南极假丝酵母脂肪酶B在毕赤酵母细胞表面高效展示体系的构建与定向进化研究
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摘要
随着基因工程的发展,微生物细胞表面展示系统已发展出噬菌体展示系统、细菌表面展示系统、酵母表面展示系统等,广泛应用在生物学研究的许多领域,例如研究蛋白质-蛋白质之间的相互识别与相互作用、由多肽构象或蛋白质突变库中获取特定功能蛋白、生产抗体、生产口服疫苗、开发全细胞催化剂、开发生物传感器和开发细胞吸附剂等等,由此成为最近几年生物技术领域的研究的热点之一
毕赤酵母(Pichia pastoris)表达系统是近年来发展迅速、应用广泛的一种真核表达系统,被认为是微生物表面展示系统极有应用前景的宿主菌之一。目前已有的毕赤酵母表面展示系统主要是利用来自酿酒酵母Flol蛋白的絮凝结构域为锚定蛋白将外源蛋白展示于菌体表面,融合蛋白非共价连接于毕赤酵母细胞壁上。为进一步的丰富毕赤酵母展示系统,并应用于南极假丝酵母脂肪酶B,本论文一方面通过构建不同锚定蛋白的毕赤酵母展示体系来筛选高活力的脂肪酶全细胞催化剂,另一方面利用定向进化技术来提高脂肪酶的热稳定性和活性,并应用于非水相中短链脂肪酸酯的合成,拟降低脂肪酶酯化合成的成本,为其大规模的工业应用奠定基础,具体的研究内容如下：
1、α凝集素和絮凝素展示体系的构建
本研究首先构建了α-凝集素与絮凝素C端GPI锚定的两个新型的酵母表面展示载体,结合本实验室已构建的絮凝素N端展示载体；并以南极假丝酵母脂肪酶B作为研究对象对构建的展示体系进行验证分析,采用脂肪酶的水解活力和FLAG标签蛋白标记等手段来分析目的蛋白在酵母细胞壁的锚定情况。将经平板与荧光验证的阳性重组子进行摇瓶发酵得到3种脂肪酶全细胞催化剂,分别为KNS-CALB、KFS-CALB和KFL-CALB,进一步的对其进行了酶学性质的分析。结果表明：KFS-CALB在水解活力达到270U/g dry cell和耐热性方面保温2h后的残留酶活力是最大酶活的50%以上,KNS-CALB的水解活力为200U/g dry cell,60℃保温的半衰期为1h; KFL-CALB的的水解活力为170U/g dry cell,60℃保温的半衰期为0.5h耐热性相对较差。
2、新的Pir型和GPI型毕赤酵母展示CALB体系的构建
本研究克隆了来自酿酒酵母的Pir蛋白的成熟肽基因Pirl和Pir4,以及GPI型的Sedl蛋白的全基因序列,连接入毕赤酵母分泌表达载体pPIC9K中,构建3种不同的毕赤酵母细胞表面展示通用载体pKPir1、pKPir4和pKSedl,利用α-factor信号肽将蛋白引导分泌至细胞外。再以带有FLAG标签的南极假丝酵母脂肪酶B蛋白的C末端同3种锚定蛋白N端融合,并利用免疫荧光显微镜检测,证明新构建的毕赤酵母展示通用载体是成功的。蛋白电泳和Western blot初步证明了融合蛋白Sedl-CALB可被温和碱从细胞壁中抽提,进一步的证明成功的构建了毕赤酵母展示载体。
3、脂肪酶CALB的定向进化
本研究利用易错PCR的方法对CALB进行随机突变,利用三丁酸甘油酯乳化平板和96孔平板高通量酶活筛选的方法对约2000个酵母重组子进行筛选并得到了一株活力提高的菌株；经测序研究发现有4个氨基酸发生突变(Thr57Ala、Ala87Thr、Arg168Lys和Gly226Arg),其水解活力提高了1倍。利用生物信息学分析发现其突变位点的空间结构位于活性中心附近,可能有利于其同相关底物的结合,进而提高了其水解活性。
同时,利用定靶位点区域饱和突变的方法,并基于毕赤酵母展示技术对脂肪酶CALB进行了定向进化与改造研究。在60℃下三丁酸甘油酯乳化平板对大约5000个酵母重组突变子进行了筛选并得到2株产脂肪酶热稳定性较高的菌株,测序发现其中一个突变菌株mCALB7有7个氨基酸(Pro218Asn, Leu219Lys, Phe220Thr, Val221Ser, Leu278Gly, Ala279Met和Ala281Ile)发生突变,其在60℃保温的半衰期提高了1倍。mCALB168中也有3个氨基酸(Thr57Ala、Ala87Thr和Arg168Lys)突变产生,其在60℃保温的半衰期提高了约2倍。进一步的利用其模拟的空间结构分析发现2个突变子的分别有3和4个氢键的增加,酶性质分析其原因可能是突变子热稳定性提高的一个重要原因。
4、构建的毕赤酵母全细胞催化剂在非水相中合成活力的比较
研究分析不同类型的锚定蛋白展示的CALB全细胞催化剂在非水相中催化合成短链脂肪酸酯,确定α凝集素展示的KNS-CALB菌株在合成活力方面的优势,并初步获得其在乙酸乙酯和丙酸乙酯合成方面优于商品化的同类型的脂肪酶。
此外,还研究分析在水相中耐热性优良的突变子在有机相中的耐热稳定性,结果发现突变子的耐热性并没有显著的提高,说明定向改造的脂肪酶耐热性在水相中与有机相中并不体现一定的相关性。因此,有必要建立一种在有机相中高通量筛选耐热性突变子的方法体系,为脂肪酶在有机合成中的应用奠定基础。
With the application of recombinant DNA technology, microbial cell-surface display systems have been used in various microorganisms, such as in bacteria, in phage and especially in yeast. Microbial cell-surface display system has been widely used in biotechnologieal research and industrial applications, including:identifying Protein-Protein inieraetions, displaying polypeptide libraries as selection devices, mapping functional protein epitopes, immobilizing proteins and enzymes as whole-cell biocataysts, producing antibodies and live vaccines, making bioadsorbents for the removal of harmful chemicals and heavy metals, therefore, which has becoming a research hotspot in the field of biotechnology in recent years.
The lipase B from Candida antarctica is one of a very important lipase, which have some good propertirs of biocatalysis and postion selection. In this paper, we cloned the gene calb and expressed the functional protein in Pichia pastoris systems including the P. pastoris cell-surface display system. The methylotrophic yeast is a sigal-cell eukaryote microorganism and has been widely used to express various recombinant proteins for both basic laboratory research and industrial manufacture. It became a host for cell-surface display system. For the rich yeast display system, using P. pastoris cell-surface to display Candida antarctica lipase B. While this papper by constructing different yeast cell-surface diplay system by various anchor proteins, then, using these systems to screen of some higher activity whole-cell catalysts. On the other hand, using directed evolution technology to improve the thermal stability and activity, furthermore, using the whole-cell biocatalysts to synthsize the short chain fatty acid esters in non-aqueous phase. And then to lower the production cost of enzyme preparation for its large-scale lay the foundation for industrial applications. Specific study as follows:
PartⅠConstructing and comparing the yeast display systems by usingα-agglutinin and flocculation functional domain(flolp)
This part constructed two new type yeast display systems, including theα-agglutinin system and flolp system in Phichia pastoris cell surface. Combined with Our laboratory had constructed the FS system. Then using the lipase B from Candida antarctica as a model protein to check the 3 dispalying vectors in yeast. Lipase hydrolysis activity and FLAG tags and other means to detect the protein in the yeast cell wall anchoring conditions. FLAG tag sequence and the N-terminal protein CALB fusion, when the recombinant yeast cells can be detected when the green fluorescent CALB has been successfully verified the anchoring on the surface. Will be verified by the positive plate and the fluorescent recombinant shake flask fermentation by three kinds of whole-cell catalyst, was carried out further analysis of enzymatic properties.
Part II Constructing and analysizing the Pir-type and GPI-type displaying CALB on Pichia cell surface
This part has cloned the gene Pir1, Pir4 and Sedl from Saccharomyces cerevisiae and ligated into Pichia pastoris expression vectors pPIC9K to construct 3 different yeast cell surface display systems, namely, pKPirl, pKPir4 and pKSed1 which all using-factor secretion signal peptide. Then tagged with a FLAG Candida antarctica lipase B protein with three kinds of C-terminal N-anchored fusion protein and used immunofluorescence microscopy, that the newly constructed yeast display common carrier is successful. Protein electrophoresis and Western blot demonstrated an initial Sedl-CALB fusion protein can be mild-alkali extraction from the cell wall, further evidence of the successful construction of a yeast display system.
Part III Improving the thermostability and activity of lipase CALB by use of directed evolution technology
Using the method of error-prone PCR to random mutation CALB,then, using 96 plate high-throughput screening method of activity of about 2,000 recombinant yeasts. The outcome of screening is obtained a strain which has higher hydrolytic activity than the wild type lipase.The sequence of the mutant was found 4 amino acids mutant (Thr57Ala、Ala87Thr Arg168Lys and Gly226Arg), using bioinformatics analysis shows that the spatial structure of point mutations around the active site, which may be beneficial to its combination with the relevant substrate, thereby improving its hydrolytic activity.
Then, using target sites of regional saturation mutagenesis in the yeast cell-surface system, the lipase CALB was redesigned by using directed evolution technology. At 60℃,using tributyrin emulsion plate reorganization of about 5,000 yeast mutants were screened and received two higher thermal stability of lipase producing strains. Sequencing indicated that KFS-mCALB7 mutation of 7 amino acids (Pro218Asn,Leu219Lys,Phe220Thr, Val221Ser,Leu278Gly,Ala279Met and Ala281Ile),KFS-mCALB168 also has three amino acid (Thr57Ala, Ala87Thr and Arg168Lys) mutations arise. Further use of its simulation of the spatial structure analysis revealed that two strains were 3 and 4 hydrogen bonds increases, which may be mutants of thermal stability is an important reason.
Part IV Comparing the yeast whole-cell catalysts in non-aqueous synthetic activity
This section, we analyzed the different types of anchoring proteins CALB-displayed whole-cell catalysts in non-aqueous phase, which can catalyze the synthesis of short chain fatty acid ester. The data shows the KNS-CALB strain that has advantage in the synthesis and initial access to ethyl acetate. Some synthetic activty of KNS-CALB is superior to the commercialization of the same type of lipase. In addtion, in the aqueous phase of the excellent heat-resistant mutants in the organic phase of thermal stability of the mutants were found that the thermostability has no significant increase. The outcome indicated that directional transformation of thermal lipase in the aqueous phase does not reflect the organic phase with a certain correlation. Therefore, it is necessary to establish a high-throughput screening in the organic phase, thermal mutants of the methodology for lipase in organic synthesis basis.
毕赤酵母(Pichia pastoris)表达系统是近年来发展迅速、应用广泛的一种真核表达系统,被认为是微生物表面展示系统极有应用前景的宿主菌之一。目前已有的毕赤酵母表面展示系统主要是利用来自酿酒酵母Flol蛋白的絮凝结构域为锚定蛋白将外源蛋白展示于菌体表面,融合蛋白非共价连接于毕赤酵母细胞壁上。为进一步的丰富毕赤酵母展示系统,并应用于南极假丝酵母脂肪酶B,本论文一方面通过构建不同锚定蛋白的毕赤酵母展示体系来筛选高活力的脂肪酶全细胞催化剂,另一方面利用定向进化技术来提高脂肪酶的热稳定性和活性,并应用于非水相中短链脂肪酸酯的合成,拟降低脂肪酶酯化合成的成本,为其大规模的工业应用奠定基础,具体的研究内容如下：
1、α凝集素和絮凝素展示体系的构建
本研究首先构建了α-凝集素与絮凝素C端GPI锚定的两个新型的酵母表面展示载体,结合本实验室已构建的絮凝素N端展示载体；并以南极假丝酵母脂肪酶B作为研究对象对构建的展示体系进行验证分析,采用脂肪酶的水解活力和FLAG标签蛋白标记等手段来分析目的蛋白在酵母细胞壁的锚定情况。将经平板与荧光验证的阳性重组子进行摇瓶发酵得到3种脂肪酶全细胞催化剂,分别为KNS-CALB、KFS-CALB和KFL-CALB,进一步的对其进行了酶学性质的分析。结果表明：KFS-CALB在水解活力达到270U/g dry cell和耐热性方面保温2h后的残留酶活力是最大酶活的50%以上,KNS-CALB的水解活力为200U/g dry cell,60℃保温的半衰期为1h; KFL-CALB的的水解活力为170U/g dry cell,60℃保温的半衰期为0.5h耐热性相对较差。
2、新的Pir型和GPI型毕赤酵母展示CALB体系的构建
本研究克隆了来自酿酒酵母的Pir蛋白的成熟肽基因Pirl和Pir4,以及GPI型的Sedl蛋白的全基因序列,连接入毕赤酵母分泌表达载体pPIC9K中,构建3种不同的毕赤酵母细胞表面展示通用载体pKPir1、pKPir4和pKSedl,利用α-factor信号肽将蛋白引导分泌至细胞外。再以带有FLAG标签的南极假丝酵母脂肪酶B蛋白的C末端同3种锚定蛋白N端融合,并利用免疫荧光显微镜检测,证明新构建的毕赤酵母展示通用载体是成功的。蛋白电泳和Western blot初步证明了融合蛋白Sedl-CALB可被温和碱从细胞壁中抽提,进一步的证明成功的构建了毕赤酵母展示载体。
3、脂肪酶CALB的定向进化
本研究利用易错PCR的方法对CALB进行随机突变,利用三丁酸甘油酯乳化平板和96孔平板高通量酶活筛选的方法对约2000个酵母重组子进行筛选并得到了一株活力提高的菌株；经测序研究发现有4个氨基酸发生突变(Thr57Ala、Ala87Thr、Arg168Lys和Gly226Arg),其水解活力提高了1倍。利用生物信息学分析发现其突变位点的空间结构位于活性中心附近,可能有利于其同相关底物的结合,进而提高了其水解活性。
同时,利用定靶位点区域饱和突变的方法,并基于毕赤酵母展示技术对脂肪酶CALB进行了定向进化与改造研究。在60℃下三丁酸甘油酯乳化平板对大约5000个酵母重组突变子进行了筛选并得到2株产脂肪酶热稳定性较高的菌株,测序发现其中一个突变菌株mCALB7有7个氨基酸(Pro218Asn, Leu219Lys, Phe220Thr, Val221Ser, Leu278Gly, Ala279Met和Ala281Ile)发生突变,其在60℃保温的半衰期提高了1倍。mCALB168中也有3个氨基酸(Thr57Ala、Ala87Thr和Arg168Lys)突变产生,其在60℃保温的半衰期提高了约2倍。进一步的利用其模拟的空间结构分析发现2个突变子的分别有3和4个氢键的增加,酶性质分析其原因可能是突变子热稳定性提高的一个重要原因。
4、构建的毕赤酵母全细胞催化剂在非水相中合成活力的比较
研究分析不同类型的锚定蛋白展示的CALB全细胞催化剂在非水相中催化合成短链脂肪酸酯,确定α凝集素展示的KNS-CALB菌株在合成活力方面的优势,并初步获得其在乙酸乙酯和丙酸乙酯合成方面优于商品化的同类型的脂肪酶。
此外,还研究分析在水相中耐热性优良的突变子在有机相中的耐热稳定性,结果发现突变子的耐热性并没有显著的提高,说明定向改造的脂肪酶耐热性在水相中与有机相中并不体现一定的相关性。因此,有必要建立一种在有机相中高通量筛选耐热性突变子的方法体系,为脂肪酶在有机合成中的应用奠定基础。
With the application of recombinant DNA technology, microbial cell-surface display systems have been used in various microorganisms, such as in bacteria, in phage and especially in yeast. Microbial cell-surface display system has been widely used in biotechnologieal research and industrial applications, including:identifying Protein-Protein inieraetions, displaying polypeptide libraries as selection devices, mapping functional protein epitopes, immobilizing proteins and enzymes as whole-cell biocataysts, producing antibodies and live vaccines, making bioadsorbents for the removal of harmful chemicals and heavy metals, therefore, which has becoming a research hotspot in the field of biotechnology in recent years.
The lipase B from Candida antarctica is one of a very important lipase, which have some good propertirs of biocatalysis and postion selection. In this paper, we cloned the gene calb and expressed the functional protein in Pichia pastoris systems including the P. pastoris cell-surface display system. The methylotrophic yeast is a sigal-cell eukaryote microorganism and has been widely used to express various recombinant proteins for both basic laboratory research and industrial manufacture. It became a host for cell-surface display system. For the rich yeast display system, using P. pastoris cell-surface to display Candida antarctica lipase B. While this papper by constructing different yeast cell-surface diplay system by various anchor proteins, then, using these systems to screen of some higher activity whole-cell catalysts. On the other hand, using directed evolution technology to improve the thermal stability and activity, furthermore, using the whole-cell biocatalysts to synthsize the short chain fatty acid esters in non-aqueous phase. And then to lower the production cost of enzyme preparation for its large-scale lay the foundation for industrial applications. Specific study as follows:
PartⅠConstructing and comparing the yeast display systems by usingα-agglutinin and flocculation functional domain(flolp)
This part constructed two new type yeast display systems, including theα-agglutinin system and flolp system in Phichia pastoris cell surface. Combined with Our laboratory had constructed the FS system. Then using the lipase B from Candida antarctica as a model protein to check the 3 dispalying vectors in yeast. Lipase hydrolysis activity and FLAG tags and other means to detect the protein in the yeast cell wall anchoring conditions. FLAG tag sequence and the N-terminal protein CALB fusion, when the recombinant yeast cells can be detected when the green fluorescent CALB has been successfully verified the anchoring on the surface. Will be verified by the positive plate and the fluorescent recombinant shake flask fermentation by three kinds of whole-cell catalyst, was carried out further analysis of enzymatic properties.
Part II Constructing and analysizing the Pir-type and GPI-type displaying CALB on Pichia cell surface
This part has cloned the gene Pir1, Pir4 and Sedl from Saccharomyces cerevisiae and ligated into Pichia pastoris expression vectors pPIC9K to construct 3 different yeast cell surface display systems, namely, pKPirl, pKPir4 and pKSed1 which all using-factor secretion signal peptide. Then tagged with a FLAG Candida antarctica lipase B protein with three kinds of C-terminal N-anchored fusion protein and used immunofluorescence microscopy, that the newly constructed yeast display common carrier is successful. Protein electrophoresis and Western blot demonstrated an initial Sedl-CALB fusion protein can be mild-alkali extraction from the cell wall, further evidence of the successful construction of a yeast display system.
Part III Improving the thermostability and activity of lipase CALB by use of directed evolution technology
Using the method of error-prone PCR to random mutation CALB,then, using 96 plate high-throughput screening method of activity of about 2,000 recombinant yeasts. The outcome of screening is obtained a strain which has higher hydrolytic activity than the wild type lipase.The sequence of the mutant was found 4 amino acids mutant (Thr57Ala、Ala87Thr Arg168Lys and Gly226Arg), using bioinformatics analysis shows that the spatial structure of point mutations around the active site, which may be beneficial to its combination with the relevant substrate, thereby improving its hydrolytic activity.
Then, using target sites of regional saturation mutagenesis in the yeast cell-surface system, the lipase CALB was redesigned by using directed evolution technology. At 60℃,using tributyrin emulsion plate reorganization of about 5,000 yeast mutants were screened and received two higher thermal stability of lipase producing strains. Sequencing indicated that KFS-mCALB7 mutation of 7 amino acids (Pro218Asn,Leu219Lys,Phe220Thr, Val221Ser,Leu278Gly,Ala279Met and Ala281Ile),KFS-mCALB168 also has three amino acid (Thr57Ala, Ala87Thr and Arg168Lys) mutations arise. Further use of its simulation of the spatial structure analysis revealed that two strains were 3 and 4 hydrogen bonds increases, which may be mutants of thermal stability is an important reason.
Part IV Comparing the yeast whole-cell catalysts in non-aqueous synthetic activity
This section, we analyzed the different types of anchoring proteins CALB-displayed whole-cell catalysts in non-aqueous phase, which can catalyze the synthesis of short chain fatty acid ester. The data shows the KNS-CALB strain that has advantage in the synthesis and initial access to ethyl acetate. Some synthetic activty of KNS-CALB is superior to the commercialization of the same type of lipase. In addtion, in the aqueous phase of the excellent heat-resistant mutants in the organic phase of thermal stability of the mutants were found that the thermostability has no significant increase. The outcome indicated that directional transformation of thermal lipase in the aqueous phase does not reflect the organic phase with a certain correlation. Therefore, it is necessary to establish a high-throughput screening in the organic phase, thermal mutants of the methodology for lipase in organic synthesis basis.
引文
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