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 productionUsed 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 enzymatic processes implemented on industrial scale
0
100
200
300
400
500
600
700
1 2 3 4
Num
ber o
f enz
ymes
DSM plus partnersBiocatalyticsSigma-AldrichCodexis
oxidoreductases
transferases
hydrolaseslyases
supplier 1supplier 2supplier 3
0
100
200
300
400
500
600
700
1 2 3 4
Num
ber o
f enz
ymes
DSM plus partnersBiocatalyticsSigma-AldrichCodexis
oxidoreductases
transferases
hydrolaseslyases
supplier 1supplier 2supplier 3
oxidoreductases
transferases
hydrolaseslyases
supplier 1supplier 2supplier 3
Number of accessible enzymes for rapid screening at DSM
Continuous e
xpan
sion
> 1500 enzymes in DSM enzyme collection > 3000
Mutation atcontact pointof subunits
Mutation atcontact pointof subunits
PharmaPLE®
Candida sp.2CorynebacteriumRhodococcus
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
Alcohol
dehyd
rogen
ases
P450
Enoate
redu
ctase
s
Oxidas
es
BVMOs
S-omeg
a-Tran
samina
ses
R-omeg
a-Trans
amina
ses
L-Amino
acid
trans
amina
ses
D-Amino
acid
trans
aminas
es
Transam
inase
s (tot
al)
Lipas
es / E
steras
es
Protea
ses /
Peptid
ases
/ Amida
ses
Amidase
s
Nitrilas
es
Epoxid
ases
Haloalc
ohol
deha
logen
ases
D-Hyd
antoi
nase
s
L-Hyd
antoina
se
R-HNL
S-HNL
Ammonia ly
ases
Halohy
drine
deha
logen
ase
Decarb
oxyla
ses
Nitrile
hydra
tases
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!
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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
PretreatedWheat Straw suspension
PretreatedWheat Straw suspension
Incubation:pH 4.5time = 20 hrsTemp 65 oC
Incubation:pH 4.5time = 20 hrsTemp 65 oC
Addition of NMR standard + spin down
Addition of NMR standard + spin down
Flow NMR analysis
Cellulases activity assay
Recipe for TEC assay:200 µL Enzyme supernatant700 µL 3 % Pre Treated Wheat Straw 100 µL Citrate buffer pH 4.5
• Combined screening of several compounds
•Automated data work out and reporting possible
• Reporting format compatible with CHTS
RankingRanking
Example of Flow NMR Screening
DSM Cytochrome P450 Platforms
Source Number of enzymes
Bacillus (WTs and mutants) 20
Pseudomonas 2
Marine 2
Ralstonia 2
Saccharopolyspora 1
Burkholderia 1
Nocardia sp. 22
Fusarium 2
Rhodococcus 2
Bacillus BM3 Library
Amycolatopsis 4 libraries
Streptomyces work in progress
DSM P450 platform is: • continuously growing collection of enzymes • available in ready to screen format • offers a broad array of enzymes from diverse sources
1st generation platform
Pig Liver Esterase
pH indicator plate
Pig Liver Esterase synthetic applications
Kinetic resolution Desymmetrization
50% yield 100% yield
e.g. M. Ohno, M. Otsuka, Org. Reactions, 37, (1989)
• Animal derived • Not kosher, not halal • Undefined mixture of isoforms
Pharma PLE® the need for non-animal derived biocatalysts
PharmaPLE® γ-PLE
pH indicator plate
Various hosts PLE genes
Pichler et al. (2005), DSM patent application
R1O
O
R2 R1O
O
R2 R1O
O
H+X
