Nigel Mouncey

  • View
    224

  • Download
    0

Embed Size (px)

Text 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

  • | 2

    Dow AgroSciences Contributes Through Key Crop Sciences

    Plant Genetics and Seeds

    Crop Protection

    Urban Pest Management

    Vegetation Management

    Range and Pasture

    DOW AGROSCIENCES RESTRICTED

  • | 33

    2013 Sales by Operating Segment(dollars in millions)

  • | 4

    Fermentation Plays a Key Role in Dows Product Portfolio

    Spinosyns: Spinosad, Spinetoram

    Secondary Metabolite Pesticide

    Acrylic acid (OPX)

    Propionic acid, Propanol

    Lactic acid (PLA)

    Ethanol (Algenol)

    Other commodity and specialty chemicals

  • | 5

    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

  • | 6

    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

    ColonyPicker

    Microarray

    Akta ProteinPurification

  • Rationally-Guided Approaches for Secondary Metabolite Production

  • | 8

    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 & antsApplications on vegetables, cotton, tree fruit and nutsSelectivity 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

    O

    O

    O H

    H

    H

    R

    H

    O

    O

    H

    N

    O

    O

    O

  • | 9

    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

    m0 5 10 15 20 25 30

    mAU

    0

    500

    1000

    1500

    2000

    2500

    3000

    DAD1 B, Sig=230,4 Ref=off (M:\LEWER'~1\HP1050~1\CDMDAT~1\MDGR2193\042407AB.D)

    8.2

    92 8

    .601

    9.3

    85 1

    0.01

    4 1

    0.44

    6 1

    0.64

    9 1

    1.05

    8 1

    2.19

    7 1

    2.75

    4 16.

    771

    17.

    535

  • | 10

    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

  • | 11

    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, 3UTR- Regulation (RBS,

    Terminator)- Upstream- Downstream- Intergenic

  • | 12

    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

  • | 13

    Development of an Industrializable Fermentation Process for Propionic Acid

  • | 14

    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 worlds 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

  • | 15

    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

  • | 16

    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.

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    0 24 48 72 96 120

    orga

    nic

    acid

    con

    cent

    ratio

    n (g

    /L)

    time (h)

    propionicaceticlacticsuccinic

    Headspace Nitrogen = solid Sparged Nitrogen = dashed

  • | 17

    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

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    0 12 24 36 48 60 72 84 96

    orga

    nic

    acid

    and

    sug

    ar c

    once

    ntra

    tion

    (g/L

    )

    time (h)

    ECM = dashed

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    0 12 24 36 48 60 72 84 96

    orga

    nic

    acid

    and

    sug

    ar c

    once

    ntra

    tion

    (g/L

    )

    time (h)

    ECM = dashedECM + vitamins = solid

    glucose acetic propionic succinic

  • | 18

    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

  • | 19

    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