A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

A

plate

method

for

rapid

screening

of

Ketogulonicigenium

vulgare

mutants

for

enhanced

2-keto-l-gulonic

acid

production

Weichao

Yang

_,

_Litao

_Han

_,

_Mandlaa

_Mandlaa

_,

_Haihong

_Zhang

_,

_Zhongze

_Zhang

_,

Hui

Xu

a_{ChineseAcademyofSciences,InstituteofAppliedEcology,Shenyang,China}

b_{NortheastPharmaceuticalGroupCo.,Ltd.,Fermentation&BiotechnologyLaboratory,Shenyang,China}

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Articlehistory: Received16April2014 Accepted5December2016 Availableonline21February2017 AssociateEditor:RosaneFreitas Schwan Keywords: Ketogulonicigeniumvulgare pHindicator Rapidscreening Spaceflightmutation 2-Keto-l-gulonicacid

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AnewplatemethodwasdevelopedforrapidscreeningofKetogulonicigeniumvulgaremutants overproducing2-keto-l-gulonicacid(2-KLG).Thescreeningmethodologytookthe advan-tage oftheaciditycausedby2-KLG,whichchangesthecolor ofbromothymolblue(pH indicator)frombluetoyellow.Usingtheproposedmethod,amutant,K.vulgare65,was selectedfrom20,000coloniesproducedbyastrainsubjectedtospaceflightmutagenesis. Whenco-culturedwithBacillusmegaterium2980in20-Lfermenters,K.vulgare65showeda highconversionrate(94.45%)ofl-sorboseto2-KLG.Incontrasttothetraditionalscreening method,thisonesignificantlyimprovedthefrequencyofobtainingpositivemutants.The proposedplatescreeningmethodiscost-effectiveandeasytorunandisthususefulforthe isolationandscreeningofK.vulgaremutantsoverproducing2-KLG.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

2-Keto-l-gulonicacid (2-KLG)isanimportantprecursor for the synthesis of vitamin C (l-ascorbic acid) by a two-step fermentationprocess,whichaccountsfor90%oftheglobal vitaminCproduction.1 _{Researchhasshownthat2-KLGcan} bebioconvertedfroml-sorbosebyamixedcultureof Ketogu-lonicigeniumvulgare andBacillus spp.2,3 K.vulgare contains a completeenzymesystemforconvertingl-sorboseto2-KLG,4 butitsgrowthispoorinthemonoculture,resultinginavery lowyieldof2-KLG.5_{Acompanionstrain,Bacillusmegaterium,}

∗ _{Correspondingauthor.}

E-mails:[email protected],[email protected](H.Xu).

is generallyconsidered togenerate andrelease, inthe fer-mentation broth, the metabolites that stimulateK. vulgare propagationand2-KLGaccumulation.6–10_{Thesemetabolites} contain activeproteins,6 amino acids,8 antioxidants,10 and otheractivesubstrates.7,9_{Inrecentyears,K.vulgarehasbeen} considered tobethe keymicroorganism forthe bioconver-sionofl-sorboseto2-KLGandofgreaterimportancethanthe companionbacteriumin2-KLGproduction.11,12

Random mutagenesisand selectionstrategies are effec-tiveforthescreeningofK.vulgaremutantscapableof2-KLG overproduction.13,14_{Whenobtaininghigh-efficiencymutants} ofK.vulgare,itiscrucialtoscreenalargenumberofmutants

http://dx.doi.org/10.1016/j.bjm.2017.02.002

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

immediately after a mutagenic treatment. Thus, easy and efficient screening methods are desired for the screening andisolationof2-KLG-overproducingmutantsofK.vulgare. Sincemixedfermentationinvolvesco-cultivationoftwo bac-terialstrains, the associatedscreeningprocess isrelatively laborious and complicated.7 _{Currently,only onemethod is} availableforthescreeningofK.vulgaremutants,i.e.,the 2-KLGdetermination method.13 _{In this method, the primary} screeningisdone byrandomlyselectingK. vulgarecolonies onplates.Then,thecoloniesareusedforfermentationwith Bacillus spp. inglass tubes, followed bythe determination of the amount of 2-KLG produced. After that, K. vulgare mutantswithhigh yields of2-KLG are selectedand trans-ferred toflasks forfurtherfermentation. Finally, K. vulgare mutantscapableofhighly efficientconversionofl-sorbose to 2-KLG are selected as potential strains for industrial applications.As it involvesrandom colonyselectioninthe primary screening step, followed by laborious in-tube fer-mentationand determinationof2-KLGconcentrations,this conventionalscreeningmethodisconsideredinefficientand time-consuming.

Inthepresentstudy,anewplatescreeningmethodwas developedfor screening ofK. vulgare mutants witha high conversionrateofl-sorboseto2-KLG.Theproposedmethod isbasedon the followingpoints:(1) theactive metabolites (activators) secreted by B. megaterium enhance the growth ofK.vulgare5,6,8–10_{; and(2) the2-KLG secretedbyK.vulgare} mutantslowers the mediumpH ina certain zoneon agar plates,therebychangingthecolorofthebromothymolblue indicator from blue to yellow. The proposed method was designedforrapidscreeningofK.vulgaremutantstodetect thosecapableof2-KLGoverproduction.Itwasfurtherapplied tothescreeningof20,000coloniesofK.vulgareobtainedafter spaceflightmutagenesis,amongwhichthemutantK.vulgare 65wasselected.K.vulgare65wasco-culturedwithB. mega-teriumunderlaboratoryconditions,anditsconversionrateof l-sorboseto2-KLGwasmonitored.Theresultswereusedto evaluatetheefficiencyoftheindicatorplatemethodforthe isolationandscreeningof2-KLG-overproducingmutantsof K.vulgare.

Materials

and

methods

Bacterialstrains

Thecandidate2-KLG-producingstrains,K.vulgare07andB. megaterium2980,wereobtainedfromNortheast Pharmaceuti-calGroupCo.,Ltd.(Shenyang,China).

Laboratorymedia

Threemediawerepreparedforthemaintenance,growthtest, andisolation,respectively,ofK.vulgare07andB.megaterium 2980.Theseincludedl-sorbose-limitingmedium(20.0gof l-sorbose, 3.0g ofcorn steep liquor,1.0gofcarbamide, 1.0g ofKH2PO4,0.2gofMgSO4·7H2O,and1.0gofCaCO3perliter;

pH6.7–7.0),fermentationmedium(80.0gofl-sorbose,15.0g of corn steep liquor, 12.0g of carbamide, 1.0g of KH2PO4,

0.2gofMgSO4·7H2O,and1.0gofCaCO3perliter;pH6.7–7.0;

l-sorboseandcarbamideweresterilizedseparately),and iso-lationmedium(20.0gofl-sorbose,5.0gofyeastextract,5.0g ofcornsteepliquor,10.0gofpeptone,3.0gofbeefextract,1.0g ofcarbamide,1.0gofKH2PO4,0.2gofMgSO4·7H2O,and18gof

agarperliter;pH7.0–7.2).Themediawereautoclavedpriorto use.

Spaceflightmutagenesis

Spaceflightmutagenesishasbeenshowntobemoreefficient thanothermutationmethodsformicrobes.15–17 _Theunique environmentsintheouterspace,suchasmicrogravity,strong radiation,thepresenceofhigh-energyparticles,and alternat-ingmagneticfields,caninducegenemutations.18_Inthisstudy, the spaceflightmutagenesistechnologywas usedtoobtain moremutants.

The two candidate strains, K. vulgare 07 and B. mega-terium2980,wereco-culturedonl-sorbose-limitingmediumin Eppendorftubesfor48handthentransferredtothereturning moduleofthemannedspacecraftShenzhouVIII.The space-craftwaslaunched at5:58a.m.(Beijingtime)onNovember 1,2011fromtheJiuquanSatelliteLaunchingCenter(Gansu, China).Aftera397-hflightinouterspace,thereturning mod-ulelandedintheInnerMongoliaAutonomousRegion,China. The Eppendorf tubes with anintact package were keptin aniceboxandtransportedimmediatelytothelaboratoryfor use.

EffectsofB.megaterium-secretedactivatorsonthegrowth

ofK.vulgare

ThecultureofK.vulgare07waspreparedas10-foldserial dilu-tions.A0.1-mLaliquotofthedilutedculture,containing30–50 colony-formingunits(CFU),wasspreadonanagarplate con-taining30mLofsolidifiedisolationmedium.Twoagarplates wereinoculated.Tooneplate,0.1mLofco-culturebrothofB. megaterium2980andK.vulgare07(30-hfermentationat29◦C, OD650=0.935), pre-filteredthrough a sterile0.22-␮m

mem-branefilter,wasaddedandspread.Theotheragarplatewas usedasacontrol.Thetwoplateswereincubatedat29◦Cfor 96h.Thediametersof40coloniesoneachplatewere deter-mined using a Vernier caliper (0–150, Harbin Measuring & CuttingToolGroupCo.,Ltd.,Heilongjiang,China).

ChoiceofpHindicator

Three pH indicators were tested for the direct determina-tion of 2-KLG production on plates, including methyl red [2-(4-dimethylaminophenylazo)benzoicacid;Tianjin Yongda ChemicalReagentCo.,Ltd.,Tianjin,China],bromocresol pur-ple (5,5-dibromo-O-cresolsulfonphthalein; Shenyang Xinxi Reagent Factory, Liaoning, China), and bromothymol blue (3,3-dibromothymolsulfonphthalein;TianjinBodiChemicals Co.,Ltd.,Tianjin,China).Eachindicatorwasdissolvedin dis-tilledwatercontaining5%(v/v)ethanol,andtheconcentration was adjusted to0.1% (w/v).One milliliterof each solution wasaddeddropwisetoeachagarplatetoindicatethecolor changesaroundthecoloniesofK.vulgare.Theindicatorthat showedanobviouscolorchangewaschosenasasuitablepH indicator.

Newplatescreeningmethod

Theschematicoftheplatescreeningprocedureisshownin Fig.1.First,10-foldserialdilutionsofacultureofK.vulgare07 subjectedtospaceflightmutagenesiswereprepared.A0.1-mL aliquotofthedilutedculturecontaining30–50CFU/mLwas thenspread onanagarplatecontaining30mLofsolidified isolation medium.Second, 0.1mLofco-culture broth ofB. megaterium2980andK.vulgare07(30-hfermentationat29◦C, OD650=0.800–1.000), pre-filtered through a sterile 0.22-␮m

membrane filter, was added to each agar plate inoculated withthespaceflight-inducedK.vulgaremutants.After96hof incubationat29◦C,coloniesofK.vulgaremutantsappeared on each plate. Third, to obtain pure mutant isolates, cells fromeach colonygrownonagar plateswere transferredto fresh agar plates using velvets and incubated at 29◦C for 96h. Finally, 1mL of a bromothymol blue solution (0.1%,

w/v)wasaddeddropwisetoeachagarplate,andtheplates were incubated at 29◦C for 30–60min. Yellow zones were observedaroundthecoloniesofK.vulgare07mutants,andthe diametersoftheyellowzonesweremeasuredusingaVernier caliper.Colonieswiththelargestdiametersofyellowzoneson agarplateswereselectedaspotentialmutantsofinterestfor furtherflask fermentationscreening. Approximately20,000 colonies ofK. vulgare 07 were screened, and 500 potential mutantswereselectedforfurtherscreening.

Shake-flaskfermentationscreeningandfermentationtests

Aftertheprimaryagarplatescreening,theselectedmutants werefurtherscreenedbyshake-flaskfermentation.TheK. vul-gare07mutantswereco-culturedwithB.megaterium2980in triplicate250-mLErlenmeyerflaskscontaining20mLof fer-mentationmedium.After48hoffermentationat29◦Cona

1. K. vulgare was induced by space flight and incubated in shake flask.

2. K. vulgare was isolated by serial dilution and growth on agar plates. The agar plates contained the growth activators from B. megaterium

3. Cells from each colony on agar plates were transferred from a velvet to new plates.

4. After addition of bromothymol blue solution, the yellow zones around the colonies of K. vulgare mutants were discerned.

5. The colony, which showed the largest diameter of the yellow zone on one plate, was regarded as mutant.

6. The mutants were further screened by flask fermentation.

The efficient K. vulgare mutants for 2-KLG production Mutant

rotatingplatform shaker(HZQ-R, DonglianGroup Co.,Ltd., Heilongjiang,China)at220rpm,aK.vulgare07mutantwith thehighestproductionof2-KLG(K.vulgare65)waschosento evaluatethefermentationstabilityin20-Lfermenters.The 2-KLGconcentrationinthefermentationbrothwasdetermined byiodometry.19_{Thesorboseconcentrationwasmeasuredby} thesulfuricanthronereactionanddetectedat620nmusing a UV–Visspectrophotometer (VIS-7220, Rayleigh Analytical InstrumentCo.,Ltd., Beijing, China).20 _{The conversionrate} wasdefinedastheratioofthe2-KLGyieldinthefermentation brothtoatheoreticalvalue.

ComparisonoftraditionalandpHindicatorscreening

methods

ThecultureofK. vulgare 07 subjected tospaceflight muta-genesiswaspreparedas10-foldserialdilutionsandspread onagarplates.Forthetraditionalscreeningmethod,colonies ofK.vulgare were selected randomly,then inoculated with B. megaterium 2980 in 250-mL flasks containing 20mL of thefermentationmedium,andincubatedat29◦Cfor38h.21 Thecoloniesthatproduced higherconcentrationsof2-KLG (≥62.0mg/mL)wereregardedaspositivemutants.Forthenew screeningmethod, the agar-plate colonieswith the largest diametersofzoneswhosecolorchangedaftertheadditionof 1mLofabromothymolbluesolution(0.1%,w/v)wereselected forfurtherflaskfermentationscreening,similar tothe tra-ditionalscreeningmethod.Fiftycolonieswerechosenfrom agarplatesbyeachscreeningmethodforflaskfermentation. Thenumberofpositivemutantsobtainedbyeachmethodwas counted.

Statisticalanalysis

Thedifferencein2-KLGproductionbetweengroupswas eval-uatedusingtheStudent’st-testintheStatisticalProgramfor SocialSciences(SPSS13.0forWindows,SPSS,Inc.,Chicago, IL,USA).Ap-valueof<0.05wasconsideredstatistically signif-icant.

Results

EffectsofB.megateriumfiltrateongrowthofK.vulgare

Theresultsofthebacterialgrowthtestshowedthatthe aver-agecolonydiameter(4.209mm)ofK.vulgare07incubatedona platewithaB.megaterium2980filtratewassignificantlygreater (p<0.05)thanthat(3.019mm)ofK.vulgare07incubated with-outtheB.megaterium2980filtrate.AfurthergrowthtestofK. vulgare07withgradientvolumes(0.1,0.2,and0.3mL)ofthe B.megaterium2980filtrateshowednostatisticallysignificant differences(p>0.05)inthecolonydiameters(datanotshown). Theseobservationswereconsistentwithreportsshowingthat B.megateriumactsasacompanionbacteriumandsecretsone ormultipleactivatorspromotingthegrowthofK.vulgare.5,22 Becauseof the increasedcell number of K. vulgare 07,the corresponding2-KLGconcentrationwasimprovedinthe fer-mentationbroth.21_{Together,thesefindingsindicatethatthe}

activatorssecretedbyB.megateriumincreasethegrowthrate ofK.vulgare07,enhancingtheproductionof2-KLG.

ChoiceofpHindicator

Whenselectingbacterialmutantswithenhanced2-KLG pro-ductivity, simple and reliable methods are desired for the qualitativeanalysisof2-KLGproduction.Here,wedeveloped aplatemethodforthescreeningandisolationofK.vulgare mutantswithaltered2-KLG productivity.Therationalewas based onthefactthatthe 2-KLGsecretedbycoloniesofK. vulgare 07 lowers the surrounding pH on anagar plate.To this end,aproperindicatorisneededtoshowthechanges inthepHofthemedium.UpontheadditionofapH indica-tor,thecolorofthezonearoundamutantcolonygrownon anagarplatewouldchange.Theoretically,thediameterofthe zonewithachangedcolorshouldincreasewiththeincrease intheproductionof2-KLG.Inthisstudy,threepHindicators weretestedforthedirectdeterminationof2-KLGproduction onaplate.Theresultsshowedthatbromothymolbluewas superiortotheothertwoindicatorsintermsoftheobvious color changeandasuitablepHrange(bluetoyellowatpH 6.5–7.5). Uponthe additionofabromothymolbluesolution (0.1%,w/v)toanagarplate,zonessurroundingcoloniesofK. vulgare 07mutantschangedtheircolor frombluetoyellow becauseK.vulgare07mutantsreleased2-KLGonagarplates, therebyacidifyingsurroundingareasaroundcolonies.

Relationshipbetweenthesizeofcolor-changingzoneand

2-KLGconcentration

Whenagivenvolumeofa2-KLGsolutionwaspipettedonto anagar plate,yellowzoneswere observed.Toevaluatethe correlationbetweenthesizeoftheyellowcolorzoneandthe concentration ofthebacterially produced2-KLG, 50␮Lofa 2-KLG solution (0–20g/L) was added toanagar plate.With an increasing dose of2-KLG, the yellowzones accordingly increased(Fig.2).Thecorrelationanalysisshowedthat the diameterofthecolor-changingzonewaslinearlycorrelated (R2_{=0.984)withthequantityof2-KLGreleasedintotheagar.}

Newlyproposedagarplatescreeningmethod

Basedontheaboveexperimentalresults,asimpleandrapid agar platescreeningmethodwasdevelopedforthe prelim-inary screeningandisolationofK.vulgare mutantscapable ofefficientproductionof2-KLG.Theschematicoftheplate screening procedure is shown in Fig. 1, and the detailed descriptionofthenewmethodisprovidedinMaterialsand Methods.

ComparisonofconventionalandpHindicatorscreening

methods

ThecoloniesofK.vulgare07grownafterspaceflight mutagen-esiswerescreenedbythetwodifferentscreeningmethodsto comparetheirscreeningefficiencies.Theresultsshowedthat byusingthetraditionalscreeningmethod,onlythreepositive mutantswereobtainedafterscreening50coloniesrandomly selectedfrom50agarplateseach(about2000coloniesintotal).

22 20 18 16 14 12 10 8 6 4 2 0 0 2 4 6 8 10

2-keto-L-gulonic acid concentration (g/I)

Changed color z

one diameter (mm)

12 14 16 18 20 22

Fig.2–Relationshipbetween2-keto-l-gulonicacid concentrationandthechangeinthediameterofcolorzone ofbromothymolbluepHindicatoronagarplates.

Onthecontrary,byusingthenewscreeningmethod,19 posi-tivemutantswereobtainedafterscreening50coloniesfrom50 agarplateseach.Theresultsindicatedthatthenewscreening methodgreatlyimprovedtheprobabilityofobtainingpositive mutants.

ScreeningofK.vulgaremutantsusingproposedpH

indicatorplatemethod

InordertoverifytheefficiencyofthenewlyestablishedpH indicatorplatemethod,wescreenedmorethan20,000 post-spaceflightmutagenesiscoloniesofK.vulgare07.Afteragar platescreeningandflaskfermentationscreening,nine effi-cientmutantswere finallyobtained.Eachofthesemutants displayedasignificantlyhigher(p<0.05)2-KLGyieldthanthat oftheparentstrain(Table1).Thehighest2-KLG concentra-tion(73.75g/L) wasachievedinaco-cultureofK.vugare65 andB.megaterium2980,withtherateincreaseof12.75% rela-tivetothatinaco-cultureoftheparentstrain(K.vulgare07)

Table1–Determinationof2-KLGproducedbyscreened mutantsinflaskfermentation.

Screenedmutants 2-KLG(gL−1) KB06-2980 72.54±0.16a KB10-2980 68.10±0.29a KB21-2980 71.44±0.22a KB22-2980 69.83±0.30a KB23-2980 72.42±0.71a KB27-2980 70.40±0.25a KB37-2980 70.10±0.56a KB38-2980 70.52±0.28a KB65-2980 73.75±0.24a KB07-2980(parentstrains) 65.04±0.44 Allthedataarepresentedasmean±S.D.

a _{Statisticallysignificantcomparedwithparentstrains.}

Table2–FermentationofKB65-2980in20Lfermenters for10batches. KB65-2980 (mutantstrains) KB07-2980 (parentstrains) 2-KLG(gL−1) 91.63±1.40a _84.74±1.38 Conversionrate(%) 94.45±1.44a _87.34±1.42 Period(h) 41.0±2.7a _44.0±2.8 Productivity(gL−1_h−1_{) 2.24±0.13}a _1.93±0.13

Allthedataarepresentedasmean±S.D.

a _{Statisticallysignificantcomparedwithparentstrains.}

and B.megaterium 2980.Furthermore,anaverage l-sorbose-to-2-KLG conversionrate of94.45% and 2-KLGproductivity of2.24gL−1h−1wereachievedbyco-cultivationofK.vulgare 65andB.megaterium2980(KB65-2980)in10batchesin20-L fermenters(Table2).

Discussion

Despitetherapiddevelopmentofnoveltechniques,23 conven-tionalmethodssuchasmutagenesisandselectionbasedon directmeasurementsstillplayacentralroleasreliableand cost-effectiveproceduresforstrainimprovement.13,22,24 How-ever,theneedtouseamixedfermentationsystemfor2-KLG productionbyK.vulgareandB.megateriummakestraditional screeningmethodsforK.vulgaremutantscapableof2-KLG overproductionlaboriousandcomplicated,7_{leadingtoamuch} lowerscreeningefficiency.

Inthepresentstudy,wedescribedarapidscreening pro-cedureforhigh-efficiency2-KLG-overproducingmutantsofK. vulgare.Theproposedmethodavoidsthenegativeeffectsof theactivecompanionbacteriumonthegrowthand conver-sionactivitiesofK.vulgare,thussignificantlyincreasingthe probability ofobtaining highlyefficientK. vulgare mutants. Thenewmethodisalsoadvantageousbecause ofthe ease of operationand the possibility of screeninga large num-ber(thousands) ofmutantsbyoneoperatorwithinashort period(<30days).Comparedwiththeconventionalscreening method,ourproposedpHindicatorplatemethodshortensthe screeningtimeby90%andneedsnolargeinstruments.

Despitethatthenewmethodallowsscreeningofalarge number ofK.vulgare mutantsinthe preliminaryscreening stepmuchfaster,theselectedmutantsshouldbefurther con-firmedbyco-fermentationwithB.megateriumforthefollowing tworeasons.Ononehand,theindicatormethodusedinthis study cannot differentiatebetween secretionof 2-KLG and other acidic metabolites byK. vulgaremutants, which may influencetheaccuracyoftheplatescreeningresults.Onthe otherhand,moreimportantly,industrialproductionof2-KLG iscarriedoutinamixedfermentationmode;hence,the2-KLG productionefficiencyofK.vulgaremutantsmustbetestedin aco-culturewithB.megaterium.

Thescreeningmethodprovidesanapproachtoscreening forefficientmutantsforothermixedfermentationsystems. Withaslightadjustmentofcultureconditionsandselection of an appropriatepH indicator, the proposed pH indicator plate methodcaneasilybeusedforthescreeningofother

bacterialstrainssuitablefortheproductionofacidicproducts inamixedfermentationsystem.

Conclusions

Inthisstudy,wedevelopedanovelpHindicatorplatemethod fortheisolationandscreeningofK.vulgaremutantscapableof overproductionof2-KLG.Comparedwiththeexisting conven-tionalmethod,theproposedscreeningmethodismorerapid, sensitive,andreliable.Inaddition,itiscost-effectiveandeasy touse.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisstudywassupportedbytheProgramofNationalKeyNew DrugCreationofChina(No.2011ZX09203-001-14).Theauthors wouldliketothankthestaff,Fermentation&Biotechnology LaboratoryofNEPGfortheirtechnicalsupport.

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1. BremusC,HerrmannU,Bringer-MeyerS,SahmH.Theuseof microorganismsinl-ascorbicacidproduction.JBiotechnol. 2006;124:196–205.

2. YinGL,HeJM,RenSX,etal.ProductionofvitaminC precursor-2-keto-l-gulonicacidfroml-sorbosebyanovel bacterialcomponentsystemofSCB329-SCB933.IndMicrobiol. 1997;27:1–7(inChinese).

3. ZhangJ,LiuJ,ShiZ,LiuL,ChenJ.ManipulationofB. megateriumgrowthforefficient2-KLGproductionbyK. vulgare.ProcessBiochem.2010;45:602–606.

4. LiuL,LiY,ZhangJ,etal.Completegenomesequenceofthe industrialstrainKetogulonicigeniumvulgareWSH-001.J Bacteriol.2011;193:6108–6109.

5. LuSJ,WangJ,YaoJM,YuZL.Studyontheeffectofmutated Bacillusmegateriumintwo-stagefermentationofvitaminC. PlasmaSciTechnol.2003;5:2011–2016.

6. FengS,ZhangZ,ZhangCG,ZhangZZ.EffectofBacillus megateriumonGluconobacteroxydansinmixedculture.ChinJ ApplEcol.2000;11:119–122(inChinese).

7. MaQ,ZhouJ,ZhangW,MengX,SunJ,YuanYJ.Integrated proteomicandmetabolomicanalysisofanartificial microbialcommunityfortwo-stepproductionofvitaminC. PLoSONE.2011;6:e26108.

8. ZhangJ,ZhouJ,LiuJ,ChenK,LiuL,ChenJ.Developmentof chemicallydefinedmediasupportinghighcelldensity growthofKetogulonicigeniumvulgareandBacillusmegaterium. BioresourTechnol.2011;102:4807–4814.

9.ZhouJ,MaQ,YiH,WangL,SongH,YuanYJ.Metabolome profilingrevealsmetaboliccooperationbetweenBacillus megateriumandKetogulonicigeniumvulgareduringinduced swarmmotility.ApplEnvironMicrob.2011;77:7023–7030. 10.ZhuY,LiuJ,DuG,ZhouJ,ChenJ.Sporulationandspore

stabilityofBacillusmegateriumenhanceKetogulonigenium vulgarepropagationand2-keto-l-gulonicacidbiosynthesis. BioresourTechnol.2012;107:399–404.

11.GiridharR,SrivastavaAK.L-sorboseproductionina continuousfermenterwithandwithoutcellrecycle.WorldJ MicrobBiot.2001;17:185–189.

12.TakagiY,SugisawaT,HoshinoT.Continuous

2-keto-L-gulonicacidfermentationbymixedcultureof KetogulonicigeniumvulgareDSM4025andBacillusmegaterium orXanthomonasmaltophilia.ApplMicrobiolBiotechnol. 2010;86:469–480.

13.LuX,GuoH,LiB.Screeningofassociatedbacteria Gluconobacteroxydansintwo-stepfermentationofVC. Biotechnology.2005;15:23–25(inChinese).

14.ZhaoSG,YaoLM,SuCX,etal.Purificationandpropertiesof anewl-sorbosedehydrogenaseaccelerativeproteinfrom Bacillusmegateriumbredbyion-beamimplantation.Plasma SciTechnol.2008;10:398–402.

15.GuoL,GuoL,LuY,ZhangL.Breedingofassociatedbacteria Bacillusthuringiensisin“two-stepfermentation”ofVc.JHebei Univ(NatSci).2006;26:108–111(inChinese).

16.GaoH,LiuM,ZhuoY,etal.Assessingthepotentialofan induced-mutationstrategyforavermectinoverproducers. ApplEnvironMicrob.2010;76:4583–4586.

17.VaishampayanPA,RabbowE,HorneckG,VenkateswaranKJ. SurvivalofBacilluspumilussporesforaprolongedperiodof timeinrealspaceconditions.Astrobiology.2012;12:487–497. 18.WangJ,LiuC,LiuJ,etal.Spacemutagenesisofgenetically

engineeredbacteriaexpressingrecombinanthuman interferon␣1bandscreeningofhigheryieldingstrains. WorldJMicrobBiot.2014;30:943–949.

19.XuA,YaoJ,YuL,etal.MutationofGluconobacteroxydansand Bacillusmegateriuminatwo-stepprocessofl-ascorbicacid manufacturebyionbeam.JApplMicrobiol.2004;96: 1317–1323.

20.YemmEW,WillisAJ.Theestimationofcarbohydratesin plantextractsbyanthrone.BiochemJ.1954;57:508–514. 21.MandlaaYangW,HanL,WangZ,XuH.Two-helper-strain

co-culturesystem:anovelmethodforenhancementof 2-keto-l-gulonicacidproduction.BiotechnolLett. 2013;35:1853–1857.

22.ZhuY,LiuJ,LiuJ,DuG,ZhouJ,ChenJ.Ahighthroughput methodtoscreencompanionbacteriumfor

2-keto-l-gulonicacidbiosynthesisbyco-culturing Ketogulonicigeniumvulgare.ProcessBiochem.2012;47: 1428–1432.

23.CaiL,YuanMQ,LiZJ,ChenJC,ChenGQ.Geneticengineering ofKetogulonigeniumvulgareforenhancedproductionof 2-keto-l-gulonicacid.JBiotechnol.2012;157:320–325. 24.ParkCS,YangHJ,KimDH,KangDO,KimMS,ChoiNS.A

screeningmethodforbeta-glucanhydrolaseemploying TrypanBlue-coupledbeta-glucanagarplateandbeta-glucan zymography.BiotechnolLett.2012;34:1073–1077.