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Engineering Conferences International ECI Digital Archives 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 Follow this and additional works at: http://dc.engconfintl.org/metabolic_ix Part of the Biomedical Engineering and Bioengineering Commons 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 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