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*

OO

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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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Headspace Nitrogen = solid Sparged Nitrogen = dashed

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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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glucose acetic propionic succinic

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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