细菌抗菌肽的分类、作用机理及基因工程研究进展.doc

抗菌肽的分类、作用机理及基因工程研究进展细菌抗菌肽的分类、作用机理及基因工程研究进展 刘小雨2016年10月 目录1 抗菌肽的分类21.1 来源于细菌的抗菌肽21.1.1来源于芽孢杆菌只抗革兰氏阴性菌（G-）的抗菌肽71.1.2来源于芽孢杆菌抗真菌的的抗菌肽71.1.2来源于其它物种抗真菌同时抗的革兰氏阴性菌（G-）的抗菌肽102 抗菌肽的作用机理112.1 细胞膜电势依赖通道的形成112.2 抑制细胞呼吸作用112.3 抑制细胞外膜蛋白的合成112.4 抑制细胞壁的形成112.5 进入细胞核结合到DNA或RNA上影响细胞113抗菌肽在各个表达系统中的表达特点及研究进展123.1 抗菌肽在大肠杆菌表达系统中的研究进展123.1.1密码子偏爱性改造123.1.2 串联表达123.1.3 融合表达133.1.4 杂合表达133.2 抗菌肽在酵母表达系统中的研究进展134 国内基因工程改造的相关专利技术144.1 国内专利技术144.2 外源蛋白在枯草芽孢杆菌中表达可供选择的技术路线154.2.1 复制质粒融合表达路线164.2.2 代谢途径调控路线164.2.3 基因定点突变路线165 抗菌肽及其工业应用前景16目前，由于抗生素在临床治疗中的不规范使用，不仅造成经济上的巨大损失和药物不良反应；而且导致细菌和真菌等病原微生物正在以惊人的速度对抗生素产生强耐药性。特别是多重耐药菌和超级耐药菌的出现，经常在很短时间内出现全世界范围内的流行，给人们的生命安全和经济带来巨大损失。因此对具有高抗菌活性和安全性抗菌肽的研发日益受到重视，人们期望抗菌肽成为抗生素的理想替代品之一。抗菌肽（AMPS）一般由1060个氨基酸残基组成，分子量约为20007000Da，并且热稳定性较好。抗菌肽的抗菌机制不同于传统抗生素，其作用靶点大致分为细胞膜作用靶点和胞内作用靶点(DNA，RNA，酶等)两大类，抗菌肽的抗菌活性和毒性与抗菌肽的二级结构、净电荷数量和疏水性等理化参数密切相关。迄今为止，大约有2700多种抗菌肽已经被分离出来，部分已经通过化学合成和基因工程表达大规模制备。在实际应用方面，部分抗菌肽已成功应用于医疗卫生和食品加工等行业，其中尤其引人注意的是多种抗菌肽已经进入二、三期临床试验阶段【】 Neetoo, H., M. Ye, and H.Q. Chen, Potential antimicrobials to control Listeria monocytogenes in vacuum-packaged cold-smoked salmon pate and fillets. International Journal of Food Microbiology, 2008. 123(3): p. 220-227.【】Lazarev, V.N. and V.M. Govorun, Antimicrobial peptides and their use in medicine. Applied Biochemistry and Microbiology, 2010. 46(9): p. 803-814】。 抗菌肽的来源与制备大致分为三条途径：天然产物(微生物，植物，动物，海洋生物来源)提取，化学合成（液相合成法和固相合成法）和基因工程（大肠杆菌，枯草芽孢杆菌和毕赤酵母表达载体等）制备。天然产物提取易受来源限制，特别是来自珍稀动植物资源的抗菌肽，提取更易受到原材料来源限制。化学合成的生产成本较高，且有多种剧毒性试剂（氢氟酸、氰化钾等）需要大量应用，产生的“三废”对环境造成的污染问题也难以解决。通过基因工程和发酵工程的方法制备抗菌肽可以有效降低生产成本，生产工艺容易放大；产生的废液处理工艺较为成熟。目前使用原核表达系统制备抗菌肽占所有抗菌肽基因表达系统的80 %左右。人们使用原核表达系统表达抗菌肽已经积累了许多经验，对抗菌肽表达质粒构建，生产工艺的中试和放大也有所进展。但是抗菌肽的原核表达依然具有很大技术挑战性，其不仅对宿主菌有毒性，而且由于抗菌肽分子量较少，易被宿主菌各种内源性蛋白酶降解。以往研究表明抗菌肽采用与融合标签表达是实现抗菌肽高效表达的有效策略之一。目前常用的融合标签有硫氧还蛋白（Trx）、类固醇异构酶（KSI）及谷胱甘肽-S-转移酶（GST）等，但在实际应用中，发现上述标签存在标签蛋白过大、促可溶性效果差、表达产物剪切效率低等问题，因此寻找提高抗菌肽的可溶性表达和产率方法依然是目前研究的重要方向。1 抗菌肽的分类按照抗菌肽的来源分类，截止到2016年7月，共有2717种抗菌肽被公布，其中来源于细菌的有269种，来源于古生菌的有4种，来源于原生生物的有8种，来源于真菌的有13种，来源于植物的有333种，来源于动物的有2042种。由于在芽孢杆菌表达系统中，缺少真核以及动物、植物等相应的修饰系统，来源于动物、植物以及真菌的基因在原核表达系统中很难得到高效表达，所以本综述中重点关注来自于细菌的抗菌肽及其抗菌活性；其具体信息如下：1.1 来源于细菌的抗菌肽来源于细菌的抗菌肽一共有269种，其部分信息如下：NameSourceActivityNameSourceActivityHominicinStaphylococcus hominisMBBL 2-9anti-Gram+anti-Gram-Tricyclic peptide RP 71955Streptomyces strainsAA3891Antiviralanti-HIVPlantaricin ALactobacillus plantarumC11anti-Gram+ anti-Gram-Cancer cellsCarnobacteriocin B2Carnobacterium piscicolaanti-Gram+ anti-Gram-Leucocin ALeuconostoc gelidumUAL-187anti-Gram+anti-Gram-Nisin ZLactococcus lactisSIK-83anti-Gram+ Cancer cellsNisin AStreptococcus lactisanti-Gram+SpermicidalAntibiofilmWound healing, Cancer cellsSubtilinBacillus subtilisanti-Gram+Pep5Staphylococcus epidermidisstrain 5anti-Gram+Microcin J25Escherichia coliAY25anti-Gram-Gramicidin Asoil bacterium,Bacillus brevis（短杆菌）anti-Gram+ anti-Gram-antiviralSpermicidalanti-HIVEricin SBacillus subtilisA1/3anti-Gram+Siamycin IIStreptomyces strainsAA3891（链霉菌）Antiviralanti-HIVLactococcin 972Lactococcus lactisIPLA 972（乳酸乳球菌）anti-Gram+Mundticin ATO6Enterococcus mundtiiATO6（蒙氏肠球菌）anti-Gram+Piscicolin 126 / Piscicocin ViaCarnobacterium piscicolaJG126（栖鱼肉杆菌）anti-Gram+Sakacin PLactobacillus sakeLb674anti-Gram+Pediocin PA-1Pediococcus acidilacticiPAC-1.0anti-Gram+SpermicidalMesentericin Y105Leuconostoc mesenteroidesanti-Gram+Carnobacteriocin BM1Carnobacterium piscicolaLV17Banti-Gram+anti-Gram-Curvacin A / sakacin ALactobacillus sakeLb706anti-Gram+PlanosporicinPlanomonospora albaanti-Gram+anti-Gram-Gassericin ALactobacillus gasseriLactobacillus reuteriLA6anti-Gram+anti-Gram-Circularin AClostridium beijerinckiiATCC 25752anti-Gram+anti-Gram-Closticin 574Clostridium tyrobutyricumADRIAT 932anti-Gram+anti-Gram-Enterocin E-760Enterococcus durans/faecium/hiraeanti-Gram+anti-Gram-Enterocin AEnterococcus faeciumanti-Gram+Divercin V41Carnobacterium divergensV41anti-Gram+Divergicin M35Carnobacterium divergensM35anti-Gram+CoagulinBacillus coagulansI-4anti-Gram+Listeriocin 743AListeria innocua743anti-Gram+Mundticin KSEnterococcus mundtiiNFRI 7397anti-Gram+antiviralLeucocin CLactococcus lactisMMFIanti-Gram+Lactococcin MMFIILactococcus lactisMMFIanti-Gram+Sakacin G-anti-Gram+Plantaricin 423Lactobacillus plantarum423anti-Gram+Plantaricin C19Lactobacillus plantarumC19anti-Gram+Enterocin PEnterococcus faeciumP13anti-Gram+Bacteriocin 31Enterococcus faecalisanti-Gram+Carnobacteriocin B1Carnobacterium piscicola LV17Banti-Gram+anti-Gram-Butyrivibriocin AR10Butyrivibrium fibrisolvens AR10anti-Gram+anti-Gram-Subtilosin ABacillus subtilisBacillus amyloliquefaciens, orBacillus atrophaeusanti-Gram+anti-GramantiviralSpermicidalEnterocin AS-48Enterococcus faecalis S-48anti-Gram+anti-Gram-Plantaricin ASM1Lactobacillus plantarumA-1anti-Gram+UberolysinStreptococcus uberis 42anti-Gram+anti-Gram-Acidocin BLactobacillus acidophilus M46anti-Gram+anti-Gram-Carnocin UI49Carnobacterium piscicolaUI49anti-Gram+anti-Gram-LicheninBacillus licheniformisanti-Gram+anti-Gram-Nisin QHalobacterium strain AS7092anti-Gram+Serracin-P 23 kDa subunitSerratia plymuthicaanti-Gram+anti-Gram-Serracin-P 43 kDa subunitSerratia plymuthicaanti-Gram+anti-Gram-VariacinKocuria variansanti-Gram+anti-Gram-Mutacin IIStreptococcus mutansanti-Gram+anti-Gram-Mutacin IIIStreptococcus mutansanti-Gram+Mutacin, or Mutacin B-Ny266Streptococcus mutansanti-Gram+anti-Gram-Thuricin SBacillus thuringiensis serovar entomocidusanti-Gram+Aureocin A53Staphylococcus aureusA53anti-Gram+Siamycin IStreptomyces strain AA6532Antiviralanti-HIVNP-06Streptomyces strain AA6532Antiviralanti-HIVLactococcin G-aLactococcus lactis LMGT2081anti-Gram+anti-Gram-Lactococcin QLactococcus lactis QU 4anti-Gram+LCIBacillus subtilisanti-Gram+anti-Gram-Enterocin 1071Enterococcus faecalis BFE 1071anti-Gram+anti-Gram-Plantaricin SLactobacillus plantarum LPCO10anti-Gram+anti-Gram-AFP1Streptomyces tendae Tu901（唐德链霉菌）antifungalPlantaricin NC8Lactobacillus plantarumNC8anti-Gram+Carnocyclin ACarnobacterium maltaromaticum UAL307anti-Gram+Lactacin FLactobacillus johnsonii VPI11088 (Laf+)anti-Gram+Brochocin CBrochothrix campestris ATCC 43754anti-Gram+Thermophilin 13Streptococcus thermophilusanti-Gram+ABP-118Lactobacillus salivarius subsp. salivarius UCC118anti-Gram+anti-Gram-Salivaricin PLactobacillus salivarius DPC6005anti-Gram+anti-Gram-Mutacin IVStreptococcus mutans UA140anti-Gram+anti-Gram-Lactocin 705Lactobacillus casei CRL 705anti-Gram+anti-Gram-CytolysinEnterococcus faecali（粪肠球菌）anti-Gram+anti-Gram-Mammalian cellsPlantaricin EFLactobacillus plantarumC1anti-Gram+anti-Gram-Plantaricin JKLactobacillus plantarum C1anti-Gram+anti-Gram-Enterocin SE-K4Enterococcus faeciumanti-Gram+Acidocin J1132Lactobacillus acidophilus JCM 1132anti-Gram+anti-Gram-Curvaticin L442Lactobacillus curvatus L442anti-Gram+Bacteriocin 32Enterococcus faeciumanti-Gram+Bacteriocin 43Enterococcus faeciumanti-Gram+Bacteriocin T8Enterococcus faecium T8anti-Gram+Enterocin BEnterococcus faeciumT136;Enterococcus faeciumW3anti-Gram+Acidocin ALactobacillus acidophilus TK9201anti-Gram+Enterocin QEnterococcus faecium L50anti-Gram+Enterocin RJ-11Enterococcus faecalis RJ-11anti-Gram+Enterocin L50Enterococcus faecium L50anti-Gram+MR10AEnterococcus faecalisMRR 10-3;Enterococcus faecalis710Canti-Gram+anti-Gram-Lacticin 3147Lactococcus lactis DPC3147anti-Gram+SpermicidalSalivaricin AStreptococcus salivarius 20P3anti-Gram+Microcin E492Klebsiella pneumoniaeRYC492anti-Gram-Cancer cellsHiracin JM79Enterococcus hirae DCH5anti-Gram+Thermophilin 9Streptococcus thermophilus LMD-9anti-Gram+Penocin APediococcus pentosaceus ATCC 25745anti-Gram+Salivaricin BStreptococcus salivarius K12anti-Gram+Lacticin 481Lactococcus lactis CNRZ 481anti-Gram+Bacteriocin J46Lactococcus lactis J46anti-Gram+Nukacin ISK-1Staphylococcus warneri ISK-1, or Staphylococcus simulans 3299anti-Gram+Streptococcin A-FF22streptococcus strain FF22anti-Gram+Lactocin SLactobacillus sake L45anti-Gram+MersacidinBacillus sp. strain HIL Y-85 54728anti-Gram+Gramicidin BBacillus brevisanti-Gram+anti-Gram-Microcin C7Escherichia colianti-Gram-Microcin B17Escherichia colianti-Gram-Microcin LEscherichia coli LR05anti-Gram-Microcin MEscherichia coli MC4100anti-Gram-Microcin I47Escherichia coli H47anti-Gram-Pep27Streptococcus pneumoniae（肺炎双球菌）anti-Gram+anti-Gram-Cancer cellsMicrocin 24E. coli 2424anti-Gram-BTLB. subtilis B-TL2antifungalAdDLPAnaeromyxobacter dehalogenansantiparasitic AntimalarialPaenibacillinPaenibacillus polymyxaanti-Gram+EpiderminStaphylococcus epidermidisT 3298anti-Gram+LichenicidinBacillus licheniformisATCC 14580 or VK21anti-Gram+Enterocin XEnterococcus faeciumKU-B5anti-Gram+Thuricin CDBacillus thuringiensis DPC 6431anti-Gram+DuramycinStreptoverticillium griseoverticillatum PA-48009anti-Gram+Duramycin BStreptoverticillium strain R2075anti-Gram+Duramycin CStreptomyces griseoluteus R2107anti-Gram+CinnamycinStreptomyces cinnamoneus cinnamoneus DSM 40005anti-Gram+ antifungalMammalian cellsPAMPPropionibacterium jensenii LMG 3032anti-Gram+Michiganin AOmato pathogen, Clavibacter michiganensis subsp. michiganensisanti-Gram+Ipomicinsweet potato pathogen,Streptomyces ipomoeaeanti-Gram+ActagardineActinoplanes garbadinensis ATCC31048 and 31049anti-Gram+Sublancin 168Bacillus subtilis 168anti-Gram+Bovicin HJ50Streptococcus bovis HJ50anti-Gram+Thermophilin 1277Streptococcus thermophilus SBT1277anti-Gram+Plantaricin WLactobacillus plantarumanti-Gram+Staphylococcin C55Staphylococcus aureus C55anti-Gram+GalliderminStaphylococcus gallinarum (F16/P57) T3928anti-Gram+anti-Gram-Epilancin 15XStaphylococcus epidermidis15X154anti-Gram+Epilancin K7Staphylococcus epidermidis K7anti-Gram+Epicidin 280Staphylococcus epidermidis BN 280anti-Gram+Microbisporicin A1Microbispora sp., strain 107891anti-Gram+SmbBStreptococcus mutans GS5anti-Gram+HaloduracinBacillus haloduransC-125anti-Gram+CypemycinStreptomyces sp. OH-4156anti-Gram+PlantazolicinBacillus amyloliquefaciens FZB42anti-Gram+Garvicin MLLactococcus garvieaeDCC43anti-Gram+Lactocyclicin QLactococcus sp. strain QU 12anti-Gram+anti-Gram-EntianinBacillus subtilis DSM 15029Tanti-Gram+CapistruinBurkholderia thailandensisE264anti-Gram-Lariatin ARhodococcus jostii K01-B0171anti-Gram+Lariatin BRhodococcus jostii K01-B0171anti-Gram+Glycocin FLactobacillus plantarum KW30anti-Gram+Leucocyclicin QLeuconostoc mesenteroides TK41401anti-Gram+anti-Gram-LaterosporulinBrevibacillus laterosporus Strain GI-9anti-Gram+ anti-Gram-antifungalGP-19Xenorhabdus budapestensisNMC-10anti-Gram+ anti-Gram-antifungalEP-20Xenorhabdus budapestensisNMC-10antifungalGeobacillin IGeobacillus thermodenitrificansNG80-2anti-Gram+Geobacillin IIGeobacillus thermodenitrificansNG80-2anti-Gram+Bacteriocin LS2Lactobacillus salivariusBGHO1anti-Gram+PatellamideLissoclinum patella-Blp1Lactobacillus salivariusBGHO1anti-Gram+TrichamideTrichodesmium erythraeum-Enterocin NKR-5-3DEnterococcus faeciumanti-Gram+Microcin SEscherichia coliG3/10anti-Gram-Microcin PDIEscherichia coli25anti-Gram-m2163Lactobacillus casei ATCC 334anti-Gram+m2386Lactobacillus caseiATCC 334anti-Gram+SclerosinPseudomonas sp. strain DF41anti-Gram+antifungalAstexin-1Asticcacaulis excentricusCB 48anti-Gram-Enterocin AS-48RJEnterococcus faeciumRJ16anti-Gram+ anti-Gram-Warnericin RKStaphylococcus warneriRKanti-Gram-Mammalian cellsdelta-lysin IStaphylococcus warneriRKanti-Gram-Mammalian cellPaenicidin APaenibacillus polymyxaNRRL B-30509anti-Gram+Antiviral lectin scytovirincyanobacterium,Scytonema variumAntiviralanti-HIVCyanovirin-NcyanobacteriumAntiviralanti-HIVMicrocystis viridis lectinblue-green algae,Microcystis viridisAntiviralanti-HIVPln149Lactobacillus plantarumNRIC 149anti-Gram+antifungalNAI-802Actinoplanes spanti-Gram+BacCH91Staphylococcus aureusCH-91anti-Gram+Fusaricidin ABacillus polymyxaKT-8anti-Gram+antifungalFusaricidin BBacillus polymyxaKT-8anti-Gram+antifungalFusaricidinBacillus polymyxaKT-8anti-Gram+antifungalFusaricidin DBacillus polymyxaKT-8anti-Gram+antifungalPeptide AN5-1Paenibacillus alveiAN5anti-Gram+ anti-Gram-NRWCBacillus subtilisATCC 6633anti-Gram-PE1Paenibacillus ehimensisB7anti-Gram+ anti-Gram-antifungalDaptomycinStreptomyces roseosporusNRRL11379anti-Gram+ColistinPaenibacillus polymyxavar. colistinusanti-Gram-, AntibiofilmSubtilosin A1Bacillus subtilisanti-Gram+AP02221Streptococcus macedonicusACA-DC 198anti-Gram+SubtilomycinBacillus subtilisMMA7anti-Gram+ anti-Gram-antifungalGramicidin SBacillus brevisanti-Gram+ anti-Gram-antifungalMammalian cellsEnterocin RM6Enterococcus faecalisOSY-RM6anti-Gram+lacticin QLactococcus lactisQU 5anti-Gram+LsbALactococcus lactisanti-Gram+LsbBLactococcus lactisanti-Gram+BHT-BStreptococcus rattusBHTanti-Gram+BHT-A1Streptococcus rattusBHTanti-Gram+Enterocin 7BEnterococcus faecalis 710C;anti-Gram+CmmAMP-Iplant pathogenanti-Gram+Brevifactina marine actinobacteriumanti-Gram+ anti-Gram-antifungalAntioxidant,Salivaricin 9Streptococcus salivariusNU10anti-Gram+Bacteriocin OR-7Lactobacillus salivariusstrain NRRL B-30514anti-Gram-Bacteriocin L-1077Lactobacillus salivarius1077 (NRRL B-50053)anti-Gram+ anti-Gram-Bactofencin ALactobacillus salivariusDPC6502anti-Gram+Plantaricin 163Lactobacillus plantarum163anti-Gram+ anti-Gram-Champacyclinmarine sediments of the Gotland Deep (Baltic Sea)anti-Gram-antifungalFengycin B2Bacillus thuringiensisantifungalBacFL31Enterococcus faeciumFL31anti-Gram+PaenibacterinPaenibacillus thiaminolyticusOSY-SEanti-Gram+anti- Gram-Cerecidin A1Bacillus cereusanti-Gram+Cerecidin A7Bacillus cereusanti-Gram+Microcin BEscherichia colianti-Gram-BsaA2community-acquired methicillin-resistantSanti-Gram+PepG1 longStaphylococcus aureusanti-Gram+ anti-Gram-PepG1 shortStaphylococcus aureusanti-Gram+ anti-Gram-AN5-2Paenibacillus alveiAN5anti-Gram+ anti-Gram-Baceridinplant-associated Bacillus strainanti-Gram+Cancer cellsLassomycin-anti-Gram+Gageostatin ABacillus subtilisanti-Gram+ anti-Gram-antifungalCancer cellsGageostatin BBacillus subtilisanti-Gram+ anti-Gram-antifungalCancer cellsGageostatin CBacillus subtilisanti-Gram+ anti-Gram-antifungalCancer cellsGageotetrin ABacillus subtilisantifungalGageotetrin BBacillus subtilisantifungalGageotetrin CBacillus subtilisantifungalThurincin HBacillus thuringiensisSF361anti-Gram+Bac-GM100Brevibacillus brevisstrain GM100anti-Gram+ anti-Gram-antifungalAmylocyclicinBacillus amyloliquefaciensFZB42anti-Gram+SonorensinBacillus sonorensisMT93anti-Gram+PerfrinClostridium perfringensanti-Gram+SilStreptococcus iniaeSF1anti-Gram+Garvicin ALactococcus garvieae21881anti-Gram+Taromycin Athe marine actinomyceteanti-Gram+Microbisporicin A2Microbispora ATCC PTA-5024anti-Gram+Tolworthcin 524Bacillus thuringiensissubspanti-Gram+ anti-Gram-Plantaricin ZJ008Lactobacillus plantarumZJ008anti-Gram+ anti-Gram-Plantaricin ZJ5Lactobacillus plantarumZJ5anti-Gram+ anti-Gram-RLID 12.1Bacillus subtilisRLID 12.1anti-Gram+ anti-Gram-antifungalLMW peptidePediococcus pentosaceusIE-3anti-Gram+ anti-Gram-CarnolysinCarnobacterium maltaromaticumC2anti-Gram+Micrococcin P1Staphylococcus epidermidisstrain 115anti-Gram+antiparasiticAntimalarialS. amritsarensis lipopeptideStreptomyces amritsarensisanti-Gram+Ala-6-fenycinBacillus sp. strain P34anti-Gram+ anti-Gram-antiviralEnterocin O16Enterococcus faecalisanti-Gram+PseABacillus pseudomycoidesDSM 12442anti-Gram+Labyrinthopeptin A2Actinomadura namibiensisantiviral注：不同的颜色对应不同的抗性1.1.1来源于芽孢杆菌只抗革兰氏阴性菌（G-）的抗菌肽针对细菌中的抗菌肽，其中同源性最高的芽孢杆菌来源的抗菌肽更容易在芽孢杆菌表达系统中进行表达，故优先统计来源于芽孢杆菌只抗革兰氏阴性菌（G-）的抗菌肽（AMP）；共有3种，具体信息如下：a：NameNRWCSourceBacillus subtilisATCC 6633Activityanti-Gram-3D StructureUnknownSequenceNRWCFAGDDTitleCharacterization of an antimicrobial peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity on Haemophilus parasuis【】Teixeira ML, Dalla Rosa A, Brandelli A.Characterization of an antimicrobial peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity on Haemophilus parasuis;Microbiology. 2013 May;159(Pt 5):980-8.Pub-Med;2013】AuthorTeixeira ML, Dalla Rosa A, Brandelli A.2013ReferenceMicrobiology. 2013 May;159(Pt 5):980-8.Pub-Medb：NameColistinSourceBacillus polymyxa（多粘芽孢杆菌）Activit