海洋酵母新型菊粉酶基因的克隆及表达
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摘要
菊粉(inulin)是D-呋喃果糖分子由β-2,1-糖苷键连接组成的链状多糖，其末端有一个葡萄糖残基以α-2,1-糖苷键与之相连。菊粉广泛存在于菊芋(Jerusalemartichoke)、雪莲果(Yacon)、菊苣(Chicory tubes)和牛蒡(Arctium)等多种植物中。菊粉酶(inulinase)是一种呋喃果糖基水解酶，它靶向作用于菊粉中的β-2,1糖苷键并将其水解为果糖和葡萄糖。菊粉酶可以分为外切菊粉酶和内切菊粉酶。外切菊粉酶可以从菊粉分子的非还原末端催化水解下-D-呋喃果糖残基,其底物可为菊粉、蔗糖、果聚糖；内切菊粉酶可以水解菊粉内部的β-2,1糖苷键使其变成菊糖三糖、菊糖四糖、菊糖五糖为主的低聚果糖。在本研究中为了了解海洋酵母菌菊粉酶基因及其功能和应用，分别克隆了海洋隐球酵母Cryptococcus aureus HYA菌株的菊粉酶基因和海洋酵母Candida membranifaciens subsp.flavinogenie W14-3菌株的菊粉酶基因，同时将从C.aureus HYA菌株克隆得到的菊粉酶基因HYAINU1分别在Pichia pastoris和Saccharomyces cerevisiae中表达。
C. aureus HYA菌株具有菊粉酶活性，从C.aureus HYA菌株中克隆的菊粉酶基因HYAINU1基因登录号为JX073660，该菊粉酶基因的ORF框共1653bp，基因中不含内含子，翻译550个氨基酸，氨基酸序列中的前23个为信号肽部分，蛋白预测分子量大小为59.5kDa。在该菊粉酶氨基酸序列中含有酵母外切菊粉酶的保守序列WMNDPNGL、RDP、ECP、FS和Q，同时含有2个N-糖基化位点。在启动子中含有1个TATA框、2个5′-SYGGRG-3′序列，若干5′-CGG-3′序列。5′-SYGGRG-3′序列是Mig1中C2H2锌指蛋白的锚定位点，说明该基因表达受到葡萄糖阻遏。而5′-CGG-3′序列是转录激活蛋白Zn(II)2Cys6的锚定位点，说明该基因表达受到底物诱导。
将菊粉酶基因HYAINU1去掉信号肽部分，连接到pPICZaA表达载体中，在巴斯德毕赤酵母Pichia pastoris X-33中表达。表达的融合蛋白通过不连续SDS-PAGE凝胶电泳和Western Blot分析验证，得到蛋白分子量大小为60.0kDa左右的目的条带。含有HYAINU1基因的P. pastoris X-33转化子在培养过程中，培养液中测得的最高菊粉酶酶活力单位为16.3±0.24U/mL。该重组菊粉酶最适反应温度为50℃，最适pH值为5.0。纯化的重组菊粉酶水解菊粉后发现大量单糖，说明C. aureus HYA菌株分泌的菊粉酶为外切菊粉酶。
将菊粉酶基因HYAINU1连接到pMIRSC11表达载体中在高产酒精酵母W0菌株的尿嘧啶缺陷型菌株W12d中表达，筛选后得到转化子MHYAINU1-69。该转化子培养到72h时酶活力为19.2±0.3U/mL。经150mL体系的酒精发酵研究确认最佳接种量和最适菊粉浓度分别为10.0%(v/v)和25.0%(w/v)，当发酵到120h时，酒精浓度可达到11.9%(v/v，20°C)。当扩大到3-L发酵体系，发酵时间为120h时发酵液酒精浓度为13.1±0.6%(v/v，20°C)。
分离自中国东海表层海水样品的海洋酵母C.membranifacienssubsp.flavinogenie W14-3菌株可分泌核黄素，但也具有菊粉酶活性，W14-3菌株菊粉酶基因的基因登录号为KC576811。该菊粉酶基因的ORF框共1536bp，翻译512个氨基酸，通过软件EditSeq预测的蛋白分子量大小为57.8kDa。W14-3菌株的菊粉酶基因5′端上游序列的非编码区中除含启动子外还存在2个5′-SYGGRG-3′序列及若干5′-CGG-3′序列，说明W14-3菌株菊粉酶基因的表达同样受到葡萄糖的阻遏和底物的诱导。
Inulin consists of linear chains of β-2,1-linked D-fructofuranose moleculesterminated by a glucose residue through a sucrose-type linkage at the reducing end.Inulin mainly exsits in jerusalem artichoke, yacon, chicory tubes, and arctium.Inulinases are fructofuranosyl hydrolases that target on the β-2,1linkage of inulin andhydrolyze it into fructose and glucose. They can be divided into exo-inulinase andendo-inulinase. The exo-inulinase catalyzes the removal of the terminal fructoseresidues from the non-reducing end of the inulin molecule while the endo-inulinasehydrolyzes the internal linkages in inulin to yield inulotriose, inulotetraose, andinulopentaose. In this study, the full-length of the inulinase gene was cloned andcharacterized from C. aureus HYA and C. membranifaciens subsp.flavinogenieW14-3, respectively. At the same time, the cloned HYAINU1gene from C. aureusHYA was expressed in P. pastoris and S. cerevisiae, respectively.
The marine yeast C. aureus HYA had inulinases activity.The HYAINU1genefrom C. aureus HYA was cloned and characterized (Accession number: JX073660).The gene had an open reading frame (ORF) of1653bp long encoding an inulinase.The coding region of the gene was not interrupted by any intron. It encoded551amino acid residues of a protein with a putative signal peptide of23amino acids andthe calculated molecular mass of59.5kDa. The protein sequence deduced from theinulinase structural gene contained the inulinase consensus sequences(WMNDPNGL),(RDP), ECP, FS and Q.It also had two conserved putativeN-glycosylation sites. After analysis of the promoter of the HYAINU1gene, it wasfound that it had one TATA box and several sequences such as5′-SYGGRG-3′and5′-CGG-3′. It has been reported that Mig1, the main effector in glucose repression, isa C2H2zinc finger protein that is able to bind the promoters of a variety of genesrepressed by glucose and the binding site in the promoter should have the sequence5′-SYGGRG-3′. It also has been documented that the transcriptional activator, Zn(II)2Cys6protein is able to bind the promoters of a variety of genes induced by thesubstrates and the binding site in the promoter has the sequence5′-CGG-3′. Thismeans that expression of the HYAINU1gene cloned from C. aureus HYA can berepressed by glucose and fructose and induced by the substrates, such inulin andsucrose.
The inulinase gene without the signal sequence was subcloned into pPICZaAexpression vector and expressed in P. pastoris X-33. After the expressed fusionprotein was analyzed by SDS-PAGE and Western Blot, a specific band withmolecular mass of about60kDa was found. Enzyme activity assay verified therecombinant protein as an inulinase. A maximum inulinase activity of16.3±0.24U/mL was obtained from the culture supernatant of P. pastoris X-33harboring the inulinase gene. The optimal temperature and pH for action of theenzyme were50°C and5.0, respectively. A large amount of monosaccharides weredetected after the hydrolysis of inulin with the purified recombinant inulinase. Theresults suggested that the recombinant inulinase produced by P. pastoris X-33carrying the HYAINU1gene had exo-inulinase activity.
The HYAINU1gene with the signal sequence was subcloned into pMIRSC11expression vector and expressed in Saccharomyces sp. W12d (a diploid uracilauxotroph strain of the high ethanol producing yeast Saccharomyces sp. W0). Thetransformant MHYAINU1-69obtained produced19.2±0.3U/mL of extracellularinulinase within72h of cultivation. During the150-mL fermentation,11.9%(v/v,20°C)ethanol was produced within120h and the inoculum size and inulin concentrationwere10.0%(v/v) and25.0%(w/v), respectively. During the3-L fermentation,13.1±0.6%(v/v,20°C) of ethanol was produced from inulin.
The marine yeast strain W14-3was isolated from seawater of China Eastern Seaand identified to be Candida membranifaciens subsp. flavinogenie W14-3. The strainW14-3was found to have inulinases activity and produce riboflavin.The inulinasegene from the strain W14-3was cloned by degenerate PCR and genomic walking.The cloned gene had an open reading frame (ORF) of1536bp long encoding aninulinase (Accession number: KC576811). It encoded512amino acid residues with the calculated molecular mass of57.8kDa. After analysis of the5′-flanking region ofthe inulinase gene, it was found that in its promoter there were two sequences of5′-SYGGRG-3′and several sequences of5′-CGG-3′, which had the same function asconsensus sequences of the promoter of the HYAINU1gene from C. aureus HYA,suggesting that expression of the inulinase gene cloned from C. membranifacienssubsp. flavinogenie W14-3can be repressed by glucose and fructose and induced bythe substrates, such inulin and sucrose.
C. aureus HYA菌株具有菊粉酶活性，从C.aureus HYA菌株中克隆的菊粉酶基因HYAINU1基因登录号为JX073660，该菊粉酶基因的ORF框共1653bp，基因中不含内含子，翻译550个氨基酸，氨基酸序列中的前23个为信号肽部分，蛋白预测分子量大小为59.5kDa。在该菊粉酶氨基酸序列中含有酵母外切菊粉酶的保守序列WMNDPNGL、RDP、ECP、FS和Q，同时含有2个N-糖基化位点。在启动子中含有1个TATA框、2个5′-SYGGRG-3′序列，若干5′-CGG-3′序列。5′-SYGGRG-3′序列是Mig1中C2H2锌指蛋白的锚定位点，说明该基因表达受到葡萄糖阻遏。而5′-CGG-3′序列是转录激活蛋白Zn(II)2Cys6的锚定位点，说明该基因表达受到底物诱导。
将菊粉酶基因HYAINU1去掉信号肽部分，连接到pPICZaA表达载体中，在巴斯德毕赤酵母Pichia pastoris X-33中表达。表达的融合蛋白通过不连续SDS-PAGE凝胶电泳和Western Blot分析验证，得到蛋白分子量大小为60.0kDa左右的目的条带。含有HYAINU1基因的P. pastoris X-33转化子在培养过程中，培养液中测得的最高菊粉酶酶活力单位为16.3±0.24U/mL。该重组菊粉酶最适反应温度为50℃，最适pH值为5.0。纯化的重组菊粉酶水解菊粉后发现大量单糖，说明C. aureus HYA菌株分泌的菊粉酶为外切菊粉酶。
将菊粉酶基因HYAINU1连接到pMIRSC11表达载体中在高产酒精酵母W0菌株的尿嘧啶缺陷型菌株W12d中表达，筛选后得到转化子MHYAINU1-69。该转化子培养到72h时酶活力为19.2±0.3U/mL。经150mL体系的酒精发酵研究确认最佳接种量和最适菊粉浓度分别为10.0%(v/v)和25.0%(w/v)，当发酵到120h时，酒精浓度可达到11.9%(v/v，20°C)。当扩大到3-L发酵体系，发酵时间为120h时发酵液酒精浓度为13.1±0.6%(v/v，20°C)。
分离自中国东海表层海水样品的海洋酵母C.membranifacienssubsp.flavinogenie W14-3菌株可分泌核黄素，但也具有菊粉酶活性，W14-3菌株菊粉酶基因的基因登录号为KC576811。该菊粉酶基因的ORF框共1536bp，翻译512个氨基酸，通过软件EditSeq预测的蛋白分子量大小为57.8kDa。W14-3菌株的菊粉酶基因5′端上游序列的非编码区中除含启动子外还存在2个5′-SYGGRG-3′序列及若干5′-CGG-3′序列，说明W14-3菌株菊粉酶基因的表达同样受到葡萄糖的阻遏和底物的诱导。
Inulin consists of linear chains of β-2,1-linked D-fructofuranose moleculesterminated by a glucose residue through a sucrose-type linkage at the reducing end.Inulin mainly exsits in jerusalem artichoke, yacon, chicory tubes, and arctium.Inulinases are fructofuranosyl hydrolases that target on the β-2,1linkage of inulin andhydrolyze it into fructose and glucose. They can be divided into exo-inulinase andendo-inulinase. The exo-inulinase catalyzes the removal of the terminal fructoseresidues from the non-reducing end of the inulin molecule while the endo-inulinasehydrolyzes the internal linkages in inulin to yield inulotriose, inulotetraose, andinulopentaose. In this study, the full-length of the inulinase gene was cloned andcharacterized from C. aureus HYA and C. membranifaciens subsp.flavinogenieW14-3, respectively. At the same time, the cloned HYAINU1gene from C. aureusHYA was expressed in P. pastoris and S. cerevisiae, respectively.
The marine yeast C. aureus HYA had inulinases activity.The HYAINU1genefrom C. aureus HYA was cloned and characterized (Accession number: JX073660).The gene had an open reading frame (ORF) of1653bp long encoding an inulinase.The coding region of the gene was not interrupted by any intron. It encoded551amino acid residues of a protein with a putative signal peptide of23amino acids andthe calculated molecular mass of59.5kDa. The protein sequence deduced from theinulinase structural gene contained the inulinase consensus sequences(WMNDPNGL),(RDP), ECP, FS and Q.It also had two conserved putativeN-glycosylation sites. After analysis of the promoter of the HYAINU1gene, it wasfound that it had one TATA box and several sequences such as5′-SYGGRG-3′and5′-CGG-3′. It has been reported that Mig1, the main effector in glucose repression, isa C2H2zinc finger protein that is able to bind the promoters of a variety of genesrepressed by glucose and the binding site in the promoter should have the sequence5′-SYGGRG-3′. It also has been documented that the transcriptional activator, Zn(II)2Cys6protein is able to bind the promoters of a variety of genes induced by thesubstrates and the binding site in the promoter has the sequence5′-CGG-3′. Thismeans that expression of the HYAINU1gene cloned from C. aureus HYA can berepressed by glucose and fructose and induced by the substrates, such inulin andsucrose.
The inulinase gene without the signal sequence was subcloned into pPICZaAexpression vector and expressed in P. pastoris X-33. After the expressed fusionprotein was analyzed by SDS-PAGE and Western Blot, a specific band withmolecular mass of about60kDa was found. Enzyme activity assay verified therecombinant protein as an inulinase. A maximum inulinase activity of16.3±0.24U/mL was obtained from the culture supernatant of P. pastoris X-33harboring the inulinase gene. The optimal temperature and pH for action of theenzyme were50°C and5.0, respectively. A large amount of monosaccharides weredetected after the hydrolysis of inulin with the purified recombinant inulinase. Theresults suggested that the recombinant inulinase produced by P. pastoris X-33carrying the HYAINU1gene had exo-inulinase activity.
The HYAINU1gene with the signal sequence was subcloned into pMIRSC11expression vector and expressed in Saccharomyces sp. W12d (a diploid uracilauxotroph strain of the high ethanol producing yeast Saccharomyces sp. W0). Thetransformant MHYAINU1-69obtained produced19.2±0.3U/mL of extracellularinulinase within72h of cultivation. During the150-mL fermentation,11.9%(v/v,20°C)ethanol was produced within120h and the inoculum size and inulin concentrationwere10.0%(v/v) and25.0%(w/v), respectively. During the3-L fermentation,13.1±0.6%(v/v,20°C) of ethanol was produced from inulin.
The marine yeast strain W14-3was isolated from seawater of China Eastern Seaand identified to be Candida membranifaciens subsp. flavinogenie W14-3. The strainW14-3was found to have inulinases activity and produce riboflavin.The inulinasegene from the strain W14-3was cloned by degenerate PCR and genomic walking.The cloned gene had an open reading frame (ORF) of1536bp long encoding aninulinase (Accession number: KC576811). It encoded512amino acid residues with the calculated molecular mass of57.8kDa. After analysis of the5′-flanking region ofthe inulinase gene, it was found that in its promoter there were two sequences of5′-SYGGRG-3′and several sequences of5′-CGG-3′, which had the same function asconsensus sequences of the promoter of the HYAINU1gene from C. aureus HYA,suggesting that expression of the inulinase gene cloned from C. membranifacienssubsp. flavinogenie W14-3can be repressed by glucose and fructose and induced bythe substrates, such inulin and sucrose.
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