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
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
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
DDGS
Storagetank
Distillation & dehydration
Alcoholrecovery
FuelblendingAmylolytic
Yeast!
Water
Grinding
MaizeWheat
TriticaleRye
Slurrytank
Water
Slurrytank
Saccharification& Fermentation
Ethanol production from starch
4
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
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
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
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
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
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
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
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
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
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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
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
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
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
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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
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
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
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
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
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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!
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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
25
Introduction: xylan CBP
Colins et al, 2005
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
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