Nigel Mouncey
Transcript of Nigel Mouncey
Industrial Biotechnology at Dow AgroSciences
Nigel J. Mouncey, PhDAssociate R&D Director,Bioengineering and Bioprocessing R&D Leader
BIO World Congress on Industrial BiotechnologyPhiladelphia, May 15 2014
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Dow AgroSciences Contributes Through Key Crop Sciences
Plant Genetics and Seeds
Crop Protection
Urban Pest Management
Vegetation Management
Range and Pasture
DOW AGROSCIENCES RESTRICTED
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2013 Sales by Operating Segment(dollars in millions)
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Fermentation Plays a Key Role in Dow’s Product Portfolio
● Spinosyns: Spinosad, Spinetoram
● Secondary Metabolite Pesticide
● Acrylic acid (OPX)
● Propionic acid, Propanol
● Lactic acid (PLA)
● Ethanol (Algenol)
● Other commodity and specialty chemicals
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Comprehensive Industrial Biotechnology R&D Platform
Host Strain Selection
Economics, Systems Biology, Analytics, Bioinformatics
Scale-Up and Tech Transfer
Strain Improvement
Fermentation Process Design
and Optimization
Product Recovery
Design and Optimization
Manufacturing Support
Collaboration, Cross-Functional Integration
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• Fermentation Process Development
• High Throughput Screening and Selection
• Metabolic Engineering
• Microbial Physiology
• Enzyme Engineering
• Protein Expression/Purification
• Synthetic Biology
• Systems Biology
• Oilseed Processing
• Downstream Recovery
• Analytical Chemistry
Bioengineering and Bioprocessing R&D Areas of Expertise
Colony Picker
Microarray
Akta Protein Purification
Rationally-Guided Approaches for Secondary Metabolite Production
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The Spinosyns Family of Insecticides
●Spinosad: Naturally derived fermentation product Mixture of two primary factors
Major – spinosyn A, R = HMinor – spinosyn D, R = CH3
●Trade names: Tracer*, Conserve*, Success*, SpinTor*● Insecticidal Spectrum: Lepidoptera, Diptera, thrips,
termites and some Coleoptera & ants●Applications on vegetables, cotton, tree fruit and nuts●Selectivity for many beneficials, excellent mammalian and environmental profile
●Spinetoram: Second generation spinosyn Mixture of chemically modified spinosyns J and L
Major – 3’-O-ethyl-5,6-dihydro spinosyn JMinor – 3’-O-ethyl spinosyn L
●Trade names: Delegate*, Radiant*, Exalt*
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Improved Spinosyn Production Strains
The Spinosyns Systems Biology Program
Improved Spinosyn Production Process
Fluxomics(metabolic flux analysis of entire
biochemical reaction network)
Metabolomics(analysis of secondary metabolite
profile,structure elucidation of compounds)
Proteomics(analysis of protein spectrum dependent upon conditions)
Genomics(Genome Sequencing & Resequencing,in silico analysis of genome sequence,mapping & identification of mutations)
Transcriptomics(gene expression analysis,
investigation of regulatory mechanisms)
Targets
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DAD1 B, Sig=230,4 Ref=off (M:\LEWER'~1\HP1050~1\CDMDAT~1\MDGR2193\042407AB.D)
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Genome Mapping to Identify Variants
WT
Strain 1
Strain 2
Reference # Variants
Strain 3
Strain 4
Strain 5
WT
Strain 1
Strain 2
Strain 4Strain 3
Strain 5
Reference
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Genomics Enables Mutation Effect Prediction
HIGH- Frame-shift- Start lost - Stop gained - Stop lost
MODERATE- Non-synonymous- Codon deletion- Codon change plus
insertion/deletion
LOW- Synonymous coding- Synonymous start- Start gained
MODIFIER- 5’ UTR, 3’UTR- Regulation (RBS,
Terminator)- Upstream- Downstream- Intergenic
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BBRD Synthetic Biology Program
● Leveraging both internal and external capabilities to develop a full Design-Build-Test-Analyse program for Spinosyns Strain Improvement
AnalyticsKnowledgeManagement
Strain Evaluation
Rational StrainDesign
Mutagenesis & Screening
StrainEngineering
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Development of an Industrializable Fermentation Process for Propionic Acid
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Propionic Acid(PA) and Propionibacteria
● Commonly used as a food preservative due to anti-fungal activity
● Precursor for other chemicals Herbicides, Polymers, Pharmaceuticals
● Currently produced from petrochemicals (ethylene) by hydrocarboxylation (“oxo” process).
● Dow is the world’s second largest producer of PA – second to Eastman Chemical
● 400 KTA global market with steady growth (2.5% per year)
● Propionibacteria naturally produce propionic acid Facultative anaerobic bacteria
15 species described that can utilize a wide range of substrates
H2C=CH2 + H2O + CO → CH3CH2CO2H
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Propionibacterium acidipropionici ATCC 4875 as a Platform Strain for Industrialization
● Despite years of research in academia, fermentation metrics are far short of industrial targets
● Initial development work improvedproductivity 2-fold, yield 12% and titer ~20%,but little progress was made on reducingbyproducts or media costs
● Productivity must be doubled to achieve target
ATCC 4875 Fermentation Metrics
Metric Literature Process Phase 1 Optimization Target
Productivity (g/L/h) 0.28± 0.1 0.53 ± 0.1 >1Yield (g/g) 0.48 ± 0.05 0.54 ± 0.1 >0.55Titer (g/L) 40.5 ± 1.5 49.5 ± 0.45 50PA/(SA+AA+LA) 1.1 ± 0.1 1.2 ± 0.1 >3Media Cost ($/ kg of PA) $38.27 $34.02 <$1.40
Stowers, C. et al..JIMB. (2014). 41:837-852
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Head Space Nitrogen Reduces Byproduct Accumulation
● Sparging nitrogen increases lactate accumulation and reduces succinate accumulation. Byproduct ratio (PA/(LA+AA+SA)) of 1.90 achieved.
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Advantaged PA Production With Cheap Substrates
● Switch to Pure Enzymatically-Treated Corn Mash
● 25 g/L of PA produced at a rate of ~0.5 g/L/h with vitamin supplementation
● Yield of 0.60 g/g
● Byproducts reduced to give PA:byproduct of 2.4:1
● Media cost reduced to $0.96/kg PA
*Vitamins: riboflavin, pantothenic acid, biotin, thiamine and cyanocobalamin at 2 mg/L each
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Key Take Home Messages
● Strain and fermentation development is a highly integrated process requiring strong collaboration across functional groups (strain development, fermentation optimization, scale-up, economic evaluation)
● An integrated strain and fermentation development capability is effective across a wide variety of products and technical challenges
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Acknowledgements
Dow PA TeamChris Stowers, Brandon Rodriguez, Brad Cox, Viet Pham, Barry Fish, Yomaira Pagan-Torres, Sanjib Biswas, Paul Larsen, Vishesh Shah, Babu Raman, Patrick Reifel, Matt Roach, Amudhan Venkateswaran, Paul Speakman, Amy Keeney, Kyle McFerran, Craig Finnegan, Paul Swanson, Paul Lewer, Scott Greenwalt, Bryan Ward, Karan Bansal, Prasanth Maddipati, Allison Lutocka, Samantha Hall, Paul Ketterer, Joe Brunson, Derek Jamrog, Kelly Hill , Mike Harris, Josh Watson, and Emma Patterson
External CollaboratorsOhio State University: Professor S.T. Yang Laboratory
University of Queensland:Professor Lars NielsenProfessor Jens KromerDr. Esteban MarcellinDr. Jennifer SteenFrauke KrackeNicolas Lekieffre
THANK YOU!
Dow Spinosyn TeamPatrick Adu-Peasah, Michael Alley, Karan Bansal, Sheena Becker, Prakash Bhosale, Erik Blackburn, Joe Brunson, Stefanie Casada, Matt Chase, Brad Cox, Sarah Delaplane, Maia Donahue, Clive Evans, Craig Finnegan, Ute Galm, Robbi Garrison, Mike Harris, Samantha Hall, Kelly Hill, Elizabeth Ibwe, Owen Jamison, Derek Jamrog, Paul Ketterer, Esha Khullar, Allison Lutocka, Prasanth Maddipati, Shrinivasrao Mane, Jessica Marty, Patrick McCarthy, Nigel Mouncey, Nadaraj Palaniappan, Emma Patterson, Babu Raman, Tom Ramseier, Patrick Reifel, Mireya Rivera, Matt Roach, Matt Robinson, Jeff Running, Paul Speakman, Shreedharan Sriram, Paul Swanson, Ashley Thomas, Eric Traub, Brittani Truelove, Josh Watson, Bryan Ward, Steve Wensing