Engineering of Metabolic Pathways and Global Regulators of Yarrowia Lipolytica to Produce High Value Commercial Products Ethal Jackson Du Pont

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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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Recommended Citation 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

This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Metabolic Engineering IX by an authorized administrator of ECI Digital Archives. For more information, please contact [email protected]. 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 : LandLand--basedbased Renewable Source of Omega--33Omega

Current Wild Harvest of Ocean Fish Unsustainable

Future Renewable LandLand--basedbased Fermentation 3

Essential OmegaOmega--33 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--33 program

Goal: 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.

Yarrowia lipolytica Sugar 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 : 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 Oleaginous Yarrowia

• Useful molecular genetic tools 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 forfor Production of OmegaOmega--33 & OmegaOmega--66 Fatty Acids

∆9D C E C E Oleic Acid Stearic Acid 16/18 Palmitate 14/16 Myristic Acid [C18:1] [C18:0] [C16:0] [C14:0]

∆12D

Linoleic Acid ∆∆∆9E ∆∆∆8D ∆∆∆5D [LA, C18:2] 20:2 (EDA) 20:3 (DGLA) 20:4 ( ARA ) ∆∆∆17D ∆15D ∆17D ∆∆∆9E ∆∆∆8D ∆5D 18:3 (ALA) 20:3 (ETrA) 20:4 (ETA) 20:520:5 (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 βββ-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 StrainStrain

Each Step: 1,500 GC

ATCC 20362 Y2224 Y4001 Y4001U1 Y4036 Y4036U Wild type Y. lipolytica Integrate multiple genes into host’s genome by homologous & non-homologous recombination

Y4070 Y4086 Y4086U1 Y4128 Y4128U3 Y4217

Y4217U Y4259 Y4259U2 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

Pentose phosphate shunt Glucose NADPH

ER EPA-CoA TAGs (Oil) EPA/TAGs Lipid Pyruvate Fatty Acids Acyl (C16)-CoA Body

Fatty Acids Acetyl-CoA X Malonyl-CoA Acetyl-CoA Acetyl-CoA

Oxaloacetate Citrate Citrate Oxaloacetate Citrate Isocitrate Malate Oxaloacetate Malate Acetyl Isocitrate ααα-Ketoglutarate -CoA Fumarate Glyoxylate Succinate Succinate Mitochondrion Peroxisome

Cytoplasm 12

Pex10 Gene Function

• Member of peroxin class of proteins involved in peroxisome biogenesis • Identified Pex10 knockout that blocked βββ-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 Isocitrate Glyoxylate P peroxisome Electron micrograph M mitochondrion Succinate N nucleus of Yarrowia lipolytica NE nuclear envelope Peroxisome CW cell wall V vacuole 13

Peroxisome Mutations Increase EPA Titer Knock out of the Pex10 gene resulted in an EPA titer increase >2X

40 Pex10Pex10-- 38 30

20 PEX10+ 17 10 EPA% ofof EPA% EPA% FAME FAME

0 Y4127 Y4128 Strains 14

Control the Carbon Flux for Enhanced EPA Production

60 Pushing Pulling 56 50

40 30 20 C18: <24% 18 10 C16: 4% % of Total Lipids Lipids of of Total Total % % 5 3 3 1 2 2 1.5 0 1 1 0.5 EPA TAG Fatty Acids Sink 15 Searching for A Global Regulator of Lipid Metabolism in Yarrowia

Pentose phosphate shunt Glucose NADPH

ER EPA-CoA TAGs EPA/TAGs (Oil) Lipid Pyruvate Fatty Acids Acyl (C14)-CoA Body

Fatty Acids Acetyl -CoA Malonyl-CoA Acetyl-CoA Acetyl-CoA

Oxaloacetate Citrate Citrate Oxaloacetate Citrate Isocitrate Acetyl-CoA Malate Oxaloacetate Malate Isocitrate a-Ketoglutarate Fumarate Glyoxylate Succinate Peroxisome Succinate Mitochondrion Global Regulator?

Cytoplasm 16 Yarrowia SNF1 :: Global Regulator of Lipid Accumulation

snf1 ∆ High

Medium Control

LipidLipid %DCW %DCW Low

0 0 1 2 3 4 5 Days in oleaginous phase 17 Three DAG Acyltransferases for Oil Biosynthesis

R R R R R R TAG biosynthesis R E P E P E P E E R G3PLPA PA DAG TAG

G3PAT LPAAT PDAT DGAT2 PC-DAG Glycerol exchange DGAT1 CPT1 EPA biosynthesis PC pool .R E R 18:1 18:2 20:2 20:3 20:4 20:5 PC

FAS LPCAT/ LPAAT PC recycling EPA 18:2 20:2 20:5 16:0 18:0 18:1 CoA pool

PEX GPAT Glycerol-3-phosphate acyltransferase DGAT Diacylglycerol acyltransferase βββ-oxidation CPT CDP-choline:diacylglycerol cholinephosphotransferase LPAAT Lysophosphatidic Acid Acyltransferase LPCAT acyl-CoA::lyso-phosphatidylcholine acltransferase PDAT phopholipid::diacylglycerol acyltransferase 18

Oil Content In Y . lipolytica DAG AT Mutants

TAG

100 100 90 85 83 80 70 60 49 50 39 40 30 23 20 13 Oil content,content, OilOil 10 2

TF % dcw (% (% WT) (% WT) dcwdcw% % TF TF 0

ATCC 90812 StdOil WTS ∆∆∆ S-DT1d1 ∆∆∆ S-DT2d2 S-PT∆∆∆p S-DT1∆∆∆d1 S-DT1∆∆∆d1 S-DT2∆∆∆d2 S-DT1∆∆∆d1 Std S-DT2∆∆∆d2 S-PT∆∆∆p S-PT∆∆∆p S-DT2∆∆∆d2 S-PT∆∆∆p Single mutants Double mutants 19

OverOver--ExpressionExpression of DGAT and PDAT Improves Oil

60 58

41 40

32 30

10 Total Lipids as Dry Cell Weight (%) (%) Cell Cell Weight Weight as as LipidsLipidsDry Dry Total Total 0 Y9502 Y9502 Y9502 + + PDAT DGAT2

Strain Comparison 20

OmegaOmega--33 Fermentation Research: Strain Evaluation and Process Development

Glucose feed Lipid (g/L)

Cell Density and EPA

Byproducts Byproducts Lipid Content (% DCW) content (% DCW) (% content and and Excreted Byproducts + + NH 4 EPA Content (% DCW) 4 Cell Density Cell

Fermentation Time

Air 21

Process Flow for Strain Evaluation by Fermentation

1,500 50,000

600

Micro-24 200 9 Air Bioreactor

* Numbers refer to the strains tested 22

MicroMicro--2424 Bioreactor vs. Lab--ScaleLab Scale Fermentor

Vessel/ Controllability Experimental Data Work Capacity Reactor

Online process- T, 24 Individual pH, pO2 1000 individual reactors, experiments T, pH, pO Final point – titer, 3-7 mL 2 /year/person rate, yield

Online process- T, Single 40 individual T, pH, pO 2 pH, pO2, feeding reactor, experiments feeding Time course – 2-10 L /year/person titer, rate, yield 23

MicroMicro--2424 Bioreactor -- bridge between shake flask and fermentor

70 Strain #1 Flask 60 mico-24 50 2-L fermentor 40

0 cell density product#1 product #2 product #3

60 Strain #2 Flask 50 mico-24 40 2-L fermentor

0 cell density product#1 product #2 product #3 24

MicroMicro--2424 Bioreactor -- help identify the best strain and condition

Identify the best production strain

Identify the best fermentation conditions 25 Products: Current OmegaOmega--33 Market

Oil Pharma- Concentrates ceuticals

Dietary Supplements

Yarrowia Processed Extracted Functional Biomass Biomass Oil Foods

Medical Foods & Pharma

Aquaculture

Terrestrial Animal Agriculture

Pet Foods

Industrial Applications

Omega-3 market growing 12-15%/year 26

new harvest ™ • 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 Natural Channel Ingredient Marketer 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 – NewHarvet TM – EPA Oil – Verlasso TM Salmon – EPA Biomass

• DuPont will continue to drive lower costs and broaden the platform