bloom 1 , W.H. van Zyl 1 - PowerPoint PPT Presentation

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Page 1: Engineering yeasts for next generation ethanol production

Engineering yeasts for next generation ethanol production 

Riaan den Haan1, D.C. la Grange1, M. Mert1, H. Kroukamp1, M. Saayman1, M. Viktor1, J.E. McBride3, L.R. Lynd3, M. Ilmen4, M. Penttilä4, J.F. Görgens2, M. Bloom1,

W.H. van Zyl1 

(1) Depts. of Microbiology, and (2) Process Engineering Stellenbosch University, South Africa (3) Mascoma Corporation, Lebanon, NH(4) VTT Technical Research Centre of Finland

Page 2: Engineering yeasts for next generation ethanol production

2

Introduction

• Biofuels such as ethanol have gained significant interest due to environmental concerns and issues such as energy security - resulting in the current first generation ethanol market

• Most of the ethanol produced worldwide is produced from starch• The development of a yeast that converts raw starch to ethanol in 

one step (CBP) could yield significant cost reductions in 1st generation bioethanol production from corn starch

• 2nd Generation bioethanol produced from lignocellulosic biomass has great benefits in terms of energy balance, food security,  etc.

• Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies

Page 3: Engineering yeasts for next generation ethanol production

Ethanol production from starch

DDGS

Liquefaction

SecondaryLiquefaction

95ºC, ~90 min

Grinding

CornWheat

TriticaleRye

Water

Slurrytank

Jet Cooker>100ºC

>5 - 8 min

Thermostableα-amylase Glucoamylase Yeast Alcohol

recovery

Fuelblending

Saccharification Fermentation Distillation & dehydration

Storagetank

3

Page 4: Engineering yeasts for next generation ethanol production

DDGS

Storagetank

Distillation & dehydration

Alcoholrecovery

FuelblendingAmylolytic

Yeast!

Water

Grinding

MaizeWheat

TriticaleRye

Slurrytank

Water

Slurrytank

Saccharification& Fermentation

Ethanol production from starch

4

Page 5: Engineering yeasts for next generation ethanol production

5

Introduction: starch CBP

-amylaseCH OH2

O

OH

OH

CH OH2O

OH

OH

CH OH 2O

OH

OH

CH OH2O

OH

OH

CH OH2O

OH

OH

OO O OOH O

-amylaseglucoamylase

Amylose

pullulanase isoamylase

CH OH2O

OH

OH

CH OH2O

OH

OH

CH OH2O

OH

OH

CH OH2O

OH

OH

OO O O

OH

Amylopectin

CH OH2O

OH

OH

CH OH2O

OH

OH

CH 2O

OH

OH

O O O

CH OH2O

OH

OH

CH OH2O

OH

OH

OOH O

-amylaseglucoamylase

-amylase

Page 6: Engineering yeasts for next generation ethanol production

6

Results: Screening amylolytic genes

• Glucoamylases Aspergillus awamori (glaA) Rhizopus oryzae (glaR) Humicola grisea thermoidea (gla1) Saccharomycopsis fibuligera (gluI) Thermomyces lanuginosis (TLG)

• α-Amylases Aspergillus oryzae (AMYLIII) Lipomyces kononenkoae (LKA) Saccharomycopsis fibuligera (SFA)

• Genes were cloned into episomal plasmids and activity assayed in lab strains

• Best candidates were cloned into vectors to allow multicopy chromosomal integration in industrial yeast strains

Patent nr. WO 2011/128712 A1

Page 7: Engineering yeasts for next generation ethanol production

7

Results: Screening amylolytic genes

Glucoamylase(Soluble Starch)

Glucoamylase(Raw Starch)

Alpha-amylase

AGA/AOA 0.52 U/ml 0.047 U/ml 0.15 U/ml

TLG/SFA 1.187 U/ml 0.576 U/ml 0.812 U/ml

Soluble Starch Raw Starch

Page 8: Engineering yeasts for next generation ethanol production

Results: Raw starch batch fermentations

• 2% Raw Starch• 0.05% Glucose• Inoculate with 0.3 g/L  Dry Weight Cells

0 24 48 72 96 120144168192216240264288312336360384408

0

1

2

3

4

5

6

7

8

9

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

SFA/TLG (EtOH) MH1000 (EtOH)SFA/TLG (Weight Loss) MH1000 (Weight Loss)

TIME (h)

EtO

H (g

/L)

Wei

ght L

oss (

g)

Weight loss – 0.94 gEtOH production – 8.08 g/L87.85% conversion 

Page 9: Engineering yeasts for next generation ethanol production

Results: Raw starch batch fermentations

• 10% Raw Starch• 0.05% Glucose• Inoculate with 20 g/L  Wet Weight Cells

0 48 96 144 192 240 288 336 384 4320

10

20

30

40

50

60

MH1000Stargen-10mg/gMH1000-10mg/gSFA/TLGSFA/TLG-2.5mg/gSFA/TLG-5mg/gSFA/TLG-7.5mg/gSFA/TLG-10mg/g

Time (h)

Etha

nol (

g/L)

Max EtOH produced – 56.596 g/L , thus ~95% conversion 

Page 10: Engineering yeasts for next generation ethanol production

10

Discussion: Starch CBP

• Raw starch conversion was possible with no added enzymes or with reduced enzyme loadings; fermentation times must be improved

• Current and future prospects:• Screen yeast strains with superior fermentation capacities• Screen a wider array of α-amylase encoding genes• Create strain with higher copy numbers of genes

Page 11: Engineering yeasts for next generation ethanol production

11

Introduction: Lignocellulose CBP

• Lignocellulosic biomass consisting of mainly lignin, cellulose and hemicellulose, is an abundant, renewable & sustainable source of fuels etc.

• The main barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose

Page 12: Engineering yeasts for next generation ethanol production

12

• Conversion of biomass to ethanol is a complex process and advances are required at several stages for efficiency and cost effectiveness

• The CBP microbe thus converts pretreated biomass directly to ethanol

• “Widely considered to be the ultimate low-cost configuration of cellulose hydrolysis and fermentation” – DOE/USDA Joint research Agenda

• No ideal CBP organisms exists

Enzyme production

Feedstock hydrolysis

Hexose fermentation(mainly glucose)

Pentose fermentation(mainly xylose)

Biomass pretreatment

ETHANOL

CBP

Introduction: Lignocellulose CBP

Page 13: Engineering yeasts for next generation ethanol production

13

Elements required for CBP with S. cerevisiae

• EG and BGL expression  successful

• CBH expression problematic• This study: screen several 

CBH candidates for expressibility in S. cerevisiae

• Genes were cloned into episomal plasmids and activity assayed in lab strains

Page 14: Engineering yeasts for next generation ethanol production

14

Results: CBH expression screening

CBH1 CBH1 (modified) CBH2H. grisea cbh1T. aurantiacus cbh1 T. emersonii cbh1 N. fischerii cbh1 P. janthinellum cbh1 G. zeae cbh1N. haematococca cbh1F. poae cbh1As. terreus cbh1P. chrysogenum cbh1 N. crassa cbh1C. thermophilum cbh1 Ac. thermophilun cbh1 T. reesei cbh1

Tecbh1-TrCBM-CTecbh1-HgCBM-CTecbh1-CtCBM-CTecbh1-TrCBM-NTecbh1-TrCBM-N2Tecbh1-TrCBM-C2

C. heterostrophus cbh2G. zeae cbh2I. lacteus cbh2 V. volvacea cbh2 Piromyces sp. cbh2 T. emersonii cbh2T. reesei cbh2C. lucknowense cbh2A. cellulolyticus cbh2C. thermophilum cbh2 

Page 15: Engineering yeasts for next generation ethanol production

15

Results: CBH expression

• Growth of strains in minimal media to examine secreted proteins:• N-glycosylation observed• Large variation in protein levels produced• Protein levels not necessarily reflecting activity 

levels – not all produced protein active• Candidate producing superior levels identified

Page 16: Engineering yeasts for next generation ethanol production

16

% Avicel degradatio

n       μM

 MU re

leased per minute

0

5

10

15

20

25

30

35

0

5

10

15

20

25 24 Hours48 Hours

Results: CBH1 & CBH2 co-expression

• Several well expressed CBH1s and CBH2s  combined in the same strain

• Though lower levels of either CBHs were observed in co-expression, higher levels of crystalline cellulose hydrolysis resulted – likely due to synergy

Page 17: Engineering yeasts for next generation ethanol production

17

Figure 12

3.5No added BGL External BGL added

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Cello

bios

eg/

L

0

0.5

1

1.5

2

2.5

3

Etha

nol p

rodu

ced

(g/L

)

48 H96 H168 H

Results: Avicel conversion

• To test conversion of avicel to ethanol by CBH producing yeasts:• Strains cultured in YPD• 2% Avicel added• Novozyme 188 (BGL) added

• Cultures producing CBHs converted Avicel to cellobiose in the absence of BGL

• Cultures producing CBHs converted Avicel to ethanol in the presence of BGL

• ~30% of theoretical maximum

Figure 12

3.5No added BGL External BGL added

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Cello

bios

eg/

L

0

0.5

1

1.5

2

2.5

3

Etha

nol p

rodu

ced

(g/L

)

48 H96 H168 H

Page 18: Engineering yeasts for next generation ethanol production

18

Discussion: cellulose CBP

• High level secretion of exoglucanases is required for crystalline cellulose utilization - major hurdle in CBP yeast development

• Indentified gene candidates compatible with expression in yeast  T.e.CBH1 and its T.r.CBM attached derivative yielded 100-200 

mg/liter in shake flasks and ~300 mg/liter in HCD conditions The highest CBH level secreted, ~1 g/liter C.l.CBH2b (~4% tcp) 

exceeded any previous reports on CBH production in S. cerevisiae• Thus S. cerevisiae is capable of secreting CBHs at high levels that 

compare well with the highest heterologous protein production levels described for S. cerevisiae

Page 19: Engineering yeasts for next generation ethanol production

19

Introduction: strain engineering

• The innate low secretion capacity of S. cerevisiae, even when compared to other yeast species represents a drawback in its development as a CBP organism

• Over-expression of genes encoding foldases, chaperones or other parts of the secretion pathways or knockouts of genes encoding negative regulators have been shown to increase secretion capacity in fungi

• We aimed to improve the secretion of hydrolases by S. cerevisiae through strain engineering

Page 20: Engineering yeasts for next generation ethanol production

20

β-G

luco

sida

se a

ctivi

tyU

/mg

DCW

0

10

20

30

40

50

60

70

80

90

100

RefCel3A

Cel3A-SOD1

Cel3A-PSE1

Cel3A-PSE1/S

OD1

Results: strain engineering

• Enhanced secretion of native proteins was reported when the protein secretion enhancer 1 protein (PSE1) of S. cerevisiae was overexpressed

• Pse1 was overproduced in a strain expressing S.f.bgl1

• Pse1 overproduction yielded an almost 4-fold improvement of BGL activity

• Sod1 co-overproduction yielded a further ~20% increase

• The effect of these genes were reporter protein specific as less effect was seen on T.r.Cel7B and N.p.Cel6A

Page 21: Engineering yeasts for next generation ethanol production

21

0

1

2

3

4

5

6

Reference Parental Δmnn10 Δmnn11

Rel

ativ

e in

vert

ase

activi

ty p

er D

CW

Constructed Cel7A secreting strains

Relative invertaseactivities of constructed strains

24h48h72h

0

0.5

1

1.5

2

2.5

Reference Parental Δmnn10 Δmnn11

Rel

ativ

e ce

llobio

hydro

lase

Iac

tivi

ty p

er D

CW

Constructed Cel7A secreting strains

Relative Cel7A activities of constructed strains

0

1

2

3

4

5

6

Reference Parental Δmnn10 Δmnn11 Rel

ativ

e in

vert

ase

activi

ty p

er D

CW

Constructed Cel7A secreting strains

Relative invertaseactivities of constructed strains

24h48h72h

0

0.5

1

1.5

2

2.5

Reference Parental Δmnn10 Δmnn11

Rel

ativ

e ce

llobio

hydro

lase

Iac

tivi

ty p

er D

CW

Constructed Cel7A secreting strains

Relative Cel7A activities of constructed strains

Results: strain engineering• Knock-out of MNN-genes in S. cerevisiae have been shown to have a 

general effect on secretion enhancement

• Two N-glycosylation mutants, ΔMNN10 and ΔMNN11 had significantly higher extracellular enzyme activity for both Cel7A and invertase

• Changes in cell wall structure or the degree of enzyme glycosylation may have contributed to this enhanced secretion phenotype

Page 22: Engineering yeasts for next generation ethanol production

22

Conclusion

• Fermentation of raw starch by recombinant S. cerevisiae strains was demonstrated without the addition of commercial enzymes

• S. cerevisiae was shown to be capable of expression of levels of CBHs that would overcome the barrier of sufficiency for conversion of cellulosic biomass to ethanol Simultaneous expression of CBHs with EG and β-glucosidase 

enabled S. cerevisiae to directly convert cellulosic substrates to ethanol and to grow on cellulose under CBP conditions

• S. cerevisiae strains could be manipulated to allow improved secretion of hydrolase enzymes

• Combining optimal gene candidates in enhanced host strains will lead to improved strains for CBP applications

Page 23: Engineering yeasts for next generation ethanol production

23

Acknowledgments: Lee LyndJohn McBrideElena BrevnovaAllan FroehlichAlan GilbertHeidi HauErin WiswallHoowen Xu

Merja PenttiläMarja IlmenAnu Koivula Sanni Voutilainen

Emile van ZylRiaan den HaanMarlin MertDanie La GrangeMaryna SaaymanMarko ViktorHeinrich Kroukamp

Thank you!

Page 24: Engineering yeasts for next generation ethanol production

24

Barriers to lignocellulose CBP with S. cerevisiae

1. Consumption of all major sugar constituents of biomass2. High level expression of cellulases, especially 

cellobiohydrolases3. Expression of the diverse enzymes required to hydrolyze 

biomass4. Production of enzymes and consumption of sugars in 

toxic process conditions

Page 25: Engineering yeasts for next generation ethanol production

25

Introduction: xylan CBP

Colins et al, 2005

Page 26: Engineering yeasts for next generation ethanol production

26

Control

Xylanase

Xylanase/Xylo

Marker

Control

Xylanase

Xylanase/Xylo

Marker

Control

Xylanase

Xylanase/Xylo

Marker

48 h 72 h 136 h

XyloseXylobioseXylotrose

Time (hours)

Biom

ass (OD

600)

0   1    2   3    4   5   6   7   8   9  10

      24    48      72       96     120    144    168    192   216

Introduction: xylan CBP• Xylanase & xylosidase

T. reesei xyn2 and A. niger xlnD Demonstrated degradation of 

birchwood xylan to D-xylose

• Xylose isomerase Synthetic codon optimised        

B. thetaiotaomicron xylA Xylose used as sole carbon 

source

• Construct strain YMX1• xylA integrated• xyn2 & xlnD episomal

Page 27: Engineering yeasts for next generation ethanol production

Results: xylan CBP

0 5 10 15 20 25 30050100150200250300350400450

Biomass

YMX1 YMXR DLG56

Time (days)

Cell

dens

ity

(101

0 ce

lls p

er m

l)• YP-Xylan (50 g/L beechwood)• YMX1 strain pre-culture grown on xylose• 10% innoculum

• Growth of S. cerevisiae on xylan as sole carbohydrate was achieved but growth rate has to be improved