Applications in the pharmaceutical industry of ARTP and MMC

Applications in the pharmaceutical industry of ARTP and MMC1.ARTPIn recent years,ARTP(Atmospheric Room Temperature Plasma)has developed rapidly.ARTP is a new glow discharge technology.It uses inert gas discharge to produce a large number of high-energy active particles at Room Temperature and Atmospheric pressure.The technology can generate the plasma including a large amount of chemically reactive species causing intensive damage of genetic material. These species can permeate into the cell,and cause the DNA damage,then induce SOS repair mechanism of microbial cells.SOS is a high fault rate repair process,which can cause varied mismatch sites,and thus form the final stable genetic mutants,which target strains can be screened.Compared with the traditional mutagenesis methods,ARTP is not only discharge safe and pollution-free,but also has a variety of damage mechanisms to genetic materials,so the diversity of acquired mutagenesis also increases.The characteristics of ARTP mutagenesis show unique advantages in dealing with microbial mutagenesis breeding.At present,ARTP has been widely used in the breeding of bacteria,actinomycetes,fungi,yeast and other microorganisms,and has been more and more widely used in the fields of antibiotics,drug intermediates,raw materials and health care products.2.MMCMicrofluidic chip is used to generate droplets and exchange liquid of microbial culture in MMC (Microbial microdroplet culture system).The volume of liquid droplets is2microns,and200independent microbial droplets can be cultured and continuously passed in parallel.In particular,because of the excellent performance of dissolved oxygen and mixing in the droplets in the water-in-oil micro-system,the microbial growth performance is close to or better than that of shaker.In addition,based on the advantage of chip system,MMC can also meet various experimental requirements,such as fluorescence,visible light,dissolved oxygen and pH online detection,microbial colony count,high-throughput screening,adaptive evolution and other different functions,showing great application potential in antibiotics,drug intermediates,raw materials,health care products and other fields.Fig.1Microbial microdroplet culture(MMC)system.(A)The outer design of MMC.(B)The inner design of MMC.The millifluidic chip is indicated with red rectangle.(C)Millifluidic chip for droplet generation,droplet coalescence,droplet segregation and detection.3.Application of ARTP and MMCThe following table shows partial application of ARTP and MMC in medicine and other related fields.Tab1Applications of ARTP and MMC in medicine and other related fields（partial）NO.Title Author Institution Journal Strain Target Product Result1Inhibition and trendanalysis of carotenoidmetabolic flux ofRhodosporidiumtoruloideWANGYafang,LIUYan,LIXianzhen,YANG FanSchool of BiologicalEngineering,DalianPolytechnic UniversityJournal of DalianPolytechnicUniversitRhodosporidum toruloidesCarotenoidDetermined.The results showed that the defectof the carotenoid synthesis pathway couldreduce the contents of Carotenoid,biomass andlipid by70%,48%and40%,spectively2Enhancement ofascomycin productionvia a combination ofatmospheric and roomtemperature plasmamutagenesis inStreptomyceshygroscopicus andmedium optimizationZhituo Yu，XiaofangShen，Yuanjie Wu，SongbaiYang，Dianwen Ju，Shaoxin ChenDepartment ofMicrobiological andBiochemical Pharmacy,The Key Laboratory ofSmart Drug Delivery,Ministry of Education,School of Pharmacy,Fudan University，Shanghai Institute ofPharmaceutical Industry,China State Institute ofPharmaceutical Industry,ShanghaiAMB ExpressStreptomyceshygroscopicus AscomycinIn order to improve the strain production,theoriginal S.hygroscopicus ATCC14891strainwas treated here with atmospheric and roomtemperature plasma to obtain a stablehigh-producing S.hygroscopicus SFK-36strainwhich produced495.3mg/L ascomycin,a32.5%increase in ascomycin compared tothe ATCC14891.3Mutagenesis ofRhodobactersphaeroides usingatmospheric and roomtemperature plasmatreatment for efficientRong-SongZou,SuyanLi,Le-LeZhang,Yi-JunHan,GeGao,XiangyanCollege of Forestry,Beijing ForestryUniversity,Center,Shenzhou Biology&Technology,BiobreedingResearch Center,WuxiJournal ofBioscience andBioengineeringRhodobactersphaeroidesCoenzyme Q10In the first round of screening in24-well plates,three mutants were obtained,with theproduction of CoQ10at311mg/L,307mg/L,and309mg/L,which were increasedfrom the parent’s production at265mg/L.Furthermore,a second round of mutation and3NO.Title Author Institution Journal Strain Target Product Resultproduction of coenzyme Q10g Sun,andXiao qiangGongResearch Institute ofApplied Technologies,Tsinghua Universityscreening was performed based on the mutantstrain with the highest production in the firstround,leading to the identification of a mutantAR01with the production of CoQ10at w330mg/L.Finally,590mg/L CoQ10was obtainedfor AR01after100h fermentation,whichwas w25.5%higher than that of the originalparent strain.4Astaxanthinoverproduction in yeastby strain engineeringand new gene targetuncoveringJin Jin，YingWang，MingdongYao，XiaoliGu，Bo Li，Hong Liu，MingzhuDing，WenhaiXiao，YingjinYuanKey Laboratory ofSystems Bioengineering(Ministry of Education),School of Chemical&EngineeringTianjinUniversity2.SynBioResearch Platform,Collaborative InnovationCenter of ChemicalScience and Engineering(Tianjin)TianjinUniversityBiotechnology forBiofuelsSaccharomycescerevisiaeAstaxanthinAstaxanthin yield was further increased by0.83-fold(to10.1mg/g DCW)via ARTPmutagen esis,which is the highest reportedyield at shake-fask level in yeast so far.5Enhanced doxorubicinproduction byStreptomyces peucetiususing a combination ofclassical strainmutation and mediumWang X,TianX,Wu Y,ShenX,YangS,Chen Sa Shanghai Institute ofPharmaceutical IndustryPreparativeBiochemistry andBiotechnologyStreptomycespeucetiusdoxorubicinI n this study,a DXR-resistance screeningmethod was developed to screen for DXRhigh-producing mutants.Then,S.peucetiusSIPI-11was treated several times with UV andARTP(atmospheric and room tempera tureplasma)to induce mutations.Treated strains4NO.Title Author Institution Journal Strain Target Product Result optimization.were screened by spreading on aDXR-containing plate,isolating a mutant(S.peucetius33-24)with enhanced DXR yield(570mg/L vs.119mg/L for the original strain).6Breeding andfermentationoptimization ofL-arginineproducing strainsCHENGGong XUJian-ZhongGUOYan-Feng XUKai ZHANGWei-Guo*The Key Laboratory ofIndustrialBiotechnology,Ministryof Education,School ofBiotechnology,JiangnanUniversityMicrobiologyChinaCorynebacterium.glutamicumL-arginineAfter several rounds of screening,a strain wasselected resistant to15g/L L-homoarginine and0.7g/L8-azaguaine,and designated asC.glutamicum ARG3-16(L-HAr,8-AZr,SGr,L-His−).L-arginine production of ARG3-16was49.79%higher than that of the originalstrain.7Mutation Breeding ofγ-Polyglutamic AcidProducing Strain byAtmospheric and RoomTemperature PlasmaCHENShuangxi,ZHANGErchao,ZHANG Lele,XIA QihaoInstitute ofBioengineering,Collegeof Life Science,HenanUniversityChinese Journal ofPharmaceuticalsBacillussubtilisγ-polyglutamicacidOne mutant named HNCL1266was obtainedwith the yield ofγ-polyglutamic acid in shakingflask of26g/L,which was30％higher thanthat of HD11.The mutant strain showed highhereditary stability.8The Screening andCharacterization ofHigh-yield-carotenoidStrain fromDeinococcuswulumuqiensis R12byARTPJin WeiyueSong MingkaiJiao WenhaoJiang LingLi ShuangXu XianState key laboratory ofmaterials-orientedchemical engineering,College ofBiotechnology andPharmaceuticalengineering,NanjingTech University:StateBiotechnologyBulletinD.wulumuqiensiscarotenoidThe strain M1produced612μg/g dry cellweight（DCW）of carotenoid after fermenting72h,2.8times of carotenoid by original D.wulumuqiensis R12（212μg/g DCW）,andheredity was stable.The carotenoid of the strainM1showed stronger activity in high ironion reducing power（OD700=0.52）than that（OD700=0.34）of original strain,and higher5NO.Title Author Institution Journal Strain Target Product Resultkey laboratory of materials-oriented chemical engineering, College of food science and light industry Nanjing Tech University DPPH scavenging rate（33.33%）than23.09%, indicating the mutant strain M1possessed better anti-oxidative capacity.9Screening of BacillusLincheniformis withhigh-yieldtetramethylpyrazine byatmospheric roomtemperature plasmamutationMeng Wu,Ding Xuemei,WangRuiming,XiaoDongguangCollege of biologicalEngineering,TianjinUniversity of Scienceand TechnologyFaculty of light industry,Qilu University ofTechnology,JinanChina BrewingBaclicuslincheniformistetramethylpyrazine TTMPCompared to the original stain,the maximumTTMP yield increased from37.89g/L to43.16g/L,which incrcased by13.91％．10Mutation Breeding ofUridine-ProducingStrains by MethodcombiningAtmospheric and RoomTemperature PlasmaandHigh-throughputScreeningHeyun Wu,HongchaoZhang,HuiYuan,Guoliang Li,XiaoguangFan,NingChen,XixianXieNational and LocalUnited Engineering Labof Metabolic ControlFermentationTechnology,TianjinCollege ofBiotechnology,TianjinUniversity of Scienceand Technology,TianjinBacillussubtilisUridineAs a result,four mutant strains,B.subtilisA219,B.subtilis A260,B.subtilis A566and B.subtilis F126,were isolated.Compared with the original strain,B.subtilis TD131,which accumulated3.5g/L of uridine in a5L fermentor,the fourmutants had significant improvements inuridine productivity,amounted to12g/L,14.5g/L,16g/L and18g/L respectively.6NO.Title Author Institution Journal Strain Target Product Result11ARTP mutagenesisbreeding high yieldbacteria of bacillus.ZhaoGuozhong,Wang Fengya,Xu Tingting,Li LixiaNorth ChinaPharmaceutical GroupCorporation;Science andtechnologyresearchBaclicuslincheniformisbacitracinThe mutant strain B-78was obtained byatmospheric pressure room temperature plasma(ARTP)mutagenesis,and its shake flask titerwas increased by38.5%compared with theoriginal strain.After5passages,it still hasstable antibiotic production characteristics.12Screening of HighL-AsparaginaseActivity Mutants ofRecombinant BacteriaWB600byAtmospheric and RoomTemperature PlasmasMutation SystemCHENXuan,LIUSong,FENGYue,DUGuocheng,CHEN JianKey Laboratory ofIndustrialBiotechnology，Ministryof Education，JiangnanJournal of foodand biotechnologyBacillussubtilisL-asparaginasea mutant of B.subtilis WB600（pMA5-wapA-ansZ）with a activity of48.4U/mL wasobtained，the activity value was increased by30%.13Isolation,Identificationand Mutation Breedingof Strain Producingα-Glucosidase InhibitorfromMulberry LeafZHUMengfeng,XU Wei,SHAO Rong,WEI PingCollege of Pharmacy,Nanjing Tech UniversityFood ScienceBacillusatrophaeusα-glucosidaseinhibitorCombining ARTP mutation breeding withhigh-throughput screening,a mutant strainnamed T-690was obtained from880mutantstrains,whose inhibition ratio(73.25%)wasincreased by40.61%compared with the originalstrain.7NO.Title Author Institution Journal Strain Target Product Result14Mutation breeding ofhigh9α-hydroxy-androst-4-ene-3,17-dionetransforming strainsfrom phytosterols andtheir conversionprocess optimizationYang Ma,XiangdongWang,MenghuiWang,Hui Li,Jinsong Shi,andZhenghongXuPharmaceutical Science,Jiangnan UniversityChinese Journal ofBiotechnologyMycobacterium9α-OH-ADThe high production strain named C33with agood genetic stability was selected and theproduct molar yield achieved to15.5%,whichwas34.8%higher than that of original strainwith15g/L phytosterols.Furthermore,thefermentation medium was optimized throughthe design of orthogonal experiment.Besides,oil-water bidirectional transformation systemwas set up to improve the9α-OH-AD molaryield of mutant strain C33.With adding12mLsoybean oil to each1g phytosterols,the molaryield of9α-OH-AD reached47.0%,whichincreased by2times than that of control(15.5%).15Screening of anacid-resistantSaccharomycesboulardii andoptimization of its highdensity cultivationconditionsLi Feilong,ChenXiaohua,Zheng Xin,XieYongzhen,Tan Zhilei,JiaShiruKey Laboratory ofIndustrial FermentationMicrobiology,Ministryof Education,College ofBiotechnology,TianjinUniversity of Science&Technology,TianjinChina brewingSaromycesboulardiiAcid-resistantThe results of stability of the mutant strainresistant to low pH showed that after20timegenemtion，mutant YB-3had good geneticstability and its survival rate was51.79％.In5Lfermentor,the high density cultvation of mutantYB-3were optimized．The dry cell weight ofS.boulardii obtained was58.79%，which wasincreased by55.52％than that of the originalstrain．8NO.Title Author Institution Journal Strain Target Product Result16Mutation screening ofKluyveromyces lactisusing ARTP andcatalysis characteristicsof the whole cell ofmutant strainCao Gang,LiuYun,GuoJinling,LvYucai,YuHuashun,Gong DachunLaboratory,China ThreeGorgesUniversity;2.Departmentof EnzymeProduction,Angel YeastCo.,LtdChinaBrewing,05,2018Kluyveromyceslactisketo-reducetaseThe conversion rate of mutant strain K15toacetophenone was up to91.8%,which was2.5time higher than that of the starting strain.Thestrain had a wide application prospect inbiocatalysis field.17Screening andBreeding of HighProducing Strain for［γ-HyMeLeu4］CyA byARTP-UV CompositeMutagenesisZHANG Li，DAI Meng，ZHENGGui-zhen，ZHAO Ying，LIU Jing，ZHANG Jia，WANGFu-qiangNew Drug R&DCo.Ltd.of N.ChinaPharm.Group(NCPG),Nat＇lEngin.Res.Ctr.ofMicrob.Med．JOURNAL OFMICROBIOLOGY Feb,，2014，34(1)：68-71NonomuraeadietziaecyclosporinderivativesThe results showed that a high yielding strainpossessing stable genetic performance wasobtained using the mutagenesis withtransformation efficiency increased by32%overthe starting strain．18Application of PlasmaTechnology in theMutation Breeding ofStreptomyces fradiaeand Pleurotus sp.WANG Zhong-zhong,ZHANG Ping,SHIYan-pengNingxiatairuiPharmaceuticalCo.,Ltd.Strains InstituteChineseVeterinary DrugJournaltreptomycesfradiae,Pleurotus sp.antibioticBesides，one strain of Streptomyces fradiae andthat had10.4%higher ability and one strainof Pleurotus sp．that had9.6%higher ability ofproducing antibiotic than original strains wereselected．9NO.Title Author Institution Journal Strain Target Product Result19Mutation ofStreptomycesaureofaciens with HighYieldDemethylchlortetracycline by Atmospheric andRoom TemperaturePlasmaLIN GuizhenYE RuifangCHENGLintongMAOQuanguiNational key laboratoryof bioreactor engineeringof east China universityof science andtechnology;Henan tianfangpharmaceutical co.LTD.Chinese Journal ofPharmaceuticalsStreptomycesaureofaciensdemethylchlortetracyclineproductivity.A mutant A6-6-9was screenedand showed high genetic stability after fourpared with the original strainunder the same fl ask fermentation process,themutant A6-6-9showed Faster aminonitrogenconsumption,slower growth rate and glucoseconsumption rate,and enhanced productivityof1with an increase of22.5％..20A Mutant strain withhigh Erythromucinyield obtained by usingnovel ARTP and UVMutationShenXiaojing,Zhang Ping,Shi YanpengNingxia TairuiPharmaceutical Co.LtdStrains Institute,YinchuanChineseveterinarymedicinemagazine.Saccharopolyspora erythraeaErythromycinAtmospheric and room temperatureplasma(ARTP)-UV composite mutagenesiswere adopted to treat witherythromycin-producing strain,fourhigh-efficient producing strains were obtainedby screening,which was increased infermentation potency by25.2%over the originalstrain.The mutant12#with genetic stability wasObtained.Its yield of erythromycin was10029U/mL,28.6%higher than the original strain.ARTP-UV composite Mutation was a simpleand efficient method for screening.10NO.Title Author Institution Journal Strain Target Product Result21Production ofOritavancinIntermediate A82846Bby Strain Improvementand MediumOptimizationZHENGLinghui,HONG Yun,CHENXiaojing,TENG Yun,BAI HuaZhejiang HisunPharmaceutical Co.,Ltd.,TaizhouChinese Journal ofPharmaceuticalsNocardiaorientalisOllie'sintermediateNocardia orientalis HS807-N-1287was treatedwith atmospheric and room temperature plasma(ARTP)mutation for production of oritavancinintermediate A82846B(1),and screened in theselective plates containing1and tyrosine.Amutant HS807-A-639with high productivity of1was obtained.22Rapid MutationBreedingSchizochytrium StrainsProducing High-yieldDocosahexenoic Acidby Atmospheric andRoom TemperaturePlasmasYuan JunZhao Ben SunMengyuWang WuYang HailinThe Key Laboratory ofIndustrial Biotechnologyof Ministry ofEducation，JiangnanUniversityBIOTECHNOLOGY BULLETINSchizochytriumsp.DHAThe DHA-yield of mutant D32increasedsignificantly up to7.31g/L,29.8%higher thanthose by the original pared with theoriginal strain,the main saturated fatty acid（C14∶0and C16∶0）by D32decreasedsignificantly,while the unsaturated fatty acidcontent increased significantly.23Breeding Strains withCompound Mutationfor High ConversionRate of HydrocortisoneXUEWei-ying,SHENYan-bing,HUANG Wei,WANG Min,DENGMing-qianLaboratory of IndustrialFermentationMicrobiology,Ministryof Education,TianjinKey Laboratory ofIndustrial Microbiology,College ofBiotechnology/TianjinUniversity of Science&Journal ofShandongAgriculturalUniversity(Natural ScienceEdition)AbsidiacoeruleaHydrocortisoneThe primary screening method was establishedon the basis of colony morphology.After6daysof culture,the colony color was darker,therewas cricoid markings on the back,HCconversion was rescreened in the bacterial strainwith dense mycelium,and the mutant AL-172with high genetic stability was obtained.Whenthe RSA concentration was3.5g/L,theconversion rate of HC reached72.52%.NO.Title Author Institution Journal Strain Target Product ResultTechnology Substrate feed concentration and HC conversionrate increased by40%and16.20%respectivelycompared with laboratory preservation strains.Ithas a good prospect of application.24Mutation by UsingAtmospheric Pressureand Room TemperaturePlasmas andHigh-throughputScreening Method forImproving VitaminB12ProductionCAI-Yingying,XIA-Miaomiao,DONG-Huina,ZHANGTongcun,ZHANG DaweiCollege ofBiotechnology,TianjinUniversity of Science&Technology；Tianjin Institute ofIndustrialBiotechnology,ChineseAcademy of SciencesJournal of TianjinUniversity ofScience&TechnologyPseudomonasdenitrificansVitamin B12Through four rounds of ARTP mutation andhigh-throughput screening，PA320-M4-1B1mutants(103.2±2.1)mg/L were obtained，whichincreased the contents of VB12by43.8%,compared with the control strainPA320(71.9±1.8)mg/L，when the strains werecultivated on a rotary shaker at30℃for6,d.25High throughputscreening of high-yieldcephalosporin Cproducing stain bymutagenesis usingatmospheric and roomtemperatureplasma(ARTP)Ying-ying,HangHai-feng,ZhuangYing-ping andChu JuState Key Laboratory ofBioreactor Engineering,East China University ofScience and TechnologyChinese journal ofantibioticsCephalosporium Acremoniumcephalosporin C,CPCThe high-yield mixture strain G6wassuccessfully screened out and the CPC titer wasnearly19.75%,higher than that of the parentalstrain W-6in the shake flask.Conclusion Thiswork provides an appropriate strategy forobtaining high CPC-yield strains by using ARTPrandom mutation methods with an efficientscreening assay.The ARTP random mutationmethod has a high positive mutation rate and theyield of cephalosporin C is significantlyincreased．NO.Title Author Institution Journal Strain Target Product Result26Screening of highechinocandin Bdeacylase-producingstrain and optimizationof biotransformationconditionsQie Li-ping,WangXiao-lian,MaJie,QianSi-yu,DaiMing-wei,Zhang WeiNCPC New DrugResearch andDevelopmentCo.,Ltd.,NationalEngineering ResearchCenter of MicrobialMedicine,HebeiIndustry MicrobialMetabolic Engineering&Technology ResearchCenterChinese Journal ofAntibioticsStreptomyces EchinocandinA stable mutant strain ZH-6-78was obtainedand its conversion efficiency was26.7%higherthan that of the original strain.The optimumconversion conditions were determined asfollows:the conversion temperature was35℃,and the conversion time was24h.27Breeding of high yieldStreptomyceslinconinensisproducing strainHuangWen-fu,Zhang Ping,Niu Chun andBi Guo-dongNingxiatairuiPhamaceutical Co LtdStrains InstituteChinese journal ofantibiotics.StreptomyceslinconinensislinconmycinThe results showed that the average yield oflincomycin was increased by30.0%via flasksfermentation and by21.4%via180-tonindustrial fermenters.Passage experimentsshowed that the mutant strain of high yieldperformance was genetically stable.。