MOL.911 HNL Expression - TU Graz

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W I S S E N T E C H N I K L E I D E N S C H A F T1MOL.911HNL Expressionuwww.tugraz.at

MOL.911 Molecular Biotechnology I2Hydroxynitrile lyase (Hnl)R1HCNCR1OR2C*R2S-selective: Hevea brasiliensisR-selective: Prunus spp.OHCN

MOL.911 Molecular Biotechnology I3(S)-Hnl of Hevea brasiliensis and(R)-Hnl of Prunus amygdalusPam HnlHb Hnl Type II Hnlintracellular protein29.2 kDahomodimer / hydrolase fold proteincatalytic triad(S)-selektive Type I Hnlsecretory protein61 kDa ( 57.9 kDa)Homology to oxidasesFADN-glycosylatedisoenzymes(R)-selektive

MOL.911 Molecular Biotechnology I43-D structure of Hb HNLK236C81S80H235D207

MOL.911 Molecular Biotechnology IIntracellular Hnl Expressionin Escherichia coli5Translational nLysGluGlu *MetAlaPheHnl ORFmini-cistronPtrcHbHnl ORFpHNL-200TrrnBpSE420

MOL.911 Molecular Biotechnology I6Soluble

MOL.911 Molecular Biotechnology I7Intracellular Hnl Expression inSaccharomyces cerevisiae and Pichia l ORFAAAGATCCCCCGGGCTGCAGGAATTCCATGGCATTCHnl ORFMetAlaPheBamHI/BglIpHILD2PAOXPGKHnl ORFpHNL-400pHNL-300T AOXPGKpMA91

MOL.911 Molecular Biotechnology I8Secretory Hnl Expression inSaccharomyces cerevisiae and Pichia pastorisXbaIGGGGTATCTCTCGAGAAAAGAGAGGCATTCMF 1-leader GlyValSerLeuGluLysArgGluAlaPheHnl ORFpHILD2PAOXPGKHnl ORFP.pastoris AOX1 PromoterpHNL-401S.cerevisiae PGK PromoterpHNL-309T AOXPGKpMA91Vectorbasis

MOL.911 Molecular Biotechnology I9Secretion-targeted Hb Hnl accumulates in thecell peripheryintracellularsecretory Direction of a naturally intracellularly expressed protein into thesecretory pathway leads to accumolation in the cell membrane

MOL.911 Molecular Biotechnology I10Hb-Hnl expression in filamentous fungiP gpdP glapANHNLT trpCHnl ORFpGLAHNLT trpCHnl ORFpUCEHNLP uceaoxHnl ORFpAOXHNLT trpCpGLASS1HNLPglaSSPgpd PaoxPgla Pgla SSgla24Hnl ORFT trpCp***HNL-pyrGgla24Hnl ORFT trpCgpd: A.niger glyceraldehydephosphate dehydrogenasegla: A.awamori glucoamylaseuce: unknown constitutively expressed gene, P.chrysogenumaox: P.chrysogenum alcohol oxidasepyrG

MOL.911 Molecular Biotechnology I11Intracellular Hnl Expression inPenicillium chrysogenum under control of PAOXTermination ProblemAOX: Penicillium chrysogenum Alcohol OxidasepAOXHNLP AOXHnl ORFTTrpCpAOXHNL-TaP AOXHnl ORFTAOXpAOXHNL-TaTP AOXHnl ORFTAOXTTrpC

MOL.911 Molecular Biotechnology I12Southern analysis ofPenicillium chrysogenum PAOX transformants1 2 3 4 5 6 7 8 9 10 11123456789 10 11pAOXHNL-TaTBMultiple ectopicintegrationpAOXHNL-Ta

MOL.911 Molecular Biotechnology INorthern blot analysis of13Penicillium chrysogenum PAOX transformantspAOXHNL-TaT1 2C34 5 6 7 8 9 10 11 12 13 14 151234 5 6 7 8 9 10 11 12 13 14 15Hnl probeCActin probe

MOL.911 Molecular Biotechnology IWestern blot analysis ofPenicillium chrysogenum PAOX transformants14 Protease ProblemsWestern blot with Hnl AntibodyTotal cellular proteins - SDS PAGE11234567823456789 109B97 kD66 kD45 kD31 kD21 kDApAOXHNL-TaT66 kD45 kD31 kD21 kD14 kD97 kD66 kD45 kD31 kD21 kDA14 kD14 kDHnlpAOXHNL-Ta97 kD66 kD45 kD31 kD21 kD14 kDDegradation!

MOL.911 Molecular Biotechnology IExpression analysis of15Penicillium chrysogenum PAOX transformantsHnl ExpressionAOX Promoter Activity TestpAOXHNL-TaTnegative controluidA: Reporter gene,ß-glucuronidaseAOX::uidA fusion2% lactose pH 8Blue colour cleavage of X-Glucoseby ß-glucuronidasev.I.2345678910purified HNL

MOL.911 Molecular Biotechnology I16Gene replacement in Pichia pastoris at AOX1NotI AOX1 promoter HIS4 selection marker AOX1 transcriptiontermination signal 3’ AOX1 region AmpR F1 ori ColE1 replicon5‘PAOX1Gene o.i. TT5‘AOX1NotIHIS4TT3‘AOX13‘Muts

MOL.911 Molecular Biotechnology IPichia pastoris Hb Hnl expression strain 1-17Fed-batch fermentation404003030020200Cell densityMethanolHnl-activity1000050100150time [h]200250100300specific Hnl-activity [U/mg]500Cell density [OD600]17

MOL.911 Molecular Biotechnology IPichia pastoris Hb Hnl expression strain 1-17Fed-batch fermentation18Fermentation time (hours after induction)015 27 29 4963 7987 97 ST 111 119 135 145 151 159 169 194 STSoluble proteins in cell extracts

MOL.911 Molecular Biotechnology IHeterologous Hnl Expression19(shake flask experiments)Construct Hostcytosol purified enzyme total per(U/mg)(U/mg)cultureHevea brasiliensis0.4215-200.5 U/g (leaves)pHNL-200E. coli0.6-0.1 U/ml OD 4pHNL-300S. cerevisiae4.6201.2 U/ml OD 4pHNL-400P. pastoris15.7406.2 U/ml OD 4pANHNLA. niger0.6- 0.1 U/ml nd

20Production of Hb Hnlwith Pichia pastoris Expression System( Fed-Batch Fermentation)Cell wet weight400 g / lCell dry weight100 g / lTotal proteinHnl proteinHnl units56 g / l23 g / l1.4 x 106 / l

21(S)-Hnl of Hevea brasiliensis and(R)-Hnl of Prunus amygdalusPam HnlHb Hnl Type II Hnlintracellular protein29.2 kDahomodimer / hydrolase fold proteincatalytic triad(S)-selektive Type I Hnlsecretory protein61 kDa ( 57.9 kDa)Homology to oxidasesFADN-glycosylatedisoenzymes(R)-selektive

MOL.911 Molecular Biotechnology IPam Hnl5: Secretory Expression ofPrunus amygdalus (R)- Hnl in Pichia pastoris22PAOX1SSHnl ORF Alpha factor signal sequence Muts andMut TransformantsEndo Hd n u3 AOX1HIS4EndoF Host: Pichia pastoris GS115TTuEndo Fn d97 kDa66 kDa39 kDa Functional secretoryexpression27 kDa highly glycosylated21 kDa14 kDa

MOL.911 Molecular Biotechnology I23High-level Expression Clone D1-17 ?Hnl wt great success with expressionSuper expression strain D1-17Standard expression cloneEngineered Hnl Proteins do it the same way !High expression levels wereNot Reproducible !!

MOL.911 Molecular Biotechnology IHigh-level Expression Clone D1-17 ?24ATTATTCGAACGAGGCCATGGCATTCMetAlaPheEcoRIHnl ORFpHNL-400pHILD2P AOXHnl ORFIntracellular Hnl ExpressioninPichia pastorisNot the Problem !Hasslacher et al., Prot.Expr.Purif., 1997T AOX

25Molecular Analysis of Expression StrainSouthern blottingHNL probeAOX1 ProbeStrange Fragments More than one Copy Integrated How ?

MOL.911 Molecular Biotechnology IMolecular Analysis of Expression Strain26 400 bp DeletionHnlΔ 383 bpΔ 29 bpHnlTandem Integration – head to head (divergent)P’AOX1PAOX13 copies of Hnl, 1 standard Integration in AOX1 Locus 2 truncated, in a head to head oriented AOX1 Promoter Fragments

MOL.911 Molecular Biotechnology IExpression Ztotal(-HbHNLwt) single copyD1.17Expression

MOL.911 Molecular Biotechnology I28Specific genomic setupHartner et al., Nucleic Acids Research, 2008, Vol. 36, Specific complex ? Concerted action of activators ? Repression-active site deleted ?

MOL.911 Molecular Biotechnology I29“hh”- Expression vectorGOIColE1 oriCYC1 transcription terminationBglII (2)EcoRV (7578)HindIII (74)1.17 hhaox1 longBglI (7445)ZeocinBglI (676)EM7 promoter1.17 hhaox1 shortpAOX1-“hh“HindIII (1413)TEF1 promoterEcoRV (6610)3 AOX1EcoRI (1484)NdeI (1527)phhAOX 561-HbHNL wtHbHNL wt8568 bpNotI (2269)XmnI (6164)XmnI (2280)3 AOX transcription terminationEheI (5801)EheI (5687)HindIII (2615)EheI (5666)HindIII (2627)XmnI (5160)EcoRV (2785)KpnI (3225)HIS4BglI (3622)XmnI (3728)EheI (3766)KpnI (3837)SalI (4211)

MOL.911 Molecular Biotechnology I30Expression of Hb Hnl mutants in Pichia pastorisSuper expression strain D1-17Standard expression cloneNovel expression clonesModifiedhh-AOX1-based promoterSystemIntracellular ExpressionHnl

MOL.911 Molecular Biotechnology IScreening for High-level Expression31Screening systems based on principle of translational couplingDoes it work in Eukaryotes ?Well known in ProkayotesPAOX1Hb HNLSh ble3’-AOX1ZeocinR5’ CapStopATGStop3’ Poly AStart NNNUGAATGNNN .Hb HnlBicistronic system1 mRNA, 2 ProteinsSh BleVeeresh Juturu, PhD Thesis

MOL.911 Molecular Biotechnology I32Correlating Hnl expression to zeocin resistanceVeeresh Juturu,PhD ThesisIncreasingZeocinConcentrationMD 4.9x104BMMS 4.9x10450 µg/mlBMMS 4.9x104BMMS 4.9x104100 µg/mlBMMS 4.9x104 ,150 µg/ml

MOL.911 Molecular Biotechnology I33Hnl expressionScreen for High Resistance123Check for Expression Level45678A50 mg/L ZeocinB150 mg/L ZeocinCDEControlsF- Single copy D1-17Row A Clones from BMMS 50 µg/ml ZeoRow C Clones from BMMS 150 µg/ml ZeoLane 1D-1F GFP expressing strain (- control)Lane 4D-4F P. pastoris HNL single copy strain ( control)Lane 8D-8F P. pastoris HNL multi copy strain ( control)

343.11.16

10 Hb-Hnl expression in filamentous fungi P gla Hnl ORF T trpC pGLAHNL aox Hnl ORFP T trpC pAOXHNL uce pUCEHNL gla SS gla24 Hnl ORFP T trpC pGLASS1HNL P gpd T trpC pANHNL Hnl ORF P p***HNL T pyrG trpCP gpd P gla aox Hnl ORF gla SS gla24 P -pyrG gpd: A.niger glyceraldehydephosphate dehydrogenase gla: A.awamori glucoamylase uce: unknown constitutively expressed gene, P.chrysogenum

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