biocatalysis @ dsm ager.pdf biocatalysis @ dsm david ager principal scientist dsm chemtech r&d...

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