Weiße Biotechnologie – Anwendung von mikrobiellen … · ncp_967849ae78842 z...

Bio tec hn o lo gy Re se arc h

An d In fo rm a t ion N etwo rk A GB io te ch no log y Re se arc h

A nd In fo rm at io n N etwo rk A G

Weiße Biotechnologie –Anwendung von mikrobiellen Enzymen, Biokatalysatoren und bioaktiven Stoffen in industriellen Prozessen

WiTechWi / Arbeitskreis Wissenschaft-Technologie-Wirtschaft in der Kulturkreisgesellschaft Schwalbach

AES / Albert-Einstein Schule Schwalbach10.03.2010

PD Dr. Stefan PelzerCorporate Development Microbiology

BRAIN Aktiengesellschaft

EvoSolution®: for novel BioActivesand novel Enzymes

Weiße Biotechnologie

Definition und Bedeutung der Weißen Biotechnologie

Screening und Anwendung von Enzymen• Enzyme im Alltag / das Metagenom als Quelle• Fallbeispiel: Alkoholdehydrogenasen

Neue Produkte der Weißen Biotechnologie

1

2

4

©B

RAI

N A

G

Designermikroorganismen• Das Paradabeispiel: Fermentative Vitamin B2 Produktion• 1,3 Propandiol Biosynthese in E. coli• Mikrobielle Produktion von Vorstufen des Artemisinins

3

Zusammenfassung5


1. Definition und Bedeutung der Weißen Biotechnologie

„Biotechnologie umfasst alle innovativen Methoden,

Verfahren oder Produkte, die die Nutzung von lebenden

Organismen oder ihrer zellulären und subzellulären

Bestandteile beinhalten, sowie die kommerzielle

Umsetzung von Erkenntnissen der Molekularbiologie,

Virologie, Mikrobiologie und Zellbiologie.“

Deutscher Biotech Report 2002

„Biotechnologie umfasst alle innovativen Methoden,

Verfahren oder Produkte, die die Nutzung von lebenden

Organismen oder ihrer zellulären und subzellulären

Bestandteile beinhalten, sowie die kommerzielle

Umsetzung von Erkenntnissen der Molekularbiologie,

Virologie, Mikrobiologie und Zellbiologie.“

Deutscher Biotech Report 2002



White Biotechnology:

Application of nature´stoolbox

in industrial production[EuropaBio, Bruxelles 2003]

Rote (medizinische)Biotechnologie

weiße(industrielle)

Biotechnologie

grüne (agrar)Biotechnologie

Biotreibstoff/Bioenergie

Nahrungsmittel/FutterIndustrie

Feinchemikalien KörperpflegeReinigungsmittel

”Bulk”Chemikalien

Polymere

Spezialchemikalien

Aminoacids, Vitamins, Aspartam, Phytase, etc.

Bioethanol, 2G-fuels, 3G-fuels, Biogas, etc.

Enzymes, Detergents, Pulp, Paper, Textile, etc.

PLA, PHAs, PHB,SORONA, etc.

1,3 PD, Acrylamide,Succinic Acid, Lactic

Acid etc.

7-ACS, building blocksetc.

Cyclodextrine, Building Blocks, Hyaluronsäure,

etc.



Fermentationen mit Hilfe von Mikroorganismen Otto Röhm1879-1939

1907: „enzymatische Lederbeize“1909: OROPON (Proteasen für die Ledergerbung)

statt Hundekot und Taubenmist„Röhm und Haas“, Darmstadt

Biotechnologie in der industrielle Produktion

Weiße Biotechnologie: Keine neue Disziplin !



Source: McKinsey 2009: White Biotechnology, Press Briefing

Growth rates:

Total Chemical Industry: ca. 2.5%

White Biotech: ca. 9%

McKinseys aktuelle Prognose für die Weiße Biotechnologie






1

2

4

©B

RAI

N A

G


3

Zusammenfassung5


Enzyme für industrielle Anwendung sind Teil unseres Alltags !

2. Screening und Anwendung von Enzymen

Global market for Industrial Enzymes(2007): $ 2.3 Bln (CAGR 4%)

Novozymes A/S (Denmark)Danisco A/S (Denmark)DSM N.V. (Netherlands)

Animal Feed

Baking, Beverage, DairyDetergents

Pulp and Paper

Cellulase LaccaseAmylase

Textiles and Leather

Amylases, Proteases, Lipases, Pectate Lyases…

Chymosin, Laktase, Pectinase, Xylanase, Asparaginase, Lipase, Transglutaminase etc…

Phytases

Amylases, Xylanase, Lipases, Katalases, etc.

Only 130 – 200 enzymes in industrial application !!

OHO

OH

OH

Chemicals

Oxidoreduktase



temperature stability

substrate range

substrate selectivity (regio-, enantio-)

turnover frequency (kcat)

pH profile

temperature profile

product inhibition

solvent stability

substrate specificity(KM,kcat/KM)

pH stability

byproduct/ingredient inhibition

space-time yield

Act

ivity

Effic

ienc

y

Spec

ifici

tySt

abili

ty

ingredient / byproduct stability

producibility / expression yield substrate conversion (%), yield

specific activity (kat/kg, U/mg)

Lorenz & Eck (2005) Nature Mirobio Rev 3: 510-516

A Bdesired Reaction ?reaction constraints


Sequence-basedScreening

• sequence-motive driven• use of structure-function

knowledge

Activity-basedScreening

• activity driven • independent from known

sequences, genes,...

candidatesof specified functional group

candidatesof specified activity

Sequencing &Data-/Gene-mining

• Habitat or clone driven• use of annotation

knowledge

candidatesfrom genome-fragments

Strain- / Habitat-Collections

NP_854323NP_215160CAD93527CAB07104NP_335086AAK44900YP_264476AAZ19042CAA61351AAF87665NP_149260AAK76842BAD13383BAD10980AAM18212BAD10977BAD10974AAB16939AAA99002NP_746151AAN69615AAB35246BAA86922YP_260562AAY92726AAB60168YP_079396AAU23758YP_171472BAD78952NP_484155BAB77635NP_440460BAA17140YP_172292BAD79772NP_923742NP_949068CAE29172NP_662200AAM72542ZP_00237655EAL14590NP_371139CAG42358YP_042710BAB56777YP_040069CAG39642NP_763944AAO03986YP_160332CAI09431NP_840542CAD84367YP_035278AAT59258AAU19314YP_082533ZP_00236030EAL16683NP_830813AAP08014NP_834057AAP11258BAD12779AAG06337AAF89744NP_251639CAA47949CAA37863YP_045825CAG68003AAK81864NP_975515CAE77157AAA95966CAA83733NP_975514CAE77156AAA95965AAA95964AAZ43794NP_975361CAE77003YP_115761AAV27481AAZ44221NP_072996AAC71551NP_110161AAB96016NP_325868CAC13210NP_325867CAC13209NP_325869CAC13211NP_325866CAC13208NP_757778BAC44182NP_072977AAC71532NP_110133AAB96044NP_758313BAC44717NP_757932BAC44336YP_015897AAT27686

NP_073014AAC71569NP_110207AAB95971NP_077851AAF30426YP_189619AAW52917

YP_015763AAT27552AAZ44336AAM09096NP_325864CAC13206NP_326526CAC13868YP_274221AAZ36304YP_095481AAU27534YP_094512AAU26565AAM73852YP_267930AAZ24762NP_832278AAP09479NP_831724AAP08925

NP_347452AAK78792ZP_00229382EAL10642AAT03436YP_013259ZP_00233885EAL06269YP_053724AAT75840YP_265740AAZ21137

YP_083832AAU18015YP_036612AAT61445NP_832211AAP09412YP_019137NP_844875AAT31612AAP26361NP_978840AAS41448ZP_00240205EAL12154YP_028582AAT54633

ZP_00670109EAO17307NP_840654CAD84481NP_250306AAG05004

NP_418033AAC76600ZP_00230895AAT03306EAL09249YP_013129ZP_00232335EAL07778YP_169099CAG44656BAC96634AAO07075NP_936664NP_762085NP_233140AAF96652NP_800507BAC62340ZP_00634424EAN71086

ZP_00638408EAN74783NP_936270BAC96240

ZP_00637221EAN76091ZP_00675753EAO25482YP_268512AAZ24140NP_867010CAD74552

ZP_00551941EAM70544YP_269124AAZ27340ZP_00513728EAM52931

YP_112615AAU90766ZP_00395190NP_847303YP_021770YP_038906YP_031000AAT59606AAT34245AAT57050AAP28789NP_981315AAS43923AAU15657YP_086191AAP11762NP_834561

ZP_00238748EAL13690

ZP_00426848EAM26562

ZP_00485260ZP_00478656ZP_00494699ZP_00488379ZP_00442325ZP_00643568ZP_00447313ZP_00451894ZP_00436711ZP_00429015ZP_00470102ZP_00500501ZP_00216580ZP_00277743ZP_00168705YP_157959CAI07058ZP_00334481

ZP_00243085

YP_218113AAX67032NP_462097AAL22056NP_994284AAS63161NP_933380BAC93351NP_759595AAO09122ZP_00265883ZP_00141440ZP_00318079

YP_048010CAG70188ZP_00152476ZP_00564372EAN03964NP_460180AAL20139AAX65066YP_216147NP_993567AAS62444

YP_059606AAT86423AAZ50879AAX71366

NP_834045AAP11246

NP_372122YP_005592AAS81965

ZP_00558191EAM97967NP_347168AAK78508YP_206894AAW88006

AAZ51456AAX71927YP_060154AAT86971

NP_459729AAX64654YP_215735AAL19688

YP_095604AAU27657YP_215483AAL19409AAX64402NP_459450

YP_005463AAS81836NP_348633AAK79973NP_833338AAP10539ZP_00238596EAL13711YP_004527AAS80900NP_346912AAK78252YP_204236AAW85348

NP_968776CAE79769YP_274281AAZ37093NP_792112AAO55807YP_235172AAY37134YP_258989AAY91158NP_744233AAN67697

NP_250462AAG05160NP_900850AAQ58855YP_000530AAS69167BAC69260BAB69216NP_822725

ZP_00411904EAL97227ZP_00468303ZP_00444139ZP_00493732ZP_00486708ZP_00450667ZP_00482603ZP_00475865ZP_00499118ZP_00441203ZP_00429591ZP_00433997ZP_00211620ZP_00282428ZP_00167310

ZP_00644507ZP_00641897

ZP_00403707ZP_00284730ZP_00053150AAC45583

ZP_00644213ZP_00050689

ZP_00655483EAO09720

ZP_00644470ZP_00428089

ZP_00387401

ZP_00209910

ZP_00644306YP_241382AAY47362NP_635661AAM39585

ZP_00636610EAN68883

ZP_00130496

ZP_00447384ZP_00441185ZP_00429086ZP_00452047ZP_00433728

ZP_00641594

ZP_00644554

ZP_00602617EAN10964

ZP_00266891

AAW38859

YP_185282

ZP_00403422ZP_00008247

ZP_00644800ZP_00641555

YP_028583AAT54634

ZP_00652817EAO12446EAO31763

ZP_00387402AAX31560

NP_721207AAN58513

ZP_00349271ZP_00284165

ZP_00652815EAO12444EAO31765

ZP_00382490YP_246758AAY61593

ZP_00340383AAY82369ZP_00644436

AAA25879AAA17487

AAB24833AAB22135

AAB32393

AAB20588

AAB20587AAB49525

NP_826954BAC73489

AAB47150AAB17907

NP_826955BAC73490ZP_00616225EAM75965

AAB31769

NP_834224AAP11425

AAB32002ZP_00392985AAP30025

AAB34665ZP_00480083AAB34664

NP_635660AAM39584

ZP_00652814EAO12443

ZP_00507187EAM38495

ZP_00145040EAA23356NP_602681AAL93980ZP_00498321ZP_00481316ZP_00466597ZP_00476549ZP_00433851ZP_00450076ZP_00444978ZP_00432407ZP_00491464ZP_00212156

ZP_00151210

ZP_00166041ZP_00284028ZP_00216910ZP_00243362

ZP_00281512

ZP_00310159ZP_00217610

ZP_00317930

ZP_00303829

ZP_00262866

ZP_00140625

ZP_00497995ZP_00493911ZP_00467933ZP_00487507ZP_00477006ZP_00482780ZP_00212945

ZP_00278129

ZP_00167989

NP_762632AAO07622YP_047176CAG69354

ZP_00244096AAP51116ZP_00350744

ZP_00440632ZP_00049091

ZP_00602616EAN10963

NP_599274

ZP_00652816EAO12445EAO31764

ZP_00050653

ZP_00641899

AAL82804AAL82803

CAH94363ZP_00645813ZP_00121958

AAT35584

ZP_00642837ZP_00387048

ZP_00655482EAO09719

ZP_00642729ZP_00005488

NP_854791NP_215621CAA17221CAD93996YP_225189CAF19603

NP_338084AAK47898

NP_336829AAK46643NP_338083AAK47897

NP_336498AAK46312AAF74988NP_338382AAK48196NP_336264AAK46078

YP_056804AAT83846AAA26744AAA26743NP_418004AAC76572AAG58697NP_290133BAB37856AAP19155NP_709326AAN45033NP_839344NP_312460YP_218560AAX67479NP_462542AAL22501

YP_168499AAV96530

YP_275953AAZ34855NP_791384AAO55079YP_258332AAY90488ZP_00266489NP_743774AAN67238YP_160269CAI09368NP_252318AAG07016ZP_00137017ZP_00268548NP_900409AAQ58415

ZP_00005972

NP_720476AAN53076YP_267568AAZ25779NP_799580BAC61413

ZP_00318409

YP_220901AAX73540

YP_156155AAV82606

NP_421318AAK24486

NP_717658

AAN55102NP_933633BAC93604

NP_772826BAC51451

NP_759345AAO08872

ZP_00303372

NP_416659NP_288737NP_311073BAB36469AAG57292AAC75215YP_149968AAV76656CAD02576NP_456753NP_804512AAO68361

NP_461139AAL21098YP_070044CAH20755NP_405087CAC90324AAS61632NP_669970NP_992755AAM86221YP_050806CAG75615YP_171770BAD79250ZP_00163462CAD55618

NP_720593AAN57899ZP_00134188

YP_241875AAY47855NP_638733AAM42657YP_202505AAW77120NP_641089AAM35625NP_440483BAA17163NP_286098BAB33833NP_308437AAG54706NP_639973BAB93575NP_274324AAF41680NP_284235CAB84747

NP_967849CAE78842

ZP_00154697AAC21853NP_438352ZP_00156025

YP_207741AAW89329

NP_107485BAB53271NP_532166AAL42482NP_697168AAN29083ZP_00375498EAL76137

ZP_00645605ZP_00433225ZP_00429764YP_102145ZP_00451720ZP_00445668ZP_00439982AAU49104ZP_00497549ZP_00469602ZP_00489821ZP_00483189ZP_00478192ZP_00641932ZP_00492526ZP_00281306ZP_00168898

CAE39529CAE35200NP_886379NP_891370CAE44007NP_882253

YP_128998CAG19196

ZP_00213945ZP_00161264ZP_00107860YP_192411AAW61755YP_045419CAG67597

YP_133228CAG23428

ZP_00419664EAM03998YP_259769AAY91935ZP_00632044EAN64545ZP_00472279EAM24286

ZP_00415509EAM07363

YP_085073AAU16775YP_037799AAT61634

YP_080981AAU25343ZP_00634757EAN70658ZP_00635054EAN70471ZP_00635973EAN69548

ZP_00587265EAN38281

ZP_00668099EAO19372

ZP_00519804EAM61362ZP_00514941EAM51776

ZP_00539855EAM86938NP_348583AAK79923

ZP_00588695EAN26035ZP_00512527EAM42874ZP_00660535EAO16011ZP_00592218EAN22562ZP_00528005EAM35833ZP_00401241EAL78118ZP_00520502EAM60367ZP_00303838ZP_00317665ZP_00316528

ZP_00633685EAN71527

YP_270178AAZ24550ZP_00524794EAM56129ZP_00527460EAM53506ZP_00520499EAM60364ZP_00520563EAM60428ZP_00520564EAM60429ZP_00520496EAM60361

ZP_00520691EAM60103

ZP_00412981EAL96232ZP_00413157EAL95906

YP_121945BAD60581YP_225260CAF19674ZP_00572816EAN12938ZP_00547783EAM82850

NP_335773AAK45587

ZP_00351711ZP_00109832ZP_00161957ZP_00109831ZP_00490359ZP_00450401ZP_00431748ZP_00439004ZP_00434872AAU46424YP_105911ZP_00498705ZP_00468887ZP_00482255ZP_00479657ZP_00495817ZP_00446546ZP_00455075EAM12284ZP_00460677EAM20075ZP_00215143ZP_00422589EAM30790ZP_00278615AAR38335AAR37932

NP_532679AAL42995

ZP_00268216YP_213443CAH09535

ZP_00460266ZP_00452619EAM20502EAM14892ZP_00213415ZP_00421236EAM32214ZP_00283090

AAG34997YP_210999BAD48154CAH07054YP_098688NP_809065AAO75259NP_904726AAQ65625

YP_120038BAD58674YP_116930BAD55566YP_119453BAD58089

YP_120593BAD59229

YP_119237BAD57873YP_118588BAD57224YP_120771BAD59407NP_599594

YP_118722BAD57358YP_116770BAD55406NP_338463AAK48277

NP_336393AAK46207

NP_334547AAK44361

NP_602069BAC00272

NP_338462AAK48276

YP_060091AAT86908BAC64290AAX71840NP_802457NP_664462NP_607143AAM79265AAL97642AAK33917AAZ51366NP_269196ZP_00365873ZP_00388074AAV60822YP_141546AAV62731YP_139637NP_721797AAN59103NP_688194AAN00067

ZP_00332514NP_267928AAK05869AAK84012AAF62859AAM45148AAF62860BAB69464AAF02201ZP_00319041ZP_00064003ZP_00319390NP_814531AAO80601

AAK99343AAK74766NP_358133NP_345126ZP_00403421YP_042050CAG41686YP_187439AAW38649CAG44332NP_373153YP_044629BAB58791NP_765768AAO05855YP_189796AAW53104NP_763781AAO03823YP_189902AAW53151ZP_00231778YP_014993EAL08379AAT05170EAL05749ZP_00234401YP_177325BAD66364

BAC15556AAA23060

NP_116810AAK62844

ZP_00316105

ZP_00568180EAN17574ZP_00412824EAL96075

ZP_00384405ZP_00341777

YP_028769AAT54820NP_845051YP_019335AAT31810AAP26537

YP_084024AAU17824

YP_036796AAT60036ZP_00526388EAM54600

NP_624807CAB53320

YP_144380BAD70937YP_004722AAS81095ZP_00396441EAL82772NP_296226AAF12047

ZP_00522297EAM58722

NP_870096CAD79251ZP_00524193EAM56845

ZP_00239847EAL12496AAT59349YP_035466YP_082724AAU19122NP_831010AAP08211

YP_175238BAD64277

ZP_00608109EAN27483ZP_00519810EAM61368

NP_822479BAC69014CAA78842NP_107250BAB53036NP_419074AAK22242NP_856137AAK46838NP_337024CAD97351NP_216979CAA16040AAC60471AAA25813AAQ02789NP_249738AAG04436NP_627378CAB59658NP_824788BAC71323YP_117676BAD56312

NP_855186CAB02047CAD96201NP_216013AAK45811NP_335997AAT47740NP_855608CAD94660NP_216439CAB06503AAK46246NP_336432YP_118016BAD56652

YP_046621CAG68799

YP_118187BAD56823YP_116705BAD55341

NP_624653CAB56134NP_857441NP_218292CAD95990CAB02482AAB06505YP_121062BAD59698

NP_607915AAL98414AAM80204AAK34568NP_665401NP_269847NP_801532BAC63365NP_720848AAN58154NP_785847CAD64698

YP_193653AAV42622

NP_786562CAD65434

NP_107604BAB53390YP_162628AAV89517

NP_285539AAF12416NP_631604CAC16441

NP_626531CAB61729NP_864506CAD72187

YP_111320CAH38781AAU46165YP_105648AAF59826

CAA43847BAD69792YP_102158AAU49131NP_792753AAO56448AAG42401

NP_945813CAE25904

NP_773623BAA78097

NP_630302CAB36607NP_630669CAA19783

NP_627274CAC44518

YP_056763AAT83805

NP_599580NP_630216CAC33953CAA90431CAA90432

NP_639796CAC36720

NP_822324BAC68859

NP_936808BAC96778NP_800369BAC62202

NP_233249AAF96761YP_130755CAG20953

BAD90677BAD90676

AAB52231AAB28083YP_097708BAD47174

ZP_00467355ZP_00434153ZP_00450301ZP_00431522ZP_00441040ZP_00444413ZP_00498093ZP_00492893ZP_00480375ZP_00476906ZP_00486667

ZP_00281185ZP_00213606ZP_00350732NP_622726AAM24330ZP_00320263

ZP_00447439ZP_00442409ZP_00432017ZP_00437503ZP_00486217ZP_00470002ZP_00476780ZP_00482061ZP_00501278ZP_00495283ZP_00643598ZP_00215515ZP_00139117ZP_00267453NP_601715BAB99906YP_226755CAF21176

NP_623384AAM24988ZP_00357529

ZP_00381057

ZP_00397050EAL82371YP_192510AAW61854

NP_638713AAY47872YP_241892AAM42637NP_643806AAM38342

YP_199730AAW74345

NP_779608

AAO29257NP_299674AAF85194NP_626539CAB61737

NP_487153BAB74812ZP_00161104ZP_00110330NP_440538BAA17218

AAN03557AAB30793ZP_00163468YP_171777CAD55625BAD79257NP_682828BAC09590

NP_895685CAE22033

NP_874693AAP99345NP_896343CAE06763

NP_892386CAE18726ZP_00501509ZP_00491742ZP_00488195ZP_00430561ZP_00437585ZP_00469732ZP_00448242ZP_00443959ZP_00438044ZP_00484080ZP_00479581ZP_00642485ZP_00283178ZP_00169193

ZP_00348589ZP_00241597ZP_00316613BAA00727AAC60403ZP_00266340ZP_00137283

NP_970265CAE78324

NP_876554NP_445035AAP98211AAD18420BAA98481AAF38318NP_300330NP_224476NP_296790AAF39269NP_219639AAC67727NP_829376AAP05254NP_969967CAE78026NP_869620CAD76998

ZP_00154106ZP_00142733EAA26142NP_360784AAL03685YP_247205AAY62040ZP_00340737YP_067668AAU04186CAA72452YP_265842AAZ21239ZP_00270328ZP_00004792ZP_00055736ZP_00281957YP_045747CAG67925

YP_198376AAW71134

ZP_00210381NP_883921CAE36944CAE33577NP_889621YP_168590AAV96621

ZP_00511650EAM43721ZP_00529629EAM34085ZP_00588769EAN25913NP_662962AAM73304

ZP_00591169EAN23539ZP_00530775EAM64819NP_662734AAM73076

ZP_00356011NP_970041CAE78100

ZP_00213721ZP_00149493YP_075731BAD40887ZP_00048910ZP_00007111ZP_00602076EAN34864YP_175521BAD64560ZP_00393873

ZP_00384474

ZP_00121273ZP_00121130ZP_00412088EAL97411

NP_695980AAN24616ZP_00120292NP_865863CAD73548

NP_337926CAD95418NP_217815AAK47740NP_856971CAB07062NP_335111AAK44925

NP_939473CAE49636ZP_00209186ZP_00169066

ZP_00522358EAM58497

NP_294380AAF10236

ZP_00110920ZP_00464886ZP_00454162EAM15936EAM13026ZP_00424448EAM29051

ZP_00593581EAN53871ZP_00597775EAN50346YP_158469CAI07568ZP_00626493EAN60082ZP_00628081EAN58839ZP_00419507EAM04163ZP_00505690EAM40550

ZP_00629988EAN66905

ZP_00554651EAM67600ZP_00638552EAN74557ZP_00585859EAN39852ZP_00627941EAN58905

NP_626380CAB51971NP_827255BAC73790

ZP_00547846EAM82790ZP_00657860EAO07366ZP_00378580ZP_00414045EAL95154

ZP_00414044EAL95153

YP_147678BAD76110BAD18327NP_691221BAC12256YP_075874BAD41030ZP_00538480EAM88489NP_228828CAA04932AAD36099BAA02182AAG53982ZP_00390038YP_080734AAU25096

ZP_00588830EAN25974

NP_691527BAC12562YP_077784AAU22146YP_144971BAD71528YP_074080BAD39236

ZP_00495250ZP_00476812ZP_00482029ZP_00501246ZP_00469948ZP_00434328ZP_00486249ZP_00458926EAM21983ZP_00218153ZP_00169555

ZP_00579859EAN45667ZP_00424701EAM28695

NP_631032CAB89027NP_296242AAF12060NP_295260AAF11100NP_891093NP_886226CAE39369CAE34922NP_229151AAD36421

ZP_00669447EAO17949NP_841100CAD84938ZP_00399840EAL79504NP_785330CAD64178YP_078404AAU22766

NP_786041CAD64892

NP_244192BAB07045AAF70202NP_562512BAB81302ZP_00402549NP_631119CAC01555ZP_00050100ZP_00383721

ZP_00567731EAN18030NP_628561CAB95903NP_773254ZP_00568061EAN17720NP_828631BAC75166

YP_046113CAG68291YP_259981AAY92147YP_106611AAU45418

YP_112343CAH39827YP_165260AAV97564BAD61058NP_631650CAC16711ZP_00460265EAM20501

NP_523043CAD18635NP_856873NP_217719CAB08320CAD95320YP_165986AAV94038

AAV97951AAQ83881NP_102005BAB47791YP_260040AAY92206CAC34851AAC36352

NP_104840BAB50626NP_774185NP_946993CAE27088YP_166968AAV95010NP_894894NP_893203YP_074367BAD39523YP_151259AAV77947NP_455257CAD05159NP_805929AAO69789

AAQ56595

NP_752703AAN79246YP_247909AAX87249AAC21862NP_438361NP_873706AAP96095NP_797216BAC59100NP_231729AAF95243NP_933811BAC93782YP_204197AAW85309YP_129251CAG19449YP_191386AAW60730YP_055806AAT82848NP_696071AAN24707NP_628538CAC08324

NP_627433CAB38590NP_821994BAC68529

NP_821644BAC68179YP_079254AAU23616NP_219389AAC65907NP_625018CAB69685AAD09315AAA53485NP_894218CAE20560

YP_275494AAZ34423ZP_00660505EAO15981ZP_00529749EAM33996NP_894493CAE20835YP_224388CAF18659

NP_659950AAM54963EAM18737EAM14590ZP_00462160ZP_00452317YP_119470BAD58106YP_121699BAD60335YP_118613BAD57249YP_117775BAD56411AAR90147NP_940346CAE50546YP_120497BAD59133YP_225767CAF21491YP_121674BAD60310YP_110757CAH38206YP_105345AAU46806YP_233439AAY35401ZP_00458724EAM21781YP_190954AAW60298YP_048539CAG73336ZP_00568583EAN17130ZP_00549236EAM81382ZP_00612761EAN05959ZP_00658051EAO07177AAF87663ZP_00669700EAO17663NP_842165CAD86072AAZ47165YP_159461CAI08560ZP_00577215EAN48224ZP_00663505EAO23983NP_786288CAD65143YP_206398AAW87510ZP_00669701EAO17664NP_842166CAD86073AAZ47166YP_159460CAI08559ZP_00669996EAO17535NP_841897CAD85786YP_276252AAZ34194 YP_121027BAD59663

ZP_00488867ZP_00477779ZP_00470500ZP_00449567ZP_00484379ZP_00494373ZP_00501759ZP_00446086ZP_00439043ZP_00437780ZP_00430822AAU47639YP_103084YP_108053CAH35433ZP_00463877ZP_00457181EAM16987EAM10150ZP_00216007ZP_00421743EAM31718ZP_00280298NP_519893CAD15474ZP_00170595AAC41424ZP_00509483EAM36399ZP_00245626AAN69799AAM16269NP_746335NP_635770AAY47472YP_241492AAM39694NP_640731AAM35267NP_107583BAB53369YP_157044CAI06143NP_217486CAB05441CAD96681NP_856639NP_337560AAK47374ZP_00657495EAO07685YP_250326CAI36708NP_419588AAK22756ZP_00053313ZP_00376368EAL75098ZP_00303727ZP_00579272EAN46234ZP_00397382EAL81983NP_295208AAF11051ZP_00215124ZP_00302205ZP_00215452ZP_00170489AAC38151YP_075954BAD41110ZP_00166178AAS77247NP_947767CAE27866BAA82510ZP_00243004ZP_00242348NP_521793CAD17383NP_855087NP_215916CAB02181CAD94296NP_335895AAK45709CAD94295CAB02180NP_855086NP_215915YP_120309BAD58945ZP_00214276NP_766870AAQ08176ZP_00527143EAM53839ZP_00218166ZP_00651227EAO33477EAO14385EAO31491NP_778743AAO28392NP_298542AAF84062YP_275789AAZ35718NP_791591AAO55286ZP_00262504NP_522669CAD18259NP_745942AAN69406ZP_00265514ZP_00461108ZP_00454019EAM19729EAM13349ZP_00216404ZP_00426378EAM27061ZP_00283177ZP_00463505ZP_00456565EAM17349EAM10791ZP_00564943EAN03081ZP_00616266EAM76006YP_119473BAD58109ZP_00615504EAM76579NP_631192CAC04237YP_095906AAU27959NP_867424CAD74970ZP_00309317ZP_00282137ZP_00233403AAT04890EAL06730YP_014713EAL10506ZP_00229552

YP_175635BAD64674NP_243114BAB05967NP_887295CAE31245ZP_00202741ZP_00502277EAM41080YP_056976AAT84018CAD47862ZP_00280503ZP_00215128ZP_00618279EAM73995ZP_00572277EAN13479

YP_119596BAD58232YP_119587BAD58223CAD10803ZP_00659722EAO05477YP_190596AAW59940YP_117488BAD56124CAF18686BAB97511YP_224415NP_599371ZP_00230038EAL10189ZP_00232793EAL07447YP_012739AAT02916NP_784685CAD63532AAS77245AAS77242NP_286217BAB33952NP_308556AAG54825AAC73578NP_415009NP_752530AAN79074

AAP15953AAN42076NP_706369NP_836147BAA14305NP_459485AAL19444NP_806101CAD04975NP_455086AAO69961AAV78118YP_151430AAX64438YP_215519ZP_00379897AAM45391ZP_00267829NP_886976CAE30925CAE40664NP_879165NP_882777CAE36009ZP_00553732EAM68725AAL17934AAG09918ZP_00654972EAO10232BAD06009AAF70342

CAA37862ZP_00638174EAN74947YP_048101CAG70279AAM34295AAQ75424NP_521431CAD17100ZP_00167220ZP_00214274YP_046115CAG68293

ZP_00162774AAT38586AAS73030AAG10492AAY82592

YP_169934CAG45574NP_807232NP_458019CAD09594AAO71092YP_152952AAV79640NP_462912AAL22871NP_756691AAN83265

YP_218907AAX67826NP_232882AAF96394ZP_00505273EAM45672ZP_00379884

NP_696280AAN24916ZP_00206573CAF19872BAB98561YP_225458NP_600394

YP_267690AAZ28637ZP_00481647ZP_00446417ZP_00438759ZP_00436109ZP_00499736ZP_00493749ZP_00477033ZP_00433016ZP_00488254ZP_00644195ZP_00462512ZP_00452872EAM18260EAM14288AAG21825ZP_00420065EAM33121ZP_00218537ZP_00279971

ZP_00203732AAZ48558ZP_00564666EAN03200ZP_00284199

NP_522672CAD18262

ZP_00595484EAN52277ZP_00169229ZP_00508112EAM37930ZP_00241496NP_384534CAC41865

NP_533152AAL43468ZP_00572229EAN13501

ZP_00615316EAM77052ZP_00380853ZP_00568198EAN17592YP_082339AAU19508NP_979556AAS42164ZP_00570356EAN15469ZP_00218391YP_273516AAZ36096ZP_00572164EAN13582

ZP_00573804EAN11955YP_118331BAD56967AAC45283AAC01724NP_851538BAC76574

ZP_00567275EAN18330ZP_00570319EAN15432

NP_853888CAD93087CAB07012NP_214731NP_334634AAK44448NP_794033AAO57728

ZP_00377886

ZP_00481895ZP_00478555ZP_00494463AAU46624ZP_00448345ZP_00432102ZP_00445722ZP_00438735ZP_00433587YP_105266ZP_00487107ZP_00469529ZP_00498975ZP_00460872EAM20002ZP_00213846ZP_00279948YP_259829AAY91995ZP_00140207

ZP_00623226EAN55073YP_168208AAV96240NP_105354BAB51140ZP_00628884EAN68421NP_535720AAL46036NP_386368CAC46841NP_993197CAC90820AAS62074NP_405562CAF28545

NP_522988CAD18580

NP_856769CAD96811AAB18414CAE55547YP_177924

AAC12774

AAA19698AAY45707ZP_00615229EAM76965ZP_00441234AAU48993ZP_00477610ZP_00445903ZP_00466928ZP_00450698ZP_00429622ZP_00496022ZP_00483025ZP_00435631ZP_00500817YP_102010ZP_00489649ZP_00642562ZP_00464665EAM16193ZP_00215798ZP_00282691NP_518560CAD13967AAS77236AAS77233AAS77238

ZP_00679008EAO35481NP_102082BAB47868NP_250788AAG05486ZP_00139790NP_627621CAB42768ZP_00052730

AAF87664

NP_891018CAE34847NP_886157CAE39294CAE40524NP_879040

NP_103349BAB49135ZP_00506988EAM38940NP_880757CAE42375NP_889678CAE33634CAD96798CAA16142NP_217600NP_856756

YP_120918BAD59554NP_215592CAD93965NP_854760CAA17192NP_335548AAK45362NP_301342CAC29822

NP_218004CAD95703NP_857156CAB08710NP_338136AAK47950

ZP_00560689EAM95511YP_116414BAD55050ZP_00279645BAD07370NP_336934AAK46748

ZP_00582602EAN42929NP_716249AAN53694ZP_00585337EAN40527YP_130084CAG20282

YP_047995CAG70173AAB17013AAA02895ZP_00620925EAN57263ZP_00569850EAN15885ZP_00569835EAN15870ZP_00569839EAN15874ZP_00215385NP_440291BAA16971NP_299032AAF84552NP_635475AAM39399YP_241193AAY47173

ZP_00302166ZP_00525711EAM55284

YP_276486AAZ35417ZP_00262960

NP_816581AAO82651BAC63469AAM80100NP_801636NP_665297YP_060773AAT87590NP_607757AAL98256AAZ52025NP_269743AAK34464

ZP_00365884AAX72560YP_189665AAW52979

YP_187341AAW37325ZP_00366248AAX72163NP_269431AAZ51687NP_607425AAK34152AAL97924AAM79597NP_664794AAT87174YP_060357BAC63963NP_802130NP_372874BAB58512YP_187151AAW37173YP_189499AAO05570AAW52814NP_765484

ZP_00659635EAO05573

ZP_00568181EAN17575ZP_00660049EAO05151ZP_00375838EAL75948

ZP_00615255EAM76991ZP_00615936EAM76230ZP_00379418

NP_771629NP_102879BAB48665ZP_00414352EAL94798YP_249948CAI36330

NP_372975BAB58613YP_187257AAW38548

NP_765577AAO05663

NP_854790NP_215620CAD93995CAA17220

AAO67500

ZP_00480964ZP_00476678ZP_00493429ZP_00470898ZP_00490582ZP_00435890ZP_00502212ZP_00446132ZP_00448986ZP_00432556ZP_00440501ZP_00642326ZP_00420938EAM32421

ZP_00579539EAN46059NP_294545AAF10396

ZP_00395517EAL84099YP_276542AAZ35780YP_237451AAY39413NP_794577AAO58272ZP_00242599ZP_00304441

ZP_00268970

YP_045838CAG68016

BAB68337

ZP_00578993EAN46473

ZP_00303142YP_079964AAU24326

CAE39233CAE34786NP_890957NP_886098CAE40678NP_879178

ZP_00509612EAM36278ZP_00168533ZP_00105982

ZP_00278752ZP_00622541EAN55762ZP_00007522

YP_213918YP_101848CAH10029BAD51314NP_811996AAO78190

NP_868091CAD75643

ZP_00283556ZP_00216391ZP_00596074EAN51870

ZP_00459421EAM21356ZP_00302279AAG59804ZP_00398061EAL81319

NP_371179BAB56817YP_185594YP_042748CAG42396AAW37776YP_040107CAG39682NP_763979AAO04021AAW53664YP_187905BAD90560

NP_561095BAB79885BAA81642NP_967631CAE78624NP_979490AAS42098ZP_00602753EAN10898

ZP_00604939EAN08735NP_816794AAO82864

ZP_00385382AAP80677

YP_194213AAV43182

ZP_00318715ZP_00046495

YP_194473AAV43442ZP_00046348ZP_00387664ZP_00385595ZP_00385630ZP_00323306ZP_00319787

ZP_00488059ZP_00434242ZP_00430362ZP_00479264ZP_00446005ZP_00449275ZP_00495217ZP_00483793ZP_00500399ZP_00469819

ZP_00212144

ZP_00440720

ZP_00447889ZP_00170075ZP_00052265NP_562497BAB81287

NP_855113CAB09244CAD94322NP_215942

CAD97166CAB00984NP_855954NP_216800

NP_853891NP_334637NP_214734AAK44451CAD93090CAB07015

ZP_00615247EAM76983

ZP_00379415YP_055745AAT82787

NP_870225CAD77300

NP_865495CAD73179NP_870537CAD77614NP_484123BAB77603ZP_00158051

ZP_00108194

ZP_00573564EAN12188

ZP_00547797EAM82864

ZP_00628472EAN68009NP_867885CAD75432

YP_080470AAU24832

ZP_00520812EAM60224ZP_00525697EAM55270NP_293859AAF09724

ZP_00540581EAM86207

NP_870897CAD77975

NP_561223BAB80013NP_782124AAO36061NP_228966AAD36236CAA04934

NP_870895CAD77973

NP_864836CAD72520

NP_812012AAO78206ZP_00121894NP_695404AAN24040NP_786764CAD65642NP_786763CAD65641NP_786266CAD65121

NP_784706CAD63553

ZP_00323987YP_193073AAV42042

ZP_00047031ZP_00387524

NP_349519AAK80859ZP_00121135

NP_721071AAN58377ZP_00490528ZP_00482080ZP_00476882ZP_00447458ZP_00437484ZP_00442428ZP_00431998ZP_00470021ZP_00495302ZP_00501297

ZP_00428278

ZP_00050255YP_046054CAG68232ZP_00302384ZP_00305160ZP_00303758

ZP_00613615EAN05396

NP_627838CAB42031

NP_825708BAC72243ZP_00356199ZP_00660449EAO04759ZP_00527387EAM53568ZP_00411365EAL97607

ZP_00319571

YP_156559AAV83010

YP_226545CAF20644

YP_202009AAW76624NP_643607AAM38143AAP49217

YP_242111AAY48091NP_638494AAM42418NP_780062AAO29711NP_298071AAF83591

YP_272949AAZ34625NP_790416AAO54111NP_253799AAG08497AAB61674BAC21259

NP_742584AAN66048YP_108832CAH36239

NP_949194CAE29298

NP_459562AAC38796AAL19521YP_151361AAV78049YP_215595AAX64514NP_455154CAD05053NP_806032AAO69892NP_930727CAE15883CAA47020NP_900668AAQ58672YP_066730CAG37723

ZP_00462138ZP_00452296EAM18715EAM14569ZP_00422366EAM30942YP_105387AAU46955YP_046384CAG68562AAL54855ZP_00638131EAN74904

ZP_00597789EAN50360

YP_235937AAY37899ZP_00526104EAM54876ZP_00457370EAM09916ZP_00463570EAM17310

ZP_00569870EAN15905CAC93710AAN01204BAC10671

ZP_00416797EAM06437

YP_104109AAU48228

ZP_00461193EAM19563

NP_518701CAD14110

BAC98499BAC98498BAC98496BAA84997AAG09700AAG22559BAC98500AAU25837AAT48728BAC98501

AAQ62779AAD22743

BAC98502

BAA02519AAA81002

BAC98497BAC98503

AAF80996AAA25882AAU10321AAD09856BAA36468AAU13930AAP80239AAA25881BAB64913AAU12351

BAA02012AAC60402AAF66684

AAF01331NP_896093CAE22443

ZP_00668817EAO18323NP_840953CAD84790

ZP_00536918EAM78516NP_628904CAA20402BAD44773

NP_841338CAD85200AAZ48028NP_968049CAE79042

ZP_00616819EAM75485

NP_866254CAD73940AAL47055

CAA41727BAA77240ZP_00380194BAA76305

NP_770840

ZP_00107868NP_773103

ZP_00121887ZP_00526955EAM54019ZP_00206623ZP_00121628ZP_00333123

ZP_00134960CAA01212

NP_599275AAQ13462

ZP_00318640AAB94505

AAQ01679

AAQ88181ZP_00316511

AAN28935

AAW82873

NP_923900ZP_00056432

AAB53648

ZP_00152228ZP_00130014

ZP_00357726ZP_00317670CAA63420ZP_00310620AAS78630AAO65812

ZP_00314998

ZP_00356754

ZP_00170353AAN38719ZP_00303896

ZP_00316065AAN64190

CAD61042

CAE52328

NP_893961AAF87662

ZP_00120759AAR37801

ZP_00218223

ZP_00120290NP_892475CAB63926

ZP_00063556AAZ46340ZP_00279379NP_894064ZP_00642360AAC43262

ZP_00062707

ZP_00357418AAK84028

ZP_00335841AAD22079

BAA03399AAM90952

BAB68338

NP_847799AAP29285YP_022328AAT34803AAT57541AAT63507YP_031491YP_039392YP_086668AAU20230NP_981822AAS44430ZP_00239215EAL13110NP_835062AAP12263

YP_013383AAT03560ZP_00229502EAL10762ZP_00232928EAL07310

YP_060563AAX72274AAT87380AAZ51843NP_687533AAM99405

NP_815391AAO81461YP_014483AAT04660ZP_00231033EAL09154ZP_00234482EAL05672YP_085459AAU16389YP_020992YP_038184YP_030282NP_846579AAT63110AAT33467AAT56333AAP28065AAS43096ZP_00238889EAL13522NP_980488NP_833840AAP11041

YP_220861NP_697126AAX73500AAN29041NP_767099BAC45724

NP_533344AAL43660

NP_953461AAR35788NP_967341CAE77995

YP_160921CAI10020NP_744467AAN67931

NP_519838CAD15419AAK81865

YP_269028AAZ26246

YP_170001CAG45649NP_274679CAB85152NP_284637AAF42023YP_011142AAS96401

ZP_00502048CAH39215YP_111746AAU46108YP_105215ZP_00432756ZP_00644928ZP_00434618ZP_00441375ZP_00447831ZP_00490748ZP_00468688ZP_00494526ZP_00480799ZP_00478886

ZP_00462322EAM18551BAD13380CAA10510AAT85573ZP_00213135ZP_00426665EAM26788

ZP_00457061EAM10323

NP_778696AAO28345EAO31310NP_298472AAF83992ZP_00639059EAN74112ZP_00597747EAN50318

AAR13273ZP_00654617EAO10599

AAP44576AAD29442CAA56779NP_902383AAQ60383ZP_00136191BAA09136AAT85571

NP_251553AAG06251NP_214744CAB06997CAD93099NP_853900

YP_206047AAW87159

AAZ46883YP_045773CAG67951

YP_056707AAT83749NP_710578AAN47596YP_000336AAS68973ZP_00235580EAL17010AAU17144YP_084705NP_979727AAS42335NP_833148AAP10349YP_173740BAD62779NP_813971AAO80043

YP_192979AAV41948

ZP_00443696ZP_00430965ZP_00466681ZP_00476371ZP_00440390ZP_00437924ZP_00484529ZP_00501909ZP_00451262ZP_00487923

ZP_00493354

ZP_00212676ZP_00283713ZP_00243802ZP_00167978ZP_00152189

YP_047498CAG69676

YP_045530CAG67708ZP_00279747

ZP_00346371ZP_00334775ZP_00279276

ZP_00642250NP_623425AAM25029

ZP_00264184ZP_00139269

YP_175871BAD64910ZP_00394281

ZP_00217877ZP_00169361ZP_00388841ZP_00133902ZP_00387770ZP_00045972

ZP_00391128ZP_00322708

NP_600371CAF19849YP_225435BAB98536ZP_00379064

YP_047522CAG69700

NP_934849BAC94820ZP_00392960

ZP_00497783ZP_00448855ZP_00440824ZP_00494154ZP_00444534ZP_00468067ZP_00486597ZP_00481389ZP_00476751ZP_00432480ZP_00434097ZP_00218237ZP_00279492ZP_00171380ZP_00214656ZP_00171584

ZP_00214227ZP_00166675ZP_00170529ZP_00269705ZP_00241619ZP_00152184

ZP_00052826ZP_00496640ZP_00487558ZP_00466447ZP_00482832ZP_00491063ZP_00474651ZP_00215959ZP_00452046ZP_00447383ZP_00429085ZP_00433727ZP_00441186ZP_00245180ZP_00149593ZP_00167442ZP_00265745ZP_00136590

ZP_00316903

ZP_00284161CAE92890YP_175982BAD65021

ZP_00381258ZP_00392264

NP_629559CAB70650NP_824004BAC70539

AAC60485YP_251854CAI38236NP_823346BAC69881

YP_246025AAY60860

ZP_00349270

ZP_00349391NP_761196AAO10723AAQ04476NP_934783BAC94754NP_797560BAC59444YP_133605CAG23805NP_232620AAF96133

CAA68639AAG47649AAD22078NP_902384AAQ60384BAA23128BAA01035BAA09135NP_251552AAT85570AAG06250CAA44997AAB53647YP_047804CAG69982CAA56780AAP44577AAD29441

ZP_00654618EAO10600

ZP_00639058EAN74111ZP_00636179EAN69301NP_253500AAG08198AAC34733AAV34204AAT85572AAV34203AAA50466BAA00960AAC60400ZP_00462321ZP_00458702EAM18550EAM08623BAD13379ZP_00426667EAM26790AAW30196AAC05510YP_111747CAH39216CAA49812CAA02073ZP_00426666EAM26789YP_112321CAH39805YP_106588AAU45438NP_778695AAO28344EAO31309

NP_298471AAF83991AAR13272

YP_257759AAY96024AAC15585NP_746959AAN70423ZP_00418427EAM05214CAC07191

AAB01071NP_710768AAN47786AAS71538YP_002901

NP_840851CAD84688BAB58833BAB43769NP_373195NP_375790YP_042090CAG41728NP_647407CAG44373YP_044670BAB96455YP_187480AAW37342YP_189847AAW53186AAC38597NP_763836AAO03878AAA19729

BAD90561AAT34964AAG35726NP_763800AAO03842AAW53119YP_189885

BAD90565AAG35723YP_042422CAG42068AAK29127AAA26633YP_039776CAG39341YP_185209AAW37524BAD90562AAF21294YP_187618AAW53417AAP02960

YP_189935AAW53221AAC67547

CAC84594CAC83747YP_083942AAU17907NP_978931AAS41539YP_036713AAT59991ZP_00239430EAL12983AAT54743AAT31724YP_028692AAP26455YP_019249NP_844969NP_781602AAO35539

AAW47928AAO92067AAV35102CAA64621AAF40217AAN72417AAD30278AAM21775AAM21774AAC12257YP_147839AAL28099BAD76271AAF63229NP_294057AAF09912ZP_00237638EAL14882NP_831907AAP09108YP_074438BAD39594

YP_174487BAD63526ZP_00547494EAM83113

ZP_00370997EAL55885NP_347664AAK79004ZP_00520498EAM60363

NP_868074CAD75621

ZP_00525679EAM55252BAC00500ZP_00455145EAM12038ZP_00616915EAM75386ZP_00520717EAM60129ZP_00569559EAN16151NP_787201AAO44170NP_561658BAB80448ZP_00522347EAM58486ZP_00523965EAM56997ZP_00524310EAM56621ZP_00676420EAO38014

YP_016219AAT28008NP_110095AAB96079AAA79183

ZP_00529227EAM34515NP_970967AAS10848NP_967701CAE78694NP_757987BAC44391

YP_262937AAY95069ZP_00573400EAN12331YP_118521BAD57157

YP_119822BAD58458CAA67627AAT83812YP_056770YP_056483AAT83525NP_626008CAB50950NP_827735BAC74270NP_625552CAB99145ZP_00657678EAO07564ZP_00600062EAN36909ZP_00412504EAL96348AAB71210YP_250218CAI36600YP_250217CAI36599YP_251647CAI38029YP_224378CAF18649YP_224377CAF18648NP_938513CAE48621AAR90148ZP_00572051EAN13693NP_828265BAC74800NP_295801AAF11628NP_628956CAB92662NP_824638BAC71173ZP_00573685EAN12059ZP_00550432EAM72102ZP_00594932EAN53020NP_520203CAD15789ZP_00506377EAM39667AAU27243AAM73853YP_095190BAA22231AAA22574CAB12064NP_388152CAB95850AAR84668AAL36938CAD24006CAB12664NP_388716CAD23620CAA02196YP_079680AAU24042YP_176491BAD65530YP_095141AAU27194ZP_00144893EAA23506NP_603381AAL94680YP_166143AAV94195NP_894267CAE20609NP_897577CAE07999NP_875461AAQ00114NP_892710CAE19051ZP_00675352EAO25921ZP_00515821EAM51130ZP_00387403ZP_00284166ZP_00339732ZP_00320271ZP_00133753AAP12531ZP_00357257ZP_00308314ZP_00280801ZP_00006618ZP_00161632ZP_00266727ZP_00169589

NP_682772ZP_00151895AAB71813ZP_00346514ZP_00121263ZP_00384787ZP_00382590ZP_00053734ZP_00380884ZP_00108555AAM77996ZP_00151799NP_104647ZP_00051870CAA79652

0.10




Prokaryoten: Die unsichtbare Mehrheit und das Problem des Zugangs


unspecific dye (DAPI)

Natures Toolbox: optimiert im Verlauf von 3.5 Mrd. Jahre der Evolution



FISH: Fluoreszenz in situ Hybridisierung


Habitat Population size Productivity(cells/yr x 1029)

Marine heterotrophs Above 200m 3.6 x 1028 8.2

Below 200m 8.2 x 1028 1.1

Marine autotrophs 2.9 x 1027 7.1

Soil 2.6 x 1029 1Subsurface 4.9 x 1030 0.03

Domestic animals 4.3 x 1024 0.02

Whitman et al. (1998) Proc. Natl. Acad. Sci. USA 95, pp.6578-6583

2. Stammentwicklung: Voraussetzungen und Strategien


Amann et al. (1995) Microbiol. Rev. 59, pp.143-169

Habitat Culturability (cfu)seawater 0,001-0,1%

freshwater 0,25%

mesotrophic lake 0,1-1%

unpolluted estuarine waters 0,1-3%

activated sludge 1-15%

sediments 0,25%

soil 0,30%„The great plate count anomaly“

Gesamtzahl: 4 - 6 x 1030

60% der globalen Biomasse50% des elementaren Sauerstoffs



Anzahl prokaryotischer Organismen in 1 cm³ Boden

DNA source Abundance (cells cm-3)

Community genome complexity (bp)

Genome equivalents *

Forest soil 4,8 x 109 2,5 x 1010 6000

Forest soil, cultivated prokaryotes

1,4 x 107 1,4 x 108 35

Pasture soil (Weide) 1,8 x 1010 (1,5 x 1010) - (3,5 x 1010) 3500 - 8800

Arable soil (Acker) 2,1 x 1010 (5,7 x 108) - (1,4 x 109) 140 - 350

Pristine marine sediment (unberührt)

3,1 x 109 4,8 x 1010 11400

Marine fish-farm sediment

7,7 x 109 2,0 x 108 50

Salt crystallizing pond, 22% salinity

6,0 x 107 2,9 x 107 7

Torsvik et al. (2002) Science 296, 1064

* Genome equivalents relative to E. coli genome (4,1 x 106 bp)

Gans et al., 2005:

~ 1.000 000genome equivalents~ 2 billion ORFs per cm3 soil

Gans et al. (2005) Science 309, 1387



Der Baum des Lebens – enorme Diversität


EUCARYA

ARCHAEABACTERIA

1 change / 10 nt

Homo = manZea = maize


Das Potential des Metagenoms

Kultivierte und bisher nicht kultivierte Organismen (=100%) verdanken ihre Existenz der DNA als Träger der genetischen Information

Metagenom: = Gesamtheit der genomischen Information von Mikroorganismen einer Lebensgemeinschaft oder eines Biotops

Der Zugang zum Metagenom erlaubt die Nutzung der gesamten BiodiversitätMetagenomics: Genomtechnologien, mit denen das Metagenom eines Habitats analysiert

werden kann

Potential:

• neue Enzyme und Biokatalysatoren (ca. 130 Enzyme in Produktionsverfahren)

• neue Stoffwechselwege (IP, höhere Ausbeute)

• neue bioaktive Wirkstoffe und Biomaterialien



Kb

610555450

375

295225

DNA M1 M2

Schleper C, Kletzin A, Lorenz P, Eck J et al. WO2004/018673

• Direkt extrahierte genomische DNA• DNA Aufreinigung zur Klonierung


Herstellung von Metagenomgenbanken



Herstellung von Metagenomgenbanken

Daniel, 2005, Nat. Rev. Micro. 3, 470


Case study: Alcohol Dehydrogenases (E.C.1.1.1.)

• Enzyme Library of novel and diverse Alcohol-Dehydrogenases

Platform for synthesis of chiral alcohols from ketonesby enantioselective reduction

Kataoka et al., 2003 AMB 62:437



Why are Alcohol Dehydrogenases important Enzymes for Chemical/Pharma Industry ?

• produce chiral alcohols through the asymmetric reduction of prochiral compoundsunder mild conditions (chemo-, regio- and stereo-selective, theoretical yield 100%)

• chiral alcohols are building blocks for pharmaceutical, agrochemical and chemicalindustries

• worldwide sales for enatiomer drugs (2009): > US$ 150 billion(biocatalysis up to 15-20%)

HIV ProteaseInhibitor

Patel 2004, Food Technol Biotechnol. 42: 305

• diastereoselective reduction

• reaction yield 95%

• diastereomeric purity of 98,2%

• enantiomeric excess of 99,4 %



Whole-cell biocatalystcontaining: - ADH, - GDH or FDH- NAD(P)

O OH

>100 g/Lsubstrate input

<24h,No / very low amount

of cofactor,Pure aqueous reaction media

Broad substrate range

Robust and technicallyfeasible process

>95% conversion>99% ee

High productconcentration

• High expression• HCD fermentation• Lowest costs!

Novel Alcohol-Dehydrogenase platform



Example from the new ADH-platform

R. Schulze, P. Lorenz, J. Eck, O. May, H. Gröger, H. Trauthwein, PCT Pat. Appl. WO2005103239, 2006.

specific activity substrate spectrumexpression levelinducednon-induced

ADH

R

>>100 U/mg(p-Cl-1-Phenylethanol)

R

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

p-Cl-A

cetoph

enon

m-Cl-A

cetop

henon

a,m-D

i-Cl-A

cetoph

enon

2-Octa

non

2-Dec

anon

Methyla

cetoac

etat

Ethylace

toace

tat

Cinnam

y alco

hol

Rel

. Act

ivity

[%]

R

Substrates



O NAD(P)H OH

containingADH,GDH,

whole cell catalyst

Substrate concentration:>150 g/L >98% conversion

>75% yield>99.4% ee

4 Months from inquiry to delivery; Successful production campaigns

multi-ton process

A customer process







1

2

4

©B

RAI

N A

G

Designermikroorganismen• Das Paradebeispiel: Fermentative Vitamin B2 Produktion• 1,3 Propandiol Biosynthese in E. coli• Mikrobielle Produktion von Vorstufen des Artemisinins

3

Zusammenfassung5


3. Designermikroorganismen

• Wildtypstämme(Wirkstoffe, Vitamine, AS)

Klassische(Mikro)biologie

• Stammentwicklungund Optimierung durchunspezifische Mutation und Selektion

• Precursor-gerichteteBiosynthese

• Biotransformation

• HeterologeExpressionvon Genen

• Pathway/Metabolic-engineering

• KünstlicheStoffwechselwege

• Host-optimisation byknock-in / knock-out

• WirtsstämmeWirkstoffe, Vitamine, AS, Fein- und Basischemikalien

• Strain DevelopmentGenetic engineering

Molekular-biologie

Ganzzellbiokatalysatoren, Designer BugsGMO

ProduktionsorganismenNon-GMO


StärkeHydrolysate

MolkeUltrafiltrate

Melasse

Zucker

Silage

Agro-IndustrialNebenprodukte

als nachwachsendeRohstoffe

Screening geeigneter Mikroorganismen

Erzeugung von „Designer Bugs“

Fermentative Produktion von• Chemikalien

specialty / fine / bulk• Wirkstoffen• Materialien• Energiequellen

Prom enzyme1 enzyme2 enzyme3 enzyme4

Pathway Engineering• Engineering existierender Stoffwechselwege

Künstliche Pathways• Kombination existierender Pathways



Sources: BRAIN AG, Schmid (2002); Biehl et al. (2004); www.biotechnologie.de

Product Segment BioProcess Production Market value Volume [t/a] [mill. €]

Cyclodextrins Speciality BioCatalysis 5.000 50Glutamate Food Fermentation 1.500.000 1.800

Lactic acid Food/Textile Fermentation 150.000 270Phenylalanine Food/API Fermentation 10.000 100Vitamin B12 Food/Cosmetics Fermentation 20 500Vitamin C Food/Feed Fermentation 80.000 640Penicilline API/Feed Fermentation 45.000 13.500Threonine Feed Fermentation 30.000 180

Lysine Feed Fermentation 700.000 1.400Enzymes Detergent Fermentation 2.500 500Acrylamide Base Chem. BioCatalysis 100.000 28PLA Polymers Fermentation 140.000 315

Selected Marketed White Biotech Products


http://www.biotechnologie.de



Das Paradebeispiel – Fermentative Vitamin B2 Produktion

Historie der Vitaminforschung

• Beginn 20. Jahrhundert: Biochemiker C. FunkForschungsgebiet: Mangelkrankheiten BeriBeri, Pellagra, Skorbut und Rachitis

• Vita + Amin (das Leben und Amine)

• Vitamin: sind für Mensch und Tier essentiell - Erfüllen als niedermolekulare prosthetische Gruppen im aktiven Zentrum katalytische Funktion

• Nomenklatur:

Fettlösliche Vitamin A

Antiskorbut Faktor Vitamin C

Wasserlösliches Vitamin B

• Bis 1941 wurden alle 13 heute bekannten Vitamine entdeckt



1928-1967: 20 Nobelpreisträger in Zusammenhang mit Erforschung von Vitaminen ausgezeichnet

Fermentative Vitamin B2 Produktion


Fermentative Vitamin B2 Produktion


Synonyme: Riboflavin, Lactoflavin, Ovoflavin, Lebensmittelfarbstoff E101, 7,8-Dimethyl-10-ribityl-isoalloxazin

Integraler Bestandteil der Coenzyme FMN (flavin adenine mononucleotide) und FAD (flavin adenine dinucleotide)

• hitzestabil

• lichtempfindlich

• als wasserlöslich beschrieben, ist aber relativ schlecht löslich(65 mg/l)

• Flavin (flavus = gelb)E106

Isoalloxazinring

Ribitolseitenkette



Vitamin B2: Chemische Synthese

N

N

NH3C

H3C

O

ON

HH

OHH

OHH

OHH

CH2OH

NH2H3C

H3C

HH

OHH

OHH

OHH

CH2OH

NH

NH

NH

O

O O

O

NH

NH

O

O O

H

H

H3C

H3C

HH

OHH

OHH

OHH

CH2OH

NH

H3C

H3C NH2

H3C

H3C

HH

OHH

OHH

OHH

CH2OH

NH

N NC6H5

H2N

1 D-ribose2 H2/Pd C6H5-N2Cl

3,4-dimethylaniline

barbituric acid

alloxan

N-(2-amino-4,5-dimethylphenyl)D-1'-ribitylamine

aniline

riboflavin

1. Möglichkeit „Labormethode“(Kuhn und Karrer 1934/1935)

2. Möglichkeit „Technische Methode“(Tishler, Pfister et al. 1947)

DimethylanilineRibose

>60% yield


Vitamin B2: Chemische Synthese

Bacillus transketolasemutant




Mikrobielle Riboflavin Produktion

1940: Hefen als natürliche Hochproduzenten: Eremothecium ashbyi und Ashbya gossypii

1968: Stilllegung der fermentativen Anlage

1990: Fermentative Herstellung mit A. gossypii (Übernahme BASF von MSD)

2000: Roche Vitamins / DSM NP Fermentative Herstellung in B. subtilis

A. gossypii: WT produziert bereits 100 mg/g Biomasse Riboflavin-Kristalle in einer B. subtilis ü/n Kultur

20 µm


First known example for a recombinant bug producing a small organic compound

The riboflavin biosynthesis pathway in bacteria. Bacillus gene names are underlined.

Recombinant production strains - Target: gene copy number

1983: B. subtilis VNIIGenetika 304/pMX45,

• deregulated purin metabolism

• deregulated riboflavin metabolism (ribC-mutation)

• Plasmid pMX45: encoding 10 kb rib-cluster from B. subtilis ribO-mutant

ribO ribTribAribD ribE ribHdereg.

ribO ribTribAribD ribE ribHdereg.

=> 4,5 g/l Riboflavin(25 h fermentation)



First and prime example for a recombinant bug producing a small organic compound

Riboflavin crystals in aB. subtilis o/n culture

20 µm

Structure of Riboflavin (Vitamin B2)

Vitamin B2 Fact Sheet:• 1940: Ashbya gossypii: a natural overproducer

• 1983: First recombinant production in B. subtilis (4,5 g/l)

• 1990: A. gossypii fermentation process (BASF)

• 1996: Closure of chemical process (BASF)

• 2000: Roche Vitamins / DSM NP Fermentation in B. subtilis(Yield: >30 g/l)

• Production: 3.000 t / a

• 70% Feed, 30% Food

• Market leader

DSM NP (B. subtilis, Grenzach, Germany)

BASF (Ashbya gossypii, South Korea)

Hubei Guangji Pharmaceuticals (China)




Mikrobielle Riboflavin Produktion

0

20

40

60

80

100

120

140

160

Life

Cyc

le Im

pact

%

Global WarmingPotential

AcidificationPotential

EutrophicationPotential

Ozone CreationPotential

Cumulated EnergyConsumption

0

20

40

60

80

100

120

140

160

Life

Cyc

le Im

pact

%

Global WarmingPotential

AcidificationPotential

EutrophicationPotential

Ozone CreationPotential

Cumulated EnergyConsumption

Hoppenheidt, K., W. Mücke, et al. (2004)

Chemisch-technisch FermentationGlukose Ausgangsstoff Glukose, Biomasse, Öle

23% Zuckergehalt, insgesamt 87%

8 Synthese-Stufen 1

Luftbelastung/Abfälle bis 36% geringer

Energieverbrauch bis 25% geringer

Abwasserbelastung um 2/3 reduziert

reichlich Organische Lösungsmittel keine

Max. 60% Ausbeute 80%


1,3 Propanediol Biosynthesis in E. coli

Glucose

1,3 Propanediol

Glycerin

1,3 Propanediol:building block in production of polymers

OH

OHOH

OH

OH



1,3 Propanediol

Glycerin

OH

OHOH

OH

OH

Microorganisms producing 1,3 PD fromGlycerol feedstock anaerobically (70 g/l):

KlebsiellaCitrobacterClostridumetc.

Nakamura and Whited, 2003, Curr. Opi. Biotechnol. 14, 454-459





Saccharomyces cerevisiae Nakamura and Whited, 2003, Curr. Opi. Biotechnol. 14, 454-459




Source: G. Whited, Genencor, BioTrans 2003

Metabolic / Pathway engineering of E. colilimit growthstreamline carbon flow by engineering/optimisationof pathway and enzymology> 36 target enzymes targeted for yield optimisation

1,3-PD Producer strain in 2003:135 g/ltr 1,3-PD from Glucose50% of carbon source (glucose) is used for 1,3-PD

0,55 mol/mol 1,3-PD from Glycerin0,63 mol/mol 1,3-PD from Glycerin with Glucose cofermentation

250 FTE

Microbial chemical factories !




(DuPont, 2009)

Sorona®

Produktionskapazität pro Jahr: 1,3-Propandiol (PDO)

0

25

50

75

100

98 00 02 04 06 08 Jahr

Prod

uktio

n [ k

t /a]

Shell PDO (Gas, Öl) DuPont PDO (Öl - Degussa)DuPont Bio-PDO (Mais)

Terephtalic acid



Microbial Production of Artemisinin-Precursors

Malaria: fact sheet• WHO: 247 million cases in 2006• kills more the 1 million people (every 30 sec a child)• parasite: Plasmodium falciparum• transmitter: infective female Anopheles mosquito• problem: resistant strains to almost all currently used drug therapies

Plasmodium falciparumfemale Anopheles mosquito




Treatment option: Artemisinin (sesquiterpen-lactone-endoperoxide) => problem: high price (factor 10 - 20), suppy (planting cycles)

Artemisia annuaEinjähriger Beifuss(China, India, Europe)

Artemisinin

Extracted from leave:content 0,1-0,4% DW


S. cerevisiae

??

E. coli



Ca. 150 mg/l AmorphadieneCa. 100 mg/l Artemisinic acid

Ro et al., 2006, Nature, 440, 940-943

1. Increase of Farnesyl-Pyrophosphate (FPP)- leveland decrease of sterol biosynthesis

2. Introduction of the amorphadiene synthase gene(ADS) from A. annua

3. Cloning of the Cytochrom P450 MO from A. annuaà 3-Step-Oxidation of amorphadieneleading to artemisinic acid

1.

2.

3.

JD. Keasling(Berkeley)

Three Steps to Artemisinic acid production in S. cerevisiae




Tsuruta et al., 2009, PLOS 4, e4489

E. coli S. cerevisiae A. annua

E. coli:+ process optimization=> 27,4 g/l amorpha-4,11-diene

mvaS mvaA

S. aureus



Micobial Cellulose (MC) in Biomedical Application

4. Neue Produkte der Weißen Biotechnologie

Acetobacter/Gluconacetobacter xylinum

TEM, Hirai et al., 1999, ICR Annual Report 6, 28-29

Fact sheet:

• A. xylinum (1962), gram -, obligative aerob

• A. xylinum cell polymerizes up to 200.000 Glucose unit /s

• ribbon contains ca. 1000 glucan chains

• metabolically inert, highly pure extracellular deposit

• 4 enzyme functions for cellulose biosynthesisGlucosekinase: Glucose => Glucose 6PPhosphoglucomutase: Glucose 6 P => Glucose 1 PPyrophosphorylase: Glucose 1 P => UDP-GlucoseCellulose-Symthase (membrane): ß-1-4 Glucan

• substrates: glucose, mannitol, xylose

• static culture produce cellulosic surface mat


Micobial Cellulose (MC) in Biomedical Application


Due to physical and mechanical properties MC is an ideal material for high quality wound dressing

Czaja, et al., 2007 Biomacromolecules, 8, 1-12


Micobial Cellulose (MC): Treatment of chronic wounds and burns



Study in Poland (second degree burns)

MC treated wounds:

• healed faster

• excellent molding to the wound surface

• moist environment

• reduces pain

• reduces scar formation


Micobial Cellulose (MC): Cellulose tubes as blood vessel substitute


Fermentation:

• cellulose yield increases withoxygen ratio

• 7 days of fermentation

• silicone tube determines shape


Bodin et al., 2007, Biotech. & Bioeng. 97, 1-12

Mechanical Properties:

• tubes sustain blood pressure of 250 mmHg (up to 880 mm at 100% O2)

Cell seeding:

• confluent layer of endothelial cellsonto lumen after 7 days


5. Zusammenfassung

• Weisse Biotechnologie ist eine der Zukunftstechnologien und kann füreine Zukunft auch mit limitierten fossilen Rohstoffen sorgen(“Biologisierung der Chemieindustrie”)

• Neue Enzyme/Biokatalysatoren werden benötigt und die Metagenomtechnologie erlaubt den Zugang zur vollen Biodiversität

• Moderne Stammentwicklung kann zur effizienten Produktion von Wirkstoffen, Feinchemikalien und Basischemikalien eingesetzt werden

• Der weitere Erfolg der Weißen Biotechnologie hängt von derWirtschaftlichkeit bestimmter Prozesse und der Umsetzung wirklicherInnovationen ab


PD Dr. Stefan Pelzer

[email protected]

www.brain-biotech.de

Vielen Dank für Ihre Aufmerksamkeit!

mailto:[email protected]

http://www.brain-biotech.de

Weiße Biotechnologie – Anwendung von mikrobiellen … · ncp_967849ae78842 z...

Documents

Transcript of Weiße Biotechnologie – Anwendung von mikrobiellen … · ncp_967849ae78842 z...