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Metabolic Engineering IX
Proceedings
Summer 6-7-2012
Engineering of Metabolic Pathways and Global
Regulators of Yarrowia lipolytica to Produce High
Value Commercial Products
Ethal Jackson
Du Pont
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Ethal Jackson, "Engineering of Metabolic Pathways and Global Regulators of Yarrowia lipolytica to Produce High Value Commercial
Products" in "Metabolic Engineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium
Series, (2013). http://dc.engconfintl.org/metabolic_ix/18
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Engineering of Metabolic Pathways
and Global Regulators of Yarrowia
lipolytica to Produce High Value
Commercial Products
Ethel Jackson
CR&D, E.I. du Pont de Nemours and Company, USA
Metabolic Engineering IX, Biarritz, France 2012
2
Metabolic Engineering of Yarrowia
Yarrowia::
Land--based Renewable Source of OmegaLand
Omega-3
Current Wild Harvest of Ocean Fish Unsustainable
Future Renewable LandLand-based Fermentation
3
Essential OmegaOmega-3 Fatty Acids: EPA and DHA
• EPA & DHA required in diet of humans & animals
Total Omega-3 market ~$7B, growing 10-12%/yr.
Markets include aquaculture, human nutrition,
pharmaceuticals, animal feed, pet food
• Primary source is wild-caught ocean
fish
Only synthesized in nature by plankton and algae
Small amount DHA from algae fermentation
• Critical for salmon aquaculture
85% of world market for fish oil
• No highly productive land-based source of EPA available
4
DuPont’s OmegaOmega-3 program
Develop a clean and sustainable source of
Omega-3 and Omega-6 fatty acids by fermentation
Approach: Metabolically engineered oleaginous yeast to
produce EPA and/or DHA by fermentation.
Goal:
Sugar
Yarrowia lipolytica
Omega-3 oil
O
OH
Eicosapentaenoic Acid (EPA)
5
TOPICS
•
Yarrowia lipolytica – good production host for
products of metabolic engineering
•
Omega-3 metabolic engineering
•
Omega-3 fermentation process
•
First commercial products
•
Lessons Learned
•
Summary
6
Yarrowia lipolytica
lipolytica:: A Good Production Host
•
Safe – FDA approved for food-grade citric acid
production
•
Good Fermentation Organism
- Robust growth
- High oil content
- Growth on Sugars
•
Oil body
Conventional strain breeding
- Mating types
•
Useful molecular genetic tools
Oleaginous Yarrowia
7
Development of a Comprehensive Genetic
Toolbox for Yarrowia lipolytica
•
Genome Sequence Information available
21 Mb; 6 chromosome; 6650 ORFs
•
Transformation System
Stable integration into genome or replicating plasmids
•
Selectable Markers
No antibiotic resistance genes needed
•
Strategy for Chromosomal Integration of Multiple Foreign Genes
•
Genetic Tools Based on Homologous Recombination
Targeted gene deletions
Targeted gene integrations
•
Genetic Tools Based on Non-Homologous End Joining
Random gene disruptions
Random gene integrations
•
Method for Identifying the Chromosomal Locations of Integrated Genes
8
Metabolic Engineering of Yarrowia for
Production of OmegaOmega-3 & Omega
Omega--6 Fatty Acids
Oleic Acid
[C18:1]
∆9D
Stearic Acid
[C18:0]
C16/18E
Palmitate
[C16:0]
C14/16E
Myristic Acid
[C14:0]
∆12D
Linoleic Acid
[LA, C18:2]
∆9E
20:2 (EDA)
∆8D
20:3 (DGLA)
∆5D
20:4 (ARA)
∆17D
∆17D
∆15D
18:3 (ALA)
∆9E
20:3 (ETrA)
∆8D
20:4 (ETA)
∆5D
20:5 (EPA)
(EPA)
Eicosapentaenoic Acid
Yarrowia native pathway
Engineered pathway options
9
Strategies to Build an EPA Production Strain
• Build an efficient EPA biosynthetic pathway
- Use of different strong promototers
- Codon-optimization of heterologous genes
- Multiple copies of structural genes
- Focus on limiting steps
- Push and pull carbon flux
• Screen for high oil and EPA productivity
• Eliminate fatty acid β-oxidation and increase oil content
- Modification of Peroxisome
- Generate mutants with key enzymes of β-oxidation knocked-out
• Control fatty acid transportation
- Fine regulation of acyltransferases
- Direct fatty acid flux for increased EPA productivity
• Manipulating global regulators
- Alter nitrogen control of lipid synthesis by Snf1 mutation
10
Metabolic Engineering
Generation of The First Commercial Production Strain
Each Step: 1,500 GC
ATCC 20362
Wild typeY. lipolytica
Y2224
Y4001
Y4001U1
Y4036
Y4036U
Y4128U3
Y4217
Integrate multiple genes into host’s genome by
homologous & non-homologous recombination
Y4070
Y4217U
Y4086
Y4259
Y4086U1
Y4259U2
Y4128
Y4305
56% EPA
Y4305 (56% EPA)
15 Steps, 8 Generations
32 copies of 9 different genes
No antibiotic marker
11
EPA/Oil Biosynthesis
A Complex Process in Engineered Yarrowia Cell
Glucose
Pentose phosphate shunt
NADPH
ER
EPA-CoA
Pyruvate
Acyl (C16)-CoA
TAGs
(Oil)
EPA/TAGs
X
Fatty Acids
Acetyl-CoA
Acetyl-CoA
Oxaloacetate
Citrate
Lipid
Body
Fatty Acids
Malonyl-CoA
Citrate
Acetyl-CoA
Oxaloacetate
Citrate
Isocitrate
Oxaloacetate
Malate
Fumarate
α-Ketoglutarate
Succinate
Malate
Acetyl
-CoA
Glyoxylate
Peroxisome
Mitochondrion
Cytoplasm
Isocitrate
Succinate
12
Pex10 Gene Function
• Member of peroxin class of proteins involved in peroxisome biogenesis
• Identified Pex10 knockout that blocked β-oxidation pathway (key to
keeping oil and EPA high)
Lipid Body
EPA/TAGs
Fatty Acids
X
Fatty Acids
Acetyl-CoA
Oxaloacetate
Citrate
Acetyl-CoA
Malate
P
M
N
NE
CW
V
peroxisome
Electron micrograph
mitochondrion
of Yarrowia lipolytica
nucleus
nuclear envelope
cell wall
vacuole
Isocitrate
Glyoxylate
Peroxisome
Succinate
13
Peroxisome Mutations Increase EPA Titer
Knock out of the Pex10 gene resulted in an EPA titer increase >2X
EPA% of FAME
50
Pex10-Pex10
40
38
30
20
PEX10+
17
10
0
Y4127
Y4128
Strains
14
% of Total Lipids
Control the Carbon Flux for
Enhanced EPA Production
60
Pushing
50
Pulling
56
40
30
C18: <24%
20
10
3
0
18
C16: 4%
1
1
5
2
3
2
Fatty Acids
1
0.5
1.5
EPA
TAG
Sink
15
Searching for A Global Regulator
of Lipid Metabolism in Yarrowia
Glucose
Pentose phosphate shunt
NADPH
ER
EPA-CoA
Pyruvate
TAGs
(Oil)
Acyl (C14)-CoA
Acetyl-CoA
Citrate
Malonyl-CoA
Citrate
Isocitrate
Oxaloacetate
Malate
Fumarate
Lipid
Body
Fatty Acids
Fatty Acids
Acetyl-CoA
Oxaloacetate
EPA/TAGs
Acetyl-CoA
Oxaloacetate
Acetyl-CoA
Malate
a-Ketoglutarate
Isocitrate
Glyoxylate
Succinate
Mitochondrion
Citrate
Global
Regulator?
Cytoplasm
Peroxisome
Succinate
16
Yarrowia SNF1:
SNF1:
Global Regulator of Lipid Accumulation
Lipid %DCW
snf1∆
High
Control
Medium
Low
0
0
1
2
3
4
5
Days in oleaginous phase
17
Three DAG Acyltransferases for Oil Biosynthesis
E
E
P
G3P
R
E
P
LPA
G3PAT
R
R
P
E
PA
R
R
TAG biosynthesis
DAG
LPAAT
TAG
PDAT
PC-DAG
exchange
Glycerol
E
R
R
R
DGAT2
DGAT1
CPT1
EPA biosynthesis
PC pool
18:1
18:2
20:2
20:3
20:4
20:5
E
.R
R
PC
LPCAT/
FAS
LPAAT
PC recycling
EPA
16:0
18:0
18:1
18:2
20:5
20:2
PEX
β-oxidation
CoA pool
GPAT
DGAT
CPT
LPAAT
LPCAT
PDAT
Glycerol-3-phosphate acyltransferase
Diacylglycerol acyltransferase
CDP-choline:diacylglycerol cholinephosphotransferase
Lysophosphatidic Acid Acyltransferase
acyl-CoA::lyso-phosphatidylcholine acltransferase
phopholipid::diacylglycerol acyltransferase
18
Oil Content In Y.
Y. lipolytica DAG AT Mutants
Oil content,
TF % dcw (% WT)
TAG
ATCC
90812
100
100
85
90
83
80
70
60
49
50
39
40
30
23
20
13
10
2
0
Oil
Std
Std
WT
S
∆
d1
S-DT1
∆S-DT2
d2
∆p
S-PT
Single mutants
∆d1
S-DT1
∆d2
S-DT2
∆d1
S-DT1
∆p
S-PT
∆d2
S-DT2
∆p
S-PT
Double mutants
∆d1
S-DT1
∆d2
S-DT2
∆p
S-PT
19
Total Lipids as Dry Cell Weight (%)
Over--Expression of DGAT and PDAT Improves Oil
Over
70
60
58
50
41
40
32
30
20
10
0
Y9502
Y9502
+
PDAT
Y9502
+
DGAT2
Strain Comparison
20
Omega-3 Fermentation Research:
OmegaStrain Evaluation and Process Development
Cell Density
Lipid Content (% DCW)
Excreted
Byproducts
EPA Content (% DCW)
Lipid and EPA content (% DCW)
Cell Density and Byproducts (g/L)
Glucose feed
NH4 +
NH
4
+
Fermentation Time
Air
21
Process Flow for Strain Evaluation by Fermentation
50,000
1,500
600
Micro-24
Bioreactor
200
* Numbers refer to the strains tested
9
Air
22
Micro--24 Bioreactor vs. LabMicro
Lab-Scale Fermentor
Vessel/
Reactor
24
reactors,
3-7 mL
Single
reactor,
2-10 L
Controllability
Experimental Data
Individual
Online process- T,
pH, pO2
T, pH, pO2
T, pH, pO2
feeding
Final point – titer,
rate, yield
Online process- T,
pH, pO2, feeding
Time course –
titer, rate, yield
Work Capacity
1000 individual
experiments
/year/person
40 individual
experiments
/year/person
23
Micro-24 Bioreactor
Micro- bridge between shake flask and fermentor
70
60
Strain #1
Flask
mico-24
50
2-L fermentor
40
30
20
10
0
cell density
product#1
product #2
product #3
60
Strain #2
Flask
50
mico-24
40
2-L fermentor
30
20
10
0
cell density
product#1
product #2
product #3
24
Micro-24 Bioreactor
Micro- help identify the best strain and condition
Identify the best
production strain
Identify the best
fermentation conditions
25
Products: Current OmegaOmega-3 Market
Oil
Concentrates
Pharmaceuticals
Dietary
Supplements
Yarrowia
Biomass
Processed
Biomass
Extracted
Oil
Functional
Foods
Medical
Foods &
Pharma
Aquaculture
Terrestrial
Animal
Agriculture
Pet Foods
Industrial
Applications
Omega-3 market growing 12-15%/year
26
newharvest™
•
•
•
•
Vegetarian, renewable source - made from yeast,
not fish
Highly concentrated for convenient dosage
Avoids fishy taste and “burps”
No cholesterol. No PCBs.
www.newharvest.com
Strategic
Partners
Active
Ingredient
Natural
Marketer
Channel
Partner
27
Verlasso’s Harmoniously Raised Fish (Salmon)
• A new category of premium farmed salmon
that is beyond sustainable
farming practices
Raised on a diet rich in omega-3
Fish in Fish out ratio 1 to 1 vs. 4 to 1
One of the lowest pen densities
No hormones or preventative antibiotics
• Partnership between DuPont and
AquaChile
• Validated through extensive market research
National Market Test in 5 US Cities
Marketing Support
Building the Brand
Partnership with the retailers
27
28
MY LESSONS LEARNED
• Project choice: make sure the view is worth the climb
- No well-established chemical process
- Multiple different products possible
• Collaboration is key: integrate metabolic engineering and
fermentation engineering research
- Strain, construction and process research should be integrated
from the start
- Results determine production strain attributes and
process parameters
• Cost is king
- Capital investment barrier – must optimize fermentation productivity
- Raw material cost
• There’s no substitute for good science
29
Summary
• Yarrowia lipolytica is a useful host for metabolic engineering
– Powerful genetic tool box for metabolic engineering
– Robust, safe fermentation production organism
– Engineered production strains are stable without antibiotic selection
• DuPont’s process is a sustainable alternative for Omega-3s
– Land-based fermentation from renewable resources at commercial scale
– Naturally contaminant free, no DHA
– Versatile technology enables production of tailored oil compositions, different
PUFA’s and multiple product applications
• Launched two commercial products
– NewHarvetTM – EPA Oil
– VerlassoTM Salmon – EPA Biomass
• DuPont will continue to drive lower costs and broaden the platform