地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证
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摘要
地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。
Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene(amyL), an protease gene(aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene(vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4%respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.
Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene(amyL), an protease gene(aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene(vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4%respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.
引文
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[7]Li KF,Cai DB,Wang ZQ,et al.Development of an efficient genome editing tool in Bacillus licheniformis using CRISPR-Cas9 nickase.Appl Environ Microbiol,2018,84(6):e02608-17.
[8]Nahrstedt H,Waldeck J,Gr?ne M,et al.Strain development in Bacillus licheniformis:construction of biologically contained mutants deficient in sporulation and DNA repair.J Biotechnol,2005,119(3):245-254.
[9]Wang QZ,Tao C,Zhao XM,et al.Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production.Biotechnol Bioeng,2012,109(7):1610-1621.
[10]Qi GF,Kang YF,Li L,et al.Deletion of meso-2,3-butanediol dehydrogenase gene bud C for enhanced D-2,3-butanediol production in Bacillus licheniformis.Biotechnol Biofuels,2014,7:16.
[11]Meyer-Leon L,Senecoff JF,Bruckner RC,et al.Site-specific genetic recombination promoted by the FLP protein of the yeast 2-micron plasmid in vitro.Cold Spring Harb Symp Quant Biol,1984,49:797-804.
[12]Sadowski PD.The FLP recombinase of the 2-μm plasmid of Saccharomyces cerevisiae.Prog Nucleic Acid Res Mol Biol,1995,51:53-91.
[13]Teng Y,Yang X.Gene targeting:the beginning of a new era in genetics.Hereditas(Beijing),2007,29(11):1291-1298(in Chinese).滕艳,杨晓.基因打靶技术:开启遗传学新纪元.遗传,2007,29(11):1291-1298.
[14]Sánchez-Martínez C,Pérez-Martín J.Site-specific targeting of exogenous DNA into the genome of Candida albicans using the FLP recombinase.Mol Genet Genomics,2002,268(3):418-424.
[15]Tan XM,Liang FY,Cai K,et al.Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants.Appl Microbiol Biotechnol,2013,97(4):6373-6382.
[16]Li YR,Gu ZH,Zhang L,et al.Gene Knockout of Bacillus licheniformis mediated by CRISPR/Cas9system.Genomics Appl Biol,2017,36(10):4188-4196(in Chinese).李由然,顾正华,张梁,等.CRISPR/Cas9系统介导的地衣芽孢杆菌基因敲除.基因组学与应用生物学,2017,36(10):4188-4196.
[17]Kim IC,Cha JH,Kim JR,et al.Catalytic properties of the cloned amylase from Bacillus licheniformis.J Biol Chem,1992,267(31):22108-22114.
[18]Ministry of Light Industry of PRC.QB/T 1803-1993General methods of determination for industral enzymes.Beijing:China Light Industry Press,1994(in Chinese).中华人民共和国轻工业部.QB/T 1803-1993工业酶制剂通用试验方法.北京:中国轻工业出版社,1994.
[19]Hoffmann K,Wollherr A,Larsen M,et al.Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence.Appl Environ Microbiol,2010,76(15):5046-5057.
[20]Jakobs M,Hoffmann K,Grabke A,et al.Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains.Microbiology,2014,160:2136-2147.
[21]Zakataeva NP,Nikitina OV,Gronskiy SV,et al.A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains.Appl Microbiol Biotechnol,2010,85(4):1201-1209.
[22]Shatalin KY,Neyfakh AA.Efficient gene inactivation in Bacillus anthracis.FEMS Microbiol Lett,2005,245(2):315-319.
[23]Li YR.Gene cloning,identification and expression of isoamylase from Bacillus lentus CICIM3Q4[D].Wuxi:Jiangnan University,2013(in Chinese).李由然.Bacillus lentus CICIM304异淀粉酶的基因克隆、鉴定与表达[D].无锡:江南大学,2013.
[24]Yan X,Yu HJ,Hong Q,et al.Cre/loxP system and PCR-based genome engineering in Bacillus subtilis.Appl Environ Microbiol,2008,74(17):5556-5562.
[25]Zhao AC,Long DP,Tan B,et al.Progress in research of FLP/FRT site-specific recombination system in higher eukaryotes.Sci Agric Sin,2011,44(15):3252-3263(in Chinese).赵爱春,龙定沛,谭兵,等.FLP/FRT位点特异性重组系统在高等真核生物中的研究进展.中国农业科学,2011,44(15):3252-3263.
[2]Mo JY.Preparation,regeneration and genetic transformation of Bacillus licheniformis protoplasts[D].Wuxi:Jiangnan University,2009(in Chinese).莫静燕.地衣芽孢杆菌原生质体的制备、再生及转化研究[D].无锡:江南大学,2009.
[3]Waschkau B,Waldeck J,Wieland S,et al.Generation of readily transformable Bacillus licheniformis mutants.Appl Microbiol Biotechnol,2008,78(1):181-188.
[4]Yu HM,Ma YC.Gene knockout strategies for metabolic pathway regulation in industrial microbes.Chin JBiotech,2010,26(9):1199-1208(in Chinese).于慧敏,马玉超.工业微生物代谢途径调控的基因敲除策略.生物工程学报,2010,26(9):1199-1208.
[5]Rey MW,Ramaiya P,Nelson BA,et al.Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species.Genome Biol,2004,5(10):R77.
[6]Liu BN.Effects of Bacillus licheniformis in converting potato starch waste and its mechanism of metabolism[D].Harbin:Harbin Institute of Technology,2015(in Chinese).刘冰南.地衣芽孢杆菌转化薯渣与汁水效能及其代谢机理研究[D].哈尔滨:哈尔滨工业大学,2015.
[7]Li KF,Cai DB,Wang ZQ,et al.Development of an efficient genome editing tool in Bacillus licheniformis using CRISPR-Cas9 nickase.Appl Environ Microbiol,2018,84(6):e02608-17.
[8]Nahrstedt H,Waldeck J,Gr?ne M,et al.Strain development in Bacillus licheniformis:construction of biologically contained mutants deficient in sporulation and DNA repair.J Biotechnol,2005,119(3):245-254.
[9]Wang QZ,Tao C,Zhao XM,et al.Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production.Biotechnol Bioeng,2012,109(7):1610-1621.
[10]Qi GF,Kang YF,Li L,et al.Deletion of meso-2,3-butanediol dehydrogenase gene bud C for enhanced D-2,3-butanediol production in Bacillus licheniformis.Biotechnol Biofuels,2014,7:16.
[11]Meyer-Leon L,Senecoff JF,Bruckner RC,et al.Site-specific genetic recombination promoted by the FLP protein of the yeast 2-micron plasmid in vitro.Cold Spring Harb Symp Quant Biol,1984,49:797-804.
[12]Sadowski PD.The FLP recombinase of the 2-μm plasmid of Saccharomyces cerevisiae.Prog Nucleic Acid Res Mol Biol,1995,51:53-91.
[13]Teng Y,Yang X.Gene targeting:the beginning of a new era in genetics.Hereditas(Beijing),2007,29(11):1291-1298(in Chinese).滕艳,杨晓.基因打靶技术:开启遗传学新纪元.遗传,2007,29(11):1291-1298.
[14]Sánchez-Martínez C,Pérez-Martín J.Site-specific targeting of exogenous DNA into the genome of Candida albicans using the FLP recombinase.Mol Genet Genomics,2002,268(3):418-424.
[15]Tan XM,Liang FY,Cai K,et al.Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants.Appl Microbiol Biotechnol,2013,97(4):6373-6382.
[16]Li YR,Gu ZH,Zhang L,et al.Gene Knockout of Bacillus licheniformis mediated by CRISPR/Cas9system.Genomics Appl Biol,2017,36(10):4188-4196(in Chinese).李由然,顾正华,张梁,等.CRISPR/Cas9系统介导的地衣芽孢杆菌基因敲除.基因组学与应用生物学,2017,36(10):4188-4196.
[17]Kim IC,Cha JH,Kim JR,et al.Catalytic properties of the cloned amylase from Bacillus licheniformis.J Biol Chem,1992,267(31):22108-22114.
[18]Ministry of Light Industry of PRC.QB/T 1803-1993General methods of determination for industral enzymes.Beijing:China Light Industry Press,1994(in Chinese).中华人民共和国轻工业部.QB/T 1803-1993工业酶制剂通用试验方法.北京:中国轻工业出版社,1994.
[19]Hoffmann K,Wollherr A,Larsen M,et al.Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence.Appl Environ Microbiol,2010,76(15):5046-5057.
[20]Jakobs M,Hoffmann K,Grabke A,et al.Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains.Microbiology,2014,160:2136-2147.
[21]Zakataeva NP,Nikitina OV,Gronskiy SV,et al.A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains.Appl Microbiol Biotechnol,2010,85(4):1201-1209.
[22]Shatalin KY,Neyfakh AA.Efficient gene inactivation in Bacillus anthracis.FEMS Microbiol Lett,2005,245(2):315-319.
[23]Li YR.Gene cloning,identification and expression of isoamylase from Bacillus lentus CICIM3Q4[D].Wuxi:Jiangnan University,2013(in Chinese).李由然.Bacillus lentus CICIM304异淀粉酶的基因克隆、鉴定与表达[D].无锡:江南大学,2013.
[24]Yan X,Yu HJ,Hong Q,et al.Cre/loxP system and PCR-based genome engineering in Bacillus subtilis.Appl Environ Microbiol,2008,74(17):5556-5562.
[25]Zhao AC,Long DP,Tan B,et al.Progress in research of FLP/FRT site-specific recombination system in higher eukaryotes.Sci Agric Sin,2011,44(15):3252-3263(in Chinese).赵爱春,龙定沛,谭兵,等.FLP/FRT位点特异性重组系统在高等真核生物中的研究进展.中国农业科学,2011,44(15):3252-3263.