1. 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

3. Distillation & dehydration
Ethanol production from starch
Thermostable
α-amylase
Alcohol
recovery
Glucoamylase
Yeast
Liquefaction
Saccharification
Fermentation
Distillation & dehydration
Grinding
Corn
Wheat
Triticale
Rye
Jet Cooker
>100ºC
>5 - 8 min
Storage
tank
Water
Slurry
tank
Secondary
Liquefaction
95ºC, ~90 min
Fuel
blending
DDGS

4. Distillation & dehydration
Ethanol production from starch
Alcohol
recovery
Saccharification
& Fermentation
Distillation & dehydration
Amylolytic
Yeast!
Water
Grinding
Maize
Wheat
Triticale
Rye
Slurry
tank
Storage
tank
Fuel
blending
DDGS

5. Introduction: starch CBP
-amylase
CH OH 2 O OH
-amylase
glucoamylase
Amylose
pullulanase isoamylase
CH OH 2 O OH
Amylopectin CH
-amylase
glucoamylase
-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/ A1

7. Results: Screening amylolytic genes
Soluble Starch
Raw Starch
Glucoamylase
(Soluble Starch)
(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

8. Results: Raw starch batch fermentations
0.05% Glucose
Inoculate with 0.3 g/L Dry Weight Cells
Weight loss – 0.94 g
EtOH production – 8.08 g/L
87.85% conversion

9. Results: Raw starch batch fermentations
0.05% Glucose
Inoculate with 20 g/L Wet Weight Cells
Max EtOH produced – 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. Introduction: Lignocellulose CBP
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
Biomass pretreatment
Enzyme production
Feedstock hydrolysis
CBP
Hexose fermentation (mainly glucose)
Pentose fermentation (mainly xylose)
ETHANOL

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

14. Results: CBH expression screening
CBH1 (modified)
CBH2
H. grisea cbh1
T. aurantiacus cbh1
T. emersonii cbh1
N. fischerii cbh1
P. janthinellum cbh1
G. zeae cbh1
N. haematococca cbh1
F. poae cbh1
As. terreus cbh1
P. chrysogenum cbh1
N. crassa cbh1
C. thermophilum cbh1
Ac. thermophilun cbh1
T. reesei cbh1
Tecbh1-TrCBM-C
Tecbh1-HgCBM-C
Tecbh1-CtCBM-C
Tecbh1-TrCBM-N
Tecbh1-TrCBM-N2
Tecbh1-TrCBM-C2
C. heterostrophus cbh2
G. zeae cbh2
I. lacteus cbh2
V. volvacea cbh2
Piromyces sp. cbh2
T. emersonii cbh2
T. reesei cbh2
C. lucknowense cbh2
A. cellulolyticus cbh2
C. 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. 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
% Avicel degradation μM MU released per minute

17. 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

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 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. 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
β-Glucosidase activity
U/mg DCW 10 20 30 40 50 60 70 80 90
100
Ref
Cel3A
Cel3A-SOD1
Cel3A-PSE1
Cel3A-PSE1/SOD1
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. 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

23. Thank you! Acknowledgments: Lee Lynd John McBride Elena Brevnova
Allan Froehlich
Alan Gilbert
Heidi Hau
Erin Wiswall
Hoowen Xu
Merja Penttilä
Marja Ilmen
Anu Koivula
Sanni Voutilainen
Emile van Zyl
Riaan den Haan
Marlin Mert
Danie La Grange
Maryna Saayman
Marko Viktor
Heinrich Kroukamp

24. Barriers to lignocellulose CBP with S. cerevisiae
Consumption of all major sugar constituents of biomass
High level expression of cellulases, especially cellobiohydrolases
Expression of the diverse enzymes required to hydrolyze biomass
Production of enzymes and consumption of sugars in toxic process conditions

25. Introduction: xylan CBP
Colins et al, 2005

26. Introduction: xylan CBP
Control
Xylanase
Xylanase/Xylo
Marker
48 h
72 h
136 h
Xylose
Xylobiose
Xylotrose
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
Time (hours)
Biomass (OD600)

27. Results: xylan CBP 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