St r ept omycesi ncar nat us のリファンピシン耐性とシネフンギン生産性の向上

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I mpr ovementofSi nef ungi n- Pr oduci ngStr ai nof St r ept omycesi ncar nat us byConf er r i ng

Ri f ampi ci n- Resi stancethr oughUl tr avi ol et Li ghtI r r adi ati onandPr otopl astRegener ati on

TakashiTamur a,LiYi nShu,Hi dehi koTanaka andKenj iI nagaki

(DepartmentofBioresourcesChemistry)

Secondarymetaboliteproductionbygram-positivebacteriaisstrictlyregulated atthetranscriptionofthebiosyntheticgenestomRNA inresponsetocertain stringentconditions.Therefore,somemutationaldisruptionofregulatorydomains ofthebacterialRNApolymerasemightincreasetheproductionoftheantibiotics.

Inthisstudy,wehaveattemptedtoimprovethe sinefungin-producing strain of StreptomycesincarnatusNRRL8057 byirradiatingultravioletlightontheproto- plast,andselectingmutantsthatacquiredtheresistancetorifampicin,theantibi- oticwhichspecificallybindstotheβ‑subunitofbacterialRNA polymerase.After three roundsofmutation,10 strainswere obtained with varied resistance to rifampicin.A mutantwhichshowedthehighestresistancewasfoundtohavethe highestsinefunginproduction,whichwas2.4timeshigher(0.45±0.11μg/ml)than the wild type strain (0.19±0.07μg/ml).The breeding approach byrifampicin- resistance may be advantageous overthe classicalrandom screening since it requiresmuchsmallernumberofcandidatestobeexamined.

Keywords:Sinefungin,Protoplastregeneration,Rifampicin,RNApolymerase, Streptomycesincarnatus

Introduction

Sinefunginisanucleosideantibiotic,inwhicha moleculeofornithineislinkedtothe5ʼ‑endof adenosinethroughaC‑C bond(Fig.1).Thenu- cleosideantibioticwasisolatedatRhone-Poulenc Laboratories from the culture broth ofStre- ptomycesincarnatusNRRL8089 andatEliLilly Research Laboratories from the fermentation brothofStreptomycesgriseolus.Theantibiotic hasastronginhibitoryeffectonvariousfungi andparasites ,butisconsideredasnephrotox- icinvivo.Ithasbeenreportedthatthecell-free extractpreparedfrom ahighproducingvariant ofS.incarnatusproduced sinefungin from L‑

arginineandATPinthepresenceofpyridoxal‑5ʼ

‑phosphate,dithiothreitolandmagnesium ion . The enzymes thatare involved in sinefungin productionhavenotbeencharacterizeddueto theinstabilityandlowexpressionoftheenzymes. Thus,theimprovementin theyield ofthemi- crobialproductionofsinefunginwouldbeneces- saryandshouldallow ustopurifyandcharacter- izetheintriguingenzymes,whichcondenseATP andL‑arginine.Ithasbeenreportedrecentlythat certainmutationsconferringresistancetostrep- tomycin,gantamicin,and rifampicin resultin increasedantibioticsproductionbyStreptomyces

ReceivedOctober1,2001

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coelicolor and other Bacilli . This paper describes the effect of the introduction of rifampicin-resistanceto the sinefugin‑producer strainofS.incarnatusNRRL8057.

MaterialsandMethods Organismsand Medium

The sinefungin producerStreptomyces incar- natusNRRL8057wasobtainedfrom theAgricul- turalResearch Service,United StatesDepart- ment of Agriculture. The R6 regeneration medium contained200gofsucrose,10gofdex- trin,1gofcasaminoacid,50mgofMgSO 7H O, 11g of monosodium L‑glutamate,100mg of K SO ,7g ofCaCl 2H O,100mL of100mM sodium 3‑( ‑Morphorino)propanesulfonate buffer(pH 7.2),20gagaranddistilledwaterto 1000ml.TSB agar medium contained 3g of trypticsoybroth(DIFCO)and1.5gofagarin100 mLofdeionizedwater.

UltravioletLightIrradiationonProtoplast Conditionsforcultivationandpreparationof protoplasts were as described previously . Protoplastsweresedimentedbycentrifugationat 500to600× gfor10min,andspreadonR6regen- erationmedium.Theplatewasirradiatedwith ultravioletlight(ToshibaGL15lamp)inaclean bench (3.31× 10 J/cm /sec),quickly contained inadarkplace,andincubatedat30°Cfor2days untilcoloniesgrew visible.Theplateswerethen coveredwith3mlofsoftagarcontaining60μg/

mlrifampicin,andfurtherincubatedat30°Cfor3

days.

SamplePreparation

Theregeneratedprogenywassubculturedon 1.0mlofTSB‑agarmedium ina24‑holeassay plate,andwasallowedtogrow for5daysat30

°C.Theculturemedium wascutinhalveswitha toothpick,andonehalfwasdippedin1.0mlof3.0 M sodium acetatebuffer,pH 5.3andboiledfor20 minwith0.1gofactivatedcharcoal.Then,char- coalpowderwasseparatedfrom thesupernatant fluidaftercentrifugationat10,000rpm for10min, and washed twicewith 1mlofwater.Compo- nentsabsorbedoncharcoalwereelutedbysoak- ingthecharcoalpowderin1mlof50% aqueous acetoneovernight,thenca.20mgofmagnesium phosphate was added to the mixture and centrifugedat10,000rpm for10min.Clearsuper- natantsolutionwastransferredtoanew sample tubeandconcentratedtodrynessonspeed-vac concentrator.Theresiduewasdissolvedin50μl ofsterilewater.

EcoRIMethylaseInhibitorassay

Aliquotof10μloftheabovesamplewasmixed with1.2μgoflambdaDNA (TaKaRa),3.2nmolS

‑adenosyl‑L‑methionine,2μlofEcoRImethylase buffer(× 10),and10unitsofEcoRImethylase (totalvolume20μl)andincubatedat37°Cfor1h.

Twomicroliterof3M sodium acetate,pH 5.3, and20μlofisopropanolwereaddedtothesolu- tion.Afterthesolutionwasstandatroom tem- peraturefor10min,DNA wasprecipitated by centrifugationat14,000rpm for20min.Thepellet waswashed with cold 70% ethanol,and dried under the reduced pressure.The residue was dissolvedin8μlofsterile,deionizedwaterand mixedwith1μlof(× 10)bufferforEcoRIrestric- tionenzyme.EcoRIrestrictionenzyme(10U)was addedandincubatedat37°Cfor2h.Thesolution wasmixedwith1μlofsampleloadingbuffer(× 10)andrunon0.7%‑agarosegel.Whensinefun- ginwascontainedinthesamplesolution,lambda DNA wasdigestedbyEcoRIrestrictionenzyme, andthecharacteristicladderwasobservedonthe Fig.1 Sinefungin.

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gel.Whenthestraindidnotproducesinefungin theagarose-gelelectrophoresiswouldshow the intactlambdaDNA.

Determination of Sinefungin Production by HPLC

Sinefungin production was detected by the EcoRImethylaseinhibition assay asdescribed above,andtheamountofsinefunginproducedin themedium wasdetermined by HPLC system implementedwithacation-exchangecolumn.A strainofS.incarnatuswassubculturedonthree TSB agarmedia (1μl)contained in a 24‑hole assay plate,and grown at30°C for 5 days. Sinefungin was extracted from the culture medium asdescribedaboveusingactivatedchar- coal,anddeterminedbyanalyticalHPLCusinga CAPCELL PAK SCX column(4.6mm I.D.× 150 mm,ShiseidoFineChemicals),ShimadzuLC‑6A liquid chromatography pump,and a Shimadzu SPD‑6A uv spectrophotometric detector.The column was developed with 0.5M ammonium formate(pH 4.0)ataflow rateof0.5μl/min.The chromatogram wasmonitoredbyabsorptionat 260nm.Thesinefungin contentin each sample wasestimatedfrom thepeakareaofsinefungin elutedat13.8min.Theproductivityofeachstrain wasdesignatedasthemeananddeviationcalcu- latedfrom thethreeseparatecultures.

ResultsandDiscussion

MembersofthegeneraStreptomyces,Bacillus, andPseudomonasaresoilbacteriathatproducea highproportionofagriculturallyandmedically important antibiotics. The development of rationalapproachestoimprovetheproductionof antibioticsfrom theseorganismsisthereforeof considerable industrial and economic impor- tance.IthasbeenreportedthatrpsLmutationis likely to enhance the antibiotic production of streptomycetes by inducing the mutations on RNA binding site ofribosomalprotein .We firsttriedtoobtainstreptomycinresistantstrains ofS.incarnatus,butwecouldnotobtainthose

mutantsundervariousmutagenesisconditions includingvariedlengthofultravioletlightirradi- ationandvariedstreptomycinconcentrations.

Becausethesinefunginproducer,S.incarnatus NRRL8089,didnotform sporesonseveralmedia tested,weattemptedtoseparateeachcellfrom themyceliabydigestingthecellwallandprepar- ingprotoplast.Conditionssuitableforprepara- tion and regeneration ofstreptomycete proto- plastshavebeendevelopedbyOkanishietal , and theprocedurehasbeen modified and im- provedbyShirahataetal andBaltzandMatsu- shima . The protoplasts ofS. incarnatus regeneratedthemyceliaform mostefficientlyon R6medium.Ultravioletlighthasbeenirradiated on the regeneration medium shortly afterthe protoplastswerespreadontheplate.Theirradia- tiontimehasbeenoptimizedtobe4min,where thesurvivalratewasintherangeof0.1to0.5%.

Ourresultssuggestthattheprotoplastregenera- tioncanbeanalternativemethodforusingcopi- oushaploidspores,whichisusuallyrecommend- edtominimizetheriskoftherevertantappear- anceandunstablephenotype.

Thesinefungin production by the wild type strainvariedintherangeof0.13to0.30μg/μl, andthemeanproductivityofthesixwildtype strainswas0.19± 0.07μg/μl.Thewildstrainwas resistanttorifampicinuptotheconcentrationof 16μg/mlwhengrownontheTSB‑agarmedium.

Thesinefunginproductionbywildtypestrains andmutantsaresummarizedinFig.2.Thefirst roundofmutagenesishasyielded3strains,which increasedtherifampicinresistanceupto64μg/

ml.Oneofthemutantsdecreasedthesinefungin production to 0.16± 0.13μg/mlwhiletheother tworetainedtheproductivityat0.23± 0.11μg/ml and 0.24± 0.08μg/ml.Thus,thefirstround of mutationdidnotincreasedthesinefunginproduc- tivitynotablyalthoughtheantibioticresistance hasincreasedalmost4‑times.Thesecondround ofmutationhasyieldedonlyonemutantstrain, whichwasresistantto250μg/mlrifampicin,but

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thesinefunginproductivitywas0.25± 0.09μg/ml. Again,thesecondarymetaboliteproductiondid notincreaseastheincreased rifampicin‑resistance,whichwasalmost16‑foldofthewildtype strain.A strain thatincreased the sinefungin- productionwasfoundamongmutantsobtained by thethird round ofmutation.Among the6 mutants,whichsurvivedwhentherifampicinwas added in the concentration of500μg/ml,one strainproducedsinefunginattheconcentration of 0.45± 0.11μg/ml. The other five mutant strainsproducedsinefunginintherangeof0.18to 0.28μg/ml.

Ourprincipalfinding in thisstudy indicates thatthe introduction of rifampicin‑resistance resultedinapproximately2‑to3‑foldincreaseof thesinefungin-productionbyS.incarnatus,but this is normally the case found in classical mutagenesisandrandom screening.Althoughthe sinefunginproductiondidnotincreasesoremark- ably,the three roundsofmutation finally in- creasedtheproductivityby2.4‑foldofthepro- ductionbythewildtypestrain.Itshouldbenoted thatthetotalnumberofmutantsweassayedthe productivitybyHPLC wasonly10throughthe three round ofmutation.Searching for high- producer strains after ultraviolet light- irradiationnormallytakesthetroubleofassay- inghundredsofstrainssincesuch therandom mutation by physical or even by chemical mutagenesisnormallyresultsin2‑to3‑foldof

productivity enhancement only at the small chancesof0.1‑0.5%.Inthispointofview,the screening for antibiotic-resistance may be an effectivemethodtoreducethetroubleofexamin- inghundredsofstrains.Furthermutationconfer- ring theresistanceto ribosome-binding antibi- oticssuchasgentamicin totherifampicin‑resis- tantmutantmayfurtherincreasethesinefungin productionofS.incarnatus.

Acknowledgements

TheauthorsaregratefultoDr.KozoOchi,National Food Research Institutes,forhelpfulsuggestions.The presentworkwassupportedinpartbyagrant-in-aidfor ScientificResearch(07680688)from theMinistryofEduca- tion,ScienceandCultureofJapantoK.I.

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UV照射とプロトプラスト再生法による