CN1763176A - 一种提高酵母菌甲醇耐受性的方法 - Google Patents
一种提高酵母菌甲醇耐受性的方法 Download PDFInfo
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- CN1763176A CN1763176A CN 200510098410 CN200510098410A CN1763176A CN 1763176 A CN1763176 A CN 1763176A CN 200510098410 CN200510098410 CN 200510098410 CN 200510098410 A CN200510098410 A CN 200510098410A CN 1763176 A CN1763176 A CN 1763176A
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Abstract
本发明公开了一种提高酵母菌甲醇耐受性的方法。该提高酵母菌甲醇耐受性的方法,是将聚羟基脂肪酸酯合成途径相关基因导入酵母菌。本发明通过向巴斯德毕赤氏酵母(Pichia pastoris) GS115中导入聚羟基脂肪酸酯合成途径相关基因,获得了能够快速生长和更高甲醇耐受性的酵母表型突变株。本发明运用基因工程及微生物发酵相结合的方法,创造性地优化改造了酵母菌的生理代谢途径,从而促进酵母菌快速生长,为实现更低成本更高蛋白质产量提供了优良的宿主菌株。本发明进一步表明PHA的产生对宿主代谢过程及生理功能的维持有重要的意义,而且外源导入PHA生物合成途径对宿主是有益的。
Description
技术领域
本发明涉及生物技术领域中一种提高酵母菌甲醇耐受性的方法。
背景技术
聚羟基脂肪酸酯(Polyhydroxyalkanoate或PHA)是许多种类的细菌都能合成的一种细胞内聚酯,在生物体内主要是作为细胞内碳源和能源的贮藏物而存在(Lemoigne M.Products of dehydration and of polymerization of β-hydroxybutyric acid.Bull.Soc.Chem.Biol.,1926,8:770-782)。近年来研究发现PHB(聚羟基丁酸酯)也存在于原核生物和真核生物的膜中。在原核生物中,它存在于其原生质膜中;而在真核生物中,则以在线粒体和微体膜中的比例为最多。相比于胞内的高分子量的聚合物,存在于膜上的PHB分子较小,只有100-200个单体。对这种小分子量PHB的研究表明,它可能起着膜上的离子通道的作用。最近,在人的血细胞中也发现较多量的这种低分子量PHB。PHA作为新型的生物可降解塑料不但可应用于低值易耗日用品行业,而且由于它的一些特殊性质,如生物相容性、光学活性、压电性、气体相隔性等,有可能在某些高附加值领域(如组织工程)得以应用,对PHA的研究已经成为一个基础理论与实践应用结合得非常紧密的分支领域。
根据PHA单体组分的碳链长短,可将PHA分为三种类型:
(1)短链PHA(short-chain-length PHA,SCL-PHA),单体含3-5个碳原子,如聚羟基丁酸酯(polyhydroxybutyrate,PHB)、羟基丁酸与羟基戊酸共聚的聚羟基丁酸羟基戊酸酯PHBV,[poly(hydroxybutyrate-co-hydroxyvalerate)]。
(2)中长链PHA(medium-chain-length PHA,MCL-PHA),单体含6个以上碳原子,如聚羟基己酸酯(PHHx)、聚羟基辛酸酯(PHO)。
(3)含短链与中长链单体的PHA,多是两种或两种以上单体的随机共聚物(Lageveen RG,Huisman GW,Preusting H,Ketelaar H,Eggink G and WitholtB.Formation of Polyesters by Pseudomonas oleovorans:Effect of Substrateson formation and composition of poly-(R)-3-hydroxyalkanoates andpoly-(R)-3-hydroxyalkenoates.Appl.Environ.Microbiol,1988,54:2924-2932)。
PHA的积累对于产生菌本身适应外界环境的变化及逆境生存具有重要的意义。以PHB为例,有些细菌胞内的PHB可以减缓细胞内重要成分-RNA和蛋白质在饥饿条件下的降解,从而可以增加细菌对生存逆境的抵抗能力。在某些芽孢杆菌属中,尽管孢子的形成并不必需PHB,但PHB可以作为孢子形成时的能源和碳源从而增加这些菌的生存机会。此外,PHB还可以为某些固氮菌属的包囊形成提供必要的碳源和能源。对Rhizobium sp.ORS 571的固氮与PHB的形成及降解的研究表明,当节结中的氧浓度增加时,可以通过PHB降解保护固氮酶不受损伤(Anderson AJ andDawes EA.Occurense,Metabolism,Metabolic Role,and Industrial Use ofBacterial Polyhydroxyalkanoates.Microb.Rev.,1990,54:450-472)。不同类型PHA在细菌内的合成途迳是不同的(Lee SY.BacterialPolyhydroxyalkanoates.Biotech.Bioeng.1996,49:1-14),目前已知的合成途径主要有:
1.由乙酰辅酶A直接合成PHB途径:以罗氏真氧菌(Wautersia eutropha H16,又名Ralstonia eutropha,Alcaligenes eutrophus)为代表的PHB生物合成系统包含三种酶:由一个合成操纵子基因phbCAB编码β-酮基硫解酶(β-ketothiolase)、NADPH依赖的乙酰乙酰辅酶A还原酶(acetoacyl-CoA reductase)和PHB合成酶(PHB synthase);三种酶分别由phbA、phbB和phbC基因编码,由同一个启动子调控表达。细胞内的代谢中间体乙酰辅酶A在β-酮基硫解酶(β-ketothiolase,PhaA)的催化作用下形成乙酰乙酰辅酶A,然后在NADPH依赖的还原酶(PhaB)的作用下还原为(R)-羟基丁酰辅酶A,(R)-羟基丁酰辅酶A在PHB合成酶的作用下聚合成PHB,合成过程需要消耗还原力NADPH,产生NADP(Schubert P,SteinbüchelA,Schlegel HG.Cloning of Alcaligenes eutrophuspoly-β-hydroxybutyrate synthetic pathway and synthesis of PHB inEscherichia coli.J.Bacteriol.1988,170:5837-5847;Peoples O P,Sinskey A J.Poly-β-hydroxybutyrate biosynthesis in Alcaligeneseutrophus H16.Identification and characterization of the PHBpolymerase gene(phbC).J.Biol.Chem.1989,264:15298-15303)。
2.脂肪酸从头合成途径:以恶臭假单胞菌(Pseudomonas putita)为代表,该条途径合成PHA需要两种酶的催化:phaG编码的3-羟酰基-酰基转移蛋白-辅酶A转移酶(3-Hydroxyacyl-ACP-CoA transferase)和phaC编码的PHA合酶。脂肪酸从头合成途径的中间体3-羟酰基-酰基转移蛋白由3-羟酰基-酰基转移蛋白-辅酶A转移酶(PhaG)催化形成PHA的前体3-羟基酯酰辅酶A,再由PHA合酶聚合成PHA。恶臭假单胞菌(Pseudomonas putita)的3-羟酰基-酰基转移蛋白-辅酶A转移酶PhaG的编码基因phaG的Genbank登录号是AF052507(Rehm BH,KrugerN and SteinbuchelA.A new metabolic link between fatty acid de novosynthesis and polyhydroxyalkanoic acid synthesis.The phaG gene fromPseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrierprotein-coenzyme A transferase.1998.J.Biol.Chem.273(37),24044-24051)。
3.β-氧化途径:以嗜水气单胞菌(Aeromonas hydrophila)为代表,从这条途径合成PHA需要一种结构蛋白和另外两种酶的催化作用:phaP编码一种结合PHA颗粒表面的结构蛋白,phaJ编码(R)-烯酰基-水合酶((R)-enoyl-CoA hydratase)和phaC编码PHA合酶。脂肪酸β-氧化的中间体烯酰基-酰基转移蛋白由(R)-烯酰基-水合酶(PhaJ)催化形成PHA的前体3-羟基酯酰辅酶A,再由PHA聚合酶聚合成PHA。嗜水气单胞菌(Aeromonas hydrophila)的三个PHA合成相关基因组成一个操纵子(PHA synthase operon),操纵子编码基因的Genbank检索号是AY093685,包括PhaP,PhaJ和PhaC的编码基因(Lu XY,Wu Q,Chen JY,ZhangWJ,Zhang G and Chen GQ.Molecular cloning of polyhydroxyalkanoatessynthesis operon from Aeromonas hydrophila and its expression in E.coli.Biotech Prog 20(2004)1332-1336)。
在PHA的代谢途径中,既有能量的代谢(NAD/NADH,NADP/NADPH等),也有物质的代谢(乙酰辅酶A,脂肪酸等),因此PHA的代谢过程,实际上是一个细胞内能量和物质的改变过程。由于PHA代谢有益于宿主细胞,能够广泛存在于各种原核或真核细胞中,因此可以将各种PHA合成机制作为调节手段应用于不同的微生物,从而调节其物质和能量的代谢流和代谢水平,进一步影响细胞生长代谢(张晋宇。(2005)表达phbCAB基因对兽疫链球菌中乳酸和透明质酸产量的影响。清华大学硕士学位论文。)。
酵母是基因工程最常用的外源基因真核表达系统,但常规酵母质粒表达系统,即酿酒酵母(S accharomyces cerevisiae)质粒不稳定导致表达效率低。80年代末,国外一些科研工作者开始对非常规酵母(non-conventional yeast)表达体系进行了探索,其中包括巴斯德毕赤氏酵母(Pichia pastoris)、乳酸克鲁维酵母(Kluyveromyces lactis)以及多形汉逊酵母(Hansenula polymorpha)等(Wegner,G.Emerging applications of the methylotrophic yeasts.FEMS Microbiol.Rev.1990,7,279-283)。其中,以甲醇作为唯一碳源的巴斯德毕赤氏酵母外源基因表达体系最引人注目。这是由于巴斯德毕赤氏酵母体系的独特优点:(1)高表达,它能利用很强的启动子,具有极快的细胞生长速度,所以表达量相对很高。(2)高稳定,该系统的载体能和细胞基因组进行整合,所以构建的重组菌株遗传性状稳定,一般不会出现外源基因随生长繁殖而丢失的现象。(3)严紧调控,外源基因在醇氧化酶启动子的严紧调节控制下,可通过加入甲醇而诱导外源基因表达(Cregg JM,Ceregino JL,Shi J,et al.Recombinant protein expressioninPichia Pastoris.Molecular Biotechnology 2000,16:23-52)。
乙醇氧化酶(alcohol oxygenase,AOX)催化甲醇代谢的第一步,甲醇被氧化成为甲醛和H2O2,部分甲醛进一步被胞浆脱氧酶氧化成甲酸和CO2,同时这一过程为细胞生长提供能量。乙醇氧化酶只有在细胞在甲醇培养基中生长才高水平表达(占总可溶性蛋白的30%),在其他碳源如葡萄糖、甘油或乙醇中几乎无法检测到(Couderc,R.and Baratti,J.Oxidation of methanol by the yeast Pichiapastoris:purification and properties of alcohol oxidase.Agric.Biol.Chem.1980,44,2279-2289)。毕赤酵母表达载体含有乙醇氧化酶基因5′AOX1启动子、3′AOX1终止子,这两段序列之间是供外源基因插入的多克隆位点。通常以组氨醇脱氢酶(Histidinol dehydrogenase,HIS)基因HIS4作为营养互补型筛选标志或Zeocin及卡那霉素基因(kanamycin,Kana)赋予酵母的遗传霉素(G418)抗性作为筛选标志。作为一个能在大肠杆菌中繁殖、扩增的穿梭质粒,它还含有pBR322质粒的部分序列和氨苄青霉素(Amp,Ampicillin)抗性基因的筛选标志,表达载体通过同源重组整合到酵母细胞染色体DNA中。重组时用DNA内切酶Bg1 II、Dra I和Sal I等线性化表达载体,通过电转化等方法使之整合于酵母基因组内的AOX1基因的位置,整合后的外源基因可随酵母的生长稳定地传代(Cregg JM,Ceregino JL,Shi J,et al.Recombinant protein expressionin PichiaPastoris.Molecular Biotechnology 2000,16:23-52)。
目前应用最广泛的甲醇快速利用型宿主是Cregg等1985年构建的GS115菌株,它含有AOX1和AOX2基因,在含有甲醇的培养基中生长迅速(Mut+)(Cregg JM,Vedvick TS,Raschke WC,Recent advances in the expression of foreign genesin Pichia pastoris.Bio/Technology.11(1993)905-910)。载体转化入酵母后主要以插入或置换两种方式与酵母基因组发生重组。含有AOX1基因序列的表达载体既可以在酵母基因组AOX1或HIS4位点发生单交换(Single crossover),插入到酵母基因组中;也可以发生双交换(Double crossover)而取代AOX1基因,以何种方式发生重组都可以同时有多个拷贝整合到基因组中,而且整合的基因拷贝数不同,表达量也有很大差异,通常情况下,拷贝数越多表达量越高。AOX1启动子(PAOX1)受甲醇的严格调控,在碳源为非甲醇的条件下,AOX1基因转录的mRNA检测不到,但在甲醇为唯一碳源的培养基中,AOX1基因编码的mRNA可达到总RNA的5%,AOX1则占可溶性蛋白的30%,由此可见甲醇的添加可以诱导AOX1基因转录水平的提高。在PAOX1调控下表达外源基因时,可通过调节甲醇而适时地控制表达过程,这样可以有效地减轻宿主选择压力,保证外源基因的稳定存在。
发明内容
本发明的目的是提供一种提高酵母菌甲醇耐受性的方法。
本发明所提供的提高酵母菌甲醇耐受性的方法,是将聚羟基脂肪酸酯合成途径相关基因导入酵母菌。
所述聚羟基脂肪酸酯合成途径相关基因选自下述1),2)和3)中的任意一种:1)由phbC,phbB和phbA组成的phbCAB基因;2)phaG和phaC基因;3)phaPCJ。
所述聚羟基脂肪酸酯合成途径相关基因优选为phaPCJ。
所述phbC具有Genbank登录号为J05003的自5′端第842位至2611位脱氧核苷酸组成的核苷酸序列;所述phbA具有Genbank登录号为J04987的自5′端第40位至1221位脱氧核苷酸组成的核苷酸序列;所述phbB具有Genbank登录号为J04987的自5′端第1296位至2036位脱氧核苷酸组成的核苷酸序列。
所述phaPCJ基因优选为嗜水气单胞菌(Aeromonas hydrophila 4AK4)的phaPCJ(序列1),Genbank登录号为AY093685。
序列1由2920个脱氧核苷酸组成,其编码序列为自序列1的5′端第292位至2920位脱氧核苷酸。
所述phaG基因具有序列表中序列2的核苷酸序列(Genbank登录号为AF052507),其编码序列为自序列2的5′端第912位至1799位脱氧核苷酸;所述phaC基因具有序列1的自5′端第735位至2519位脱氧核苷酸组成的序列。
上述PHA合成途径相关基因可以通过多种途径导入酵母菌中,例如,利用表达载体如质粒、粘粒、噬菌体等,通过转化、转导、接合等手段导入酵母菌中;也可以利用原生质体融合等手段来实现。具体可将PHA合成途径相关基因克隆到针对于目的菌的质粒载体上,将构建的质粒导入到目的菌中获得重组菌。PHA合成途径相关基因可以在质粒上或整合到染色体上。
所述phaPCJ可通过pPIC3.5K-phaPCJ导入酵母菌;所述pPIC3.5K-phaPCJ是将phaPCJ插入pPIC3.5K的BamHI和EcoRI识别位点间得到的重组表达载体。
本发明优选电击转化的方法将构建在酵母胞内高效表达载体pPIC3.5K上的重组质粒pPIC3.5K-phaPCJ导入优选的宿主细胞巴斯德毕赤酵母(Pichia pastorisGS115)。为提高转化效率,将重组质粒表达载体导入目的菌中的方法优选为电转化法,但也可以采用其它生物工程领域中常用的方法,包括热激法、原生质体转化法和接合转化法。
上述酵母菌为嗜甲醇酵母。
所述嗜甲醇酵母可为巴斯德毕赤氏酵母(Pichia pastoris)或多形汉逊酵母(Hansenula polymopha)。
所述巴斯德毕赤氏酵母(Pichia pastoris)优选为巴斯德毕赤氏酵母(Pichiapastoris)GS115。
虽然甲醇对于毕赤酵母诱导表达蛋白是必需和有益的,但是由于酵母对于甲醇的耐受性有限,因此当甲醇浓度高时将抑制菌体生长(抑制菌体生物量,细胞干重),从而进一步限制了目的蛋白的表达(赵菁菁,茹炳根.山蛭素(haemadin)在毕赤甲醇酵母中的表达及其性质鉴定.北京大学学报(自然科学版),Vol.41,No.4,2005.p506-512)。本发明通过向巴斯德毕赤氏酵母(Pichia pastoris)GS115中导入聚羟基脂肪酸酯合成途径相关基因,获得了能够快速生长和更高甲醇耐受性的酵母表型突变株。本发明运用基因工程及微生物发酵相结合的方法,创造性地优化改造了酵母菌的生理代谢途径,从而促进酵母菌快速生长,为实现更低成本更高蛋白质产量提供了优良的宿主菌株。本发明进一步表明PHA的产生对宿主代谢过程及生理功能的维持有重要的意义,而且外源导入PHA生物合成途径对宿主是有益的。
本发明所提供的提高酵母菌甲醇耐受性的方法,是通过基因工程的手段在甲醇酵母中导入异源PHA合成途径的相关基因,实现在甲醇酵母中建立PHA合成途径,营造PHA代谢途径与宿主固有途径的交互作用(cross-talking),从而影响细胞的整个代谢网络。将本发明的方法应用到各种酵母菌中,可以有效的影响酵母菌的代谢途径,增加宿主酵母菌的抗逆性,提高目标产物的生物合成产量。
在不偏离本发明的精神和范围的情况下,本领域的普通技术人员可以在形式和细节上对其做出各种改变和改进,这些均在本发明的保护范围之内,如利用密码子简并原则或碱基互补原则所获得的与本发明的聚羟基脂肪酸酯合成途径的相关基因功能基本相同的核酸分子,对于聚羟基脂肪酸酯合成途径的相关酶,在其不起主要作用的位点进行氨基酸插入和/或缺失和/或替换所得到的功能基本相同的蛋白质或多肽的编码基因,来实现本发明的目的,均被认为落入了本发明的保护范围。
附图说明
图1为pPIC3.5K-phaPCJ的物理图谱
图2为转pPIC3.5K-phaPCJ的宿主菌与转pPIC3.5K的宿主菌用2%甲醇诱导48小时的细胞干重柱状图
图3为转pPIC3.5K-phaPCJ的宿主菌与转pPIC3.5K的宿主菌用2%甲醇诱导不同时间的细胞干重柱状图
图4为转pPIC3.5K-phaPCJ的宿主菌与转pPIC3.5K的宿主菌的蛋白质印迹分析图谱
具体实施方式
下述实施例中的实验方法,如无特别说明,均为常规方法。
下述实施例中的百分含量,如无特别说明,均为质量百分含量。
实施例1、提高巴斯德毕赤氏酵母(Pichia pastoris)GS115的甲醇耐受性
1、酵母胞内表达重组质粒pPIC3.5K-phaPCJ的构建
构建重组质粒pPIC3.5K-phaPCJ包括以下步骤:
1)目的基因的获得:用细菌基因组DNA提取试剂从嗜水气单胞菌(Aeromonashydrophila)(购自中国科学院微生物研究所“中国普通微生物纯培养物保藏中心”)中提取基因组DNA作为模板,以phaPCJ基因特异性上、下游引物(phaPCJ-upper-BamHI:5’>TT
G GAT CCT GGA GAC CGA TGA TGA ATA TG<3’;phaPCJ-lower-Eco RI:5’>AT
G AAT TCT TAA GGC AGC TTG ACC ACG G<3’)进行目的基因片段(2641bp)的PCR扩增、纯化,结果得到2641bp的PCR片段。其中,50μl PCR反应体系包括:1μl基因组DNA模板,1μl上游引物,1μl下游引物,5μl PCR反应缓冲液(10×),6μl dNTP混合物,35.5μl灭菌去离子水,0.5μl高保真聚合酶(pyrobest polymerase);PCR条件如下:先95℃预变性8min;然后94℃变性1min,65℃退火1min,72℃延伸2min,30个循环后,72℃后延伸10min。
2)目的基因与载体的双酶切和连接:
从美国Invitrogen公司购买到酵母胞内表达载体pPIC3.5K。用TaKaRa公司的限制性内切酶BamHI和EcoRI在37℃8小时酶切载体与步骤1)得到的2641bp的PCR片段后,用T4 DNA连接酶连接已酶切纯化的载体和目的基因DNA,载体与插入片段的摩尔比为1∶3,10ul连接体系,16℃保温24h。
3)重组质粒的转化和验证:
将步骤2)的10μl连接混合物与50μl大肠杆菌感受态细胞E.coli JM109混合,预冷,加入1mm电击杯中,1.25Kv电击转化,迅速加入1ml LB培养基,37℃振荡培养1h,取200μl涂布含有50μg/ml氨苄青霉素和50μg/ml卡那霉素双抗LB平板,37℃培养12~16h挑取阳性克隆,接入4ml含双抗(50μg/ml氨苄青霉素和50μg/ml卡那霉素)LB液体培养基中培养8小时,取3ml提取质粒,进行PCR验证,以阳性克隆质粒为模板,使用目的基因phaPCJ基因特异性引物phaPCJ-upper-BamHI和phaPCJ-lower-EcoRI进行PCR扩增。将PCR扩增能得到2.6kb DNA片段的阳性克隆质粒进行DNA测序分析,确保翻译读码框正确。将具有自序列1的5′端第292位至2920位脱氧核苷酸的phaPCJ基因片段的质粒命名为pPIC3.5K-phaPCJ(图1)。
2、重组酵母阳性转化子的获得
1)重组质粒线性化:采用Sal I酶切线性化重组质粒pPIC3.5K-phaPCJ。
2)毕赤酵母感受态细胞的制备和转化实验操作程序:
a)从美国Invitrogen公司购买到甘油管保存的酵母宿主菌株(Pichiapastori5)GS115,划线于YPD琼脂平板上,于30℃活化培养2天。挑取单菌落接种于装有1ml YPD液体培养基的试管中,于30℃震荡培养1天。
b)把新鲜培养的酵母按10%(体积比)的接种量转移到装有10ml YPD液体培养基的150ml三角瓶中,于30℃继续震荡培养至OD600值为1.4-1.5。
c)移取1ml培养液于EP离心管中,于4℃低温离心5分钟(5000rpm),弃去上清液。以1ml含有20mM HEPES和37.5μl的1M DTT的YPD溶液悬浮酵母细胞沉淀,于30℃培养15分钟。于4℃低温离心5分钟(5000rpm),弃去上清液。
d)取1.5ml1M山梨醇悬浮酵母细胞沉淀,于4℃低温离心5分钟(5000rpm),弃去上清液,共进行3次;也就是用1M山梨醇洗酵母细胞三次。
e)用20μl 1M山梨醇悬浮酵母细胞,合并2管,得到40μl的电感受态酵母GS115,取5μl经限制性内切酶Sac I线性化的表达载体pPIC3.5K-phaPCJ和40μl的电感受态酵母GS115混合。
f)电击杯(0.2cm)放置冰上预冷,将转化混合物转移到电击杯中,吸干杯的外表面,放入电转化仪的样品槽中,进行电转化;
g)取出电击杯,迅速加入1毫升含有180.2mg山梨醇的YPD培养基,并用灭菌枪头转移到灭菌的Eppendorf管中,30℃培养1.5小时。
h)取培养液涂布于含有1mg/mL抗生素G418(遗传霉素,Geneticin)的YPD平板上,于30℃培养2-5天后出现抗性菌落,挑取抗性菌落,用phaPCJ基因特异性上、下游引物(phaPCJ-upper-BamH I:5’>TT
G GAT CCT GGA GAC CGA TGA TGAATA TG<3’;和phaPCJ-lower-EcoR I:5’>AT
G AAT TCT TAA GGC AGC TTG ACCACG G<3’)进行菌落PCR,结果获得四个可扩增到2.6kb的PCR片段的阳性酵母菌落,说明pPIC3.5K-phaPCJ整合到基因组DNA上了。将阳性酵母菌分别命名为P7-I GS115/pPIC3.5K-phaPCJ,P12-H GS115/pPIC3.5K-phaPCJ,P13-1 GS115/pPIC3.5K-phaPCJ,P18-2 GS115/pPIC3.5K-phaPCJ。
按照上述步骤2的方法,用pPIC3.5K转化酵母宿主菌株(Pichia pastoris)GS115,得到含有pPIC3.5K的重组酵母WT GS115,作为对照宿主菌。
3、阳性重组毕赤酵母的诱导培养:
贮备液、培养基配制如下:
磷酸缓冲液(200ml):45.6g K2HPO4,7ml H3PO4。
13.4%酵母氮素碱基(10×YNB):0.45μm滤器过滤除菌。
500×生物素(0.02%Biotin):0.45μm滤器过滤除菌。
甲醇:0.45μm滤器过滤除菌。
A)生长培养基(改良BMGY)配制:
酵母浸出物 1g
胰蛋白胨 2g
甘油 2ml
蒸馏水定容至78ml,121℃灭菌20min
再加入
磷酸缓冲液 10ml,
10×YNB 10ml,
500×生物素 2ml。
总体积 100ml
B)诱导表达培养基(改良BMYY)配制:
酵母浸出物 1g
胰蛋白胨 2g
蒸馏水定容至76ml,121℃灭菌20min
再加入
磷酸缓冲液 10ml
10×YNB 10ml
甲醇 2ml
500×生物素 2ml
总体积 100ml
将含phaPCJ基因的重组酵母P7-I GS115/pPIC3.5K-phaPCJ,P12-HGS115/pPIC3.5K-phaPCJ,P13-1 GS115/pPIC3.5K-phaPCJ,P18-2GS115/pPIC3.5K-phaPCJ,只含pPIC3.5K的重组酵母WT GS115,分别在生长培养基中生长2天(48h)后,在无菌条件下离心,更换甲醇诱导培养基,继续培养2天(48h)后,收获细胞,低温冷冻干燥细胞,称量细胞干重,结果如图2所示,表明重组酵母P7-I GS115/pPIC3.5K-phaPCJ,P12-H GS115/pPIC3.5K-phaPCJ,P13-1 GS115/pPIC3.5K-phaPCJ和P18-2 GS115/pPIC3.5K-phaPCJ的细胞干重均明显高于只含pPIC3.5K的重组酵母WT GS115,与WT GS115相比有更加优越的生长特性。
将重组酵母菌P7-I GS115/pPIC3.5K-phaPCJ,P12-H GS115/pPIC3.5K-phaPCJ,P13-1 GS115/pPIC3.5K-phaPCJ,P18-2 GS115/pPIC3.5K-phaPCJ和WT GS115分别在生长培养基中生长2天(48h)后,在无菌条件下离心,更换甲醇诱导培养基,继续培养48h、96h、144h后,收获细胞,低温冷冻干燥细胞,称量细胞干重,结果如图3所示,不同甲醇诱导时间(48h,96h,144h,甲醇浓度v/v 2%)的细胞干重,进一步证实重组酵母菌P7-I GS115/pPIC3.5K-phaPCJ,P12-HGS115/pPIC3.5K-phaPCJ,P13-1 GS115/pPIC3.5K-phaPCJ和P18-2 GS115/pPIC3.5K-phaPCJ比对照宿主菌WT GS115拥有更加优越的细胞生长特性和更高的甲醇耐受性。一般在重组毕赤酵母中诱导表达外源蛋白质,为了提高蛋白质表达量,在诱导培养二天后通过补加0.5%(V/V)的甲醇以达到目的。在本发明的实验中,补加的甲醇量可以达到2%(V/V)而不影响细胞生长。图3中,P7-I GS115,P12-H GS115,P13-I GS115,P13-I GS115,P18-II分别表示P7-I GS115/pPIC3.5K-phaPCJ,P12-H GS115/pPIC3.5K-phaPCJ,P13-1 GS115/pPIC3.5K-phaPCJ和P18-2 GS115/pPIC3.5K-phaPCJ。
实施例2、Western blott ing检测含有phaPCJ的重组酵母表达phaP和phaJ异源基因的情况
1、PhaP和PhaJ抗血清的制备
(1)制备纯化的可溶蛋白质PhaP和PhaJ
设计PhaP基因特异性引物phaP-upper-BamH I:5’>TT
G GAT CCT GGA GAC CGATGA TGA ATA TG<3’、phaP-lower-HindIII:5’>AAT
AAG CTT GGC CTT GCC CGTGCT CTT C<3’;和PhaJ基因特异性引物phaJ-upper-BamH I:5’>TAT T
GG ATC CATGAG CGC ACA ACC CCT TG<3’、phaJ-lower-Hind III:5’>AAT
AAG CTT AGG CAGCTT GAC CAC GGC<3’,分别以酵母重组质粒pPIC3.5K-phaPCJ为模板,PCR扩增获得特异的DNA片段phaP和phaJ。从德国NOVAGEN公司购得原核表达载体pET28a(+),将载体pET28a(+)用BamH I和HindIII酶切后与特异的DNA片段phaP和phaJ分别进行连接和转化大肠杆菌感受态细胞E.coJi BL21(DE3)。在卡那霉素抗性平板上筛选阳性克隆,小量表达实验筛选过量表达带有组氨酸融合标签蛋白质的重组菌。纯化带组氨酸标签的蛋白质的亲和层析方法参照镍柱(Ni-NTAResin,Qiagen公司)使用说明书进行,得到纯化的带有组氨酸融合标签的PhaP和PhaJ蛋白质。
(2)小鼠的免疫及PhaP和PhaJ多克隆抗血清的制备
实验动物:BALB/C小白鼠,注射途径为腹腔注射。
初次免疫,5-10μg步骤(1)制备的PhaP和PhaJ分别溶解于磷酸(PBS)缓冲液中,加入等体积弗氏完全佐剂混合乳化,乳化好后立即使用。为了获取高质量(高滴度和高亲和力)的抗血清,有必要对实验动物作加强注射。初次免疫后第15天进行第一次加强免疫,加强免疫注射的剂量为初次免疫剂量的1/2(一半),隔两周加强免疫一次,共进行3次。第三次加强注射后第三天,分别从免疫的BALB/C小白鼠尾部取血50μL,室温放置2小时后离心,取血清即获得抗PhaP的多克隆抗血清和抗PhaJ的多克隆抗血清。对每一动物作免疫接种前先采集血样,得到的正常血清作免疫印迹时的阴性对照。
2、Western blotting检测含有phaPCJ的重组酵母表达phaP和phaJ的情况
根据抗原-抗体分子间特异性识别、结合的原理设计重组酵母菌表达PHA合成相关蛋白质的免疫检测实验。
分别取实施例1中甲醇诱导48小时的1ml诱导表达的酵母转化子P7-IGS115/pPIC3.5K-phaPCJ和WT GS115菌液离心,收集菌体,加入100μl 0.5MTris(pH6.8)缓冲液悬浮细胞后,加入25μl 4×SDS-PAGE上样缓冲液(4×SDS-PAGE上样缓冲液的配方为:50mg SDS,4mg溴酚蓝,1mlβ-巯基乙醇,3ml甘油,2ml 0.5M Tris(pH6.8)缓冲液,4ml去离子水,总体积10ml),煮沸裂解。取25μl细胞裂解液制备成的胞内蛋白样品进行SDS-PAGE和Western blotting。SDS-PAGE后,电转移胶上的表达蛋白质到NC膜上(电转条件:Tris-glycine缓冲液(Tris-glycine缓冲液的配方为:5.8g Tris,2.9g glycine,0.37g SDS溶于800ml去离子水,加入200ml甲醇),20伏,4℃,2小时),对固定于电转膜上的蛋白质进行丽春红S染色[(具体方法见分子克隆实验指南第二版P893)Ponceau S,Sigma P3504;0.1%丽春红溶解于5%(v/v)的冰乙酸,丽春红S染色法可与后续免疫学检测方法兼容],该染料只会短暂显色而且在进行印迹分析时可被洗去。染色后,用蒸馏水洗去染液。加入1%无钙的脱脂奶粉封闭液(1克无钙的脱脂奶粉溶于磷酸缓冲液,pH7.4),在转速为60rpm的摇床上室温处理2-4小时,封闭膜上剩余的疏水结合位点;弃去封闭液,分别以步骤1制备的PhaP多克隆抗血清和phaJ多克隆抗血清作为一抗血清,用磷酸缓冲液以1∶400的体积比稀释,在缓慢摇床上室温处理45分钟后,用磷酸缓冲液洗三次,每次10分钟;加入二抗(兔抗鼠IgG,偶联HRP),以1∶2000体积比稀释(5微升二抗加入10毫升的磷酸缓冲液中),在缓慢摇床上室温处理45分钟后,用磷酸缓冲液洗三次,每次10分钟;加入0.6mg/mL DAB的底物溶液和15uL的过氧化氢(6mg DAB溶于10mL的磷酸缓冲液)显色。结果如图4所示,含有pPIC3.5K-phaPCJ的酵母转化子P7-IGS115/pPIC3.5K-phaPCJ表达了14kDa的PhaP,19kDa的PhaJ。而转化了空载体的对照宿主菌WT GS115中没有PhaP和PhaJ的表达。图4中,1为含有pPIC3.5K-phaPCJ的酵母转化子P7-I GS115/pPIC3.5K-phaPCJ的总细胞蛋白质与phaJ多克隆抗血清的杂交结果,2为转空载体的对照宿主菌WT GS115与phaJ多克隆抗血清的杂交结果,3为转空载体的对照宿主菌WT GS115与phaP多克隆抗血清的杂交结果,4为含有pPIC3.5K-phaPCJ的酵母转化子P7-IGS115/pPIC3.5K-phaPCJ的总细胞蛋白质与phaP多克隆抗血清的杂交结果。
该结果表明PHA生物合成相关基因在含有pPIC3.5K-phaPCJ的重组酵母中获得了准确转录和正确翻译,同时说明PHA生物合成代谢赋予了含有pPIC3.5K-phaPCJ的重组酵母更快的生长优越性和更高的甲醇耐受性(如前所述,补加甲醇从0.5%提高到2%)。对甲醇的耐受性提高的机理可能是含phaPCJ基因的重组毕赤酵母由于更加快速的生长和芽殖,需要更多的甲醇作为碳源;反之,更高的甲醇浓度提供了更多的碳源供重组毕赤酵母更加快速的生长和芽殖以产生更大量的外源蛋白质,从而形成良性循环。
序列表
<160>2
<210>1
<211>2920
<212>DNA
<213>嗜水气单胞菌(Aeromonnas hydrophila)
<400>1
gcagtctgaa taagcttcca gcctatcagc gcggcgccag cacggcgagg gagcatcggg 60
tcccgtgcgt cacctctcgt ctgacccacg cccccgacgg aggtcgctga caaaaaaatt 120
caaacagaaa ttaacattta tgtcatttac accaaaccgc atttggttgc agaatgctca 180
aacgtgtgtt tgaacagagc aagcaacacg taaacaggga tgacatgcag tacccgtaaa 240
aagggccgat tggcccacaa caacactgtt ctgccgaact ggagaccgat gatgaatatg 300
gacgtgatca agagctttac cgagcagatg caaggcttcg ccgcccccct cacccgctac 360
aaccaactgc tggccagcaa catcgagcag ctgacccggt tgcagctggc ctccgccaac 420
gcctacgccg aactgggcct caaccagttg caggccgtga gcaaggtgca ggacacccag 480
agtctggctg ccctcggcac agtgcagctg gagaccgcca gccagctctc ccgccagatg 540
ctggacgaca tccagaagct gagcgccctc ggccagcagt tcaaggaaga gctggatgtc 600
ctgaccgcgg acggcatcaa gaagagcacg ggcaaggcct gataacccct ggctgcccgt 660
tcgggcagcc acatctcccc acgactcgac gctacgggct agctcccgcc tcgggtgtgg 720
gtgaaggaga gcacatgagc caaccatctt atggcccgct gttcgaggcc ctggcccact 780
acaatgacaa gctgctggcc atggccaagg cccagacaga gcgcaccgcc caggcgctgc 840
tgcagaccaa tctggacgat ctgggccagg tgctggagca gggcagccag cagccctggc 900
agctgatcca ggcccagatg aactggtggc aggatcagct caagctgatg cagcacaccc 960
tgctgaaaag cgcaggccag cagagcgagc cggtgatcac cccggagcgc agcgatcgcc 1020
gcttcaaggc cgaggcctgg agcgaacaac ccatctatga ctacctcaag cagtcctacc 1080
tgctcaccgc caggcacctg ctggcctcgg tggatgccct ggacggcgtc ccccagaaga 1140
gccgggagcg gctgcgtttc ttcacccgtc agtacgtcaa cgccatggca cccagcaact 1200
tcctggccac caacccggag ctgctcaagc tcaccctgga gtccgacggc cagaacctgg 1260
tgcgcgggct ggccctcttg gccgaggatc tggagcgcag cgccgatcag ctcaacatcc 1320
gcctgaccga cgaatccgcc ttcgagctcg gccgggatct ggcgaccacc ccgggccggg 1380
tggtgcagcg caccgagctc tatgagctga tccagtacag ccccaccacg gaaaccgtgg 1440
gcaagacgcc tgtgctgatc gtgcccccct tcatcaacaa gtactacatc atggacatgc 1500
ggccccagaa ctccctggtc gcctggctgg tcgcccaggg ccagacggtg ttcatgatct 1560
cctggcgcaa cccgagcgta gcccaggccc aaatcgatct cgacgactac gtggtggatg 1620
gcgtcatcgc cgccctggac ggcgtggaag cggccaccgg cgagcgggag gtgcacggca 1680
tcggctactg catcgacggc accgccctgt cgctcgccat gggctggctg gcggcgcggc 1740
gccagaagca gcgggtgcgc actgccaccc tgttcaccac cctgctggac ttctcccagc 1800
cgggggagct tggcatcttc attcacgagc ccatcatagc ggcgctcgag gcgcaaaatg 1860
aggccaaggg catcatggac gggcgccagc tggctgtctc tttcagcctg ctgcgggaga 1920
acagcctcta ctggaactac tacatcgaca gctacctcaa gggtcagagc ccggtggcct 1980
tcgatctgct gcactggaac agcgacagca ccaatgtggc gggcaagacc cacaacagcc 2040
tgccgcgccg tctctatctg gagaaccagc tggtgaaggg ggagctcaag atccgcaaca 2100
cccgcatcga tcttggcaag gtgaagaccc ctgtgctgct ggtgtcggcg gtggacgatc 2160
acatcgccct ctggcagggc acctggcagg gcatgaagct gtttggcggg gagcagcgct 2220
tcctcctggc agagtccggc cacatcgccg gcatcatcaa cccgccggtc gccaacaagt 2280
acggcttctg gcacaacggg gccgaggccg atagcccgga gagctggctg gcaggggcga 2340
cgcatcagag cggctcctgg tggcccgaga tgatgggctt tatccagagc cgtgacgaag 2400
ggtcagagcc cgtccccgca cgggtgcccg aggaggggct ggcccccgcc cccggccact 2460
atgtcaaggt gcggctcaac cccgtgtttg ccagcgccac agaggaggac gccgcatgag 2520
cgcacaaccc cttgaagtgg gccagaaggc ccgtctcagc aagcggttcg gggcggcaga 2580
ggtggccgcc ttcgccgcgc tctcggagga tttcaaccct ctgcaccttg atccggcctt 2640
cgccgccacc acggcgttcg agcgacccat cgtccacggc atgctgctgg ccagcctctt 2700
ctccgggctg ctgggccagc agttgccggg caaggggagc atctatctgg gccagagcct 2760
cagcttcaag ctgccggtct ttgtcgggga tgaggtgacg gccgaggtgg aggtgaccgc 2820
ccttcgcagc gacaagccca tcgccaccct gaccacccgc atcttcactt caaacggcgc 2880
cctcgccgtg acgggggaag ccgtggtcaa gctgccttaa 2920
<210>2
<211>3061
<212>DNA
<213>恶臭假单胞菌(Pseudomonas putida)
<400>2
gaattcagcc gcgtagagct ggacgagcaa ctgctgcagg ccggccgccc ggcccgctac 60
ctgatgctgt acaagcccac tggctgcgta acggccaccc acgatccgca acaccgtacc 120
gttctcgacc tgctgccagc ggcgttgcga gatgacctgc acatagccgg cgcgcctgga 180
cttcaacacc accggcctga tgatcctgac caacgatggc caatggtcac ggcggcctga 240
ccagcctgcc accaagctgc ccaagcatta tctggtggac accgaggacg agattggcga 300
gcactatgtg gccaagtttc gcgagggttt ctattttgcc ttcgaagacc tcaccaccca 360
acctgcccag ctggacatcc tcggccccca ccgagcccgg ctggcgatcg tcgaggggcg 420
ttaccaccag gtcaagcgca tgttcgggca tttcaacaac aaggtgatcg ggctgcatcg 480
ggagagcatg ggggcgatcc ggctggatcc ggggttggcg ccgggggagt atcgtgaact 540
gacggccaat gagatagcca ctgtctaggc cgtgacagac agcccgtgtc gtcatacgac 600
cgctcagcga caaaagtcac attacttacc gaacggcact tgcgcgatcc ccaacccact 660
gcttgaatcc aaatcgtcag tctgcatgtg actaccaagt cacacctgca gccgatgaca 720
ctttttgccg gccacccaaa gcctagatgc cttggggcac ggcaaattgc ccgccaaaaa 780
caataccgtc gacgcaagtg ccaaggatcg acacagggcc cccggattat cttcaggcaa 840
atgcctacct gtcataaaga acgtgcaccc taggtgacgc gaataccctt tttgcgccag 900
gagtcgatga catgaggcca gaaatcgctg tacttgatat ccaaggtcag tatcgggttt 960
acacggagtt ctatcgcgcg gatgcggccg aaaacacgat catcctgatc aacggctcgc 1020
tggccaccac ggcctcgttc gcccagacgg tacgtaacct gcacccacag ttcaacgtgg 1080
ttctgttcga ccagccgtat tcaggcaagt ccaagccgca caaccgtcag gaacggctga 1140
tcagcaagga gaccgaggcg catatcctcc ttgagctgat cgagcacttc caggcagacc 1200
acgtgatgtc tttttcgtgg ggtggcgcaa gcacgctgct ggcgctggcg caccagccgc 1260
ggtacgtgaa gaaggcagtg gtgagttcgt tctcgccagt gatcaacgag ccgatgcgcg 1320
actatctgga ccgtggctgc cagtacctgg ccgcctgcga ccgttatcag gtcggcaacc 1380
tggtcaatga caccatcggc aagcacttgc cgtcgctgtt caaacgcttc aactaccgcc 1440
atgtgagcag cctggacagc cacgagtacg cacagatgca cttccacatc aaccaggtgc 1500
tggagcacga cctggaacgt gcgctgcaag gcgcgcgcaa tatcaacatc ccggtgctgt 1560
tcatcaacgg cgagcgcgac gagtacacca cagtcgagga tgcgcggcag ttcagcaagc 1620
atgtgggcag aagccagttc agcgtgatcc gcgatgcggg ccacttcctg gacatggaga 1680
acaagaccgc ctgcgagaac acccgcaatg tcatgctggg cttcctcaag ccaaccgtgc 1740
gtgaaccccg ccaacgttac caacccgtgc agcaggggca gcatgcattt gccatctgag 1800
cggctcggcg ccttgtagcc aatacccgca ggccacgggg cgccgacaag cttttttata 1860
acttgggctt ctaattcgct gaaggttctg gtaaaaagtc gagctcagat gcgggtatag 1920
tttagtggca aaacgaaagc ttcccaagct ttagttgagg gttcgattcc ctctacccgc 1980
tccacatcgc agtcccgcat ggcgttccag caacgtcatc gcagtcaaaa ggagccttgg 2040
ctcctttttt cgtttttcat cctgccttga cctggcccat ggccaattac ccaccgatcc 2100
gcttcaatgc gcatcgggcc tttgcgtgcg caagcgaaac ggcttgtggc ggatatgctc 2160
actggattcg tgaaactatt cgaaaggaca acgcatgttt ctctcccgct ggctaccggg 2220
ccttgccaac ctgctgcact accgccgtga atggttccac gccgatctgc aagcgggcct 2280
gtcggtagcc gcgatccaga ttcccattgc cattgcctat gcgcagatcg tcgggctgcc 2340
gccgcaatat ggcctgtacg cctgtgtgct accgatgatg gtctacgcgc tgatcggtag 2400
ctcgcgccag ctgatggtcg gccccgacgc cgccacctgc gcgatgatcg ccggtgccgt 2460
ggcaccgctg gccatgggtg acccgcagcg catcgtcgaa ctgtcggtga tcgtcaccgt 2520
gctggtcggc gtgatgctga ttgccgcggg cctggcgcgg gccgggttca tcgccagctt 2580
cttctcgcgg ccgatcctga tcggctacct caacggtatc ggcctgagcc tgatcgccgg 2640
gcagctgtcc aaggtggtgg gcttcaagat cgagggcgac ggtttcatcc tcagcctgat 2700
caacttcttc cagcgcctgg gggaaattca ctgggtcaca ttgatcatcg gcctggccgc 2760
cctgggcctg ctcatctggc tgccacggcg ctacccgcgc ctgcccgcag ccctcacggt 2820
agtggcgctg ttcatgctgc tggttggcct gttcggcctc gaccgcttcg gcgttgccgt 2880
ccttgggccg gtacctgcag gcatcccgca actggcctgg ccacacagca acctggcgaa 2940
atgaagagcc tgctgcggcg ncgccctggg tatcgccacc gtcagcttct gcagcgccat 3000
gcttaccgca cgcagctttg ccgcccggca tggctatgcg atcaacgcca accacgaatt 3060
c 3061
Claims (10)
1、一种提高酵母菌甲醇耐受性的方法,是将聚羟基脂肪酸酯合成途径相关基因导入酵母菌。
2、根据权利要求1所述的方法,其特征在于:所述聚羟基脂肪酸酯合成途径相关基因选自下述1),2)和3)中的任意一种:1)phbC,phbB和phbA;2)phaG和phaC;3)phaPCJ。
3、根据权利要求2所述的方法,其特征在于:所述聚羟基脂肪酸酯合成途径相关基因为phaPCJ。
4、根据权利要求2所述的方法,其特征在于:所述phbC具有Genbank登录号为J05003的自5′端第842位至2611位脱氧核苷酸组成的核苷酸序列;所述phbA具有Genbank登录号为J04987的自5′端第40位至1221位脱氧核苷酸组成的核苷酸序列;所述phbB具有Genbank登录号为J04987的自5′端第1296位至2036位脱氧核苷酸组成的核苷酸序列。
5、根据权利要求2所述的方法,其特征在于:所述phaG基因具有序列表中序列2的核苷酸序列;所述phaC基因具有序列1的自5′端第735位至2519位脱氧核苷酸组成的序列。
6、根据权利要求3所述的方法,其特征在于:所述phaPCJ具有序列表中序列1的核苷酸序列。
7、根据权利要求3所述的方法,其特征在于:所述phaPCJ通过pPIC3.5K-phaPCJ导入酵母菌;所述pPIC3.5K-phaPCJ是将phaPCJ插入pPIC3.5K的BamHI和EcoRI识别位点间得到的重组表达载体。
8、根据权利要求1至7中任一所述的方法,其特征在于:所述酵母菌为嗜甲醇酵母。
9、根据权利要求8所述的方法,其特征在于:所述甲醇酵母为巴斯德毕赤氏酵母(Pichia pastoris)或多形汉逊酵母(Hansenula polymopha)。
10、根据权利要求9所述的方法,其特征在于:所述巴斯德毕赤氏酵母(Pichiapastoris)为巴斯德毕赤氏酵母(Pichia pastoris)GS115。
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