微滴数字PCR技术在多拷贝木聚糖酶酿酒酵母工程菌筛选中的应用
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摘要
为获得高产木聚糖酶酿酒酵母工程菌,利用rDNA整合法构建木聚糖酶酿酒酵母整合表达载体,实现木聚糖酶基因的多拷贝表达,并利用微滴数字聚合酶链式反应技术对转化子拷贝数进行检测,分析木聚糖酶基因拷贝数与酶活力之间的关系。结果表明:利用rDNA整合法获得了10株不同拷贝数木聚糖酶酿酒酵母工程菌,对其酶活力进行测定,发现当拷贝数小于9时,菌株产酶能力随拷贝数增加而增强,9拷贝数时菌株产酶能力最强,酶活力为308 U/m L,超过9拷贝,产酶能力降低。
In order to obtain a high yield of xylanase from genetically engineered Saccharomyces cerevisiae, the S. cerevisiaeexpression vector for the multicopy expression of the xylanase gene was constructed by r DNA integration method. Then, the copy number of transformants was detected by droplet digital PCR, and the relationship between the copy number of the xylanase gene and the enzyme activity was analyzed. The results indicated that 10 strains of S. cerevisiae were obtained by r DNA integration method. Their enzymatic activity was measured. The xylanase production capacity was increased to the maximum of 308 U/m L with copy number up to 9, but decreased when the copy number was more than 9.
In order to obtain a high yield of xylanase from genetically engineered Saccharomyces cerevisiae, the S. cerevisiaeexpression vector for the multicopy expression of the xylanase gene was constructed by r DNA integration method. Then, the copy number of transformants was detected by droplet digital PCR, and the relationship between the copy number of the xylanase gene and the enzyme activity was analyzed. The results indicated that 10 strains of S. cerevisiae were obtained by r DNA integration method. Their enzymatic activity was measured. The xylanase production capacity was increased to the maximum of 308 U/m L with copy number up to 9, but decreased when the copy number was more than 9.
引文
[1]单志琼,周峻岗,周宇飞,等.产碱性木聚糖酶菌株的筛选及酶学性质[J].遗传,2012,34(3):356-365.DOI:10.3724/SP.J.1005.2012.00356.
[2]孙振涛,赵祥颖,刘建军,等.微生物木聚糖酶及其应用[J].生物技术,2007,17(2):93-97.DOI:10.3969/j.issn.1004-311X.2007.02.033.
[3]李杰,张会,张莹莹,等.食品级木聚糖酶黑曲霉工程菌的构建[J].东北农业大学学报,2013,44(11):7-13.DOI:10.3969/j.issn.1005-9369.2013.11.002.
[4]LU Y,ZHONG H,TANG Q,et al.Construction and verification of CYP3A5 gene polymorphisms using a Saccharomyces cerevisiae expression system to predict drug metabolism[J].Molecular Medicine Reports,2017,15(4):1593-1600.DOI:10.3892/mmr.2017.6214.
[5]PAYNE T,HANFREY C,BISHOP A L,et al.Transcript-specific translational regulation in the unfolded protein response of Saccharomyces cerevisiae[J].FEBS Letters,2008,582(4):503-509.DOI:10.1016/j.febslet.2008.01.009.
[6]WANG H Y,XIAO D F,ZHOU C,et al.YLL056C,from Saccharomyces cerevisiae,encodes a novel protein with aldehyde reductase activity[J].Applied Microbiology&Biotechnology,2017,101(11):4507-4520.DOI:10.1007/s00253-017-8209-5.
[7]KWAN E X,WANG X S,AMEMIYA H M,et al.rDNA Copy number variants are frequent passenger mutations in Saccharomyces cerevisiae deletion collections and de novo transformants[J].G3(Bethesda),2016,6(9):2829-2838.DOI:10.1534/g3.116.030296.
[8]JAMES S A,WEST C,DAVEY R P,et al.Prevalence and dynamics of ribosomal DNA micro-heterogeneity are linked to population history in two contrasting yeast species[J].Scientific Reports,2016,6:28555.DOI:10.1038/srep28555.
[9]武志强,贾耐兵,李娜,等.酵母整合型载体的构建及其功能分析[J].生物学通报,2008,43(5):47-50.DOI:10.3969/j.issn.0006-3193.2008.05.020.
[10]程诚,熊亮,李勇昊,等.混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究[J].微生物学通报,2016,43(7):1411-1418.DOI:10.13344/j.microbiol.china.150576.
[11]周巍,李月华,孙勇,等.微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌[J].食品科学,2017,38(16):287-291.DOI:10.7506/spkx1002-6630-201716046.
[12]詹成,燕丽,王琳,等.数字PCR技术的发展和应用[J].复旦学报(医学版),2015,42(6):786-789.DOI:10.3969/j.issn.1672-8467.2015.06.017.
[13]王杰,王全,田娜,等.不同植物组织RNA提取方法的比较分析[J].北京农学院学报,2015,30(1):76-80.DOI:10.13473/j.cnki.issn.1002-3186.2014.0093.
[14]周晨妍,刘振华,王丹丹,等.木聚糖酶Xyn43A基因在大肠杆菌及毕赤酵母中的表达比较[J].食品与发酵工业,2016,42(6):15-19.DOI:10.13995/j.cnki.11-1802/ts.201606003.
[15]唐天乐,高炳淼,长孙东亭,等.高质量毕赤酵母基因组DNA提取方法比较[J].生物技术通报,2010,2010(1):196-199.DOI:10.13560/j.cnki.biotech.bull.1985.2010.01.021.
[16]高丽丽,王庆华,梁会超,等.酿酒酵母羊毛甾醇合酶基因单倍体缺陷型突变株的构建[J].药学学报,2014,49(5):742-746.DOI:10.16438/j.0513-4870.2014.05.001.
[17]GIETZ R D,WOODS R A.Yeast transformation by the LiAc/SScarrier DNA/PEG method[J].Methods in Molecular Biology,2006,313:107-120.DOI:10.1007/978-1-4939-1363-3_1.
[18]SRISUTHAM S,SARALAMBA N,MALLERET B,et al.Four human Plasmodium species quantification using droplet digital PCR[J].PLoSONE,2017,12(4):e0175771.DOI:10.1371/journal.pone.0175771.
[19]CAVALLI M,DE NOVI L A,DELLA S I,et al.Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma[J].British Journal of Haematology,2017,177(4):588.DOI:10.1111/bjh.14616.
[20]ARVIA R,SOLLAI M,PIERUCCI F,et al.Droplet digital PCR(ddPCR)vs quantitative real-time PCR(qPCR)approach for detection and quantification of Merkel cell polyomavirus(MCPyV)DNA in formalin fixed paraffin embedded(FFPE)cutaneous biopsies[J].Journal of Virological Methods,2017,246:15-20.DOI:10.1016/j.jviromet.2017.04.003.
[21]SUZAWA K,YAMAMOTO H,OHASHI K,et al.Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system[J].Oncology Reports,2017,37(5):3100-3106.DOI:10.3892/or.2017.5567.
[22]张居作,徐君飞,陈汉忠,等.BE-91菌株木聚糖酶活力测定条件的优化[J].湖北农业科学,2010,49(4):950-953.DOI:10.14088/j.cnki.issn0439-8114.2010.04.003.
[23]史红玲,汪俊卿,邬敏辰,等.毕赤酵母产重组木聚糖酶发酵条件的优化及其酶学性质[J].中国生物制品学杂志,2012,25(10):1362-1365.DOI:10.13200/j.cjb.2012.10.123.shihl.016.
[24]JIA P,PURCELL M K,PAN G,et al.Analytical validation of a reverse transcriptase droplet digital PCR(RT-ddPCR)for quantitative detection of infectious hematopoietic necrosis virus[J].Journal of Virological Methods,2017,245:73-80.DOI:10.1016/j.jviromet.2017.03.010.
[25]KLINE M C,DUEWER D L.Evaluating droplet digital PCR for the quantification of human genomic DNA:lifting the traceability fog[J].Analytical Chemistry,2017,89(8):4648-4654.DOI:10.1021/acs.analchem.7b00240.
[26]姜羽,胡佳莹,杨立桃.利用微滴数字PCR分析转基因生物外源基因拷贝数[J].农业生物技术学报,2014,22(10):1298-1305.DOI:10.3969/j.issn.1674-7968.2014.10.013.
[27]于晓帆,高宏伟,孙敏,等.荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆[J].食品科学,2016,37(16):235-241.DOI:10.7506/spkx1002-6630-201616038.
[28]BRUNETTI C,ANELLI L,ZAGARIA A,et al.Droplet digital PCRis a reliable tool for monitoring minimal residual disease in acute promyelocytic leukemia[J].Journal of Molecular Diagnostics,2017,19(3):437-444.DOI:10.1016/j.jmoldx.2017.01.004.
[29]GITTINS J R,D’ANGELO C,OSWALD F,et al.Fluorescent protein-mediated colour polymorphism in reef corals:multicopy genes extend the adaptation/acclimatization potential to variable light environments[J].Molecular Ecology,2015,24(2):453-465.DOI:10.1111/mec.13041.
[30]黄贞杰,陈玲,张积森,等.ScINO1基因克隆及酵母多基因多拷贝整合表达载体的构建[J].福建师大学报(自然科学版),2012,28(6):100-105.DOI:10.3969/j.issn.1000-5277.2012.06.019.
[31]LIN X Q,LIANG S L,HAN S Y,et al.Quantitative iTRAQ LCMS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris[J].Journal of Proteomics,2013,91:58-72.DOI:10.1016/j.jprot.2013.06.031.
[32]MARX H,MECKLENBR腢KER 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.DOI:10.1111/j.1567-1364.2009.00561.x.
[33]吴志伟,徐立新,佟金,等.多拷贝策略在增强目的基因表达中的应用[J].生命科学研究,2016,20(2):166-170.DOI:10.16605/j.cnki.1007-7847.2016.02.014.
[2]孙振涛,赵祥颖,刘建军,等.微生物木聚糖酶及其应用[J].生物技术,2007,17(2):93-97.DOI:10.3969/j.issn.1004-311X.2007.02.033.
[3]李杰,张会,张莹莹,等.食品级木聚糖酶黑曲霉工程菌的构建[J].东北农业大学学报,2013,44(11):7-13.DOI:10.3969/j.issn.1005-9369.2013.11.002.
[4]LU Y,ZHONG H,TANG Q,et al.Construction and verification of CYP3A5 gene polymorphisms using a Saccharomyces cerevisiae expression system to predict drug metabolism[J].Molecular Medicine Reports,2017,15(4):1593-1600.DOI:10.3892/mmr.2017.6214.
[5]PAYNE T,HANFREY C,BISHOP A L,et al.Transcript-specific translational regulation in the unfolded protein response of Saccharomyces cerevisiae[J].FEBS Letters,2008,582(4):503-509.DOI:10.1016/j.febslet.2008.01.009.
[6]WANG H Y,XIAO D F,ZHOU C,et al.YLL056C,from Saccharomyces cerevisiae,encodes a novel protein with aldehyde reductase activity[J].Applied Microbiology&Biotechnology,2017,101(11):4507-4520.DOI:10.1007/s00253-017-8209-5.
[7]KWAN E X,WANG X S,AMEMIYA H M,et al.rDNA Copy number variants are frequent passenger mutations in Saccharomyces cerevisiae deletion collections and de novo transformants[J].G3(Bethesda),2016,6(9):2829-2838.DOI:10.1534/g3.116.030296.
[8]JAMES S A,WEST C,DAVEY R P,et al.Prevalence and dynamics of ribosomal DNA micro-heterogeneity are linked to population history in two contrasting yeast species[J].Scientific Reports,2016,6:28555.DOI:10.1038/srep28555.
[9]武志强,贾耐兵,李娜,等.酵母整合型载体的构建及其功能分析[J].生物学通报,2008,43(5):47-50.DOI:10.3969/j.issn.0006-3193.2008.05.020.
[10]程诚,熊亮,李勇昊,等.混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究[J].微生物学通报,2016,43(7):1411-1418.DOI:10.13344/j.microbiol.china.150576.
[11]周巍,李月华,孙勇,等.微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌[J].食品科学,2017,38(16):287-291.DOI:10.7506/spkx1002-6630-201716046.
[12]詹成,燕丽,王琳,等.数字PCR技术的发展和应用[J].复旦学报(医学版),2015,42(6):786-789.DOI:10.3969/j.issn.1672-8467.2015.06.017.
[13]王杰,王全,田娜,等.不同植物组织RNA提取方法的比较分析[J].北京农学院学报,2015,30(1):76-80.DOI:10.13473/j.cnki.issn.1002-3186.2014.0093.
[14]周晨妍,刘振华,王丹丹,等.木聚糖酶Xyn43A基因在大肠杆菌及毕赤酵母中的表达比较[J].食品与发酵工业,2016,42(6):15-19.DOI:10.13995/j.cnki.11-1802/ts.201606003.
[15]唐天乐,高炳淼,长孙东亭,等.高质量毕赤酵母基因组DNA提取方法比较[J].生物技术通报,2010,2010(1):196-199.DOI:10.13560/j.cnki.biotech.bull.1985.2010.01.021.
[16]高丽丽,王庆华,梁会超,等.酿酒酵母羊毛甾醇合酶基因单倍体缺陷型突变株的构建[J].药学学报,2014,49(5):742-746.DOI:10.16438/j.0513-4870.2014.05.001.
[17]GIETZ R D,WOODS R A.Yeast transformation by the LiAc/SScarrier DNA/PEG method[J].Methods in Molecular Biology,2006,313:107-120.DOI:10.1007/978-1-4939-1363-3_1.
[18]SRISUTHAM S,SARALAMBA N,MALLERET B,et al.Four human Plasmodium species quantification using droplet digital PCR[J].PLoSONE,2017,12(4):e0175771.DOI:10.1371/journal.pone.0175771.
[19]CAVALLI M,DE NOVI L A,DELLA S I,et al.Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma[J].British Journal of Haematology,2017,177(4):588.DOI:10.1111/bjh.14616.
[20]ARVIA R,SOLLAI M,PIERUCCI F,et al.Droplet digital PCR(ddPCR)vs quantitative real-time PCR(qPCR)approach for detection and quantification of Merkel cell polyomavirus(MCPyV)DNA in formalin fixed paraffin embedded(FFPE)cutaneous biopsies[J].Journal of Virological Methods,2017,246:15-20.DOI:10.1016/j.jviromet.2017.04.003.
[21]SUZAWA K,YAMAMOTO H,OHASHI K,et al.Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system[J].Oncology Reports,2017,37(5):3100-3106.DOI:10.3892/or.2017.5567.
[22]张居作,徐君飞,陈汉忠,等.BE-91菌株木聚糖酶活力测定条件的优化[J].湖北农业科学,2010,49(4):950-953.DOI:10.14088/j.cnki.issn0439-8114.2010.04.003.
[23]史红玲,汪俊卿,邬敏辰,等.毕赤酵母产重组木聚糖酶发酵条件的优化及其酶学性质[J].中国生物制品学杂志,2012,25(10):1362-1365.DOI:10.13200/j.cjb.2012.10.123.shihl.016.
[24]JIA P,PURCELL M K,PAN G,et al.Analytical validation of a reverse transcriptase droplet digital PCR(RT-ddPCR)for quantitative detection of infectious hematopoietic necrosis virus[J].Journal of Virological Methods,2017,245:73-80.DOI:10.1016/j.jviromet.2017.03.010.
[25]KLINE M C,DUEWER D L.Evaluating droplet digital PCR for the quantification of human genomic DNA:lifting the traceability fog[J].Analytical Chemistry,2017,89(8):4648-4654.DOI:10.1021/acs.analchem.7b00240.
[26]姜羽,胡佳莹,杨立桃.利用微滴数字PCR分析转基因生物外源基因拷贝数[J].农业生物技术学报,2014,22(10):1298-1305.DOI:10.3969/j.issn.1674-7968.2014.10.013.
[27]于晓帆,高宏伟,孙敏,等.荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆[J].食品科学,2016,37(16):235-241.DOI:10.7506/spkx1002-6630-201616038.
[28]BRUNETTI C,ANELLI L,ZAGARIA A,et al.Droplet digital PCRis a reliable tool for monitoring minimal residual disease in acute promyelocytic leukemia[J].Journal of Molecular Diagnostics,2017,19(3):437-444.DOI:10.1016/j.jmoldx.2017.01.004.
[29]GITTINS J R,D’ANGELO C,OSWALD F,et al.Fluorescent protein-mediated colour polymorphism in reef corals:multicopy genes extend the adaptation/acclimatization potential to variable light environments[J].Molecular Ecology,2015,24(2):453-465.DOI:10.1111/mec.13041.
[30]黄贞杰,陈玲,张积森,等.ScINO1基因克隆及酵母多基因多拷贝整合表达载体的构建[J].福建师大学报(自然科学版),2012,28(6):100-105.DOI:10.3969/j.issn.1000-5277.2012.06.019.
[31]LIN X Q,LIANG S L,HAN S Y,et al.Quantitative iTRAQ LCMS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris[J].Journal of Proteomics,2013,91:58-72.DOI:10.1016/j.jprot.2013.06.031.
[32]MARX H,MECKLENBR腢KER 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.DOI:10.1111/j.1567-1364.2009.00561.x.
[33]吴志伟,徐立新,佟金,等.多拷贝策略在增强目的基因表达中的应用[J].生命科学研究,2016,20(2):166-170.DOI:10.16605/j.cnki.1007-7847.2016.02.014.