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WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en %SCOLA D%NGINYERIA $EPARTAMENT D%NGINYERIA 1UĿMICA "IOLGICA I !MBIENTAL
,IPIDOMICS STUDIES OF RECOMBINANT 0ICHIA PASTORIS FOR IMPROVED RECOMBINANT PROTEIN SECRETION THROUGH CELL ENGINEERING
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!UTHOR .ďRIA !DELANTADO
3UPERVISORS 0AU &ERRER &RANCISCO 6ALERO
+EYWORDS 0ICHIA PASTORIS LIPIDOMICS HYPOXIA CHEMOSTAT CELL ENGINEERING
0ROGRAMA DE $OCTORAT EN "IOTECNOLOGIA $EPARTAMENT D%NGINYERIA 1UĿMICA "IOLGICA I !MBIENTAL %SCOLA D%NGINYERIA 5NIVERSITAT !UTNOMA DE "ARCELONA "ELLATERRA $ESEMBRE
4HIS WORK WAS SUPPORTED BY THE PROJECT #41 2 OF THE 3PANISH -INISTRY OF %CONOMY AND #OMPETITIVENESS AND THE GRANT 0)& OF THE !UTONOMUS 5NIVERSITY OF "ARCE LONA 5!" 4HE GROUP IS MEMBER OF 3'2 AND THE 2EFERENCE .ETWORK IN "IOTECHNOLOGY 82" 'ENERALITAT DE #ATALUNYA
0ART OF THE MANUSCRIPT ANALYSIS WERE PERFORMED IN THE #ELLULAR "IOLOGY ,ABORATORY OF THE )NSTITUTE OF "IOCHEMISTRY AT 'RAZ 5NIVERSITY OF 4ECHNOLOGY UNDER THE SUPERVISION OF 0ROF $R 'ķNTHER $AUM WHO WE WOULD LIKE TO THANK HIS COLLABORATION 7E WOULD ALSO LIKE TO THANK )VO &EUSSNER FOR THE POSSIBILITY TO ANALYSE THE SPHINGOLIPID CONTENT AND 0ABLO 4ARAZONA FOR KINDLY PERFORM THE ANALYSES JJJ
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4HE INCREASING AVAILABILITY OF OMICS DATABASES PROVIDES AN IMPORTANT KNOWLEDGE BASE FOR THE DESIGN OF NOVEL YEAST ENGINEERING STRATEGIES SINCE THEY OFFER SYSTEMS LEVEL INFORMATION ON THE PHYSIOLOGY OF THE CELLS UNDER DIVERSE GROWTH CONDITIONS AND GENETIC BACKGROUNDS )N A PREVIOUS STUDY A BENElCIAL EFFECT OF LOW OXYGEN AVAILABILITY ON THE EXPRESSION OF A HUMAN &AB FRAGMENT IN 0ICHIA PASTORIS HAS BEEN IDENTIlED 4RANSCRIPTOMIC ANALYSES POINTED OUT IMPORTANT REGULATION OF THE 5NFOLDED 0ROTEIN 2ESPONSE 502 AND LIPID METABOLISM WITH A SIGNIlCANT TRANSCRIPTIONAL UP REGULATION OF ERGOSTEROL AND SPHINGOLIPID BIOSYNTHETIC PATHWAYS &URTHERMORE CELLS CULTURED IN THE PRESENCE OF mUCONAZOLE AN ANTIFUNGAL AGENT THAT INHIBITS STEROL # DEMETHYLASE %RGP ACTIVITY IN THE ERGOSTEROL BIOSYNTHETIC PATHWAY SHOWED A REDUCED AMOUNT OF ERGOSTEROL IN THE PLASMA MEMBRANE RESULTING IN INCREASED PROTEIN SECRETION LEVELS -OREOVER IT IS KNOWN THAT THE TRANSCRIPTION FACTOR (AC WHICH PLAYS A KEY ROLE BY REGULATING THE 502 REGULATES LIPID BIOSYNTHESIS )N THE PRESENT STUDY A REFERENCE STRAIN OF 0 PASTORIS SECRETING THE & &AB ANTIBODY FRAGMENT AS A MODEL RECOMBINANT PROTEIN WAS GENETICALLY MODIlED IN ORDER TO CHANGE ITS LIPID COMPOSITION ! SET OF STRAINS HARBOURING EITHER DELETED $%3 352 OR OVEREXPRESSED $%3 GENES OF THE SPHINGOLIPID PATHWAY WERE GENERATED )N ADDITION A &AB PRODUCING STRAIN OVEREXPRESSING %2' WAS CONSTRUCTED AND AS AN ALTERNATIVE TO THE ERGOSTEROL PATHWAY KNOCKOUT THE REFERENCE STRAIN WAS TREATED WITH mUCONAZOLE &INALLY THE SPLICED FORM OF (!# WAS OVEREXPRESSED AS A STRATEGY TO DEREGULATE THE LIPID METABOLISM NETWORK 4HE SERIES OF STRAINS WERE CULTIVATED IN CHEMOSTAT CULTURES UNDER NORMOXIC AND HYPOXIC CONDITIONS 3TRAINS WERE CHARACTERISED IN TERMS OF &AB PRODUCTIVITY INTRAEXTRACELLULAR PRODUCT DISTRIBUTION CELL MORPHOLOGY BY TRANSMISSION ELECTRON MICROSCOPY 4%- AND LIPID CONTENT IN TERMS OF PHOSPHOLIPIDS FATTY ACIDS STEROLS NON POLAR LIPIDS AND SPHINGOLIPIDS IN ORDER TO FULLY UNDERSTAND HOW CELLS ADAPT TO GENETIC AND ENVIRONMENTAL CHANGES INVOLVING LIPID CHANGES 4HE OBTAINED RESULTS WERE COMBINED WITH PREVIOUSLY PUBLISHED TRANSCRIPTIONAL DATA ALLOWING US TO DEMONSTRATE AN IMPORTANT REMODELLING OF THE LIPID METABOLISM UNDER LIMITED OXYGEN AVAILABILITY
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5SBOTDSJQUPNJD BOBMZTJT )NTRODUCTION $.! -ICROARRAYS #LUSTER ANALYSIS 4HE IMPACT OF OXYGEN ON THE TRANSCRIPTOME OF RECOMBINANT 0 PASTORIS 2ESULTS AND DISCUSSION K MEANS CLUSTERING 'ENE MAPPING 'ENE SELECTION #ONCLUSIONS
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2ESULTS AND DISCUSSION $ETERMINATION THE OPTIMAL mUCONAZOLE TREATMENT FOR &AB & SECRETION #ELL DISRUPTION AND PROTEIN EXTRACTION 3CREENING FOR &AB & EXPRESSION ,IPID COMPOSITION #ONCLUSIONS
#JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO )NTRODUCTION "IOREACTOR CULTIVATIONS 9EAST LIPID COMPOSITION 2ESULTS AND DISCUSSION %STABLISHING HYPOXIC CONDITIONS IN CHEMOSTAT CULTURES #HEMOSTAT CULTIVATIONS )NTRA EXTRACELLULAR DISTRIBUTION OF THE &AB #ELL LIPID COMPOSITION &ED "ATCH CULTURES #ONCLUSIONS
*OUFHSBUFE BOBMZTJT )NTRODUCTION %FFECT OF HYPOXIA IN &AB& STRAIN %FFECT OF HYPOXIA ON ENGINEERED STRAINS %FFECT OF STRAIN ENGINEERING THROUGH SPHINGOLIPID PATHWAY %NGINEERING NEUTRAL SPHINGOLIPID PATHWAY THROUGH $%3 !LTERING INOSITOL CONTAINING SPHINGOLIPIDS PATHWAY 352 %FFECT OF STRAIN ENGINEERING THROUGH ERGOSTEROL PATHWAY %FFECT OF STRAIN ENGINEERING THROUGH (AC TRANSCRIPTION FACTOR #ONCLUSIONS
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᎙ SPECIlC GROWTH RATE HᎽᎳ ᎽᎳ ᎙ᑞᑒᑩ MAXIMUM SPECIlC GROWTH RATE H !/8 !LCOHOL OXIDASE GENE BP BASE PAIR #(!03 ; #HOLAMIDOPROPYL DIMETHYLAMMONIO= PROPANESULFONATE #, CARDIOLIPIN $ DILUTION RATE $!' DIACYLGLYCEROL $#7 DRY CELL WEIGHT G ,ᎽᎳ $-3/ DIMETHYL SULFOXIDE $/ DISSOLVED OXYGEN $0( DIPHENYL HEXATRIENE $44 $ITHIOTHREITOL %$4! ETHYENDIAMINETETRAACETATE %,)3! IMMUNOASSAY %2 ENDOPLASMIC RETICULUM %2!$ ENDOPLASMATIC RETICULUM ASSOCIATED DEGRADATION %RG ERGOSTEROL EXP EXPONENTIAL &! FATTY ACID &AB ANTIGEN BINDING FRAGMENT &LUCO mUCONAZOLE &3 FREE STEROL '!0 'LYCERALDEHYDE PHOSPHATE GENE '# GAS CHROMATOGRAPHY '# -3 GAS CHROMATOGRAPHY MASS SPECTROMETRY 'LC GLUCOSE 'LY GLYCEROL (%0%3 ; HYDROXYETHYL PIPERAZIN YL=ETHANESULFONIC ACID (0,# HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (YG HYGROMYCIN )-& INSOLUBLE MEMBRANE FRACTION )0# INOSITOL 0 CERAMIDE +/ KNOCK OUT ," ,URIA "ROTH ,$ LIPID DROPLETS -)0# MANNOSYL INOSITOL 0 CERAMIDE
-)0 Ꮄ# DIMANNOSYL INOSITOL 0 CERAMIDE /% OVEREXPRESSION /$ OPTICAL DENSITY 0! PHOSPHATIDIC ACID 0#2 POLYMERASE CHAIN REACTION 0% PHOSPHATIDYLETHANOLAMINE 0) PHOSPHATIDYLINOSITOL
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0, PHOSPHOLIPID 0-3& PHENYLMETHYLSULFONYL mUORIDE 03 PHOSPHATIDYLSERINE ᎽᎳ ᎽᎳ QP SPECIlC PRODUCT PRODUCTION RATE MMOLᑇ G$#7 H 21 RESPIRATORY COEFlCIENT 23$ RELATIVE STANDARD DEVIATION 24 ROOM TEMPERATURE 3#& SOLUBLE CYTOSOLIC FRACTION 3$ STANDARD DEVIATION 3$3 SODIUM DODECYL SULFATE 3% STERYL ESTER 3. SUPERNATANT 3, SPHINGOLIPID 4!% 4RIS !CETATE %$4! 4%- TRANSMISSION ELECTRON MICROSCOPY 4' TRIACYLGLYCEROL 4,# 4HIN ,AYER #HROMATOGRAPHY 4RIS 4RISHYDROXYMETHYL AMINOMETHANE 502 5NFOLDED 0ROTEIN 2ESPONSE 9." YEAST NITROGEN BASE 90$ YEAST PEPTONE DEXTROSE :EO :EOCIN
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1JDIJB QBTUPSJT BT B DFMM GBDUPSZ GPS SFDPNCJOBOU QSPUFJO QSPEVDUJPO 4HE METHYLOTROPHIC YEAST 0ICHIA PASTORIS IS ONE OF THE MOST EFFECTIVE AND VERSATILE SYSTEMS FOR THE EXPRESSION OF HETEROLOGOUS PROTEINS ;n= 0 PASTORIS EXPRESSION SYSTEM HAS GAINED IMPORTANCE FOR INDUSTRIAL APPLICATION AS STAND OUT BY THE NUMBER OF PATENTS PUBLISHED ;= AND THE WIDE RANGE OF PROTEIN PRODUCTS THAT HAD ENTERED THE MARKET FOR THERAPEUTIC AND INDUSTRIAL USES IN THE LAST YEARS ;= 0 PASTORIS AS A #RABTREE NEGATIVE YEAST HAS A STRONG PREFERENCE FOR RESPIRATORY GROWTH EVEN UNDER GLUCOSE EXCESS 4HIS FACT LIMITS THE FORMATION OF ETHANOL AND ACETATE INCREASES THE YIELD IN BIOMASS FROM GLYCEROL OR GLUCOSE AND ENABLES OBTAINING HIGHLY CONCENTRATED CELL BROTHS G ,ዅኻ $#7 ; = 4HIS YEAST HAS ALSO THE ABILITY TO PERFORM SOME EUKARYOTIC POST TRANSLATIONAL MODIlCATIONS SUCH AS PROCESSING SIGNAL SEQUENCES FOLDING DISULPHIDE BOND FORMATION PROTEOLYTIC PROCESSING SOME TYPES OF LIPID ADDITION AND / AND . LINKED GLYCOSILATION 0 PASTORIS HAS BEEN ENGINEERED IN ORDER TO MIMIC THE HUMAN . GLYCOSYLATION PATHWAY AND MORE RECENTLY SPECIlC TYPES OF / GLYCOSYLATION BECOMING A POTENTIAL ALTERNATIVE FOR MAMMALIAN CELL CULTURE FOR THE PRODUCTION OF RECOMBINANT THERAPEUTIC GLYCOPROTEINS FOR HUMAN USE ; = 4ECHNIQUES FOR GENETIC MODIlCATIONS SIMILAR TO THOSE DESCRIBED FOR 3 CEREVISIAE ARE AVAILABLE AND EXPRESSION VECTORS METHODS FOR TRANSFORMATION SELECTABLE MARKERS AND FERMENTATION PROCESSES HAVE BEEN DEVELOPED TO EXPLOIT ITS PRODUCTIVE POTENTIAL ; = !LONG WITH THESE FAVOURABLE PHYSIOLOGIC CHARACTERISTICS THE AVAILABILITY OF STRONG AND TIGHTLY REGULATED PROMOTERS MAKES THIS YEAST AN INTERESTING HOST FOR RECOMBINANT PROTEIN PRODUCTION 4HE PROMOTER OF THE ALCOHOL OXIDASE ) GENE !/8 HAS BEEN MAINLY EMPLOYED DUE TO ITS STRENGTH AND TIGHT REGULATION BY CARBON SOURCES ;= 4HIS PROMOTER IS REPRESSED BY GLUCOSE AND GLYCEROL AND HIGHLY INDUCED BY METHANOL )NTERESTING ALTERNATIVES TO THE !/8 PROMOTER FOR RECOMBINANT PROTEIN PRODUCTION ARE THE GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE '!0 ;= AND GLUTATHIONE DEPENDENT FORMALDEHYDE DEHYDROGENASE &,$ ;= PROMOTERS '!0 IS INVOLVED IN GLYCOLYSIS AND GLUCONEOGENESIS AND IS A STRONG CONSTITUTIVE PROMOTER ;= THAT
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POSSESSES ITS HIGHEST EXPRESSION LEVELS WHEN USING GLYCEROL AND GLUCOSE AS SUBSTRATES ; = 4HE '!0 PROMOTER SYSTEM IS SUITABLE FOR LARGE SCALE PRODUCTION AS THE NEED TO CHANGE FEEDING SOLUTIONS AND lRE AND SAFETY CONCERNS RELATED TO METHANOL ARE AVOIDED
2ECOMBINANT PROTEIN PRODUCTION IN 0 PASTORIS CAN BE PERFORMED IN SALT BASED MEDIA WITH LITTLE RISK OF CONTAMINATION ; = .OWADAYS 0 PASTORIS HAS GAINED MORE AND MORE SUCCESS FOR RECOMBINANT PROTEIN PRODUCTION BY MEANS OF QUANTITY AND QUALITY ESPECIALLY AFTER THE ACHIEVEMENT IN GENETIC ENGINEERING TO MAKE IT ABLE TO PRODUCE HUMAN TYPE . GLYCOSYLATED PROTEINS ;n= 0ROTEINS CAN BE PRODUCED INTRACELLULARLY OR SECRETED 0ROTEIN SECRETION PERMITS SIMPLE PURIlCATION STRATEGIES DUE TO THE RELATIVELY LOW LEVELS OF ENDOGENOUS PROTEINS IN THE EXTRACELLULAR MEDIUM ;= ! VARIETY OF PROTEINS HAVE BEEN EXPRESSED WITH YIELDS UP TO G ,ዅኻ OF SECRETED PROTEIN ;= AND UP TO G ,ዅኻ INTRACELLULAR PROTEIN ;= )N THE lRST BIOPHARMACEUTICAL PROTEIN A +ALLIKREIN INHIBITOR +ALBITORLj PRODUCED IN 0 PASTORIS WAS APPROVED BY THE &$! ;= 3INCE THEN SEVERAL BIOPHARMACEUTICAL PRODUCTS PRODUCED IN 0 PASTORIS HAVE BEEN APPROVED FOR HUMAN USE ; =
2ECENTLY 0 PASTORIS GENOME SEQUENCE HAS BEEN PUBLISHED ;n= INCREASING THE GENETIC AND PHYSIOLOGIC UNDERSTANDING OF THIS YEAST AND ENABLING NEW STUDIES FOR STRAIN IMPROVEMENT USING MORE RATIONAL APPROACHES
#PUUMFOFDLT EVSJOH SFDPNCJOBOU QSPUFJO TFDSFUJPO 4HE PROCESS OF PROTEIN SECRETION INVOLVES PROTEIN CONVERSION TO MATURE FORM AND ITS TRAFlC TO THE CORRECT LOCATION 4HIS PROCESS INVOLVES SEVERAL STEPS CATALYSED BY A LARGE NUMBER OF PROTEINS AND THE PASS THROUGH SEVERAL DIFFERENT ORGANELLES UNTIL THE lNAL DESTINATION &IGURE ; =
&IGURE /VERVIEW OF THE SECRETORY MACHINERY IN YEAST ;= 3OME TARGET GENES FOR 0 PASTORIS ENGINEERING FOR ENHANCEMENT PROTEIN SECRETION ARE REPRESENTED
%XPRESSION OF HETEROLOGOUS PROTEINS CAN LEAD TO SATURATION OR OVERLOADING OF THE SECRETORY PATHWAY ; = 7HEN RECOMBINANT PROTEIN FAILS TO FOLD INTO ITS NATIVE STATE OR PROTEIN EXPRESSION EXCEEDS THE FOLDING CAPACITY OF THE ENDOPLASMIC RETICULUM %2 ;= UNFOLDED PROTEINS MAY START TO AGGREGATE TRIGGERING THE UNFOLDED PROTEIN RESPONSE 502 PATHWAY WHICH IS RESPONSIBLE FOR INDUCTION OF GENES THAT ARE INVOLVED IN PROTEIN FOLDING )N PARALLEL %2 ASSOCIATED DEGRADATION %2!$ BY THE PROTEASOME IS ALSO ACTIVATED TO ELIMINATE MISFOLDED PROTEINS;= *NQBDU PG PYZHFO BWBJMBCJMJUZ PO DFMM QIZTJPMPHZ BOE SFDPNCJOBOU QSPUFJO QSPEVDUJPO
4HE MOST IMPORTANT BOTTLENECKS IN TERMS OF RECOMBINANT PROTEIN PRODUCTION AND SECRETION ARE MEMBRANE TRANSLOCATION SIGNAL PEPTIDE PROCESSING AND FOLDING WITHIN THE %2 ;= #OMMONLY USED STRATEGIES TO OVERCOME SUCH SECRETION BOTTLENECKS COMPRISE ENGINEERING THE PROTEIN FOLDING AND QUALITY CONTROL SYSTEMS IN THE %2 ;n= ENGINEERING THE INTRACELLULAR PROTEIN TRAFlCKING PATHWAY ; = MINIMISATION OF POST SECRETORY DEGRADATION ;= ENGINEERING GLYCOSYLATION ;= AND THE ENERGY METABOLISM ;=
! WIDE RANGE OF STRATEGIES HAS BEEN IMPLEMENTED TO ENGINEER 0 PASTORIS AND TO OPTIMISE PROTEIN SYNTHESIS AND SECRETION 3OME OF THEM CONSIST OF NEW INDUCTION METHODS TO TIGHTLY TUNE EXPRESSION LEVELS USING NEW PROMOTERS ; = CELL ENGINEERING METHODS SUCH AS THE INCREMENT OF COPY NUMBER ;= DELETION OF PROTEASES ; = COEXPRESSION OF HELPER FACTORS ; n= OR GLYCOSILATION ;= !PPROACHES OF CODON OPTIMISATION OF THE EXPRESSED GENES HAVE ALSO BEEN USED ;= !DDITIONALLY OPERATIONAL BIOREACTOR STRATEGIES HAVE ALSO BEEN DEVELOPED TO INCREASE PROTEIN PRODUCTION ;n= LIKE GROWTH CONTROL ; = MEDIUM SUPPLEMENTATION WITH CASAMINOACIDS ;= SURFACTANTS ;= OR TRACE SALTS ;= LOWER CULTURE TEMPERATURE ; = OR OXYGEN LIMITATION ; = %ACH OF THE FACTORS COULD ENHANCE PRODUCTION LEVELS OF PROTEIN AND A PROPER COMBINATION WILL REACH AN OPTIMAL PRODUCTION THROUGH REPEATED TRIALS
*NQBDU PG PYZHFO BWBJMBCJMJUZ PO DFMM QIZTJPMPHZ BOE SFDPNCJOBOU QSPUFJO QSPEVDUJPO %NVIRONMENTAL CONDITIONS HAVE A SIGNIlCANT IMPACT ON RECOMBINANT PROTEIN LEVELS )T HAS BEEN DESCRIBED THAT LOWER CULTURE TEMPERATURES ; = LOWER OXYGEN AVAILABILITY ;= AS WELL AS ADEQUATE SUBSTRATE FEEDING STRATEGIES IN HIGH CELL DENSITY CULTURES ; n= AND TYPE OF CARBON SOURCES ; n= RESULT IN A POSITIVE EFFECT ON PROTEIN SECRETION 7ITH THE PUBLISHED SEQUENCE OF 0PASTORIS TRANSCRIPTOMIC AND PROTEOMIC RESPONSE TO SOME OF THESE ENVIRONMENTAL FACTORS HAVE BEEN INVESTIGATED ; = /XYGEN AVAILABILITY IS CRITICAL FOR MANY BIOCHEMICAL REACTIONS IN EUKARYOTIC CELLS INCLUDING YEASTS 4HE ABILITY TO ADAPT TO OXYGEN LIMITATION IS ESSENTIAL FOR CELL SURVIVAL BUT ALSO PRODUCES IMPORTANT METABOLIC FUNCTIONAL AND STRUCTURAL CHANGES IN THE CELL ;= #ELLS CAN ADAPT TO GROWTH UNDER SEVERE OXYGEN LIMITATION TERMED HYPOXIA OR MICROAEROBIC CONDITIONS ;= 7HEN CELLS ARE GROWN AEROBICALLY MOLECULAR OXYGEN SERVES AS THE lNAL ELECTRON ACCEPTOR FOR RESPIRATION WHILE IT IS ALSO USED IN SYNTHESIS OF METABOLITES EG STEROLS OR UNSATURATED FATTY ACIDS ;= )N THE PRESENCE OF LOW AMOUNTS OF OXYGEN RESPIRATION IS DRASTICALLY REDUCED AND METABOLISM IS THEN REPROGRAMMED TO OPTIMISE YEAST CELLS FOR FERMENTATIVE DISSIMILATION OF THE CARBON SOURCE IN ORDER TO CONSERVE ENERGY AND MAINTAIN A CLOSED REDOX BALANCE ;= 4HESE METABOLIC REARRANGEMENTS ARE EASILY DETECTABLE BY THE EXCRETION OF METABOLITES IN THE CULTURE SUPERNATANT SUCH AS ETHANOL AND ARABITOL IN THE CASE OF 0 PASTORIS ;= 4HE IMPACT OF OXYGEN SUPPLY ON THE HETEROLOGOUS PRODUCTION HAS BEEN STUDIED FOR DIFFERENT HOSTS /XYGEN LIMITATION IN % COLI CULTIVATIONS TRIGGERS CELLULAR STRESS AND FORMATION OF BY PRODUCT SUCH AS ACETATE WHICH INHIBITS CELL GROWTH AND RECOMBINANT PROTEIN PRODUCTION ; = )N 3ACCHAROMYCES CEREVISIAE IT AN INCREASE ON RECOMBINANT PROTEIN PRODUCTION UNDER OXYGEN LIMITATION WAS OBSERVED ; = 4HE IMPACT OF OXYGEN LIMITATION ON 0 PASTORIS RECOMBINANT PROTEIN PRODUCTION USING THE '!0 PROMOTER AND GLUCOSE AS CARBON SOURCE WAS lRST STUDIED BY "AUMANN AND CO WORKERS ;= SHOWING A SIGNIlCANT INCREASE OF THE SPECIlC PRODUCTION RATE )N SUBSEQUENT STUDIES THE IMPACT OF OXYGEN AVAILABILITY ON THE PHYSIOLOGY OF RECOMBINANT 0 PASTORIS WAS STUDIED INTEGRATING (FOFSBM *OUSPEVDUJPO
TRANSCRIPTOMIC PROTEOMIC METABOLIC mUX AND METABOLOMIC ANALYSES ; = 4HESE STUDIES POINTED OUT TO POTENTIAL GENES AND METABOLIC PATHWAYS THAT MIGHT BE POTENTIAL TARGETS FOR STRAIN IMPROVEMENT )T WAS OBSERVED A STRONG TRANSCRIPTIONAL INDUCTION OF GLYCOLYSIS AND THE NON OXIDATIVE PENTOSE PHOSPHATE PATHWAYS AS WELL AS THE DOWNREGULATION OF THE 4#! CYCLE UNDER HYPOXIC CONDITIONS 4HESE TRANSCRIPTIONAL CHANGES WERE CONSISTENT WITH PROTEOMIC DATA AND METABOLIC mUXES #ELL STRESS RESPONSES SPECIALLY 502 AND LIPID METABOLISM WERE ALSO STRONGLY AFFECTED IN HYPOXIA 4HE BIOSYNTHESIS OF MEMBRANE COMPONENTS LIKE ERGOSTEROL AND SPHINGOLIPIDS INCLUDE A WIDE RANGE OF OXYGEN DEPENDENT REACTIONS SO THAT ALTERATIONS IN THE LIPID BALANCE UNDER OXYGEN SCARCITY WOULD BE EXPECTED
:FBTU MJQJET 4ODAY LIPIDS ARE CONSIDERED AS IMPORTANT DYNAMIC MOLECULES THAT PLAY VITAL ROLES THAT EXTEND BEYOND THEIR ROLES IN MEMBRANE STRUCTURE &OR THIS REASON THEY ARE THE FOCUS OF INTEREST OF SEVERAL STUDIES 4HE ULTIMATE GOAL IS TO EVOLVE AN INTEGRATED OMICS PICTURE THE vINTERACTOMEv OF THE GENES TRANSCRIPTS PROTEINS AND METABOLITES TO FULLY DESCRIBE CELLULAR FUNCTIONING 4HE GOAL OF LIPIDOMICS IS TO DElNE AND QUANTIFY ALL THE LIPID SPECIES IN A CELL 4HIS IS DIFlCULT TO ACHIEVE DUE TO THE HIGH NUMBER OF COMBINATIONS THAT EXIST AND HIGH VARIABILITY OF LIPID MOIETIES THAT ARE KNOWN ;= 4HE USE OF ADVANCED MASS SPECTROMETRY -3 TECHNIQUES ;= ALLOWED ANALYSING HUNDREDS OF LIPID MOLECULAR SPECIES IDENTIFYING AND QUANTIFYING THEM 3TUDIES OF CELL LIPIDOME PROVIDE A NEW PLATFORM TO BE INTEGRATED WITH THE EXISTING OMICS PLATFORMS EG PROTEOME TRANSCRIPTOME METABOLOME TO STUDY CELLULAR FUNCTIONS AND REGULATION 5P TO DATE SOME STUDIES INTEGRATING SOME OF THESE PLATFORMS ARE ALREADY AVAILABLE IN YEAST WITH PROMISING RESULTS ;n=
'BUUZ BDJET 'BUUZ BDJE TUSVDUVSF BOE GVODUJPO &ATTY ACIDS ARE THE BASIC COMPONENTS OF COMPLEX LIPIDS &REE FATTY ACIDS &! ARE CARBOXYLIC ACIDS WITH HYDROCARBON CHAINS VARYING IN CHAIN LENGTH )N SOME FATTY ACIDS THIS CHAIN IS UNBRANCHED AND FULLY SATURATED IE CONTAINS NO DOUBLE BONDS WHILE IN OTHERS THE CHAIN IS UNSATURATED IE CONTAINS ONE OR MORE DOUBLE BONDS &IGURE ;= 4HESE VARIATIONS LEAD TO THE LARGE VARIETY OF FATTY ACID MOLECULES TO SYNTHESISE MANY OTHER DIFFERENT LIPID SPECIES
&IGURE 2EPRESENTATION OF SATURATED AND UNSATURATED FATTY ACIDS
4HE FATTY ACID COMPOSITION OF 0 PASTORIS DIFFERS FROM THE ONE OBSERVED IN 3 CEREVISIAE AS IT POSSESSES POLYUNSATURATED FATTY ACIDS 3 CEREVISIAE MAJOR FATTY ACID IS PALMITOLEIC ACID # AND OLEIC ACID # FOLLOWED BY PALMITIC ACID # AND STEARIC ACID # ;= /LEIC ACID # LINOLEIC ACID # AND LINOLENIC ACID # AND PALMITIC ACID # ARE THE MOST ABUNDANT FATTY ACIDS IN 0 PASTORIS ;= :FBTU MJQJET
&UNCTIONALITY OF CELLULAR MEMBRANES AND ACTIVITY OF MEMBRANE BOUND PROTEINS ARE DEPENDENT ON THE &! COMPOSITION OF MEMBRANE LIPIDS 4O ESTABLISH AND MAINTAIN THE CORRECT COMPOSITION # AND # SATURATED &! 3&! DERIVED FROM DE NOVO SYNTHESIS ARE DESATURATED YIELDING MONO OR POLYUNSATURATED &! 5&! AND ELONGATED TO VERY LONG CHAIN FATTY ACIDS UP TO # ;= &ATTY ACID mUXES ARE IMPORTANT TO MAINTAIN MEMBRANE LIPID HOMEOSTASIS
4HE MAIN FUNCTION OF UNSATURATED FATTY ACIDS IS TO MODULATE THE PHYSICAL PROPERTIES OF MEMBRANES ESPECIALLY mUIDITY 4HUS THE FATTY ACYL COMPOSITION OF MEMBRANES VARIES IN RESPONSE TO ENVIRONMENTAL STRESS SUCH AS TEMPERATURE ETHANOL CONCENTRATION ;= NUTRITION STATUS AND OTHER FACTORS ;= 3EVERAL FUNCTIONS ARE ASSOCIATED TO FATTY ACIDS INCLUDING A ROLE AS SIGNALLING MOLECULES ;= TRANSCRIPTIONAL REGULATORS ;= AND POSTTRANSLATIONAL MODIlERS OF PROTEINS ; = &!S PLAY ALSO A ROLE ON CELL SECRETION AND PROVIDING ENERGY TROUGH THE OXIDATION PATHWAY ;= 6ERY LONG CHAIN FATTY ACIDS 6,#&! ACCOUNT ONLY FOR A OF THE CELLULAR FATTY ACID CONTENT IN 3 CEREVISIAE ;= BUT PLAY ESSENTIAL ROLES IN THE CELL ESTABLISHING OR LENGTHENING CURVED MEMBRANE STRUCTURES AND ALSO DElNING THE MEMBRANE CURVATURE AT THE NUCLEAR PORE COMPLEX ;= 4HEY ALSO SERVE SPECIlC STRUCTURAL FUNCTIONS AS COMPONENTS OF SPHINGOLIPIDS AND '0) ANCHORS
'BUUZ BDJE CJPTZOUIFUJD QBUIXBZ 9EASTS CAN GET THEIR FATTY ACIDS BY DIFFERENT WAYS THAT ARE REPRESENTED IN &IGURE $E NOVO SYNTHESIS OF FATTY ACIDS TAKES PLACE IN THE CYTOSOL AND IN MITOCHONDRIA WHERE AN ACETYL #O! DERIVED FROM CITRATE DEGRADATION OR FROM ACETATE IS CARBOXYLATED TO FORM MALONYL #O! WHICH SERVES AS A TWO CARBON BUILDING BLOCK IN THE FOLLOWING &! SYNTHESIS REACTIONS &URTHER ELONGATION STEPS TO VERY LONG CHAIN &! 6,#&! UP TO # IS CARRIED OUT IN THE ENDOPLASMIC RETICULUM $ESATURATION AND HYDROXYLATION OF &! ALSO TAKE PLACE IN THE %2 &ATTY ACID SYNTHESIS IS RESTRICTED TO CONDITIONS OF HIGH ENERGY LOAD OF THE CELLS ;=
exogenous fatty acid uptake
lipid free fatty acid lipid turnover turnover
protein SE acylation acyl-CoA TG SL elongation PL degradation de novo FA desaturation synthesis hydroxylation
&IGURE 3CHEMATIC REPRESENTATION OF FATTY ACID METABOLISM !DAPTED FROM ;= 3% STERYL ESTER 4' TRIACYLGLYCEROL 3, SPHINGOLIPID 0, PHOSPHOLIPID &! FATTY ACID
9EAST CELLS CAN ALSO TAKE UP FATTY ACIDS FROM THE ENVIRONMENT BY DIFFUSION OR THROUGH SPECIlC TRANSPORTERS 4HE UPTAKE STEP IS FOLLOWED BY THEIR ACTIVATION AND ACTIVE &! CAN BE USED FOR THE SYNTHESIS OF COMPLEX LIPIDS STORED IN FORM OF TRIACYLGLYCEROLS OR DEGRADED IN PEROXISOMES BY OXIDATION &INALLY LIPID AND PROTEIN TURNOVER ALSO PROVIDE A POOL OF FREE FATTY ACIDS THAT SHOULD BE ACTIVATED TO ACYL #O! IN ORDER TO BE FUNCTIONAL FOR THE CELL (FOFSBM *OUSPEVDUJPO
1IPTQIPMJQJET 1IPTQIPMJQJE TUSVDUVSF BOE GVODUJPO 0HOSPHOLIPIDS 0, ARE THE MOST ABUNDANT MEMBRANE LIPIDS ;= 0, ARE FORMED BY AN HYDROPHOBIC MOIETY COMPOSED BY TWO FATTY ACIDS ATTACHED THROUGH AN ESTER BOND TO THE lRST AND SECOND CARBONS OF GLYCEROL AND BY AN HYDROPHILIC MOIETY THAT CONTAINS A HIGHLY POLAR OR CHARGED GROUP ATTACHED THROUGH A PHOSPHODIESTER LINKAGE TO THE THIRD CARBON OF GLYCEROL &IGURE ;=
Polar head Non-polar tails
Phosphate group
&IGURE 'ENERAL REPRESENTATION OF PHOSPHOLIPID 0, STRUCTURE AND THE SHAPE OF EACH 0, CLASS ;=
!S AMPHIPATHIC MOLECULES 0, CAN FORM LIPID BILAYERS AND ARE THE PRECURSORS OF MEMBRANES 0, ARE SUBDIVIDED ACCORDING TO THEIR POLAR HEAD GROUP WHICH DETERMINES THEIR PHYSICAL PROPERTIES AND ALSO CONFERS A STRUCTURAL SHAPE 4HE HEAD GROUP OF PHOSPHATIDYLCHOLINE 0# AND PHOSPHATIDYLETHANOLAMINE 0% IS ZWITTERIONIC AND ANIONIC IN PHOSPHATIDYLINOSITOL 0) PHOSPHATIDYLSERINE 03 PHOSPHATIDIC ACID 0! AND CARDIOLIPIN #, 4HE DISTRIBUTION OF THESE 0, VARIES DEPENDING ON THE ORGANELLE -AJOR PHOSPHOLIPIDS IN TOTAL CELL EXTRACTS FROM 3 CEREVISIAE ARE PHOSPHATIDYLCHOLINE 0# PHOSPHATIDYLETHANOLAMINE 0% PHOSPHATIDYLINOSITOL 0) AND PHOSPHATIDYLSERINE 03 ;= WHILE IN 0 PASTORIS 0# 0% AND SIMILAR LEVELS OF 03 AND 0) ARE THE MAJOR PHOSPHOLIPIDS ;= 0HOSPHOLIPID COMPOSITION CAN VARY DRAMATICALLY ACCORDING TO CULTURE CONDITIONS SUCH AS TEMPERATURE ;= CARBON SOURCE ; = OR OXYGEN SUPPLY TO THE CULTURE ;= )N ADDITION TO ITS STRUCTURAL ROLE AS COMPONENTS OF CELLULAR MEMBRANES 0, ARE INVOLVED IN MULTITUDE OF CELLULAR FUNCTIONS 4HEY ACT AS RESERVOIR OF LIPID MESSENGERS ;= PROVIDE PRECURSORS FOR THE SYNTHESIS OF OTHER MOLECULES ; = ARE INVOLVED IN ACTIVITY OF MEMBRANE PROTEINS ;n = IN lSSION AND FUSION EVENTS ;= THE TRANSPORT TO PLASMA MEMBRANE ;= MAINTAINING THE MITOCHONDRIAL STRUCTURE AND FUNCTION ;= AND M2.! LOCALISATION ;=
1IPTQIPMJQJE CJPTZOUIFUJD QBUIXBZ 0HOSPHOLIPID BIOSYNTHESIS IS SIMILAR FOR ALL EUKARYOTES &ATTY ACIDS ARE INCORPORATED INTO PHOSPHOLIPIDS BY SEQUENTIAL TRANS ESTERIlCATION OF TWO ACYL #O! MOLECULES TO GLYCEROL PHOSPHATE '0 TO PRODUCE LYSO PHOSPHATIDIC ACID LYSO 0! FURTHER TRANSFORMED TO PHOSPHATIDIC ACID 0! &IGURE ;= 0HOSPHATIDIC ACID SERVES AS A CENTRAL METABOLITE IN THE DE NOVO SYNTHESIS OF 0, )T CAN EITHER BE DEPHOSPHORILATED TO DIACYLGLYCEROL $' TO ENTER THE LIPID STORAGE PATHWAY OR CONVERTED TO CYTYDINE DIPHOSPHATE DIACYLGLYCEROL #$0 $' ;= $' IS THE SUBSTRATE FOR TRIACYLGLYCEROL SYNTHESIS AND CONTRIBUTES TO 0% AND 0# SYNTHESIS THROUGH THE #$0 ETHANOLAMINE AND #$0 CHOLINE BRANCHES OF THE SO CALLED +ENNEDY PATHWAY WHICH USES EXOGENOUS ETHANOLAMINE AND :FBTU MJQJET
TAZ1 CLD1 CL MLCL CL PG CRD1 ISC1 GEP4
acyl-CoA PGP Glu-6P DHAP G3P SCT1, GPT2 PGS1 INO1
SLC1, ALE1 CDS1, TAM41 PIS1 1-acyl-DHAP lyso-PA PA CDP-DG Ins-3P External AYR1 inositol Serine CHO1 INM1 DGK1 PAH1 APP1 ITR1, ITR2 DPP1 external Etn LPP1 Inositol HMN1 PS GUT1 YJU3 TGL3 Etn G3P G MG DG EKI1 PSD1 PSD2 Etn-P PI ECT1 IPC STT4 CDP-Etn INP52 sphingolipids EPT1 LSB6 INP53 PIK1 SAC1 PE VPS34 YMR1
PIP external Cho CHO2 HMN1 INP51 INP54 Cho INP52 SAC1 CKI1 INP53 FIG4 OPI3 Cho-P PLC1 PCT1 OPI3 PIP2 CDP-Cho CPT1 PC
PCT1 lyso PL
TG
&IGURE 3CHEMATIC REPRESENTATION OF THE PHOSPHOLIPID METABOLIC PATHWAY IN YEAST #, CARDIOLIPIN -,#, MONOLYSOCARDIOLIPIN 0' PHOSPHATIDYLGLYCEROL 0'0 PHOSPHATIDYLGLYCEROPHOSPHATE #$0 $' CYTIDINE DIPHOSPHATE DIACYLGLYCEROL 0) PHOSPHATIDYLINOSITOL 0)0 PHOSPHATIDYLINOSITOL PHOSPHATE 0)0 PHOSPHATIDYLINOSITOL BIPHOSPHATE 03 PHOSPHATIDYLSERINE 0% PHOSPHATIDYLETHANOLAMINE 0# PHOSPHATIDYLCHOLINE %TN ETHANOLAMINE #HO CHOLINE 0! PHOSPHATIDIC ACID $' DIACYLGLYCEROL 4' TRIACYLGLYCEROL -' MONOACYLGLYCEROL ' GLYCEROL '0 GLYCEROL PHOSPHATE
CHOLINE AS SUBSTRATE FOR 0% AND 0# SYNTHESIS ;= #$0 $' CAN BE CONVERTED TO 0) OR TO 03 IN THE %2 AND TO PHOSPHATIDYLGLYCEROL PHOSPHATE 0'0 IN THE MITOCHONDRIA 03 CAN BE DECARBOXYLATED TO 0% WHICH CAN SUBSEQUENTLY BE METHYLATED TO FORM 0# ;= 0'0 IN THE MITOCHONDRIA IS DEPHOSPHORYLATED TO PRODUCE 0' WHICH REACTS WITH ANOTHER #$0 $' MOLECULE OBTAINING #,
0, SYNTHESIS IS REGULATED AT LEVELS OF GENE EXPRESSION POSTTRANSLATIONAL MODIlCATION ENZYME ACTIVITY ; = AND CONTACT SITES ;= 'ENETIC AND BIOCHEMICAL MECHANISMS INCLUDE INOSITOL ZINC #40 CYTIDINE TRIPHOSPHATE #$0 $' AND 3 !DENOSYL , HOMOCYSTEINE MEDIATED REGULATION %NZYME ACTIVITY MECHANISMS ARE MOSTLY BASED ON ENZYME PHOSPHORYLATION !DDITIONALLY PHOSPHOLIPID REGULATION IS INTERRELATED WITH THE SYNTHESIS AND REGULATION OF OTHER MAJOR LIPID CLASSES THAT INCLUDE FATTY ACIDS TRIACYLGLYCEROLS STEROLS AND SPHINGOLIPIDS
4UFSPMT 4UFSPM TUSVDUVSF BOE GVODUJPO 3TEROLS ARE ESSENTIAL LIPID CONSTITUENTS OF MEMBRANES 4HE MAJOR STEROL IN YEAST IS ERGOSTEROL WHICH IS THE END PRODUCT OF THE YEAST STEROL BIOSYNTHETIC PATHWAY %RGOSTEROL CHARACTERISTIC STRUCTURE (FOFSBM *OUSPEVDUJPO
CONSISTS OF FOUR FUSED RINGS AN ACYL SIDE CHAIN AND A HYDROPHILIC HYDROXYL GROUP THAT FACILITATES INSERTION INTO MEMBRANES &IGURE ;= 4HE STRUCTURE OF ERGOSTEROL DIFFERS FROM CHOLESTEROL BY TWO ADDITIONAL DOUBLE BONDS AND AN ADDITIONAL METHYL GROUP AT # OF THE SIDE CHAIN ;=
&REE STEROLS ARE PREDOMINANTLY PRESENT IN THE PLASMA MEMBRANE WHERE CONTROL ITS PHYSICAL STATE BY MODULATING ITS BILAYER mUIDITY AND PERMEABILITY ; = 4HEY ALSO CONSTITUTE TOGETHER WITH SPHINGOLIPIDS MEMBRANE MICRODOMAINS )N CONTRAST TO 3 CEREVISIAE A STEROL GRADIENT ALONG THE SECRETORY ROUTE IS NOT FOUND IN 0ICHIA PASTORIS ; = DUE TO THE SMALL AMOUNTS OF ERGOSTEROL PRESENT IN &IGURE 3TRUCTURE OF ERGOSTEROL PLASMA MEMBRANE ࣵ G PER MG PROTEIN IN 0 PASTORIS COMPARED TO THE ࣵ G PER MG PROTEIN IN 3 CEREVISIAE ;= )N ADDITION TO THEIR ROLE AS MEMBRANE CONSTITUENTS STEROLS ALSO HARBOUR MULTIPLE ESSENTIAL FUNCTIONS LIKE SIGNAL TRANSDUCTION ;= PROTEIN LIPIDATION IE MODIlCATION USUALLY PROTEINS BY ASSOCIATION WITH A LIPID ;= OR ENDOCYTOSIS ; =
&SHPTUFSPM CJPTZOUIFUJD QBUIXBZ %RGOSTEROL IS SYNTHESISED IN THE %2 THROUGH A COMPLICATED PATHWAY INVOLVING MORE THAN ENZYMES %RGOSTEROL PATHWAY CAN BE DIVIDED INTO THE PRE AND POST SQUALENE PATHWAYS &IGURE 3OME STEPS OF STEROL BIOSYNTHETIC PATHWAY REQUIRE OXYGEN
acetyl-CoA acetyl-CoA
ERG10 steryl ester sterol acetate acetoacetyl-CoA (LD) (secretion)
ERG13
HMG-CoA ergosterol HMG1 HMG2 ERG4
mevalonate ergosta-5,7,22,24(28)- tetraenol ERG12 ERG5 mevalonate-5P ergosta-5,7,24(28)- ERG8 trienol MVD1 ERG3
IPP episterol IDI1 ERG20 ERG2 ERG9 fecosterol squalene
ERG6 ERG1 zymosterol squalene epoxide ERG27 ERG7 ERG28 ERG11 ERG24 ERG25 lanosterol ERG26
&IGURE %RGOSTEROL BIOSYNTHETIC PATHWAY IN YEAST (-' #O! HYDROXY METHYLGLUTARYL #O! )00 ISOPENTENYL PYROPHOSPHATE :FBTU MJQJET
9EAST STEROL BIOSYNTHESIS STARTS WITH CONDENSATION OF TWO ACETYL #O! MOLECULES THAT YIELD ACETOACETYL #O! #ONDENSATION OF A THIRD ACETYL #O! TO ACETOACETYL #O! GIVE HYDROXY METHYLGLUTARYL #O! (-' #O! THAT IS FURTHER REDUCED TO MEVALONATE -EVALONATE IS PHOSPHORYLATED TWO TIMES LEADING TO MEVALONATE PHYROPHOSPHATE WHICH IS DECARBOXYLATED TO FORM ISOPENTENYL PYROPHOSPHATE )00 )00 IS THEN CONVERTED INTO DIMETHYLALLYL PYROPHOSPHATE $00 WHICH IS CONDENSED WITH AN ADDITIONAL )00 TO GERANYL PYROPHOSPHATE '00 )F AN ADDITIONAL )00 MOLECULE IS ADDED FARNESYL PYROPHOSPHATE &00 IS FORMED 3QUALENE IS FORMED FROM TWO MOLECULES OF &00 0OST SQUALENE PATHWAY INCLUDES SEVERAL OXYGEN DEPENDENT REACTIONS 3QUALENE IS EPOXIDISED TO SQUALENE EXPOXIDE AND FURTHER TRANSFORMED TO LANOSTEROL 3EVERAL DEMETHYLATION REDUCTION AND DESATURATION STEPS LEAD TO ZYMOSTEROL WHICH IS FURTHER CONVERTED TO FECOSTEROL EPISTEROL AND REDUCED TO ERGOSTEROL /NCE SYNTHESISED FREE ERGOSTEROL IS TRANSPORTED TO THE PLASMA MEMBRANE TROUGH BOTH VESICULAR AND NON VESICULAR TRANSPORT TO PREVENT ITS ACCUMULATION WHICH MAY BECOME TOXIC FOR THE CELL ;=
9EAST CELLS SYNTHESISE STEROLS IN EXCESS SO THAT MECHANISMS TO MAINTAIN STEROL HOMEOSTASIS SHOULD BE PRESENT 4HEY INCLUDE THE ESTERIlCATION OF FREE STEROLS TOGETHER WITH FATTY ACIDS AND ITS STORAGE IN LIPID DROPLETS DOWNREGULATION OF STEROL BIOSYNTHESIS AT TRANSCRIPTIONAL LEVEL ;= AND STEROL ACETYLATION TO BE SECRETED INTO THE MEDIUM AS STEROL ACETATE ;=
/POQPMBS MJQJET 9EAST CELLS ARE ABLE TO OVERCOME A POSSIBLE TOXIC EFFECT OF STEROLS AND FREE FATTY ACIDS BY CONVERTING THEM INTO MOLECULES THAT LACK CHARGED GROUPS &REE FATTY ACIDS ARE CONVERTED TO TRIACYLGLYCEROLS 4' AND STEROLS TO STERYL ESTERS 3% AND THEY CLUSTER AND FORM AN HYDROPHOBIC CORE CALLED LIPID DROPLET ,$ 4HESE NON POLAR LIPIDS ARE NOT SUITABLE AS MEMBRANE CONSTITUENTS BUT SERVE AS ENERGY SOURCE AND PRECURSORS FOR MEMBRANE LIPID SYNTHESIS
4'S CONSIST OF A GLYCEROL BACKBONE ESTERIlED WITH THREE LONG CHAIN FATTY ACIDS 4HEIR BIOGENESIS TAKES PLACE IN THE %2 WITH THE ESTERIlCATION OF THEIR PRECURSOR DIACYLGLYCEROL $' &IGURE ;=
PL
Pi PLC PL lyso-PL
DPP1 LRO1 LPP1 PA DG TG PAH1 DGA1 APP1
Pi acyl-CoA CoA Kennedy pathway
PE PC
&IGURE 3YNTHESIS OF TRIACYLGLYCEROLS 4' AND DIACYLGLYCEROLS $' IN YEAST !DAPTED FROM ;=
$' CAN RESULT FROM DEPHOSPHORILATION OF DE NOVO SYNTHESISED PHOSPHATIC ACID 0! DEGRADATION OF 0, 4' DEACYLATION ;= OR DURING SPHINGOLIPID SYNTHESIS WHERE ONE MOLECULE OF $' IS PRODUCED EACH TIME A 0, HEAD GROUP IS TRANSFERRED ONTO A CERAMIDE BACKBONE BY SPHINGOLIPID SYNTHASES ;= $' CAN BE FURTHER CONVERTED TO 4' BY ACYL #O! DEPENDENT OR ACYL #O! (FOFSBM *OUSPEVDUJPO
INDEPENDENT ACYLATION WHICH REQUIRES A 0, AS AN ACYL DONOR USUALLY 0% OR 0#;= )N CONTRAST TO 3 CEREVISIAE WHERE THE ACYL #O! DEPENDENT PATHWAY IS THE MAJOR CONTRIBUTOR TO 4' SYNTHESIS THE ACYL #O! INDEPENDENT PATHWAY SEEMS TO BE THE PREFERRED IN0 PASTORIS ;= 3TERYL ESTERS ARE SYNTHESISED BY THE ADDITION OF AN ACTIVATED FATTY ACID TO THE HYDROXYL GROUP AT # OF A STEROL MOLECULE &IGURE ;=
acyl-CoA ERG proteins
ARE2 Sterol SE
acyl-CoA CoA
&IGURE 3YNTHESIS OF STERYL ESTERS 3% IN YEAST !DAPTED FROM ;=
3%S PROVIDE A WAY TO MAINTAIN STEROL HOMEOSTASIS I E THE BALANCE BETWEEN THE INACTIVE STORAGE 3% AND THE BIOLOGICALLY ACTIVE FREE STEROLS ; = 3TERYL ESTERS ARE PRESENT IN 0 PASTORIS AT A MUCH LOWER AMOUNT THAN IN 3 CEREVISIAE 4HEY ONLY ACCOUNT FOR OF TOTAL NON POLAR LIPIDS IN 0 PASTORIS WHEREAS 3 CEREVISIAE ARE ALMOST ;= 4HIS CAN BE A CONSEQUENCE OF THE SMALL AMOUNTS OF STEROLS PRESENT IN THIS ORGANISM 3%S IN LIPID DROPLETS FROM 0 PASTORIS ARE PRESENT AS ESTERS OF ERGOSTEROL AND ERGOSTEROL PRECURSORS IN COMPARABLE AMOUNTS ;= ,IPID DROPLETS ARE COMPOSED BY A CORE OF RANDOMLY PACKED 4' SURROUNDED BY 3% 4HIS CORE IS HIGHLY HYDROPHOBIC AND IS COVERED BY A PHOSPHOLIPID MONOLAYER WITH A SPECIlC SET OF PROTEINS EMBEDDED -OST OF THESE PROTEINS ARE INVOLVED IN LIPID METABOLISM AND HOMEOSTASIS ;= 4HE AMOUNT OF PROTEINS FOUND IN ,$ FROM 0 PASTORIS WAS LOWER COMPARED TO THE PROTEOME OF ,$ IN 3 CEREVISIAE ;=
)F REQUIRED YEAST CELLS CAN MOBILISE 4' AND 3% TO PROVIDE STEROLS &! AND $' FOR THE SYNTHESIS OF MEMBRANE LIPIDS OR FOR THE PRODUCTION OF ENERGY $EGRADATION OF 4' IS MAINLY ACHIEVED BY THE 4' LIPASES YIELDING FATTY ACIDS THAN CAN BE USED TO PROVIDE PRECURSORS FOR MEMBRANE LIPID SYNTHESIS ;= $' RELEASED BY DEGRADATION OF 4' CAN BE USED AS A SUBSTRATE FOR THE SYNTHESIS OF 0% AND 0# VIA THE +ENNEDY PATHWAY ; = BUT ALSO AS A SECOND MESSENGER IN SIGNAL TRANSDUCTION ;= 3TEROLS RELEASED FROM 3% BY HYDROLISATION AND CAN BE INCORPORATED INTO MEMBRANES TO REGULATE MEMBRANE STABILITY mUIDITY AND PERMEABILITY ;= !PART FROM ITS FUNCTION AS STORAGE MOLECULES THEY HAVE OTHER FUNCTIONS ESPECIALLY 4' WHICH IS INVOLVED IN IRON 0, AND 3, METABOLISM LIPOTOXICITY AND CELL CYCLE PROGRESSION ;=
4QIJOHPMJQJET 4QIJOHPMJQJE TUSVDUVSF BOE GVODUJPO 3PHINGOLIPIDS SHARE THE COMMON STRUCTURAL FEATURE THAT ALL ARE COMPRISED OF BACKBONES CALLED LONG CHAIN BASES ,#" 4HEY ARE AMIDE LINKED WITH A LONG OR VERY LONG CHAIN FATTY ACID TO FORM CERAMIDES #ERAMIDES ARE THE SIMPLEST SPHINGOLIPIDS AND THE PRECURSOR ONTO WHICH DIFFERENT HEAD GROUPS CAN BE ADDED TO FORM MORE COMPLEX SPHINGOLIPIDS ;= 5NLIKE 3 CEREVISIAE WHERE ONLY INOSITOL CONTAINING PHOSPHORYLCERAMIDES EXIST IN 0ICHIA PASTORIS TWO CERAMIDE SYNTHASES COEXIST SHOWING DIFFERENT AFlNITIES FOR SPECIlC ,#" ANDOR ACYL #O! SUBSTRATES ;= 4HEIR SPECIlCITY ALLOWS THEM TO CATALYSE TWO CLASSES OF CERAMIDE PRECURSORS THAT WILL BE SPECIlCALLY DESTINED TO THE PRODUCTION OF EITHER INOSITOL CONTAINING PHOSPHORYLCERAMIDES :FBTU MJQJET
)0#S OR THE NEUTRAL SPHINGOLIPIDS CERAMIDES #ER AND HEXOSYLCERAMIDES (EX#ER &IGURE 4HESE SPHINGOLIPIDS HAVE DIFFERENT POLAR HEAD GROUPS AND CERAMIDE BACKBONES )0#S ARE CERAMIDES LINKED TO GLYCOSYL MOIETIES VIA INOSITOL PHOSPHATE WHICH OCCUR AS FREE MEMBRANE LIPIDS AND AS MEMBRANE ANCHORS OF COVALENTLY BOUND PROTEINS '0) ANCHORS WHILE HEXOSYLCERAMIDES CONTAIN MAINLY $ GLUCOSE OR $ GALACTOSE AS SUBSTITUENTS OF THE # HYDROXY GROUP OF THE CERAMIDE AND SERVE AS PRECURSORS FOR A LARGE VARIETY OF HIGHER GLYCOSILATED SPHINGOLIPID SPECIES 'LC#ER ;=
A B
&IGURE 3PHINGOLIPIDS PRESENT IN 0 PASTORIS A .EUTRAL SPHINGOLIPIDS 'LC#ER B )NOSITOL CONTAINING PHOSPHORYLCERAMIDE )0# ;=
!DDITIONAL DIFFERENCES BETWEEN THESE TWO ORGANISMS COMPRISE THE LENGTH OF THE FATTY ACIDS OF THEIR )0# SPHINGOLIPIDS 7HILE 3 CEREVISIAE CONSIST BASICALLY ON # FATTY ACID AND A SMALL PERCENTAGE OF # AND # 0 PASTORIS CONTAINS MOSTLY # FATTY ACIDS 3TUDIES OF LIPID COMPOSITION OF 0 PASTORIS ORGANELLES PERFORMED BY 'RILLITSCH ET AL ; = SHOWED AN ORGANELLE DEPENDENT DISTRIBUTION OF SPHINGOLIPIDS #ERAMIDES AND HEXOSYLCERAMIDES WERE PREDOMINANT IN 'OLGI FRACTIONS WHEREAS INOSITOL CONTAINING PHOSPHORYLCERAMIDES WITH # # BACKBONES WERE ALMOST EXCLUSIVELY ENRICHED IN PLASMA MEMBRANE FRACTIONS
3PHINGOLIPIDS 3, TOGETHER WITH PHOSPHOLIPIDS ARE ESSENTIAL STRUCTURAL COMPONENTS OF MEMBRANES 3PHINGOLIPIDS HAVE SATURATED OR TRANS UNSATURATED TAILS SO THEY ARE ABLE TO FORM TALLER NARROWER CYLINDERS THAN 0# LIPIDS OF THE SAME CHAIN LENGTH AND THEY PACK MORE TIGHTLY ADOPTING THE SOLID GEL PHASE WHICH IS mUIDISED BY STEROLS ;= !PART FROM THE WELL KNOWN STRUCTURAL FUNCTION SPHINGOLIPIDS ALSO PLAY ADDITIONAL ROLES AS SECOND MESSENGERS FOR REGULATING SIGNAL TRANSDUCTION PATHWAYS ;= 3PHINGOLIPIDS PLAYING ROLES IN HEAT STRESS ; = ENDOCYTOSIS ; = INTRACELLULAR PROTEIN TRAFlCKING ; = LIFESPAN ; = &URTHERMORE IT HAS BEEN ALSO DESCRIBED THE ROLE OF ,#" AS SIGNALLING MOLECULES ; =
4QIJOHPMJQJE CJPTZOUIFUJD QBUIXBZ $E NOVO SPHINGOLIPID SYNTHESIS &IGURE BEGINS IN THE ENDOPLASMIC RETICULUM %2 WITH THE CONDENSATION OF SERINE AND PALMITOYL #O! INTO KETODIHYDROSPHINGOSINE BY SERINE PALMITOYLTRANSFERASE 304 2EDUCTION OF KETODIHYDROSPHINGOSINE BY AN .!$0( REQUIRING REACTION RESULTS IN THE LONG CHAIN BASE ,#" DIHYDROSPHYNGOSINE $(3 ;= $(3 CAN BE HYROXYLATED AT # TO GIVE PHYTOSPHINGOSINE 0(3 $(3 AND 0(3 CAN ENTER TWO POSSIBLE ROUTES 4HEY CAN BE PHOSPHORYLATED TO FORM PHYTO OR DIHYDROSPHINGOSINE PHOSPHATE 0(3 0 AND $(3 0 WHICH CAN BE CLEAVED AND GENERATE FATTY ALDEHYDES AND ETHANOLAMINE PHOSPHATE 4HIS DEGRADATION PATHWAY MAY SERVE TO REGULATE OVERALL SPHINGOLPID LEVELS %THANOLAMINE PHOSPHATE CAN BE RECYCLED FOR PHOSPHATIDYLETHANOLAMNIE 0% BIOSYNTHESIS THROUGH THE +ENNEDY PATHWAY ;= 4HIS PHOSPHORYLATION STEP IS REVERSIBLE 4HE SECOND ROUTE PRODUCES COMPLEX SPHINGOLIPIDS $(3 AND 0(3 ARE AMIDE LINKED TO A VERY LONG (FOFSBM *OUSPEVDUJPO
CHAIN FATTY ACID USUALLY # TO YIELD DIHYDROCERMAIDE $(# AND PHYTOCERAMIDE 0(# AND FURTHER HYDROXYLATED &ATTY ACIDS ARE ELONGATED FROM # # TO # TO VERY LONG CHAIN FATTY ACIDS 6,#&! BY ELONGASES IN THE %2
palmitoyl-CoA
Serine LCB1 LCB2 Kennedy pathway 3-ketodihydrosphingosine
TSC10 VLCFA LAG1 LAC1 SCS7 ISC1 ethanolamine- DPL1 DHS-1P LCB5 DHS DHC 1-P YDC1 PHS-1P LCB3 SUR2 SUR2
LAG1 BAR1 LAC1 SCS7 Δ4-desaturase PHS PHC YPC1 C18:0-CoA AUR1 VLCFA SCS7 IPC DHS-Δ4 CSH1 SUR1 Δ8-desaturase CSG2 GCS GlcCer MIPC DHS-Δ4,8
C9-methyltransferase IPT1
Cer M(IP)2C
&IGURE 3PHINGOLIPID METABOLIC PATHWAY OF 0 PASTORIS
#ERAMIDES ARE SIMPLE 3, MOLECULES WITHOUT HEAD GROUPS THAT SERVE AS SUBSTRATES FOR THE SYNTHESIS OF COMPLEX LIPIDS &URTHER STEPS INTO COMPLEX SPHINGOLIPIDS INCLUDE THE ADDITION OF A PHOSPHOINOSITOL HEADGROUP TO THE CERAMIDE FORMING INOSITOLPHOSPHORYLCERAMIDES )0#S WHICH CAN UNDERGO MANNOSYLATION TO GENERATE MANNOSYL INOSITOLPHOSPHORYLCERAMIDE -)0# !FTER MANNOSYLATION ANOTHER INOSITOL
PHOSPHATE GROUP CAN BE ADDED FORMING MANNOSYLINOSITOL ኼPHOSPHORYLCERAMIDE -)0 ኼ# !DDITIONALLY 0 PASTORIS UNLIKE 3 CEREVISIAE CAN SYNTHESISE CERAMIDES AND HEXOSYLCERAMIDES )N THIS PATHWAY A MOLECULE OF $(3 IS USED BY AN ALTERNATIVE CERAMIDASE DESATURATED HYDORXYLATED AND DESATURATED AGAIN &URTHER METHYLATION CAN YIELD ADDITIONAL SPECIES 4HESE CERAMIDES CAN BE TRANSFORMED IN GLUCOCERAMIDES BY THE ADDITION OF A GLUCOSE MOIETY ;=
!BOUT THREE FOURTHS OF THE MASS OF THE SPHINGOLIPIDS IN 3 CEREVISIAE CELLS IS -)0 ኼ# WITH THE REST BEING EQUAL PARTS OF )0# AND -)0# ;= 4HE SHEAR MASS OF THESE SPHINGOLIPIDS AND THEIR NEGATIVE CHARGE ARE LIKELY TO AFFECT PROCESSES DEPENDENT UPON THE PLASMA MEMBRANE ;= 0LASMA MEMBRANE OF 0 PASTORIS IS ENRICHED IN HEXOSYLCERAMIDES AND INOSITOL CONTAINING SPHINGOLIPIDS WITH # # BACKBONES ;= 4HE INITIAL STEPS OF SPHINGOLIPID BIOSYNTHESIS INCLUDING SPHINGOID BASE AND CERAMIDE FORMATION TAKE PLACE IN THE ENDOPLASMIC RETICULUM &IGURE #OMPLEX SPHINGOLIPIDS ARE PRODUCED BY THE ADDITION OF THE POLAR HEAD GROUPS IN THE 'OLGI APPARATUS WHERE THEY ARE TRANSPORTED BY BOTH VESICULAR AND NON VESICULAR TRANSPORT ;= %NZYMES INVOLVED IN DE NOVO SPHINGOLIPID :FBTU MJQJET
&IGURE /VERVIEW OF THE 3 CEREVISIAE SPHINGOLIPID METABOLISM !DAPTED FROM ;= &ILLED ARROWS INDICATE BIOSYNTHETIC STEPS DASHED ARROWS INDICATE DEGRADATIVE AND RECYCLING STEPS
SYNTHESIS ARE TIGHTLY REGULATED THROUGH THE ACTION OF MECHANISMS THAT MODULATE THEIR ACTIVITY IN RESPONSE TO ENVIRONMENT SUBSTRATE AVAILABILITY AND THE PRESENCE OF COFACTORS CONNECTING SPHINGOLIPIDS TO OTHER METABOLITES SUCH AS LIPIDS AMINO ACIDS OR SUGARS ;= 3PHINGOLIPIDS CAN BE CATABOLISED BY HYDROLYSING THEIR POLAR HEAD GROUP TO YIELD THE CERAMIDES $(# OR 0(# WHICH CAN BE FURTHER CLEAVED TO FREE FATTY ACIDS AND ,#" $(3 AND 0(3 ;= ,#"S AS MENTIONED BEFORE CAN BE PHOSPHORYLATED AND CLEAVED TO GENERATE ETHANOLAMINE PHOSPHATE TO GENERATE PHOSPHATIDYLETHANOLAMINE 0% %XTENDED REVIEWS ABOUT SPHINGOLIPID REGULATION HAVE BEEN PUBLISHED ; =
$SPTTUBML CFUXFFO TQIJOHPMJQJE CJPTZOUIFTJT BOE PUIFS MJQJE QBUIXBZT 4HE CROSSTALK BETWEEN SPHINGOLIPIDS AND FATTY ACID METABOLISM STARTS IN THE lRST STEP OF SPHINGOLIPID BIOSYNTHESIS WHERE THE ENZYME PALMITOYLTRANSFERASE 304 CONDENSES A SERINE AND THE LONG FATTY ACID PALMITOYL #O! &URTHERMORE THE VERY LONG CHAIN FATTY ACID CONTENT OF SPHINGOLIPIDS IS OF GREAT IMPORTANCE FOR CELL GROWTH AND DEVELOPMENT
9EAST CELLS ADJUST THEIR SPHINGOLIPID PROlLE IN RESPONSE TO CHANGES IN STEROL STRUCTURES AND GENETIC EXPERIMENTS SHOW THAT STEROLS AND SPHINGOLIPIDS FUNCTIONALLY INTERACT IN BIOLOGICAL MEMBRANES ; = 3PHINGOLIPIDS ALONG WITH STEROLS ARE CRITICAL FOR FORMATION OF MICRODOMAINS WITHIN MEMBRANES THAT HAVE BEEN REFERRED TO AS LIPID RAFTS )N 3 CEREVISIAE RAFTS HAVE BEEN REFERRED TO BE INVOLVED IN SEVERAL CELLULAR FUNCTIONS WHICH ARE SUMMARISED IN &IGURE 3PHINGOLIPIDS ARE CLOSELY RELATED TO PHOSPHOLIPIDS BECAUSE THEY SHARE SOME PARTS OF THEIR METABOLIC PATHWAYS SUCH AS THE SYNTHESIS OF POLAR HEAD GROUPS OR FATTY ACYL #O!S "OTH LIPID CLASSES CAN FEED EACH OTHER AND IMPACT THEIR CONCENTRATION
-OREOVER TRIACYLGLYCEROLS 4' SYNTHESIS AND BREAKDOWN DIRECTLY IMPACTS THE ACYL COMPOSITION OF SPHINGOLIPIDS ;= $IACYLGLYCEROLS ALSO PLAY A PIVOTAL ROLE IN THE CONVERGENCE OF SPHINGOLIPIDS PHOSPHOLIPIDS AND NON POLAR LIPID METABOLIC PATHWAYS (FOFSBM *OUSPEVDUJPO
Yeast lipid rafts ! "! ! ! " # ! # # # #
&IGURE 0UTATIVE INVOLVEMENT OF LIPID RAFTS IN DIFFERENT YEAST FUNCTIONS !DAPTEDFROM;=
"OUJCPEZ GSBHNFOU BT NPEFM QSPUFJO )N THE CURRENT WORK 0 PASTORIS STRAINS PRODUCING THE ANTIGEN BINDING FRAGMENT &AB & A NEUTRALISING ANTIBODY AGAINST ()6 HAVE BEEN USED AS A MODEL PROTEIN TO CHARACTERISE THE HETEROLOGOUS PROTEIN PRODUCTION IMPACT OVER CHANGES ON CELL PHYSIOLOGY &AB FRAGMENTS ARE THE ANTIGEN BINDING REGIONS OF AN ANTIBODY MOLECULE WITH A MOLECULAR WEIGHT AROUND K$A ;= 4HEY CAN BE USED AS MODEL FOR COMPLEX PROTEIN PRODUCTION AS CONTAIN lVE DISULPHIDE BONDS ONE THEREOF REQUIRED TO CONNECT THE HEAVY CHAIN LIGHT CHAIN DIMER "ECAUSE OF THEIR PROPERTIES ANTIBODY FRAGMENTS HAVE IMPORTANT INDUSTRIAL APPLICATIONS AS THERAPEUTIC AND DIAGNOSTIC MOLECULES BUT ALSO IN PURIlCATION AND BIOSEPARATION APPLICATIONS ;= 4HE ABSENCE OF GLYCOSYLATION ON &AB REGIONS ALLOWS THEIR PRODUCTION TO BE LESS COMPLEX AND ENABLES EASIER ENGINEERING AND CULTIVATION OF MICROBIAL HOST ORGANISMS SUCH AS BACTERIA AND YEASTS ;= $ESCRIBED PRODUCT YIELDS OF &AB IN 0 PASTORIS AREUPTOMG,ዅኻ IN FED BATCH '!0 CULTIVATIONS ;= AND MG ,ዅኻ IN METHANOL INDUCED FED BATCHES ;n= WHILE MG ,ዅኻ WERE DESCRIBED IN CHEMOSTAT CULTURES UNDER '!0 PROMOTER 'ARCIA /RTEGA SUBMITTED "JNT PG UIF TUVEZ
4HE AIM OF THE PRESENT WORK WAS THE CHARACTERISATION OF A RECOMBINANT 0ICHIA PASTORIS STRAIN PRODUCING A HUMAN ANTIBODY &AB FRAGMENT IN TERMS OF LIPID COMPOSITION "ASED ON PREVIOUS RESULTS SHOWING A STRONG REGULATION OF LIPID METABOLISM WHEN CELLS ARE CULTURED UNDER LOW OXYGEN CONDITIONS ONE OF THE OBJECTIVES WAS TO STUDY THE IMPACT OF OXYGEN AVAILABILITY ON THE LIPIDOME OF THE RECOMBINANT STRAIN 4RANSCRIPTOMIC DATA UNDER THESE OXYGEN CONDITIONS ALLOWED FOR SELECTING TARGET GENES INVOLVED IN THE ERGOSTEROL AND SPHINGOLIPID PATHWAY IN ORDER TO STUDY THE EFFECT OF GENETIC MODIlCATIONS ON TWO OF THE MOST REGULATED PATHWAYS WHOSE PRODUCTS ARE ESSENTIAL ELEMENTS OF THE PLASMA MEMBRANE 0UNCTUAL MODIlCATIONS WOULD GIVE INFORMATION ABOUT MEMBRANE MODIlCATIONS RELATED TO INCREASED PROTEIN SECRETION
4VNNBSZ
4HE AIM OF THIS THESIS WAS TO CHARACTERISE THE LIPID COMPOSITION OF 0ICHIA PASTORIS CELLS PRODUCING A RECOMBINANT PROTEIN UNDER LOW OXYGEN CONDITIONS HYPOXIA UNDER WHICH A BENElCIAL EFFECT ON PROTEIN PRODUCTION HAS BEEN PREVIOUSLY OBSERVED ! 0 PASTORIS STRAIN EXPRESSING THE ANTIBODY FRAGMENT & UNDER THE CONSTITUTIVE '!0 PROMOTER WAS CHOSEN AS A CASE STUDY 4HE INVESTIGATION FOCUSED ON THE ANALYSIS OF INmUENCE OF LIPID COMPOSITION ON PROTEIN SECRETION )N #HAPTER TRANSCRIPTOMIC DATA OF GENES RELATED TO LIPID METABOLISM OF 0 PASTORIS GROWING UNDER NORMOXIC AND HYPOXIC CONDITIONS WAS USED TO PERFORM A CLUSTER ANALYSIS IN ORDER TO SELECT THE MOST REGULATED GENES )T WAS OBSERVED THAT ERGOSTEROL AND SPHINGOLIPID PATHWAYS WERE MANLY UPREGULATED UNDER HYPOXIA .OTABLY MOST OF THE CORRESPONDING ENZYMES CATALYSE OXYGEN DEPENDENT REACTIONS &OLLOWING THIS ANALYSIS SOME OF THE GENES INVOLVED IN THESE BIOSYNTHETIC PATHWAYS WERE SELECTED AS ENGINEERING TARGETS )N #HAPTER A BATTERY OF STRAINS WITH THE SELECTED GENES DELETED OR OVEREXPRESSED WAS OBTAINED 4HE AIM OF THESE MODIlCATIONS WAS TO EVALUATE THEIR IMPACT ON THE OVERALL LIPID CLASS COMPOSITION OF THE CELL AS WELL AS ON PROTEIN SECRETION )N #HAPTER SMALL SCALE CULTURES WERE PERFORMED IN ORDER TO CHARACTERISE THE OBTAINED STRAINS AND SELECT CLONES FOR FURTHER CHARACTERISATION IN CHEMOSTAT CULTURES #HAPTER UNDER BOTH NORMOXIC AND HYPOXIC CONDITIONS #ELL BEHAVIOUR IN TERMS OF PROTEIN PRODUCTION CELL MORPHOLOGY AND LIPID COMPOSITION WERE DETERMINED FOR ALL THE STRAINS IN BOTH OXYGEN CONDITIONS &INALLY IN #HAPTER LIPIDOMIC RESULTS WERE INTEGRATED WITH PREVIOUSLY PUBLISHED TRANSCRIPTOMICS DATA IN ORDER TO GAIN FURTHER INSIGHTS ON THE PHYSIOLOGICAL RESPONSE OF 0 PASTORIS TO HYPOXIC CONDITIONS AND ITS INTERCONNECTIONS WITH PROTEIN SECRETION 4HE ULTIMATE GOAL OF THESE STUDIES IS TO ENLARGE THE KNOWLEDGE BASE FOR THE SELECTION OF NEW POTENTIAL TARGETS TO IMPROVE RECOMBINANT PROTEIN SECRETION THROUGH CELL ENGINEERING
4VNNBSZ
1. Transcriptional 2. Gene analysis selection 3. Gene deletion/ overexpression
9. Integration of 4. Strain results for rational battery design
8. Cell 5. Shake Flask analyses Cultures
6. Chemostat cultures
7. Fed-Batch cultures
&IGURE 'RAPHIC SUMMARY INCLUDING THE PRINCIPAL STEPS OF THE PRESENT WORK
.BUFSJBMT BOE .FUIPET
4USBJOT %SCHERICHIA COLI % COLI WAS USED TO AMPLIFY PLASMID $.! FOR TRANSFORMATION INTO 0 PASTORIS AND SUBCLONING TASKS % COLI $( )NVITROGEN WITH GENOTYPE &ዅኻ SUP% THI GLN6 REC! REL! GYR! DEO2 NUP' ዅኻ ዅኻ ዅኻ ͒D LAC: ́ - ́ LAC:9! ARG& 5 HSD 2RK MK WAS EMPLOYED
0ICHIA PASTORIS 4HE 0 PASTORIS STARTING STRAIN WAS A 8 P'!0! &AB& STRAIN 4HE EXPRESSION CASSETTES FOR THE LIGHT AND HEAVY CHAIN OF THE HUMAN & ANTIGEN BINDING FRAGMENT &AB WERE SEPARATELY PLACED UNDER THE CONTROL OF THE 0 PASTORIS '!0 GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE CONSTITUTIVE PROMOTER AND THE 3 CEREVISIAE MATING FACTOR LEADER AND COMBINED ON ONE PLASMID 4HIS VECTOR CONFERRING RESISTANCE TO :EOCINÃ WAS INTEGRATED INTO THE GENOME OF 0 PASTORIS HOST STRAIN 8 HISዄ DERIVATIVE OF THE '3 WILD TYPE PHENOTYPE FROM )NVITROGEN
3TRAIN MAINTENANCE &OR LONG TERM STOCKS CELLS WERE GROWN IN 90$ MEDIA UNTIL EXPONENTIAL PHASE WAS REACHED HARVESTED RESUSPENDED IN 90 MEDIUM WITH 'LYCEROL VV AND FROZEN STORED AT # &OR SHORT TERM CELL MAINTENANCE CELLS WERE GROWN ON 90$ PLATES CONTAINING G M,ዅኻ :EO AT #
.PMFDVMBS #JPMPHZ .FEJB $PNQPTJUJPO ," ,URIA "ERTANI -EDIUM 4RYPTONE 9EAST %XTRACT SODIUM CHLORIDE .A#L 3TORE AT 24 4O PREPARE ," !GAR PLATES ADD !GAR 3TORE AT #
3/# -EDIUM 3/# MEDIUM IS USED IN THE lNAL STEP OF BACTERIAL CELL TRANSFORMATION TO OBTAIN MAXIMAL
.BUFSJBMT BOE .FUIPET
TRANSFORMATION EFlCIENCY OF % COLI -EDIUM COMPOSITION TRYPTONE YEAST EXTRACT M- SODIUM CHLORIDE .A#L M- POTASSIUM CHLORIDE +#L M- MAGNESIUM CHLORIDE
-G#Lኼ M- MAGNESIUM SULPHATE -G3/ኾ M- GLUCOSE !UTOCLAVE AT # FOR MIN 3TORE ALIQUOTED AT #
90$ -EDIUM WV YEAST EXTRACT WV PEPTONE !UTOCLAVE AT # FOR MIN !DD WV GLUCOSE AUTOCLAVED SEPARATELY 3TORE AT 24 !DD !GAR TO PREPARE 90$ !GAR PLATES 3TORE AT #
#VGGFST 4!% 3OLUTION ! STOCK SOLUTION OF X 4!% WAS PREPARED X "UFFER COMPOSITION M- 4RIS P( M- !CETIC ACID M- %$4!
"%$3 SOLUTION M- "ICINE .A/( P( VV ETHYLENE GLYCOL VV DIMETHYL SULFOXIDE $-3/ AND - 3ORBITOL 3TORE AT #
7FDUPST P"2 &IGURE P"2 WAS A KINDLY PROVIDED BY $R -ANSUR AND CONSISTS ON A P0)#:! VECTOR WITH THE HYGROMYCIN RESISTANCE CASSETTE THAT REPLACES THE ORIGINAL :EOCIN PRESENT IN THE COMMERCIAL VECTOR
&IGURE 3CHEMATIC VIEW OF THE EXPRESSION VECTOR P0" COMPOSED BY THE !/8 INDUCIBLE PROMOTER THE 3 CEREVISIAE ᎎ FACTOR SECRETION SEQUENCE A MULTI CLONING SITE -#3 AND SOME TAGS FOR PURIlCATION !N ORIGIN OF REPLICATION FOR % COLI IS ALSO PRESENT AS WELL AS THE :EOCIN RESISTANCE CASSETTE FOR SELECTION
P'!0(IS &IGURE P'!0(IS HAS BEEN USED FOR INTRACELLULAR CONSTITUTIVE GENE EXPRESSION AS IT CARRIES THE '!0 PROMOTER AND NO SECRETION SIGNAL IS PRESENT !PART FROM THE CONVENTIONAL REPLICATION ORIGIN IT CARRIES TWO SELECTION SYSTEMS THE HISTIDINE GENE HIS FOR AUXOTROPHY SELECTION WHEN HISዅ STRAINS .PMFDVMBS #JPMPHZ
ARE EMPLOYED AS WELL AS THE !MPICILLIN ANTIBIOTIC RESISTANCE CASSETTE P'!0(IS WAS USED TOGETHER WITH P"2 TO GENERATE THE WORKING VECTOR P'!0(!
&IGURE 3CHEMATIC VIEW OF THE EXPRESSION VECTOR P'!0(IS COMPOSED BY THE '!0 CONSTITUTIVE PROMOTER A MULTI CLONING SITE -#3 AND THE HIS AND !MPICILLIN RESISTANCE /2&S !N ORIGIN OF REPLICATION FOR % COLI IS ALSO PRESENT
P05::,% &IGURE P05::,% WAS USED FOR INTRACELLULAR EXPRESSION )T IS A VERSATILE VECTOR BACKBONE BASED ON A MODULAR DESIGN ;= )T CONTAINS THE '!0 PROMOTER THE +ANAMYCIN RESISTANCE WHICH CONFERS KANAMYCIN RESISTANCE IN % COLI AND 'ENETICIN ' RESISTANCE WHEN USED IN 0 PASTORIS
&IGURE 3CHEMATIC VIEW OF THE EXPRESSION VECTOR P05::,% COMPOSED BY THE '!0 CONSTITUTIVE PROMOTER RED A MULTI CLONING SITE -#3 AND THE +ANAMYCIN RESISTANCE /2&S GREEN !N ORIGIN OF REPLICATION FOR % COLI IS ALSO PRESENT BLUE .BUFSJBMT BOE .FUIPET
"OUJCJPUJDT :EOCINÃ 4HE 3H BLE GENE CONFERS RESISTANCE TO THIS ANTIBIOTIC )T WAS PURCHASED FROM )NVIVO'EN 7ORKING CONCENTRATIONS WERE GM,ዅኻ FOR % COLI AND GM,ዅኻ FOR 0 PASTORIS 7HEN USED IT WAS WRAPPED IN FOIL DUE TO LIGHT SENSITIVITY 3TORED AT #
(YGROMYCIN " (YGRO'OLDÃ )NVIVO'EN WAS USED AS ANTIBIOTIC FOR DUAL SELECTION EXPERIMENTS INCLUDING GENE KNOCKOUT AND OVEREXPRESSION AS AN ALTERNATIVE MODE OF ACTION THAN :EOCINÃ IS USED 4HE HPH GENE FROM 3TREPTOMYCES HYGROSCOPICUS CONFERS HYGROMYCIN RESISTANCE 7ORKING CONCENTRATIONS FOR % COLI WERE GM,ዅኻ AS INDICATED BY THE MANUFACTURER 0 PASTORIS OPTIMAL WORKING CONCENTRATION OF ANTIBIOTIC WAS TESTED BY CULTURING CELLS IN DIFFERENT ANTIBIOTIC CONCENTRATIONS !N INITIAL CULTIVATION TEST WAS PERFORMED IN M, OF 90$ MEDIA WITH A HYGROMYCIN RANGE BETWEEN AND GM,ዅኻ &IGURE ! #ELLS WERE CULTURED FOR H AND
SAMPLES WERE TAKEN TO DETERMINE THE /$ዀኺኺ OF THE CULTURES ! SECOND ROUND WITH A NARROWER RANGE OF ANTIBIOTIC CONCENTRATIONS ; GM,ዅኻ= WAS CARRIED OUT IN SHAKE mASK CULTURES FOR H &IGURE " &INALLY GM,ዅኻ WAS SELECTED AS WORKING CONCENTRATION IN 0 PASTORIS CULTURES 7HEN USED IT WAS WRAPPED IN FOIL DUE TO LIGHT SENSITIVITY 3TORED AT #
&IGURE 0 PASTORIS 8 &AB& GROWING IN 90$ WITH DIFFERENT (YGROMYCIN CONCENTRATIONS ! )NITIAL SCREENING IN M, CULTURES " 3ECOND SCREENING PERFORMED IN M, 3HAKE &LASKS .PMFDVMBS #JPMPHZ
&O[ZNFT 2ESTRICTION ENZYMES 3EVERAL RESTRICTION ENZYMES WERE USED FOR $.! DIGESTION AND ALL WERE PURCHASED FROM &ERMENTAS IN THE &AST$IGEST VERSION WHEN AVAILABLE AND ENZYME DETAILS AND REACTION CONDITIONS WERE PERFORMED ACCORDING THE PROVIDERS GUIDELINES
0HOSPHATASE $EPHOSPHORILATION OF ENDS OF $.! TO PREVENT SELF LIGATION OF FRAGMENTS BY REMOVING THE PHOSPHORYL TERMINI REQUIRED BY LIGASES WAS CARRIED OUT USING R!0ID !LKALINE 0HOSPHATASE 2OCHE ACCORDING TO THE STANDARD DEPHOSPHORILATION PROCEDURE "RIEmY UP TO G OF VECTOR $.! WAS MIXED WITH , OF R!0ID !LKALINE 0HOSPHATASE "UFFER X , OF THE ENZYME AND lLLED WITH DISTILLED WATER UP TO , 4HE MIXTURE WAS INCUBATED MIN AT # THEN INCUBATED FOR MIN AT # TO INACTIVATE THE ENZYME AND DIRECTLY USED IN THE LIGATION REACTION
,IGASE ,IGATIONS WERE PERFORMED USING 4 $.! ,IGASE 2OCHE ACCORDING TO MANUFACTURERS STANDARD ASSAY ! VECTOR TO INSERT RATIO OF WAS USED FOR OPTIMAL LIGATION RESULTS AND THE AMOUNT OF NEEDED $.! WAS CALCULATED USING %QUATION
൬൦ VECTOR q ൩ൡ INSTERT INSERT w MOLAR RATIO ൬൦INSERT ൩ൡ VECTOR VECTOR 5P TO G OF TOTAL $.! VECTOR AND INSERT WAS MIXED WITH , OF X ,IGATION "UFFER ,OF4 $.! LIGASE AND lLLED UP TO , WITH DISTILLED WATER AND INCUBATED OVERNIGHT AT # ,IGATION MIXTURE WAS USED DIRECTLY TO TRANSFORM % COLI COMPETENT CELLS ;=
$.! POLYMERASES $IFFERENT $.! POLYMERASES WERE USED DEPENDING ON THE 0#2 USE &OR KNOCK OUT CASSETTE GENERATION AND GENE AMPLIlCATION FOR CO EXPRESSION (IGH &IDELITY 3!(!2!Ã AND 2!.'%2 $.! POLYMERASES "IOLINE WERE USED 7HEN 0#2 WAS USED FOR DETECTION PURPOSES 'O4AQLj 'REEN -ASTER -IX 0ROMEGA WAS CHOSEN
%/" FYUSBDUJPOT 'EL "ANDS $.! FRAGMENTS FROM AGAROSE GELS AND ENZYMATIC REACTIONS IE 0#2 $.! DIGESTION $.! LIGATION WERE RECOVERED USING THE 7IZARDLj 36 'EL AND 0#2 #LEAN 5P 3YSTEM 0ROMEGA !LL METHODS USED FOR $.! RECOVERY ARE BASED ON THE CAPACITY OF $.! TO BIND SILICA IN THE PRESENCE OF HIGH CONCENTRATIONS OF CHAOTROPHIC SALTS THAT WILL BE REMOVED LATER WITH AN ALCOHOL BASED WASH AND $.! WILL BE lNALLY ELUTED IN A LOW IONIC STRENGTH SOLUTION SUCH AS WATER
0LASMIDIC $.! 0LASMIDIC $.! EXTRACTIONS WERE PERFORMED USING BOTH 0URE9IELDÃ 0LASMID -INIPREP 3YSTEM AND 0URE9IELDÃ 0LASMID -IDI PREP 3YSTEM 0ROMEGA &OR PLASMID $.! PURIlCATION AN INITIAL STEP TO GENERATE A CLEARED LYSATE THAT REMOVES PROTEIN LIPIDS AND CHROMOSOMAL $.! FROM PLASMID $.! IS REQUIRED 4HE 3$3 ALKALINE DENATURATION METHOD IS THE ONE USED IN THE KITS $.! IS DENATURED IN ALKALINE CONDITIONS AND THE RAPID NEUTRALISATION CAUSES THAT CHROMOSOMAL $.! FORMS AN INSOLUBLE AGGREGATE THAT PRECIPITATES OUT OF SOLUTION WHILE PLASMID $.! REHYBRIDISES AND REMAINS IN .BUFSJBMT BOE .FUIPET
SOLUTION -OREOVER POTASSIUM SALT OF 3$3 LEADS TO PROTEIN PRECIPITATION AND AGGREGATION ASSISTING IN THE ENTRAPMENT OF THE CHROMOSOMAL $.! -IDIPREP 3YSTEM ALSO INCORPORATES AN ENDOTOXIN REMOVAL WASH TO REMOVE SUBSTANTIAL AMOUNTS OF PROTEIN 2.! AND ENDOTOXIN CONTAMINANTS
'ENOMIC $.! 'ENOMID $.! FROM 0 PASTORIS WAS EXTRACTED USING THE 7IZARDLj 'ENOMIC $.! 0URIlCATION +IT 0ROMEGA FOLLOWING THE 9EAST )SOLATION 0ROTOCOL 4HE ISOLATION SYSTEM IS BASED ON SAMPLE ENZYMATIC LYSIS AND PURIlCATION BY SILICA
1SJNFS EFTJHO 0RIMERS 4ABLE WERE ALL DESIGNED USING #LONE -ANAGER 3CI %D 3OFTWARE AND PURCHASED FROM "IOMERSNET 0RIMERS WERE DESIGNED TO HAVE A '# CONTENT OF REDUCED INTRA OR INTERMOLECULAR COMPLEMENTARY SEQUENCES TO MINIMISE THE PRODUCTION OF PRIMER DIMER AND REDUCED INTRAMOLECULAR REGIONS OF SECONDARY STRUCTURE TO FAVOUR PRIMER ANNEALING 0AIRS OF PRIMERS
WERE DESIGNED WITH SIMILAR MELTING TEMPERATURE 4፦ q #
4ABLE 0RIMERS USED FOR GENE AMPLIlCATION FROM GENOMIC $.! FOR CO EXPRESSION IN 0 PASTORIS &AB & KNOCK OUT GENERATION SEQUENCING AND CLONE SELECTION
0RIMER NAME 0RIMER SEQUENCE 5SE & P'!0 '!44!44''!!!##!##!'!!4#' SEQUENCING AND 2 !/844 '#!!!4''#!44#4'!#!4## CLONE SELECTION 2 P05::,% ''#'4'!!4'4!!'#'4'!#4 & $%3?3l) #'4!4#''###!'##''##!4'!4!!#4#!#!'!4##!!4!!#' 2 $%3?.OT) #4'!#'#''##'#44!!!!'4#!4'4'#4!4''44#4# & 352?3l) #4!#!''###!'##''##!4''!#!!'44#!'4#!'4!4!4## 2 352?.OT) '##4!'#''##'#44!#4'44#444#4444''4'!4## OVEREXPRESSION & %2'?3l) #4!#!''###!'##''##!4'!'4#4''4##!''!'44' 2 %2'?.OT) #44!'#''##'##4!!44!4#'#'4#444###!4 & (ACS?3BF) '#4!'###4'#!''!##!4'###'4!'!44#44# 2 (ACS?3l) ##4!'!#''###!'''''##'##4!44##4''!!'!!4!# & (YG P4%& ''!4#####!#!#!##!4!' 2 (YG #9#44 4#4##!'#44'#!!!44!!!'##44# (YG )NT 2 ##'###44!'!44!'!44'# +NOCK OUT & $OWN 4#4#444#!'4'!##4##!44' (YG )NT & #'!'''#!!!'!!!4!'!'' $%3 5P & !4'!4!!#4#!#!'!4##!!4!!#'44# $%3 $OWN 2 #4!!#444'''4444''4''44444' $%3 50 .ESTED & 4!''###!#!'444'!'44# $%3 5P 2 (YG & #4!4''4'4'4'''''!4## 4'!!4'##!''44!4''#4#!' $%3 $OWN .ESTED 2 '!444'4##444'#''4#4# $%3 KNOCK OUT $%3 $OWN & (YG 2 '!!''#444!!444'#!!'#4''!'! '44##44#444'#'''44#' $%3 )NT & !'##44#44#'##!#444# $%3 )NT 2 '4''#'!!'!!''#444!## 2 $OWNCM '!444'4##444'#''4#4#4#4# 352 5P & !4''!#!!'44#!'4#!'4!4!4## 352 $OWN 2 44!#4'44#444#4444''4'!4##' 352 5P .EST & #44'!44#!!''#'44##4' 352 5P 2 (YG & #4!4''4'4'4'''''!4## ''444'#4!4#'4'4#!44##!444' 352 KNOCK OUT 352 $OWN & (YG 2 '!!''#444!!444'#!!'#4''!'! '##4!'#4'##!4!44#!# 352 $OWN .EST 2 4#4'4#4!'!''##!!'!!# 352 )NT & '!###!444'!!''!444#4#44' 352 )NT 2 4!#'''!#!4!#!'#44'4' .PMFDVMBS #JPMPHZ
1$3 'O4AQLj 'REEN -ASTER -IX )T WAS USED FOR DETECTION PURPOSES )T IS A PREMIXED READY TO USE
SOLUTION CONTAINING A MODIlED FORM OF 4AQ $.! POLYMERASE D.40S -G#Lኼ REACTION BUFFER AND TWO DYES BLUE AND YELLOW THAT ALLOW MONITORING OF PROGRESS DURING ELECTROPHORESIS 4HE STANDARD AMPLIlCATION PROGRAM FOR THIS POLYMERASE IS DESCRIBED IN 4ABLE
4ABLE 3TANDARD 0#2 AMPLIlCATION PROGRAM FOR 'O4AQ 'REEN -ASTER -IX 0ROMEGA
3TEP 4EMPERATURE 4IME #YCLES )NITIAL DENATURATION # MIN $ENATURATION # MIN
!NNEALING # BELOW 4ᑞ MIN %XTENSION # MINKB &INAL EXTENSION # MIN (OLD # )NDElNITE
2!.'%2 $.! 0OLYMERASE 7AS USED TO AMPLIFY THE KNOCK OUT CASSETTE AS IT IS DESIGNED TO AMPLIFY LONG TEMPLATES WITH
EXTREME SENSITIVITY )T IS SUPPLIED WITH A 2EACTION "UFFER CONTAINING D.40S -G#Lኼ AND ENHANCERS AT OPTIMAL CONCENTRATIONS 3TANDARD AMPLIlCATION PROTOCOL IS DESCRIBED AT 4ABLE
4ABLE 3TANDARD 0#2 AMPLIlCATION PROGRAM FOR 2!.'%2 $.! POLYMERASE "IOLINE
3TEP 4EMPERATURE 4IME #YCLES )NITIAL DENATURATION # MIN $ENATURATION # SEC
!NNEALING%XTENSION # BELOW 4ᑞ MINKB (OLD # )NDElNITE
3!(!2! $.! 0OLYMERASE 7AS USED TO AMPLIFY GENES FROM GENOMIC $.! FOR PLASMID INSERTION AND FURTHER GENE CO EXPRESSION 4ABLE
4ABLE 3TANDARD 0#2 AMPLIlCATION PROGRAM FOR 3!(!2! $.! POLYMERASE "IOLINE
3TEP 4EMPERATURE 4IME #YCLES )NITIAL DENATURATION # MIN $ENATURATION # MIN
!NNEALING # BELOW 4ᑞ MIN %XTENSION # MINKB &INAL EXTENSION # MIN (OLD # )NDElNITE
%/" RVBOUJmDBUJPO 4HE CONCENTRATION OF $.! WAS DETERMINED BY USING A .ANO$ROP 3PECTOPHOTOMETER 4HERMO 3CIENTIlC , OF WATER WERE LOADED TO BLANK IT , OF SAMPLE WERE LOADED AND THE SOFTWARE CALCULATED THE ABSORBANCE CORRESPONDING TO THE CONCENTRATION OF $.! PRESENT IN THE SAMPLE AND VALUES WERE ALSO MONITORED IN ORDER TO DETERMINE $.! PURITY .BUFSJBMT BOE .FUIPET
"HBSPTF (FM &MFDUSPQIPSFTJT WV AGAROSE GELS WERE RUN TO VERIFY THE REQUIRED $.! AFTER AMPLIlCATION OR RESTRICTION AS WELL AS TO PURIFY THE BANDS FOR FURTHER WORK WITH THE SELECTED $.! !GAROSE GELS WERE PERFORMED BY HEATING THE DESIRED AMOUNT OF AGAROSE IN 4!% BUFFER UNTIL DISSOLVED /NCE COOLED 39"2Lj 3AFE $.! 'EL 3TAIN )NVITROGEN WAS ADDED 3AMPLES WERE SET UP BY ADDING , OF X LOADING BUFFER BEFORE DISPENSING INTO THE WELLS OF THE GEL 'ELS WERE LOADED WITH THE 'ENE2ULERÃ KB $.! ,ADDER READY TO USE &ERMENTAS TO DETERMINE $.! FRAGMENTS SIZE 'EL IMAGES WERE OBTAINED WITH 'EL $OCÃ %: 3YSTEM FROM "IO 2AD
& DPMJ 5SBOTGPSNBUJPO #HEMICAL COMPETENT % COLI CELLS WERE PREPARED ACCORDING TO .ISHIMURAS PROTOCOL ;= &OR THE TRANSFORMATION NG OF LIGATION MIXTURE WERE ADDED TO , OF COMPETENT CELLS AND INCUBATED ON ICE FOR MIN 4HE MIXTURE WAS HEAT SHOCKED AT # FOR SEC CHILLED ON ICE FOR MIN AND RESUSPENDED INTO PRE WARMED 3/# MEDIUM 2ESUSPENDED CELLS WERE INCUBATED AT # FOR H TO ALLOW EXPRESSION OF THE ANTIBODY RESISTANCE &INALLY VOLUMES OF , , AND , WERE PLATED ONTO ," AGAR PLATES CONTAINING THE REQUIRED ANTIBIOTIC AND INCUBATED OVERNIGHT AT #
4DSFFOJOH PG USBOTGPSNBOUT !FTER TRANSFORMATION INTO % COLI $( CELLS CLONES WERE SCREENED TO DETERMINE IF THE INSERT WAS SUCCESSFULLY CLONED BY COLONY 0#2 ;= OR PLASMID PURIlCATION FOLLOWED BY RESTRICTION ENZYME DIGESTION 3ELECTED PLASMIDS WERE SEQUENCED WITH THE PRIMERS & P'!0 AND 2 !/844 FOR P'!0(! AND & P'!0 AND 2 P05::,% FOR P05::,% 3EQUENCING WAS PERFORMED AT 5!"S 'ENOMICS AND "IOINFORMATICS 3ERVICE 4HE SEQUENCES WERE ALIGNED USING #LONE -ANAGER SOFTWARE 6ERIlED PLASMID CONSTRUCTS WERE THEN LINEARISED WITH !VR)) FOR INTEGRATION INTO THE TRANSCRIPTION TERMINATION LOCUS OF !/8 !/844 AND TRANSFORMED INTO 8 P'!0:! &AB& COMPETENT CELLS
1 QBTUPSJT USBOTGPSNBUJPO %LECTROCOMPETENT 0 PASTORIS CELLS WERE PREPARED ACCORDING TO ,IN #EREGHINOS PROTOCOL ;=AS FOLLOWS CELLS WERE GROWN OVERNIGHT IN M, 90$ MEDIUM IN A # SHAKING INCUBATOR DILUTED TO
!BS OF IN M, 90$ AND GROWN UNTIL AN !BS OF WAS REACHED #ELLS WERE HARVESTED AT G FOR MIN AT 24 AND THE SUPERNATANT DISCARDED 0ELLET WAS RESUSPENDED IN M, OF ICE COLD "%$3 SOLUTION SUPPLEMENTED WITH M, - $ITHIOTHREITOL $44 AND INCUBATED FOR MIN AT RPM AT # #ELL SUSPENSION WAS CENTRIFUGED AT G FOR MIN AT 24 AND RESUSPENDED IN M, OF "%$3 &RESH COMPETENT CELLS , WERE MIXED WITH NG OF $.! IN AN ELECTROPORATION CUVETTE "IO 2AD 'ENE 0ULSER CUVETTE GAP MM AND INCUBATED ON ICE FOR MIN 3AMPLES WERE ELECTROPORATED USING A 'ENE0ULSERLj )) ELECTROPORATOR "IO 2AD USING A CHARGING VOLTAGE OF 6 A RESISTANCE OF ͕ AND A CAPACITANCE OF & !FTER ELECTROPORATION SAMPLES WERE RESUSPENDED IN A COLD MIXTURE OF M, - 3ORBITOL AND M, 90$ INCUBATED AT # IN A M, &ALCON TUBE FOR H AND , OF THE CELL SUSPENSION WERE PLATED ON 90$ AGAR PLATES CONTAINING GM,ዅኻ :EOCIN AND (YGROMYCIN AND INCUBATED AT # UNTIL COLONIES APPEAR DAYS $FMM $VMUJWBUJPO
1 QBTUPSJT DMPOF TFMFDUJPO #OLONIES WERE PICKED EXTRACTED THE GENOMIC $.! AND CHECKED FOR CORRECT INTEGRATION OR DELETION BY 0#2 SCREENING 0RIMERS FOR INTEGRATION P'!0 AND !/844 0 PASTORIS COLONY 0#2 $%3 KNOCK OUT TRANSFORMANT COLONIES WERE CHECKED FOR GENE DELETION BY COLONY 0#2 ACCORDING TO 4HE -URRAY ,AB PROTOCOL AS FOLLOWS 0#2 TUBES lLLED WITH , OF DISTILLED WATER WERE MIXED WITH CELLS FROM A COLONY 4UBES WERE FROZEN FOR MIN MICROWAVED FOR MIN ON HIGH POWER AND FROZEN AGAIN FOR MIN 4HE 0#2 MIX WAS ADDED TO THE TUBE TO lNAL , VOLUME MIXED SPINNED DOWN AND 0#2 WAS PERFORMED AS USUAL FOR 'O4AQ POLYMERASE
$FMM $VMUJWBUJPO 4UPDL TPMVUJPOT X 9." WV 9EAST .ITROGEN "ASE WITH AMMONIUM SULPHATE AND WITHOUT AMINO ACIDS &ILTER STERILISED 3TORED AT #
X "IOTIN WV "IOTIN &ILTER STERILISED 3TORED AT #
X 'LUCOSE WV 'LUCOSE !UTOCLAVED AT # FOR MIN AND COOLED DOWN TO 24 3TORED AT 24
X 'LYCEROL WV 'LYCEROL !UTOCLAVED AT # FOR MIN AND COOLED DOWN TO 24 3TOREDAT24
.FEJB 90' MEDIUM WV PEPTONE WV YEAST EXTRACT WV GLYCEROL P(
"-' "UFFERED -INIMAL 'LYCEROL AND "-$ "UFFERED -INIMAL $EXTROSE -EDIUM M- 0OTASSIUM 0HOSPHATE P( WV 9." qዅ WV "IOTIN WV 'LYCEROL OR WV $EXTROSE #OMPONENTS ARE PREPARED AS STOCK SOLUTIONS SEPARATELY STERILISED AND MIXED TOGETHER 3TORED AT 24
"ATCH -EDIUM WV GLYCEROL WV CITRIC ACID WV AMMONIUM PHOSPHATE DIBASIC
.(ኾ ኼ(0/ኾ WV CALCIUM CHLORIDE DIHYDRATE #A#Lኼ q(ኼ/ WV POTASSIUM
CHLORIDE +#L WV MAGNESIUM SULPHATE HEPTAHYDRATE -G3/ኾ q(ኼ/ M, "IOTIN M, 04- TRACE SALTS STOCK SOLUTION 4HE P( WAS SET TO WITH HYDROGEN CHLORIDE (#L
#HEMOSTAT -EDIUM WV GLUCOSE WV CITRIC ACID WV AMMONIUM PHOSPHATE DIBASIC
.(ኾ ኼ(0/ኾ WV CALCIUM CHLORIDE DIHYDRATE #A#Lኼ q(ኼ/ WV POTASSIUM
CHLORIDE +#L WV MAGNESIUM SULPHATE HEPTAHYDRATE -G3/ኾ q(ኼ/ M, "IOTIN AND M, 04- TRACE SALTS STOCK SOLUTION 4HE P( WAS SET TO WITH HYDROGEN CHLORIDE (#L .BUFSJBMT BOE .FUIPET
&ED "ATCH &EEDING -EDIUM WV GLUCOSE WV POTASSIUM CHLORIDE +#L MAGNESIUM SULPHATE
HEPTAHYDRATE -G3/ኾ q(ኼ/ WV CALCIUM CHLORIDE DIHYDRATE #A#Lኼ q(ኼ/ M, "IOTIN M, 04- TRACE SALTS SOLUTION AND M, OF ANTIFOAM 3TRUKTOL *
4RACE 3ALT 3TOCK 3OLUTION 04-
WV CUPRIC SULPHATE PENTAHYDRATE #U3/ኾ q(ኼ/ WV SODIUM IODIDE .A)
WV MANGANESE SULPHATE MONOHYDRATE -N3/ኾ q(ኼ/ SODIUM MOLYBDATE DIHYDRATE
.Aኼ-O/ኾ q(ኼ/ BORIC ACID (ኽ"/ኽ COBALT CHLORIDE #O#Lኼ q(ኼ/ ZINC
CHLORIDE :N#Lኼ FERROUS SULPHATE HEPTAHYDRATE &E3/ኾ q(ኼ/ AND M, SULPHURIC ACID
(ኼ3/ኾ
#VGGFST 0"3 M- SODIUM CHLORIDE .A#L M- POTASSIUM CHLORIDE +#L M- DISODIUM PHOSPHATE
.Aኼ(0/ኾ M- MONOPOTASSIUM PHOSPHATE +(ኼ0/ኾ !DJUST THE P( TO WITH HYDROGEN CHLORIDE (#L
0OTASSIUM 0HOSPHATE "UFFER
&IRST - STOCK SOLUTIONS OF DIPOTASSIUM PHOSPHATE +ኼ(0/ኾ AND MONOPOTASSIUM PHOSPHATE
+(ኼ0/ኾ WERE PREPARED 4O OBTAIN A M- BUFFER AT P( M, OF - +ኼ(0/ኾ AND M,
OF - +(ኼ0/ኾ WERE COMBINED AND DILUTED TO , WITH DISTILLED WATER P( IS CALCULATED ACCORDING THE (ENDERSON (ASSELBALCH EQUATION
PROTON ACCEPTOR P( P+ LOG ᗟ ᗠ PROTON DONOR
WHERE P+ AT #
'MVDPOB[PMF USFBUNFOU ,YOPHILISED &LUCONAZOLE WAS DILUTED TO MG M,ዅኻ IN $-3/ 4HE STOCK SOLUTION WAS DILUTED TO GM,ዅኻ IN STERILE DISTILLED WATER AND ADDED TO THE CULTURE MEDIUM "-$ AT lNAL CONCENTRATION OF GM,ዅኻ
4IBLF 'MBTL $VMUJWBUJPO &OR SMALL SCALE CULTIVATIONS lRST A FRESH COLONY WAS GROWN OVERNIGHT IN M, 90$ :EO (YG AT
# AND RPM AND USED TO INOCULATE A M, "-' CULTURE AT AN INITIAL /$ዀኺኺ OF #ELLS WERE INCUBATED AT # RPM UNTIL THEY REACH EXPONENTIAL PHASE -INIMAL MEDIUM ADAPTATION 4HE SCREENING CULTURES WERE PERFORMED IN , BAFmED SHAKE mASKS WITH M, OF "-$ MEDIUM
INOCULATED TO AN INITIAL /$ዀኺኺ OF AND INCUBATED AS PREVIOUSLY UNTIL CELLS REACHED THE END OF EXPONENTIAL PHASE $RY CELL WEIGHT $#7 &AB YIELD CELL WALL POROSITY AND MEMBRANE mUIDITY WERE DETERMINED FROM SHAKE mASK CULTURES
*OPDVMVN )NOCULUM CULTURES WERE GROWN IN , BAFmED SHAKE mASKS WITH M, 90' GM,ዅኻ :EOCIN GM,ዅኻ (YGROMYCIN FOR MUTANT STRAINS CULTURES #ELLS WERE GROWN FOR H AT # AND $FMM $VMUJWBUJPO
RPM IN A +HUNER ,3 8 INCUBATOR 3HAKE mASKS WERE INOCULATED FROM CRYOSTOCKS OF THE STRAINS GROWN HARVESTED BY CENTRIFUGATION AND RESUSPENDED IN BATCH MEDIUM TO BE INOCULATED INTO THE BIOREACTOR
$IFNPTUBU DVMUJWBUJPOT
#HEMOSTAT CULTIVATIONS WERE PERFORMED AT A WORKING VOLUME OF , IN A , BENCH TOP BIOREACTOR "IOSTAT " OR "IOSTAT " "RAUN "IOTECH CONNECTED TO A $#5 DIGITAL CONTROL UNIT AND THE -WIN PROCESS SUPERVISORY SYSTEM "RAUN "IOTECH )NTERNATIONAL #ELLS WERE GROWN UNDER GLUCOSE LIMITED CONDITIONS AT A CONSTANT DILUTION RATE OF Ĝ Hዅኻ USING A PERISTALTIC PUMP )SMATEC TO CONTROL THE FEEDING #ULTIVATIONS WERE CARRIED OUT USING THE BATCH MEDIUM AND CHEMOSTAT MEDIUM WITH THE ANTIFOAM 'LANAPON M, PER LITRE OF CHEMOSTAT MEDIUM 4HE P( VALUE WAS MAINTAINED AT BY ADDITION OF VV AMMONIUM HYDROXIDE THE TEMPERATURE WAS SET TO # AND AN OVERPRESSURE OF BARS 4HE AERATION RATE WAS VVM CONTROLLED BY MEANS OF THERMAL MASS mOW CONTROLLERS 4-&# "RONKHORST (I 4ECH 4HE OXYGEN SUPPLY WAS VARIED BY VARYING THE PERCENTAGE
OF OXYGEN IN A GAS MIXTURE OF AIR AND .ኼ WHILE STIRRING THE CULTURE AT A CONSTANT SPEED OF RPM $ISSOLVED OXYGEN $/ SATURATION WAS MONITORED ONLINE WITH AN OXYGEN PROBE -ETTLER 4OLEDO 3AMPLES WERE TAKEN FOR EACH PHYSIOLOGICAL STEADY STATE CONDITION AFTER AT LEAST lVE RESIDENCE TIMES
'FECBUDI DVMUJWBUJPOT
&ED BATCH CULTIVATIONS WERE CARRIED OUT IN A , "IOSTAT " "IOREACTOR "RAUN "IOTECH WORKING AT INITIAL VOLUME OF , AND lNISHING THE PROCESS AT APPROXIMATELY , !LL FED BATCH CULTIVATIONS WERE CARRIED OUT AT A SPECIlC GROWTH RATE OF Hዅኻ 4HE CULTURE CONDITIONS WERE MONITORED AND CONTROLLED AT THE FOLLOWING VALUES TEMPERATURE # P( WITH ADDITION OF VV
AMMONIUM HYDROXIDE .(ኾ/( P/ኼ ABOVE SATURATION BY CONTROLLING THE STIRRING SPEED
BETWEEN AND RPM AND USING MIXTURES OF AIR AND /ኼ AT AERATION WITHIN AND VVM 7ATER EVAPORATION LOSSES WERE MINIMISED DURING THE PROCESSES USING AN EXHAUST GAS CONDENSER WITH COOLING WATER AT # 4HE STANDARD FERMENTATION STRATEGY CONSISTED OF TWO PHASES &IRST THE BATCH PHASE STARTS WITH A STANDARD CARBON SOURCE CONCENTRATION OF G ,ዅኻ AND IT IS PERFORMED USING THE BATCH MEDIUM $URING THIS STEP YEAST GROWS AT MAXIMUM SPECIlC GROWTH RATE UNTIL THE DEPLETION OF THE # SUBSTRATE ACHIEVING A MODERATE CONCENTRATION OF BIOMASS ࣵ G ,ዅኻ 4HEREAFTER THE FED BATCH PHASE STARTS WHERE THE CULTURE REACHES HIGH CONCENTRATIONS OF BIOMASS AND PRODUCT 4HE FED BATCH PHASE WAS CARRIED OUT BY ADDING FEEDING MEDIUM 4HE FERMENTATION STRATEGY DURING THE FED BATCH PHASE AIMED TO ACHIEVE PSEUDO STEADY STATE CONDITIONS FOR SPECIlC RATES DURING CARBON LIMITING GROWTH ! PRE PROGRAMMED EXPONENTIAL FEEDING RATE PROlLE FOR SUBSTRATE ADDITION DERIVED FROM MASS BALANCE EQUATIONS TO MAINTAIN A CONSTANT SPECIlC GROWTH RATE WAS IMPLEMENTED ;= 4HIS OPEN LOOP CONTROL STRUCTURE ALLOWS MAINTAINING A CONSTANT WHICH ENHANCES PROCESS REPRODUCIBILITY AND FACILITATES THE SYSTEMATIC STUDY OF GROWTH RATE RELATED EFFECTS ON HETEROLOGOUS PROTEIN PRODUCTION 4HE EQUATIONS DERIVED FROM THE FED BATCH SUBSTRATE BALANCE USED TO APPLY THIS STRATEGY WERE DESCRIBED ELSEWHERE ; = ! CONSTANT BIOMASS TO SUBSTRATE ዅኻ YIELD 9ፗፒ G$#7 G3 WAS CONSIDERED TO DETERMINE THE INITIAL FEED RATE 4HE FEEDING MEDIUM WAS ADDED BY AUTOMATIC MICRO BURETTES -ICO"5 #RISON .BUFSJBMT BOE .FUIPET
"OBMZUJDBM NFUIPET $FMM DVMUVSF BOBMZUJDBM NFUIPET #JPNBTT EFUFSNJOBUJPO #ELL DENSITY ANALYSES WERE ALWAYS PERFORMED BY TRIPLICATE AND THE RELATIVE STANDARD DEVIATION 23$ WAS NO HIGHER THAN
$RY #ELL 7EIGHT $#7 $#7 WAS DETERMINED BY lLTERING A KNOWN VOLUME OF CULTIVATION BROTH USING PRE WEIGHTED DRIED GLASS MICROlBRE lLTERS M PORE SIZE -ILLIPORE 4HEN THE lLTERS WERE WASHED WITH TWO VOLUMES OF DISTILLED WATER AND DRIED TO CONSTANT WEIGHT AT # FOR H #ELL WEIGHT WAS DElNED AS THE WEIGHT lLTER DIFFERENCE BEFORE AND AFTER CELL lLTRATION
/PTICAL DENSITY !LTERNATIVELY BIOMASS WAS MEASURED BY OPTICAL DENSITY AT NM 3AMPLES WERE DILUTED WITH DISTILLED WATER TO BE ON THE RANGE OF ABSORBANCE
5PUBM QSPUFJO DPODFOUSBUJPO 4OTAL PROTEIN WAS DETERMINED WITH THE BICINCHONINIC ACID PROTEIN ASSAY KIT 0IERCE "#! 0ROTEIN !SSAY ACCORDING TO THE MANUFACTURERS INSTRUCTIONS "OVINE SERUM ALBUMIN "3! WAS USED AS THE PROTEIN STANDARD FOR THE CALIBRATION CURVE
2VBOUJmDBUJPO PG FYUSBDFMMVMBS NFUBCPMJUFT 3AMPLES WERE CLARIlED AT RPM FOR MIN IN A MICRO CENTRIFUGE -INISPIN %PPENDORF AND SUBSEQUENTLY lLTERED THROUGH M lLTERS -ILLIPORE 'LUCOSE GLYCEROL ETHANOL ARABITOL AND ORGANIC ACIDS CONCENTRATIONS WERE DETERMINED BY (0,# WITH A (0 LIQUID CHROMATOGRAPHY $IONEX #ORPORATION USING A IONIC EXCHANGE COLUMN )#3EP )#% #/2%'%, ( COLUMN 4RANSGENOMIC )NC !S MOBILE PHASE M- SULPHURIC ACID WAS USED -ETABOLITES WERE QUANTIlED WITH THE #HROMELEON 3OFTWARE $IONEX #ORPORATION
0GGHBT BOBMZTJT !CCURATE ESTIMATION OF OXYGEN UPTAKE RATE /52 CARBON DIOXIDE PRODUCTION RATE #02 AND
RESPIRATORY QUOTIENT 21 WAS CARRIED OUT THROUGH /ኼ AND #/ኼ BALANCES ;=
"LUE3ENS SENSORS
7HEN CHEMOSTAT CULTIVATIONS WERE PERFORMED IN A "IOSTAT " ONLINE CONCENTRATIONS OF THE /ኼ AND
#/ኼ IN THE EXHAUST GAS OF THE BIOREACTOR CULTIVATIONS WERE DETERMINED AFTER BEING COOLED IN A
CONDENSER # DRIED WITH TWO SILICA GEL COLUMNS AND SUBSEQUENTLY ANALYSED USING SPECIlC /ኼ
AND #/ኼ SENSORS "#0 #/ AND "#0 / "LUE3ENS
-ASS SPECTROMETRY "IOSTAT " CHEMOSTAT CULTIVATIONS USED A QUADRUPOLE MASS SPECTROMETER "ALZERS 1UADSTAR 0FEIFFER 6ACUUM FOR ON LINE EXHAUST GAS ANALYSIS %XHAUST GAS HUMIDITY WAS REDUCED BY USING A CONDENSER WATER AT # AND TWO SILICA GEL COLUMNS 4HE &ARADAY CUP DETECTOR WAS USED FOR ITS SIMPLICITY STABILITY AND RELIABILITY DETERMINING RESPONSES OF MZ CORRESPONDING TO THE MAJOR
GAS PEAKS .ኼ /ኼ #/ኼ !R .ORMALISED MASS SPECTROMETER SIGNALS WERE USED TO REDUCE ERRORS CAUSED BY VARIATIONS ON OPERATING CONDITIONS SUCH PRESSURE AND TEMPERATURE AS WELL "OBMZUJDBM NFUIPET
AS OTHERS THAT CAN GENERATE SOME DRIFT AND NOISE OF SIGNALS -ULTIVARIATE CALIBRATION WAS PERFORMED BY ORDINARY LEAST SQUARES /,3 MINIMISATION WITH SUITABLE STANDARD CALIBRATION MIXTURES ACCORDING TO THE COMPONENTS TO BE ANALYSED AND ITS CONCENTRATION RANGE 4HE TOTAL HUMID OFF GAS mOW RATE WAS NOT MEASURED DIRECTLY AND IT WAS CALCULATED BY INERT BALANCE AROUND THE REACTOR 4O PROPERLY ESTIMATE THIS mOW RATE DETERMINATION OF WATER COMPOSITION IS NECESSARY AND IT CAN BE CALCULATED
FROM THE QUOTIENT BETWEEN THE OFF GAS /ኼ COMPOSITION WITHOUT BIOREACTION DUE TO WATER STRIPPING
AND THE CORRESPONDING INLET /ኼ MOLAR FRACTION )NLET AIR COMPOSITION WAS OBTAINED FROM A H MEASUREMENT AVERAGE BEFORE INOCULATING
1SPDFTT QBSBNFUFST EFUFSNJOBUJPO DPOTJTUFODZ DIFDL BOE EBUB SFDPODJMJBUJPO
3PECIlC RATES Q። AND YIELDS ARE PARAMETERS OF CAPITAL IMPORTANCE FOR COMPARING DIFFERENT CULTURE CONDITIONS AND ALLOW THE IDENTIlCATION OF CHANGES IN THE PHYSIOLOGICAL CELL STATE THAT CAN IMPACT
INTO PRODUCT QUALITY ;= 4HEIR CALCULATION IS BASED ON THE CONVERSION RATES R። DETERMINED IN THE GENERAL MASS BALANCE OF THE CULTIVATION 3PECIlC RATES ARE TYPICALLY CONVERSIONS RATES RELATED TO THE BIOMASS CONCENTRATION 9IELDS ARE DElNED AS RATIOS BETWEEN RATES AND POSITIVE 4HE CONSISTENCY OF THE MEASUREMENTS WAS CHECKED BY STANDARD STATISTICAL TESTS CONSIDERING ELEMENTAL BALANCES AS CONSTRAINTS ;= 7ITH THE CURRENT EXPERIMENTAL SET UP IT WAS POSSIBLE THE MEASUREMENT OF THE lVE KEY SPECIlC RATES IN THE BLACK BOX PROCESS MODEL BIOMASS GENERATION
GLUCOSE UPTAKE Qፒ PRODUCT FORMATION Qፏ OXYGEN UPTAKE Qፎኼ AND CARBON DIOXIDE
PRODUCTION Qፂፎኼ 4HE CARBON BALANCE AND THE REDOX BALANCE WERE USED AS CONSTRAINTS AND PROTEIN PRODUCTION CONSIDERED NEGLIGIBLE IN THESE BALANCES 3O THE SYSTEM IS OVERDETERMINED AND THE DEGREE OF REDUNDANCY IS THE SAME AS THE NUMBER OF CONSTRAINTS 4HIS FACT CAN BE USED TO CHECK THE MEASUREMENTS FOR GROSS ERRORS OR UNIDENTIlED METABOLITES RESPECTING THE COVARIANCE FOR EACH MEASUREMENT ;= AND TO IMPROVE THE ACCURACY OF THE MEASURED CONVERSION RATES BY DATA RECONCILIATION METHODS ;= 4HE H VALUE GIVEN BY THE SUM OF THE WEIGHTED SQUARES OF THE RESIDUALS IS THE OUTPUT OF THE STATISTICAL TEST FOR THE PRESENCE OF GROSS ERRORS OR NEGLECTED COMPONENTS ߶ ൕዅኻ
)F H EXCEEDS THE THRESHOLD VALUE THAT DEPENDS ON THE SIGNIlCANCE LEVEL IN THIS WORK AND DEGREE OF REDUNDANCY ACCORDING TO THE ဉኼ DISTRIBUTION IT IS CONCLUDED THAT THERE ARE SIGNIlCANT ERRORS IN THE MEASUREMENTS OR ANY COMPOUND HAS NOT BEEN TAKEN INTO ACCOUNT IN THE BLACK BOX PROCESS MODEL 4HE VARIANCES OF ALL SPECIlC RATE MEASUREMENTS WERE CONSIDERED UNCORRELATED AND ESTIMATED BY REPLICATES ANDOR ERROR PROPAGATION 4HE ဉኼ TEST PERFORMED FOR ALL THE EXPERIMENTAL DATA OBTAINED FROM CHEMOSTAT CULTIVATIONS SHOWED THE MEASUREMENTS SATISlED MOSTLY THE STOICHIOMETRIC MODEL AND HENCE BOTH # BALANCE AND E BALANCE $ATA RECONCILIATION PROCEDURES ARE ALSO BASED ON THE USE OF ELEMENTAL BALANCES ACCORDING TO A BLACK BOX REACTION SCHEME TO IMPROVE THE ACCURACY OF THE MEASURED SPECIlC RATES OR YIELDS AND ALSO TO DETERMINATE THE UNKNOWN SPECIlC RATES ;= ! MEASUREMENT ERROR VECTOR IS FOUND BY USING A LEAST SQUARES APPROACH TO CALCULATE THE RECONCILED VECTOR WHICH INCLUDES THE BEST ESTIMATES OF REACTION RATES TO lT ALL CONSTRAINTS IMPOSED
$FMM EJTSVQUJPO BOE QSPUFJO FYUSBDUJPO (IGH PRESSURE HOMOGENIZATION #ELLS FROM THE CULTURES WERE HARVESTED BY CENTRIFUGATION G MIN # AND PELLETS WERE WASHED TWICE IN COLD 0"3 P( IN ORDER TO REMOVE ALL MEDIA COMPONENTS AND OTHER CONTAMINANTS #ELLS WERE THEN RESUSPENDED IN M, OF COLD BREAKING BUFFER 0"3 P( M- .BUFSJBMT BOE .FUIPET
0-3& AND DISRUPTED BY HIGH PRESSURE HOMOGENISATION USING A /NE 3HOT #ELL $ISRUPTER #ONSTANT 3YSTEMS /NCE DISRUPTED HOMOGENATES WERE CLARIlED BY CENTRIFUGATION G MIN # 3UPERNATANTS WERE COLLECTED AND STORED AS SOLUBLE CYTOSOLIC FRACTION 3#& WHILE PELLETS WERE KEPT AS THE INSOLUBLE MEMBRANE FRACTION )-& 4HE PROTEASE INHIBITOR M- 0-3& AND CHILLING CONDITIONS WERE ALWAYS USED DURING THE WHOLE PROCESS TO MINIMISE THE RISK OF PROTEIN DEGRADATION BY ENDOGENOUS PROTEASES AS WELL AS TO PRESERVE PROTEIN PROPERTIES IN THE CASE OF LOW TEMPERATURE
%VALUATION OF CELL DISRUPTION EFlCIENCY #ELL NUMBER WAS DETERMINED AFTER EACH DISRUPTION PASS BY MEANS OF mOW CYTOMETRY ASSAYS 'UAVA %ASY#UTEÃ -INI CYTOMETER -ILLIPORE 4HE DEGREE OF DISRUPTION WAS EXPRESSED AS %QUATION
N N። 8 q N
WHERE 8 IS THE DEGREE OF DISRUPTION N THE INITIAL NUMBER OF CELLS BEFORE DISRUPTION N። THE NUMBER OF NON DISRUPTED CELLS AFTER EACH PASS
0ROTEIN EXTRACTION FROM INSOLUBLE MEMBRANE FRACTION )-& 0ROTEIN EXTRACTION WAS CARRIED OUT IN CHILLY CONDITIONS &RACTION PELLETS WERE RESUSPENDED BY PIPETTING IN M, EXTRACTION BUFFER SUPPLEMENTED WITH DETERGENT AND THEN VORTEXED %XTRACTS WERE INCUBATED IN GENTLY SHAKING AT # CLARIlED BY CENTRIFUGATION G MIN # AND SUPERNATANTS WERE STORED AS INSOLUBLE MEMBRANE FRACTION )-& EXTRACTS
%XTRACTION DETERGENT AND BUFFERS &OR THE INITIAL STUDY FOCUSED ON A SCREENING FOR THE DETERGENT AND BUFFER OPTIMISATION THREE DIFFERENT BUFFERS WERE COMPARED FOR THE TARGET PROTEIN EXTRACTION
s "UFFER ! M- 4RIS (#L P( M- .A#L M- %$4! M- 0-3&
s "UFFER " GLYCEROL M- (%0%3 P( M- .A#L M- 0-3&
s "UFFER # GLYCEROL M- SODIUM PHOSPHATE P( M- %$4! M- .A#L M- 0-3&
)N ADDITION ALL THE EXTRACTION BUFFERS WERE ALSO SUPPLEMENTED WITHOFTHREEDIFFERENT DETERGENTS 4WEEN 4RITON 8 AND #(!03 FOR THE DETERGENT AND BUFFER SCREENING /NCE THE OPTIMAL COMBINATION OF BUFFER AND DETERGENT WAS SELECTED DIFFERENT CONCENTRATIONS OF #(!03 WERE TESTED FOR AN IMPROVED &AB EXTRACTION 4HEY WERE ALSO SCREENED DIFFERENT INCUBATION TIMES )N THIS SENSE EXTRACTIONS COMBINING #(!03 AND H H AND H OVERNIGHT INCUBATION TIMES WERE PERFORMED AND COMPARED
&-*4" &AB & WAS QUANTIlED BY SANDWICH %,)3! IN THE SECRETED FRACTIONS AS WELL AS IN THE 3#& AND THE )-& WELL MICROTITER PLATES -AXI3ORB WERE COATED WITH ANTI H)G' &AB SPECIlC IN 0"3 P( OVERNIGHT AT 24 3AMPLES WERE SERIALLY DILUTED IN DILUTION BUFFER 0"3 P( VV 4WEEN WV BOVINE SERUM ALBUMIN "3! AND INCUBATED IN GENTLY AGITATION FOR H AT 24 ! PAPAIN DIGESTED PURIlED NORMAL HUMAN &ABKAPPA FROM )G' WAS USED AS A STANDARD PROTEIN AT A STARTING CONCENTRATION OF NG M,ዅኻ !FTER EACH INCUBATION STEP PLATES WERE WASHED THREE TIMES WITH WASHING BUFFER 0"3 P( 4WEEN !NTI KAPPA LIGHT CHAIN ALKALINE PHOSPHATASE CONJUGATE WAS ADDED AS SECONDARY ANTIBODY IN DILUTION BUFFER AND INCUBATED FOR H AT "OBMZUJDBM NFUIPET
24 &INALLY PLATES WERE STAINED WITH P NITROPHENYL PHOSPHATE P.00 MG M,ዅኻ IN COATING BUFFER
..Aኼ#/ኽ.A(#/ኽ P( AND READ AT NM REFERENCE WAVELENGTH NM $ATA ANALYSIS WAS PERFORMED BY USING THE STANDARD CURVE H&AB STANDARD AND A POLYNOMIAL FUNCTION
1MBTNB NFNCSBOF nVJEJUZ EFUFSNJOBUJPO CZ BOJTPUSPQZ NFBTVSFNFOUT $YNAMIC AND STRUCTURAL CHARACTERISTICS OF MEMBRANES CAN BE CHANGED EITHER BY CHANGING THE ENVIRONMENTAL CONDITIONS THE MOLECULAR COMPOSITION OF THE MEMBRANE OR BY ADDING FOREIGN MOLECULES THAT INTERACT WITH MEMBRANE CONSTITUENTS #ELLS CAN CONTROL THEIR mUIDITY BY MODULATION OF THEIR MEMBRANE COMPOSITION IN ORDER TO MAINTAIN AN OPTIMAL LEVEL OF mUIDITY ;= &LUIDITY IS A VITAL PARAMETER OF MEMBRANE FUNCTION AFFECTING PERMEABILITY AND FUNCTIONS OF MEMBRANE ASSOCIATED PROTEINS SUCH AS RECEPTORS ENZYMES AND TRANSPORTERS ;= 5SUALLY THE mUIDITY OF A MEMBRANE IS MEASURED AS ANISOTROPY BY mUORESCENCE POLARISATION OF A PROBE ;= ! LIPOPHILIC mUORESCENT MOLECULE DIPHENYL HEXATRIENE $0( IS COMMONLY USED IN SUCH EXPERIMENTS AND THE MEASUREMENT OF THE STEADY STATE ANISOTROPY OF $0( IS THE MOST COMMON TECHNIQUE TO ESTIMATE ORDERING OF THE MEMBRANE LIPID CONSTITUENTS WHICH IS INVERSELY PROPORTIONAL TO MEMBRANE mUIDITY ;= 4HE ORIGIN OF ANISOTROPY IS THE EXISTENCE OF TRANSITION MOMENTS FOR ABSORPTION AND EMISSION THAT LIE ALONG SPECIlC DIRECTIONS WITHIN THE mUOROPHORE STRUCTURE )N HOMOGENEOUS SOLUTION THE GROUND STATE mUOROPHORES ARE ALL RANDOMLY ORIENTED 7HEN EXPOSED TO POLARISED LIGHT THOSE mUOROPHORES THAT HAVE THEIR ABSORPTION TRANSITION MOMENTS ORIENTED ALONG THE VECTOR OF THE INCIDENT LIGHT ARE PREFERENTIALLY EXCITED (ENCE THE EXCITED STATE POPULATION IS PARTIALLY ORIENTED
&IGURE 'ENERAL SCHEME OF !NISOTROPY DETERMINATION &ROM ;=
4HE MEASUREMENT OF mUORESCENCE ANISOTROPY IS ILLUSTRATED IN &IGURE 4HE SAMPLE IS EXCITED WITH VERTICALLY POLARISED LIGHT AND THE EMISSION IS OBSERVED $IFFERENT TRANSMISSION EFlCIENCIES FOR VERTICALLY AND HORIZONTALLY POLARISED LIGHT ARE DESCRIBED THUS THE ROTATION OF THE EMISSION POLARISER CHANGES THE MEASURED INTENSITIES 4HE MEASURED INTENSITY RATIO IS DIFFERENT FROM THE TRUE VALUE BY A FACTOR OF ' 4O CALCULATE THE ACTUAL INTENSITY RATIO THE ' FACTOR IS DESCRIBED AS THE RATIO .BUFSJBMT BOE .FUIPET
OF SENSITIVITIES OF THE DETECTION SYSTEM FOR VERTICALLY AND HORIZONTALLY POLARISED LIGHT 4HE ' FACTOR IS EASILY MEASURED USING HORIZONTALLY POLARISED EXCITATION WHERE THE EXCITED STATE DISTRIBUTION IS ROTATED TO LIE ALONG THE OBSERVATION AXIS AND THE HORIZONTALLY AND VERTICALLY POLARISED COMPONENTS ARE EQUAL ;= 4HE ANISOTROPY IS CALCULATED AS %QUATION
-EMBRANE mUIDITY WAS ASSESSED BY MEASURING mUORESCENCE ANISOTROPY OF DIPHENYL HEXATRIENE $(0 FOLLOWING INCORPORATION INTO THE PLASMA MEMBRANE ! ,3 ,UMINESCENCE 3PECTROMETER 0ERKIN %LMER WITH lXED EXCITATION AND EMISSION POLARISATION lLTERS WAS USED TO MEASURE mUORESCENCE INTENSITY PARALLEL AND PERPENDICULAR TO THE POLARISATION PHASE OF THE EXCITING LIGHT $(0 WAS EXCITED AT q NM AND EMISSION WAS MEASURED AT q NM 4HE MEASURED mUORESCENCE INTENSITIES WERE CORRECTED FOR BACKGROUND mUORESCENCE AND LIGHT SCATTERING FROM THE UNLABELLED SAMPLE 4HE DEGREE OF mUORESCENCE ANISOTROPY R WAS CALCULATED ACCORDING TO %QUATION ) 'X) ) ൰ 66 6( ' (6 )66 'X)6( )((
)66 AND )6( ARE THE mUORESCENCE INTENSITIES DETERMINED AT VERTICAL AND HORIZONTAL ORIENTATIONS OF
THE EMISSION POLARISER WHEN THE EXCITATION POLARISER IS SET IN THE VERTICAL POSITION )ፇፕ AND )ፇፇ ARE THE mUORESCENCE INTENSITIES DETERMINED AT VERTICAL AND HORIZONTAL POSITIONS OF THE EMISSION POLARISER WHEN THE EXCITATION POLARISER IS SET HORIZONTALLY ' IS A CORRECTION FACTOR FOR BACKGROUND mUORESCENCE AND LIGHT SCATTERING
#ELLS WERE SUSPENDED IN M- POTASSIUM PHOSPHATE BUFFER P( TO A lNAL /$ዀኺኺ OF G $#7 M,ዅኻ $(0 STOCK SOLUTION - IN TETRAHYDROFURAN WAS ADDED AT A lNAL CONCENTRATION OF - THE SUSPENSION WAS INCUBATED IN THE DARK AT # AND RPM FOR MIN CLARIlED RPM MIN # AND RESUSPENDED AGAIN IN M, OF POTASSIUM PHOSPHATE BUFFER &OLLOWING LABELLING M, OF CELLS WERE PLACED IN CM PATH LENGTH QUARTZ CUVETTES &LUORESCENCE ANISOTROPY WAS DETERMINED IN TRIPLICATES
&MFDUSPO NJDSPTDPQZ !N ELECTRON MICROSCOPE IS SIMILAR TO A LIGHT MICROSCOPE BUT INSTEAD OF USING ELECTRONS AT A WIDE RANGE OF WAVELENGTHS A HIGH ENERGY BEAM OF ELECTRONS AT THE SAME WAVELENGTH IS USED )N 4%- ELECTRONS EMITTED ARE FOCUSED BY A SET OF CONDENSER LENSES INTO A COHERENT PARALLEL BEAM THAT PASS THROUGH THE SAMPLE TO CREATE THE IMAGE %LECTRON DENSE AREAS IN THE SAMPLE DEmECT THE ELECTRONS FROM THEIR STRAIGHT PATHS PRODUCING DARKENED AREAS THAT FORM THE IMAGE !NALYSIS OF SUBCELLULAR STRUCTURES IN LIVING OR lXED YEAST CELLS USING LIGHT MICROSCOPIC TECHNIQUES IS A PARTICULAR EXPERIMENTAL CHALLENGE DUE TO THE SMALL SIZE OF THE CELLS 2ESOLUTION IS RATHER LIMITED AND MICROSCOPIC RESULTS ARE OFTEN NOT VERY SATISFYING &URTHERMORE THE THICK YEAST CELL WALL IS HIGHLY DIFFRACTIVE AND CAUSES INTERFERENCE PATTERNS ;= -ANY OF THESE LIMITATIONS CAN BE PARTIALLY OVERCOME BY USING SPECIALISED EQUIPMENT AND CAREFUL SAMPLE PREPARATION ! MAJOR ADVANTAGE OF A SMALL CELL IS THE EXCELLENT STATISTICS OF LOCALISATION PATTERNS THE HOMOGENEITY OF A STAINING PATTERN AND THE SIGNIlCANCE OF STRUCTURAL FEATURES CAN BE ASSESSED BY VISUAL INSPECTION OF A LARGE POPULATION OF CELLS WHICH IS EASY TO ACHIEVE WITH YEAST LIKE 0 PASTORIS 7HEN PREPARING CELLS FOR 4%- STUDIES DIFFERENT STEPS OF SAMPLE PREPARATION ARE INVOLVED ;=
s &IXATION 4HE MOST COMMON lXATION METHOD USED FOR 4%- OF YEAST IS BASED ON CHEMICAL CROSS LINKING &OR ULTRASTRUCTURAL ANALYSIS lXATION IS ACCOMPLISHED IN TWO STEPS TREATMENT WITH AN ALDEHYDE CROSS LINKER FOLLOWED BY OSMIUM OR POTASSIUM PERMANGANATE "OBMZUJDBM NFUIPET
s 0OST &IXATION )NITIAL lXATION WITH ALDEHYDE CROSS LINKERS PRESERVES OVERALL CELL STRUCTURE BUT DOES NOT lX LIPIDS /SMIUM REACTS STRONGLY WITH UNSATURATED FATS PROVIDING GOOD lXATION OF MEMBRANES AND LIPIDS
s $EHYDRATION #ELLS ARE DEHYDRATED BY INCUBATION IN INCREASINGLY CONCENTRATED ETHANOL OR ACETONE SOLUTIONS IN ORDER TO REMOVE WATER BEFORE THE RESIN IS INlLTRATED
s )NlLTRATION EMBEDDING THIN SECTIONING 0REPARATION OF CELLS FOR 4%- DERIVES FROM THE NEED TO PRODUCE ULTRA THIN SECTIONS THAT WILL ALLOW PASSAGE OF SOME ELECTRONS #UTTING THIN SECTIONS REQUIRES SUPPORT OF CELL STRUCTURE BY RESINS THAT INlLTRATE THE MOLECULAR MATRIX OF THE CELL AND SOLIDIFY ALLOWING SECTIONS AS THIN AS TO NM TO BE CUT 5NLESS CELLS ARE WELL INlLTRATED WITH RESIN THE THIN SECTIONS WILL HAVE LARGE HOLES THAT PREVENT OR LIMIT OBTAINING ULTRASTRUCTURAL DATA 9EAST IS NOTORIOUSLY DIFlCULT TO INlLTRATE AND THE USE OF LOW VISCOSITY RESINS SUCH AS 3PURRS RESIN ;= OR ULTRA LOW VISCOSITY FORMULAS MAY HELP TO PRODUCE WELL INlLTRATED SAMPLES
s 0OST STAINING #UT SAMPLES IN THE GRIDS ARE STAINED WITH ACETATE BEFORE VIEWING ;=
#ELL MORPHOLOGY STUDIES WERE CARRIED OUT AT THE -ICROSCOPY 3ERVICE OF THE 5!" USING A SIMILAR PROTOCOL AS DESCRIBED BY ,UNSDORF ET AL ;= 3AMPLES FROM BIOREACTOR CULTIVATIONS WERE IMMEDIATELY lXED AT 24 IN VV GLUTARDIALDEHYDE IN MMOL ,ዅኻ (%0%3 BUFFER P( FOR MIN #ELLS WERE STORED UNTIL EMBEDMENT FOR SEVERAL DAYS AT ș# 4HEN CELLS WERE lXED IN WV OSMIUMTETROXIDE IN M- CACODYLATE BUFFER P( #ELLS WERE DEHYDRATED ON ICE IN ETHANOL SERIES STAINED WITH WV URANYLACETATE IN VV ETHANOL INlLTRATED WITH 3PURR RESIN AND lNALLY POLYMERIZED AT ș# FOR HOURS 5LTRATHIN SECTIONS NM WERE CUT WITH A DIAMOND KNIFE USING AN ULTRAMICROTOME %- 5# ,EICA PICKED WITH &ORMVAR COATED MESH COPPER GRIDS AND POST STAINED WITH URANYL ACETATE AND LEAD CITRATE AS DESCRIBED PREVIOUSLY ;= 5LTRATHIN SECTIONS WERE ANALYSED WITH AN ENERGY lLTERED TRANSMISSION ELECTRON MICROSCOPE *%- *EOL AND IMAGES WERE CAPTURED WITH A ##$ %37 %RLANGSHEN CAMERA 'ATAN USING AS SOFTWARE THE $IGITAL -ICROGRAPH 'ATAN
-JQJE BOBMZTJT $FMM IPNPHFOJTBUJPO QSPUFJO RVBOUJmDBUJPO BOE MJQJE FYUSBDUJPO #ELL HOMOGENATES WERE OBTAINED BY SHAKING CELL PELLETS IN PRESENCE OF GLASS BEADS USING A $ISRUPTOR 'ENIE 3CIENTIlC )NDUSTRIES )NC AT # FOR MIN 0ROTEINS WERE PRECIPITATED WITH TRICHLOROACETIC ACID 4#! AT A lNAL CONCENTRATION OF TO CIRCUMVENT DISTURBING COMPONENTS AND QUANTIlED BY THE METHOD OF ,OWRY ET AL ;= "RIEmY PROTEINS WERE DILUTED TO A lNAL CONCENTRATION OF IN A CHILLED MIX OF VV 4#! IN WATER INCUBATED ON ICE FOR H CENTRIFUGED AT MAX SPEED AND # FOR MIN 3UPERNATANT WAS DISCARDED BY ASPIRATION AND THE PELLET RESUSPENDED IN , - SODIUM HYDOXIDE .A/( 3$3 SOLUTION 3AMPLES WERE HEATED AT # FOR MIN AND MIXED WITH , OF WATER AND M, OF FRESH PREPARED BUFFER
! POTASSIUM SODIUM TARTRATE COPPER SULPHATE PENTAHYDRATE #U3/ኾ q(ኼ/ 3$3
SODIUM CARBONATE .Aኼ#/ኽ IN - SODIUM HYDROXIDE .A/( AND INCUBATED AT 24 FOR MIN , OF FOLIN CIOCALTEUS REAGENT WERE ADDED VORTEXED AND INCUBATED AT 24 FOR AT LEAST MIN BEFORE ITS !BSORBANCE AT NM WAS DETERMINED ,IPIDS FROM HOMOGENATES WERE EXTRACTED ACCORDING TO &OLCH ET AL ;= #ELL FRACTION .BUFSJBMT BOE .FUIPET
CORRESPONDING TO MG TOTAL CELL PROTEIN WAS MIXED WITH M, CHLOROFORM METHANOL #(#Lኽ -E/( IN CLEAN 0YREX TUBES 3AMPLES WERE EXTRACTED BY VIGOROUS SHAKING IN A 6IBRAXLj ORBITAL
)+! FOR MIN M, OF WV MAGNESIUM CHLORIDE -G#Lኼ WAS ADDED AND EXTRACTION WAS CONTINUED FOR MIN 4HEN SAMPLES WERE CENTRIFUGED RPM MIN 24 THE AQUEOUS PHASE WAS REMOVED AND THE ORGANIC PHASE WASHED WITH M, . POTASSIUM CHLORIDE METHANOL +#L -E/( 0HASE WAS SHAKEN FOR MIN CENTRIFUGED AQUEOUS PHASE DISCARDED AND THE
REMAINING ORGANIC PHASE WAS WASHED WITH M, METHANOL WATER CHLOROFORM -E/( (ኼ/
#(#Lኽ VVV AND SHAKEN AGAIN FOR MIN 4HE lNAL ORGANIC PHASE WAS TRANSFERRED TO A NEW PYREX TUBE AND TAKEN TO DRYNESS UNDER A STREAM OF NITROGEN 4HE OBTAINED AMOUNTS FOR ALL LIPIDS WERE RELATED TO MG TOTAL CELL PROTEIN
(MZDFSPMJQJE BOBMZTJT &ATTY ACIDS WERE CONVERTED TO METHYL ESTERS BY METHANOLYSIS ADDING SULPHURIC ACID IN METHANOL AND HEATING AT # FOR MIN &ATTY ACID METHYL ESTERS WERE EXTRACTED TWO TIMES IN A MIXTURE OF LIGHT PETROLEUM AND WATER VV CENTRIFUGED RPM MIN 24 AND THE ORGANIC PHASE TRANSFERRED TO A NEW 0YREX TUBE 4HE SOLVENT WAS REMOVED UNDER A STREAM OF NITROGEN 3AMPLES WERE ANALYSED BY GAS CHROMATOGRAPHY (0 'AS CHROMATOGRAPH !GILENT 4ECHNOLOGIES USING AN (0 )../7AX CAPILLARY COLUMN M X MM ID X ĘM lLM THICKNESS WITH HELIUM AS CARRIER GAS &ATTY ACIDS WERE IDENTIlED BY COMPARISON TO THE FATTY ACID METHYL ESTER STANDARD MIX ',# " .U#HECK )NC
1IPTQIPMJQJE BOBMZTJT ,IPID EXTRACTS WERE LOADED MANUALLY ONTO !,5'2!-Lj 3), ' PLATES -ACHEREY .AGEL )NDIVIDUAL PHOSPHOLIPIDS WERE SEPARATED BY TWO DIMENSIONAL THIN LAYER CHROMATOGRAPHY #HLOROFORM METHANOL AMMONIA VVV WAS USED AS lRST AND CHLOROFORM ACETONE METHANOL ACETIC ACID WATER VVVVV AS SECOND DIMENSION SOLVENT 0HOSPHOLIPIDS WERE VISUALISED BY STAINING WITH IODINE VAPOUR SCRAPED OFF AND QUANTIlED BY THE METHOD OF "ROEKHUYSE ;= "RIEmY SAMPLES WERE INCUBATED AT # FOR MIN M, OF AN ACID MIXTURE
VV SULPHURIC ACID (ኼ3/ኾ VV PERCHLORIC ACID (#L/ኾ WERE ADDED AND HYDROLYSED AT # INTO HEATING PLATES UNDER THE HOOD )N THIS STEP DANGEROUS ACIDIC FUMES ARE FORMED 3AMPLES WERE COOLED DOWN AT 24 UNDER THE HOOD THEN M, OF FRESHLY PREPARED MOLYBDATE !MINONAPHTHOL SULPHONIC ACID !.3! MIXTURE VV WAS ADDED VORTEXED AND INCUBATED AT # FOR MIN 3AMPLES WERE CLARIlED RPM MIN 24 AND THE SAMPLES WERE MEASURED SPECTROPHOTOMETRICALLY AT NM 4HE AMOUNT OF PHOSPHATE FOR EACH SAMPLE WAS CALCULATED AS A RELATIVE AMOUNT OF THE TOTAL PHOSPHATE 4OTAL PHOSPHOLIPIDS WERE ESTIMATED BY THE SAME PROCEDURE USING A STANDARD CURVE WITH INORGANIC PHOSPHATE AT KNOWN AMOUNTS
4UFSPM BOBMZTJT )NDIVIDUAL STEROLS WERE ANALYSED BY GAS LIQUID CHROMATOGRAPHY MASS SPECTROMETRY '# -3 AFTER ALKALINE HYDROLYSIS AND SUBSEQUENT LIPID EXTRACTION ;= ,IPID EXTRACTS WERE RESUSPENDED IN M, METHANOL M, WV PYROGALLOL DISSOLVED IN METHANOL AND M, WV AQUEOUS POTASSIUM HYDROXIDE SOLUTION , OF A CHOLESTEROL SOLUTION MG M,ዅኻ IN ETHANOL WERE ADDED AS AN INTERNAL STANDARD 4UBES WERE HEATED IN A SAND BATH FOR H AT # EXTRACTED THREE TIMES BY ADDING M, N HEPTANE VORTEXING AND CENTRIFUGING RPM MIN 24 AND THE UPPER PHASE TRANSFERRED INTO A NEW TUBE 3AMPLES WERE DERIVATISED BY ADDING ,OF./ BISTRIMETHYLSILYL TRImUORACETILAMIDE "OBMZUJDBM NFUIPET
',# -3 WAS PERFORMED ON A (EWLETT 0ACKARD 'AS #HROMATOGRAPH EQUIP PED WITH A MASS SELECTIVE DETECTOR (0 USING AN (0 -3 CAPILLARY COLUMN M X MM ID X M lLM THICKNESS 3AMPLE ALIQUOTS OF , WERE INJECTED IN THE SPLITLESS MODE AT ș# INJECTION TEMPERATURE WITH HELIUM AS CARRIER GAS AND A mOW RATE SET TO M, MIN IN CONSTANT mOW MODE 4HE TEMPERATURE PROGRAM WAS ș# FOR MIN ș# MINዅኻ TO ș# AND ș# MINዅኻ TO ș# 3TEROLS WERE IDENTIlED BY THEIR MASS FRAGMENTATION PATTERN
/FVUSBM MJQJE BOBMZTJT &OR NEUTRAL LIPID ANALYSIS LIPID EXTRACTS WERE APPLIED TO !,5'2!-Lj 3), ' PLATES -ACHEREY .AGEL AND CHROMATOGRAMS WERE DEVELOPED IN AN ASCENDING MANNER TO HALF OF THE PLATE USING THE SOLVENT SYSTEM LIGHT PETROLEUM DIETHYL ETHER ACETIC ACID VVV 4HEN PLATES WERE BRIEmY DRIED AND FURTHER DEVELOPED TO THE TOP USING THE SOLVENT SYSTEM LIGHT PETROLEUM DIETHYL ETHER VV 4O VISUALISE THE BANDS PLATES WERE DIPPED INTO A DEVELOPING SOLUTION CONSISTING
OF G MANGANESE CHLORIDE TETRAHYDRATE -N#Lኼ q(ኼ/ M, WATER M, OF METHANOL AND M, OF CONCENTRATED SULPHURIC ACID BRIEmY DRIED AND HEATED AT # FOR MIN "ANDS WERE SCANNED AT NM WITH A 4,# 3CANNER WIN#!43 3OFTWARE #!-!' AND QUANTIlED USING TRIOLEIN CHOLESTERYL OLEATE AND ERGOSTEROL AS STANDARDS
4QIJOHPMJQJE BOBMZTJT &OR LIPID EXTRACTION MG FROZEN ALIQUOTS WERE PROCESSED 3AMPLES CONTAINING G PROTEIN WERE SPIKED WITH , OF A SPHINGOLIPID INTERNAL STANDARD MIX NMOL . DODECANOYL SPHING ENINE NMOL . DODECANOYL GLUCOSYL SPHING ENINE NMOL # SPHINGANINE !VANTI 0OLAR ,IPIDS )NC SUSPENDED IN M, PROPAN OL HEXANE WATER VV AND INCUBATED AT ș# FOR MIN SLIGHTLY MODIFYING A PROTOCOL OF -ARKHAM ET AL ;= $URING THE INCUBATION SAMPLES WERE BRIEmY VORTEXED AND SONICATED AFTER AND MIN 4HEN EXTRACTS WERE CLEARED FROM CELL DEBRIS BY CENTRIFUGATION DRIED UNDER A STREAM OF NITROGEN DISSOLVED IN , TETRAHYDROFURANMETHANOLWATER VV AND STORED UNDER ARGON AT ș# &OR ANALYSIS SAMPLES WERE SOLUBILISED BY GENTLE HEATING AND SONICATION -OLECULAR SPECIES SEPARATION WAS ACHIEVED BY 5LTRA 0ERFORMANCE ,IQUID #HROMATOGRAPHY 50,# USING AN !#15)49 50,#Lj SYSTEM 7ATERS #ORP EQUIPPED WITH AN !#15)49 50,#Lj (33 4 #OLUMN MM X MM M 7ATERS #ORP !LIQUOTS OF , WERE INJECTED IN PARTIAL LOOP WITH NEEDLE OVERlLL MODE 4HE mOW RATE WAS M, MINዅኻ AND THE SEPARATION TEMPERATURE WAS ș# 3OLVENT ! WAS METHANOL M- AMMONIUM ACETATE VV CONTAINING VV ACETIC ACID AND SOLVENT " WAS TETRAHYDROFURANMETHANOL M- AMMONIUM ACETATE VVV CONTAINING VV ACETIC ACID !LL LIPIDS WERE SEPARATED WITH LINEAR BINARY GRADIENTS FOLLOWING THE SAME SCHEME START CONDITIONS OR SOLVENT " HELD FOR MIN LINEAR INCREASE TO SOLVENT " FOR MIN SOLVENT " HELD FOR MIN AND RE EQUILIBRATION TO START CONDITIONS IN MIN 4HE START CONDITIONS WERE SOLVENT " FOR CERAMIDES #ER AND HEXOSYLCERAMIDES (EX#ER AND SOLVENT " FOR INOSITOL CONTAINING SPHINGOLIPIDS #HIP BASED NANOELECTROSPRAY IONISATION WAS ACHIEVED WITH A 4RI6ERSA .ANOMATELj !DVION )NC IN BOTH POSITIVE AND NEGATIVE ION MODES WITH INTERNAL DIAMETER NOZZLES A mOW RATE OF N, MINዅኻ AND A VOLTAGE OF 6 4HE ION SOURCE WAS CONTROLLED WITH THE !DVION #HIP3OFT-ANAGER SOFTWARE &UNGAL SPHINGOLIPID MOLECULAR SPECIES WERE DETECTED WITH 142!0n TANDEM MASS SPECTROMETER !" 3CIEX BY MONITORING .BUFSJBMT BOE .FUIPET
s THE TRANSITION FROM ;- (=ዄ MOLECULAR IONS TO DEHYDRATED LONG CHAIN BASE ,#" FRAGMENTS FOR #ER (EX#ER AND ,#"
s THE LOSS OF PHOSPHOINOSITOL CONTAINING HEAD GROUPS FOR INOSITOL CONTAINING SPHINGOLIPIDS ; =
$WELL TIME WAS MS AND -3 PARAMETERS WERE OPTIMISED TO MAXIMISE DETECTOR RESPONSE
5SBOTDSJQUPNJD BOBMZTJT
*OUSPEVDUJPO
4HE AVAILABILITY OF COMPLETELY SEQUENCED AND WELL ANNOTATED GENOMES OF ORGANISMS HAS CREATED OPPORTUNITIES FOR STUDYING NUMEROUS ASPECTS OF GENE FUNCTION AT THE GENOMIC LEVEL 'ENE EXPRESSION DElNED BY STEADY STATE LEVELS OF CELLULAR M2.! WAS ONE OF THE lRST ASPECTS OF GENE FUNCTION AMENABLE TO GENOME SCALE MEASUREMENT ;= 'ENOME WIDE ANALYTICAL TOOLS LIKE $.! MICROARRAYS ARE REGARDED AS DATA MINING SOURCES FOR PHYSIOLOGICAL EFFECTS STRESS REGULATION AND HOST ENGINEERING
%/" .JDSPBSSBZT
-ICROARRAYS INVOLVE THE PRODUCTION OF A SERIES OF 0#2 PRODUCTS OR SYNTHETIC OLIGONUCLEOTIDES WHICH CORRESPOND TO ALL THE PREDICTED GENES FROM A SPECIlC ORGANISM 4HEY ASSESS THE AMOUNT OF A SPECIlC TRANSCRIPT IN A SPECIlC SAMPLE AT A SPECIlC TIME ;= &LUORESCENT C$.! MICROARRAY ASSAYS USE DIRECT INCORPORATION OF TAGGED NUCLEOTIDES IN THE C$.! PREPARED BY REVERSE TRANSCRIPTION OF M2.! FROM THE SOURCE OF INTEREST &IGURE $ATABASE ANALYSIS MAKE IT POSSIBLE TO DElNE PATTERNS OF GENES EXPRESSED CONSISTENTLY ACROSS EXPERIMENTS ;= )NITIAL PROCESSING OF DATA REQUIRES THE APPLICATION OF SIGNIlCANCE lLTERS TO THE RAW DATA SET SO THAT ONLY MEANINGFUL MEASUREMENTS ENTER INTO DOWNSTREAM ANALYSIS )T HAS TO BE CONSIDERED THAT THE AMOUNT OF M2.! PRESENT DOES NOT NECESSARILY CORRELATE WITH THE AMOUNT OF PROTEIN MADE ;=
5SBOTDSJQUPNJD BOBMZTJT
&IGURE 3CHEME OF MICROARRAY PROCEEDING !DAPTED FROM ;= -ICROARRAYS ARE ARTIlCIALLY CONSTRUCTED GRIDS OF $.! SUCH THAT EACH ELEMENT OF THE GRID PROBES FOR A SPECIlC 2.! SEQUENCE 4HE BASIC TECHNIQUE INVOLVES EXTRACTION OF 2.! FROM BIOLOGICAL SAMPLES COPIED WHILE INCORPORATING EITHER mUORESCENT NUCLEOTIDES OR A TAG LATER STAINED WITH mUORESCENCE 4HE LABELLED 2.! IS THEN HYBRIDISED TO A MICROARRAY FOR A PERIOD OF TIME AFTER WHICH THE EXCESS IS WASHED OFF AND THE MICROARRAY IS SCANNED UNDER LASER LIGHT
$MVTUFS BOBMZTJT
4HE GOAL OF CLUSTER ANALYSIS IS TO IDENTIFY PATTERNS OF SIMILARITIES AND DIFFERENCES AMONG SAMPLES )F TWO GENES ARE SIMILARLY EXPRESSED IT CAN BE HYPOTHESISED THAT THE RESPECTIVE GENES ARE CO REGULATED AND POSSIBLY FUNCTIONALLY RELATED ;= #LUSTERING CAN BE DElNED AS THE PROCESS OF SEPARATING A SET OF OBJECTS INTO SEVERAL SUBSETS CLUSTERS ON THE BASIS OF THEIR SIMILARITY 4HE AIM IS TO lND CLUSTERS WHICH MEMBERS ARE SIMILAR TO EACH OTHER BUT DISTANT TO MEMBERS OF OTHER CLUSTERS IN TERMS OF GENE EXPRESSION ! NUMBER OF TOOLS FOR CLUSTERING HAVE BEEN DEVELOPED WHICH ARE FREELY AVAILABLE )N GENERAL THESE ARE BASED ON CONVENTIONAL STATISTICAL TECHNIQUES ;= + MEANS ALGORITHM ;= IS A COMMONLY USED CLUSTERING METHOD BECAUSE IT IS BASED ON A VERY SIMPLE PRINCIPLE AND PROVIDES GOOD RESULTS 4HIS ALGORITHM PARTITIONS . GENES INTO + CLUSTERS WHERE + IS PRE DETERMINED BY THE USER ;=
5IF JNQBDU PG PYZHFO PO UIF USBOTDSJQUPNF PG SFDPNCJOBOU 1 QBTUPSJT
4HE IMPACT OF OXYGEN LIMITATION ON RECOMBINANT PROTEIN PRODUCTION IN 0 PASTORIS WAS lRST STUDIED BY "AUMANN AND CO WORKERS ;= SHOWING A SIGNIlCANT INCREASE OF THE SPECIlC PRODUCTION RATE OF AN ANTIBODY FRAGMENT )N A SUBSEQUENT STUDY THE IMPACT OF OXYGEN AVAILABILITY ON THE PHYSIOLOGY OF RECOMBINANT 0 PASTORIS WAS STUDIED INTEGRATING TRANSCRIPTOMIC PROTEOMIC METABOLIC mUX AND METABOLOMIC ANALYSES ; = )N RESPONSE TO OXYGEN LIMITATION A WIDE RANGE OF TRANSCRIPTIONAL MODIlCATIONS OCCUR RESULTING IN EXTENSIVE CHANGES IN CELLULAR PROTEIN LEVELS AND ACTIVITIES HIGHLIGHTING THE ONES RELATED TO CELL RESPIRATION LIPID METABOLISM CELL MEMBRANE AND CELL WALL STRUCTURE ;= )NCREASED TRANSCRIPT LEVELS WERE OBSERVED FOR A NUMBER OF ENZYMES THAT CATALYSE OXYGEN CONSUMING REACTIONS OF THE ERGOSTEROL PATHWAY %2' %2' %2' %2' AND %2' 3IMILARLY SPHINGOLIPID SYNTHESIS GENES 352 3#3 $%3 AND 3,$ WERE ALSO UPREGULATED UNDER HYPOXIC CONDITIONS AS ALL THESE ENZYMES NEED MOLECULAR OXYGEN AS SUBSTRATE ;= 3FTVMUT BOE EJTDVTTJPO
3FTVMUT BOE EJTDVTTJPO ! STUDY OF THE TRANSCRIPTOMIC CHANGES UNDER HYPOXIC CONDITIONS FOCUSED ON THE GENES RELATED TO LIPID METABOLIC PATHWAYS WAS CARRIED OUT USING TRANSCRIPTOMIC DATA FROM "AUMANN ET AL ;=IN ORDER TO SELECT THE GENES TO FURTHER INVESTIGATE ITS ROLE ON MEMBRANE COMPOSITION AND IT EFFECT ON RECOMBINANT PROTEIN SECRETION
LNFBOT DMVTUFSJOH 4RANSCRIPTIONAL DATA CORRESPONDING TO GENES RELATED TO LIPID METABOLISM WERE EXTRACTED FROM THE TRANSCRIPTIONAL DATASET FROM "AUMANN ET AL ;= AND DATA FROM CONTROL STRAIN 0 PASTORIS 8 WILD TYPE AND THE &AB PRODUCING STRAIN WAS ANALYSED IN TERMS OF TRANSCRIPTIONAL DIFFERENCES BETWEEN HYPOXIC AND NORMOXIC CONDITIONS
&IGURE #LUSTER ANALYSIS OF THE GENES RELATED TO LIPID METABOLISM FROM ;= USING %UCLIDEAN DISTANCE ANDK ,OG &# OF THE CONTROL STRAIN 8 LEFT AND THE REFERENCE STRAIN 8 &AB RIGHT WERE USED 2ED LABELS DENOTE HIGHER LEVELS OF HYPOXIA VERSUS NORMOXIA AND GREEN LABELS LOWER LEVELS OF TRANSCRIPT IN HYPOXIA COMPARED TO NORMOXIA
&IRST A THRESHOLD OF P VALUES ऑ AND FOLD CHANGE &# ऒq WERE CHOSEN TO IDENTIFY SIGNIlCANTLY REGULATED GENES 4HEN SELECTED GENES WERE SUBJECTED TO K MEANS CLUSTER ANALYSIS USING THE 'ENESIS TOOL KWWS JHQRPHWXJUD]DW ! K MEANS CLUSTERING USING THE LOG OF THE FOLD CHANGE AS INPUT lLE WAS PERFORMED AND THE %UCLIDEAN DISTANCE AND A K WERE SELECTED AS RUNNING SETTINGS /BTAINED RESULTS ARE SHOWN IN &IGURE #LUSTER CONTAINED GENES FROM FATTY ACID PHOSPHOLIPID STEROL AND NON POLAR LIPID PATHWAYS WITH REPRESSED TRANSCRIPTS IN HYPOXIC COMPARED TO NORMOXIC CONDITIONS AND WITH SIMILAR LEVELS OF REPRESSION FOR THE TWO STRAINS #LUSTER WAS MAINLY COMPOSED OF GENES FROM THE FATTY ACID METABOLISM WHICH WERE DOWNREGULATED IN HYPOXIC CONDITIONS BUT WITH A NOTABLE EFFECT ON THE RECOMBINANT STRAIN 5SBOTDSJQUPNJD BOBMZTJT
'ENES FROM CLUSTER WITH INDUCED TRANSCRIPTS IN HYPOXIC COMPARED TO NORMOXIC CONDITIONS WERE POTENTIALLY CANDIDATES TO BE USED FOR GENE MODIlCATION 4HIS CLUSTER WAS MAINLY ENRICHED WITH GENES FROM THE ERGOSTEROL AND SPHINGOLIPID PATHWAYS AS SOME OF THEIR ENZYMES ARE OXYGEN DEPENDENT
(FOF NBQQJOH
4RANSCRIPTIONAL CHANGES OF THE LIPID BIOSYNTHESIS PATHWAYS WERE MAPPED IN &IGURE WERE SIGNIlCANT P VALUE ऑ REGULATED GENES UNDER HYPOXIC CONDITIONS ARE ILLUSTRATED
FAT1, FAA1 external FA FAA1, FAA2, FAT1 FFA acyl-CoA Phospholipids Glu-6P FAA2 DHAP G3P INO1 SCT1, GPT2 C26:0-CoA Ins-3P acyl-CoA SLC1, ALE1 C24:0-CoA CDS1, TAM41 lyso-PA INM1 ECI1 1-acyl-DHAP PA CDP-DAG C22:0-CoA AYR1 SPS19 POX1 C20:0-CoA PGS1 external Inositol DGK1 PAH1 Serine TGL3 inositol FOX2 C18:1-CoA C18:0-CoA GUT1 YJU3 APP1 OLE1 TGL4 G3P G MG DPP1 C16:1-CoA C16:0-CoA PGP ITR1, ITR2 POT1 LPP1 TGL3 PIS1 GEP4 ELO1 DAG PS
acyl(n-2)-CoA acetyl-CoA C18:0-ACP PG ECT1 PSD1 C16:0-ACP TGL3 EKI1 PSD2 ISC1 PI TGL4 Etn Etn-P CDP-Etn C14:0-ACP LRO1 CRD1 STT4 INP52 EPT1 C12:0-ACP HMN1 PE LSB6 INP53 CL PIK1 SAC1 C10:0ACP external VPS34 YMR1 C8:0-ACP Etn CLD1 Fatty Acids CHO2 PIP C6:0-ACP INP51 MLCL C4:0-ACP INP52 INP53 FAS1 TAZ1 INP54 FAS1 SAC1 PLC1 FAS2 OPI3 FIG4 PIP 3-ketoacyl-ACP CL 2 PCT1 acyl-CoA CKI1 SPO14 FAS2 Cho Cho-P CDP-Cho CPT1 DGA1 HMN1 PC acetyl-ACP malonyl-ACP external Cho
FAS1 PCT1 FAS1 lyso PL malonyl-CoA C16:0-CoA TG ACC1 Serine LCB1 LCB2
acetyl-CoA Non-polar lipids LCB 18:1;2
ERG10 Sterols TSC10 steryl esters palmitaldehyde DPL1 acetoacetyl-CoA DHS-1P Etn PHS-1P C26:0-CoA TGL1 DHS ERG13 YEH1 FA YEH2 ARE2 LCB3 HMG-CoA SUR2 LAG1 LCB5 LAC1 ergosterol YDC1 HMG1 YPC1 HMG2 ERG4 PHS C18:0-CoA mevalonate Cer ergosta-5,7,22,24(28)- BAR1 ERG12 tetraenol delta4- desaturase SCS7 mevalonate-5P ERG5 SCS7 ERG8 alpha- MVD1 ergosta-5,7,24(28)- DHS-Δ4 trienol hydroxy-Cer IPP delta8- desaturase ERG3 AUR1 IDI1 ERG20 ERG9 episterol DHS-Δ4,8 IPC squalene ERG2 C9- methyltransferase CSH1 ERG1 fecosterol SUR1 CSG2 GlcCer squalene epoxide ERG6 MIPC
ERG7 zymosterol IPT1
lanosterol ERG27 Sphingolipids ISC1 ERG28 M(IP) C ERG11 ERG25 2 ERG26 4,4- dimethylcholesta-8,14,2 ERG24 4,4-dimethyl 4-trienol zymosterol
&IGURE 4RANSCRIPTOME MAP OF MAIN LIPID BIOSYNTHETIC PATHWAYS 'ENES UPREGULATED ARE IN RED AND THE DOWNREGULATED ONES IN GREEN $ATA WAS FROM PREVIOUS STUDIES OF THE GROUP ;= 3FTVMUT BOE EJTDVTTJPO
%RGOSTEROL BIOSYNTHETIC PATHWAY IS AN OXYGEN REQUIRING PROCESS 4HEREFORE MOST OF THE GENES WHOSE PRODUCTS CATALYSE OXYGEN DEPENDING REACTIONS WERE STRONGLY UPREGULATED IN HYPOXIA ! SIMILAR BEHAVIOUR WAS OBSERVED FOR THE SPHINGOLIPID PATHWAY WHERE MOST OF THE BIOSYNTHETIC STEPS REQUIRE MOLECULAR OXYGEN AND WERE HIGHLY UPREGULATED UNDER HYPOXIC CONDITIONS 'ENES INVOLVED IN THE DE NOVO SYNTHESIS OF FATTY ACIDS !## &!3 AND FATTY ACID ACTIVATION &!! &!! WERE DOWNREGULATED 4HE METABOLIC PATHWAY INVOLVED IN THE BREAKDOWN OF TRIACYLGLYCERIDES 4'S FROM THE LIPID DROPLETS TO OBTAIN FREE FATTY ACIDS4', AND 4', DID NOT CHANGE AT TRANSCRIPTOMIC LEVEL (OWEVER 3% BREAKDOWN PATHWAY 4', 9%( 9%( TO OBTAIN FREE STEROL AND FATTY ACIDS WAS UPREGULATED 4HE OXYGEN DEPENDENT OXIDATION PROCESS TO OBTAIN ENERGY FROM FATTY ACIDS WAS STRONGLY DOWNREGULATED 4HE PHOSPHOLIPID PATHWAY DID NOT SHOW ANY SIGNIlCANT CHANGES AT LEVEL OF TRANSCRIPTOMIC PROlLE /NLY SOME GENES INVOLVED IN THE SYNTHESIS OF PHOSPHATIDYLCHOLINE 0# THROUGH THE SO CALLED +ENNEDY PATHWAY 0#4 #04 WERE UPREGULATED IN HYPOXIA 3,# INVOLVED IN THE GENERATION OF PHOSPHATIDIC ACID 0! FROM LYSO 0! WAS UPREGULATED ALONG WITH THE GENES TRANSFORMING 0! TO DIACYLGLYCEROL $!' ! SIGNIlCANT DOWNREGULATION OF )42 WAS OBSERVED MEANING THAT A LOWER UPTAKE OF EXTERNAL INOSITOL COULD BE OCCURRING %FFECT OF HYPOXIC CONDITIONS ON TRANSCRIPTIONAL FACTORS INVOLVED IN THE LIPID METABOLISM WERE ALSO ANALYSED AND (AC RESULTED TO BE THE MOST REGULATED TRANSCRIPTIONAL FACTOR &IGURE
glucose pyruvate
Mitochondria acetaldehyde ethanol
pyruvate Mga2 β-oxidation acetate Adr1 Acetyl-CoA OLE1 Peroxisome Malonyl-CoA FAs TCA cycle ACC1 FAS1, FAS2 Snf1 PL, TAG Acetyl-CoA
Cytosol Upc2 Ino4
Acetoacetyl-CoA Ergosterol Opi1
Hac1
&IGURE 4RANSCRIPTIONAL FACTORS RELATED TO LIPID METABOLISM AND ITS TRANSCRIPTIONAL REGULATION UNDER HYPOXIC CONDITIONS !DAPTED FROM ; =
(!# REGULATION COULD ALSO REASONABLY BE LINKED TO CHANGES IN THE LIPID BALANCE SINCE 502 HAS BEEN SUGGESTED TO BE ACTIVATED UPON LIPID DEPRIVATION IN ORDER TO COORDINATE MEMBRANE SYNTHESIS WITH (AC AS IMPORTANT REGULATOR ;= !DDITIONALLY ALTERATIONS IN MEMBRANE mUIDITY COULD TRIGGER THE UPREGULATION OF 0 PASTORIS (!# (AC TRIGGERS DOWNREGULATION OF ERGOSTEROL SYNTHESIS AND IT ALSO HAS AN EFFECT ON MEMBRANE PROLIFERATION AND MORPHOLOGY ; = !$2 TOGETHER WITH ALL THE GENES INVOLVED IN OXIDATION WAS TIGHTLY DOWNREGULATED UNDER HYPOXIC CONDITIONS
(FOF TFMFDUJPO )T HAS BEEN PREVIOUSLY OBSERVED A BENElCIAL EFFECT OF HYPOXIC CONDITIONS IN RECOMBINANT PROTEIN PRODUCTION 4HE CURRENT WORK IS FOCUSED ON DETERMINING WHETHER CHANGES TRIGGERED BY HYPOXIC CONDITIONS HAVE AN IMPACT ON PROTEIN SECRETION AND TO DETERMINE WHICH ARE THOSE CHANGES &OR THIS REASON AFTER A TRANSCRIPTOMIC ANALYSIS FOCUSED ON LIPID METABOLISM A SET OF GENES WERE 5SBOTDSJQUPNJD BOBMZTJT
SELECTED IN ORDER TO FURTHER INVESTIGATE ITS ROLE ON DElNING MEMBRANE PROPERTIES AND THEIR EFFECT ON RECOMBINANT PROTEIN SECRETION )T IS KNOWN THAT STEROLS AND SPHINGOLIPIDS ARE IMPORTANT COMPONENTS OF MEMBRANES AND THAT CHANGES ON THEIR LEVELS CAN DIRECTLY AFFECT MEMBRANE MORPHOLOGY AND ITS PROPERTIES &URTHERMORE SINCE HESE BIOSYNTHETIC PATHWAYS ARE OXYGEN DEPENDENT A TIGHT REGULATION OF THE INVOLVED GENES HAVE BEEN OBSERVED &OCUSED ON SPHINGOLIPID METABOLISM GENES INVOLVED IN THE EARLY STEPS OF SPHINGOLIPID BIOSYNTHESIS WERE PREFERRED AS THEIR ALTERATION CAN LEAD TO DEEPER CHANGES ON LIPID COMPOSITION 4WO MAJOR SPHINGOLIPIDS ARE PRESENT IN 0 PASTORIS CERAMIDES AND INOSITOL CONTAINING PHOSPHOLIPIDS 4WO DIFFERENT GENES RELATED TO THESE TWO DIFFERENT PATHWAYS WERE SELECTED $%3 WHICH CODIlES FOR A ́ DESATURATE INVOLVED IN CERAMIDE BIOSYNTHESIS AND 352 A # HYDOXYLASE THAT PLAYS A ROLE IN INOSITOL CONTAINING SPHINGOLIPIDS %RGOSTEROL IS AN ESSENTIAL COMPOUND OF THE CELL #ONSEQUENTLY NO GENE DELETION CAN BE PERFORMED IN ITS BIOSYNTHETIC PATHWAY !S IT WILL BE DESCRIBED LATER IN MORE DETAIL mUCONAZOLE IS AN ANTIFUNGAL THAT INHIBITS %RGP AND IT HAS BEEN DESCRIBED TO ALTER ERGOSTEROL LEVELS ;= &OR THIS REASON mUCONAZOLE TREATED CELLS AND %2' WERE ALSO SELECTED AS TARGET GENE &INALLY (AC THE ONLY TRANSCRIPTION FACTOR INVOLVED IN LIPID METABOLISM THAT WAS REGULATED IN HYPOXIC CONDITIONS WAS ALSO SELECTED )N THIS CASE ONLY GENE OVEREXPRESSION WAS CONSIDERED CONSIDERING THAT A GENE DELETION COULD LEAD TO NOT VIABLE CELLS AS (AC IS INVOLVED IN SEVERAL CELLULAR PROCESSES ! STRAIN BATTERY 4ABLE WAS lNALLY DElNED WITH THE SELECTED GENES WITH BOTH DELETION AND OVEREXPRESSION IN ORDER TO DETERMINE ITS IMPLICATION ON RECOMBINANT PROTEIN SECRETION
4ABLE "ATTERY OF GENES THAT WILL BE USED IN THIS STUDY WITH THE GENE SELECTED AND THE EXPECTED PHENOTYPE
STRAIN GENE EXPECTED PHENOTYPE 8 &AB& REFERENCE STRAIN 8 ጂ$%3 &AB& $%3 NO CERAMIDES 8 &AB& $%3 $%3 OVEREXPRESSED CERAMIDES 8 ጂ352 &AB& 352 ALTERED INOSITOL SPHINGOLIPIDS 8 &AB& 352 352 ALTERED INOSITOL SPHINGOLIPIDS 8 &AB& &LUCONAZOLE %2' LOW LEVELS OF ERGOSTEROL 8 &AB& %2' %2' HIGHER LEVELS OF ERGOSTEROLINTERMEDIATES 8 &AB& (AC (!# ALTERED LIPID COMPOSITION
$PODMVTJPOT
s ! DEEP ANALYSIS OF THE TRANSCRIPTOMIC DATA FROM HYPOXIC CULTURE CONDITIONS FOCUSED ON LIPID METABOLISM WAS CARRIED OUT
s #LUSTER ANALYSIS PROVIDED CANDIDATE GENES WITH SIGNIlCANT REGULATION IN HYPOXIA
s 'ENES $%3 352 %2' (!# WERE SELECTED FOR FURTHER INVESTIGATIONS OF THE ROLE OF LIPIDS ON RECOMBINANT PROTEIN SECRETION
4USBJO HFOFSBUJPO
*OUSPEVDUJPO (FOFT TFMFDUFE GPS DFMM FOHJOFFSJOH 4QIJOHPMJQJE ́EFTBUVSBTF %&4 0 PASTORIS LIKE MANY FUNGI SYNTHESISES GLUCOSYLCERAMIDES 'LC#ERS PREDOMINANTLY IN THE FORM OF METHYL SPHINGA DIENINE THAT CONTAIN % DOUBLE BOUNDS AT THE ́ AND ́ POSITIONS OF ,#"S LONG CHAIN BASES AND A METHYL BRANCH AT THE # POSITION ;= &IRST EXAMPLES OF SPHINGOLIPID ́ DESATURASE WERE ISOLATED BY 4ERNES ET AL ;= WHO FUNCTIONALLY IDENTIlED CANDIDATE DESATURASES BY HETEROLOGOUS EXPRESSION IN 3 CEREVISIAE ́352 MUTANTS 0REVIOUS STUDIES INDICATE THAT 0 PASTORIS STRAINS WITH THE ENDOGENOUS SPHINGOLIPID ́ DESATURASE DISRUPTED DO NOT CONTAIN ́ UNSATURATED ,#" AND ARE COMPLETELY DEVOID OF 'LC#ERS ; = 4HE REQUIREMENT FOR ́ UNSATURATED ,#"S TO INITIATE THE SYNTHESIS OF 'LC#ERS IN 0 PASTORIS IMPLIES THAT THIS MODIlCATION IS THE lRST COMMITTED STEP FOR THE BIOSYNTHESIS OF THIS CLASS OF SPHINGOLIPIDS IN THIS ORGANISM ;= 4HE ABSENCE OF 'LC#ERS IN 0 PASTORIS IS NOT ESSENTIAL FOR ITS NORMAL GROWTH ;=
4QIJOHPMJQJE $IZESPYZMBTF 463 4HE # HYDROXYLASE ENCODED BY THE 352 GENE HYDROXYLATES SPHINGOLIPIDS AT THE # POSITION OF THE SPHINGOID BASE RESULTING IN THE FORMATION OF HYDROXY SPHINGANINE PHYTOSPHINGOSINE )T IS INVOLVED IN THE )0# BIOSYNTHETIC PATHWAY REGULATING RELATIVE LEVELS OF DIHYDROSPHINGOSINE $(3 AND PHYTOSPHINGOSINE 0(3 ;= ́352 STRAINS OF 3 CEREVISIAE ARE VIABLE AND # HYDOXYLATION OF THE ,#" IS NOT REQUIRED FOR )0# BIOSYNTHESIS ; = (OWEVER 4ERNES ET AL ;= COULD NOT DELETE THIS GENE 4HE AUTHORS SUGGEST THAT THE REQUIREMENTS FOR # HYDROXYLATION OF THE ,#" DIFFER BETWEEN FUNGAL SPECIES AND THAT IN 0 PASTORIS THE # HYDROXYLATION IS REQUIRED FOR )0# BIOSYNTHESIS WHICH ARE ESSENTIAL FOR VIABILITY IN THIS ORGANISM
-BOPTUFSPM EFNFUIZMBTF &3( ,ANOSTEROL DEMETHYLASE IS THE MAJOR CYTOCHROME 0 IN 3 CEREVISIAE )T IS ENCODED BY THE GENE %2' AND CATALYSES THE OXIDATIVE REMOVAL OF THE # METHYL GROUP OF LANOSTEROL
4USBJO HFOFSBUJPO
AN ESSENTIAL REACTION IN THE BIOSYNTHESIS OF ERGOSTEROL %2' KNOCKOUTS AND STRAINS WITH NON FUNCTIONAL %RGP ENZYME ARE NON VIABLE UNLESS EXOGENOUS ERGOSTEROL IS SUPPLIED IN THE MEDIUM ;= 'ENE OVER EXPRESSION STUDIES SUGGEST THAT %RGP AND %RGP ARE THE MAJOR REGULATORY STEPS FOR THE ERGOSTEROL PATHWAY ;= )T HAS ALSO BEEN REPORTED %RG AS THE JUNCTURE FOR FUNCTIONAL STEROL MOLECULES AS DISRUPTIONS AT THIS POINT OR BEFORE RESULT IN NON VIABLE CELLS WHILE GENE KNOCKOUT AFTER THIS POINT OF THE ROUTE ARE VIABLE EVEN EVEN SIGNIlCANT CHANGES ARE OBSERVED IN THE MEMBRANES OF THESE STRAINS ;= /VEREXPRESSION OF %2' RESULTS IN A DECREASE OF LANOSTEROL AND AN INCREASE OF DOWNSTREAM STEROLS ;= 0ROTEIN LEVELS OF THIS PROTEIN ARE KNOWN TO BE AFFECTED BY CARBON SOURCE OXYGEN HEME AND GROWTH STATE OF THE CULTURE ;= %2' TRANSCRIPT LEVELS HAVE BEEN DETERMINED TO INCREASE DURING GROWTH ON GLUCOSE PRESENCE OF HEME AND DURING OXYGEN LIMITING GROWTH CONDITIONS ;= 4HESE RESULTS ARE IN CONCORDANCE WITH THE ONES OF 0 PASTORIS CULTURED IN LOW HYPOXIA WHERE %2' WAS ALSO UPREGULATED ;=
)BD 5SBOTDSJQUJPO 'BDUPS )"$ (ACP IS A TRANSCRIPTION FACTOR THAT IS REQUIRED FOR THE UNFOLDED PROTEIN RESPONSE 502 A STRESS RESPONSE PATHWAY TRIGGERED WHEN UNFOLDED OR MISFOLDED PROTEINS ACCUMULATE IN THE %2 ;= )T INDUCES THE EXPRESSION OF 502 RESPONSIVE GENES LIKE CHAPERONES AND OTHER ENZYMES INVOLVED IN THE SECRETION PROCESS EG FOLDASES PROTEINS INVOLVED IN GLYCOSYLATION ;= 4HE PRINCIPAL COMPONENTS REGULATING 502 ARE THE )REP A KINASE2.ASE AND (ACP &IGURE 5NDER NON STRESS CONDITIONS )REP IS A PROTEIN ASSOCIATED WITH "I0+ARP !S UNFOLDED PROTEINS
&IGURE 4HE UNFOLDED PROTEIN RESPONSE 502 &ROM ;=2EPRODUCED WITH PERMISSION OF *OHN 7ILEY AND 3ONS
ACCUMULATE IN THE %2 LUMEN "I0 IS RELEASED FROM )REP TO INTERACT WITH UNFOLDED PROTEINS WHICH INDUCES AUTOPHOSPHORILATION OF )REP AND ACTIVATES ITS 2.ASE FUNCTION !CTIVE )REP SPLICES THE CONSTITUTIVELY EXPRESSED (!# M2.! THE SPLICED FORM OF WHICH PRODUCES (ACP (ACP IS TRANSLOCATED TO THE NUCLEUS WHERE IT BINDS TO 502 RESPONSIVE ELEMENTS 502% IN THE PROMOTER OF 502 RESPONSIVE GENES ;= /THER STUDIES REPORTED THAT APART FROM SPLICING THE LAST lVE AMINO ACID RESIDUES OF 0P(ACP ARE ESSENTIAL FOR ACTIVATION OF THE 502 ;= 4HE EFFECT OF (!# OVEREXPRESSION IS PROTEIN ANDOR HOST SPECIlC )N 0 PASTORIS IT ALSO DEPENDS ON THE INDUCIBLECONSTITUTIVE EXPRESSION OF THE (!# ; = (OWEVER 'ASSER ET AL ;= PROVED THE BENElCIAL EFFECT OF CONSTITUTIVE OVEREXPRESSED 3C(ACP FROM IN 0 PASTORIS FOR THE PRODUCTION OF A &AB FRAGMENT (!# KNOCKOUT STRAINS OF 3 CEREVISIAE AND 0 PASTORIS ARE VIABLE AND THEY HAVE BEEN CHARACTERISED AS BEING INOSITOL AUXOTROPHS DUE TO THE INVOLVEMENT OF THE 502 *OUSPEVDUJPO
IN LIPID BIOGENESIS ; =
,OPDLPVU HFOFSBUJPO JO 1JDIJB QBTUPSJT 'ENE TARGETING IS ONE OF THE MAIN MOLECULAR TOOLS USED IN YEAST SCIENCE WHICH HELPS IN UNDERSTANDING GENE FUNCTIONS AND INTERACTIONS AS WELL AS MOLECULAR PROCESSES ;= 4ARGETED CHROMOSOMAL INTEGRATION OF FOREIGN $.! IS MAINLY DEPENDENT ON $.! DOUBLE STRANDED BREAKS $3" AND THEIR REPAIR MECHANISMS ;= )N YEAST TWO MAJOR PATHWAYS COEXIST TO REPAIR THESE $3" HOMOLOGOUS RECOMBINATION (2 AND THE NON HOMOLOGOUS END JOINING .(%* 4HE (2 PATHWAY REQUIRES INTERACTION BETWEEN HOMOLOGOUS SEQUENCES AND IS RESPONSIBLE FOR THE TARGETED INTEGRATION OF $.! ;= WHEREAS .(%* INTEGRATES $.! RANDOMLY WITH LITTLE OR NO SEQUENCE INTEGRATION 7HEN FOREIGN $.!S ARE TRANSFORMED INTO CELLS THEY ARE COMPETED BY THESE TWO RECOMBINANT PATHWAYS 4HEREFORE EFlCIENT GENE TARGETING IS DETERMINED BY THE RELATIVE STRENGTH OF THE (2 PATHWAY COMPARED WITH THAT OF THE .(%* PATHWAY ;= 5NLIKE MOST OF THE YEAST 3 CEREVISIAE HAS A VERY EFlCIENT GENE TARGETING SYSTEM $ISRUPTION CASSETTES WITH SHORT mANKING REGIONS THAT RANGE FROM BP TO BP CAN INTEGRATE WITH HIGH EFlCIENCIES VIA HOMOLOGOUS INTEGRATION AT THE CORRECT GENOMIC LOCUS ;= )N CONTRAST THE GENE TARGETING EFlCIENCIES OF SOME vNON CONVENTIONALv YEAST IE 0ICHIA PASTORIS (ANSENULA POLYMORPHA 9ARROWIA LIPOLYTICA 0ICHIA STIPITIS CAN BE EXTREMELY LOW WITH THE SAME LENGTH OF mANKING REGIONS )N THESE CASES HOMOLOGOUS ARMS VARYING FROM BP TO BP ARE REQUIRED TO ENSURE EFlCIENT GENE REPLACEMENT ;= &OR 0 PASTORIS IT HAS BEEN ESTIMATED A TARGETED GENE REPLACEMENT EFlCIENCY OF WHEN THE TOTAL LENGTH OF THE TARGET FRAGMENTS IS BP ;= AND ; =ORUPTO;= WHEN EXTENSIVE ࣩ KB REGIONS OF HOMOLOGY ARE USED (OWEVER .(%* PATHWAY IS NOT THE ONLY FACTOR THAT CONTRIBUTES TO THE LOW TARGETING EFlCIENCY OF NON CONVENTIONAL YEASTS 4HE EFlCIENCY OF HOMOLOGOUS INTEGRATION CAN BE VERY LOCUS SPECIlC ;n= 4HIS LOCUS SPECIlC PHENOMENON CAN BE DUE TO THE PRESENCE OF vHOTSPOTv REGIONS FOR YEAST GENOME OR MAYBE THE LOSS OF FUNCTION EFFECT &OR SOME GENES THE PROBABILITY OF OBTAINING THE DESIRED GENE REPLACEMENT EVENT IS SO LOW THAT TRANSFORMATION AND SCREENING PROCEDURES HAVE TO BE ITERATIVELY PERFORMED BEING LABORIOUS AND TIME CONSUMING ;= 4HE KNOCKOUT OF GENES WITH IMPORTANT PHYSIOLOGICAL FUNCTIONS OFTEN MEANS GREAT LOSS OF CELLULAR lTNESS WHICH WOULD LEAD TO A DELAYED OR FAILED APPEARANCE OF CORRECT DISRUPTANTS ;= -OREOVER STUDIES WITH (ANSENULA POLYMORPHA SHOWED DIFFERENT GENE DELETION EFlCIENCY DEPENDING ON THE ANTIBIOTIC MARKERS USED BEING (YGROMYCIN " THE ONE WITH THE BEST EFlCIENCY ;= 4HE FREQUENCY OF TARGETED GENE DISRUPTION CAN BE IMPROVED BY A SPLIT MARKER DISRUPTION STRATEGY ;= )T CONSISTS ON FUSING THE TARGET $.! FRAGMENTS WITH TRUNCATED BUT OVERLAPPING WITHIN THE SELECTABLE MARKER GENE /NLY AFTER A SUCCESSFUL (2 OF THE TWO FRAGMENT THE SELECTION MARKER BECOME ACTIVE AND CELLS CAN GROW ON A MEDIUM CONTAINING THE SELECTION AGENT ;= 3PLIT MARKER BASED TRANSFORMATION DECREASES MULTIPLE AND TANDEM INTEGRATION EVENTS DECREASING THE TOTAL NUMBER OF TRANSFORMANTS BEING SCREENED !T THE SAME TIME IT HAS BEEN SHOWN TO INCREASE THE FREQUENCY OF TARGETED GENE DISRUPTION AND HOMOLOGOUS INTEGRATION AT IN SOME FUNGI ; = 4HIS METHODOLOGY HAS BEEN ALREADY USED TO DELETE GENES IN 0 PASTORIS ; = 4USBJO HFOFSBUJPO
3FTVMUT BOE EJTDVTTJPO $POTUSVDUJPO PG UIF LOPDLPVU DBTTFUUF 4HE METHOD USED FOR KNOCK OUT THE TARGET GENES WAS BASED ON THE METHOD USED BY 4ERNES ET AL ;= BUT APPLYING THE CONCEPT OF THE SPLIT MARKER APPROACH ;= "ECAUSE THE DOMINANT MARKER GENE IS SPLIT IN SEPARATE FRAGMENTS THE GENE IS NOT FUNCTIONAL UNLESS HOMOLOGOUS RECOMBINATION OCCURS BETWEEN TWO OVERLAPPING FRAGMENTS 4RANSFORMANTS WILL NOT GROW ON A MEDIUM CONTAINING THE SELECTION AGENT UNLESS HOMOLOGOUS RECOMBINATION OCCURS BETWEEN THE OVERLAPPING REGIONS OF THE DOMINANT MARKER GENE 4HE MARKER GENE CASSETTE FUSED WITH HOMOLOGOUS mANKING SEQUENCES IS INTEGRATED INTO THE TARGET LOCUS VIA DOUBLE CROSS OVER RECOMBINATION 4HE SPLIT MARKER GENE FRAGMENTS mANKED WITH THE GENE OF INTEREST WERE CONSTRUCTED USING A FUSING 0#2 APPROACH WHICH ELIMINATES TEDIOUS CLONING PROCEDURES AND ALLOWS QUICK GENERATION OF SPLIT MARKER FRAGMENTS FOR TARGETED GENE DISRUPTION !S ILLUSTRATED IN &IGURE THE TARGET GENE WAS AMPLIlED FROM THE 0 PASTORIS 8 CHROMOSOME USING THE PRIMER PAIR 5P & AND $OWN 2 !T THE SAME TIME HYGROMYCIN RESISTANCE WAS AMPLIlED
&IGURE 3PLIT MARKER APPROACH 4HE SELECTION MARKER (YGROMYCIN IS SPLIT INTO TWO FRAGMENTS (OMOLOGOUS RECOMBINATION IS NEEDED TO ACTIVATE THE RESISTANCE GENE )T ALSO FAVOURS THE HOMOLOGOUS RECOMBINATION AT THE ADJACENT SITES IN THE mANKING REGIONS OF THE TARGETED GENE
FROM P"2 VECTOR WITH PRIMERS & (YG 4%& AND 2 (YG #9#44 BP FRAGMENT IN ORDER TO BE USED A SELECTION MARKER OF THE DISRUPTION CASSETTE .ESTED PLASMIDS 5P .ESTED &5P 2 (YG & AND $OWN & (YG 2$OWN .ESTED 2 WERE USED TO AMPLIFY THE AND THE mANKING REGIONS OF AROUND BP OF THE TARGET GENE 4HEN PRIMERS 5P .ESTED & AND (YG )NT 2 AND $OWN & AND $OWN .ESTED 2 WERE USED IN A SECOND REACTION WITH ONE OF THE lRST ROUND TEMPLATES AND THE HYGROMYCIN CASSETTE TO GENERATE AN OVERLAP PRODUCT THAT CONTAINED THE AND A TRUNCATED PART OF THE SELECTION MARKER BP AND THE WITH THE REST OF THE SELECTION MARKER WITH AN OVERLAPPING FRAGMENT OF BP 4HOSE 0#2 PRODUCTS WERE THEN DIRECTLY EMPLOYED FOR TRANSFORMATION WITH SELECTION ON MEDIUM CONTAINING GM,ዅኻ HYGROMYCIN 3FTVMUT BOE EJTDVTTJPO
0RIMERS 5P & AND (YG )NT 2 AND (YG )NT & AND $OWN 2 WERE USED TO CONlRM PROPER INTEGRATION OF THE CASSETTE AT THE AND JUNCTIONS RESPECTIVELY AND PRIMERS 5P & AND )NT 2 AND )NT & AND $OWN 2 WERE USED TO CONlRM THE ABSENCE OF THE WILD TYPE ALLELE
%FMFUJPO PG %&4 4HE WHOLE GENE $%3 WAS AMPLIlED FROM A GENOMIC $.! EXTRACTION OF 0 PASTORIS 8 USING THE PRIMERS $ES 5P & AND $ES $OWN 2 AND THE SAHARA ! PRODUCT OF BP WAS OBTAINED )N A SEPARATE 0#2 REACTION HYGROMYCIN RESISTANCE CASSETTE WAS AMPLIlED USING THE PRIMERS & (YG P4%& AND 2 (YG #9#44 &IGURE
&IGURE !MPLIlCATION OF $%3 AND HYGROMYCIN RESISTANCE
4HE PURIlED GENE WAS USED TO AMPLIFY NESTED UPSTREAM BP AND DOWNSTREAM BP REGIONS OF THE TARGET GENE IN TWO DIFFERENT 0#2 REACTIONS &IGURE A &OR THESE REACTIONS PRIMERS $ES 5P .ESTED & PAIRED WITH $ES 5P 2 (YG & AND $ES $OWN & (YG 2 WITH $ES $OWN .ESTED 2 WERE USED
DES1 DES1 UP DOWN DES1 DES1 Split-Up Split-Down
A $%3 UPSTREAM AND DOWNSTREAM FRAGMENT B $%3 SPLIT MARKER UP AND DOWN FRAGMENTS AMPLIlCATION
&IGURE !MPLIlCATION OF $%3 FRAGMENTS TO GENERATE THE KNOCKOUT CASSETTE 4USBJO HFOFSBUJPO
)N ORDER TO GENERATE THE SPLIT MARKER THE UPSTREAM FRAGMENT OF $%3 AND BP OF THE HYGROMYCIN CASSETTE WERE FUSED IN A TWO STEP 0#2 REACTION )N THE lRST ONE $ES 5P AND (YG FRAGMENTS WERE COMBINED IN A RATIO OF AND AMPLIlED FOR CYCLES WITHOUT PRIMERS IN ORDER TO FUSE BOTH FRAGMENTS 4HE SECOND 0#2 STEP ADDED THE PRIMERS $ES 5P .ESTED & AND (YG )NT 2 AND A CONVENTIONAL 0#2 WAS PERFORMED FOR CYCLES ! FRAGMENT OF ࣵ BP WAS OBTAINED NAMED $ES 3PLIT 5P &IGURE B 4HE SAME STRATEGY WAS USED TO OBTAIN THE $ES 3PLIT $OWN FRAGMENT BP AND USE OF & $OWN AND 2 $OWNCM &RAGMENTS $ES 3PLIT 5P AND $ES 3PLIT $OWN SHARED AN OVERLAPPING FRAGMENT OF THE MARKER GENE OF BP !BOUT NG OF EACH FRAGMENT WERE TRANSFORMED BY ELECTROPORATION -ATERIALS AND METHODS AND A TRIPLE RECOMBINATION EVENT SHOULD TAKE PLACE IN ORDER TO INTEGRATE THE CORRECT DELETION CASSETTE IN THE TARGET GENE #LONES WERE SELECTED IN HYGROMYCIN PLATES AND CHECKED FOR GENE DISRUPTION BY COLONY 0#2 USING PRIMERS $ES 5P & AND $ES $OWN 2 WHERE NO AMPLIlCATION WAS RELATED TO GENE DELETION
/NLY TWO CLONES OUT OF MORE THAN SCREENED WERE SELECTED AS POTENTIAL POSITIVE KNOCKOUTS &URTHER CHARACTERISATION OF THESE CLONES INCLUDED GENOMIC $.! EXTRACTION AND DIFFERENT 0#2 AMPLIlCATIONS FOR CORRECT INTEGRATION OF THE DELETION CASSETTE AND LACK OF PRESENCE OF THE TARGET $%3 GENE &IGURE #LONES # AND # SHOWED TO BE POSITIVE KNOCKOUT AS AMPLIlED FRAGMENTS WERE OBSERVED FOR THE /+ 0#2 AMPLIlCATIONS
&IGURE $ES KNOCKOUT CHARACTERISATION 5P/K REACTIONS USED $ES 5P &(YG )NT 2 TO CONlRM THE UPSTREAM PART OF THE GENE WHILE $OWN/+ REACTIONS USED (YG )NT &$ES $OWN 2 TO CONlRM THE DOWNSTREAM PART OF THE GENE 2EACTIONS 5P./ AND $OWN./ USED $ES 5P &$ES )NT 2 AND $ES )NT &$ES $OWN 2 RESPECTIVELY TO CONlRM NO WILD TYPE GENE
WAS PRESENT IN THE CHROMOSOME 8 WILD TYPE STRAIN WAS USED AS A CONTROL AND (Ꮄ/ AS A NEGATIVE AMPLIlCATION CONTROL
%FMFUJPO PG 463 4HE STRATEGY TO GENERATE 352 KNOCKOUT CLONES WAS SIMILAR TO THE ONE FOR $%3 WITH SOME MODIlCATIONS &IRST HYGROMYCIN RESISTANCE CASSETTE WAS AMPLIlED AS PREVIOUSLY DESCRIBED 352 3FTVMUT BOE EJTDVTTJPO
SUR2 SUR2 Split-Up Split-Down
A 352 UP AND DOWN FRAGMENTS B 352 SPLIT MARKER UP AND DOWN FRAGMENTS
&IGURE !MPLIlCATION OF 352 FRAGMENTS TO CONSTRUCT THE KNOCKOUT CASSETTE
GENE WAS AMPLIlED BY 0#2 USING PRIMERS 3UR 5P & AND 3UR $OWN 2 4HIS FRAGMENT WAS USED TO AMPLIFY THE NESTED FRAGMENTS OF ࣵ BP AT AND OF THE GENE &IGURE A 4HESE FRAGMENTS WERE COMBINED IN SEPARATE 0#2 REACTIONS TOGETHER WITH HYGROMYCIN RESISTANCE AND A STEP 0#2 REACTION WAS PERFORMED AS FOR $%3 KNOCKOUTS )N THIS CASE 352 3PLIT $OWN FRAGMENTS WERE COMBINED IN EQUIMOLAR AMOUNTS WITH A TOTAL $.! QUANTITY OF NG IN ORDER TO OBTAIN PURE AMPLIlCATION BANDS &IGURE B /NCE BOTH 5P AND $OWN FRAGMENTS OF THE 3PLIT MARKER WERE OBTAINED CELLS WERE TRANSFORMED AND SELECTED FOR GENE DELETION WITH HYGROMYCIN AGAR PLATES 5NLIKE WITH $%3 COLONY 0#2 LEAD TO VERY FAINT BANDS WHICH MADE THIS METHOD NOT VIABLE FOR THE SELECTION FOR 352 KNOCKOUTS 4HUS CONVENTIONAL GENOMIC $.! EXTRACTION OF THE CLONES FOLLOWED BY 0#2 AMPLIlCATION WAS PERFORMED /UT OF SCREENED CLONES ONE CLONE WAS SELECTED AS A KNOCKOUT CANDIDATE ( &URTHER CONlRMATION WAS PERFORMED BY DIFFERENT 0#2 REACTIONS &IGURE
UpNo DownNo UpOK DownOK C-
&IGURE 0#2 AMPLIlCATION OF THE FRAGMENTS TO CONlRM THE CLONE ( AS A POSITIVE KNOCKOUT FOR 352
$POTUSVDUJPO PG UIF WFDUPS Q("1)" ! NEW VECTOR CARRYING THE '!0 PROMOTER FOR CONSTITUTIVE INTRACELLULAR EXPRESSION WAS GENERATED BY MIXING TWO VECTORS AVAILABLE P'!0(IS AND P"2 P'!0(IS WAS DIGESTED WITH "GL)) AND %CO2) AND FRAGMENTS AND BP WERE OBTAINED 4HE BAND OF BP CORRESPONDING TO P'!0 PROMOTER WAS EXTRACTED AND PURIlED !T THE SAME TIME THE P"2 VECTOR WAS ALSO DIGESTED WITH "GL)) AND %CO2) OBTAINING A BAND OF BP THAT CONTAINS THE !/8 PROMOTER INDUCIBLE BY METHANOL FOLLOWED BY A SECRETION SIGNAL FACTOR FORM 3 CEREVISIAE AND A SECOND BAND OF BP CORRESPONDING TO THE HYGROMYCIN 4USBJO HFOFSBUJPO
RESISTANCE CASSETTE AND THE % COLI REPLICATION ORIGIN 4HE LAST FRAGMENT WAS PURIlED AND LIGATED WITH THE P'!0 PROMOTER FROM P'!0(IS AS REPRESENTED IN &IGURE 4HE NEW P'!0(! VECTOR WAS OBTAINED AND CORRECT VECTOR CONSTRUCTION WAS CONlRMED BY DIFFERENT RESTRICTION ASSAYS
BglII AleI MslI BmrI Van91I BanII NsiINNssii EcoICRI BlpI AvrIIAvr II PmlI SacI PmeI PasIPPasI SfiI AlwNI SacI PshAIPshAI PspXI PmeI EcoICRI EcoRIEcE AvaI DraI AleI AgeIAgA eII BmeT110I ZraI XhoI AatII SacII pAOX1' MCS' NotI BlpI pGAP 'MCS pUCpUC ororii BtgZI PAOX1 NsiI BstXI FspAI XcmI PciI ScaI AOX1TT XbaI MluI PvuI Bsu36I BsrGI 4000 HpaI MunI HindIII PstI 8000 Am pR CYC1TTC1TT BstBI EcoRV StuI pBR326_NupBR326_Nu 1000 a-factor pGAPHis ScaI 3000 6000 4220 bps EcoRI 2000 PsiI BsaAI BsrDIBsrDI PmlI 8017 bps SfiI MCS NaeI NgoMIV ori Acc65I 2000 BglII KpnI HIS AccIAI PspXI TsoI AOX1 TTT SalI SacII 4000 BspMI HYG_RHYG_R NotI SalI BspHI AvrII XbaI StuI StyI NmeAIII PciI PasIPasI BsiWI AflIII AlwNI PshAI PvuII BspHI NdeI PTEF1 NdeI RsrII PEM7 BamHI Bst1107I 3'AOX1 DraIII TsoI Tth111I NcoI PAOX1' PvuI 'pGAP AsiSI AccIII BspMI ZraI PshAI pUC ori AatII BsrGI AccI StyI SphI SgrAI NcoI MCS'' SalI
3500 'MCS
PciI 3000 500 MluI BsrGI BsiWI pGAPHA AOX1 TT PvuII CYC1TT 2500 3507 bps 1000
BamHI EcoRV StuI 2000 1500 ScaI PTEF1
PEM7 HYG_R
NcoI BstAPI StyI NmeAIII AatII NdeI PshAI RsrII ZraI TsoI BstAPI PvuI AsiSI BspMI
&IGURE 3CHEMATIC REPRESENTATION OF THE STRATEGY USED TO CONSTRUCT THE VECTOR P'!0(! WHERE THE PROMOTER OF P'!0(IS AND THE BACKBONE OF P"2 WERE COMBINED
(FOF PWFSFYQSFTTJPO %&4 463 &3( PWFSFYQSFTTJPO P'!0(! WAS USED AS A VECTOR FOR CONSTITUTIVE INTRACELLULAR OVEREXPRESSIONOF$%3 352 AND %2' !LL GENES WERE AMPLIlED BY 0#2 USING FORWARD PRIMERS HARBOURING THE .OT) RESTRICTION SEQUENCE AND REVERSE PRIMERS THAT INCLUDE THE 3l) RESTRICTION SEQUENCE 0RIMERS 'ENE AMPLIlCATIONS TOGETHER WITH P'!0(! WERE DIGESTED WITH .OT) AND 3l) AND PURIlED 4HE DIGESTED VECTOR WAS DEHOSPHORYATED BEFORE THE LIGATION WITH THE GENE INSERTS TO AVOID RELIGATION EVENTS ,IGATIONS WERE THEN TRANSFORMED TO % COLI $( CELLS AND TRANSFORMANTS WERE SELECTED BY THEIR ABILITY FOR GROWING INTO AGAR PLATES CONTAINING HYGROMYCIN 0LASMID EXTRACTIONS -INI0REPS FROM RESULTING COLONIES WERE EXAMINED FOR CORRECT INTEGRATION BY COLONY 0#2 %2 DIGESTION USING %CO26 FOR P'!0(! $ES AND P'!0(! 3UR AND .CO) FOR P'!0(! %RG AND lNALLY BY SEQUENCING #ORRECT VECTORS WERE LINEARISED WITH !VR)) AND USED TO ELECTROPORATE 0PASTORIS &AB& COMPETENT CELLS 4RANSFORMANTS WERE SELECTED IN 90$ AGAR PLATES CONTAINING :EOCIN AND (YGROMYCIN AND CORRECT VECTOR INTEGRATION WAS DETERMINED BY GENOMIC $.! EXTRACTION AND GENE AMPLIlCATION BY 0#2 !ROUND POSITIVE CLONES CARRYING EACH OF THE GENES WERE SELECTED FOR FURTHER CLONE SCREENING
)"$ PWFSFYQSFTTJPO 4HE $.! SEQUENCE CORRESPONDING TO THE FUNCTIONAL (ACP WAS CLONED INTO THE P05::,% VECTOR ALLOWING THE CONSTITUTIVE EXPRESSION OF THE TRANSCRIPTIONAL FACTOR 4HE VECTOR WAS USED TO TRANSFORM 0 PASTORIS &AB& COMPETENT CELLS )N THIS CASE CLONE SELECTION WAS PERFORMED USING 90$ AGAR PLATES CONTAINING :EOCIN AND 'ENETICIN $PODMVTJPOT
$PODMVTJPOT )N THIS CHAPTER A BATTERY OF STRAINS ALTERING THE GENES RELATED TO LIPID META BOLISM THAT WERE SELECTED FROM THE TRANSCRIPTOMIC ANALYSIS HAS BEEN GENERATED )N ORDER TO UNDERSTAND THE EFFECT OF THESE GENES ON ALTERING THE MEMBRANE AND THUS AFFECTING THE RECOMBINANT PROTEIN SECRETION STRAINS WITH BOTH DELETION AND OVEREXPRESSING THE TARGET GENES WERE GENERATED 4HE MAIN CONCLUSIONS OF THIS CHAPTER ARE
s +NOCKOUT STRAINS OF TWO GENES INVOLVED IN THE SPHINGOLIPID BIOSYNTHESIS PATHWAY $%3 AND 352 WERE OBTAINED FOR THE 0 PASTORIS STRAIN PRODUCING RECOMBINANT &AB & !S PREVIOUSLY DESCRIBED IN THE BIBLIOGRAPHY AN EFlCIENCY OFq WAS OBTAINED s ! KNOCKOUT OF 352 IN 0 PASTORIS HAS BEEN DESCRIBED FOR THE lRST TIME
s 6ECTORS FOR THE EXPRESSION OF $%3 352 AND %2' WERE CONSTRUCTED AND USED TO OBTAIN A BATTERY OF CLONES OVEREXPRESSING THESE GENES IN A 0 PASTORIS STRAIN PRODUCING RECOMBINANT &AB &
4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
*OUSPEVDUJPO
#MPDLJOH UIF FSHPTUFSPM CJPTZOUIFUJD QBUIXBZ 'MVDPOB[PMF USFBUNFOU
&LUCONAZOLE &IGURE IS ONE OF THE AZOLE ANTINFUNGALS THAT INHIBIT THE SYNTHESIS OF ERGOSTEROL IN YEAST BY DIRECT BINDING TO LANOSTEROL DEMETHYLASE %RGP 4HIS INHIBITION RESULTS IN A DEPLETION OF ERGOSTEROL FROM CELLULAR MEMBRANES THAT ARE REPLACED WITH METHYLATED STEROLS 0RESENCE OF AN ADDITIONAL /( GROUP DISTURBS MEMBRANE PACKING INCREASES MEMBRANE mUIDITY AND DRUG PERMEABILITY WHICH lNALLY INTERFERES ON &IGURE &LUCONAZOLE STRUCTURE ;= CELL VIABILITY ;= 0REVIOUS STUDIES OF THE GROUP USED mUCONAZOLE AS A MODULATOR OF %RGP LEVELS IN THE CELL AND THEREFORE CELLULAR AMOUNTS OF ERGOSTEROL ;= #ELLS WERE CULTURED IN PRESENCE OF DIFFERENT CONCENTRATIONS OF mUCONAZOLE GM,ዅኻ IN ORDER TO DETERMINE THE CONCENTRATION OF ANTIFUNGAL NOT AFFECTING CELLULAR GROWTH &IGURE AND A RANGE BETWEEN AND GM,ዅኻ TURNED OUT TO BE THE OPTIMAL
4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
20
16
12 600 OD
8
4
0 0 5 10 15 20 25 time (h)
Control Fluco 0.2 Fluco 0.4 Fluco 0.6 Fluco 0.8 Fluco 1 Fluco 2
&IGURE %XTRACTED FROM ;= 'ROWTH CURVES OF 0 PASTORIS EXPRESSING THE &AB & GROWN ON "-$ MEDIUM AND TREATED WITH mUCONAZOLE IN A RANGE OF CONCENTRATIONS BETWEEN AND ᎙GM,ᎽᎳ #ELL GROWTH WAS NOT SIGNIlCANTLY AFFECTED IN CONCENTRATIONS UP TO ᎙GM,ᎽᎳ
&URTHER CULTIVATIONS WITH THE REFERENCE &AB & STRAIN SHOWED A BENElCIAL EFFECT ON &AB SECRETION WHEN CELLS WERE TREATED WITH mUCONAZOLE CONCENTRATIONS UP TO GM,ዅኻ WHILE HIGHER CONCENTRATIONS LEAD TO A NEGATIVE EFFECT &IGURE
1.6
1.4
1.2
1 relative Fab product yields 0.8
0.6 Fluco 0.2 Fluco 0.4 Fluco 0.6 Fluco 0.8 Fluco 1.0
&IGURE %XTRACTED FROM ;= &AB & PRODUCTIVITIES OF CULTURES TREATED WITH mUCONAZOLE IN A RANGE OF CONCENTRATIONS BETWEEN AND ᎙GM,ᎽᎳ WERE NORMALISED TO THE VALUES OBTAINED FROM THE NON TREATED REFERENCE CELLS &LUCONAZOLE CONCENTRATIONS UP TO ᎙GM,ᎽᎳ HAD A BENElCIAL IMPACT ON &AB SECRETION %RROR BARS INDICATE THE STANDARD ERROR OF THE MEANS
$FMM EJTSVQUJPO BOE JOUSBFYUSBDFMMVMBS QSPUFJO EJTUSJCVUJPO ! KEY ADVANTAGE OF 0 PASTORIS AS A HOST ORGANISM IN FRONT OF OTHER ALTERNATIVES ESPECIALLY THE PROKARYOTIC SYSTEMS IS ITS ABILITY TO SECRETE THE PRODUCT TO THE CULTIVATION BROTH WHICH FACILITATES IMPORTANTLY THE DOWNSTREAM PROCESSES ;= 4HE PASSAGE OF PROTEINS THROUGH THE SECRETORY PATHWAYS PERMITS POST TRANSLATIONAL EVENTS THAT USUALLY ARE ESSENTIAL FOR THE BIOLOGICAL ACTIVITY OF THE PROTEINS ;= .EVERTHELESS HIGH LEVELS OF HETEROLOGOUS PROTEIN EXPRESSION CAN LEAD TO SATURATION OR OVERLOADING OF THE SECRETORY PATHWAYS WHERE THE PRODUCT IS ACCUMULATED INTRACELLULARLY AND OFTEN ALSO DEGRADED RESULTING INTO AN IMPORTANT DECREASE OF THE PRODUCTION YIELD 4HIS FACT TURNS OUT TO BE A MAJOR BOTTLENECK FOR BIOTECHNOLOGICAL PROCESS DEVELOPMENT ; = 4O STUDY THE EFFECT OF THE SECRETORY PATHWAY SATURATION ON THE BIOPROCESS EFlCIENCY IT IS OF CAPITAL INTEREST THE RELIABLE QUANTIlCATION AND RECOVERY OF THE TOTAL AMOUNT OF PRODUCT ACCUMULATED *OUSPEVDUJPO
INTRACELLULARLY ALONG A CULTIVATION )N YEAST CELLS PRESENCE OF THE CELL WALL REQUIRES THE SELECTION OF DISRUPTION METHODS THAT DESTRUCTS THE WALL IN ORDER TO RELEASE ALL THE PRODUCT OF INTEREST ;= )N PARTICULAR 0 PASTORIS CELLS PRESENT IMPORTANT DIFlCULTIES TO OBTAIN REPRODUCIBLE AND RELIABLE RESULTS FOR CELL DISRUPTION PROCEDURES DUE TO THE IMPORTANT THICKNESS OF ITS CELL WALL 3EVERAL METHODS FOR CELL DISRUPTION HAVE BEEN USED WITH 0 PASTORIS LIKE SONICATION ;= BEAD MILLING ; = ENZYMATIC AND CHEMICAL LYSIS ; = CELL PERMEABILISATION ; = AND HIGH PRESSURE HOMOGENISATION ;n= )N ALL CASES A FAST AND EFlCIENT CELLULAR DISRUPTION IS DESIRABLE THAT DOES NOT ALTER THE PROTEIN CHEMICALLY OR PHYSICALLY ! HIGH PRESSURE HOMOGENISER CONSISTS ON A POSITIVE DISPLACEMENT PUMP THAT FORCES A CELL SUSPENSION THROUGH THE CENTRE OF A VALVE SEAT AND RADIALLY ACROSS THE SEAT FACE 0RESSURE IS CONTROLLED BY ADJUSTING THE FORCE OF THE VALVE WHICH IS SPRING LOADED OR HYDRAULICALLY CONTROLLED &LUID mOWS RADIALLY ACROSS THE VALVE AND STRIKES AN IMPACT RING UNTIL SUSPENSION EXITS THE VALVE ASSEMBLY ;= 0REVIOUS STUDIES IN THE GROUP WERE FOCUSED ON THE EFlCACY OF HIGH PRESSURE HOMOGENISATION DISRUPTION PROCEDURES ON METHANOL BASED CULTIVATIONS AS IT IS KNOWN THAT CELLS GROWING ON THIS SUBSTRATE PRESENT A SIGNIlCANT WIDENING OF THE CELL WALL THICKNESS ; = ! SPECIlC DISRUPTION METHOD FOR CELLS GROWING ON GLUCOSE OR GLYCEROL WAS REQUIRED 3INCE AN IMPORTANT AMOUNT OF THE PROTEIN OF INTEREST IS EXPECTED TO BE RETAINED THROUGH THE SECRETORY PATHWAY BESIDES THE SOLUBLE PART OF THE CELL LYSATES THE INSOLUBLE FRACTION MUST BE TAKEN INTO ACCOUNT IN ORDER TO AVOID A MISESTIMATION OF THE TARGET PRODUCT AS IT CONTAINS THE CELL MEMBRANES ENDOPLASMIC RETICULUM %2 'OLGI AND OTHER ORGANELLES WHERE THE PROTEIN OF INTEREST MAY BE RETAINED ;= )N ADDITION A RELIABLE QUANTIlCATION OF THE PRODUCT PRESENT IN THE INSOLUBLE FRACTION REQUIRES AN EXTRACTION PROCEDURE THAT INVOLVES THE USE OF DETERGENTS WHICH MUST BE OPTIMISED FOR EACH PROTEIN IN THIS CASE FOR THE &AB & 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
3FTVMUT BOE EJTDVTTJPO %FUFSNJOBUJPO UIF PQUJNBM nVDPOB[PMF USFBUNFOU GPS 'BC ' TFDSFUJPO /PTIMAL mUCONAZOLE CONCENTRATION REQUIRED TO INCREASE PROTEIN SECRETION WITHOUT ALTERING CELL GROWTH WAS PREVIOUSLY DETERMINED IN SHAKE mASK CULTURES ;= )N THIS WORK FURTHER STUDIES ON mUCONAZOLE TREATEMENT WERE CARRIED OUT IN SHAKE mASKS IN ORDER TO ESTABLISH THE OPTIMAL AMOUNT OF mUCONAZOLE IN RELATION TO CELL MASS ALLOWING FOR THE MAXIMAL PROTEIN SECRETION &IGURE #ELLS WERE CULTURED FOR HOURS IN PRESENCE OF DIFFERENT mUCONAZOLE CONCENTRATIONS &AB SECRETED WAS RELATED TO THE RATIO OF mUCONAZOLE TO lNAL BIOMASS VALUE FOR EACH CULTIVATION AND A VALUE OF
ĘG mUCONAZOLE PER Gፃፂፖ TURNED OUT TO BE THE OPTIMAL LEADING TO A FOLD INCREASE IN YIELD OF SECRETED &AB
2 1.6
1.2
0.8 relative Fab 2F5 yield
0.4
0 0 200 400 600 800
-1 μgFluconazole · gDCW
&IGURE 2ELATIVE &AB & YIELD AS FUNCTION OF SPECIlC AMOUNT OF mUCONAZOLE PER GRAM OF BIOMASS
(IGHER RATIOS OF mUCONAZOLE PER BIOMASS LEAD TO DECREASED &AB SECRETION LEVELS COMPARED TO THE REFERENCE STRAIN NOT TREATED WITH mUCONAZOLE &LUCONAZOLE EFFECT ON PROTEIN SECRETION OVER THE TIME WAS ALSO DETERMINED )N THIS CASE CELLS CULTURED WITH DIFFERENT AMOUNTS OF mUCONAZOLE WERE ANALYSED IN TERMS OF CELL DENSITY AND &AB SECRETED AFTER AND H OF CULTIVATION &IGURE
20h 43h 48h 2.4
2
1.6
1.2 relative Fab 2F5 yield
0.8
0.4
0 Fab 2F5 Fluco 0.4 Fluco 0.6 Fluco 0.8 Fluco 1.0
&IGURE 4IME EFFECT ON &AB PRODUCTION WHEN CULTURES ARE TREATED WITH DIFFERENT AMOUNTS OF mUCONAZOLE 4HE STUDIED RANGE WAS FROM TO ᎙GM,ᎽᎳ 3FTVMUT BOE EJTDVTTJPO
)NITIALLY A NEGATIVE EFFECT OF mUCONAZOLE TREATEMENT ON &AB SECRETION WAS OBSERVED FOR THE CULTURES WITH HIGHER INITIAL mUCONAZOLE CONCENTRATION PROBABLY CAUSED BY THE LOW BIOMASS CONCENTRATION PRESENT IN THE CULTURES THAT RESULT IN A HIGH mUCONAZOLE TO BIOMASS RATIO AND LEAD TO LOW LEVELS OF SECRETED &AB ,ONGER INCUBATION TIMES H PROMPTED INCREASED &AB SECRETION UP TO A POSITIVE EFFECT OF mUCONAZOLE AS PREVIOUSLY DESCRIBED 4HE MOST SIGNIlCANT CHANGE FOR ALL THE CONCENTRATIONS WAS AT THE LONGEST INCUBATION TIME H BEING GM,ዅኻ THE OPTIMAL mUCONAZOLE AMOUNT FOR &AB SECRETION (IGHER LEVELS OF &AB OBTAINED FOR LONGER CULTIVATION TIMES CAN BE THE RESULT OF AN IMPROVED CELL SECRETION CAPACITY DUE TO LOWER CELL GROWTH RATES DURING STATIONARY PHASE !FTER HOURS UP TO FOLD INCREMENT IN mUCONAZOLE TREATED CELLS WAS OBSERVED COMPARED TO THE FOLD OBTAINED AFTER HOURS OF CULTIVATION )T CAN BE DUE TO THE FACT THAT CELLS HAVE BEEN REACH STATIONARY PHASE WERE NO LONGER GROW AND THEY HAD LONGER TIMES TO SECRETE THE PRODUCED PROTEIN
$FMM EJTSVQUJPO BOE QSPUFJO FYUSBDUJPO $ISRUPTION OF 0 PASTORIS BY USING /NE 3HOT #ELL $ISRUPTER HAS BEEN PREVIOUSLY REPORTED FOR CELLS GROWING ON MIXED FEEDS OF GLUCOSE AND METHANOL ;= &OR THIS STUDY THE DISRUPTION PROCESS WAS OPTIMISED IN TERMS OF RECOVERY OF THE WORKING ANTIBODY FRAGMENT &AB & PRODUCED CONSTITUTIVELY BY CELLS GROWING ON GLYCEROL AND GLUCOSE
%FUFSNJOBUJPO PG UIF PQUJNBM EJTSVQUJPO QSFTTVSF BOE OVNCFS PG QBTTFT 4HE lRST STEP TO OPTIMISE CELL DISRUPTION WAS TO DETERMINE THE WORKING PRESSURE AND NUMBER OF DISRUPTION PASSES THAT LEAD TO THE BEST &AB & RECOVERY 3INCE 0 PASTORIS CELL WALL IS THICK AND RESISTANT HIGH PRESSURES KBAR WERE CONSIDERED AS INITIAL TESTING CONDITIONS TO OBTAIN HIGH AMOUNTS OF CELL DISINTEGRATION #HEMOSTAT SAMPLES WERE USED DIRECTLY WHEREAS FED BATCH SAMPLES WERE DILUTED TO CELL DENSITIES SIMILAR TO CHEMOSTAT ONES WHICH WERE USED TO OPTIMISE THE PROCESS BEFORE STARTING THE DISRUPTION ANALYSES #ELL CYTOMETRY WAS USED AS DIRECT INDICATOR OF CELL DISRUPTION #ELL NUMBER HAS BEEN DESCRIBED AS THE MOST ACCURATE AND REPRODUCIBLE MEASURE OF CELL RUPTURE BUT IT REQUIRES SIGNIlCANT ANALYSIS TIME ;= )N THIS SENSE mOW CYTOMETRY ALLOWS FOR COUNTING CELL NUMBER WHILE GATHERING SPECIlCALLY WHOLE CELLS FROM CELL DEBRIS &IGURE BEING A FAST AND RELIABLE SYSTEM FOR CELL DISRUPTION DETERMINATION
&IGURE #ELL COUNTING BY mOW CYTOMETRY USED TO DETERMINE THE EXTENT OF CELL DISRUPTION AFTER DIFFERENT PASSES ELLIPSE GATE DIFFERENTIATES THE WHOLE UNDAMAGED CELLS FROM DAMAGED AND BROKEN CELLS 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
4HE USE OF OPTICAL DENSITY AS CELL DISRUPTION INDICATOR HAS BEEN SYSTEMATICALLY USED BUT IT CAN BRING TO MISESTIMATED RESULTS AS CELL DEBRIS CAN BE QUANTIlED TOGETHER WITH WHOLE CELLS )NITIAL CELL NUMBER WAS COUNTED BY mOW CYTOMETRY AND M, OF CELL ALIQUOTS WERE USED AND DISRUPTED AT KBAR OR KBAR FOR TO PASSES &IGURE #ELL NUMBER WAS THEN DETERMINED AND PERCENTAGE OF DISRUPTION WAS CALCULATED USING %Q #ELL MEMBRANES AND DEBRIS AS WELL AS NOT DISRUPTED CELLS WERE SEPARATED FROM THE SOLUBLE CYTOSOLIC FRACTION BY CENTRIFUGING 4HE DECREASED PERCENTAGE OF CELL NUMBER WAS USED AS AN INDICATOR OF CELL DISRUPTION ,IKE OTHER INDICATORS DESCRIBED IN THE LITERATURE IT CAN BE USED TO RELATE THE DEGREE OF CELL DISRUPTION WITH THE RELEASE OF THE COMPONENT OF INTEREST WHEN DETERMINING THE DESIRED COMPONENT IS NOT POSSIBLE OR IT REQUIRES DIFlCULT AND TIME CONSUMING METHODS ;= 4HUS DETERMINATION OF CELL DISRUPTION BY CALCULATING CHANGES ON THE PERCENTAGE OF CELL NUMBER WAS DIRECTLY RELATED TO HIGHER LEVELS OF RELEASED &AB &IGURE COMPARES THE LEVELS OF CELL DISRUPTION AND &AB & RECOVERED FOR EACH PRESSURE AND NUMBER OF PASSES
Fab 2 kbar Fab 2.5 kbar cell num 2 kbar cell num 2.5 kbar 3 100 2.5 80 2 -1 mL
60 1.5
% disruption 40
1 g Fab 2F5 μ
20 0.5
0 0 01234567 Number of passes
&IGURE #OMPARISON BETWEEN EXTENT OF CELL DISRUPTION AND AMOUNT OF & &AB RELEASED AFTER DIFFERENT PASSES %RROR BARS INDICATE STANDARD DEVIATION
)T WAS OBSERVED A HUGE INCREASE OF CELL DISRUPTION FROM ONE DISRUPTION PASS TO TWO DISRUPTION PASSES WHILE FURTHER PASSES INCREASED ONLY SLIGHTLY THE CELL DISRUPTION AMOUNTS )T WAS ALSO NOTICED THAT NO MAJOR DIFFERENCES WERE OBSERVED IN TERMS OF CELL DISRUPTION BETWEEN KBAR AND KBAR )N RELATION TO &AB & AMOUNTS RECOVERED LEVELS WERE SIMILAR WHEN USING KBAR REGARDLESS DISRUPTION PASSES WHILE A DECREASE ON &AB RECOVERED AMOUNTS WAS PRESENT WHEN KBAR WERE USED /PTIMAL DISRUPTION CONDITIONS WERE DElNED AS THOSE INCLUDING A HIGH PERCENTAGE OF CELL DISRUPTION COMBINED WITH HIGH AMOUNTS OF DETECTED &AB CONSIDERING THAT IN THESE CONDITIONS NO HIGH AMOUNTS OF UNBROKEN CELLS WOULD BE PRESENT IN THE INSOLUBLE MEMBRANE FRACTION )-& AND THUS THE HIGHEST AMOUNT OF PROTEIN OF INTEREST WOULD BE QUANTIlED )T WAS ALSO IMPORTANT TO CONSIDER WORKING WITH AS FEW PASSES AS POSSIBLE TO OVERCOME PROTEASE DEGRADATION PROBLEMS AND TO AVOID TIME CONSUMING STEPS 4HEREFORE TWO PASSES AT KBARS WERE CHOSEN AS WORKING CONDITIONS FOR &AB & QUANTIlCATION 4HESE SETTINGS lT WITH THE ONES PREVIOUSLY DETERMINED USING THE SAME HIGH PRESSURE HOMOGENISER ;=
0QUJNJTBUJPO GPS UIF QSPUFJO FYUSBDUJPO PG UIF JOTPMVCMF GSBDUJPO )N ORDER TO ACHIEVE A RELIABLE QUANTIlCATION OF INTRACELLULAR PROTEINS AND THEIR RECOVERY A PROTEIN EXTRACTION STEP IS NECESSARY TO DETERMINE THE AMOUNTS OF PROTEIN ASSOCIATED TO MEMBRANE FRACTION AND CELL ORGANELLES )-& WHICH ALLOWS COMPARING CHANGES AMONG DIFFERENT CULTIVATION CONDITIONS 3FTVMUT BOE EJTDVTTJPO
AS WELL AS ALONG BIOPROCESSES "ASED ON OTHER STUDIES DIFFERENT EXTRACTION BUFFERS DETERGENTS AND INCUBATION TIMES WERE TESTED IN ORDER OBTAIN THE MAXIMUM &AB EXTRACTED FROM )-&S /NCE THE BEST COMBINATION OF BUFFER AND DETERGENT WAS SELECTED ADDITIONAL STUDIES FOR OPTIMAL DETERGENT CONCENTRATION AND INCUBATION TIME WERE PERFORMED 7ITH THESE RESULTS WHEN THE SECRETED 3#& AND )-& FRACTIONS ARE COUNTED TOGETHER IT CAN BE ASSUMED AS THE TOTAL &AB PRODUCED THAT IS NOT DEGRADED 0FEFFER ET AL ;= OBSERVED HIGH DEGRADATION LEVELS OF A RECOMBINANT SECRETED &AB IN 0PASTORIS WHICH SHOULD BE TAKEN INTO ACCOUNT AS CELL MODIlCATIONS IN THE DEGRADATION OR SECRETION PROCESSES CAN LEAD TO HIGHER DETECTED PROTEIN
4FMFDUJPO PG MZTJT CVGGFS BOE EFUFSHFOU GPS QSPUFJO FYUSBDUJPO "UFFERS ! " AND # WHERE SELECTED FROM THE BIBLIOGRAPHY ; = AND THEIR EXTRACTION EFlCIENCY WAS COMPARED WHEN COMBINED WITH OF THREE CANDIDATE DETERGENTS 4WEEN 4RITON 8 AND #(!03 $ETERGENTS ARE AMPHIPATHIC MOLECULES HAVING A HYDROPHILIC PORTION USUALLY IONIC AND A HYDROPHOBIC REGION USUALLY A HYDROCARBON WHICH MAKE THEM BIND TO THE LIPID MEMBRANE UNTIL SATURATION IS REACHED )NCREASED AMOUNTS OF SURFACTANT LEAD TO THE FORMATION OF MIXED MICELLES WITH MEMBRANE LIPIDS ;= )NITIAL EXTRACTIONS OF &AB FROM )-& USED 3$3 AS EXTRACTING DETERGENT BUT THIS ANIONIC SURFACTANT WAS DISCARDED BECAUSE OF ITS EFFECT ON THE &AB QUANTIlCATION SYSTEM ALTERING ITS DETECTED LEVELS !S IT CAN BE OBSERVED IN &IGURE #(!03 TURNED OUT TO BE THE DETERGENT SHOWING THE BEST RESULTS IN TERMS OF SOLUBILISATION OF MEMBRANES AND MEMBRANOUS STRUCTURES AND THEREFORE EXTRACTED &AB #(!03 IS A ZWITTERIONIC DETERGENT AS COMPARED TO THE OTHER NON IONIC CANDIDATE DETERGENTS
&IGURE 2ELEASE OF & &AB OBTAINED BY USING THE BUFFERS AND DETERGENTS COMPARED %RROR BARS INDICATE STANDARD DEVIATION
4WEEN AND 4RITON 8 :WITTERIONIC DETERGENTS COMBINE PROPERTIES OF IONIC AND NONIONIC DETERGENTS AS THEY ARE COMPOSED OF HEAD GROUPS BEARING ANIONIC AND CATIONIC NET CHARGES ;= (IGHER LEVELS OF SOLUBILISATION WERE OBSERVED FOR #(!03 IN ALL TESTED BUFFERS BEING "UFFER " THE ONE WITH HIGHER LEVELS OF EXTRACTED &AB 3OLUBILISATION STEP IT IS DElNED AS A PROTEIN DEPENDENT STEP WHERE THE OPTIMAL EXTRACTING DETERGENT CHANGES DEPENDING ON PROTEIN CHARACTERISTICS &OR EXAMPLE THE BEST SOLUBILISATION AGENT FOR THE HUMAN OPIOID RECEPTOR (U-/2 WAS THE ANIONIC DETERGENT . ,AUROSYL SARCOSINE ;= AND THE OPTIMAL EXTRACTION DETERGENT FOR HEPATITIS " SURFACE ANTIGEN WAS REPORTED TO BE 4WEEN ;= WHILE ' PROTEIN COUPLED RECEPTORS PREFER DECYLGLUCOPYRANOSIDE ;= BOTH NON IONIC DETERGENTS 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
%FUFSNJOBUJPO PG EFUFSHFOU DPODFOUSBUJPO BOE FYUSBDUJPO UJNF %XTRACTION TIME IS A KEY PARAMETER WHEN OPTIMISING PROTEIN EXTRACTION SINCE SHORT INCUBATION TIMES RESULT INTO AN INCOMPLETE PROTEIN EXTRACTION FROM THE MEMBRANE FRACTION AND PROLONGED EXTRACTIONS MAY RESULT IN PROTEOLYTIC ACTION FROM RESIDUAL PROTEASES IN CRUDE EXTRACT !LTHOUGH HIGHER TEMPERATURE EG ROOM TEMPERATURE ACCELERATES THE EXTRACTION STEP A TEMPERATURE OF # WAS USED IN THE PROCESS IN ORDER TO MAINTAIN PRODUCT INTEGRITY /NCE #(!03 WAS SELECTED AS THE WORKING DETERGENT FOR &AB & SOLUBILISATION DIFFERENT DETERGENT CONCENTRATIONS WERE TESTED IN ORDER TO MAXIMISE THE QUANTITY OF &AB EXTRACTED &URTHERMORE DIFFERENT INCUBATION TIMES AT # AND GENTLY AGITATION WERE ALSO TESTED 4HE AMOUNTS OF SOLUBILISED &AB &IGURE A AND TOTAL PROTEIN EXTRACTED &IGURE B WERE DETERMINED
0 h 2 h 16 h 0 h 2 h 16 h 0.8 1.5 ) -1 0.6 1.0 -1
0.4
g Fab mL 0.5 μ 0.2 total protein (mg mL
0.0 0.0 1% 1.5% 2% 2.5% 1% 1.5% 2% 2.5%
% CHAPS % CHAPS
A B
&IGURE & &AB A AND TOTAL PROTEIN B OBTAINED BY USING DIFFERENT CONCENTRATIONS OF #(!03 AND INCUBATION TIMES %RROR BARS INDICATE STANDARD DEVIATION
)NCUBATION TIME HAD AN IMPORTANT IMPACT ON &AB EXTRACTION WHERE LONG INCUBATION TIMES OVERNIGHT INCUBATION RESULTED IN A FOLD HIGHER RECOVERED &AB (OWEVER THE RANGE OF #(!03 CONCENTRATIONS TESTED DID NOT ALTER SIGNIlCANTLY THE EXTRACTED &AB AMOUNTS BEING #(!03 THE BEST CONCENTRATION 7HEN THE EFFECT OF INCUBATION TIME AND #(!03 CONCENTRATION WERE RELATED TO TOTAL PROTEIN RELEASED FROM CELLS NONE OF THESE PARAMETERS SEEM TO CHANGE THE TOTAL PROTEIN DETECTED 4HIS COULD INDICATE THAT AN IMPORTANT PERCENTAGE OF TOTAL CELLULAR PROTEIN IS RELEASED FROM THE CELL FASTER THAN THE &AB IN A SIMILAR WAS AS PREVIOUSLY DESCRIBED FOR HEPATITIS " SURFACE ANTIGEN ("S!G ;= 7HEN #(!03 WERE SELECTED TOTAL PROTEIN EXTRACTED OF THE )-& WAS CONTROVERSIALLY LOW 3ELECTED CONDITIONS FOR &AB EXTRACTION WERE #(!03 WITH "UFFER " INCUBATED DURING H OVERNIGHT AT #
4DSFFOJOH GPS 'BC ' FYQSFTTJPO %&4 ,OPDLPVU TDSFFOJOH 4HE TWO POSITIVE CLONES OF 0 PASTORIS WITH THE GENE $%3 DELETED # AND # WERE GROWN IN SHAKE mASKS CULTURES WITH MINIMAL MEDIA "-$ IN ORDER TO CHARACTERISE BOTH CELLULAR GROWTH AND
&AB PRODUCTION #ELL GROWTH WAS FOLLOWED BY MEANS OF /$ዀኺኺ AND AFTER HOURS OF CULTIVATION ALL CLONES REACHED SIMILAR OPTICAL DENSITY &IGURE 4HESE RESULTS ARE CONSISTENT WITH THE ONES OBTAINED BY 4ERNES ET AL THAT DELETED $%3 OF A 0 PASTORIS WILD TYPE STRAIN ;= AND GROWTH RATE WAS MAINTAINED ,EVELS OF SECRETED &AB WERE QUANTIlED BY %,)3! AND RELATIVE &AB YIELD OF THE CLONES WERE CALCULATED USING THE STRAIN WITHOUT DELETION 8 &AB& AS REFERENCE !S IT SHOWS 3FTVMUT BOE EJTDVTTJPO
&IGURE THE TWO KNOCKOUT CLONES SECRETED LESS &AB INTO THE CULTURE MEDIUM WITH ALMOST NO &AB SECRETED BY THE CLONE $%3#
1.2
1
0.8
0.6
0.4 relative Fab yield
0.2
0 Fab2F5 ΔDes1 #C15 ΔDes1 #C28
&IGURE 2ELATIVE &AB & YIELD OBTAINED FOR THE TWO KNOCKOUTS WHEN COMPARED TO THE REFERENCE STRAIN WITHOUT THE DELETION
#LONE # WAS SELECTED AS THE WORKING ́$%3 STRAIN SO THAT &AB LEVELS WERE NOT SO CLOSE TO THE DETECTION LIMIT &URTHER STUDIES WITH THE ́$%3 STRAIN INCLUDED CULTURES WHERE CONTROL AND MUTANT STRAIN WERE CULTURED IN PRESENCE AND ABSENCE OF mUCONAZOLE 4HE AIM OF THESE CULTURES WAS TO DETERMINE WHETHER THE COMBINATION OF CERAMIDE DEPLETION AS IT SHOULD BE EXPECTED WHEN $%3 IS DELETED ;= AND ERGOSTEROL REDUCTION CAUSED BY ERGOSTEROL LEAD TO A BENElCIAL EFFECT ON PROTEIN SECRETION &AB LEVELS OF SECRETED FRACTION CYTOSOLIC FRACTION 3#& AND MEMBRANE FRACTION )-& WERE DETERMINED FOR ALL THE CULTURES IN ORDER TO OBSERVE THE DISTRIBUTION OF THE &AB WITHIN THE CELLULAR FRACTIONS &IGURE
Secreted SCF 1.8 1.8
1.6 1.6
1.4 1.4
1.2 1.2
1.0 1.0 relative Fab yield relative Fab yield 0.8 0.8
0.6 0.6 Fab2F5 ΔDes1 Fab2F5 + ΔDes1 + Fab2F5 ΔDes1 Fab2F5 + ΔDes1 + Fluco Fluco Fluco Fluco
A 3ECRETED FRACTION B 3OLUBLE #YTOSOLIC &RACTION
IMF 1.8
1.6
1.4
1.2
1.0 relative Fab yield 0.8
0.6 Fab2F5 ΔDes1 Fab2F5 + ΔDes1 + Fluco Fluco
C )NSOLUBLE -EMBRANE FRACTION
&IGURE &AB FRACTION DISTRIBUTION OF SHAKE mASK CULTIVATIONS OF THE REFERENCE STRAIN AND THE KNOCKOUT WITH OR WITHOUT mUCONAZOLE TREATMENT 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
4HE ALREADY DESCRIBED mUCONAZOLE EFFECT ON PROTEIN SECRETION WAS OBSERVED IN THE CULTURES WITH AROUND FOLD INCREASE ON &AB YIELD 4HESE CULTURES PRESENTED ALSO FOLD &AB IN THE SOLUBLE CYTOSOLIC FRACTION AND A REDUCTION OF &AB LEVELS IN THE INSOLUBLE FRACTION COMPARED TO THE REFERENCE STRAIN +NOCK OUT STRAIN DID NOT PRESENT PRODUCTION CHANGES IN TERMS OF SECRETED &AB BUT THE LEVELS IN THE CYTOSOLIC FRACTION WERE FOLD HIGHER &INALLY THE COMBINATION OF mUCONAZOLE TREATEMENT AND $%3 DELETION RESULTED ON LOWER &AB YIELDS IN ALL FRACTIONS 4ABLE SHOWS THE PERCENTAGE OF &AB DISTRIBUTED IN THE THREE WORKING FRACTIONS 3ECRETED #YTOSOLIC 3OLUBLE AND -EMBRANE )NSOLUBLE )N ALL CASES SECRETED &AB REPRESENTED MORE THAN OF THE DETECTED &AB ́$%3 STRAIN PRESENTED SLIGHTLY HIGHER INTRACELLULAR &AB LEVELS 3#& )-& COMPARED TO THE REFERENCE STRAIN AND mUCONAZOLE TREATEMENT LEAD TO A REDUCED INTRACELLULAR AMOUNTS OF &AB
4ABLE &AB DISTRIBUTION ON CELLULAR FRACTIONS
&AB& ጂ$%3 &AB& &LUCO ጂ$%3 &LUCO 3ECRETED q q q q 3#& q q q q )-& q q q q
)N ORDER TO DETERMINE PLASMA MEMBRANE mUIDITY ANISOTROPY ANALYSIS WERE CARRIED OUT !S ANISOTROPY VALUES ARE CELL GROWTH DEPENDENT CELL DENSITY WAS DETERMINED BEFORE AND ANISOTROPY WAS ANALYSED FOR CELLS WITH SIMILAR CELL DENSITY &IGURE .O SIGNIlCANT DIFFERENCES WERE OBSERVED BETWEEN CULTURES WHICH INDICATES THAT USING THIS METHODOLOGY SIMILAR mUIDITY RESULTS ARE OBTAINED !S MEMBRANE mUIDITY CAN BE CALCULATED AS THE INVERSE OF ANISOTROPY SLIGHTLY HIGHER mUIDITY COULD DElNE ́$%3 MEMBRANE
&IGURE 6ALUES OF SNISOTROPY AND /$ OF THE CULTURES
463 ,OPDLPVU TDSFFOJOH /NCE A CLONE WITH THE 352 DELETION ́352 WAS OBTAINED IT WAS CHARACTERISED IN TERMS OF CELL GROWTH &AB PRODUCTION AND COMPARED TO THE REFERENCE STRAIN &AB & AND THE ́$%3 STRAIN ALL OF THEM TREATED AND UNTREATED WITH mUCONAZOLE 4HE MUTANT STRAIN GREW SIMILARLY TO THE REFERENCE STRAIN AND A BENElCIAL EFFECT ON &AB SECRETION WAS OBSERVED &IGURE 2ELATIVE LEVELS OF SECRETED &AB WERE FOLD HIGHER THAN THE REFERENCE STRAIN WHICH COINCIDES WITH THE PREVIOUS RESULTS OBTAINED WHEN CULTURES WHERE TREATED WITH mUCONAZOLE 3FTVMUT BOE EJTDVTTJPO
2.0
1.6
1.2 relative Fab yield
0.8
0.4 Fab2F5 Fab2F5 ΔDes1 ΔDes1 ΔSur2 ΔSur2 Fluco Fluco Fluco
&IGURE &AB NORMALIZED PRODUCTIVITIES FOR ALL THE +/ STRAINS COMBINED WITH mUCONAZOLE TREATEMENT &LUCONAZOLE TREATMENT WAS ALSO STUDIED SO THAT THE TWO STRAINS WITH A DELETION IN THE SPHINGOLIPID PATHWAY COULD BE COMPARED WITH THE REFERENCE STRAIN WITH AND WITHOUT mUCONAZOLE 4HE AIM OF THIS CULTURES WAS TO CHARACTERISE THE STRAINS WHEN COMPONENTS OF THE SO CALLED vLIPID RAFTSv WERE ALTERED AND POSSIBLY AFFECT ON PROTEIN SECRETION )N &IGURE RELATIVE SECRETION LEVELS WERE PLOTTED AND THE PRESENCE OF mUCONAZOLE IN THE CULTURE MEDIUM REDUCED THE SECRETED &AB OF ́352 CELLS ́$%3 CELLS IMPROVED ITS SECRETION LEVELS AS IT WAS OBSERVED WITH THE REFERENCE STRAIN )T CAN BE CONCLUDED THAT PRESENCE OF INOSITOL CONTAINING SPECIES WITH AN ALTERED HYDROXYLATION PATTERN DUE TO THE 352 DELETION LEAD TO INCREASED PROTEIN SECRETION WHILE THE LACK OF CERAMIDES DID NOT HAVE SIGNIlCANT CHANGES ON PROTEIN SECRETION 4ERNES ET AL ;= CHARACTERISED 0 PASTORIS SPHINGOLIPID COMPOSITION SHOWING THAT ONLY MOL OF THE TOTAL SPHINGOLIPID CONTENT OF THE CELL CORRESPOND TO CERAMIDES ,ACK OF CELLULAR CERAMIDES AND SPECIlCALLY IN THE PLASMA MEMBRANE WOULD NOT AFFECT MEMBRANE MORPHOLOGY AND mUIDITY WHEREAS CHANGES ON )0# SPECIES THAT ARE THE MAJOR PART OF MEMBRANE SPHINGOLIPIDS COULD ALTER MEMBRANE PROPERTIES AND LEAD TO INCREASED PROTEIN SECRETION EVEN WHEN THE PERFORMED ANISOTROPY STUDIES DID NOT SHOW ANY mUIDITY DIFFERENCES !S NO mUIDITY CHANGES WERE OBSERVED WITH THE ANISOTROPY ANALYSIS THEY WERE NOT FURTHER USED TO CHARACTERISE CELL MEMBRANE PROPERTIES IN NEW CULTIVATIONS
%&4 PWFSFYQSFTTJPO TDSFFOJOH 'ENE $%3 WAS CLONED INTO P'!0(! AS PREVIOUSLY DESCRIBED AND CLONES WERE OBTAINED 4EN CLONES WERE SELECTED AND SCREENED FOR CELL GROWTH AND PROTEIN PRODUCTION IN SHAKE mASK CULTURES &IGURE SHOWS HOW EXCEPT FOR CLONE ALL CLONES GREW SLOWER THAN THE REFERENCE STRAIN #HANGES ON THE INTRACELLULAR EXTRACELLULAR DISTRIBUTION OF THE &AB PRODUCED WERE ALSO OBSERVED -OST OF THE OVEREXPRESSING CLONES PRESENTED MORE &AB IN THE INSOLUBLE MEMBRANE FRACTION THAN THE REFERENCE STRAIN WITH CLONE WITH MORE THAN THE OF THE &AB IN THIS FRACTION 2EGARDING THE SOLUBLE CYTOSOLIC FRACTION MOST CLONES PRESENTED ALMOST ALL OF ITS &AB IN THIS FRACTION WHICH REPRESENTS UP TO OF THE TOTAL &AB 4HESE RESULTS COULD INDICATE CHANGES IN CELL MEMBRANE COMPOSITION BY BEING INCREASING CERAMIDE CONTENT WHICH CAN AFFECT MEMBRANE PROPERTIES IN SUCH A WAY THAT CELLS CANNOT GROW PROPERLY AND THE PROTEIN SECRETION PATHWAY IS DAMAGED AND LOW AMOUNTS OF &AB IN THE SUPERNATANT ARE DETECTED &OR FURTHER STUDIES CLONE WAS SELECTED AS THE OVEREXPRESSING $%3 /% $%3 STRAIN 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
&IGURE 3CREENING $%3 OVEREXPRESSION CLONES
&3( PWFSFYQSFTTJPO TDSFFOJOH 3AME PROCEEDING AS WITH THE OVEREXPRESSED $%3 WAS USED TO SCREEN CLONES OVEREXPRESSING THE %2' GENE &IGURE SHOWS HOW IN THIS CASE CELL GROWTH FOR ALMOST ALL SELECTED CLONES WAS SLIGHTLY LOWER TO THE REFERENCE STRAIN EXCEPT FOR THE CLONES AND THAT REACHED SIGNIlCANT LOWER CELL DENSITIES AT THE END OF THE CULTURE
&IGURE 3CREENING %2' OVEREXPRESSION CLONES
&AB DISTRIBUTION AMONG CELLULAR FRACTIONS WAS ALSO STUDIED OF THE CULTURED CLONES APPEAR TO HAVE SIMILAR &AB DISTRIBUTION WITH OF THE &AB SECRETED AND LESS THAN IN THE )-& FRACTION 4OTALLY DIFFERENT &AB DISTRIBUTION WAS OBSERVED FOR CLONES AND WITH &AB SECRETED FRACTIONS CORRESPONDING TO LESS THAN #LONE PRESENTED HIGH AMOUNTS OF &AB IN THE MEMBRANE FRACTION AND ALMOST NO &AB SECRETED /VERALL RESULTS DEMONSTRATE THAT INCREASED LEVELS OF %RGP IN THE CELL DOES NOT HAVE A BENElCIAL IMPACT ON PROTEIN SECRETION BUT &AB SECRETION LEVELS AND DISTRIBUTION AMONG THE CELLULAR FRACTIONS WAS RATHER SIMILAR FOR MOST OF THE CLONES #LONE WAS USED AS OVEREXPRESSING %2' /% %RG STRAIN FOR FURTHER ANALYSIS 3FTVMUT BOE EJTDVTTJPO
463 PWFSFYQSFTTJPO TDSFFOJOH 352 GENE WAS OVEREXPRESSED USING THE SAME APPROACH AS WITH %2' AND $%3 GENES )N THIS CASE CELL GROWTH WAS SIGNIlCANTLY AFFECTED SO THAT ONLY FOUR POSITIVE CLONES WERE OBTAINED FOR CELL CHARACTERISATION &IGURE #ELLS WERE CULTURED UNTIL SIMILAR LEVELS OF CELL DENSITY TO THE REFERENCE STRAIN WERE ACHIEVED MEANING CELL CULTURES OF AROUND DAYS AND INDICATING A REDUCED SPECIlC GROWTH RATE FOR THIS CLONES 4HE AMOUNTS OF &AB PRESENT ON THE SUPERNATANT WERE QUANTIlED AND FOR OUT OF CLONES RELATIVE &AB YIELD IN THE SECRETED FRACTION WAS LOWER
3.0 14
2.5 12
10 2.0 ) 8 -1 1.5 6
1.0 DCW (g L relative Fab yield 4
0.5 2
0.0 0 2F5 #2i #3i #4i #11i
Clones overexpressing SUR2
&IGURE 3CREENING OF THE CLONES OVEREXPRESSING 352 'REY BARS REPRESENT THE RELATIVE &AB & YIELD COMPARED TO THE REFERENCE STRAIN %MPTY DOTS REPRESENT THE BIOMASS CONCENTRATION AT THE END OF THE CULTURE
&URTHER STUDIES OF CLONES OVEREXPRESSING 352 WERE NOT FEASIBLE BECAUSE CELLS COULD NOT BE MAINTAINED IN AGAR PLATES AND NO GROWTH WAS ACHIEVED USING GLYCEROL STOCKS
4HE POSSIBLE PRESENCE OF MORE SPHINGOLIPID CONTENT IN THE STRAINS WITH OVEREXPRESSED KEY GENES OF THE SPHINGOLIPID BIOSYNTHETIC PATHWAY CAN INDICATE A NEGATIVE EFFECT ON MEMBRANE COMPOSITION THAT COULD LEAD TO A LOSS OF CELL GROWTH WHICH IS NOT VIABLE FOR CELLS IN THE CASE OF INCREASED 3URP IN THE CELL )N THIS CASE SPHINGOLIPID MOIETIES CAN BE OVER HYDROXYLATED AND THESE CHANGES CAN AFFECT DRASTICALLY MEMBRANE MORPHOLOGY +IM ET AL ;= OVEREXPRESSED 352 GENE IN 3 CEREVISIAE AND OBTAINED ࣵ HIGHER CERAMIDE PRODUCTION THAN THE CONTROL STRAIN MEANING THAT AN INCREASE OF CERAMIDES IN THIS ORGANISM IS VIABLE 4HESE RESULTS ARE IN CONTRAST WITH OUR RESULTS WHERE 0 PASTORIS CELLS OVEREXPRESSING 352 RESULTED IN NON VIABLE CLONES 4HE REASON OF THIS NON VIABILITY CAN BE THE RESULT OF AN ORGANISM DEPENDENCE EG 0 PASTORIS NEEDS A TIGHTER BALANCE OF CERAMIDE TO SURVIVE THAN 3 CEREVISIAE OR DUE TO AN EFFECT OF THE RECOMBINANT PROTEIN PRODUCTION WHICH RESULTS IN CELL STRESS AND REQUIRES A MORE BALANCED MEMBRANE COMPOSITION TO SUSTAIN CELL VIABILITY
)BD PWFSFYQSFTTJPO TDSFFOJOH &OR (AC THE SEQUENCE OF THE TRANSCRIPTION FACTOR WAS CLONED INTO THE P05::,% VECTOR AND SELECTED FOR 'ENETICIN RESISTANCE #ELL GROWTH WAS NOT AFFECTED BECAUSE OF OVEREXPRESSION WITH ALL THE CLONES PRESENTING SIMILAR GROWTH RATE OR EVEN HIGHER THAN THE REFERENCE STRAIN )N TERMS OF SECRETED &AB A NEGATIVE EFFECT ON PROTEIN SECRETION WAS OBSERVED FOR MOST OF THE CLONES &IGURE 4HESE RESULTS ARE NOT CONSONANT WITH PREVIOUS RESULTS FROM 'UERFAL ET AL ;= 4HEY OBSERVED THAT WHEN (AC WAS COEXPRESSED UNDER THE CONTROL OF THE '!0 PROMOTER NO EFFECT 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
Fab 2F5 #A1 #B1 #B2 #B3 #B4 #B5 #B6 1.2 10.0
9.0 1.0 8.0
7.0 ) 0.8 -1 6.0
0.6 5.0 DCW (g L
relative Fab yield 4.0 0.4 3.0
2.0 0.2 1.0
0.0 0.0
&IGURE 3CREENING (!# OVEREXPRESSION CLONES "LACK BARS REPRESENT THE RELATIVE &AB & YIELD COMPARED TO THE REFERENCE STRAIN %MPTY DOTS REPRESENT THE BIOMASS CONCENTRATION AT THE END OF THE CULTURE
ON RECOMBINANT PROTEIN PRODUCTION WAS OBSERVED WHILE WHEN IT WAS COEXPRESSED UNDER THE METHANOL INDUCIBLE !/8 PROMOTER IT AFFECTED THE RECOMBINANT PROTEIN PRODUCTION 4HE EFFECT COULD BE POSITIVE OR NEGATIVE IN A PROTEIN DEPENDENT WAY ! NEGATIVE EFFECT RATHER THAN NO EFFECT WAS OBSERVED FOR MOST OF THE OBTAINED CLONES IN THIS STUDY #LONE " WAS SELECTED FOR FURTHER ANALYSIS AS OVEREXPRESSING (AC STRAIN /% (AC 4HIS CLONES WAS SELECTED AS ITS GROWTH RATE IS SIMILAR TO THE REFERENCE STRAIN AND THEIR PRODUCTION LEVELS WERE LOW REPRESENTING THE MAJOR PART OF THE CLONES BUT STILL NOT CLOSE TO THE DETECTION LIMIT OF THE QUANTIlCATION METHOD
-JQJE DPNQPTJUJPO )N ORDER TO HAVE AN INITIAL OVERVIEW OF THE LIPID PATTERN OF THE MUTANT STRAINS GENERATED SHAKE mASK CULTURES IN 90$ WERE PERFORMED FOLLOWED BY PELLET HOMOGENISATION AND LIPID ANALYSIS !LTHOUGH A CONSTANT CELL DENSITY WAS OBTAINED FOR ALL DE CULTURED STRAINS LIPID COMPOSITION DEPENDS ON CELL GROWTH AND ENVIRONMENT COMPOSITION SO THAT SMALL CHANGES IN THE CULTURE ENVIRONMENT COULD LEAD TO CHANGES ON THE LIPID PATTERN 3TEROLS FATTY ACIDS PHOSPHOLIPIDS AND NON POLAR LIPIDS WERE QUANTIlED
4UFSPMT 4HE TOTAL STEROL CONTENT &IGURE OF THE ANALYSED STRAINS mUCTUATED UP TO FOLD HIGHER /% (AC OR LOWER ́352 THAN THE REFERENCE STRAIN
20
16 -1
12 total prot
mg 8 sterol g μ 4
0 Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1
&IGURE 4OTAL STEROL CONTENT OF SHAKE mASK CULTURES 3FTVMUT BOE EJTDVTTJPO
)N TERMS OF SINGLE STEROL CONTENT &IGURE ERGOSTEROL WAS THE MAJOR STEROL IN ALL CELL HOMOGENATES WHILE STEROL INTERMEDIATES WERE PRESENT IN SMALL AMOUNTS ! REDUCTION OF ERGOSTEROL WAS OBSERVED FOR ́352 STRAIN WHILE OTHER MUTANT STRAINS PRESENT JUST A SMALL REDUCTION OF ERGOSTEROL CONTENT COMPARED WITH THE REFERENCE ONE /THER STEROL INTERMEDIATES PRESENTING LEVELS ABOVE THE DETECTION LIMIT WERE ZYMOSTEROL EPISTEROL -ETHYLZYMOSTEROL WERE SOME DIFFERENCES WERE OBSERVED :YMOSTEROL WAS FOLD INCREASED IN THE OVEREXPRESSING (AC STRAIN WHILE ́352 AND /% $ES STRAINS DECREASED ZYMOSTEROL LEVELS BY AND RESPECTIVELY 2EDUCED LEVELS OF METHYLZYMOSTEROL WERE ALSO OBSERVED FOR ́352 AND /% $ES STRAINS
Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1 12
-1 10
8 6
gsterol mg total prot 4 μ
2
0 zymosterol ergosterol 4-methylzymosterol episterol
&IGURE 3TEROL CONTENT OF SHAKE mASK CULTURES
'BUUZ BDJET &IGURE SHOWS HOW OLEIC ACID AND LINOLENIC ACID WERE THE TWO MAJOR COMPONENTS OF THE CELL HOMOGENATES FOR ALL THE STRAINS 4HESE RESULTS ARE CONSISTENT WITH THE ONES PREVIOUSLY DESCRIBED BY 'RILLITSCH ET AL ;= WHERE THEY ANALYSED LIPID COMPOSITION OF THE PLASMA MEMBRANE AND THE WHOLE CELL HOMOGENATE OF THE STRAIN #"3
Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 45 40 35 30 25 20 % total fatty acids 15 10 5 0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
&IGURE &ATTY ACID CONTENT OF SHAKE mASK CULTIVATIONS
,EVELS OF PALMITIC ACID # PALMITOLEIC ACID # STEARIC ACID # AND LINOLENIC ACID # WERE QUITE SIMILAR AMONG THE STRAINS WHILE SOME CHANGES WERE PRESENT FOR OLEIC ACID # AND LINOLEIC ACID # WHERE ́352 STRAIN PRESENTED INCREASED LINOLEIC ACID AND A REDUCTION OF OF OLEIC ACID )T SEEMS THAT ́352 STRAIN COMPENSATES THE REDUCTION OF OLEIC ACID BY INCREASING THE LEVEL OF LINOLENIC ACID 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
1IPTQIPMJQJET 4OTAL PHOSPHOLIPID 0, LEVELS WERE SLIGHTLY DECREASED FOR THE $%3 OVEREXPRESSING STRAIN AND (AC OVEREXPRESSING ONE 3IMILAR LEVELS WERE OBTAINED FOR THE REST OF THE CLONES &IGURE
0.25
0.20 -1
0.15 total prot mg
PL 0.10 mg
0.05
0.00 Fab 2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1
&IGURE 4OTAL PHOSPHOLIPID CONTENT OF SHAKE mASK CULTIVATIONS
&IGURE SHOWS INDIVIDUAL PHOSPHOLIPID COMPOSITION OF THE HOMOGENATES )T CAN BE OBSERVED THAT PHOSPHATIDYLCHOLINE 0# WAS THE MAJOR PHOSPHOLIPID FOR ALL THE STRAINS CORRESPONDING TO OF THE TOTAL 0, CONTENT 4HE PRESENCE OF PHOSPHATIDYLETHANOLAMINE 0% WAS AROUND BEING THE SECOND MAJOR 0, IN ALL CELL HOMOGENATES 0, AMOUNTS WERE SIMILAR TO THOSE PREVIOUSLY DESCRIBED FOR YEAST BY +LUG ET AL ;= 0# LEVELS WERE LOWER IN THE ́352 STRAIN AND THE OVEREXPRESSED %2' $ECREASED AMOUNTS OF 0# IN THESE STRAINS WERE COMPENSATED BY THE CELL BY INCREASED AMOUNTS OF 0) FOR ́352 STRAIN HIGHER AND 03 HIGHER AND LYSO 0! FOLD HIGHER FOR THE STRAIN COEXPRESSING %2' )T WAS ALSO RELEVANT THE ABSENCE OF 0! IN THE STRAIN OVEREXPRESSING $%3 SINCE 0! PLAYS AN IMPORTANT ROLE IN LIPID METABOLISM BY BEING A CENTRAL METABOLITE IN THE DE NOVO SYNTHESIS OF PHOSPHOLIPIDS 4HE ABSENCE OF THIS PHOSPHOLIPID CAN BE DUE TO THE FACT THAT ITS USED FOR THE SYNTHESIS OF OTHER PHOSPHOLIPIDS NOT BEING ACCUMULATED INTO THE CELL
Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1
60
50
40
30 % total phospholipids 20
10
0 PC PA PI PS Lyso-PL PE CL DMPE
&IGURE 0HOSPHOLIPID CONTENT OF SHAKE mASK CULTIVATIONS
/POQPMBS -JQJET .ON POLAR LIPIDS ARE COMPOSED BY STEROL ESTERS 3% TRIACYLGLYCEROLS 4!' AND FREE STEROLS &3 MAINLY ERGOSTEROL 4!'S AND 3%S SERVE AS INTRACELLULAR STORAGE MOLECULES FOR STEROLS FREE FATTY 3FTVMUT BOE EJTDVTTJPO
ACIDS AND DIACYLGLYCEROLS $!'S 4HE AMOUNTS OF 3%S WERE LOWER FOR ALL MUTANT STRAINS COMPARED TO THE REFERENCE STRAIN &IGURE B ! FOLD REDUCTION WAS PRESENT ON THE STRAIN COEXPRESSING $%3 ,EVELS OF FREE ERGOSTEROL WERE ALSO REDUCED FOR ALL THE MUTANT STRAINS &IGURE C WITH LEVELS RANGING FROM 4RIACYLGLYCEROL &IGURE A AMOUNTS WERE ALSO LOWER IN ALL THE STRAINS FOLD REDUCTION WAS PRESENT IN THE ́$%3 STRAIN FOLD LOWER FOR ́352 AND FOLD LOWER IN THE OVEREXPRESSIONG $%3 STRAIN 'LOBALLY TOTAL LEVELS OF NON POLAR LIPIDS IN CELL HOMOGENATES HAD A REDUCTION IN ALL MUTANT STRAINS WHEN COMPARED TO THE REFERENCE STRAIN
1000
-1 800 total prot 600 mg
TGA
mg 400
200
0 Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1
A 4!' CONTENT OF SHAKE mASK CULTIVATIONS
20
16 -1 12 total prot
mg 8 SE g μ
4
0 Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1
B 3% CONTENT OF SHAKE mASK CULTIVATIONS
30
25 -1
total prot 20 mg 15 Ergosterol
mg 10
5
0 Fab2F5 ΔDes1 ΔSur2 OEDes1 OEErg11 OEHac1
C &REE 3TEROL CONTENT OF SHAKE mASK CULTIVATIONS
&IGURE .ON POLAR LIPID CONTENT OF SHAKE mASK CULTIVATIONS 4NBMMTDBMF DVMUJWBUJPOT GPS TUSBJO DIBSBDUFSJTBUJPO
$PODMVTJPOT
s ! RATIO OF mUCONAZOLE PER BIOMASS OF G&LUCO G$#7 ï WAS DElNED AS THE OPTIMAL FOR &AB SECRETION ! mUCONAZOLE RATIO PER BIOMASS WAS DElNED AS THE OPTIMAL TO OBTAIN MAXIMAL
SECRETION OF &AB INTO THE CULTURE MEDIUM 4HIS RATIO WAS G&LUCO G$#7 ï
s /PTIMISED DISRUPTION PARAMETERS WERE DElNED IN ORDER TO DETECT HIGH QUANTITIES OF &AB CORRESPONDING TO THE INTRACELLULAR FRACTION 4WO PASSES AT KBAR WERE THE BEST DISRUPTION SETTINGS AND THE USE OF A BUFFER CONTAINING GLYCEROL M- (%0%3P( M- .A#L AND M- 0-3& COMBINED WITH #(!03 DURING H OVERNIGHT WAS SELECTEDAS THE BEST COMBINATION TO EXTRACT THE &AB FROM THE INSOLUBLE FRACTION ! NEW "UFFER DETERGENT AND INCUBATION TIMES WERE STABLISHED AS THE OPTIMAL FOR &AB RECOVERY FROM THE INTRACELLULAR MEMBRANE FRACTION s +NOCKOUT CLONES OF DELETED $%3 AND 352 WERE CULTURED AND AND THE EFFECT ON CELLULAR GROWTH AND &AB PRODUCTION WERE DETERMINED
s #LONES WITH OVEREXPRESSED $%3 %2' AND (!# WERE CULTURED AND THE EFFECT ON &AB PRODUCTION AND CELLULAR GROWTH WAS DETERMINED ! CLONE REPRESENTING THE MAJOR PART OF THE CLONES OBTAINED WAS SELECTED AS A STRAIN REPRESENTATIVE TO BE FURTHER USED
s 352 OVEREXPRESSION RESULTED INTO NON VIABLE CLONES
s -AJOR DIFFERENCES COMPARED TO REFERENCE STRAIN IN TERMS OF LIPID COMPOSITION WERE OBSERVED FOR THE CLONE ́352 IN SHAKE mASK CULTURES ,IPID COMPOSITION OF ALL CLONES WAS DETERMINED AND SOME DIFFERENCES WERE OBSERVED MAINLY FOR THE ́352 STRAIN
#JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
*OUSPEVDUJPO #JPSFBDUPS DVMUJWBUJPOT 5NLIKE SMALL SCALE SHAKE mASKS BIOREACTORS PROVIDE A SYSTEM TO PERFORM CELL CULTURES IN A WELL MONITORED AND CONTROLLED ENVIRONMENT 4HIS SYSTEM ALLOWS MAINTAINING CONTROLLED MOST OF THE CULTURE CONDITIONS SUCH AS TEMPERATURE P( DISSOLVED OXYGEN OR VOLUME /NE OF THE MAIN DIFFERENCES FROM SMALL SCALE CULTIVATION SYSTEMS IS THAT THE OXYGEN TRANSFER RATE CAN BE CONTROLLED IN A BIOREACTOR ALLOWING ACHIEVING HIGHER CELL DENSITIES AND PRODUCTION RATES $EPENDING ON THE MODE OF OPERATION BIOREACTORS CAN BE CLASSIlED AS BATCH FED BATCH OR CONTINUOUS 4HE PREFERENCE OF 0 PASTORIS FOR RESPIRATORY GROWTH ALLOWS REACHING VERY HIGH CELL DENSITIES WHEN CULTURED IN A CONTROLLED ENVIRONMENT OF A BIOREACTOR WHICH CAN IMPROVE OVERALL PROTEIN YIELDS 3OME GENERAL GUIDELINES TO PERFORM BIOREACTOR CULTURES WITH THIS ORGANISM ARE AVAILABLE FROM )NVITROGEN KWWSV WRROVWKHUPRILVKHUFRPFRQWHQWVIVPDQXDOV SLFKLDIHUPBSURWSGI "IOREACTOR CULTIVATIONS ARE NORMALLY PERFORMED IN A FED BATCH MODE WITH TWO PHASES FOR CONSTITUTIVE EXPRESSION EG WHEN USING THE '!0 PROMOTER OR THREE PHASES FOR INDUCIBLE EXPRESSION ;= 'LYCEROL IS THE PREFERRED CARBON SOURCE FOR THE BATCH PHASE ;= AS IT IS A NON FERMENTABLE CARBON SOURCE THAT ALLOWS HIGH SPECIlC GROWTH RATES 4HE OPTIMUM GROWTH TEMPERATURE FOR 0PASTORIS HAS BEEN DElNED AS # HOWEVER IT HAS BEEN ESTABLISHED THAT LOWER CULTIVATION TEMPERATURES EG # CAN IMPROVE THE YIELDS OF RECOMBINANT PROTEIN BY DECREASING FOLDING STRESS AND ENABLING MORE EFlCIENT PROTEIN SECRETION WITHOUT HAMPERING CELL GROWTH ; = 0 PASTORIS IS CAPABLE OF GROWING ACROSS A RELATIVELY BROAD P( RANGE /PTIMUM P( IS CRITICAL FOR PROTEIN FORMATION AND STABILITY 'ENERALLY THE WORKING P( RANGES BETWEEN AND ;=
$IFNPTUBU DVMUJWBUJPOT #HEMOSTAT CULTURES PROVIDE A METHOD TO GROW MICROORGANISMS IN CONSTANT CAREFULLY CONTROLLED CONDITIONS AS IT ENABLES STRICT CONTROL OF CULTURE P( DISSOLVED OXYGEN CONCENTRATION AND TEMPERATURE ALL OF WHICH ARE KNOWN TO AFFECT CELL PHYSIOLOGY 4HE NUTRIENT MEDIUM CONTAINS SATURATING CONCENTRATIONS OF ALL OF THE GROWTH COMPONENTS WITH THE EXCEPTION OF ONE THE CONCENTRATION OF WHICH DETERMINES BOTH CELL DENSITY AND THE GROWTH RATE
#JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
)N CHEMOSTAT CULTURES THE DILUTION RATE $ IS DElNED AS THE RATIO OF THE mOW RATE OF THE INGOING MEDIUM F , Hዅኻ AND THE CULTURE VOLUME 6 , 7HEN THE CULTURE VOLUME IS KEPT CONSTANT BY CONTINUOUS REMOVAL OF CULTURE BROTH A STEADY STATE IS REACHED IN WHICH THE SPECIlC GROWTH RATE Hዅኻ IS EQUAL TO THE DILUTION RATE WHEN $ IS SMALLER THAN THE MAXIMUM SPECIlC GROWTH RATE "ECAUSE CONCENTRATIONS OF ALL METABOLITES AND SUBSTRATES IN STEADY STATE CHEMOSTAT CULTURES ARE CONSTANT IN TIME THESE CULTURES PROVIDE A mEXIBLE AND REPRODUCIBLE PLATFORM FOR STUDIES ON MICROBIAL PHYSIOLOGY AND GENE EXPRESSION ALLOWING A GOOD METHODOLOGY TO PERFORM COMPARISONS BETWEEN DIFFERENT ENVIRONMENTAL CONDITIONS OR BETWEEN DIFFERENT STRAINS AT AN IDENTICAL GROWTH RATE ; =
'FE#BUDI DVMUJWBUJPOT &ED BATCH CULTIVATIONS ALLOW HIGH BIOMASS AND PRODUCT CONCENTRATIONS 0ROCESSES ARE SHORTER AND STRAIN STABILITY AND CONTAMINATION ARE MUCH LESS OF AN ISSUE THAN IN CONTINUOUS CULTURES 4HESE FEATURES MAKE THEM SUITABLE TO PRODUCE RECOMBINANT PROTEINS FOR INDUSTRIAL USES )N A FED BATCH FERMENTATION SYSTEM AN INITIAL BATCH PHASE IS USED FOR FAST INITIAL GROWTH OF BIOMASS AND A STARTING POINT FOR A CONTROLLED FEED TO THE CULTURE 4HE FEED WILL OFTEN CONSIST OF CONCENTRATED MEDIUM THAT WILL BE APPLIED IN SUCH A WAY THAT THE CARBON SOURCE IS LIMITED TO MAINTAIN AN OPTIMAL GROWTH RATE IN A PSEUDO STEADY STATE CONDITION WHICH IS SUITED FOR HETEROLOGOUS PROTEIN PRODUCTION "Y CONTINUING THE FEED THE TOTAL VOLUME BIOMASS AND PRODUCT CONCENTRATIONS INCREASE TO HIGH LEVELS /XYGEN UPTAKE AND HEAT PRODUCTION ALSO INCREASE UNTIL A POINT WHERE THESE PARAMETERS CAN NO LONGER BE CONTROLLED TO THE OPTIMUM LEVEL
:FBTU MJQJE DPNQPTJUJPO ,IPID METABOLISM HAS BECOME AN INCREASING FOCUS OF ATTENTION NOT ONLY BECAUSE OF THEIR STRUCTURAL AND FUNCTIONAL ROLE AS MEMBRANE COMPONENTS BUT ALSO FOR THEIR INTERPLAY WITH MANY IMPORTANT METABOLIC PROCESSES ;= 4HE GOAL OF LIPIDOMICS IS TO DElNE AND QUANTITATE ALL THE LIPID MOLECULAR SPECIES IN A CELL BUT THAT IS COMPLICATED BY THE HIGH NUMBER OF COMBINATIONS POSSIBLE WITH THE LARGE NUMBER OF KNOWN FATTY ACIDS THAT CAN BE OCCUPYING A POSITION ;= 4HE FULL LIPID CONTENT OF CELLS COMPRISES SEVERAL MOLECULAR LIPID SPECIES PRODUCED BY A METABOLIC NETWORK THAT INTERCONNECTS THE LIPID METABOLISM OF FATTY ACIDS GLYCEROPHOSPHOLIPIDS GLYCEROLIPIDS SPHINGOLIPIDS AND STEROL LIPIDS ; = #HANGES ON LIPID METABOLISM CAN AFFECT LIPID COMPOSITION OF THE MEMBRANE CHANGING ITS mUIDITY ; n= AND OTHER PHYSIOLOGICAL TRAITS ; n = SOME OF WHICH COULD ULTIMATELY FAVOUR RECOMBINANT PROTEIN SECRETION 5P TO DATE LIPID COMPOSITION OF 0 PASTORIS HAVE BEEN LITTLE INVESTIGATED 4HE COMPOSITION OF 0 PASTORIS ORGANELLES SUCH AS PLASMA MEMBRANE ;= PEROXISOMES ;= MITOCHONDRIA ;= LIPID DROPLETS ;= AND ENDOPLASMIC RETICULUM ;= HAVE BEEN CHARACTERISED &URTHERMORE LIPID CHANGES WITH REGARD TO CARBON SOURCE EFFECT ;= HAVE BEEN RECENTLY DESCRIBED (OWEVER SUCH FUNDAMENTAL STUDIES WERE PERFORMED EXCLUSIVELY USING WILD TYPE STRAINS )N THIS WORK THE LIPID COMPOSITION OF A RECOMBINANT 0 PASTORIS STRAIN IS USED AS A REFERENCE STRAIN AND LIPID CHANGES ARE DESCRIBED FROM ENVIRONMENTAL ALTERATIONS AS WELL AS GENETIC MODIlCATIONS ON LIPID BIOSYNTHETIC PATHWAYS IN ORDER TO DETERMINE LIPID CHANGES LEADING TO A SECRETION IMPROVEMENT *OUSPEVDUJPO
:FBTU MJQJE BOBMZTJT )NDEPENDENTLY OF THE SUBSEQUENT ANALYSIS THE lRST STEP OF LIPID ANALYSIS IS NORMALLY THE EXTRACTION OF LIPIDS "ECAUSE LIPIDS ARE WATER INSOLUBLE THEIR EXTRACTION REQUIRES A COMBINATION OF POLAR AND NON POLAR ORGANIC SOLVENTS 4HE GOAL OF THE EXTRACTION PROCEDURE GENERALLY IS A QUANTITATIVE RECOVERY OF ALL THE DIFFERENT LIPID CLASSES 4HE COMMONLY USED METHODS FOR TOTAL LIPID RECOVERY ARE &OLCH ET AL ;= AND ITS MODIlED VERSION BY "LIGH AND $YER ;= 4HE METHOD OF &OLCH INVOLVES TWO SUCCESSIVE STEPS HOMOGENISATION OF TISSUES AND EXTRACTION WITH CHLOROFORMMETHANOL FOLLOWED BY lLTRATION AND REMOVAL OF NON LIPID CONTAMINANTS BY WASHING WITH POTASSIUM CHLORIDE +#L SOLUTION OBTAINING A LAYER CONTAINING THE LIPIDS ;= )N ORDER TO EXTRACT LIPIDS FROM WHOLE YEAST CELLS A lRST STEP TO BREAK OPEN THE YEAST CELL WALL IS INCLUDED WHICH USUALLY CONSISTS ON THE DISINTEGRATION IN THE PRESENCE OF GLASS BEADS ! TYPICAL LIPID EXTRACT CONTAINS POLAR AND NON POLAR LIPIDS 4HE MAIN COMPONENTS OF THE POLAR LIPIDS ARE GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS 4HE MAIN COMPONENTS OF THE NON POLAR LIPIDS ARE FREE FATTY ACIDS &! DIACYLGLYCEROLS $!' TRIACYLGLYCEROLS 4' STEROLS AND STERYL ESTERS 3% ;= $IFFERENT METHODS ARE EMPLOYED FOR THE SEPARATION AND ANALYSIS OF DIFFERENT CLASSES OF LIPIDS -OREOVER SEVERAL TECHNIQUES OF LIPID ANALYSIS EXIST ALL OF THEM PRESENTING THEIR ADVANTAGES AND DRAWBACKS AS IT IS REPORTED IN 4ABLE 4HE COMPLEXITY OF NATURAL LIPID EXTRACTS IS SUCH THAT IT IS
4ABLE !DVANTAGES AND DRAWBACKS OF THE MOST IMPORTANT TECHNIQUES OF LIPID ANALYSIS !DAPTED FROM ;=
!DDITIONAL 0RINCIPLE !DVANTAGES $RAWBACKS COMMENTARIES 3EPARATION BY POLARITY )NEXPENSIVE FAST OXIDATION OF UNSATURATED 4HIN LAYER DIFFERENCES ON A SEPARATION OF COMPLEX LIPIDS WHEN 4,# STORED INITIAL METHOD FOR CHROMATOGRAPHY vSTATIONARY PHASEv SILICA MIXTURES DIFFERENT FOR A WHILE LIMITED COMPLEX MIXTURES 4,# GEL STAINING PREPARATIVE APPLICATIONS (IGH QUALITY SEPARATIONS (IGH PERFORMANCE 3EPARATION UNDER HIGH MORE TIME CONSUMING 2OUTINE METHOD IN APPLICABLE ON LIQUID PRESSURE BY ELUTION WITH AND EXPENSIVE THAN 4,# MANY LABORATORIES PREPARATIVE SCALE CHROMATOGRAPHY DIFFERENT SOLVENTS ON A DIFlCULT DETECTION OF #OMPOSITION MOBILE POSSIBILITY OF COUPLING (0,# vSTATIONARY PHASEv SATURATED LIPIDS PHASE CHALLENGING WITH -3 'AS 3EPARATION OF VOLATILE &ATTY ACID ANALYSIS /NLY VOLATILE COMPOUNDS 7IDELY APPLIED FOR CHROMATOGRAPHY COMPOUNDS ON A CARRIER !UTOMATED DEVICES CAN BE ANALYSED FATTY ACYL LIPIDS '# '# -3 GAS $ETECTION BY -3 COMMERCIALLY AVAILABLE $ERIVATISATION IS REQUIRED -!,$) AND %3) -3 3OFT IONISATION ENABLE LIPID HIGHLY SENSITIVE DIRECT ION SUPPRESSION %3) MOST FREQUENTLY MASS CHARACTERISATION WITHOUT ANALYTE DETECTION IMPURITIES AFFECT USED 3CREENING OF SPECTROMETRY MAJOR ANALYTE SIMPLE HANDLING SIGNIlCANTLY LIPID TISSUES POSSIBLE FRAGMENTATION $IFFERENCES IN ELECTRON #OMPLEX SPECTRA DENSITIES LEAD TO !LL LIPIDS DETECTABLE OBTAINED LIMITED .-2 CAN BE USED DIFFERENT CHEMICAL SHIFTS ï(ïǓ# .-2 INTERACTION STUDIES CAN SENSITIVITY NEED UNDER IN VIVO OF THE OBSERVED NUCLEUS BE EASILY PERFORMED DEUTERATED SOLVENT CONDITIONS WITHIN A GIVEN EXPENSIVE EQUIPMENT COMPOUND $IFFERENCES IN ELECTRON DIRECT ABSOLUTE ONLY LIPIDS WITH DENSITIES LEAD TO DETERGENTS REQUIRED QUANTITATION POSSIBLE PHOSPHORUS DETECTABLE Ǔï0 .-2 DIFFERENT CHEMICAL SHIFTS TO SUPPRESS 0, DIFFERENTIATION OF LIMITED SENSITIVITY OF THE OBSERVED AGGREGATION ISOMERIC LIPIDS EXPENSIVE EQUIPMENT COMPOUND
RARELY POSSIBLE TO CLAIM THAT ALL THE LIPID CLASSES OF A SAMPLE CAN BE SEPARATED IN ONE OPERATION )T IS THEREFORE OFTEN WORTHWHILE TO BE ABLE TO ISOLATE DISTINCT SIMPLE LIPID FRACTIONS FOR FURTHER ANALYSIS ;= #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
"Y vSHOTGUN LIPIDOMICSv WHERE LIPID EXTRACTS ARE DIRECTLY SUBJECTED TO -3 %JSING ET AL ;= PROVIDED A SYSTEM TO ANALYSE ࣵOFTHE3 CEREVISIAE LIPIDOME BY MONITORING LIPID SPECIES CONSTITUTING MAJOR LIPID CLASSES AND SEVERAL BIOSYNTHETIC INTERMEDIATES &URTHERMORE THE ANALYSIS REQUIRED LITTLE SAMPLE COMPARED TO PREVIOUS METHODS
5IJO -BZFS $ISPNBUPHSBQIZ 4HIN ,AYER CHROMATOGRAPHY 4,# HAS BEEN USED TO QUANTIFY LEVELS OF NON POLAR LIPIDS USING A $ 4,# AS WELL AS PHOSPHOLIPID CONTENT BY A $ 4,# )T IS A COMMON TECHNIQUE AS IT IS A SIMPLE VERSATILE AND HIGHLY SENSITIVE AND IT CAN BE USED AS A DETECTIVE OR QUANTITATIVE TECHNIQUE )T IS BASED ON THE SEPARATION OF LIPIDS USING ALUMINIUM FOIL SHEETS COATED WITH SILICA GEL ADSORBENT 3ILICA GEL IS A POLAR ADSORBENT SO THAT POLAR LIPIDS ARE MORE TIGHTLY ADSORBED THEN THE NON POLAR ONES $IFFERENT SOLVENT SYSTEMS ARE USED TO SEPARATE THE LIPID CLASS OF INTEREST /NCE SEPARATED PLATES MUST BE STAINED IN ORDER TO VISUALISE THE POSITION OF THE LIPIDS WHERE THE MOST COMMON USED IS IODINE VAPOUR &OR THE SEPARATION AND DETECTION OF NEUTRAL LIPIDS 4,# IS A SUITABLE TECHNIQUE BECAUSE THE EQUIPMENT USED IS VERY SIMPLE ALTHOUGH IS GENERALLY TIME CONSUMING AND DOES NOT DETERMINE MOLECULAR SPECIES LIKE $!' /N THE OTHER HAND $ 4,# HAS BEEN USED TO ANALYSE PHOSPHOLIPIDS AS IT IS A POWERFUL TOOL TO SEPARATE COMPLEX LIPID MIXTURES (OWEVER SEVERAL DISADVANTAGES LIMIT THEIR APPLICATIONS AS ONLY A SINGLE SAMPLE CAN BE INVESTIGATED BEING MUCH MORE TIME CONSUMING THAN $ 4,# AND SIMULTANEOUS APPLICATION OF STANDARDS IS IMPOSSIBLE .ORMALLY A SOLVENT MIXTURE WITH HIGH ELUTION POWER IS USED lRST FOLLOWED BY ELUENTS WITH LOWER ELUTION POWER CONCENTRATING THE ANALYTE IN EACH STEP ;= 4,# SEPARATION IS OF LOW RESOLUTION AND DOES NOT PROVIDE MOLECULAR INFORMATION OF THE DIVERSITY OF FATTY ACYL COMPOSITIONS
(BT $ISPNBUPHSBQIZ 'AS CHROMATOGRAPHY '# AND GAS CHROMATOGRAPHY MASS SPECTROMETRY '# -3 HAS BEEN USED TO QUANTIFY FATTY ACIDS AND STEROLS RESPECTIVELY '# IS A FORM OF PARTITION CHROMATOGRAPHY IN WHICH THE MOBILE PHASE IS A GAS AND THE STATIONARY PHASE IS A LIQUID ! SAMPLE IS INJECTED INTO THE GAS PHASE WHERE IT IS VOLATILISED AND PASSED ONTO THE LIQUID PHASE WHICH IS HELD IN SOME FORM IN A COLUMN COMPONENTS SPEND DIFFERENT TIMES IN THE MOBILE PHASE AND THE STATIONARY PHASE DEPENDING ON THEIR RELATIVE AFlNITIES FOR THE LATTER AND EMERGE FROM THE END OF THE COLUMN EXHIBITING PEAKS OF CONCENTRATION IDEALLY WITH A 'AUSSIAN DISTRIBUTION ;= "EFORE THE FATTY ACID COMPONENTS OF LIPIDS CAN BE ANALYSED BY '# IT IS NECESSARY TO CONVERT THEM TO LOW MOLECULAR WEIGHT NON POLAR DERIVATIVES '# -3 IS MOST FREQUENTLY USED TO QUANTIFY STEROLS IN EXTRACTS 4HE SEPARATION OF STEROLS IN '# SYSTEMS DEPENDS ON THE POLARITY AND MOLECULAR WEIGHT OF THE MOLECULE $ERIVATISATION TO TRIMETHYLSILYL 4-3 ETHERS IS REQUIRED IN ORDER TO PROPERLY ANALYSE THE STEROLS AS IT ENABLES MORE EFlCIENT VOLATILISATION AND MORE SENSITIVE ANALYSIS ;= -ASS SPECTROMETERS CAN BE COUPLED TO '# TO MONITOR MOLECULAR WEIGHT OF THE COMPONENTS AS WELL AS THE PURITY OF THE SAMPLES ;= 3FTVMUT BOE EJTDVTTJPO
3FTVMUT BOE EJTDVTTJPO &TUBCMJTIJOH IZQPYJD DPOEJUJPOT JO DIFNPTUBU DVMUVSFT &IRST HYPOXIC CHEMOSTAT STUDIES OF 0 PASTORIS REVEALED THAT STRINGENT HYPOXIA LEADS TO BIOREACTOR WASH OUT ;= )N ORDER TO ESTABLISH LESS SEVERE WORKING HYPOXIC CONDITIONS IE ALLOWING FOR A RESPIROFERMENTATIVE METABOLISM THAT WOULD PREVENT THIS PHENOMENON 0 PASTORIS PRODUCING &AB & WAS GROWN IN GLUCOSE LIMITED CHEMOSTAT CULTURES AT A DILUTION RATE OF Hዅኻ USING DIFFERENT PERCENTAGES OF OXYGEN IN THE INLET GAS RANGING FROM , MINዅኻ TO , MINዅኻ &IGURE "IOMASS CONCENTRATION AND GLUCOSE ETHANOL AND ARABITOL QUANTITIES WERE MEASURED IN EACH STEADY STATE
Ethanol DCW Specific Fab production rate 30.0 0.10
0.09
) 25.0 -1 0.08
0.07 ) 20.0 -1 h
0.06 -1 X ) / DCW (g L g
-1 15.0 0.05 Fab 0.04 (mg p
10.0 q 0.03
Ethanol (g L 0.02 5.0 0.01
0.0 0.00 0 0.2 0.4 0.6 0.8 1 1.2
Air Flow Inlet (L min-1)
&IGURE 3PECIlC &AB & PRODUCTION RATES WERE DETERMINED AT DIFFERENT OXYGEN MOLAR FRACTIONS BU CHANGING THE AIR mOW IN THE INLET GAS %THANOL PRODUCTION WAS MONITORED AS THE BY PRODUCT OF HYPOXIC CONDITIONS
4HE DESIRED WORKING HYPOXIC CONDITION WAS DElNED AS THE LOWER AIR mOW THAT PERMITTED A STABLE CELL CONCENTRATION IE NO WASHOUT IN THE BIOREACTOR WHILE SIGNIlCANT AMOUNTS OF ETHANOL AND ARABITOL WERE PRESENT IN THE MEDIA THEREBY INDICATING RESPIROFERMENTATIVE METABOLIC CONDITION "ASED ON THIS PRELIMINARY SERIES OF CHEMOSTATS A MIXTURE OF , MINዅኻ AIR AND , MINዅኻ OF
.ኼ IN THE INLET GAS WAS SELECTED CORRESPONDING TO A QP OF ETHANOL AND ARABITOL OF MMOL%T/( ዅኻ ዅኻ ዅኻ ዅኻ G$#7 H AND MMOL!RAB G$#7 H RESPECTIVELY 4ABLE $ATA WAS CONSISTENT WITH THE PREVIOUS RESULTS PRESENTED BY "AUMANN AND CO WORKERS ;= $ECREASED BIOMASS YIELD COEFlCIENT DUE TO THE PARTIALLY FERMENTATIVE METABOLISM WAS ALSO OBSERVED IN BOTH STUDIES
4ABLE #OMPARISON OF HYPOXIC PARAMETERS IN A "IOSTAT " AND A "IOSTAT " SYSTEM
&AB Q%T/( Q!RAB /XYGEN $#7 G 983 G$#7 SECRETED Q&AB MGፅፚ # #ULTURE ዅኻ ዅኻ ዅኻ ዅኻ MMOL%T/( MMOL!RAB CONDITIONS , GGLC MG&AB G$#7 H ዅኻ ዅኻ ዅኻ ዅኻ BALANCE G$#7 H G$#7 H ,ዅኻ , MINዅኻ q q q q q ND "IOSTAT " , MINዅኻ q q q q q q , MINዅኻ q q q q q ND "IOSTAT " , MINዅኻ q q q q q q
$#7 DRY CELL WEIGHT 983 YIELD OF BIOMASS ON GLUCOSE Q SPECIlC PRODUCTION RATE # BALANCE CARBON BALANCE #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
/NCE ESTABLISHED THE HYPOXIC CONDITIONS FOR THE "IOSTAT " SAME PARAMETERS WERE USED IN A "IOSTAT " WITH AN IDENTICAL VESSEL AND WHERE THE IMPELLER FOR STEERING WAS ADJUSTED AT THE SAME POSITION 4HUS SIMILAR OXYGEN TRANSFER AND HYPOXIC CONDITIONS WOULD BE EXPECTED 2ESULTING PARAMETERS FOR THE TWO REACTOR SYSTEMS USING THE SAME SETTINGS ARE SHOWN IN 4ABLE WHERE IT CAN BE OBSERVED
A FOLD INCREASE OF Q&AB WHEN "IOSTAT " IS USED UNDER HYPOXIC CONDITIONS WHILE EQUIVALENT
CONDITIONS IN "IOSTAT " LEAD TO AN INCREASE OF Q&AB UP TO FOLD )N HYPOXIC CONDITIONS Q%T/(
AND Q!RAB INCREASED COMPARED TO NORMOXIA IN BOTH BIOREACTORS BUT LOWER LEVELS WERE PRESENT IN "IOSTAT " CONSISTENT WITH THE LESS STRINGENT HYPOXIA ACHIEVED ,ESS AIR mOW IN THE INLET GAS LED TO THE WASH OUT OF THE REACTOR INDICATING THAT , MINዅኻ WAS THE MOST RESTRICTIVE AIR mOW TO WORK WITH "IOSTAT " WITHOUT WASHING OUT EVEN WHEN THEY PROVIDE LESS RESTRICTIVE HYPOXIC CONDITIONS THAN IN "IOSTAT " /NCE THE HYPOXIC CONDITIONS WERE ESTABLISHED CHEMOSTAT CULTIVATIONS WITH THE BATTERY OF STRAINS GENERATED WERE PERFORMED IN BOTH NORMOXIC AND HYPOXIC CONDITIONS
$IFNPTUBU DVMUJWBUJPOT #HEMOSTAT CULTIVATIONS WERE PERFORMED AS INDICATED IN THE -ATERIALS AND -ETHODS SECTION %XCEPT FOR /% $%3 ALL STRAINS WERE CULTURED AT A GROWTH RATE OF Hዅኻ CORRESPONDING TO THE ዅኻ HALF OF THEIR MAX /% $%3 EXHIBIT A LOWER MAXIMUM GROWTH RATE AND IT WAS CULTURED AT H
CORRESPONDING TO ITS HALF MAX WHICH ENSURES EQUAL CELL METABOLIC STATE THAN THE REST OF STRAINS 3AMPLES WERE TAKEN AFTER A MINIMUM OF lVE RESIDENCE TIMES TO ENSURE CELL METABOLIC STEADY STATE #ULTIVATIONS WERE ANALYSED IN TERMS OF GROWTH AND &AB PRODUCTIVITIES 4ABLE &OR ALL THE CULTURES IN HYPOXIC CONDITIONS LOWER BIOMASS CONCENTRATIONS WERE OBTAINED DUE TO THE LOWER BIOMASS TO SUBSTRATE YIELD OF THE PARTIALLY FERMENTATIVE METABOLISM OF CELLS CULTURED UNDER LOW OXYGEN CONDITIONS &URTHERMORE ALL STRAINS ACHIEVED HIGHER &AB PRODUCTIVITIES IN HYPOXIA WHERE THE HIGHEST INCREASE OBSERVED IN THE STRAIN OVEREXPRESSING %2' /% %2' FOLD ́$%3 FOLD AND /% (AC FOLD !LL OF THEM EXHIBITED HIGHER &AB PRODUCTIVITIES DUE TO THE CULTIVATION IN HYPOXIC CONDITIONS THAN THE REFERENCE STRAIN FOLD 5NDER NORMOXIC CONDITIONS MOST OF THE STRAINS PRESENTED SIMILAR &AB PRODUCTION RATES THAN THE REFERENCE STRAIN EXCEPT FOR ́352 AND THE mUCONAZOLE TREATED CELLS WITH FOLD AND FOLD INCREASE RESPECTIVELY 'LUCOSE AND GLYCEROL WERE MEASURED BY (0,# AND VALUES WHERE ALWAYS UNDER THE DETECTION LEVEL %THANOL AND ARABITOL WERE DETECTED IN THE CULTURE MEDIUM OF HYPOXIC CULTIVATIONS ALTHOUGH THEIR SPECIlC PRODUCTION RATES WERE LOWER THAN IN PREVIOUS HYPOXIC STUDIES ;= INDICATING LESS STRINGENT HYPOXIC STRESS AND PROVIDING TRUE STEADY STATE CONDITIONS IN THE BIOREACTOR IE NO WASHING OUT /% $%3 LEVELS OF ETHANOL AND ARABITOL WERE SIMILAR IN HYPOXIC AND NORMOXIC CONDITIONS INDICATING THAT NO CHANGE TO RESPIROFERMENTATIVE METABOLISM WAS ACHIEVED )N ORDER TO ESTABLISH HYPOXIC CONDITIONS FOR THIS STRAIN AIR mOW WAS REDUCED PROPORTIONALLY TO THE DILUTION RATE 4HUS AN AIR mOW , MINዅኻ WAS USED INSTEAD OF , MINዅኻ %THANOL AND ARABITOL PRODUCTION RATES DENOTED THE SELECTED AIR mOW WAS NOT RESTRICTIVE ENOUGH TO SHIFT TO RESPIROFERMENTATIVE METABOLISM !S EXPECTED 21 WAS ALSO INCREASED IN HYPOXIA EXCEPT FOR THE STRAIN OVEREXPRESSING (AC WHICH PRESENTED REDUCED 21 LEVELS IN HYPOXIA EVEN CELLS WERE GROWING UNDER RESPIROFERMENTATIVE CONDITIONS 4HE CARBON RECOVERY DATA OF THE CHEMOSTAT CULTIVATIONS WAS ABOVE AND THE OBTAINED DATA IS CONSISTENT WITH PREVIOUS RESULTS ;= WHICH PROVES A WELL DElNED SYSTEM AND THE REPRODUCIBILITY OF THE CULTIVATION SYSTEM 3FTVMUT BOE EJTDVTTJPO q q q q q q q q q q q q q q 21 # BALANCE STANDARD ERROR q
ዅኻ H #/ ዅኻ q q q q q q q q q q q q q q THE MEAN #/ Q $#7 MMOL G
ዅኻ H / / ዅኻ q q q q q q q q q q q q q q Q MMOL $#7 G
ዅኻ H !RAB ዅኻ q q q q q q q !RAB Q $#7 MMOL G S GROWN UNDER NORMOXIC AND HYPOXIC CONDITIONS
ዅኻ H ND ND ND ND ND ND ND %T/( ዅኻ q q q q q q q q q q q q q %T/( Q $#7 MMOL G
ዅኻ ፅፚ H ND ዅኻ q q q q q q q q q q q q q q MG Q SPECIlC PRODUCT FORMATION RATE ND NOT DETECTED 6ALUES REPRESENT &AB $#7 Q G
ዅኻ , q q q q q q q q q q q q q q &AB &AB SECRETED MG
BIOMASS TO SUBSTRATE YIELD $#7 ዅኻ G q q q q q q q q q q q q q q 83 GLC G 83 9 0HYSIOLOGICAL PARAMETERS OF THE CHEMOSTAT CULTURES OF THE WORKING STRAIN
ዅኻ q q q q q q q q q q q q q q G , 4ABLE $#7 (YPOXIA (YPOXIA (YPOXIA (YPOXIA (YPOXIA (YPOXIA (YPOXIA /XYGEN .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA CONDITIONS $#7 DRY CELL WEIGHT GLC GLUCOSE 9 352 $%3 &LUCO ́ ́ &AB& #ULTURE /% (AC /% $%3 /% %2' #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
*OUSBFYUSBDFMMVMBS EJTUSJCVUJPO PG UIF 'BC #ELLS WERE DISRUPTED USING THE METHODOLOGY DESCRIBED IN THE -ATERIALS AND -ETHODS 3ECTION &AB & AMOUNTS PRESENT IN THE SUPERNATANT 3. SOLUBLE CYTOSOLIC FRACTION 3#& AND INSOLUBLE MEMBRANE FRACTION )-& WERE QUANTIlED BY %,)3! 4HUS INTRACELLULAR AND EXTRACELLULAR DISTRIBUTION OF THE &AB & WITHIN THE CELLS WAS DETERMINED 4ABLE 4HE PERCENTAGE OF SECRETED &AB & WAS CALCULATED AS THE AMOUNT OF &AB PRESENT IN THE SUPERNATANT IN RELATION TO THE TOTAL &AB 3. 3#& )-& 5NDER ALL CONDITIONS TESTED RELATIVE SECRETION LEVELS OF &AB WERE ABOVE SUGGESTING THAT NO IMPORTANT SECRETION LIMITATIONS WERE PRESENT IN PRODUCING CELLS UNDER STEADY STATE CONDITIONS 7HEREAS &AB SPECIlC PRODUCTIVITIES WERE HIGHER UNDER REDUCED OXYGEN AVAILABILITY &AB YIELD IN THE INTRACELLULAR FRACTION WAS VERY LOW AND SIMILAR IN BOTH CONDITIONS 4HAT IS INCREASED &AB SYNTHESIS LEVELS CAUSED BY THE UPREGULATION OF THE '!0 PROMOTER UNDER HYPOXIC CONDITIONS ;= DID NOT RESULT IN AN OVERLOAD OF THE SECRETION CAPACITY !LTHOUGH SECRETION OF HETEROLOGOUS PROTEINS IS LIABLE TO SEVERAL BOTTLENECKS THAT LIMIT YIELD ; = OUR RESULTS SUGGEST THAT NO SECRETION LIMITATIONS ARE PRESENT WHEN CELLS ARE PRODUCING HIGHER AMOUNTS OF PROTEIN UNDER THE SELECTED HYPOXIC CONDITIONS 4HIS MAY INDICATE THAT MEMBRANE ALTERATIONS DUE TO HYPOXIC CULTURE CONDITIONS FAVOUR PROTEIN SECRETION AVOIDING INTRACELLULAR ACCUMULATION EVEN WHEN &AB PRODUCTION IS INCREASED )T HAS ALSO BEEN OBSERVED THAT THE INSOLUBLE FRACTION )-& WHERE THE MEMBRANE RELATED FRACTIONS ARE PRESENT IE ENDOPLASMIC RETICULUM PLASMA MEMBRANE PRESENTED ALMOST NO RECOMBINANT PROTEIN INDICATING NO PROTEIN ACCUMULATION IN THE %2 DUE TO MISFOLDING EVENTS -OREOVER NO ACCUMULATION OF &AB IN THE CYTOSOLIC FRACTION WAS DETECTED WHICH CAN BE DUE TO A HIGH DEGRADATION OF MISFOLDED PROTEIN WITHIN THE CELL WHICH 0FEFFER ET AL REPORTED TO BE AROUND OF THE TOTAL PROTEIN PRODUCED IN THEIR STUDIES PERFORMED WITH AN ANTIBODY FRAGMENT ;=
$FMM MJQJE DPNQPTJUJPO ,IPID COMPOSITION OF CELL HOMOGENATES WAS DETERMINED FOR ALL THE CULTURES AND OXYGEN CONDITIONS TO INVESTIGATE THE CORRELATION BETWEEN CHANGES ON PROTEIN PRODUCTION AND ALTERED LIPID PROlLES ,IPID CHANGES WERE EXPECTED IN ALL STRAINS AS GENETIC MODIlCATIONS IN THE MUTATED STRAINS WERE POINTED TO LIPID BIOSYNTHETIC PATHWAYS !DDITIONALLY CHANGES UNDER HYPOXIC CONDITIONS WERE ALSO PRESUMED AS BIOSYNTHESIS OF CERTAIN LIPIDS E G ERGOSTEROL UNSATURATED FATTY ACIDS REQUIRE OXYGEN AND PREVIOUS TRANSCRIPTOMIC STUDIES REVEALED THAT ERGOSTEROL AND SPHINGOLIPID BIOSYNTHETIC PATHWAYS WERE REGULATED UNDER HYPOXIA ;=
'BUUZ BDJE DPNQPTJUJPO 2ELATIVE AMOUNTS OF FATTY ACIDS OF THE CELL LIPID EXTRACTS WERE ANALYSED FOR EACH STRAIN AND CULTURE CONDITION AND ARE SHOWN IN 4ABLE )N ALL CASES OLEIC ACID # WAS THE MAJOR FATTY ACID IN THE CELL HOMOGENATES FOLLOWED BY LINOLEIC ACID # AND PALMITIC ACID # !LL STRAINS PRESENTED SIMILAR LEVELS OF FATTY ACID SPECIES WITH THE MAJOR DIFFERENCES OBSERVED FOR THE STRAIN /% (AC WHICH SHOWED DECREASE OF LINOLEIC ACID AND AN INCREASE OF PALMITIC ACID BY COMPARED TO THE REFERENCE STRAIN &LUCONAZOLE TREATED CELLS PRESENTED REDUCTION OF OLEIC ACID AMOUNTS ́352 SHOWED HIGHER AMOUNTS OF LINOLENIC ACID # AND REDUCTION OF CEROTIC ACID # 2EGARDING TOTAL AMOUNTS OF SATURATED FATTY ACIDS AN INCREASE OF WAS OBSERVED FOR /% (AC STRAIN COMPARED TO THE REFERENCE STRAIN 4OTAL MONOUNSATURATED FATTY ACIDS REMAINED SIMILAR FOR ALL THE STRAINS IN NORMOXIC CONDITIONS EXCEPT FOR mUCONAZOLE TREATED CELLS SHOWING A REDUCTION OF 3FTVMUT BOE EJTDVTTJPO q q q q q q q q q q q q q q 3ECRETION q q q q q q q q q q q q q q
ዅኻ $#7 G &AB MG q q q q q q q q q q q q q q %XPRESSION TIONS q q q q q q q q q q q q q q
q q q q q q q q q q q q q q STANDARD DEVIATION q q q q q q q q q q q q q q q MG 4OTAL &AB q q q q q q q q q q q q q q 6ALUES REPRESENT THE MEAN q q q q q q q q q q q q q q 3. 3#& )-& 4OTAL 3. 3#& )-& &AB PRODUCT DISTRIBUTION WITHIN THE CELL UNDER NORMOXIC AND HYPOXIC CONDI
ዅኻ &AB H 4ABLE q q q q q q q q q q q q q q ዅኻ MG &AB $#7 Q G
ዅኻ H $ (YPOXIA (YPOXIA (YPOXIA (YPOXIA (YPOXIA (YPOXIA (YPOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA .ORMOXIA /XYGEN CONDITIONS 352 $%3 &LUCO ́ ́ &AB& /% (AC /% $%3 #ULTURE /% %2' #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO YITCAI# PLII CD AMTLI CD # ACID PALMITOLEIC # ACID PALMITIC # ACID MYRISTIC # %%2' /% #ULTURE %$%3 /% %(AC /% &AB& ́ ́ &LUCO $%3 352 CONDITIONS /XYGEN OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA /THERS # # # # # # # # q q q q q q q q q q q q q q AL 4ABLE q q q q q q q q q q q q q q AT CDCMOIINO EEEC TANADMTN TAN UTRDIN CULTURED STRAINS MUTANT AND STRAIN REFERENCE OF COMPOSITION ACID &ATTY q q q q q q q q q q q q q q SERCAI# OECAI# LNLI CD # ACID LINOLEIC # ACID OLEIC # ACID STEARIC q q q q q q q q q q q q q q H MEAN THE FTTLFTYACIDS FATTY TOTAL OF q $FO TRIPLICATES FROM 3$ q q q q q q q q q q q q q q q q q q q q q q q q q q q q OMXCADHPXCCONDITIONS HYPOXIC AND NORMOXIC q q q q q q q q q q q q q q ᎎ LNLNCAI# CRTCAI6LE REPRESENT 6ALUES ACID CEROTIC # ACID LINOLENIC q q q q q q q q q q q q q q q q q q q q q q q q q q q q 5&!3&! ## q q q q q q q q q q q q q q 3FTVMUT BOE EJTDVTTJPO
&INALLY TOTAL LEVELS OF POLYUNSATURATED FATTY ACIDS WERE HIGHER IN THE ́352 STRAIN AND AND LOWER FOR /% %2' AND /% (AC RESPECTIVELY 4HEREFORE A CHANGE OF POLYUNSATURATION TO SATURATION OF THE FATTY ACID SPECIES IN THE STRAIN /% (AC WAS OBSERVED THAT COULD LEAD TO CHANGES IN THE MEMBRANE PROPERTIES
)N HYPOXIA A CHANGE IN THE DEGREE OF UNSATURATION WAS GIVEN BY THE INCREMENT OF OLEIC ACID AND THE DECREASE OF LINOLENIC ACID SPECIES PRESENT IN THE HOMOGENATES (IGH SATURATION OF FATTY ACIDS ACTS AS A PROTECTIVE MECHANISM BY CREATING A MORE RIGID MEMBRANE AND REDUCING THE OXIDATIVE STRESS THAT RESULTS FROM THE OXIDATION OF UNSATURATED FATTY ACIDS ;= )N THIS SENSE /% (AC STRAIN EXHIBIT THE MOST SEVERE CHANGE WITH LEVELS OF OLEIC ACID HIGHER THAN OF THE TOTAL FATTY ACID CONTENT AND LEVELS OF LINOLEIC ACID ACCOUNTING FOR THE OF THE REFERENCE STRAIN ,EVELS OF OLEIC ACID AND LINOLENIC ACID EXHIBIT MILDER CHANGES COMPARED TO THE REST OF STRAINS )N 0 PASTORIS IT HAS BEEN SHOWN THAN THE RELATIVE AMOUNT OF POLYUNSATURATED FATTY ACIDS IS MUCH HIGHER THAN IN 3 CEREVISIAE ; =
1IPTQIPMJQJE DPNQPTJUJPO 4OTAL PHOSPHOLIPID 0, CONTENT LIPID EXTRACTS WERE DETERMINED AND ARE SHOWN IN &IGURE 3LIGHTLY REDUCED TOTAL AMOUNTS WERE PRESENT IN STRAINS /% $%3 /% %2' AND /% (AC STRAINS WHILE THE REST OF STRAINS MAINTAINED SIMILAR LEVELS TO THE REFERENCE STRAIN (YPOXIC CONDITIONS WERE TRANSLATED TO DIFFERENT EFFECTS ON TOTAL 0, CONTENT DEPENDING ON THE STRAIN &AB & AND mUCONAZOLE TREATED CELLS PRESENTED REDUCED LEVELS OF TOTAL 0, IN HYPOXIA WHILE THE OPPOSITE EFFECT WAS OBSERVED FOR ́$%3 ́352 /% %2' AND /% (AC &INALLY /% $%3 STRAIN DID NOT CHANGE ITS 0, LEVELS DUE TO HYPOXIC CONDITIONS
Normoxia Hypoxia 0.30
0.25 -1
0.20 total prot mg
PL 0.15 mg 0.10
0.05
0.00 2F5 ΔDes1 OE Des1 ΔSur2 Fluco OE Erg11 OE Hac1
&IGURE 4OTAL 0, CONTENT OF THE STRAINS IN BOTH NORMOXIC AND HYPOXIC CONDITIONS
)N TERMS OF SPECIlC PHOSPHOLIPIDS 4ABLE PHOSPHATIDYLCHOLINE 0# AND PHOSPHATIDYLETHANOLAMINE 0% PHOSPHATIDYLINOSITOL 0) AND PHOSPHATODYLSERINE 03 WERE THE MAJOR PHOSPHOLIPIDS IN THE HOMOGENATES 4HIS RESULTS ARE IN CONCORDANCE WITH PREVIOUS PHOSPHOLIPID CHARACTERISATION OF 0 PASTORIS ; = (YPOXIC CONDITIONS RESULTED IN HIGHER AMOUNTS OF PHOSPHATIDYLETHANOLAMINE ࣵ EXCEPT FOR ́$%3 AND /% %2' AND A NOTABLE INCREASE OF PHOSPHATIDYLSERINE 03 WHILE LEVELS OF PHOSPHATIDYLINOSITOL 0) WERE SUBSTANTIALLY REDUCED IN ALL THE STRAINS 0HOSPHOLIPIDS ARE THE MAJOR LIPIDS OF CELLULAR MEMBRANES THUS CHANGES ON THE RELATIVE AMOUNTS OF PHOSPHOLIPIDS CAN ALTER SIGNIlCANTLY ITS PROPERTIES -OREOVER CELLULAR AMOUNTS OF 0) CAN #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO #ULTURE %%2' /% # POPAIYCOIE ! POPAII CD ) PHOSPHATIDYLINOSI 0) ACID PHOSPHATIDIC 0! PHOSPHATIDYLCHOLINE 0# %$%3 /% %(AC /% &AB& ጂ ጂ &LUCO $%3 352 CONDITIONS /XYGEN OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA OMXA .ORMOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA YOI (YPOXIA AL 4ABLE #0 )0 !# YO0 $-0% LYSO 0, #, 0! 03 0) 0% 0# q q q q q q q q q q q q q q HSHLPDCMOIINO EEEC TANADMTN TAN CULTURE STRAINS MUTANT AND STRAIN REFERENCE OF COMPOSITION 0HOSPHOLIPID IEHLPOPAIYEHNLMN6LE ERSN H MEAN THE REPRESENT 6ALUES PHOSPHATIDYLETHANOLAMINE DIMETHYL q q q q q q q q q q q q q q O0 HSHTDLEIE YO0 YOHSHLPD0 PHOSPHAT 0% LYSOPHOSPHOLIPIDS ,YSO 0, PHOSPHATIDYLSERINE 03 TOL q q q q q q q q q q q q q q q q q q q q q q q q q q q q FTTLPHOSPHOLIPIDS TOTAL OF q NNROI N YOI CONDITIONS HYPOXIC AND NORMOXIC IN D $FO DUPLICATES FROM 3$ q q q q q q q q q q q q q q q q q q q q q q q q q q q q ND ND ND ND ND ND ND ND ND ND ND ND ND ND DLTAOAIE , CRILPN -% $-0% CARDIOLIPIN #, IDYLETHANOLAMINE q q q q q q q q q q q q q q 3FTVMUT BOE EJTDVTTJPO
HAVE AN IMPORTANT IMPACT ON CELL MEMBRANE COMPOSITION AS IT IS INVOLVED IN INOSITOL CONTAINING SPHINGOLIPID BIOSYNTHESIS )T HAS BEEN REPORTED THAT BOTH 0) AND 03 ARE ANIONIC CHARGE AND 0) CAN SUBSTITUTE 03 IN ORDER TO PRESERVE 0, BALANCE BUT NOT THE INVERSE ;= WHICH MAKE THE OBSERVED RESULTS BE THE CONSEQUENCE OF MAJOR IMPORTANT CHANGES ON CELL DURING HYPOXIC CONDITIONS
/POQPMBS MJQJE DPNQPTJUJPO .ON POLAR LIPIDS WERE ANALYSED IN ORDER TO EXPLAIN THE WHOLE LIPIDOMIC PATTERN OF THE STRAINS 4RIACYLGLYCEROLS 4' STERYL ESTERS 3% AND FREE STEROLS &3 WERE QUANTIlED USING 4,# AND THE RESULTS ARE SHOWN IN 4ABLE !LMOST ALL STRAINS PRESENTED BOTH 4' AND 3% INCREASED WHEN CELLS WERE CULTURED IN HYPOXIA UP TO FOLD FOR 4' IN mUCONAZOLE TREATED CELLS AND FOLD FOR 3% IN ́$%3 )NCREASED LEVELS OF 4' UNDER HYPOXIA ARE CONSISTENT WITH PRELIMINARY LIPID ANALYSES PREVIOUSLY PERFORMED BY OUR GROUP ;= )N THE CASE OF FREE STEROLS LEVELS DECREASE FOR MOST OF THE STRAINS WHEN CHANGING FROM NORMOXIC TO HYPOXIC CONDITIONS PROBABLY DUE TO LOW LEVELS OF STEROL BIOSYNTHESIS EXCEPT FOR ́352 mUCONAZOLE TREATED CELLS AND /% (AC WERE &3 LEVELS REMAINED EQUAL OR RAISED IN HYPOXIA
4ABLE .ON POLAR LIPID COMPOSITION OF REFERENCE STRAIN AND MUTANT STRAINS CULTURED IN NORMOXIC AND HYPOXIC CONDITIONS
&REE 3TEROLS /XYGEN 4' ᎙G LIPID 3% ᎙G LIPID #ULTURE ᎽᎳ ᎽᎳ ᎙G LIPID CONDITIONS MGTOT PROT MGTOT PROT ᎽᎳ MGTOT PROT .ORMOXIA q q q &AB & (YPOXIA q q q .ORMOXIA q q q ጂ $%3 (YPOXIA q q q .ORMOXIA q q q /% $%3 (YPOXIA q q q .ORMOXIA q q q ጂ 352 (YPOXIA q q q .ORMOXIA q q q &LUCO (YPOXIA q q q .ORMOXIA q q q /% %2' (YPOXIA q q q .ORMOXIA q q q /% (AC (YPOXIA q q q 4' 4RIACYLGLYCEROLS 3% 3TEROL %STERS &3 &REE 3TEROLS 6ALUES REPRESENT THE MEAN Ĝ 3$ FROM TRIPLICATES
.ON POLAR LIPID SYNTHESIS IN YEAST IS INVOLVED IN THE MAINTENANCE OF THE EQUILIBRIUM OF SATURATED AND UNSATURATED FATTY ACIDS AND ALSO IN MEMBRANE COMPOSITION REGARDING FATTY ACIDS AND PHOSPHOLIPIDS IN ORDER TO PRESERVE MEMBRANE mUIDITY ;= #HANGES ON 4' LEVELS RELATED TO GROWTH PHASE STATIONARY VS EXPONENTIAL HAVE BEEN REPORTED ;= SUGGESTING A POSSIBLE NON POLAR LIPID STORAGE THAT DEPENDS ON CELL GROWTH AND THAT COULD EXPLAIN INCREASED LEVELS OF BOTH ዅኻ 4' AND 3% UNDER HYPOXIC CONDITIONS SINCE IT IS KNOWN THAT H CORRESPONDS TO OF MAX IN
NORMOXIC CONDITIONS BUT NO DATA FOR HYPOXIC CONDITIONS ARE AVAILABLE ! LOWER MAX UNDER HYPOXIA #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
WOULD MEAN A HIGHER RELATIVE GROWTH RATE OF CELLS CULTURED UNDER THESE CONDITIONS AND THUS ALTERED LEVELS OF STORAGE LIPIDS DUE TO A DIFFERENT CELL METABOLISM -OREOVER IT HAS BEEN DESCRIBED THAT CHANGES IN GLUCOSE METABOLISM CAUSED BY THE SHIFT FROM RESPIRATORY TO RESPIROFERMENTATIVE METABOLISM CAN IMPACT NON POLAR LIPID HOMEOSTASIS BY CHANGING ACTIVITY OF 4' LIPASES ;=
4UFSPM DPNQPTJUJPO !S SHOWN IN 4ABLE THE MAJOR STEROL FOUND UNDER ALL ANALYSED CONDITIONS WAS ERGOSTEROL #OMPARED TO THE REFERENCE STRAIN LOWER LEVELS OF ERGOSTEROL WERE PRESENT IN ́352 mUCONAZOLE TREATED CELLS /% %2' AND /% (AC )NTERMEDIATES OF THE ERGOSTEROL PATHWAY LIKE METHYL ZYMOSTEROL ZYMOSTEROL OR EPISTEROL WERE ALSO DETECTED BUT ONLY IN SMALL AMOUNTS 3TRAINS REACTED TO HYPOXIC CONDITIONS IN DIFFERENT WAYS 7HILE &AB & /% $%3 AND /% %2' REDUCED THEIR ERGOSTEROL CONTENT UP TO IN THE CASE OF THE REFERENCE STRAIN ́$%3 ́352 mUCONAZOLE TREATED CELLS AND /% (AC PRESENTED HIGHER LEVELS OF ERGOSTEROL IN THEIR CELL HOMOGENATES 2EGARDING TO ERGOSTEROL INTERMEDIATES HIGHER LEVELS OF EPISTEROL WERE PRESENT IN MOST OF THE STRAINS EXCEPT FOR ́352 DECREASE AND /% %2' WHICH PRESENTED NO CHANGES 3IGNIlCANT CHANGES WER ALSO OBSERVED FOR ZYMOSTEROL WITH LOWER LEVELS ON THE REFERENCE STRAIN /% $%3 ́352 AND /% (AC AND AND FOLD INCREASE FOR ́$%3 mUCONAZOLE TREATED CELLS AND /% %2' STRAINS RESPECTIVELY &URTHERMORE mUCONAZOLE TREATED CELLS EXHIBITED ADDITIONAL INTERMEDIATES EG LANOSTEROL DIMETHYLCHOLESTA DIENOL AS A RESULT OF ITS EFFECT ON INHIBIT %RGP #ULTIVATION UNDER HYPOXIC CONDITIONS RESULTED IN FOLD INCREASED LEVELS OF THESE INTERMEDIATES %RGOSTEROL IS AN ESSENTIAL COMPONENT OF MEMBRANES AND REGULATES mUIDITY AND PERMEABILITY OF THE PLASMA MEMBRANE ;= )T HAS A BROAD FUNCTION IN CELLULAR PROCESSES LIKE PLASMA MEMBRANE FUSION PHEROMONE SIGNALLING OR PROTEIN SORTING ; = 0RESENCE OF STEROLS IN THE MEMBRANES DECREASE MEMBRANE mUIDITY THUS THE REDUCTION OF ERGOSTEROL AMOUNTS INDUCED BY HYPOXIC CONDITIONS CAN RESULT ON HIGHER MEMBRANE mUIDITY 3HARMA ;= SUGGESTED AN ADAPTIVE RESPONSE TO ALTERED STEROL STRUCTURES THROUGH CHANGES IN LIPID COMPOSITION AND mUIDITY THAT CAN BE ALSO PRESENT WHEN STEROL IS DEPRIVED $ESPITE REDUCED AMOUNTS OF STEROLS IN HYPOXIA GENES OF THE ERGOSTEROL BIOSYNTHETIC PATHWAY WERE HIGHLY UPREGULATED IN HYPOXIA DUE TO ITS DEPENDENCE ON OXYGEN AVAILABILITY !S ERGOSTEROL IS AN ESSENTIAL COMPONENT OF THE CELLS HIGH TRANSCRIPT LEVELS OF INTERMEDIATE REACTIONS AIM FOR SUFlCIENT AMOUNTS OF ERGOSTEROL FOR CELL SURVIVAL %RGOSTEROL LEVELS IN THE CELL WOULD BE INmUENCING THE ENTIRE LIPID METABOLISM SINCE IT HAS BEEN DESCRIBED THAT %2' MUTANTS IN 3 CEREVISIAE LED TO CHANGES OF MORE THAN OF 0, AND MORE THAN OF SPHINGOLIPID SPECIES ;=
4QIJOHPMJQJE DPNQPTJUJPO 3HPINGOLIPID ANALYSES WERE KINDLY PERFORMED BY 0ABLO 4ARAZONA AT 'EORG !UGUST 5NIVERSITY '¨TTINGEN 'ERMANY FOLLOWING THE PROTOCOL DESCRIBED IN -ATERIALS AND -ETHODS 3PHINGOLIPID SPECIES WERE QUANTIlED AS THE PERCENTAGE OF RELATIVE PEAK AREA FOR EACH SPECIE AND ARE SHOWN IN &IGURE )N ADDITION TO THE TYPICAL INOSITOL CONTAINING SPHINGOLIPIDS 0 PASTORIS AS MOST YEASTS CONTAINS CERAMIDES AND HEXOSYLCERAMIDES WHICH ARE MISSING IN 3 CEREVISIAE ,ONG CHAIN BASES ,#" OF CERAMIDES WERE MOSTLY SPHING ENINE SPHINGADIENINE AND PHYTOSPHINGOSINE 4HE MAJOR CERAMIDE WAS PHYTOSPHINGOSINE COMBINED WITH MONOHYDROXYLATED LIGNOCERIC ACID FOLLOWED BY SPHING ENINE COMBINED WITH STEARIC ACID IN BOTH MONO AND UNHYDROXYLATED FORMS 3FTVMUT BOE EJTDVTTJPO q q q q q q q q q q q q q q q q q q q q q q q q q q q q %PISTEROL %RGOSTEROL q q FECOSTEROL METHYL ND ND ND ND ND ND ND ND ND ND ND ND q q q q q q q q q q q q q q
MOXIC AND HYPOXIC CONDITIONS ᎽᎳ :YMOSTEROL TOTAL PROT MG 3TEROL G q q q q q q q q q q q ᎙ METHYL ZYMOSTEROL ND ND ND q q q q DIENOL DIMETHYLCHOLESTA ND ND NOT DETECTABLE 6ALUES REPRESENT THE MEAN Ĝ 3$ FROM TRIPLICATES q q q q 3TEROL COMPOSITION OF REFERENCE STRAIN AND MUTANT STRAINS CULTURED IN NOR q 4ABLE 3QUALENE ,ANOSTEROL (YPOXIA ND ND (YPOXIA (YPOXIA ND ND ND (YPOXIA ND (YPOXIA ND ND ND (YPOXIA ND (YPOXIA ND ND ND .ORMOXIA ND ND ND .ORMOXIA ND ND ND .ORMOXIA ND ND ND .ORMOXIA ND ND ND .ORMOXIA ND ND ND .ORMOXIA ND ND ND .ORMOXIA ND /XYGEN CONDITIONS 352 $%3 &LUCO ጂ ጂ &AB& /% (AC /% $%3 /% %2' #ULTURE #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
ΔSur2 Erg11 Hac1 46:0;4 FabFab 2F5 2F5 ΔDes1Des1 Des1Des1 ΔSur2 Erg11 Fluconazole Hac1 46:0;3 44:0;5 44:0;4
C 44:0;3 2 42:0;5 42:0;4 42:0;3 M(IP) 36:0;4
M(IP)2C [mannosyl- 36:0;3
diinositolphosphorylceramide] 34:0;4 34:0;3 46:0;4 46:0;3 44:0;5 44:0;4 44:0;3 42:0;5 42:0;4
MIPC 42:0;3 36:0;4 36:0;3 MIPC [mannosyl-
inositolphosphorylceramide] 34:0;4 34:0;3 46:0;5 46:0;4 46:0;3 46:0;2 44:0;5 44:0;4 44:0;3 44:0;2
IPC 42:0;5 42:0;4 42:0;3 42:0;2 36:0;4 IPC [inositolphosphorylceramide] 36:0;3 34:0;4 34:0;3 19:2;2/18:0;1 19:2;2/16:0;1 18:2;2/18:0;1 18:2;2/16:0;1 18:1;2/18:0;1 HexCer
HexCer 18:1;2/16:0;1 18:0;2/18:0;1 [Hexosylceramide] 18:0;2/16:0;1 18:0;3/26:0;1 18:0;3/24:0;1 18:0;3/18:0;1 18:0;3/16:0;1 18:0;3/26:0;0 18:0;3/24:0;0 18:0;3/18:0;0 18:0;3/16:0;0 19:2;2/18:0;1 19:2;2/16:0;1 19:2;2/18:0;0 19:2;2/16:0;0
Cer 18:2;2/18:0;1 18:2;2/16:0;1 18:2;2/18:0;0
Cer [Ceramide] 18:2;2/16:0;0 18:1;2/18:0;1 18:1;2/16:0;1 18:1;2/18:0;0 18:1;2/16:0;0 18:0;2/18:0;1 18:0;2/16:0;1 18:0;2/18:0;0 18:0;2/16:0;0 03060900 3060900 30609003060900 30609003060900 306090
Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia
&IGURE 3TEROL COMPOSITION OF REFERENCE STRAIN AND MUTANT STRAINS CULTURED IN NORMOXIC AND HYPOXIC CONDITIONS 6ALUES ARE GIVEN AS THE RELATIVE PEAK AREA FOR EACH 3, SPECIE
&OR HEXOSYLCERAMIDES METHYLATED SPHINGADIENINE WAS THE MAJOR ,#" AND MONOHYDROXYLATED STEARIC ACID THE PREFERRED FATTY ACYL )NOSITOL CONTAINING SPHINGOLIPIDS WERE ENRICHED IN PHYTOSPHINGOSINE AS THE ,#" CONTAINING MONOHYDOXYLATED LIGNOCERIC ACID # AS THE FATTY ACYL MOIETY ́$%3 STRAIN EXHIBITED A LACK OF MOST OF THE CERAMIDE SPECIES AS IT WOULD BE EXPECTED BASED ON PREVIOUS STUDIES OF 4ERNES ET AL WHERE A WILD TYPE STRAIN OF 0 PASTORIS WITH A DELETION OF $%3 GENE WAS DESCRIBED ;= )N THIS STRAIN HEXOSYLCERAMIDES CHANGED ITS DISTRIBUTION COMPARED TO THE REFERENCE STRAIN WITH HIGHER CONTENT OF SPECIES CONTAINING SHORTER ,#" AND FATTY ACYL MOIETIES (OWEVER INOSITOL CONTAINING 3, REMAINED SIMILAR TO THE REFERENCE STRAIN )T WAS ALSO OBSERVED THAT OVEREXPRESSION OF $%3 DID NOT TRIGGER SIGNIlCANT DIFFERENCES ON SPHINGOLIPID RELATIVE AMOUNTS ́352 STRAIN CARRYING A DELETION OF THE # HYDOXYLASE PRESENTED AN ALTERED DISTRIBUTION OF RELATIVE AMOUNTS OF INOSITOL CONTAINING SPHINGOLIPIDS WITH THE PRESENCE OF SPECIES MISSING IN
THE REFERENCE STRAIN SUCH AS SPECIES FOR ALL )0# -)0# AND -)0 ኼ# SPHINGOLIPID TYPES &URTHERMORE A LACK OF CERAMIDES WITH VERY LONG FATTY ACIDS WAS ALSO OBSERVED /VEREXPRESSION OF %2' RESULTED IN A PREFERENCE FOR CERAMIDES WITH VERY LONG FATTY ACIDS AND HIGHER RELATIVE AMOUNTS OF # SPECIES OF INOSITOL CONTAINING SPHINGOLIPIDS WHILE NO SIGNIlCANT CHANGES IN mUCONAZOLE TREATED CELLS WERE OBSERVED COMPARED TO THE REFERENCE STRAIN &INALLY OVEREXPRESSION OF (AC PRESENTED SIMILAR RELATIVE AMOUNTS OF SPHINGOLIPIDS AS THE 3FTVMUT BOE EJTDVTTJPO
REFERENCE STRAIN 0 PASTORIS CERAMIDE SYNTHASE "ARP WAS REPORTED TO HAVE SPECIlCITY FOR DIHYDROXY ,#" AND
## FATTY ACIDS ;= /UR RESULTS SHOWED ELEVATED RELATIVE AMOUNTS OF TRIHYDROXY ,#" AND VERY LONG CHAIN FATTY ACIDS IN THE REFERENCE STRAIN WHICH DIFFERS FROM THE PREVIOUS RESULTS OF THE 0 PASTORIS WILDTYPE STRAIN BUT ARE SIMILAR TO THE ONES REPORTED FOR STRAINS WITH ́ DESATURASE DELETED AND THE GLUCOCERAMIDE OVEREXPRESSED ;= ! POSSIBLE REASON COULD BE THE EFFECT OF RECOMBINANT PROTEIN PRODUCTION WHICH COULD ACTIVATE 502 IN THE CELL AND THEREFORE CHANGE LIPID COMPOSITION OF THE CELLS
'FE#BUDI DVMUVSFT )N ORDER TO CHARACTERISE KNOCKOUT STRAINS FOR &AB PRODUCTION ́$%3 STRAIN WAS SELECTED AND CULTURED FOLLOWING A FED BATCH STRATEGY -ATERIALS AND -ETHODS #ELLS WERE MONITORED IN TERMS OF CELL GROWTH &AB SECRETION AND MEMBRANE mUIDITY &IGURES AND
BATCH FED-BATCH
&IGURE &ED BATCH PROlLE OF THE REFERENCE STRAIN &AB & $#7 ᎙ &AB SPECIlC PRODUCTION RATES AND ANISOTROPY WERE MONITORED
4HESE RESULTS WERE COMPARED TO THE ONES OBTAINED FOR THE REFERENCE 4HE MAIN PARAMETERS FOR EACH STRAIN ARE RESUMED IN 4ABLE .O SIGNIlCANT GROWTH DIFFERENCES WERE DETECTED BETWEEN BOTH STRAINS ́$%3 EXHIBITED SLIGHTLY LOWER &AB EXTRACELLULAR TITRES 2ESULTS DERIVED FROM FED BATCH CULTIVATIONS WERE CONSISTENT WITH SHAKE mASK CULTURES #JPSFBDUPS DVMUJWBUJPOT BOE TUSBJO DIBSBDUFSJTBUJPO
BATCH FED-BATCH
&IGURE &ED BATCH PROlLE OF THE REFERENCE STRAIN ጂ$%3 $#7 ᎙ &AB SPECIlC PRODUCTION RATES AND ANISOTROPY WERE MONITORED
4ABLE #ULTURE PARAMETERS OF TWO FED BATCH CULTURES USING GLYCEROL AS A CARBON SOURCE IN THE BATCH PHASE AND GLUCOSE IN THE FED BATCH PHASE
4OTAL Q&AB MEAN 4OTAL &AB -AX 993 "ATCH ዅኻ 983 Gፃፂፖ 908 MG&AB PROD 10 MG&AB MEAN H MGፅፚ PRODUCTION BIOMASS G ዅኻ ዅኻ ዅኻ ዅኻ 0HASE ዅኻ ዅኻ ዅኻ GGLY G$#7 MG&AB , H G$#7 H MG&AB , Hዅኻ &AB & q q q q q q q q ́$%3 q q q q q q q q 983 "IOMASS TO SUBSTRATE YIELD ᎙ SPECIlC GROWTH RATE Q&AB SPECIlC &AB PRODUCTION RATE 908 &AB TO BIOMASS
YIELD 10 VOLUMETRIC PRODUCTIVITY
$PODMVTJPOT
s (YPOXIC CONDITIONS WITHOUT WASHING OUT REACTOR HAVE BEEN DElNED AS THE ONES CORRESPONDING TO AN AIR mOW IN THE INLET OF , MINዅኻ MIXED WITH NITROGEN UP TO VVM 4HESE CONDITIONS PROVIDE HYPOXIC ENVIRONMENT IN A "IOSTAT " AND A "IOSTAT " WITH THE SAME VESSEL AND IMPELLER
s #ELLS CULTURED UNDER HYPOXIC CONDITIONS SECRETE HIGHER AMOUNTS OF &AB
s .O SIGNIlCANT MORPHOLOGICAL DIFFERENCES CAN BE OBSERVED BETWEEN STRAINS AND OXYGEN CONDITIONS
s #HANGES IN THE LIPID CONTENT OF THE STRAINS HAVE BEEN OBSERVED
s (YPOXIA HAS AN EFFECT ON ALTERING LIPID COMPOSITION OF THE CELLS AS IT HAS BEEN OBSERVED BY ALL THE STRAINS
s &AB BATCH CULTURES SHOWED THAT SHAKE mASK RESULTS ARE CONSISTENT *OUFHSBUFE BOBMZTJT PG IZQPYJD DPOEJUJPOT BOE FOHJOFFSFE MJQJE NFUBCPMJTN FGGFDU PO SFDPNCJOBOU 1 QBTUPSJT
*OUSPEVDUJPO ,IPIDS AS ESSENTIAL COMPONENTS OF CELL MEMBRANES IN LIVING ORGANISMS REQUIRE COMPLEX REGULATORY MECHANISMS TO GENERATE AND MAINTAIN LIPID COMPOSITION ; = #ELLS EMPLOY COORDINATED PATHWAYS OF SYNTHESIS AND DEGRADATION OF PHOSPHOLIPIDS SPHINGOLIPIDS AND STEROLS ; n= 4HESE INTERCONNECTED NETWORKS CONSTITUTE A HIGHLY DYNAMIC BIOLOGICAL CORE THAT ALLOWS CELLS NOT ONLY TO ADAPT THEIR LIPID PROlLE TO ENVIRONMENTAL CHANGES SUCH HEAT STRESS OSMOTIC STRESS OR NUTRIENT AND OXYGEN AVAILABILITY ; n= BUT ALSO TO SENSE THE LEVELS OF DIFFERENT LIPIDS AND ADJUST THEIR LIPID COMPOSITION TO PRESERVE CELLULAR FUNCTIONS ; n= 0UNCTUAL PERTURBATIONS COULD BE COMPENSATED BY THE INTERLINKED PATHWAYS PRESERVING CELL VIABILITY WITH NO OBVIOUS PHYSIOLOGICAL CONSEQUENCES ;= )N THE PRESENT CHAPTER PREVIOUS TRANSCRIPTIONAL DATASET OF RECOMBINANT 0 PASTORIS STRAINS IS INTEGRATED WITH LIPIDOMIC PROlLING ANALYSES AIMING AT THE IDENTIlCATION OF POTENTIAL CHANGES CORRELATING POSITIVELY WITH IMPROVED PROTEIN SECRETION
*OUFHSBUFE BOBMZTJT PG UIF IZQPYJB FGGFDU PO UIF SFGFSFODF TUSBJO 'BC' #ULTIVATION OF RECOMBINANT 0 PASTORIS UNDER HYPOXIC CONDITIONS HAS BEEN PREVIOUSLY REPORTED TO HAVE A STRONG POSITIVE EFFECT ON SPECIlC PRODUCTIVITY ; = "AUMANN ET AT ;= FOUND LIPID METABOLISM AS A HIGHLY TRANSCRIPTIONALLY REGULATED PATHWAY WHEN CELLS WERE CULTURED IN HYPOXIA )N THE PRESENT STUDY WE INTEGRATE PREVIOUS TRANSCRIPTIONAL DATASET OF RECOMBINANT 0 PASTORIS STRAIN WITH LIPIDOMIC PROlLING ANALYSES AIMING AT THE IDENTIlCATION OF POTENTIAL CHANGES CORRELATING POSITIVELY WITH IMPROVED PROTEIN SECRETION
&ATTY ACID DESATURASES ARE SUBJECT TO TIGHT CONTROL AT TRANSCRIPTIONAL LEVEL ON THE LEVEL OF M2.! STABILITY ;= AND ALSO WITH RESPECT TO PROTEIN STABILITY THROUGH THE UBIQUITIN PROTEASOME DEPENDENT %2 ASSOCIATED DEGRADATION PATHWAY ; = 4RANSCRIPT LEVELS OF OUR REFERENCE
*OUFHSBUFE BOBMZTJT
STRAIN IN HYPOXIC CONDITIONS RESULTED IN UPREGULATION OF THE / DEPENDENT ́ FATTY ACID DESATURASE ENCODED BY THE GENE /,% AND INVOLVED IN THE BIOSYNTHESIS OF UNSATURATED FATTY ACIDS (IGH SATURATION OF FATTY ACIDS ACTS AS A PROTECTIVE MECHANISM BY CREATING A MORE RIGID MEMBRANE AND REDUCING THE OXIDATIVE STRESS THAT RESULTS FROM THE OXIDATION OF UNSATURATED FATTY ACIDS ;= 9EASTS CAN REGULATE THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS 05&! TO CONTROL THE MEMBRANE mUIDITY IN STRESS CONDITIONS !DDING A DOUBLE BOND IN A FATTY ACYL SUBSTRATE IS A VERY DEMANDING PROCESS THAT INVOLVES TWO ELECTRONS AND THE CLEAVAGE OF TWO # ( BONDS K#ALMOL AND CONSUMES MOLECULAR OXYGEN ;= 4HIS OXYGEN DEPENDENT ACTIVITY COULD EXPLAIN THE INCREASED RELATIVE AMOUNTS OF MONOUNSATURATED &! AND AN OPPOSITE TREND FOR POLYUNSATURATED &! IN HYPOXIA EVEN IF LEVELS OF /,% TRANSCRIPT WERE UPREGULATED &IGURE 4HE PRESENCE OF THE MONOUNSATURATED &! OLEIC ACID AS THE MAJOR &! AND NOT SATURATED FATTY ACIDS COULD INDICATE THAT IT IS RESPONSIBLE OF MAINTAINING MEMBRANE mUIDITY ENABLING PROTEIN SECRETION
Fab 2F5 Normoxia Hypoxia 60
50 -1
40 total prot
30 % FA mg % FA 20
10
0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C26:0 Others
&IGURE 2ELATIVE AMOUNTS OF FATTY ACIDS OF THE REFERENCE STRAIN &AB & IN NORMOXIC AND HYPOXIC CONDITIONS
4RANSCRIPTOMIC DATA &IGURE REVEALED A REPRESSION OF GENES INVOLVED IN THE OXIDATION OF FATTY ACIDS 4HESE RESULTS WERE ALSO REPORTED FOR 3 CEREVISIAE GROWN IN HYPOXIC CONDITIONS AND IT IS KNOWN THAT A REPRESSION OF THIS PATHWAY EXISTS WHEN GLUCOSE IS PRESENT AS CARBON SOURCE ; =
0HOSPHATIDYLINOSITOL 0) AND PHOSPHATIDYLSERINE 03 ARE SYNTHESISED BY 0ISP AND #HOP RESPECTIVELY WHICH COMPETE FOR #$0 $!' MAKING THIS BRANCH AN IMPORTANT POINT OF REGULATION ;= .O SIGNIlCANT CHANGES AT TRANSCRIPTOMIC LEVEL WERE OBSERVED FOR THESE TWO GENES IN HYPOXIA HOWEVER AS IT CAN BE OBSERVED IN &IGURE LEVELS OF 03 INCREASED SIGNIlCANTLY WHILE 0) LEVELS DROP IN THIS CULTURE CONDITION 03 AND 0) ARE KEY DETERMINANTS OF MEMBRANE SURFACE CHARGE "OTH TYPES OF PHOSPHOLIPIDS ARE ANIONIC CHARGE BUT THEY DIFFER ON THEIR SHAPE 03 IS CYLINDRICAL SHAPED WHICH PREFERENTIALLY FORMS mAT BILAYER STRUCTURES WHILE 0) HAS AN INVERTED CONICAL SHAPE AND FORM STRUCTURES WITH POSITIVE CURVATURES ;= !DDITIONALLY DISTRIBUTION OF 03 AND 0) CAUSES THE ELECTROSTATIC PROPERTIES OF THE MEMBRANE TO VARY MAKING A HIGHLY CHARGED CYTOSOLIC LEAmET ON PLASMA MEMBRANE DUE TO ITS ABUNDANCE ;= -EMBRANE DEFORMING DOMAINS ARE CRUCIAL FOR PROTEIN MEMBRANE INTERACTIONS AND BOTH MEDIATE FUNCTIONAL INTERACTIONS WITH POSITIVELY CHARGED REGIONS OF PERIPHERAL AND INTEGRAL MEMBRANE PROTEINS ; = (OWEVER SOME DOMAINS AND PROTEINS TEND TO PREFER 0) &GGFDU PG IZQPYJB JO 'BC' TUSBJO
Fab 2F5 Normoxia Hypoxia 60
50 -1 40 total prot 30
20 % PL · mg % PL
10
0 PC PA PI PS Lyso-PL PE CL DMPE
&IGURE 2ELATIVE AMOUNTS OF PHOSPHOLIPIDS OF THE REFERENCE STRAIN &AB & IN NORMOXIC AND HYPOXIC CONDITIONS
RATHER THAN 03 MAKING 0) A MAJOR PLAYER ON CONTROLLING A VARIETY OF CELLULAR FUNCTIONS ;= )T HAS ALSO BEEN REPORTED FOR 3 CEREVISIAE THAT 0) CAN SUBSTITUTE 03 IN ORDER TO PRESERVE PHOSPHOLIPID BALANCE BUT NOT THE INVERSE ;=
)NOSITOL IS A POTENT REGULATOR OF PHOSPHOLIPID METABOLISM IN YEAST "OTH TRANSCRIPTION OF GENES ENCODING ENZYMES REQUIRED FOR SYNTHESIS OF 0, AND ACTIVITIES OF THESE ENZYMES ARE REGULATED PRIMARILY BY THE INTRACELLULAR CONCENTRATION OF FREE INOSITOL A PRECURSOR IN THE SYNTHESIS OF 0) ;=
-OST OF THE GENES THAT ARE REGULATED IN RESPONSE TO PRESENCE OF INOSITOL CONTAIN THE 5!3)./ ELEMENT WHEREAS THE MAJORITY OF THE GENES THAT RESPOND TO BOTH INOSITOL AND CHOLINE FALL INTO OTHER CATEGORIES EG UNFOLDED PROTEIN RESPONSE 502 PATHWAY LOW OXYGEN RESPONSE ELEMENT ,/2% ; = )NOSITOL USED IN 0) SYNTHESIS IS EITHER SYNTHESISED DE NOVO OR OBTAINED FROM THE GROWTH MEDIUM VIA THE )42 AND )42 ENCODED INOSITOL TRANSPORTERS ;= /UR TRANSCRIPTIONAL DATASET INDICATED THAT )42 TRANSCRIPT LEVELS WERE HIGHLY DOWNREGULATED UNDER HYPOXIC CONDITIONS WHILE )42 WAS UPREGULATED )T HAS BEEN DESCRIBED FOR 3 CEREVISIAE THAT )42 IS REPRESSED BY INOSITOL AND CHOLINE VIA /PIP WHILE )42 IS EXPRESSED CONSTITUTIVELY ;= )N ADDITION WHEN INTRACELLULAR INOSITOL LEVELS FALL THE TRANSCRIPTION OF A WIDE VARIETY OF GENES THAT ARE REQUIRED FOR DE NOVO PHOSPHOLIPID SYNTHESIS INCLUDING )./ #(/ AND /0) ARE INDUCED FAVOURING 03 AND 0% AND 0# PATHWAYS ;= /UR TRANSCRIPTOMIC DATA ONLY INDICATE UPREGULATION OF )./ WHILE NO SIGNIlCANT CHANGES ON #(/ AND /0) WERE OBSERVED (OWEVER 03 AND 0% RELATIVE AMOUNTS INCREASED IN HYPOXIA 4HIS CHANGE ON THE RELATIVE AMOUNTS OF THESE PHOSPHOLIPIDS COULD BE THE RESULT OF DECREASED INOSITOL AVAILABILITY FOR CELLS GROWING UNDER HYPOXIC CONDITIONS 4HIS THEORY WOULD BE SUPPORTED BY THE INCREMENT OF 4' LEVELS IN HYPOXIA WHICH HAS BEEN ALREADY RELATED TO THE ABSENCE OF INOSITOL ;= /N THE OTHER HAND 'ASPAR ET AL ;= STUDIED ALTERATIONS OF MEMBRANE LIPIDS OF 3 CEREVISIAE IN RESPONSE TO PRESENCE OF INOSITOL AND CHOLINE IN THE CULTURE MEDIUM AND THEY POINTED OUT THAT A LACK OF INOSITOL LEAD TO A DROP OF 0) LEVELS WHILE 03 LEVELS REMAINED CONSTANT WHICH WOULD ONLY PARTIALLY EXPLAIN OUR RESULTS *OUFHSBUFE BOBMZTJT
4HE NEGATIVE REGULATOR /PIP WHICH REPRESSES MANY OF THE GENES CONTAINING5!3)./ ELEMENTS REGULATED BY INOSITOL INCLUDING )./ ;= WAS TRANSCRIPTOMICALLY DOWNREGULATED IN HYPOXIC CONDITIONS )./ AT THE SAME TIME IS ONE OF THE MOST HIGHLY INDUCED GENES BY THE UNFOLDED PROTEIN RESPONSE 502 ;= WHICH OCCURS VIA REPRESSION OF /PIP BY (AC ;= 3TUDIES WITH PHOSPHOLIPIDS INDICATE THAT CHANGES ON THEIR LEVELS DO NOT ONLY OCCUR BY CHANGES ON TRANSCRIPTION LEVELS OF THE GENES INVOLVED IN THE PHOSPHOLIPID PATHWAY BUT ALSO BY CHANGES ON CONTACT SITES AND TRANSPORTERS ;= )T COULD EXPLAIN THE INCREMENT OF 03 AND 0% IN HYPOXIA EVEN WHEN TRANSCRIPTIONAL LEVELS OF THE GENES INVOLVED IN THEIR SYNTHESIS WERE NOT REGULATED
"RIEmY HYPOXIC CONDITIONS COULD TRIGGER LOW LEVELS OF INOSITOL IN THE CELL 4HIS FACT WOULD EXPLAIN THE DOWNREGULATION OF /0) WHICH WOULD PROMPT THE UPREGULATION OF )./ AND )42 GENES (YPOXIA ALSO CHANGED THE LEVELS OF PHOSPHOLIPIDS PRESENTING LOWER LEVELS OF 0) AND HIGHER LEVELS OF 03 AND 0% &URTHERMORE TRIACYLGLYCEROLS WERE ALSO HIGHER IN HYPOXIA PROBABLY CAUSED BY A LACK OF INOSITOL 502 COULD ALSO BE INDUCED AS RESULT OF HYPOXIC CONDITIONS AS IT HAS BEEN RECENTLY REPORTED CELLULAR STRESS AS A CONSEQUENCE OF HYPOXIA ;= WHICH WOULD EXPLAIN THE TRANSCRIPTIONAL CHANGES OCCURRING IN THE CELL IN THIS CONDITION
3URMA ET AL ;= REPORTED THE EXISTENCE OF A CROSSTALK BETWEEN UNFOLDED PROTEIN RESPONSE 502 )2% (!# THE ENDOPLASMIC RETICULUM ASSOCIATED PROTEIN DEGRADATION %2!$ MACHINERY 5"8 FATTY ACID METABOLISM /,% AND CONSEQUENTLY MEMBRANE LIPID SATURATION AND HOW 5"8 MODULATES /LEP LEVELS 4HE ACCUMULATION OF MISFOLDED PROTEINS IN THE %2 ACTIVATES THE 502 ;= AND THIS IS ESPECIALLY COMMON WHEN RECOMBINANT PROTEINS ARE EXPRESSED BUT PERTURBATIONS WITH LIPID METABOLISM CAN ALSO INDUCE 502 SIGNALLING ; =
$ESPITE REDUCED AMOUNTS OF ERGOSTEROL IN HYPOXIC CONDITIONS GENES OF THE ERGOSTEROL BIOSYNTHETIC PATHWAY WERE HIGHLY UPREGULATED DUE TO ITS DEPENDENCE ON OXYGEN AVAILABILITY -OLECULAR OXYGEN IS ESSENTIAL FOR ERGOSTEROL SYNTHESIS FROM SQUALENE REQUIRING OXYGEN MOLECULES IN THE WHOLE REACTION "IEN ET AL ;= SUGGESTED AN INTERPLAY BETWEEN HYPOXIC STRESS AND STEROL HOMEOSTASIS FOR 3 POMBE SHOWING HOW 3REP CONTROLS STEROL HOMEOSTASIS AND ALSO FUNCTIONS AS AN HYPOXIC TRANSCRIPTION FACTOR 7HEN STEROL SYNTHESIS DECREASES UNDER LOW OXYGEN 3REP IS PROTEOLYTICALLY ACTIVATED TO INCREASE TRANSCRIPTION OF GENES REQUIRED FOR ERGOSTEROL SYNTHESIS AND CELL GROWTH ;= ! SIMILAR EFFECT COULD BE OCCURRING IN 0 PASTORIS IN ORDER TO MAINTAIN LIPID HOMEOSTASIS !DDITIONALLY 3HARMA ;= SUGGESTED AN ADAPTIVE RESPONSE TO ALTERED STEROL STRUCTURES THROUGH CHANGES IN LIPID COMPOSITION AND mUIDITY THAT CAN BE ALSO PRESENT WHEN STEROL IS DEPRIVED %RGOSTEROL LEVELS IN THE CELL COULD BE INmUENCING CHANGES INTO THE ENTIRE LIPID COMPOSITION SINCE CHANGES OF MORE THAN OF 0, AND MORE THAN OF SPHINGOLIPID SPECIES HAVE BEEN REPORTED IN %2' MUTANTS OF 3 CEREVISIAE ;= POINTING OUT A CROSSTALK BETWEEN LIPID BIOSYNTHETIC PATHWAYS
%XCESS OF FATTY ACIDS THAT ARE DERIVED FROM ENDOGENOUS DE NOVO SYNTHESIS LIPID TURNOVER OR NUTRITIONAL SUPPLY ARE STORED AS TRIACYLGLYCEROLS IN CYTOSOLIC LIPID DROPLETS KEEPING THE TOTAL AMOUNT OF CELLULAR PHOSPHOLIPIDS WITHIN A NARROW RANGE ;= 4HE REGULATORY INTERPLAY AND METABOLIC INTERCONVERSION BETWEEN STORAGE LIPIDS IE TRIACYLGLYCEROLS AND MEMBRANE LIPIDS IE PHOSPHOLIPIDS HAVE BEEN RECOGNISED AS AN IMPORTANT DETERMINANT OF CELLULAR GROWTH AND PROLIFERATION IN YEAST ; = (YPOXIC CONDITIONS RESULTED IN HIGHER LEVELS OF TRIACYLGLYCEROLS AND STERYL ESTERS AS IT CAN BE OBSERVED IN &IGURE !S STATED BEFORE 4' INCREMENT CAN BE THE RESULT OF LOW LEVELS OF INOSITOL &GGFDU PG IZQPYJB JO 'BC' TUSBJO
Fab 2F5 Normoxia Hypoxia 200
-1 160
120
80
g lipid · mg total prot 40 μ
0 TG SE FS
ᎽᎳ &IGURE !MOUNTS OF NON POLAR ᎙GLIPID MGTOTAL PROT OF THE REFERENCE STRAIN &AB & IN NORMOXIC AND HYPOXIC CONDITIONS 4RIACYLGLYCEROLS 4' STERYL ESTERS 3% AND FREE STEROLS &3 ARE REPRESENTED
PRESENT IN THE CELL -OREOVER CHANGES IN GLUCOSE METABOLISM CAUSED BY THE SHIFT FROM RESPIRATORY TO RESPIROFERMENTATIVE METABOLISM CAN ALSO IMPACT NON POLAR LIPID HOMEOSTASIS BY CHANGING ACTIVITY OF 4' LIPASES ;= "OTH ENZYMES .TEP AND ,ROP WHICH TURNED OUT TO BE UPREGULATED IN HYPOXIA CATALYSE REACTIONS THAT EITHER DIRECTLY OR INDIRECTLY PROMOTE SYNTHESIS OF 4' AND CONTRIBUTE TO THE ADJUSTMENT OF THE MOLECULAR SPECIES COMPOSITION OF MEMBRANE PHOSPHOLIPIDS ;=
3PHINGOLIPIDS APART FROM ITS FUNCTION DElNING MEMBRANE STRUCTURE ASSOCIATE TO ERGOSTEROL TO FORM MICRODOMAINS vLIPID RAFTSv AND ALSO HAVE AN IMPORTANT ROLE AS SECOND MESSENGERS 4HAT IS CHANGES IN SPHINGOLIPID LEVELS CAN IMPACT ON CELL METABOLISM AS IT EXIST AN INTERCONNECTION BETWEEN SPHINGOLIPID BIOSYNTHESIS AND OTHER METABOLIC PATHWAYS ;=
Cer HexCer 50 100
40 80
30 60
20 40
Relative Peak Amount (%) Relative Peak 10 20
0 0
18:0;2/16:0;018:0;2/18:0;018:0;2/16:0;118:0;2/18:0;118:1;2/16:0;018:1;2/18:0;018:1;2/16:0;118:1;2/18:0;118:2;2/16:0;018:2;2/18:0;018:2;2/16:0;118:2;2/18:0;119:2;2/16:0;019:2;2/18:0;019:2;2/16:0;119:2;2/18:0;118:0;3/16:0;018:0;3/18:0;018:0;3/24:0;018:0;3/26:0;018:0;3/16:0;118:0;3/18:0;118:0;3/24:0;118:0;3/26:0;1 18:0;2/16:0;118:0;2/18:0;118:1;2/16:0;118:1;2/18:0;118:2;2/16:0;118:2;2/18:0;119:2;2/16:0;119:2;2/18:0;1
IPC MIPC M(IP)2C 100 100 100
80 80 80
60 60 60
40 40 40
20 20 20 Relative Peak Amount (%) Relative Peak
0 0 0
34:0;334:0;436:0;336:0;442:0;242:0;342:0;442:0;544:0;244:0;344:0;444:0;546:0;246:0;346:0;446:0;5 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4
&IGURE 2ELATIVE AMOUNTS OF SPHINGOLIPIDS RELATIVE PEAK AMOUNT OF THE REFERENCE STRAIN &AB & IN NORMOXIC AND HYPOXIC CONDITIONS 4RIACYLGLYCEROLS 4' STERYL ESTERS 3% AND FREE STEROLS &3 ARE REPRESENTED
3PHINGOLIPID PATHWAY WAS TIGHTLY UPREGULATED UNDER HYPOXIC CONDITIONS SINCE MOST OF THE REACTIONS INVOLVED IN THE PATHWAY REQUIRE OXYGEN *OUFHSBUFE BOBMZTJT
&IGURE SHOW HOW IN HYPOXIC CONDITIONS SOME CLASSES OF SPHINGOLIPIDS CHANGED THEIR RELATIVE AMOUNT IN CELL HOMOGENATES #ERAMIDES WITH POLYUNSATURATED FATTY ACID DECREASED WHICH IS CONSISTENT WITH THE FACT THAT LESS POLYUNSATURATED FATTY ACIDS WERE PRESENT IN HYPOXIA !N INCREASE OF CERAMIDES CARRYING METHYLATED MOIETIES WAS ALSO OBSERVED .OTABLY CERAMIDE DOUBLED ITS CONTENT ALTHOUGH 352 RESPONSIBLE TO TRANSFORM # INTO # FATTY ACYLS OF CERAMIDES WAS NOT UPREGULATED IN HYPOXIA (YPOXIA HAD ALSO AN EFFECT ON INOSITOL CONTAINING SPHINGOLIPIDS REDUCING RELATIVE AMOUNTS OF # IE CONTAINING # FATTY ACIDS SPECIES AND FAVOURING THE AMOUNTS OF # SPECIES IE CONTAINING # FATTY ACIDS WHICH IS IN AGREEMENT WITH THE PREVIOUS RESULTS WITH CERAMIDES 4HESE RESULTS COULD INDICATE THAT SPHINGOLIPID SPECIES WITH LONGER CHAIN FATTY ACIDS AND MORE METHYLATIONS THAT FORM MEMBRANES IN HYPOXIC CONDITIONS COULD HAVE AN IMPACT ON MEMBRANE PROPERTIES THAT RESULTS IN A BENElCIAL EFFECT ON &AB SECRETION
$ECREASED LEVELS OF ERGOSTEROL UNDER HYPOXIC CONDITIONS COULD BE INVOLVED IN CHANGES OF SPHINGOLIPID SPECIES OBSERVED IN CELL HOMOGENATES ! PLAUSIBLE EXPLANATION TO THESE CHANGES WOULD BE THAT CELLS ADAPT TO STEROL CHANGES BY ALTERING THE REST OF MEMBRANE LIPIDS IN ORDER TO MAINTAIN MEMBRANE HOMEOSTASIS 3UCH ADAPTATION WOULD BE MAINLY ACHIEVED THROUGH MODULATION OF THEIR SPHINGOLIPID COMPOSITION AND WOULD BE REmECTED IN THE OBSERVATION THAT THESE COMPOSITIONAL CHANGES WERE NOT RELATED TO AN ALTERED MEMBRANE mUIDITY MEASURED BY mUORESCENCE ANISOTROPY 3IMILAR RESULTS HAVE BEEN PREVIOUSLY REPORTED IN 3 CEREVISIAE BY 'UAN ET AL ;=
(YPOXIC CONDITIONS YIELD TO STRONG TRANSCRIPTIONAL REGULATION OF KEY PARAMETERS OF PROTEIN PRODUCTION AS ALREADY REPORTED BY "AUMANNETAL ;= )N THE CURRENT STUDY IT HAS BEEN CONlRMED THAT CHANGES IN CELLULAR PROCESSES SUCH AS LIPID METABOLISM LED TO ALTERED CHANGES ON RELATIVE AMOUNTS OF LIPIDS IN CELL HOMOGENATES WHICH IN TURN WOULD LEAD TO CHANGES IN THE COMPOSITION OF CELLULAR MEMBRANES 4HESE CHANGES MIGHT ALSO CONTRIBUTE TO THE OBSERVED ENHANCED LEVELS OF PROTEIN SECRETION THROUGH CHANGES ON MEMBRANE mUIDITY THAT PERMIT HIGHER SECRETION RATES AND NO RECOMBINANT PROTEIN ACCUMULATION WITHIN THE CELL !T THE SAME TIME IT HAS ALSO BEEN OBSERVED THAT ACTIVATION OF 502 AS A CONSEQUENCE OF BOTH RECOMBINANT PROTEIN PRODUCTION AND HYPOXIA IMPLY ALTERATION OF CELLULAR LIPID COMPOSITION I E ALTERED LIPID COMPOSITION COULD BE THE RESULT OF LOW OXYGEN ENVIRONMENTAL CONDITIONS AND PROTEIN PRODUCTION STRESSES
&GGFDU PG IZQPYJD DVMUVSF DPOEJUJPOT PO DFMM MJQJE DPNQPTJUJPO PG FOHJOFFSFE TUSBJOT )N ORDER TO DETERMINE SIGNIlCANT CHANGES ON CELL LIPID COMPOSITION AS A RESULT OF CELL ENGINEERING OF LIPID PATHWAYS AND HYPOXIC CULTURE CONDITIONS LOG OF THE FOLD CHANGE FOR EACH LIPID SPECIE COMPARING NORMOXIA VERSUS HYPOXIA WAS PERFORMED $ATA WAS PLOTTED AS A HEAT MAP WHERE REDUCTION OF LIPID LEVELS IS SHOWN IN GREEN NO CHANGES IN WHITE AND INCREASED LEVELS IN RED 4HIS METHOD ALLOWED EASILY OBSERVING SIGNIlCANT CHANGES ON LIPID COMPOSITION BETWEEN OXYGEN CONDITIONS FOR EACH STRAIN
4HE EFFECT OF HYPOXIC CONDITIONS COMPARED TO NORMOXIC CONDITIONS FOR EACH STRAIN OF THE PRESENT STUDY ARE SHOWN IN &IGURE 'ENERAL TRENDS COMMON AMONG ALL STRAINS WERE REDUCED AMOUNTS OF POLYUNSATURATED FATTY ACIDS PHOSPHATIDYLINOSITOL 0) AND SOME CERAMIDES BUILDED UP WITH &GGFDU PG IZQPYJB PO FOHJOFFSFE TUSBJOT
POLYUNSATURATED FATTY ACIDS 3OME OTHER LIPID SPECIES LIKE OLEIC ACID # TRIACYLGLYCEROLS 4' STERYL ESTERS 3% AND EPISTEROL INCREASED THEIR LEVELS WHEN STRAINS WERE CULTURED IN HYPOXIA )N ADDITION INOSITOL CONTAINING SPHINGOLIPIDS PRESENTED A DIVERSE ALTERATION OF THEIR SPECIES THAT WAS STRAIN DEPENDENT 2EDUCED AMOUNTS OF ERGOSTEROL DUE TO HYPOXIC CONDITIONS RESULTED IN HIGHER AMOUNTS OF STEROL INTERMEDIATES
4HE EFFECT OF HYPOXIA IN THE ́$ES STRAIN WHEN COMPARED TO THE EFFECT ON THE REFERENCE STRAIN &AB & EXHIBITED A HIGHER INCREMENT ON 3%S AND REDUCED LEVELS OF CERAMIDES AND HEXOSYLCERAMIDES &URTHERMORE CHANGES IN THE LEVELS OF THE -)0# WERE NOT AS SEVERE AS THE ONES OBSERVED IN THE REFERENCE STRAIN 4HE STRAIN OVEREXPRESSING $%3 /% $ES HYPOXIC CONDITIONS LED TO CHANGES ON LIPID COMPOSITION THAT APPEARED TO BE THE OPPOSITE OF THE REFERENCE STRAIN )T WAS THE CASE OF CEROTIC ACID # MOST OF THE CERAMIDES HEXOSYLCERAMIDES AND SOME INOSITOL CONTAINING
SPHINGOLIPIDS LIKE )0#S AND ALL -)0# AND -)0 ኼ# SPHINGOLIPID SPECIES -AJOR CHANGES ON ́3UR STRAIN DUE TO HYPOXIA WERE THE REDUCTION OF PHOSPHOLIPID LEVELS )0# AND ACCOMPANIED WITH A HIGH INCREASE OF CERAMIDE # HEXOSYLCERAMIDE # AND -)0# &LUCONAZOLE TREATED CELLS EXHIBITED MAJOR DIFFERENCES ON STEROLS AS A HIGH INCREMENT OF STEROL INTERMEDIATES WERE OBSERVED DUE TO HYPOXIC CONDITIONS COMPARED TO THE HYPOXIC EFFECT ON THE REFERENCE STRAIN %FFECT OF HYPOXIA ON OVEREXPRESSING %RG CELLS RESULTED IN LOWER LEVELS OF CEROTIC ACID FREE STEROLS CERAMIDE HEXOSYLCERAMIDE AND )0# -)0#
AND -)0 ኼ# SPECIES )N ADDITION LEVELS OF METYLZYMOSTEROL ZYMOSTEROL AND )0# SHOWED A HIGHER INCREMENT THAN IN THE REFERENCE STRAIN &INALLY EFFECT OF HYPOXIA ON THE STRAIN OVEREXPRESSING (AC HAD AN INCREMENT ON LYSOPHOSPHOLIPIDS METHYLZYMOSTEROL AND HEXOSYLCERAMIDE AND DECREASED LEVELS OF CEROTIC ACID ZYMOSTEROL CERAMIDE HEXOSYLCERAMIDE AND
)0# AND -)0 ኼ# *OUFHSBUFE BOBMZTJT
&IGURE (EAT MAP OF THE EFFECT OF HYPOXIA ON THE LIPID SPECIES FOR EACH STRAIN 4HE LOG OF THE &# OF HYPOXIA VERSUS NORMOXIA IS PLOTTED IN ORDER TO SEE MAJOR DIFFERENCES &GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI TQIJOHPMJQJE QBUIXBZ
&GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI TQIJOHPMJQJE QBUIXBZ
0RINCIPAL CHANGES IN TERMS OF LIPID COMPOSITION OF ALL THE STRAINS GENERATED IN THIS STUDY WHEN COMPARED TO THE REFERENCE STRAIN ARE SHOWN IN &IGURE 4HESE CHANGES WERE OBTAINED FROM NORMOXIC CONDITIONS IN ORDER TO STUDY THE EFFECT OF GENETIC MODIlCATIONS IN TERMS OF CELL PHYSIOLOGY )N GENERAL TERMS THE MOST SIGNIlCANT CHANGES WERE OBSERVED FOR SPHINGOLIPID COMPOSITION WITH THE MOST IMPORTANT CHANGES OBSERVED FOR THE STRAINS WHOSE SPHINGOLIPID PATHWAY WAS ENGINEERED )N CONTRAST STRAINS WITH ENGINEERED STEROL PATHWAY DID NOT PRESENT SIGNIlCANT CHANGES ON THE LEVELS OF THE SPECIES INVOLVED IN THE STEROL PATHWAY
/NE OF THE MOST SIGNIlCANT CHANGES WAS THAT ALL STRAINS CONTAINED LOWER LEVELS OF CARDIOLIPIN #, COMPARED TO THE REFERENCE STRAIN ,EVELS OF CARDIOLIPIN IN THE REFERENCE STRAIN WERE HIGHER THAN THE USUAL WITH LEVELS MORE SIMILAR TO THE ONES OBSERVED IN MITOCHONDRIAL MEMBRANES WHICH ARE ENRICHED IN THIS PHOSPHOLIPID #, PLAYS AN IMPORTANT ROLE NOT ONLY IN MITOCHONDRIAL BIOGENESIS BUT ALSO IN ESSENTIAL CELLULAR FRACTIONS NOT GENERALLY ASSOCIATED WITH RESPIRATORY FUNCTIONS EG MITOCHONDRIAL PROTEIN IMPORT CELL WALL BIOGENESIS TRANSLATIONAL REGULATION OF ELECTRON TRANSPORT CHAIN COMPONENT AGEING APOPTOSIS ;= )T HAS BEEN REPORTED THAT #, IS ENRICHED IN CONTACT SITES ;= (IGH LEVELS OF #, HAVE NOT BEEN REPORTED BEFORE *OUFHSBUFE BOBMZTJT
&IGURE (EAT MAP OF THE STRAINS VERSUS REFERENCE STRAIN IN NORMOXIC CONDITIONS ,OG OF THE &# WAS PLOTTED IN ORDER TO SEE MAJOR DIFFERENCES &GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI TQIJOHPMJQJE QBUIXBZ
&OHJOFFSJOH OFVUSBM TQIJOHPMJQJE QBUIXBZ UISPVHI %&4
4WO MAJOR CLASSES OF COMPLEX SPHINGOLIPIDS HAVE BEEN IDENTIlED IN 0 PASTORIS NAMELY THE
INOSITOL CONTAINING SPHINGOLIPIDS )0# -)0# AND -)0 ኼ# AND THE NEUTRAL SPHINGOLIPIDS #ER AND (EX#ER ;= 4HESE SPHINGOLIPID CLASSES VARY IN THEIR POLAR HEAD GROUPS AS WELL AS THEIR CERAMIDE BACKBONES ;= 4HE PATHWAY LEADING TO GLUCOSYLCERAMIDE BIOSYNTHESIS IS MAINLY COMPOSED BY FOUR ENZYMES "ARP WHICH OCCUPIES THE KEY BRANCH POINT PREFERRING DIHYDROXYL SPHINGOID BASES AND ## ACYL #O! AS SUBSTRATES AND PRODUCING CERAMIDE SPECIES SPHINGOLIPID ́ AND ́ DESATURASES FATTY ACID HYDROXYLASE AND A GLUCOSYLCERAMIDE SYNTHASE THAT ADDS THE GLUCOSE HEAD GROUP
4ERNES ET AL ;= REPORTED THAT DELETION OF ́ DESATURASE IN 0 PASTORIS WILD TYPE RESULTS IN ABSENCE OF GLUCOCERAMIDES (EX#ER IN THE CELL 4HE GENE ENCODING FOR THIS ENZYME WAS SELECTED AS TARGET FOR CELL ENGINEERING OF OUR RECOMBINANT PROTEIN STRAIN IN ORDER TO DETERMINE LEVELS OF PROTEIN SECRETION DUE TO CHANGES ON CERAMIDE AMOUNTS IN CELL MEMBRANES &IGURE SHOWS THE LIPID PROlLE OF THE REFERENCE STRAIN PRODUCING THE RECOMBINANT &AB AND THE STRAINS GENERATED IN THIS STUDY CARRYING A DELETION OF THE GENE ENCODING FOR THE ́ DESATURASE ́$%3 OR OVEREXPRESSING IT )T CAN BE OBSERVED THAT ONLY CERAMIDES AND HEXOSYLCERAMIDES COMPOSED BY SATURATED ,#" WERE PRESENT IN CELL HOMOGENATES OF THE ́$%3 STRAIN AS IT WOULD BE EXPECTED BY THE ABSENCE OF THE ́ DESATURASE INVOLVED IN THE UNSATURATION OF THE ,#" AND REQUIRED FOR THE ACTIVITY OF THE ́ DESATURASE #ERAMIDE SPECIES DETECTED IN ́$%3 HOMOGENATES ARE IN CONCORDANCE TO THE ONES PRESENT IN 4ERNES STUDIES ;= WHERE NEITHER MONO NOR DIUNSATURATED #ER COULD BE DETECTED IN ́$%3 STRAIN BUT LEVELS OF SATURATED #ER WERE ENHANCED IN COMPARISON TO THE WILD TYPE !CCORDING TO 4ERNES ET AL RESULTS A LACK OF HEXOSYLCERAMIDES WOULD BE EXPECTED FOR THE STRAIN ́$%3 )NSTEAD WE DETECTED THE PRESENCE OF SOME (EX#ER SPECIES IN ́$%3 CELL HOMOGENATES ALTHOUGH THE SPECIES THAT WERE PRESENT WERE TOTALLY DIFFERENT FROM THE ONES PRESENT IN THE REFERENCE STRAIN 4HE WAS THE MOST PREDOMINANT SPECIE IN ́$%3 CELLS AND ALMOST NOT DETECTED IN THE REFERENCE STRAIN WHERE THE MAJOR SPECIE WAS 4HESE RESULTS ARE IN CONTRAST WITH THE ONES OF 4ERNES ET AL PREVIOUSLY MENTIONED
)T SHOULD BE MENTIONED THAT RELATIVE AMOUNTS OF SPHINGOLIPIDS WERE DETERMINED HOWEVER TOTAL AMOUNTS OF SPHINGOLIPIDS WERE NOT QUANTIlED 4HEREFORE ONLY DIFFERENCES BETWEEN SPECIES OF THE SAME SPHINGOLIPID CLASS CAN BE NOTED (ENCE IT COULD BE POSSIBLE THAT RELATIVELY SMALL AMOUNTS OF CERAMIDES WOULD BE PRESENT IN CELL HOMOGENATES COMPARED TO THE REFERENCE STRAIN 4HE SAME SITUATION COULD BE OCCURRING TO THE STRAIN OVEREXPRESSING $%3 WHICH COULD BE CONTAINING HIGHER LEVELS OF CERAMIDES AND HEXOSYLCERAMIDES COMPARED TO THE REFERENCE STRAIN
)T WAS ALSO OBSERVED THE PRESENCE OF RELATIVE AMOUNTS OF )0# SPECIES OF AND CARBONS IN THE KNOCKOUT STRAIN WHICH WERE NOT PRESENT IN THE REFERENCE STRAIN 4ERNES ET AL INVESTIGATED A
POSSIBLE INTERCONNECTION BETWEEN BIOSYNTHESIS OF (EX#ER AND )0# -)0# AND -)0 ኼ# AND THEY DETERMINED THAT SOME CERAMIDES WERE INCORPORATED INTO )0# TO A LIMITED EXTENT BUT THAT THESE
)0# SPECIES WERE NOT PROCESSED FURTHER INTO -)0# AND -)0 ኼ# 4HIS FACT CAN BE THE REASON OF THE PRESENCE OF )0# SPECIES WITH FEWER CARBONS WHICH WOULD BE GENERATED FROM THE CERAMIDE SPECIES WITH THIS CARBON COMPOSITION *OUFHSBUFE BOBMZTJT
Fab Mutant vs Reference
2.5
2.0
1.5
1.0 c production rate fold change fi
0.5 speci ΔDes1 Des1
FA PL Fab 2F5 ΔDes1 Des1 60
50 -1 40
30
20 % FA · mgtotal prot % FA
10
0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C26:0 Others
HexCer Cer 60 100
50 80
40 60 30 40 20
Relative Peak Amount (%) Relative Peak 10 20
0 0
18:0;2/16:0;018:0;2/18:0;018:0;2/16:0;118:0;2/18:0;118:1;2/16:0;018:1;2/18:0;018:1;2/16:0;118:1;2/18:0;118:2;2/16:0;018:2;2/18:0;018:2;2/16:0;118:2;2/18:0;119:2;2/16:0;019:2;2/18:0;019:2;2/16:0;119:2;2/18:0;118:0;3/16:0;018:0;3/18:0;018:0;3/24:0;018:0;3/26:0;018:0;3/16:0;118:0;3/18:0;118:0;3/24:0;118:0;3/26:0;1 18:0;2/16:0;118:0;2/18:0;118:1;2/16:0;118:1;2/18:0;118:2;2/16:0;118:2;2/18:0;119:2;2/16:0;119:2;2/18:0;1
IPC MIPC M(IP)2C 100 100 100
80 80 80
60 60 60
40 40 40
20 20 20 Relative Peak Amount (%) Relative Peak
0 0 0
34:0;334:0;436:0;336:0;442:0;242:0;342:0;442:0;544:0;244:0;344:0;444:0;546:0;246:0;346:0;446:0;5 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4
Sterol 2F5 KO Des1 SE Des1 Non-polar 12 200 40
-1 10 160 -1 -1 30 8 120 6 20 80 4 g TG · mg total prot
μ 10 g SE/FS · mg total prot
40 μ g Sterol · mg total prot. 2 μ
SETGFS SE TG FS SE TG FS 0 0 0 Fab 2F5 ΔDes1 Des1
squalene lanosterol Episterol Zymosterol Ergosterol Ergostatraenol
4-Methylzymosterol 14- methylfecosterol ergosta5,7,22-trien3ol
ergosta 5 8 22 24 tetraenol
4, 14-dimethylcholesta-8,24-dienol
&IGURE 'ENERAL OVERVIEW OF THE RECOMBINANT PROTEIN PRODUCTION AND LIPID COMPOSITION OF THE CELLS CARRYING A DELETION OF THE GENE $%3 ORANGE OR WITH THE GENE OVEREXPRESSED GREEN AND THE VALUES OF THE REFERENCE STRAIN BLUE WITHOUT THE GENETIC MODIlCATIONS &GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI TQIJOHPMJQJE QBUIXBZ
$ISTRIBUTION OF CERAMIDES AND HEXOSYLCERAMIDES IN THE STRAIN OVEREXPRESSING $ES WAS MORE SIMILAR TO THE REFERENCE STRAIN COMPOSED BY THE SAME SPHINGOLIPID SPECIES ALTHOUGH ENRICHED WITH SPECIES FORMED WITH SATURATED VERY LONG CHAIN FATTY ACYLS !PART FROM SPHINGOLIPID ONLY NON POLAR LIPIDS HAD DIFFERENCES WITH THE REFERENCE STRAIN )N THIS SENSE LEVELS OF TRIACYLGLYCEROLS 4' FOR BOTH ́$ES AND /% $ES WERE LOWER THAN THE REFERENCE STRAIN WHILE LEVELS OF STERYL ESTERS AND FREE STEROLS WERE SLIGHTLY LOWER AND HIGHER RESPECTIVELY
!CCORDING TO THE OBSERVED RESULTS CERAMIDES ARE IMPORTANT FOR CELL SECRETION AS ABSENCE OF THIS SPHINGOLIPID CLASS LEAD TO LOWER LEVELS OF &AB SECRETION -OREOVER WE CAN NOT DETERMINE WHETHER OVEREXPRESSION OF ́ DESATURASE RESULTS IN HIGHER AMOUNTS OF NEUTRAL SPHINGOLIPIDS BUT CHANGES ON THE RELATIVE AMOUNTS OF SPHINGOLIPIDS SPECIES WERE OBSERVED AND THEY RESULT IN SLIGHT INCREASE ON THE LEVELS OF SECRETED &AB AS OBSERVED IN &IGURE &AB PANEL
"MUFSJOH JOPTJUPM DPOUBJOJOH TQIJOHPMJQJET QBUIXBZ 463
3URP IS REQUIRED FOR HYDROXYLATION OF DIHYDROSPHINGOSINE AT # POSITION TO YIELD PHYTOSPHINGOSINE IN ONE OF THE lRST STEPS OF THE INOSITOL CONTAINING SPHINGOLIPID )0# BIOSYNTHESIS $ELETION OF 352 IN 3 CEREVISIAE RESULTS IN VIABLE STRAINS LACKING COMPLETELY # HYDROXYLATED SPHINGOLIPIDS IE PHYTOSPHINGOSINE AND ACCUMULATION OF DIHYDROSPHINGOSINE AND DIHYDROSPHINGOSINE PHOSPHATE ;= 4HESE OBSERVATIONS INDICATE THAT # HYDROXYLATION OF THE ,#" IS NOT REQUIRED FOR )0# BIOSYNTHESIS IN THIS ORGANISM ; = !DDITIONALLY 352 MUTANTS WERE REPORTED TO DISPLAY ALTERED PHOSPHOLIPID COMPOSITION AND ABNORMAL MORPHOLOGY IN STATIONARY PHASE ;= +LOSE ET AL ;= OBSERVED LOWER MEMBRANE ORDER OF 352 MUTANTS WHICH HAVE BEEN SHOWN TO TRIGGER DEFECTIVE PLASMA MEMBRANE TRANSPORT OF INTEGRAL MEMBRANE PROTEINS ; =
4ERNES ET AL COULD NOT OBTAIN 0 PASTORIS STRAINS WITH DELETED 352 AND PROPOSED # HYDROXYLATION OF THE ,#" TO BE REQUIRED FOR THE ,AGP SYNTHASE TO USE IT FOR )0# BIOSYNTHESIS )N THE CURRENT STUDY A STRAIN WITH 352 WAS OBTAINED SUGGESTING THAT REQUIREMENTS OF BOTH 0 PASTORIS AND 3 CEREVISIAE ARE SIMILAR REGARDING INOSITOL CONTAINING SPHINGOLIPIDS
&IGURE #ER PANEL SHOWS HOW CERAMIDE SPECIES WITH TRIHYDOXYLATED ,#" WERE ALMOST ABSENT IN ́352 STRAIN AS A CONSEQUENCE OF THE LACK OF # HYDOXYLASE IN THE KNOCKOUT STRAIN 2ELATIVE AMOUNTS OF HEXOSYLCERAMIDES AND THE SPECIES THAT CONSTITUTED THEM WERE SIMILAR BETWEEN THE KNOCKOUT STRAIN AND THE REFERENCE STRAIN
.OTABLY THE MAIN DIFFERENCES WERE OBSERVED WITH )0# -)0# AND -)0 ኼ# WHERE SPECIES PRESENT IN THE ́352 STRAIN WERE DERIVED FROM DIHYDROSPHINGOSINE IN CONTRAST WITH THE REFERENCE STRAIN WHICH CONTAINED SPECIES DERIVED FROM PHYTOSPHINGOSINE 4HESE OBSERVATIONS ARE IN AGREEMENT WITH THE RESULTS REPORTED FOR 3 CEREVISIAE STRAINS WITH THE SAME DELETION !N INCREMENT ON THE RELATIVE AMOUNTS OF SPHINGOLIPIDS WITH SHORTER CHAINS WAS ALSO OBSERVED IN THE ́352 STRAIN 3HORTENING OF VERY LONG FATTY ACIDS AS IT WAS OBSERVED FOR THE 0 PASTORIS ́352 STRAIN COULD HAVE AN EFFECT ON SORTING AND PLASMA MEMBRANE COMPOSITION *OUFHSBUFE BOBMZTJT
Fab
FA Fab 2F5 ΔSur2 PL Fab 2F5 ΔSur2 60 70
50 -1 60
-1 50 40 40 30 30
20 · mg total prot % PL % FA · mgtotal prot % FA 20
10 10
0 0 PC PA PI PS Lyso-PL PE CL DMPE C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C26:0 Others Cer HexCer 30 100
80
20 60
40 10 20 Relative Peak Amount (%) Relative Peak
0 0
18:0;2/16:0;018:0;2/18:0;018:0;2/16:0;118:0;2/18:0;118:1;2/16:0;018:1;2/18:0;018:1;2/16:0;118:1;2/18:0;118:2;2/16:0;018:2;2/18:0;018:2;2/16:0;118:2;2/18:0;119:2;2/16:0;019:2;2/18:0;019:2;2/16:0;119:2;2/18:0;118:0;3/16:0;018:0;3/18:0;018:0;3/24:0;018:0;3/26:0;018:0;3/16:0;118:0;3/18:0;118:0;3/24:0;118:0;3/26:0;1 18:0;2/16:0;118:0;2/18:0;118:1;2/16:0;118:1;2/18:0;118:2;2/16:0;118:2;2/18:0;119:2;2/16:0;119:2;2/18:0;1
IPC MIPC M(IP)2C 100 100 100
80 80 80
60 60 60
40 40 40
20 20 20 Relative Peak Amount (%) Relative Peak
0 0 0
34:0;334:0;436:0;336:0;442:0;242:0;342:0;442:0;544:0;244:0;344:0;444:0;546:0;246:0;346:0;446:0;5 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4
Sterols 2F5 KO Sur2 Non-polar 12 200 40
10 -1
160 -1 -1 30 8 120 6 20
4 80 g TG · mg total prot
μ 10 g SE/FS · mg total prot g Sterol · mg total prot. 2 μ μ 40
0 SETGFS SE TG FS 0 0 Fab 2F5 ΔSur2 squalene lanosterol Episterol Zymosterol Ergosterol Ergostatraenol
4-Methylzymosterol 14- methylfecosterol ergosta5,7,22-trien3ol
ergosta 5 8 22 24 tetraenol
4, 14-dimethylcholesta-8,24-dienol
&IGURE 'ENERAL OVERVIEW OF THE RECOMBINANT PROTEIN PRODUCTION AND LIPID COMPOSITION OF THE CELLS CARRYING A DELETION OF THE GENE 352 PURPLE AND THE VALUES OF THE REFERENCE STRAIN BLUE WITHOUT THE GENETIC MODIlCATIONS &GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI FSHPTUFSPM QBUIXBZ
7HEREAS FATTY ACID AND PHOSPHOLIPID IN CONTRAST WITH THE RESULTS FROM 3 CEREVISAE ;= DID NOT CONTAIN SIGNIlCANT CHANGES ́352 EXHIBITED REDUCED LEVELS OF ERGOSTEROL IN CELL HOMOGENATES &URTHERMORE LEVELS OF STERYL ESTERS AND TRIACYLGLYCEROLS TURNED OUT BEING SIGNIlCANTLY HIGHER IN THE KNOCKOUT STRAIN 3IMILAR INCREMENT IN 3% AND 4' WAS ALSO OBSERVED FOR CELLS UNDER HYPOXIC CONDITIONS #HANGES ON LIPID COMPOSITION IN THE STRAIN WITH 352 DELETED FAVOURED THE SECRETION OF &AB BY FOLD COMPARED TO THE REFERENCE STRAIN &IGURE &AB PANEL 4HE AMOUNTS OF INCREASED PRODUCTIVITY ARE CLOSE TO THE FOLD INCREMENT OBSERVED AS A RESULT OF CULTIVATION UNDER HYPOXIC CONDITIONS .ONETHELESS CHANGES ON LIPID COMPOSITION IN THE KNOCKOUT STRAIN AND THE REFERENCE STRAIN IN HYPOXIA ARE DISTINCT 4HE CHARACTERISTIC PHOSPHATIDYLINOSITOL 0) REDUCTION ON HYPOXIC CONDITIONS WAS NOT PRESENT IN THE ́352 CELLS WHICH SLIGHTLY INCREASED ITS 0) RELATIVE AMOUNTS 3PHINGOLIPID COMPOSITION WAS TOTALLY DIFFERENT BETWEEN BOTH CONDITIONS &IGURE WITH THE COMMON LIPID PATTERNS OBSERVED IN REDUCED LEVELS OF ERGOSTEROL 4ABLE AND LEVELS OF NON POLAR LIPIDS )NCREASED AMOUNTS OF TRIACYLGLYCEROLS AND STERYL ESTERS WERE OBSERVED WHILE THE INCREMENT OF THE 3% IN ́352 WAS MUCH HIGHER THAN IN HYPOXIA &URTHERMORE LEVELS OF FREE STEROLS WERE REDUCED IN BOTH CASES WHEN COMPARED TO THE REFERENCE STRAIN IN NORMOXIC CONDITIONS 4ABLE
&INALLY WHEN ́352 CELLS WERE CULTURED IN HYPOXIA MOST OF THE CHANGES ALREADY DESCRIBED FOR THE REFERENCE STRAIN IN TERMS OF PHOSPHOLIPID FATTY ACIDS AND NEUTRAL LIPID COMPOSITION WERE ALSO OBSERVED (OWEVER LEVELS OF ERGOSTEROL OF THE KNOCKOUT STRAIN IN HYPOXIC CONDITIONS DID NOT DECREASE AS COMPARED TO THE CELLS IN NORMOXIC CONDITIONS BUT THEY RATHER SLIGHTLY INCREASE ! POSSIBLE EXPLANATION FOR THE ABSENCE OF SYNERGIC EFFECT ON ERGOSTEROL LEVELS DUE TO BOTH GENETIC MODIlCATIONS IE GENE DELETION AND ENVIRONMENTAL CONDITIONS IE HYPOXIA COULD BE THAT ERGOSTEROL IS ESSENTIAL FOR CELL GROWTH AND CELL WOULD MAINTAIN MINIMAL ERGOSTEROL LEVELS IN ORDER TO SURVIVE 0RESENCE OF SPHINGOLIPIDS WITH SHORTER CHAIN OF THE FATTY ACID MOIETY THAT WAS PRESENT IN ́352 CELLS INCREASED THEIR RELATIVE AMOUNTS IN THE CELLS CULTURED IN HYPOXIC CONDITIONS !LL THESE MODIlCATIONS INCREASED THE LEVELS OF &AB SECRETED IN HYPOXIA BUT IN THIS CASE THE LEVELS WERE LOWER THAN THE FOLD OBSERVED IN THE REFERENCE STRAIN POSSIBLY BECAUSE CELLS HAD A LIMITATION OF &AB SECRETION
&GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI FSHPTUFSPM QBUIXBZ &LUCONAZOLE IS AN AZOLE ANTIFUNGAL AGENT THAT BLOCKS THE ERGOSTEROL BIOSYNTHESIS PATHWAY BY INHIBITING THE %RGP ACTIVITY RESULTING IN ERGOSTEROL DEPLETION ;= &LUCONAZOLE TREATED CELLS WERE USED IN ORDER TO REDUCE LEVELS OF ERGOSTEROL AS PREVIOUSLY REPORTED BY "AUMANN ET AL ;= &URTHERMORE %2' WAS OVEREXPRESSED IN THE &AB PRODUCING STRAIN IN ORDER TO CHARACTERISE THE EFFECT OF ALTERED ERGOSTEROL PATHWAY ON THE LEVELS OF &AB SECRETED "AMMERT ET AL ;= STUDIED THE TRANSCRIPTIONAL CHANGES IN 3 CEREVISAE IN RESPONSE TO TREATMENT WITH ANTIFUNGAL AGENTS 4HESE CHANGES INCLUDED DIRECTLY RELATED GENES OF THE ERGOSTEROL PATHWAY BUT ALSO OTHER GENES RELATED TO MEMBRANE STRUCTURE AND FUNCTION INCLUDING THE DECREASE OF 352 TRANSCRIPT LEVELS !S IT CAN BE OBSERVED IN &IGURE CELL HOMOGENATES OF mUCONAZOLE TREATED CELLS PRESENTED LOWER RELATIVE AMOUNTS OF MONOHYDROXYLATED FATTY ACIDS &! PANEL WHICH WAS COMPENSATED BY A SLIGHT INCREMENT OF LINOLEIC ACID AND CEROTIC ACID 4HEY ALSO EXHIBIT INCREASED RELATIVE AMOUNTS OF PHOSPHATODYLCHOLINE PHOSPHATIDIC ACID AND PHOSPHATIDYLSERINE 0, PANEL *OUFHSBUFE BOBMZTJT
Fab Mutant vs Reference 2.5
2.0
1.5
1.0 c production rate fold change fi
0.5 speci Erg11 Fluco
FA Fab 2F5 Erg11 Fluco PL Fab 2F5 Erg11 Fluco 60 70 -1 50 60
-1 50 40 40 30 30
20 % PL · mg total prot % PL % FA · mgtotal prot % FA 20
10 10
0 0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C26:0 Others PC PA PI PS Lyso-PL PE CL DMPE
Cer HexCer 50 100 40 80 30 60
20 40
Relative Peak Amount (%) Relative Peak 10 20
0 0
18:0;2/16:0;018:0;2/18:0;018:0;2/16:0;118:0;2/18:0;118:1;2/16:0;018:1;2/18:0;018:1;2/16:0;118:1;2/18:0;118:2;2/16:0;018:2;2/18:0;018:2;2/16:0;118:2;2/18:0;119:2;2/16:0;019:2;2/18:0;019:2;2/16:0;119:2;2/18:0;118:0;3/16:0;018:0;3/18:0;018:0;3/24:0;018:0;3/26:0;018:0;3/16:0;118:0;3/18:0;118:0;3/24:0;118:0;3/26:0;1 18:0;2/16:0;118:0;2/18:0;118:1;2/16:0;118:1;2/18:0;118:2;2/16:0;118:2;2/18:0;119:2;2/16:0;119:2;2/18:0;1
IPC MIPC M(IP)2C 100 100 100
80 80 80
60 60 60
40 40 40
20 20 20 Relative Peak Amount (%) Relative Peak
0 0 0
34:0;334:0;436:0;336:0;442:0;242:0;342:0;442:0;544:0;244:0;344:0;444:0;546:0;246:0;346:0;446:0;5 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4
Sterols 2F5 SE Erg11 Fluco Non-polar 12 200 40
-1 10
160 -1 -1 30 8 120 6 20 80 4 g TG · mg total prot
μ 10 g SE/FS · mg total prot
40 μ g Sterol · mg total prot. 2 μ SETGFS SE TG FS SE TG FS 0 0 0 Fab 2F5 Erg11 Fluco
squalene lanosterol Episterol Zymosterol Ergosterol Ergostatraenol
4-Methylzymosterol 14- methylfecosterol ergosta5,7,22-trien3ol
ergosta 5 8 22 24 tetraenol
4, 14-dimethylcholesta-8,24-dienol
&IGURE 'ENERAL OVERVIEW OF THE RECOMBINANT PROTEIN PRODUCTION AND LIPID COMPOSITION OF THE CELLS OVEREXPRESSING THE GENE %2' MAROON OR TREATED WITH mUCONAZOLE KHAKI AND THE VALUES OF THE REFERENCE STRAIN BLUE WITHOUT THE GENETIC MODIlCATIONS &GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI FSHPTUFSPM QBUIXBZ
!S EXPECTED A REDUCTION OF ERGOSTEROL WAS PRESENT IN THE CELLS AND THE STEROL INTERMEDIATE LANOSTEROL WHICH IS INVOLVED IN THE %RG REACTION WAS PRESENT IN CELL HOMOGENATES !CCUMULATION OF LANOSTEROL AND OTHER STEROL PRECURSORS IN CELLS TREATED WITH mUCONAZOLE HAS BEEN PREVIOUSLY REPORTED FOR OTHER YEASTS ; = 2EGARDING SPHINGOLIPID CONTENT mUCONAZOLE TREATED CELLS PRESENTED REDUCED AMOUNTS OF CERAMIDES CONTAINING DIHYDROSPHINGOSINE AND SIGNIlCANT INCREMENT ON RELATIVE LEVELS OF CERAMIDES COMPOSED BY PHYTOSPHINGOSINE AND # # FATTY ACYLS (EXOSYLCERAMIDES AND INOSITOL CONTAINING SPHINGOLIPIDS LEVELS KEPT SIMILAR TO THE REFERENCE STRAIN !DDITIONALLY NON POLAR LIPIDS INCREASED SIGNIlCANTLY THE TRIACYLGLYCEROL CONTENT ON mUCONAZOLE TREATED CELLS ,EVELS OF STERYL ESTERS WERE SLIGHTLY REDUCED WHILE FREE STEROLS INCREASED COMPARED TO THE REFERENCE STRAIN %STERIlCATION OF STEROLS IS A REGULATORY MECHANISM WHICH PROTECTS CELL MEMBRANES AGAINST EXCESS OF LIPID COMPONENTS AND MAINTAINING STEROL HOMEOSTASIS ;=
3TERYL ESTERS AND FREE STEROLS ARE INTERCONVERTIBLE )T HAS BEEN REPORTED THAT UNDER STEROL DEPLETION STERYL ESTERS ARE MOBILISED TO FREE STEROLS AS THEY ARE REQUIRED FOR PLASMA MEMBRANE FORMATION ;= !ZOLES ARE KNOWN TO INDUCE EXPRESSION OF %2' M2.! ;= WHICH WAS ALSO OBSERVED FOR CELLS CULTURED IN HYPOXIA (OWEVER REDUCTION OF ERGOSTEROL LEVELS AND INDUCED EXPRESSION OF BOTH mUCONAZOLE TREATED AND CELLS CULTURED IN HYPOXIA DID NOT TOTALLY COINCIDE WITH THE OTHER LIPID SPECIES ALTHOUGH THESE CHANGES WERE MORE SIMILAR THAN THE ONES OBSERVED WITH 352 DELETION #HANGES IN LIPID COMPOSITION DUE TO mUCONAZOLE TREATMENT INCREASED &AB SECRETION BY FOLD 4HESE OBSERVATIONS COULD BE EXPLAINED EITHER BECAUSE ADAPTIVE RESPONSE TO LOW ERGOSTEROL CONTENT TRIGGERED BY mUCONAZOLE TREATMENT BY CHANGING LIPID COMPOSITION DIFFERS FROM THE ONE CAUSED IN HYPOXIC CONDITIONS OR BECAUSE HYPOXIC CONDITIONS LEAD TO SEVERAL CHANGES IN THE CELL BEING LOW ERGOSTEROL CONTENT ONE OF THEM AND PROVOKING A DEEPER ALTERATION OF CELL LIPIDOME )T WAS ALSO OBSERVED THAT A SYNERGIC EFFECT OF mUCONAZOLE AND HYPOXIC CONDITIONS DID NOT EXIST
/VEREXPRESSING %2' CELL HOMOGENATES ALSO PRESENT CHANGES ON LIPID COMPOSITION 3TRIKINGLY THE AMOUNTS OF ERGOSTEROL IN THIS STRAIN WERE LOWER THAN IN THE REFERENCE STRAIN 3TEROL DEMETHYLASE %2' TOGETHER WITH SQUALENE EPOXIDASE %2' ARE THE MAJOR REGULATORY STEPS IN THE POST SQUALENE PART OF THE ERGOSTEROL PATHWAY 6EEN ET AL ;= OVEREXPRESSED %2' IN 3 CEREVISIAE OBTAINING DECREASED LANOSTEROL CONTENT AND THE LATTER STEROL INTERMEDIATES INCREASED AND THE TOTAL STEROL CONTENT ROSE UP TO FOLD MAINLY CONSISTING IN PRECURSORS AND NOT ERGOSTEROL 4HIS INDICATES ADDITIONAL REGULATION MECHANISMS IN THE CELL IN ORDER TO PREVENT QUANTITATIVE CONVERSION INTO ERGOSTEROL /VEREXPRESSION STUDIES OF %2' IN 3 CEREVISIAE HAVE SHOWN THAT EVEN THE GENE WAS HIGHLY EXPRESSED ONLY OF THE PROTEIN EXISTED AS ACTIVE ENZYME ;= 4HIS IS BECAUSE STEROL DEMETHYLASE IS DEPENDENT OF .!$0( AND HEME ;= AND ITS ASSOCIATION WITH HEME IS REQUIRED TO BE ACTIVE IN VIVO )N OUR STUDIES LEVELS OF FREE STEROLS INCREASED TRIACYLGLYCEROLS DECREASED AND STERYL ESTERS REMAINED SIMILAR ! FOLD INCREMENT OF FREE STEROLS WAS OBSERVED IN THE /% %RG STRAIN AND IT CAN BE THE RESULT OF %2' OVEREXPRESSION INCREASING THE TOTAL STEROL CONTENT BUT NOT ERGOSTEROL AS IT HAS BEEN PREVIOUSLY DISCUSSED FOR 3 CEREVISIAE &URTHERMORE %2' OVEREXPRESSION LED TO HIGHER RELATIVE AMOUNTS OF PALMITIC ACID AND CEROTIC ACID AND REDUCTION OF LINOLEIC AND LINOLENIC ACID 2EGARDING PHOSPHOLIPIDS LEVELS OF 0# AND 0% INCREASED IN THE OVEREXPRESSING STRAIN COMPARED TO THE REFERENCE STRAIN 3PHINGOLIPIDS HAD ALSO ALTERED PATTERN ON THE RELATIVE AMOUNTS OF THEIR SPECIES BEING ESPECIALLY *OUFHSBUFE BOBMZTJT
REMARKABLE THE INCREMENT OF CERAMIDE COMPOSED BY PHYTOSPHINGOSINE AS ,#" AND CEROTIC ACID AS FATTY ACYL &URTHERMORE CERAMIDES WITH NONHYDROXYLATED FATTY ACIDS EG WERE DRASTICALLY REDUCED IN /% %RG CELLS (AXOSYLCERAMIDES DID CHANGE NEITHER
RELATIVE AMOUNTS NOR SPECIE COMPOSITION )0# -)0# AND -)0 ኼ# INCREASED THEIR LEVELS OF SPECIES AND COMPENSATED BY REDUCTION ON RELATIVE AMOUNTS OF SPECIES WHICH IS IN AGREEMENT WITH CERMIDE RESULTS AND MEANING THAT AN AUGMENT OF SPHINGOLIPIDS WITH CARBONS WAS OCCURRING WHEN %2' WAS OVEREXPRESSED 7HEREAS 3 CEREVISIAE HAS A STRONG PREVALENCE FOR # FATTY ACIDS ;= 'RILLITSCH ET AL ;= REPORTED ALMOST EQUAL AMOUNTS OF # AND # FATTY ACIDS FOR 0 PASTORIS )N OUR STUDIES A STRONG PREFERENCE FOR # FATTY ACIDS HAS BEEN OBSERVED 352 IS RESPONSIBLE FOR CONVERTING # FATTY ACIDS TO # 4HE OBTAINED RESULTS SUGGEST THAT %2' OVEREXPRESSION COULD INmUENCE LEVELS OF 352 IN THE CELL RESULTING ON AN INCREMENT OF # SPECIES
#HANGES ON LIPID COMPOSITION OF CELLS OVEREXPRESSING %2' WERE NOT BENElCIAL FOR &AB SECRETION &IGURE &AB PANEL AS LEVELS OF &AB WERE LOWER THAN THE OBTAINED FOR THE REFERENCE STRAIN (OWEVER FOLD INCREMENT ON &AB SECRETION WAS OBSERVED WHEN /% %RG CELLS WERE CULTURED IN HYPOXIA #HANGES ON LIPID COMPOSITION OF THESE CELLS DUE T HYPOXIC EFFECT WERE SIMILAR TO THE REFERENCE STRAIN BUT IN THIS CASE EFFECT ON &AB SECRETION WAS HIGHER /N THE OTHER HAND NO SYNERGIC EFFECT OF mUCONAZOLE TREATMENT AND HYPOXIA WAS OBSERVED LEADING TO FOLD HIGHER AMOUNTS OF &AB SECRETED
&GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI )BD USBOTDSJQUJPO GBDUPS
4HE TRANSCRIPTION FACTOR (AC OF 0 PASTORIS HAS BEEN COEXPRESSED AND THE LIPID COMPOSITION HAS BEEN DETERMINED (ACP CAN MODULATE THE LEVELS OF CHAPERONES FOLDASES AND PROTEINS THAT ARE RESPONSIBLE FOR LIPID AND INOSITOL METABOLISM #OEXPRESSION OF THE ACTIVATED SPLICED VARIANT OF THE (!# GENE HAS BEEN USED IN SEVERAL ORGANISMS AS A TARGET FOR STRAIN ENGINEERING TO IMPROVE PROTEIN SECRETION ; = !S 'UERFAL ET AL HAD SHOWN THAT CONSTITUTIVE EXPRESSION OF THE (AC HAD LITTLE TO NO EFFECT ON HETEROLOGOUS PROTEIN PRODUCTION ;= /UR RESULTS CONlRM THIS STATEMENT AS NO IMPROVEMENT ON &AB SECRETION WAS OBSERVED &IGURE &AB PANEL
6OGL ET AL ;= STUDIED EFFECT OF (AC COEXPRESSION IN 0 PASTORIS PRODUCING A MEMBRANE PROTEIN AT TRANSCRIPTOMIC LEVEL 4HEY OBSERVED DOWNREGULATION OF ERGOSTEROL BIOSYNTHESIS GENES AND UPREGULATION OF PHOSPHATIDYLINOSITOL 0) AND SPHINGOLIPID BIOSYNTHESIS 4HESE RESULTS ARE IN AGREEMENT WITH OURS AS ERGOSTEROL CONTENT OF CELLS OVEREXPRESSING (AC CONTAINED LOWER AMOUNTS OF ERGOSTEROL (OWEVER LEVELS OF FREE STEROLS WERE SIMILAR TO THE REFERENCE STRAIN 2ELATIVE LEVELS OF SATURATED FATTY ACIDS INCREASED ACCOMPANIED BY LOWER LEVELS OF MONO AND POLYUNSATURATED FATTY ACIDS EXCEPT FOR OLEIC ACID # THAT SLIGHTLY INCREASED IN THE (AC OVEREXPRESSING STRAIN ,EVELS OF 0) WERE SIMILAR TO THE REFERENCE STRAIN SO THAT THE REPORTED UPREGULATION OF 0) BIOSYNTHESIS UPON (AC EXPRESSION DID NOT LEAD TO HIGHER LEVELS OF 0) IN OUR STUDIES 2EGARDING SPHINGOLIPIDS CERAMIDE SPECIES CONTAINING # FATTY ACIDS HAD A TENDENCY TO INCREASE THEIR RELATIVE LEVELS AT THE EXPENSE OF LOWER LEVELS OF # CONTAINING SPECIES (AXOSYLCERAMIDES WERE EXHIBITED A SIMILAR PATTERN WITH HIGHER CONTENT OF SPECIES CONTAINING # FATTY ACIDS AND LOWER CONTENT OF THE ONES WITH # )NOSITOL CONTAINING SPHINGOLIPIDS MAINTAIN SIMILAR LEVELS IN THE /% (AC STRAIN COMPARED TO THE REFERENCE STRAIN .ON POLAR LIPIDS PRESENTED &GGFDU PG TUSBJO FOHJOFFSJOH UISPVHI )BD USBOTDSJQUJPO GBDUPS
LOWER LEVELS OF 4' AND 3% WHILE LEVELS OF FREE STEROLS WERE SIMILAR IN BOTH STRAINS
Fab Mutant vs Reference 2.5
2.0
1.5
1.0 c production rate fold change fi
0.5 speci Hac1
FA PL Fab 2F5 Hac1 Fab 2F5 Hac1 60 70
50 -1 60
-1 50 40 40 30 30
20 · mg total prot % PL
% FA · mgtotal prot % FA 20
10 10
0 0 PC PA PI PS Lyso-PL PE CL DMPE C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C26:0 Others Cer HexCer 40 100
80 30 60 20 40
10
Relative Peak Amount (%) Relative Peak 20
0 0
18:0;2/16:0;018:0;2/18:0;018:0;2/16:0;118:0;2/18:0;118:1;2/16:0;018:1;2/18:0;018:1;2/16:0;118:1;2/18:0;118:2;2/16:0;018:2;2/18:0;018:2;2/16:0;118:2;2/18:0;119:2;2/16:0;019:2;2/18:0;019:2;2/16:0;119:2;2/18:0;118:0;3/16:0;018:0;3/18:0;018:0;3/24:0;018:0;3/26:0;018:0;3/16:0;118:0;3/18:0;118:0;3/24:0;118:0;3/26:0;1 18:0;2/16:0;11 18:0;2/18:0;118:1;2/16:0;118:1;2/18:0;118:2;2/16:0;118:2;2/18:0;119:2;2/16:0;119:2;2/18:0;1
IPC MIPC M(IP)2C 100 100 100
80 80 80
60 60 60
40 40 40
20 20 20 Relative Peak Amount (%) Relative Peak
0 0 0
34:0;334:0;436:0;336:0;442:0;242:0;342:0;442:0;544:0;244:0;344:0;444:0;546:0;246:0;346:0;446:0;5 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4 34:0;3 34:0;4 36:0;3 36:0;4 42:0;3 42:0;4 42:0;5 44:0;3 44:0;4 44:0;5 46:0;3 46:0;4
Sterols 2F5 SE Hac1 Non-polar 12 200 40
10 -1 -1 160 -1 30 8 120 6 20
4 80 g TG · mg total prot
μ 10 g SE/FS · mg total prot μ g Sterol · mg total prot. 2 40 μ
0 SETGFS SE TG FS 0 0 Fab 2F5 Hac1
squalene lanosterol Episterol Zymosterol Ergosterol Ergostatraenol
4-Methylzymosterol 14- methylfecosterol ergosta5,7,22-trien3ol
ergosta 5 8 22 24 tetraenol
4, 14-dimethylcholesta-8,24-dienol
&IGURE 'ENERAL OVERVIEW OF THE RECOMBINANT PROTEIN PRODUCTION AND LIPID COMPOSITION OF THE CELLS OVEREXPRESSING THE GENE (!# TURQUOISE AND THE VALUES OF THE REFERENCE STRAIN BLUE WITHOUT THE GENETIC MODIlCATIONS *OUFHSBUFE BOBMZTJT
$PODMVTJPOT
s ! BENElCIAL EFFECT DUE TO CULTURING CELLS IN HYPOXIC CONDITIONS HAVE BEEN DETERMINED FOR ALL THE STRAINS USED IN THIS STUDY 3IMILAR CHANGES ON LIPID COMPOSITION WERE OBSERVED AS A RESULT OF HYPOXIA WHICH INCLUDE REDUCTION OF POLYUNSATURATED FATTY ACIDS PHOSPHATIDYLINOSITOL ERGOSTEROL AND INOSITOL CONTAINING SPHINGOLIPIDS WITH # FATTY ACIDS 4HESE CHANGES APPEARED WITH AN INCREMENT OF PHOSPHATIDYLSERINE TRIACYLGLYCEROLS AND INOSITOL CONTAINING SPHINGOLIPIDS WITH # FATTY ACIDS
s 3IGNIlCANT CHANGES ON LIPID COMPOSITION WERE OBSERVED IN THE STRAINS WITH ENGINEERED SPHINGOLIPID PATHWAY (OWEVER ANY OF THE MODIlCATIONS RESULTED IN COINCIDENT LIPID COMPOSITION TO THE ONE CORRESPONDING TO HYPOXIC CONDITIONS
s $ELETION OF THE GENE ENCODING THE # HYDOXYLASE 352 INVOLVED IN SPHINGOLIPID PATHWAY RESULTED IN MAJOR CHANGES ON LIPID COMPOSITION WHICH RESULTED IN CHANGES ON MEMBRANE COMPOSITION THAT INCREASED UP TO FOLD THE &AB SECRETION (OWEVER THESE CHANGES WERE DIFFERENT FROM THE ONES OCCURRING IN HYPOXIC CONDITIONS
s ! COMMON PATTERN AMONG THE CHANGES THAT RESULT IN HIGHER LEVELS OF &AB SECRETED INCLUDE A REDUCTION OF THE FREE STEROL CONTENT THE ERGOSTEROL CONTENT AND AN INCREMENT OF TRIACYLGLYCEROLS WITHIN THE CELL 4HIS WOULD MEAN THAT STEROLS ARE A KEY PLAYER ON MEMBRANE COMPOSITION AND RECOMBINANT PROTEIN SECRETION
s !S IT WAS EXPECTED THE EFFECT OF LOW OXYGEN CULTURE CONDITIONS RESULTED IN HIGHER PROTEIN SECRETION THAN PUNCTUAL CHANGES ON THE LIPID BIOSYNTHETIC PATHWAY
s #HANGING CELL LIPID COMPOSITION BY MEANS OF GENETIC MODIlCATIONS COULD BE A SUCCESSFUL SYSTEM TO ENHANCE RECOMBINANT PROTEIN PRODUCTION )T CAN BE COMPLEMENTED WITH PREVIOUS ATTEMPTS ON OPTIMISING RECOMBINANT PROTEIN SECRETION BY COEXPRESSING GENES INVOLVED IN PROTEIN FOLDING AND %2 STRESS (FOFSBM DPODMVTJPOT
$EEP ANALYSIS OF THE TRANSCRIPTOMIC DATA FROM CELLS CULTURED UNDER LOW OXYGEN CONDITIONS WAS PERFORMED FOCUSED ON THE GENES INVOLVED IN THE LIPID METABOLISM #ONCRETELY A CLUSTER ANALYSIS LET TO IDENTIFYING THE MOST REGULATED GENES IN HYPOXIA ! SET OF GENES INVOLVED IN THE ERGOSTEROL AND SPHINGOLIPID PATHWAYS WERE SELECTED AS TARGET GENES FOR CELL ENGINEERING $%3 352 AND %2' &URTHERMORE THE TRANSCRIPTION FACTOR (AC WAS ALSO UPREGULATED IN HYPOXIA )TS INVOLVEMENT REGULATING GENES OF THE LIPID METABOLISM MADE IT A GOOD CANDIDATE TO STUDY THE EFFECT OF LIPID MODIlCATIONS ON PROTEIN SECRETION AND IT WAS INCLUDED IN THE SET OF SELECTED GENES
4HE REFERENCE STRAIN WAS USED TO CONSTRUCT A BATTERY OF STRAINS WHERE THE SELECTED GENES WERE DELETED $%3 352 OR OVEREXPRESSED $%3 %2' (!# 4WO KNOCKOUTS OF $%3 AND ONE OF 352 WERE OBTAINED USING A SPLIT MARKER STRATEGY WHICH WAS CHOSEN TO IMPROVE THE EFlCIENCY BY FAVOURING HOMOLOGOUS RECOMBINATION INSTEAD OF NON HOMOLOGOUS END JOINING !N EFlCIENCY AROUND WAS OBTAINED $ESPITE THE EFFORTS OF 4ERNES ET AL TO OBTAIN A STRAIN OF 0 PASTORIS WITH A DELETION OF 352 THEY WERE NO SUCCESSFUL SUGGESTING THAT HYDROXYLATION OF CERAMIDES WAS REQUIRED FOR THE FURTHER STEPS TO OBTAIN INOSITOL CONTAINING SPHINGOLIPIDS )N THIS WORK A DELETION OF 352 IN 0 PASTORIS HAS BEEN DESCRIBED FOR THE lRST TIME
!LL THE STRAINS WERE CULTURED IN SHAKE mASK AND WERE CHARACTERISED IN TERMS OF CELL GROWTH AND &AB PRODUCTION AND A REPRESENTATIVE CLONE FOR EACH GENE MODIlCATION WAS SELECTED FOR FURTHER STUDIES 2EMARKABLY OVEREXPRESSION OF 352 RESULTED INTO NON VIABLE CLONES 4HE PRODUCTION OF &AB WAS MONITORED IN TERMS OF PROTEIN SECRETED AND ALSO INTRAEXTRACELLULAR DISTRIBUTION WAS DETERMINED /PTIMISED DISRUPTION PARAMETERS WERE DElNED TO MAXIMISE THE RECOVERED &AB IN EACH FRACTION AND ALLOWED TO OBSERVE A HIGH PERCENTAGE OF &AB THAT WAS SECRETED &URTHERMORE IN ORDER TO REDUCE THE AMOUNTS OF ERGOSTEROL AN ESSENTIAL ELEMENT OF CELL MEMBRANES mUCONAZOLE TREATMENT WAS USED FOR THIS PURPOSE AND THE QUANTITY OF mUCONAZOLE REQUIRED TO OPTIMISE PROTEIN SECRETION WITHOUT IMPAIRING CELL GROWTH WAS DETERMINED 4HESE RESULTS COMPLEMENT THE PREVIOUS DATA OF mUCONAZOLE TREATMENT WHERE ONLY AN INITIAL CONCENTRATION OF mUCONAZOLE WAS DElNED AND THUS HIGH VARIABILITY ON THE LEVELS OF &AB SECRETED WERE OBSERVED FROM CULTURE TO CULTURE
#HEMOSTAT CULTURES OF EACH STRAIN IN NORMOXIC AND HYPOXIC CONDITIONS WERE PERFORMED IN ORDER TO CHARACTERISE THEM IN TERMS OF &AB PRODUCTION INTRAEXTRACELLULAR DISTRIBUTION CELL MORPHOLOGY AND LIPID CONTENT OF CELL HOMOGENATES )N ALL STRAINS AN INCREASE OF &AB SPECIlC &AB PRODUCTIVITY
(FOFSBM DPODMVTJPOT
WAS OBSERVED IN HYPOXIA 4HIS INCREASE DID NOT LEAD &AB ACCUMULATION IN THE INTRACELLULAR FRACTION OF THE CELLS AND HIGH PERCENTAGES OF THE TOTAL &AB DETECTED WERE PRESENT IN THE SECRETED FRACTION 3TUDIES OF THE LIPID COMPOSITION INDICATED THAT CHANGES DUE TO HYPOXIA OCCUR AND INCLUDE REDUCTION OF POLYUNSATURATED FATTY ACIDS PHOSPHATIDYLINOSITOL ERGOSTEROL AND INOSITOL CONTAINING SPHINGOLIPIDS WITH # FATTY ACIDS 4HESE CHANGES WERE COMBINED WITH AN INCREMENT OF PHOSPHATIDYLSERINE TRIACYLGLYCEROLS AND INOSITOL CONTAINING SPHINGOLIPIDS WITH # FATTY ACIDS &URTHERMORE GENETIC CHANGES OF LIPID METABOLISM RESULT IN ALTERED LIPID PATTERN WHEN COMPARED TO THE REFERENCE STRAIN (OWEVER ANY OF THE MODIlCATIONS RESULTED IN COINCIDENT LIPID COMPOSITION TO THE ONE CORRESPONDING TO HYPOXIC CONDITIONS
4HE MOST PROMISING STRAIN WAS THE ONE HARBOURING THE DELETION OF THE GENE ENCODING THE # HYDOXYLASE 352 INVOLVED IN SPHINGOLIPID PATHWAY RESULTED IN MAJOR CHANGES ON LIPID COMPOSITION WHICH RESULTED IN CHANGES ON MEMBRANE COMPOSITION THAT INCREASED UP TO FOLD THE &AB SECRETION (OWEVER THESE CHANGES WERE DIFFERENT FROM THE ONES OCCURRING IN HYPOXIC CONDITIONS AND THE LEVELS OF &AB SECRETED DUE TO HYPOXIC CULTIVATION WERE NOT ACHIEVED ! COMMON PATTERN AMONG THE STRAINSCONDITIONS SHOWING A BENElCIAL EFFECT ON RECOMBINANT PROTEIN SECRETION WERE THE REDUCTION OF THE FREE STEROL CONTENT THE ERGOSTEROL CONTENT AND THE INCREMENT OF TRIACYLGLYCEROLS 4HESE TRENDS WOULD SUGGEST A KEY ROLE OF STEROLS DElNING MEMBRANE PROPERTIES AND ITS DIRECT IMPACT ON PROTEIN SECRETION &INALLY CHANGES ON LIPID COMPOSITION OF CELL MEMBRANES SPECIALLY FOCUSING ON THE ERGOSTEROL CONTENT COULD BE A PROMISING TARGET FOR FUTURE CELL ENGINEERING STRATEGIES IN ORDER TO INCREASE PROTEIN SECRETION 4HESE STRATEGIES COULD BE COMPLEMENTED WITH OTHER STRATEGIES FOCUSED ON OPTIMISING THE EARLY STEPS OF PROTEIN PRODUCTION 3FGFSFODFT
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3FGFSFODFT
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;= 0UXBAUM 6 -ATTANOVICH $ 'ASSER " 1UO VADIS 4HE CHALLENGES OF RECOMBINANT PROTEIN FOLDING AND SECRETION IN 0ICHIA PASTORIS !PPL -ICROBIOL "IOTECHNOL n
;= 3HA # 9U 87 ,I & 8U 9 )MPACT OF 'ENE $OSAGE ON THE 0RODUCTION OF ,IPASE FROM 2HIZOPUS CHINENSIS ##4## - IN 0ICHIA PASTORIS !PPL "IOCHEM "IOTECHNOL n
;= $AMASCENO ,- (UANG #* "ATT #A 0ROTEIN SECRETION IN 0ICHIA PASTORIS AND ADVANCES IN PROTEIN PRODUCTION !PPL -ICROBIOL "IOTECHNOL NOV n
;= $AMASCENO ,- !NDERSON +A 2ITTER ' #REGG *- /LD ,* "ATT #A #OOVEREXPRESSION OF CHAPERONES FOR ENHANCED SECRETION OF A SINGLE CHAIN ANTIBODY FRAGMENT IN 0ICHIA PASTORIS !PPL -ICROBIOL "IOTECHNOL FEB n
;= 'ASSER " -AURER - 'ACH * +UNERT 2 -ATTANOVICH $ %NGINEERING OF 0ICHIA PASTORIS FOR IMPROVED PRODUCTION OF ANTIBODY FRAGMENTS "IOTECHNOL "IOENG n
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;= 7HYTESIDE ' !LCOCER -*# +UMITA *2 $OBSON #- ,AZAROU - 0LEASS 2* ET AL .ATIVE STATE STABILITY DETERMINES THE EXTENT OF DEGRADATION RELATIVE TO SECRETION OF PROTEIN VARIANTS FROM 0ICHIA PASTORIS 0,O3 /NE JAN E
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;= )VASHOV 6A 'RILLITSCH + +OEFELER ( ,EITNER % "AEUMLISBERGER $ +ARAS - ET AL ,IPIDOME AND PROTEOME OF LIPID DROPLETS FROM THE METHYLOTROPHIC YEAST 0ICHIA PASTORIS "IOCHIM "IOPHYS !CTA -OL #ELL "IOL ,IPIDS n 3FGFSFODFT
;= :ANGHELLINI * .ATTER + *UNGREUTHMAYER # 4HALHAMMER ! +URAT #& 'OGG &ASSOLTER ' ET AL 1UANTITATIVE MODELING OF TRIACYLGLYCEROL HOMEOSTASIS IN YEASTnMETABOLIC REQUIREMENT FOR LIPOLYSIS TO PROMOTE MEMBRANE LIPID SYNTHESIS AND CELLULAR GROWTH &%"3 * NOV n
;= 4URKISH ! 3TURLEY 3, 2EGULATION OF TRIGLYCERIDE METABOLISM ) %UKARYOTIC NEUTRAL LIPID SYNTHESIS v-ANY WAYS TO SKIN !#!4 OR A $'!4v !M * 0HYSIOL 'ASTROINTEST ,IVER 0HYSIOL 'n
;= +OHLWEIN 3$ 4RIACYLGLYCEROL (OMEOSTASIS )NSIGHTS FROM 9EAST * "IOL #HEM n
;= !GUILERA 2OMERO ! 'EHIN # 2IEZMAN ( 3PHINGOLIPID HOMEOSTASIS IN THE WEB OF METABOLIC ROUTES "IOCHIM "IOPHYS !CTA -OL #ELL "IOL ,IPIDS n
;= 4ERNES 0 7OBBE 4 3CHWARZ - !LBRECHT 3 &EUSSNER + 2IEZMAN ) ET AL 4WO 0ATHWAYS OF 3PHINGOLIPID "IOSYNTHESIS !RE 3EPARATED IN THE 9EAST 0ICHIA PASTORIS * "IOL #HEM FEB n
;= 7ARNECKE $ (EINZ % 2ECENTLY DISCOVERED FUNCTIONS OF GLUCOSYLCERAMIDES IN PLANTS AND FUNGI #ELL -OL ,IFE 3CI MAY n
;= VAN -EER ' 6OELKER $2 &EIGENSON '7 -EMBRANE LIPIDS WHERE THEY ARE AND HOW THEY BEHAVE .AT 2EV -OL #ELL "IOL FEB n
;= $ICKSON 2# 3UMANASEKERA # ,ESTER 2, &UNCTIONS AND METABOLISM OF SPHINGOLIPIDS IN 3ACCHAROMYCES CEREVISIAE 0ROG ,IPID 2ES n
;= $ICKSON 2# ,ESTER 2, -ETABOLISM AND SELECTED FUNCTIONS OF SPHINGOLIPIDS IN THE YEAST 3ACCHAROMYCES CEREVISIAE "IOCHIM "IOPHYS !CTA -OL #ELL "IOL ,IPIDS n
;= #HEN 07 &ONSECA ,, (ANNUN 9! 6OIT %/ #OORDINATION OF 2APID 3PHINGOLIPID 2ESPONSES TO (EAT 3TRESS IN 9EAST 0,O3 #OMPUT "IOL MAY E
;= &UNATO + 6ALLdzE " 2IEZMAN ( "IOSYNTHESIS AND 4RAFlCKING OF 3PHINGOLIPIDS IN THE 9EAST 3ACCHAROMYCES CEREVISIAE "IOCHEMISTRY n
;= (UANG 8 ,IU * $ICKSON 2# $OWN 2EGULATING 3PHINGOLIPID 3YNTHESIS )NCREASES 9EAST ,IFESPAN 0,O3 'ENET E
;= $ICKSON 2# ,ESTER 2, 3PHINGOLIPID FUNCTIONS IN 3ACCHAROMYCES CEREVISIAE "IOCHIM "IOPHYS !CTA JUN n
;= ,IU + :HANG 8 3UMANASEKERA # ,ESTER 2, $ICKSON 2# 3IGNALLING FUNCTIONS FOR SPHINGOLIPID LONG CHAIN BASES IN 3ACCHAROMYCES CEREVISIAE "IOCHEM 3OC 4RANS 0T n
;= -ONTEFUSCO $* -ATMATI . (ANNUN 9! 4HE YEAST SPHINGOLIPID SIGNALING LANDSCAPE #HEM 0HYS ,IPIDS n
;= (ANNICH *4 5MEBAYASHI + 2IEZMAN ( $ISTRIBUTION AND FUNCTIONS OF STEROLS AND SPHINGOLIPIDS #OLD 3PRING (ARB ,AB 0ERSPECT "IOL MAY n
;= #OWART ,! /BEID ,- 9EAST SPHINGOLIPIDS RECENT DEVELOPMENTS IN UNDERSTANDING BIOSYNTHESIS REGULATION AND FUNCTION "IOCHIM "IOPHYS !CTA MAR n
;= (ECHTBERGER 0 :INSER % 3AF 2 (UMMEL + 0ALTAUF & $AUM ' #HARACTERIZATION QUANTIlCATION AND SUBCELLULAR LOCALIZATION OF INOSITOL CONTAINING SPHINGOLIPIDS OF THE YEAST 3ACCHAROMYCES CEREVISIAE %UR * "IOCHEM OCT n
;= $ICKSON 2 2OLES FOR 3PHINGOLIPIDS IN 3ACCHAROMYCES CEREVISIAE )N #HALFANT # 0OETA - EDITORS 3PHINGOLIPIDS AS 3IGNAL 2EGUL -OL 3% VOL OF !DVANCES IN %XPERIMENTAL -EDICINE AND "IOLOGY 3PRINGER .EW 9ORK P n 3FGFSFODFT
;= "RESLOW $+ 3PHINGOLIPID HOMEOSTASIS IN THE ENDOPLASMIC RETICULUM AND BEYOND #OLD 3PRING (ARB 0ERSPECT "IOL n
;= -ERRILL !( 3PHINGOLIPID AND GLYCOSPHINGOLIPID METABOLIC PATHWAYS IN THE ERA OF SPHINGOLIPIDOMICS #HEM 2EV n
;= 'ULATI 3 ,IU 9 -UNKACSI !" 7ILCOX , 3TURLEY 3, 3TEROLS AND SPHINGOLIPIDS $YNAMIC DUO OR PARTNERS IN CRIME 0ROG ,IPID 2ES n
;= -OLLINEDO & ,IPID RAFT INVOLVEMENT IN YEAST CELL GROWTH AND DEATH &RONT /NCOL JAN/CTOBER
;= *OOSTEN 6 ,OKMAN # (ONDEL #!6$ 0UNT 0* 4HE PRODUCTION OF ANTIBODY FRAGMENTS AND ANTIBODY FRAGMENTS BY YEASTS AND lLAMENTOUS FUNGI -ICROB #ELL &ACT n
;= DE -ARCO ! "IOTECHNOLOGICAL APPLICATIONS OF RECOMBINANT SINGLE DOMAIN ANTIBODY FRAGMENTS -ICROB #ELL &ACT
;= 3PADIUT / #APONE 3 +RAINER & 'LIEDER ! (ERWIG # -ICROBIALS FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES AND ANTIBODY FRAGMENTS 4RENDS "IOTECHNOL n
;= "UCHETICS - $RAGOSITS - -AURER - 2EBNEGGER # 0ORRO $ 3AUER - ET AL 2EVERSE ENGINEERING OF PROTEIN SECRETION BY UNCOUPLING OF CELL CYCLE PHASES FROM GROWTH "IOTECHNOL "IOENG MAY n
;= 4AKAHASHI + 4OSHIFUMI 9 4AKAI 4 2A # /KUMURA + 9OKOTA 4 ET AL 0RODUCTION OF HUMANIZED &AB FRAGMENT AGAINST HUMAN HIGH AFlNITY )G% RECEPTOR IN 0ICHIA PASTORIS "IOSCI "IOTECHNOL "IOCHEM n
;= ,ANGE 3 3CHMITT * 3CHMID 2$ (IGH YIELD EXPRESSION OF THE RECOMBINANT ATRAZINE SPECIlC &AB FRAGMENT +" BY THE METHYLOTROPHIC YEAST 0ICHIA PASTORIS * )MMUNOL -ETHODS n
;= .ING $ *UNJIAN 8 1ING : 3HENG 8 7ENYIN # 'UIRONG 2 ET AL 0RODUCTION OF RECOMBINANT HUMANIZED ANTI ("S!G &AB FRAGMENT FROM 0ICHIA PASTORIS BY FERMENTATION * "IOCHEM -OL "IOL n
;= 3TADLMAYR ' -ECKLENBRƊUKER ! 2OTHMķLLER - -AURER - 3AUER - -ATTANOVICH $ ET AL )DENTIlCATION AND CHARACTERISATION OF NOVEL 0ICHIA PASTORIS PROMOTERS FOR HETEROLOGOUS PROTEIN PRODUCTION * "IOTECHNOL DEC n
;= .ISHIMURA ! -ORITA - .ISHIMURA 9 3UGINO 9 ! RAPID AND HIGHLY EFlCIENT METHOD FOR PREPARATION OF COMPETENT %SCHERICHIA COLI CELLS .UCLEIC !CIDS 2ES
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;= ,IN #EREGHINO * 7ONG 77 8IONG 3 'IANG 7 ,UONG ,4 6U * ET AL #ONDENSED PROTOCOL FOR COMPETENT CELL PREPARATION AND TRANSFORMATION OF THE METHYLOTROPHIC YEAST 0ICHIA PASTORIS "IOTECHNIQUES n
;= 2ESINA $ #OS / &ERRER 0 6ALERO & $EVELOPING HIGH CELL DENSITY FED BATCH CULTIVATION STRATEGIES FOR HETEROLOGOUS PROTEIN PRODUCTION IN 0ICHIA PASTORIS USING THE NITROGEN SOURCE REGULATED &,$ 0ROMOTER "IOTECHNOL "IOENG SEP n
;= !RNAU # 2AMON 2 #ASAS # 6ALERO & /PTIMIZATION OF THE HETEROLOGOUS PRODUCTION OF A 2HIZOPUS ORYZAE LIPASE IN 0ICHIA PASTORIS SYSTEM USING MIXED SUBSTRATES ON CONTROLLED FED BATCH BIOPROCESS %NZYME -ICROB 4ECHNOL MAY n 3FGFSFODFT
;= 7ECHSELBERGER 0 3AGMEISTER 0 (ERWIG # 2EAL TIME ESTIMATION OF BIOMASS AND SPECIlC GROWTH RATE IN PHYSIOLOGICALLY VARIABLE RECOMBINANT FED BATCH PROCESSES "IOPROCESS "IOSYST %NG n
;= 6AN $ER (EIJDEN 24*- (EIJNEN ** (ELLINGA # 2OMEIN " ,UYBEN +#A- ,INEAR CONSTRAINT RELATIONS IN BIOCHEMICAL REACTION SYSTEMS ) #LASSIlCATION OF THE CALCULABILITY AND THE BALANCEABILITY OF CONVERSION RATES "IOTECHNOL "IOENG n
;= 7ANG .3 3TEPHANOPOULOS ' !PPLICATION OF MACROSCOPIC BALANCES TO THE IDENTIlCATION OF GROSS MEASUREMENT ERRORS "IOTECHNOL "IOENG n
;= 7ECHSELBERGER 0 (ERWIG # -ODEL BASED ANALYSIS ON THE RELATIONSHIP OF SIGNAL QUALITY TO REAL TIME EXTRACTION OF INFORMATION IN BIOPROCESSES "IOTECHNOL 0ROG n
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;= ,OWRY /( 2OSEBROUGH .* &ARR !, 2ANDALL 2* 0ROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT * "IOL #HEM n
;= &OLCH * ,EES - 3LO ! SIMPLE METHOD FOR THE ISOLATION AND PURIlCATION OF TOTAL LIPIDES FROM ANIMAL TISSUES * "IOL #HEM
;= "ROEKHUYSE 2- )SOLATION CHARACTERIZATION AND QUANTITATIVE ANALYSIS BY TWO DIMENSIONAL THIN LAYER CHROMATOGRAPHY OF DIACYL AND VINYL ETHER PHOSPHOLIPIDS "IOCHEM "IOPHYS !CTA n
;= 1UAIL -A +ELLY 3, 4HE EXTRACTION AND ANALYSIS OF STEROLS FROM YEAST )N %VANS EDITOR 9EAST 0ROTOC VOL 4O (UMANA 0RESS P n
;= -ARKHAM *% ,I * #AHOON %" *AWORSKI *' 3EPARATION AND )DENTIlCATION OF -AJOR 0LANT 3PHINGOLIPID #LASSES FROM ,EAVES * "IOL #HEM n
;= %JSING #3 -OEHRING 4 "AHR 5 $UCHOSLAV % +ARAS - 3IMONS + ET AL #OLLISION INDUCED DISSOCIATION PATHWAYS OF YEAST SPHINGOLIPIDS AND THEIR MOLECULAR PROlLING IN TOTAL LIPID EXTRACTS A STUDY BY QUADRUPOLE 4/& AND LINEAR ION TRAP ORBITRAP MASS SPECTROMETRY * -ASS 3PECTROM n
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;= -ELTZER 03 ,ARGE SCALE GENOME ANALYSIS )N "AXEVANIS !$ /UELLETTE "&& EDITORS "IOINFORMA ! 0RACT 'UID TO !NAL 'ENES 0ROTEINS SECOND EDI ED .EW 9ORK *OHN 7ILEY AND 3ONS )NC P n
;= $ALE *7 VON 3CHANTZ - !NALYSIS OF 'ENE %XPRESSION )N $ALE *7 VON 3CHANTZ - EDITORS &ROM 'ENES TO 'ENOMES #ONCEPTS !PPL $.! 4ECHNOL VOL 7EST 3USSEX %NGLAND *OHN 7ILEY 3ONS ,TD P n
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;= -AC1UEEN * 3OME METHODS FOR CLASSIlCATION AND ANALYSIS OF MULTIVARIATE OBSERVATIONS )N 0ROC lFTH "ERKELEY 3YMP -ATH 3TAT 0ROBAB VOL /AKLAND #! 53! P n
;= #ARNICER - #ANELAS A" TEN 0IERICK ! :ENG : VAN $AM * !LBIOL * ET AL $EVELOPMENT OF QUANTITATIVE METABOLOMICS FOR 0ICHIA PASTORIS -ETABOLOMICS n
;= 0ETRANOVIC $ 4YO + 6EMURI '. .IELSEN * 0ROSPECTS OF YEAST SYSTEMS BIOLOGY FOR HUMAN HEALTH INTEGRATING LIPID PROTEIN AND ENERGY METABOLISM &%-3 9EAST 2ES DEC n
;= #OX *3 #HAPMAN 2% 7ALTER 0 4HE UNFOLDED PROTEIN RESPONSE COORDINATES THE PRODUCTION OF ENDOPLASMIC RETICULUM PROTEIN AND ENDOPLASMIC RETICULUM MEMBRANE -OL "IOL #ELL SEP n
;= 'UERFAL - 2YCKAERT 3 *ACOBS 00 !MELOOT 0 #RAENENBROECK +6 $ERYCKE 2 ET AL 4HE (!# GENE FROM 0ICHIA PASTORIS CHARACTERIZATION AND EFFECT OF ITS OVEREXPRESSION ON THE PRODUCTION OF SECRETED SURFACE DISPLAYED AND MEMBRANE PROTEINS -ICROB #ELL &ACT n
;= "AUMANN + !DELANTADO . ,ANG # -ATTANOVICH $ &ERRER 0 0ROTEIN TRAFlCKING ERGOSTEROL BIOSYNTHESIS AND MEMBRANE PHYSICS IMPACT RECOMBINANT PROTEIN SECRETION IN 0ICHIA PASTORIS -ICROB #ELL &ACT NOV
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;= /BEID ,- /KAMOTO 9 -AO # 9EAST SPHINGOLIPIDS METABOLISM AND BIOLOGY "IOCHIM "IOPHYS !CTA DEC n
;= (AAK $ 'ABLE + "EELER 4 $UNN 4 (YDROXYLATION OF 3ACCHAROMYCES CEREVISIAE CERAMIDES REQUIRES 3URP AND 3CSP * "IOL #HEM NOV n
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;= 6EEN - 3TAHL 5 ,ANG # #OMBINED OVEREXPRESSION OF GENES OF THE ERGOSTEROL BIOSYNTHETIC PATHWAY LEADS TO ACCUMULATION OF STEROLS IN &%-3 9EAST 2ES OCT n
;= ,EES .$ "ARD - 3TEROL BIOCHEMISTRY AND REGULATION IN THE YEAST 3ACCHAROMYCES CEREVISIAE )N $AUM ' EDITOR ,IPID -ETAB -EMBR "IOG 3% VOL OF 4OPICS IN #URRENT 'ENETICS 3PRINGER "ERLIN (EIDELBERG P n !VAILABLE FROM KWWS G[GRLRUJ ^B`
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;= 3TANSlELD ) #LIFFE +2 +ELLY 3, #HEMOSTAT STUDIES OF MICROSOMAL ENZYME INDUCTION IN 3ACCHAROMYCES CEREVISIAE 9EAST n
;= #HANG (* 2OLE OF THE 5NFOLDED 0ROTEIN 2ESPONSE 0ATHWAY IN 3ECRETORY 3TRESS AND 2EGULATION OF )./ %XPRESSION IN 3ACCHAROMYCES CEREVISIAE 'ENETICS n 3FGFSFODFT
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;= !SHE -0 "ILL 2- -APPING THE YEAST HOST CELL RESPONSE TO RECOMBINANT MEMBRANE PROTEIN PRODUCTION RELIEVING THE BIOLOGICAL BOTTLENECKS "IOTECHNOL * JUN n
;= 7HYTESIDE ' .OR 2- !LCOCER -*# !RCHER $" !CTIVATION OF THE UNFOLDED PROTEIN RESPONSE IN 0ICHIA PASTORIS REQUIRES SPLICING OF A (!# M2.! INTRON AND RETENTION OF THE # TERMINAL TAIL OF (ACP &%"3 ,ETT APR n
;= 6ALKONEN - 0ENTTILƊ - 3ALOHEIMO - 0ENTTILA - %FFECTS OF )NACTIVATION AND #ONSTITUTIVE %XPRESSION OF THE 5NFOLDED 0ROTEIN 2ESPONSE 0ATHWAY ON 0ROTEIN 0RODUCTION IN THE 9EAST 3ACCHAROMYCES CEREVISIAE -OL -ICROBIOL FEB n
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;= #HEN : 3UN ( ,I 0 (E . :HU 4 ,I 9 %NHANCEMENT OF THE 'ENE 4ARGETING %FlCIENCY OF .ON #ONVENTIONAL 9EASTS BY )NCREASING 'ENETIC 2EDUNDANCY 0,O3 /NE MAR E
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;= (IGGINS $2 #REGG *- 0ICHIA 0ROTOCOLS (UMANA 0RESS )NC
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;= 3ARAYA 2 +RIKKEN !- +IEL *!+7 "AERENDS 2*3 6EENHUIS - VAN DER +LEI )* .OVEL GENETIC TOOLS FOR I (ANSENULA POLYMORPHA I &%-3 9EAST 2ES NOVP n
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;= "ENEY , 'ERVAIS 0 )NmUENCE OF THE mUIDITY OF THE MEMBRANE ON THE RESPONSE OF MICROORGANISMS TO ENVIRONMENTAL STRESSES !PPL -ICROBIOL "IOTECHNOL OCTn
;= ,EARMONTH 20 -EMBRANE &LUIDITY IN 9EAST !DAPTATION )NSIGHTS FROM &LUORESCENCE 3PECTROSCOPY AND -ICROSCOPY )N 'EDDES #$ EDITOR 2EV &LUORESC ST ED .EW 9ORK 3PRINGER P
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;= (OHENBLUM ( 'ASSER " -AURER - "ORTH . -ATTANOVICH $ %FFECTS OF GENE DOSAGE PROMOTERS AND SUBSTRATES ON UNFOLDED PROTEIN STRESS OF RECOMBINANT 0ICHIA PASTORIS "IOTECHNOL "IOENG n
;= 3PIDEN %- 3CALES 0* +ENTISH 3% -ARTIN '*/ #RITICAL ANALYSIS OF QUANTITATIVE INDICATORS OF CELL DISRUPTION APPLIED TO 3ACCHAROMYCES CEREVISIAE PROCESSED WITH AN INDUSTRIAL HIGH PRESSURE HOMOGENIZER "IOCHEM %NG * JANn
;= 0FEFFER - -AURER - +¨LLENSPERGER ' (ANN 3 'RAF !" -ATTANOVICH $ -ODELING AND MEASURING INTRACELLULAR mUXES OF SECRETED RECOMBINANT PROTEIN IN 0ICHIA PASTORIS WITH A NOVEL 3 LABELING PROCEDURE -ICROB #ELL &ACT JAN
;= :EDER ,UTZ ' #HEROUATI . 2EINHART # 0ATTUS & 7AGNER 2 $OT BLOT IMMUNODETECTION AS A VERSATILE AND HIGH THROUGHPUT ASSAY TO EVALUATE RECOMBINANT '0#2S PRODUCED IN THE YEAST 0ICHIA PASTORIS 0ROTEIN %XPR 0URIF NOV n
;= ,ÌRENZ &ONFRĿA 6 0ERĈLVAREZ -ARĿN ! 0ADRÌS % ,AZAROVA 4 3OLUBILIZATION 0URIlCATION AND #HARACTERIZATION OF )NTEGRAL -EMBRANE 0ROTEINS )N 2OBINSON !3 EDITOR 0ROD -EMBR 0ROTEINS 3TRATEG %XPR )SOL ST ED 7ILEY 6#( 6ERLAG 'MB( #O P n
;= 3ARRAMEGNA 6 -ULLER ) -OUSSEAU ' &ROMENT # -ONSARRAT " -ILON ! ET AL 3OLUBILIZATION PURIlCATION AND MASS SPECTROMETRY ANALYSIS OF THE HUMAN MU OPIOID RECEPTOR EXPRESSED IN 0ICHIA PASTORIS 0ROTEIN %XPR 0URIF n
;= #HI 7+ +U #( #HANG ## 4SAI *. 4WO STEP CELL DISRUPTION FOR THE EXTRACTION OF MEMBRANE ASSOCIATED RECOMBINANT PROTEIN FROM 3ACCHAROMYCES CEREVISIAE !NN . 9 !CAD 3CI MAYn
;= +IM 3+ %FFECT OF %XPRESSION OF 'ENES IN THE 3PHINGOLIPID 3YNTHESIS 0ATHWAY ON THE "IOSYNTHESIS OF #ERAMIDE IN 3ACCHAROMYCES CEREVISIAE * -ICROBIOL "IOTECHNOL /CTOBER n
;= ,I : 8IONG & ,IN 1 $!NJOU - $AUGULIS !* 9ANG $3# ET AL ,OW 4EMPERATURE )NCREASES THE 9IELD OF "IOLOGICALLY !CTIVE (ERRING !NTIFREEZE 0ROTEIN IN 0ICHIA PASTORIS 0ROTEIN %XPR 0URIF APR n
;= 0IPER -$7 $ARAN ,APUJADE 0 "RO # 2EGENBERG " +NUDSEN 3 .IELSEN * ET AL 2EPRODUCIBILITY OF /LIGONUCLEOTIDE -ICROARRAY 4RANSCRIPTOME !NALYSES * "IOL #HEM n
;= (OSKISSON 0! #ONTINUOUS CULTURE MAKING A COMEBACK -ICROBIOLOGY n
;= +OHLWEIN 3$ 4HE BEAUTY OF THE YEAST LIVE CELL MICROSCOPY AT THE LIMITS OF OPTICAL RESOLUTION -ICROSC 2ES 4ECH DEC n
;= 7RIGHT 2 4RANSMISSION ELECTRON MICROSCOPY OF YEAST -ICROSC 2ES 4ECH DEC n
;= 3PURR !2 ! LOW VISCOSITY EPOXY RESIN EMBEDDING MEDIUM FOR ELECTRON MICROSCOPY * 5LTRASTRUCT 2ES n
;= 3ANTOS !833 2IEZMAN ( 9EAST AS A MODEL SYSTEM FOR STUDYING LIPID HOMEOSTASIS AND FUNCTION &%"3 ,ETT AUG n
;= 2AJAKUMARI 3 2AJASEKHARAN 2 $AUM ' 4RIACYLGLYCEROL LIPOLYSIS IS LINKED TO SPHINGOLIPID AND PHOSPHOLIPID METABOLISM OF THE YEAST 3ACCHAROMYCES CEREVISIAE "IOCHIM "IOPHYS !CTA -OL #ELL "IOL ,IPIDS AUG 3FGFSFODFT
;= 'UAN 8, 3OUZA #- 0ICHLER ( 3CHAAD / +AJIWARA + 7AKABAYASHI ( ET AL &UNCTIONAL INTERACTIONS BETWEEN SPHINGOLIPIDS AND STEROLS IN BIOLOGICAL MEMBRANES REGULATING CELL PHYSIOLOGY -OL "IOL #ELL !PRIL n
;= ,OS $A -URATA . -EMBRANE mUIDITY AND ITS ROLES IN THE PERCEPTION OF ENVIRONMENTAL SIGNALS "IOCHIM "IOPHYS !CTA "IOMEMBR n
;= $AUM ' ,EES .$ "ARD - $ICKSON 2 "IOCHEMISTRY CELL BIOLOGY AND MOLECULAR BIOLOGY OF LIPIDS OF 3ACCHAROMYCES CEREVISIAE 9EAST n
;= 9U !1 3HI 4, :HANG " 8ING ,* ,I -# 4RANSCRIPTIONAL REGULATION OF DESATURASE GENES IN 0ICHIA PASTORIS '3 ,IPIDS NOV n
;= ,INDBERG , 3ANTOS !83 2IEZMAN ( /LSSON , "ETTIGA - ,IPIDOMIC 0ROlLING OF 3ACCHAROMYCES CEREVISIAE AND :YGOSACCHAROMYCES BAILII 2EVEALS #RITICAL #HANGES IN ,IPID #OMPOSITION IN 2ESPONSE TO !CETIC !CID 3TRESS 0,O3 /NE n
;= *ENKINS '- 2ICHARDS ! 7AHL 4 -AO # /BEID , (ANNUN 9 )NVOLVEMENT OF YEAST SPHINGOLIPIDS IN THE HEAT STRESS RESPONSE OF 3ACCHAROMYCES CEREVISIAE * "IOL #HEM DEC n
;= DE 'HELLINCK ! &RAGNETO ' ,AUX 6 (AERTLEIN - *OUHET * 3FERRAZZA - ET AL ,IPID POLYUNSATURATION DETERMINES THE EXTENT OF MEMBRANE STRUCTURAL CHANGES INDUCED BY !MPHOTERICIN " IN 0ICHIA PASTORIS YEAST "IOCHIM "IOPHYS !CTA "IOMEMBR
;= 7RIESSNEGGER 4 'ķBITZ ' ,EITNER % )NGOLIC % #REGG * DE LA #RUZ "* ET AL ,IPID COMPOSITION OF PEROXISOMES FROM THE YEAST 0ICHIA PASTORIS GROWN ON DIFFERENT CARBON SOURCES "IOCHIM "IOPHYS !CTA APR n
;= 7RIESSNEGGER 4 ,EITNER % "ELEGRATIS -2 )NGOLIC % $AUM ' ,IPID ANALYSIS OF MITOCHONDRIAL MEMBRANES FROM THE YEAST 0ICHIA PASTORIS "IOCHIM "IOPHYS !CTA MAR n
;= +LUG , 4ARAZONA 0 'RUBER # 'RILLITSCH + 'ASSER " 4R¨TZMķLLER - ET AL 4HE LIPIDOME AND PROTEOME OF MICROSOMES FROM THE METHYLOTROPHIC YEAST 0ICHIA PASTORIS "IOCHIM "IOPHYS !CTA NOV
;= "LIGH %' $YER 7* ! RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIlCATION #AN * "IOCHEM 0HYSIOL n
;= *ACOB : 9EAST ,IPIDS %XTRACTION 1UALITY !NALYSIS AND !CCEPTABILITY #RIT 2EV "IOTECHNOL JAN n
;= 3CHNEITER 2 $AUM ' !NALYSIS OF YEAST LIPIDS )N 8IAO 7 EDITOR -ETHODS -OL "IOL VOL ND ED 4OTOWA .* (UMANA 0RESS )NC P n
;= &UCHS " 3ķ 2 4EUBER + %IBISCH - 3CHILLER * ,IPID ANALYSIS BY THIN LAYER CHROMATOGRAPHY ! REVIEW OF THE CURRENT STATE * #HROMATOGR ! n
;= #HRISTIE 77 'AS #HROMATOGRAPHY AND ,IPIDS RD ED #HRISTIE 77 EDITOR "RIDGWATER /ILY 0RESS ,TD
;= (EUPEL 2# )SOLATION AND 0RIMARY #HARACTERIZATION OF 3TEROLS )N 0ARISH 7$.*"4!O3 3TEROIDS /"3 EDITORS !NAL 3TEROLS /THER "IOL 3IGNIF 3TEROIDS !CADEMIC 0RESS P n
;= 2OSENFELD % "EAUVOIT " 2OLE OF THE NON RESPIRATORY PATHWAYS IN THE UTILIZATION OF MOLECULAR OXYGEN BY 3ACCHAROMYCES CEREVISIAE 9EAST n
;= $E 'HELLINCK ! 3CHALLER ( ,AUX 6 (AERTLEIN - 3FERRAZZA - -ARdzCHAL % ET AL 0RODUCTION AND ANALYSIS OF PERDEUTERATED LIPIDS FROM 0ICHIA PASTORIS CELLS 0,O3 /NE n 3FGFSFODFT
;= #ARMAN '- (ENRY 3A 0HOSPHOLIPID BIOSYNTHESIS IN YEAST !NNU 2EV "IOCHEM n
;= "AGNAT - +ERƊNEN 3 3HEVCHENKO ! 3IMONS + ,IPID RAFTS FUNCTION IN BIOSYNTHETIC DELIVERY OF PROTEINS TO THE CELL SURFACE IN YEAST 0ROC .ATL !CAD 3CI53! MAR n
;= 0ROSZYNSKI 4* +LEMM 27 'RAVERT - (SU 00 'LOOR 9 7AGNER * ET AL ! GENOME WIDE VISUAL SCREEN REVEALS A ROLE FOR SPHINGOLIPIDS AND ERGOSTEROL IN CELL SURFACE DELIVERY IN YEAST 0ROC .ATL !CAD 3CI 5 3 ! DEC n
;= :HANG , $ĿAZ $ĿAZ . :ARRINGHALAM + (ERMANSSON - 3OMERHARJU 0 #HUANG * $YNAMICS OF THE ETHANOLAMINE GLYCEROPHOSPHOLIPID REMODELING NETWORK 0,O3 /NE E
;= #ARMAN '- (ENRY 3A 0HOSPHOLIPID BIOSYNTHESIS IN THE YEAST 3ACCHAROMYCES CEREVISIAE AND INTERRELATIONSHIP WITH OTHER METABOLIC PROCESSES 0ROG ,IPID 2ES n
;= .OHTURFFT ! :HANG 3# #OORDINATION OF ,IPID -ETABOLISM IN -EMBRANE "IOGENESIS !NNU 2EV #ELL $EV "IOL n
;= 'ASPAR -, (OFBAUER (& +OHLWEIN 3$ (ENRY 3! #OORDINATION OF 3TORAGE ,IPID 3YNTHESIS AND -EMBRANE "IOGENESIS %VIDENCE FOR CROSS TALK BETWEEN TRIACYLGLYCEROL METABOLISM AND PHOSPHOLIPIDYLINOSITOL SYNTHESIS * "IOL #HEM n
;= !LVAREZ 6ASQUEZ & 3IMS +* 6OIT %/ (ANNUN 9A #OORDINATION OF THE DYNAMICS OF YEAST SPHINGOLIPID METABOLISM DURING THE DIAUXIC SHIFT 4HEOR "IOL -ED -ODEL
;= :AMAN 3 ,IPPMAN 3) :HAO 8 "ROACH *2 (OW 3ACCHAROMYCES RESPONDS TO NUTRIENTS !NNU 2EV 'ENET n
;= 9OUNG "0 3HIN **( /RIJ 2 #HAO *4 ,I 3# 'UAN 8, ET AL 0HOSPHATIDIC !CID )S A P( "IOSENSOR THAT LINKS MEMBRANE BIOGENESIS TO METABOLISM 3CIENCE !UGUST n
;= 4ULLER ' .EMEC 4 (RASTNIK # $AUM ' ,IPID COMPOSITION OF SUBCELLULAR MEMBRANES OF AN &9 DERIVED HAPLOID YEAST WILD TYPE STRAIN GROWN ON DIFFERENT CARBON SOURCES 9EAST n
;= 'ASPAR -, !REGULLIN -! *ESCH 3! (ENRY 3! )NOSITOL )NDUCES A 0ROFOUND !LTERATION IN THE 0ATTERN AND 2ATE OF 3YNTHESIS AND 4URNOVER OF -EMBRANE ,IPIDS IN 3ACCHAROMYCES CEREVISIAE * "IOL #HEM n
;= $E 3MET #( 6ITTONE % 3CHERER - (OUWELING - ,IEBISCH ' "ROUWERS *& ET AL 4HE YEAST ACYLTRANSFERASE 3CTP REGULATES FATTY ACID DESATURATION BY COMPETING WITH THE DESATURASE /LEP -OL "IOL #ELL n
;= 'ONZALEZ #) -ARTIN #% &ATTY !CID RESPONSIVE #ONTROL OF M2.! 3TABILITY 5.3!452!4%$ &!449 !#)$ ).$5#%$ $%'2!$!4)/. /& 4(% 3!##(!2/-9#%3 /,% 42!.3#2)04 * "IOL #HEM n
;= +ATO ( 3AKAKI + -IHARA + 5BIQUITIN PROTEASOME DEPENDENT DEGRADATION OF MAMMALIAN %2 STEAROYL #O! DESATURASE * #ELL 3CI n
;= "RAUN 3 -ATUSCHEWSKI + 2APE - 4HOMS 3 *ENTSCH 3 2OLE OF THE UBIQUITIN SELECTIVE #$#5&$.0, CHAPERONE SEGREGASE IN %2!$ OF /,% AND OTHER SUBSTRATES %-"/ * n
;= "IGAY * !NTONNY " #URVATURE ,IPID 0ACKING AND %LECTROSTATICS OF -EMBRANE /RGANELLES $ElNING #ELLULAR 4ERRITORIES IN $ETERMINING 3PECIlCITY $EV #ELL n 3FGFSFODFT
;= 'RINSTEIN 3 )MAGING SIGNAL TRANSDUCTION DURING PHAGOCYTOSIS PHOSPHOLIPIDS SURFACE CHARGE AND ELECTROSTATIC INTERACTIONS !M * 0HYSIOL #ELL 0HYSIOL #n
;= (OLTHUIS *#- -ENON !+ ,IPID LANDSCAPES AND PIPELINES IN MEMBRANE HOMEOSTASIS .ATURE n
;= $I 0AOLO ' $E #AMILLI 0 0HOSPHOINOSITIDES IN CELL REGULATION AND MEMBRANE DYNAMICS .ATURE n
;= ,OEWEN #*2 'ASPAR -, *ESCH 3A $ELON # +TISTAKIS .4 (ENRY 3A ET AL 0HOSPHOLIPID METABOLISM REGULATED BY A TRANSCRIPTION FACTOR SENSING PHOSPHATIDIC ACID 3CIENCE n
;= *ESCH 3! ,IU 0 :HAO 8 7ELLS -4 (ENRY 3! -ULTIPLE %NDOPLASMIC 2ETICULUM TO .UCLEUS 3IGNALING 0ATHWAYS #OORDINATE 0HOSPHOLIPID -ETABOLISM WITH 'ENE %XPRESSION BY $ISTINCT -ECHANISMS * "IOL #HEM n
;= *ESCH 3! :HAO 8 7ELLS -4 (ENRY 3! 'ENOME WIDE ANALYSIS REVEALS INOSITOL NOT CHOLINE AS THE MAJOR EFFECTOR OF )NOP )NOP AND UNFOLDED PROTEIN RESPONSE TARGET GENE EXPRESSION IN YEAST * "IOL #HEM n
;= .IKAWA * (OSAKA + 9AMASHITA 3 $IFFERENTIAL REGULATION OF TWO MYO INOSITOL TRANSPORTER GENES OF 3ACCHAROMYCES CEREVISIAE -OL -ICROBIOL n
;= +IMATA 9 )SHIWATA +IMATA 9 9AMADA 3 +OHNO + 9EAST UNFOLDED PROTEIN RESPONSE PATHWAY REGULATES EXPRESSION OF GENES FOR ANTI OXIDATIVE STRESS AND FOR CELL SURFACE PROTEINS 'ENES TO #ELLS n
;= 4AVASSOLI 3 #HAO *4 9OUNG "0 #OX 2# 0RINZ 7A DE +ROON !)0- ET AL 0LASMA MEMBRANE ENDOPLASMIC RETICULUM CONTACT SITES REGULATE PHOSPHATIDYLCHOLINE SYNTHESIS %-"/ 2EP n
;= 'ARCIA /RTEGA 8 6ALERO & -ONTESINOS *, ! VERSATILE APPROACH TO IMPLEMENT OXYGEN LIMITING CONDITIONS FOR IMPROVING RECOMBINANT PROTEIN PRODUCTION IN 0ICHIA PASTORIS !PPL -ICROBIOL "IOTECHNOL
;= 3URMA -! +LOSE # 0ENG $ 3HALES - -REJEN # 3TEFANKO ! ET AL ! ,IPID % -!0 )DENTIlES 5BX AS A #RITICAL 2EGULATOR OF ,IPID 3ATURATION AND ,IPID "ILAYER 3TRESS -OL #ELL n
;= 2ON $ 7ALTER 0 3IGNAL INTEGRATION IN THE ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE .AT 2EV -OL #ELL "IOL JUL n
;= 0INEAU , #OLAS * $UPONT 3 "ENEY , &LEURAT ,ESSARD 0 "ERJEAUD *- ET AL ,IPID INDUCED %2 STRESS SYNERGISTIC EFFECTS OF STEROLS AND SATURATED FATTY ACIDS 4RAFlC JUN n
;= 6OLMER 2 VAN DER 0LOEG + 2ON $ -EMBRANE LIPID SATURATION ACTIVATES ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE TRANSDUCERS THROUGH THEIR TRANSMEMBRANE DOMAINS 0ROC .ATL !CAD 3CI 5 3 ! n
;= "IEN #- %SPENSHADE 0* 3TEROL REGULATORY ELEMENT BINDING PROTEINS IN FUNGI (YPOXIC TRANSCRIPTION FACTORS LINKED TO PATHOGENESIS %UKARYOT #ELL n
;= (UGHES !, 4ODD ", %SPENSHADE 0* 32%"0 0ATHWAY 2ESPONDS TO 3TEROLS AND &UNCTIONS AS AN /XYGEN 3ENSOR IN &ISSION 9EAST #ELL n
;= :ANGHELLINI * .ATTER + *UNGREUTHMAYER # 4HALHAMMER ! +URAT #& 'OGG &ASSOLTER ' ET AL 1UANTITATIVE MODELING OF TRIACYLGLYCEROL HOMEOSTASIS IN YEASTnMETABOLIC REQUIREMENT FOR LIPOLYSIS TO PROMOTE MEMBRANE LIPID SYNTHESIS AND CELLULAR GROWTH &%"3 * n 3FGFSFODFT
;= +URAT #& 7OLINSKI ( 0ETSCHNIGG * +ALUARACHCHI 3 !NDREWS " .ATTER + ET AL #DK#DC DEPENDENT ACTIVATION OF THE MAJOR TRIACYLGLYCEROL LIPASE 4GL IN YEAST LINKS LIPOLYSIS TO CELL CYCLE PROGRESSION -OL #ELL n
;= 2OSENBERGER 3 $AUM ' ,IPIDS OF YEAST MITOCHONDRIA )N 2ES 3IGNPOST P n
;= +IM 3 &YRST ( 3ABA * !CCUMULATION OF PHOSPHORYLATED SPHINGOID LONG CHAIN BASES RESULTS IN CELL GROWTH INHIBITION IN 3ACCHAROMYCES CEREVISIAE 'ENETICS n
;= $ESFARGES , $URRENS 0 *UGUELIN ( #ASSAGNE # "ONNEU - !IGLE - 9EAST MUTANTS AFFECTED IN VIABILITY UPON STARVATION HAVE A MODIlED PHOSPHOLIPID COMPOSITION 9EAST n
;= +LOSE # %JSING #3 'ARCĿA 3ĈEZ !* +AISER (* 3AMPAIO *, 3URMA -A ET AL 9EAST LIPIDS CAN PHASE SEPARATE INTO MICROMETER SCALE MEMBRANE DOMAINS * "IOL #HEM JUL n
;= 'AIGG " 4OULMAY ! 3CHNEITER 2 6ERY LONG CHAIN FATTY ACID CONTAINING LIPIDS RATHER THAN SPHINGOLIPIDS PER SE ARE REQUIRED FOR RAFT ASSOCIATION AND STABLE SURFACE TRANSPORT OF NEWLY SYNTHESIZED PLASMA MEMBRANE !40ASE IN YEAST * "IOL #HEM NOV n
;= "AMMERT '& &OSTEL *- 'ENOME WIDE EXPRESSION PATTERNS IN 3ACCHAROMYCES CEREVISIAE COMPARISON OF DRUG TREATMENTS AND GENETIC ALTERATIONS AFFECTING BIOSYNTHESIS OF ERGOSTEROL !NTIMICROB !GENTS #HEMOTHER MAY n
;= 'HANNOUM -A 2ICE ," !NTIFUNGAL AGENTS MODE OF ACTION MECHANISMS OF RESISTANCE AND CORRELATION OF THESE MECHANISMS WITH BACTERIAL RESISTANCE #LIN -ICROBIOL 2EV OCT n
;= (ULL #- 0ARKER *% "ADER / 7EIG - 'ROSS 5 7ARRILOW !'3 ET AL &ACULTATIVE STEROL UPTAKE IN AN ERGOSTEROL DElCIENT CLINICAL ISOLATE OF CANDIDA GLABRATA HARBORING A MISSENSE MUTATION IN %2' AND EXHIBITING CROSS RESISTANCE TO AZOLES AND AMPHOTERICIN " !NTIMICROB !GENTS #HEMOTHER n
;= 6EEN - ,ANG # 0RODUCTION OF LIPID COMPOUNDS IN THE YEAST 3ACCHAROMYCES CEREVISIAE !PPL -ICROBIOL "IOTECHNOL n
;= ,EBER 2 :INSER % (RASTNIK # 0ALTAUF & $AUM ' %XPORT OF STERYL ESTERS FROM LIPID PARTICLES AND RELEASE OF FREE STEROLS IN THE YEAST 3ACCHAROMYCES CEREVISIAE "IOCHIM "IOPHYS !CTA "IOMEMBR n
;= 3ONG *, (ARRY *" %ASTMAN 24 "RIAN ' 7HITE 4# /LIVER "' 4HE #ANDIDA ALBICANS ,ANOSTEROL ᎎ $EMETHYLASE %2' 'ENE 0ROMOTER )S -AXIMALLY )NDUCED AFTER 0ROLONGED 'ROWTH WITH !NTIFUNGAL $RUGS 4HE #ANDIDA ALBICANS ,ANOSTEROL $EMETHYLASE %2' 'ENE 0ROMOTER )S -AXIMALLY )NDUCED AFTER 0ROLONGED 'R !NTIMICROB !GENTS #HEMOTHER n
;= 6EEN - 3TAHL 5 ,ANG # #OMBINED OVEREXPRESSION OF GENES OF THE ERGOSTEROL BIOSYNTHETIC PATHWAY LEADS TO ACCUMULATION OF STEROLS IN &%-3 9EAST 2ES n
;= 7EBER *- 0ONTI #' +APPELI / 2EISER * &ACTORS AFFECTING HOMOLOGOUS OVEREXPRESSION OF THE 3ACCHAROMYCES CEREVISIAE LANOSTEROL ALPHA DEMETHYLASE GENE 9EAST n
;= "ARD - ,EES .$ 4URI 4 #RAFT $ #OFRIN , "ARBUCH 2 ET AL 3TEROL SYNTHESIS AND VIABILITY OF ERG CYTOCHROME 0 LANOSTEROL DEMETHYLASE MUTATIONS IN 3ACCHAROMYCES CEREVISIAE AND #ANDIDA ALBICANS ,IPIDS n
;= 6ALKONEN - 7ARD - 7ANG ( 0ENTTILƊ - 3ALOHEIMO - )MPROVEMENT OF &OREIGN 0ROTEIN 0RODUCTION IN !SPERGILLUS NIGER VAR AWAMORI BY #ONSTITUTIVE )NDUCTION OF THE 5NFOLDED 0ROTEIN 2ESPONSE !PPL %NVIRON -ICROBIOL DEC n 3FGFSFODFT
;= 6OGL 4 4HALLINGER '' :ELLNIG ' $REW $ #REGG *- 'LIEDER ! ET AL 4OWARDS IMPROVED MEMBRANE PROTEIN PRODUCTION IN 0ICHIA PASTORIS 'ENERAL AND SPECIlC TRANSCRIPTIONAL RESPONSE TO MEMBRANE PROTEIN OVEREXPRESSION . "IOTECHNOL MAR n "QQFOEJY
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