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  • Biocatalysis @ DSM

    David Ager Principal Scientist DSM ChemTech R&D BV PMB 150 9650 Strickland Road, Suite 103 Raleigh NC 27615 USA [email protected]

  • Biocatalysis

    •  Broadest collection of off the shelf enzymes (>3000) •  Excellent expression and enzyme design capabilities •  Secure supply of enzyme at any scale (with FTO!) •  Unmet track record (also regulatory) in large scale implementation

    (>30 processes industrialized) •  Critical mass for all important expertise •  Open innovation approach provides access to huge network

    D192

    V193

    V194

    A199

    G200

    T226

    Used in tons scale production Used in lab and pilot scale

    Epoxides, DiolsHaloalcohol dehalogenases

    Epoxides, DiolsEpoxide hydrolases

    Alcohols, SulfoxidesMonooxygenases (P450, Baeyer-Villiger)

    Amides, NitrilesNitrile hydratases

    Carboxylic acids, NitrilesNitrilases

    Alcohols and Amino acidsDehydrogenases (alcohol & amino acid)

    Alcohols, Aldehydes, Carboxylic acidsOxidases

    AminesOmega-Transaminases

    CyanohydrinsHydroxynitrile lyases

    Amino acids, N-Acetyl-Amino acidsAcylases

    Amino acids, AmidesAmidases

    Amino acidsHydantoinases, Carbamoylases, Racemases

    Amino acidsAmmonia lyases

    Alcohols, Esters, Carboxylic acidsLipases and Esterases

    Peptides, Amines, CarboxyestersProteases

    Alcohols, Diols, Amino acoholsAldolases

    Product classesEnzyme Platforms

    Epoxides, DiolsHaloalcohol dehalogenases

    Epoxides, DiolsEpoxide hydrolases

    Alcohols, SulfoxidesMonooxygenases (P450, Baeyer-Villiger)

    Amides, NitrilesNitrile hydratases

    Carboxylic acids, NitrilesNitrilases

    Alcohols and Amino acidsDehydrogenases (alcohol & amino acid)

    Alcohols, Aldehydes, Carboxylic acidsOxidases

    AminesOmega-Transaminases

    CyanohydrinsHydroxynitrile lyases

    Amino acids, N-Acetyl-Amino acidsAcylases

    Amino acids, AmidesAmidases

    Amino acidsHydantoinases, Carbamoylases, Racemases

    Amino acidsAmmonia lyases

    Alcohols, Esters, Carboxylic acidsLipases and Esterases

    Peptides, Amines, CarboxyestersProteases

    Alcohols, Diols, Amino acoholsAldolases

    Product classesEnzyme Platforms

    >30 e nzym

    atic proc

    esse s im

    plem ente

    d on indu

    stria l sca

    le

    0

    100

    200

    300

    400

    500

    600

    700

    1 2 3 4

    Nu m

    be r o

    f e nz

    ym es

    DSM plus partners Biocatalytics Sigma-Aldrich Codexis

    oxid ored

    ucta ses

    tran sfera

    ses

    hydr olas

    es lyas

    es

    supplier 1 supplier 2 supplier 3

    0

    100

    200

    300

    400

    500

    600

    700

    1 2 3 4

    Nu m

    be r o

    f e nz

    ym es

    DSM plus partners Biocatalytics Sigma-Aldrich Codexis

    oxid ored

    ucta ses

    tran sfera

    ses

    hydr olas

    es lyas

    es

    supplier 1 supplier 2 supplier 3

    oxid ored

    ucta ses

    tran sfera

    ses

    hydr olas

    es lyas

    es

    supplier 1 supplier 2 supplier 3

    Number of accessible enzymes for rapid screening at DSM

    Co nti

    nu ou

    s e xp

    an sio

    n

    > 1500 enzymes in DSM enzyme collection > 3000

    Mutation at contact point of subunits

    Mutation at contact point of subunits

    PharmaPLE®

    Candida sp.2 Corynebacterium Rhodococcus

    Moraxella

    Thermoanaerobium

    Pseudomonas Candida sp. 1

    Sporidiolobus

    >500 Enzymes

  • Enzyme classes split for industrial manufacturing

    DSM has access to all classes and has commercialized processes using all of the classes mentioned

    Straathof, A. J. J.; Panke, S.; Schmid, A. Curr. Opin. Biotechnol. 2002, 13, 548

  • 0

    10

    20

    30

    40

    50

    60

    70

    80

    90

    100

    Alc oh

    ol de

    hy dro

    ge na

    se s

    P4 50

    En oa

    te red

    uc tas

    es

    Ox ida

    se s

    BV MO

    s

    S- om

    eg a-T

    ran sa

    mi na

    se s

    R- om

    eg a-T

    ran sa

    mi na

    se s

    L-A mi

    no ac

    id tra

    ns am

    ina se

    s

    D- Am

    ino ac

    id tra

    ns am

    ina se

    s

    Tra ns

    am ina

    se s (

    tot al)

    Lip as

    es / E

    ste ras

    es

    Pr ote

    as es

    / P ep

    tid as

    es / A

    mi da

    se s

    Am ida

    se s

    Nit rila

    se s

    Ep ox

    ida se

    s

    Ha loa

    lco ho

    l d eh

    alo ge

    na se

    s

    D- Hy

    da nto

    ina se

    s

    L-H yd

    an toi

    na se

    R- HN

    L

    S- HN

    L

    Am mo

    nia ly

    as es

    Ha loh

    yd rin

    e d eh

    alo ge

    na se

    De ca

    rbo xy

    las es

    Nit rile

    hy dra

    tas es

    DSM Enzyme Collection > 3000 enzymes ready for screening

  • Industrial applications > 30 processes implemented

    Enzyme Platforms Product classes

    Aldolases Alcohols, Diols, Amino acohols

    Proteases Peptides, Amines, Carboxyesters

    Lipases and Esterases Alcohols, Esters, Carboxylic acids

    Ammonia lyases Amino acids

    Hydantoinases, Carbamoylases, Racemases Amino acids

    Amidases Amino acids, Amides

    Acylases Amino acids, N-Acetyl-Amino acids

    Hydroxynitrile lyases Cyanohydrins

    Omega-Transaminases Amines

    Oxidases Alcohols, Aldehydes, Carboxylic acids

    Dehydrogenases (alcohol & amino acid) Alcohols and Amino acids

    Nitrilases Carboxylic acids, Nitriles

    Nitrile hydratases Amides, Nitriles

    Monooxygenases (P450, Baeyer-Villiger) Alcohols, Sulfoxides

    Epoxide hydrolases Epoxides, Diols

    Haloalcohol dehalogenases Epoxides, Diols

    2009

    2009 2008 2007

    2010

    Introduced

    Used in tons scale production Used in lab and pilot scale

    2005

  • Enzymes used in large scale production

    Product Process Citric Acid Fermentation

    Vit. C Biotransformation: sorbitol à sorbose

    Vit. B2 Fermentation: Metabolic Engineering

    Aspartame Biocatalysis: Protease

    D-p-Hydroxyphenylglycine Biocatalysis: Hydantoinase / Carbamoylase

    6-APA Fermentation & Biocatalysis: Acylase

    7-ADCA Fermentation & Biocatalysis: Metabolic Engineering, Acylase

    Cephalexin Biocatalysis: Acylase

    Agro application Biocatalysis: Hydroxynitrile Lyase

    Commercial products ranging from 500 tpa to 100,000 tpa

  • DSM core competencies

    HTS cloning in PluGbug

    HTS screening & analysis

    Enzyme production

    Application (cGMP)

    Biodiversity through partnering and in-house activities

    genes

    Expansion of Enzyme Spectrum

  • P450s Regioselective hydroxylations, dealkylations

    Diverse enzymes: Diverse chemistry:

    CYP116B1 (P450-Rhf)

    CYP505B1

    CYP102A1 (P450-BM3)

    other self-

    sufficient CYP450s > 50

  • Challenges in Applications of P450s

    PdR P450

    Pdx RH2 + O2

    R-OH + H2O

    NADH + H+

    NAD+

    Example: Bacterial three-component cytochrome P450 mono-oxygenases (PdR: putidaredoxin reductase; Pdx: putida-redoxin; P450: heme-containing monooxygenase).

    Regeneration

    scale-up

    Expression (membrane bound) Stability Selectivity/Activity Uncoupling

    Challenges to be solved

  • Classical and Targeted Pravastatin Process

    Superior in terms of production costs and environment

    Secretion

    Streptomyces carbophilus

    Pravastatin

    Glucose Bioconversion

    Glucose

    Pravastatin Penicillium chrysogenum

    Compactin Penicillium citrinum

  • Conversion of Compactin to Pravastatin in P. chrysogenum •  P450 from S. carbophilus (known 2-step process) known,

    but inactive in Penicillium! •  Broad search for other P450 enzymes •  Active P450 from Amycolatopsis orientalis!

    Compactin epi-Pravastatin

    P450 A. orientalis

    the wrong epimer!

  • Page 13

    Typical Platform Screening by Flow NMR

    Ø  Soluble samples (mainly water samples) Ø  Automatic sample injection Ø  No sample equilibration Ø  Average 1 minute per sample Ø  Automatic data work out Ø  Application: High-Throughput Screening

    Supernatant in 96 well MTP format

    Supernatant in 96 well MTP format

    Pretreated Wheat Straw suspension

    Pretreated Wheat Straw suspension

    Incubation: pH 4.5 time = 20 hrs Temp 65 oC

    Incubation: pH 4.5 time = 20 hrs Temp 65 oC

    Addition of NMR standard + spin down

    Addition of NMR standard + spin down

    Flow NMR analysis

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