Biocatalysis @ DSM

17 Pages·2014·2.03 MB·English

by  David Ager

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
AEAEllddnnoozzllaayysseemmssee PPllaattffoorrmmss APAPllccrroooohhddoolluuss,, ccDDiittoo llccss,,ll AAaammssiissnnooee aassccoohhoollss Num>> b13e7750r0000 00o00f eancczeysmsiebsle i nen DzySmMe es nfozry rmapei dc oslclreecetnioinng a t DSM PharmaPLE® Pseudomonas Candida sp. 1
HEMNNDOOHAAHALPHEMNNDOOHAAHALP>iiprprcmmcmmaiieyyaiieyyxmxmooppttttoorrrroo3yyddddlhlhiinnooaaiiiiimimddtteexxllllllaaddyyrrooeeeaeaaassaa0agagiioodndnaoaoddooaasslleessssccaahhxx ttrsrseexxeessnnsseeooeeoo--yyooyyyyeeesseeii TTsshhhhssddnniiaaaaggggssssnnrrooyyrrn nneeiieeaaaallttaaddllyynnddrrnnnnttssrrddziiaaaaaa ooaallsseeeeEEeessssssllssyaassaahheesseeeeeellmm,,ssaassyyttssssm sseeeeCCllaaiioo((rrnnss((ssaaPPggaaaaaaaeerree44ssllbbssssccnn55eeteeaaooaai00ssssmmhhss,,c eeooBBoo ssllaayy p&&eellaayy rssaaeeoeemmrr--ssVViic,,nn iiRRllooelliiaagg aascceecceerriis))mmdd))eaasssee ssimplemEEAACAAACAAAAAPEEAACAAACAAAAAPpplmepplmellmmmmmlllmmmmmlayayeccccccccoopoopaarrooiiooooooiiiiiiiiiibbddnxxxxnnttnnnnnnnnnnhhhhhhhhiiooiiiieeooddeooooeooooddoddoxxtoooooossshsheeleelee yyss,,ellllllaaaaaaaassssss ssssssyyll,,NNccccccccii ,,,,cc,,dd ,, ,,dSS a a iiiiiiiiiiDDDD AAEEddddddddAArrttaauunnrr iissssssssiiiissmmiinnllccllooooddllffddoee,,,,ttiioossllll dd eeiisssseessNNAAAAnnxxssnrrhhiieemm--ssmm,,dd yyAA ss,,NNee iiddiiicc,,CCddssnnii eettneeCCeeoorraassttiissaallrr yy,,deeaabb rrllCCssccbb--oouAAiiaaooxxddmmrrxxyysssbbyylliiiiootnneeccxxoorss aayyitt ccaaeellaiiiiccrrccddsslii ddssaa ssccsiiddcssale oooxxxiiidddoooNumber of enzymesNumber of enzymesrrreeeddd123456123456000000000000uuu00000000000000ccctttaaasssttteeerrraaasss11nnnsssCfffoeeerrrnaaatisssneeehhh22sssuyyyodddurrrooos lllaaaesssxeee33psssanlllsyyyiaaaosssneee44sss DBSCDBSCsssssssssuuuiiiiSoSouuuuuuogogddpppppppppMMcmcmeepppppppppaa axaxlllllllllttppiiiiiiiiiaaii--eeeeeeeeessllAAuullrrrrrrrrryy slsl dd333tt222111 iirrccppiiccssaahhrrttnneerrss McoMcoooffuu nnssttttaauuaattbbcciioouutt nnnnpp iiooaattssttiinntt Sporidiolobus >500 EnzTyhmermesoanaerobiumMoRrCaChxoaoernydlldnoaiecdobaca sccpute.s2rium
Used in tons scale production
Used in lab and pilot scale
•  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
T226
V193
V194
D192
A199
G200
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
DSM Enzyme Collection
> 3000 enzymes ready for screening
100
90
80
70
60
50
40
30
20
10
0
Alcohol dehydrogenaEsneosate Pr4e5d0uctaseOsxidaSs-eosBmVegMRa--OosTLrm-aeAngsmai-anTmDori -anaAnacissmidae ntsrmoi ananacsisade Ttsmrriaannnasssaaesmmiinnaasseesss (eLtiso pt/ aalsP)eesp t/i dEastseers a/ seAsmidaAsemsiHdaalsoeaNlsitcriolhaEsopl eosdxiedhaalsDoe-gsHeyndaasnteL-soiHnyadsaenstoinase R-HaHlNAoLhmyS-dmriHonnNieLa ldyeahsalDeosegcearnbNaitosrxielyle ahsyedsratases
a
e
ot
Pr
Industrial applications
> 30 processes implemented
Introduced
Enzyme Platforms Product classes
Aldolases Alcohols, Diols, Amino acohols 2005
Proteases Peptides, Amines, Carboxyesters
Lipases and Esterases Alcohols, Esters, Carboxylic acids 2007
2008
Ammonia lyases Amino acids
2009
Hydantoinases, Carbamoylases, Racemases Amino acids
Amidases Amino acids, Amides
Acylases Amino acids, N-Acetyl-Amino acids
Hydroxynitrile lyases Cyanohydrins
2009
Omega-Transaminases Amines
Oxidases Alcohols, Aldehydes, Carboxylic acids
2010
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
Used in tons scale production
Used in lab and pilot scale
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
Expansion of Enzyme Spectrum
Biodiversity through partnering DSM core competencies
and in-house activities
HTS cloning Enzyme
in PluGbug production
genes
Application
HTS screening
(cGMP)
& analysis
P450s
Regioselective hydroxylations, dealkylations
Diverse enzymes: Diverse chemistry:
CYP505B1
other
self-
sufficient
> 50
CYP450s
CYP102A1
(P450-BM3)
CYP116B1
(P450-Rhf)
Challenges in Applications of
P450s
Expression (membrane bound)
Stability
Challenges to be solved
Selectivity/Activity
Uncoupling
√
scale-up
NADH
Regeneration
+ H+ RH + O
2 2
Pdx
NAD+
R-OH + H O
2
PdR
P450
Example: Bacterial three-component cytochrome P450 mono-oxygenases
(PdR: putidaredoxin reductase; Pdx: putida-redoxin; P450: heme-containing
monooxygenase).
Classical and Targeted Pravastatin Process
Bioconversion
Secretion
Glucose
Streptomyces
carbophilus
Penicillium citrinum
Compactin Pravastatin
Glucose
Pravastatin
Penicillium chrysogenum
Superior in terms of production costs
and environment