18S rDNA介导的桔青霉核酸酶P1表达载体的构建及表达分析
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摘要
本研究采用黑曲霉Bdel4菌株为宿主菌,敲除gla A,aam A,An05g02100和An12g06930四个主要的胞外分泌蛋白,为核酸酶P1的表达和分泌提供通路。通过PCR扩增得到黑曲霉Bdel4的r DNA序列,构建了以r DNA序列为整合位点的重组质粒p Rp T-nuc P1-18S和p Rp T-2A-nuc P1-18S,将构建的重组质粒p Rp T-nuc P1-18S和p Rp T-2A-nuc P1-18S转入黑曲霉Bdel4。通过半荧光定量PCR进行转化子初筛,之后测定核酸酶P1的酶活复筛,选出一株高活性菌株用于工业生产。采用SYBR Green实时荧光定量PCR法对含外源目的基因nucP1的总基因组样本重复检测,取平均值作为目的基因拷贝数,探究其与酶活表达水平之间的联系。结果显示含9个nuc P1基因拷贝数的菌株的酶活水平达到88.5 U/mL,较核酸酶P1单拷贝菌株增加了3.86倍,较之前研究中异源表达核酸酶产量提高1.20倍,再增加nuc P1的拷贝数时,酶活水平随着拷贝数的增加而降低。
In this study, the Aspergillus niger Bdel4 strain was used as the host strain, and the four major extracellular secretion proteins,gla A, aamA, An05 g02100 and An12 g06930, were knocked out to provide a pathway for the expression and secretion of nuclease P1. The rDNA sequence of A. niger Bdel4 was amplified by PCR, and the recombinant plasmids, pRpT-nucP1-18 S and pRpT-2 A-nucP1-18 S were constructed through using the rDNA sequence as integration sites. The constructed recombinant plasmids pRpT-nucP1-18 S and pRpT-2 A-nucP1-18 S were then transferred into A. niger Bdel4. Prescreening of the transformants was carried out by semi-quantitative fluorescent PCR, and the activity of nuclease P1 was then measured to screen a strain with high activity for industrial production. The total genomic samples containing the exogenous target gene of nuc P1 were repeatedly detected by SYBR Green real-time fluorescent quantitative PCR, and the average value was taken as the copy number of the target gene nuc P1 to explore the relationship between the copy number and the expression level of the enzyme.The result showed that the enzymatic activity of the strain containing 9 nuc P1 gene copies reached 88.52 U/ml, which was 3.86 times higher than that of the nuclease P1 for the single copy strain, and 1.2-fold as high as that of the heterologous nuclease P1 reported in the previous studies. When the copy number of nuc P1 was increased to over 9, the enzyme activity decreased.
In this study, the Aspergillus niger Bdel4 strain was used as the host strain, and the four major extracellular secretion proteins,gla A, aamA, An05 g02100 and An12 g06930, were knocked out to provide a pathway for the expression and secretion of nuclease P1. The rDNA sequence of A. niger Bdel4 was amplified by PCR, and the recombinant plasmids, pRpT-nucP1-18 S and pRpT-2 A-nucP1-18 S were constructed through using the rDNA sequence as integration sites. The constructed recombinant plasmids pRpT-nucP1-18 S and pRpT-2 A-nucP1-18 S were then transferred into A. niger Bdel4. Prescreening of the transformants was carried out by semi-quantitative fluorescent PCR, and the activity of nuclease P1 was then measured to screen a strain with high activity for industrial production. The total genomic samples containing the exogenous target gene of nuc P1 were repeatedly detected by SYBR Green real-time fluorescent quantitative PCR, and the average value was taken as the copy number of the target gene nuc P1 to explore the relationship between the copy number and the expression level of the enzyme.The result showed that the enzymatic activity of the strain containing 9 nuc P1 gene copies reached 88.52 U/ml, which was 3.86 times higher than that of the nuclease P1 for the single copy strain, and 1.2-fold as high as that of the heterologous nuclease P1 reported in the previous studies. When the copy number of nuc P1 was increased to over 9, the enzyme activity decreased.
引文
[1]Masao Fujimoto,Akira Kuninaka,Hiroshi Yoshino.Some physical and chemical properties of nuclease P1[J].Agr.BioI.Chem.,1975,10(39):1991-1997
[2]Gite S,Reddy G,Shankar V.Active-site characterization of S1 nuclease I.Affinity purification and influence of amino-group modification[J].Biochemical Journal,1992,285(2):489-494
[3]宋威,张芹,李欢庆,等,复合载体固定化细胞发酵生产核酸酶P1的对比研究[J].河南工业大学学报(自然科学版),2008,3:51-54SONG Wei,ZHANG Qin,LI Huan-qing,et al.Study on the immobilizing Penicillium citrirum cells for producing nuclease P1using different multipile carriers[J].Journal of Henan University of Technology(Nature Science Edition),2008,3:51-54
[4]Yanan W.Expression,Purification and activity analysis of nuclease P1 in Escherichia coli and Pichia pastoris[D].Northwest Sci-tech University of Agriculture and Forestry,2012
[5]Guo-Qing Y,Shi L E,Yu Y,et al.Production,purification and characterization of nuclease p1 from Penicillium citrinum[J].Process Biochemistry,2006,41(6):1276-1281
[6]梁剑光,顾秋忆,秦修东,等.利用常压室温等离子体(ARTP)诱变选育高产核酸酶P1菌株[J].食品工业科技,2015,21:183-186LIANG Jian-guang,GU Qiu-yi,QIN Xiu-dong,et al.Screening of high yield nuclease P1 strain by exposed under atmospheric and room temperature plasma[J].Science and Technology of Food Industry,2015,21:183-186
[7]刘向勇,沈煜,郭亭,等.r DNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用[J].山东大学学报,2005,40(3):105-109LIU Xiang-yong,SHEN Yu,GUO Ting,et al.Construction of a ribosomal DNA multi-copy integration vector and application in the industrial Saccharomyces cerevisiae strain[J].Journal of Shandong University,2005,40(3):105-109
[8]支晓慧.基于r DNA序列的酵母整合载体的构建及应用[J].中国医药生物技术,2011,5:330-335ZHI Xiao-hui.Construction and application of Saccharomyces cerevisiae integration vector based on rDNAsequence[J].Chin Med Biotechnol,2011,5:330-335
[9]Klabunde J,Kunze G,Gellissen G,et al.Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element[J].FEMS Yeast Research,2003,4(2):185-193
[10]Wim Hartingsveldt,Ineke E Mattern,Cora M J Zeijl,et al.Development of a homologous transformation system for Aspergillus niger based on the pyrG gene[J].Mol Gen Genet,1987,206(1):71-75
[11]Qinting He,Nan Li,Xiaochun Chen,et al.Mutation breeding of nuclease p1 production in Penicillium citrinum by low-energy ion beam implantation[J].Korean Journal of Chemical Engineering,2011,28(2):544-549
[12]Ye L,Pan L.A comparison of the unfolded protein response in solid-state with submerged cultures of Aspergillus oryzae[J].Bioscience,Biotechnology,and Biochemistry,2008,72(11):2998-3001
[13]Saliola,M,Mazzoni C,Solimando N,et al.Use of the Kl ADH4 promoter for ethanol-dependent production of recombinant human serum albumin in Kluyveromyces lactis[J].Appl Environ Microbiol,1999,65(1):53-60
[14]Lin L L,Ma Y J,Chien H R,et al.Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome[J].Enzyme and Microbial Technology,1998,23(6):360-365
[15]Hohenblum H,Brigitte Gasser,Michael Maurer,et al.Effects of gene dosage,promoters,and substrates on unfolded protein stress of recombinant Pichia pastoris[J].Biotechnol Bioeng,2004,85(4):367-375
[16]Kobayashi K,Kuwae S,Ohya T,et al.High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J].Biosci.Bioeng,2000,89:55-61
[17]Zhu T,Guo M,Tang Z,et al.Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris[J].Appl Microbiol,2009,107(3):954-963
[18]Jansing J,Sack M,Augustine S M,et al.CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lackingβ-1,2-xylose and coreα-1,3-fucose[J].Plant Biotechnology Journal,2019,17(2):350-361
[19]Subramanian V,Logan A Schuster,Kyle T Moore,et al.Aversatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus,Trichoderma reesei[J].Biotechnol Biofuels,2017,10:34
[20]Archer D B,D J Jeenes,D A Mackenzie.Strategies for improving heterologous protein production from filamentous fungi[J].Antonie Van Leeuwenhoek,1994,65(3):245-250
[21]Wang H,Yang YF,Wang W,et al.Construction and verification of anti-MM sc Fv-t P fusion protein expression vector[J].Journal of Southern Medical University,2017,37(9):1149-1155
[22]Souza-Moreira T M,Navarrete C,Chen X,et al.Screening of2A peptides for polycistronic gene expression in yeast[J].FEMS Yeast Research,2018,18(5):36
[23]Schuetze T,V Meyer.Polycistronic gene expression in Aspergillus niger[J].Microb Cell Fact,2017,16(1):162
[24]孙海烨.18S r DNA介导的乳酸克鲁维酵母整合型表达载体的构建及初步验证[D].无锡:江南大学,2017SUN Hai-ye.Construction and preliminary verification of a18S ribosomal DNA-mediated integrative K.lactis expression vector[D].Wuxi:Jiangnan University,2017
[25]Wery,J,Gutker D,Renniers A C,et al.High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma[J].Gene,1997,184(1):89-97
[26]Marx H,Mecklenbr?uker A,Gasser B,et al.Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus[J].FEMS Yeast Research,2009,9(8):1260-1270
[27]Bergkamp R J,Kool I M,Geerse R H,et al.Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis[J].Curr Genet,1992,21(4-5):365-370
[28]Klabunde,J,Diesel A,Waschk D,et al.Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha[J].Appl Microbiol Biotechnol,2002,58(6):797-805
[29]Cheng,R,XiangZhi Lin,ZhaoKai Wang,et al.Establishment of a transgene expression system for the marine microalga Schizochytrium by 18S r DNA-targeted homologous recombination[J].World Journal of Microbiology and Biotechnology,2011,27(3):737-741
[30]郭美锦,朱泰承,张明,等.重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响[J].中国生物工程杂志,2007,7:7-11GUO Mei-jin,ZHU Tai-cheng,ZHANG Ming,et al.Influence of ethanol utilization phenotype and gene dosage on heterologous protein expression in recombinant Pichia pastoris[J].China Biotechnology 2007,7:7-11
[2]Gite S,Reddy G,Shankar V.Active-site characterization of S1 nuclease I.Affinity purification and influence of amino-group modification[J].Biochemical Journal,1992,285(2):489-494
[3]宋威,张芹,李欢庆,等,复合载体固定化细胞发酵生产核酸酶P1的对比研究[J].河南工业大学学报(自然科学版),2008,3:51-54SONG Wei,ZHANG Qin,LI Huan-qing,et al.Study on the immobilizing Penicillium citrirum cells for producing nuclease P1using different multipile carriers[J].Journal of Henan University of Technology(Nature Science Edition),2008,3:51-54
[4]Yanan W.Expression,Purification and activity analysis of nuclease P1 in Escherichia coli and Pichia pastoris[D].Northwest Sci-tech University of Agriculture and Forestry,2012
[5]Guo-Qing Y,Shi L E,Yu Y,et al.Production,purification and characterization of nuclease p1 from Penicillium citrinum[J].Process Biochemistry,2006,41(6):1276-1281
[6]梁剑光,顾秋忆,秦修东,等.利用常压室温等离子体(ARTP)诱变选育高产核酸酶P1菌株[J].食品工业科技,2015,21:183-186LIANG Jian-guang,GU Qiu-yi,QIN Xiu-dong,et al.Screening of high yield nuclease P1 strain by exposed under atmospheric and room temperature plasma[J].Science and Technology of Food Industry,2015,21:183-186
[7]刘向勇,沈煜,郭亭,等.r DNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用[J].山东大学学报,2005,40(3):105-109LIU Xiang-yong,SHEN Yu,GUO Ting,et al.Construction of a ribosomal DNA multi-copy integration vector and application in the industrial Saccharomyces cerevisiae strain[J].Journal of Shandong University,2005,40(3):105-109
[8]支晓慧.基于r DNA序列的酵母整合载体的构建及应用[J].中国医药生物技术,2011,5:330-335ZHI Xiao-hui.Construction and application of Saccharomyces cerevisiae integration vector based on rDNAsequence[J].Chin Med Biotechnol,2011,5:330-335
[9]Klabunde J,Kunze G,Gellissen G,et al.Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element[J].FEMS Yeast Research,2003,4(2):185-193
[10]Wim Hartingsveldt,Ineke E Mattern,Cora M J Zeijl,et al.Development of a homologous transformation system for Aspergillus niger based on the pyrG gene[J].Mol Gen Genet,1987,206(1):71-75
[11]Qinting He,Nan Li,Xiaochun Chen,et al.Mutation breeding of nuclease p1 production in Penicillium citrinum by low-energy ion beam implantation[J].Korean Journal of Chemical Engineering,2011,28(2):544-549
[12]Ye L,Pan L.A comparison of the unfolded protein response in solid-state with submerged cultures of Aspergillus oryzae[J].Bioscience,Biotechnology,and Biochemistry,2008,72(11):2998-3001
[13]Saliola,M,Mazzoni C,Solimando N,et al.Use of the Kl ADH4 promoter for ethanol-dependent production of recombinant human serum albumin in Kluyveromyces lactis[J].Appl Environ Microbiol,1999,65(1):53-60
[14]Lin L L,Ma Y J,Chien H R,et al.Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome[J].Enzyme and Microbial Technology,1998,23(6):360-365
[15]Hohenblum H,Brigitte Gasser,Michael Maurer,et al.Effects of gene dosage,promoters,and substrates on unfolded protein stress of recombinant Pichia pastoris[J].Biotechnol Bioeng,2004,85(4):367-375
[16]Kobayashi K,Kuwae S,Ohya T,et al.High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J].Biosci.Bioeng,2000,89:55-61
[17]Zhu T,Guo M,Tang Z,et al.Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris[J].Appl Microbiol,2009,107(3):954-963
[18]Jansing J,Sack M,Augustine S M,et al.CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lackingβ-1,2-xylose and coreα-1,3-fucose[J].Plant Biotechnology Journal,2019,17(2):350-361
[19]Subramanian V,Logan A Schuster,Kyle T Moore,et al.Aversatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus,Trichoderma reesei[J].Biotechnol Biofuels,2017,10:34
[20]Archer D B,D J Jeenes,D A Mackenzie.Strategies for improving heterologous protein production from filamentous fungi[J].Antonie Van Leeuwenhoek,1994,65(3):245-250
[21]Wang H,Yang YF,Wang W,et al.Construction and verification of anti-MM sc Fv-t P fusion protein expression vector[J].Journal of Southern Medical University,2017,37(9):1149-1155
[22]Souza-Moreira T M,Navarrete C,Chen X,et al.Screening of2A peptides for polycistronic gene expression in yeast[J].FEMS Yeast Research,2018,18(5):36
[23]Schuetze T,V Meyer.Polycistronic gene expression in Aspergillus niger[J].Microb Cell Fact,2017,16(1):162
[24]孙海烨.18S r DNA介导的乳酸克鲁维酵母整合型表达载体的构建及初步验证[D].无锡:江南大学,2017SUN Hai-ye.Construction and preliminary verification of a18S ribosomal DNA-mediated integrative K.lactis expression vector[D].Wuxi:Jiangnan University,2017
[25]Wery,J,Gutker D,Renniers A C,et al.High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma[J].Gene,1997,184(1):89-97
[26]Marx H,Mecklenbr?uker A,Gasser B,et al.Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus[J].FEMS Yeast Research,2009,9(8):1260-1270
[27]Bergkamp R J,Kool I M,Geerse R H,et al.Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis[J].Curr Genet,1992,21(4-5):365-370
[28]Klabunde,J,Diesel A,Waschk D,et al.Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha[J].Appl Microbiol Biotechnol,2002,58(6):797-805
[29]Cheng,R,XiangZhi Lin,ZhaoKai Wang,et al.Establishment of a transgene expression system for the marine microalga Schizochytrium by 18S r DNA-targeted homologous recombination[J].World Journal of Microbiology and Biotechnology,2011,27(3):737-741
[30]郭美锦,朱泰承,张明,等.重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响[J].中国生物工程杂志,2007,7:7-11GUO Mei-jin,ZHU Tai-cheng,ZHANG Ming,et al.Influence of ethanol utilization phenotype and gene dosage on heterologous protein expression in recombinant Pichia pastoris[J].China Biotechnology 2007,7:7-11