脂肪酶lipAB操纵子在防御假单胞菌中的克隆、表达及活性研究
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摘要
对荚壳伯克氏菌PG1(Burkholderia glumae PG1)基因组中的脂肪酶操纵子lipAB片段进行直接克隆,构建含有脂肪酶基因的分泌表达载体,实现其在防御假单胞菌Pf-5(Pseudomonas protegens Pf-5)中的异源表达,并研究重组工程菌的胞外脂肪酶活性。利用Red/ET直接克隆技术获得克隆载体p15A-cm-lipAB;再通过亚克隆技术构建重组表达载体pBBR1-km-lipAB和pBBR1-km-Papra-lipAB,将这两个表达载体分别电转至Pf-5中,通过卡那霉素或者阿伯拉霉素抗性筛选得到转化子,以三丁酸甘油酯平板扩散法和对硝基苯酚法检测脂肪酶酶活,并通过实时荧光定量PCR检测启动子的替换对lipA表达的影响。本研究从PG1中成功克隆了脂肪酶操纵子lipAB(GenBank accession number:AJK49931. 1 and AJK49932. 1);成功构建了重组工程菌Pf-5/pBBR1-km-lipAB和Pf-5/pBBR1-km-Papra-lipAB,并成功检测到两株工程菌的胞外脂肪酶活性;以LB培养基培养至24 h时,启动子优化后lipA基因表达量是原始水平的2. 1倍;在LB培养基摇瓶发酵至66 h时,Pf-5/pBBR1-km-lipAB的脂肪酶酶活最高且为13. 51 U/mL,而Pf-5/pBBR1-km-Papra-lipAB的酶活为46. 85 U/mL,是Pf-5/pBBR1-km-lipAB的3. 47倍。初步实现基因lipA在Pf-5中的表达,发现组成型启动子Papra比lipAB的原始启动子PlipAB效率更高,为将来实现规模化生产奠定了技术基础。
To implement heterologous expression of Burkholderia glumae PG1 lipase operon lipAB in Pseudomonas protegens Pf-5 via Red/ET homologous recombineering. The vector p15 A-cm-lipAB was obtained using Red/ET direct cloning technology. Then, two recombinant expression vectors pBBR1-km-lipAB and pBBR1-km-Papra-lipAB with different promoters were constructed by subcloning technology,and electrotransformated the resultant expression vectors into P. protegens Pf-5. Transformants obtained by kanamycin or apramycin resistance screening. The tributyrin glyceryl trinitrate plate diffusion method and the p-nitrophenol method were used for the assay of the activities of lipase,and the effect of promoter replacement on lipA expression was examined by qRT-PCR. We successfully cloned the lipase operon lipAB( GenBank accession number: AJK49931. 1 and AJK49932. 1). After the achievement of engineering bacteria Pf-5/pBBR1-km-lipAB and Pf-5/pBBR1-km-Papra-lipAB,fermentation results indicated that the activity of extracellular lipase in Pf-5 was accomplished. Moreover,it was found that the expression level of lipA gene was 2. 1-fold the original level after promoter optimization. When the flask in LB medium was fermented to 66 h,the lipase activity of Pf-5/pBBR1-km-lipAB supernatant was 13. 51 U/mL,with that of Pf-5/pBBR1-km-Papra-lipAB supernatant was 46. 85 U/mL resulting in 3. 47-fold variation after promoter optimization. PG1 lipase gene lipA can be successfully heterologously expressed in Pf-5 via genetic engineering. Results reveal that the constitutive promoter Paprais more efficient than the original promoter PlipABin Pf-5 strain. Furthermore,the present study provides an important prerequisite for scale production and industrial application of the lipase.
To implement heterologous expression of Burkholderia glumae PG1 lipase operon lipAB in Pseudomonas protegens Pf-5 via Red/ET homologous recombineering. The vector p15 A-cm-lipAB was obtained using Red/ET direct cloning technology. Then, two recombinant expression vectors pBBR1-km-lipAB and pBBR1-km-Papra-lipAB with different promoters were constructed by subcloning technology,and electrotransformated the resultant expression vectors into P. protegens Pf-5. Transformants obtained by kanamycin or apramycin resistance screening. The tributyrin glyceryl trinitrate plate diffusion method and the p-nitrophenol method were used for the assay of the activities of lipase,and the effect of promoter replacement on lipA expression was examined by qRT-PCR. We successfully cloned the lipase operon lipAB( GenBank accession number: AJK49931. 1 and AJK49932. 1). After the achievement of engineering bacteria Pf-5/pBBR1-km-lipAB and Pf-5/pBBR1-km-Papra-lipAB,fermentation results indicated that the activity of extracellular lipase in Pf-5 was accomplished. Moreover,it was found that the expression level of lipA gene was 2. 1-fold the original level after promoter optimization. When the flask in LB medium was fermented to 66 h,the lipase activity of Pf-5/pBBR1-km-lipAB supernatant was 13. 51 U/mL,with that of Pf-5/pBBR1-km-Papra-lipAB supernatant was 46. 85 U/mL resulting in 3. 47-fold variation after promoter optimization. PG1 lipase gene lipA can be successfully heterologously expressed in Pf-5 via genetic engineering. Results reveal that the constitutive promoter Paprais more efficient than the original promoter PlipABin Pf-5 strain. Furthermore,the present study provides an important prerequisite for scale production and industrial application of the lipase.
引文
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[11] RAMETTE A,FRAPOLLI M,FISCHERLE M S,et al.Pseudomonas protegens sp. nov.,widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin[J]. Systematic and Applied Microbiology,2011,34(3):180-188.
[12] NAGARAJKUMAR M,JAYARAJ J,MUTHUKRISHNAN S,et al. Detoxific-ation of oxalic acid by Pseudomonas fluorescens strain PfMDU2:implications for the biological control of rice sheath blight caused by Rhizoctonia solani[J]. Microbiological Research,2005,160(3):291-298.
[13]孙广正,姚拓,赵桂琴,等.荧光假单胞菌防治植物病害研究现状与展望[J].草业学报,2015,24(4):174-190.SUN Guangzheng,YAO Tuo,ZHAO Guiqin,et al. Research progress and prospects for controlling plant disease susing Pseudomonas flurescens[J]. Acta Prataculturae Sinica,2015,24(4):174-190.
[14] ZHA D,XU L,ZHANG H,et al. Molecular identification of lipase LipA from Pseudomonas protegens Pf-5 and characterization of two whole-cell biocatalysts Pf-5 and Top10lipA[J]. Journal of Microbiology and Biotechnology,2014,24(5):619-628.
[15] ZHA D,ZHANG H,ZHANG H,et al. N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5:a must for its efficient folding into an active conformation[J].Biochimie,2014,105:165-171.
[16] WANG H,LI Z,JIA R,et al. RecET direct cloning and Redαβrecombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression[J]. Nature Protocols,2016,11(7):1175-1190.
[17] ZHANG Y,MUYRERS J P,TESTA G,et al. DNA cloning by homologous recombination in Escherichia coli[J]. Nature Biotechnology,2000,18(12):1314-1317.
[18] ZHANG Y,BUCHHOLZ F,MUYRERS J P,et al. A new logic for DNA engineering using recombination in Escherichia coli[J]. Nature Genetics,1998,20(2):123-128.
[19] WANG J,SAROV M,RIENTJES J,et al. An improved recombineering approach by adding RecA to lambda red recombination[J]. Journal of Microbiology and Biotechnology,2006,32(1):43-53.
[20] FU J,BIAN X,HU S,et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting[J]. Nature Biotechnology,2012,30(5):440-446.
[21]郑文韬,张友明,卞小莹. Red/ET同源重组技术及其在微生物基因组挖掘中的应用进展[J].微生物学报,2017,57(11):1735-1746.ZHENG Wentao, ZHANG Youming, BIAN Xiaoying.Advances in Red/ET recombineering and its application for microbial genome mining[J]. Acta Microbiologica Sinica,2017,57(11):1735-1746.
[22] TU Q,YIN J,FU J,et al. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency[J]. Scientific Reports,2016,20(6):246-248.
[23]邬敏辰,孙崇荣,邬显章.平板扩散法粗略确定碱性脂肪酶的活性[J].无锡轻工大学学报,2000,19(2):168-172.WU Minchen,SUN Congrong,WU Xianzhang. Diffusion plate assay for quick and rough estimation of alkaline lipase activity[J]. Journal of Wuxi University of Light Industry,2000,19(2):168-172.
[24] GAELLE P,JACQUES C B. Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase:a simple test for the determination of lipase activity in organic media[J]. Enzyme and Microbial Technology,1996,18:417-422.
[25]滕昆,张吉福,牛福星,等.脂肪酶活力测定中终止反应方法的比较分析[J].广西科学,2014,21(2):115-118.TENG Kun,ZHANG Jifu,NIU Fuxing,et al. Comparative analysis of termination reaction methods in lipase activity assay[J]. Guangxi Science,2014,21(2):115-118.
[2] VERMA M L,AZMI W,KANWAR S S. Microbial lipases:at the interface of aqueous and non-aqueous media. a review[J].Acta Microbiol Immunol Hung,2008,55(3):265-294.
[3] GHOSH P K,SAXENA R K,GUPTA R,et al. Microbial lipases:production and applications[J]. Science Program,1996,79(Pt 2):119-157.
[4] JUNG W S,LEE J,KIM M I,et al. Structural and functional analysis of phytotoxin toxoflavin-degrading enzyme[J]. PloS One,2011,6(7):e224-243.
[5] JEONG Y,KIM J,KIM S,et al. Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops[J]. Plant Diseases,2003,87(8):890-895.
[6] OLLIS D L,CHEAH E,CYGLER M,et al. The alpha/beta hydrolase fold[J]. Protein Engineerin, 1992, 5(3):197-211.
[7] LANG D,HOFMANN B,HAALCK L,et al. Crystal structure of a bacterial lipase from Chromobacterium viscosum ATCC 6918refined at 1. 6 angstroms resolution[J]. Journal of Molecular Biology,1996,259(4):704-717.
[8] JAEGER K E,RANSAC S,DIJKSTRA B W,et al. Bacterial lipases:reviews[J]. FEMS Microbiology Letters,1994,15(1):29-63.
[9] FRENKEN L G,EGMOND M R,BATENBURG A M,et al.Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues[J]. Applied and Environmental Microbiology,1992,58(12):3787-3791.
[10] BANEYX F,MUJACIC M. Recombinant protein folding and misfolding in Escherichia coli:review[J]. Nature Biotechnology,2004,22:1399-1408.
[11] RAMETTE A,FRAPOLLI M,FISCHERLE M S,et al.Pseudomonas protegens sp. nov.,widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin[J]. Systematic and Applied Microbiology,2011,34(3):180-188.
[12] NAGARAJKUMAR M,JAYARAJ J,MUTHUKRISHNAN S,et al. Detoxific-ation of oxalic acid by Pseudomonas fluorescens strain PfMDU2:implications for the biological control of rice sheath blight caused by Rhizoctonia solani[J]. Microbiological Research,2005,160(3):291-298.
[13]孙广正,姚拓,赵桂琴,等.荧光假单胞菌防治植物病害研究现状与展望[J].草业学报,2015,24(4):174-190.SUN Guangzheng,YAO Tuo,ZHAO Guiqin,et al. Research progress and prospects for controlling plant disease susing Pseudomonas flurescens[J]. Acta Prataculturae Sinica,2015,24(4):174-190.
[14] ZHA D,XU L,ZHANG H,et al. Molecular identification of lipase LipA from Pseudomonas protegens Pf-5 and characterization of two whole-cell biocatalysts Pf-5 and Top10lipA[J]. Journal of Microbiology and Biotechnology,2014,24(5):619-628.
[15] ZHA D,ZHANG H,ZHANG H,et al. N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5:a must for its efficient folding into an active conformation[J].Biochimie,2014,105:165-171.
[16] WANG H,LI Z,JIA R,et al. RecET direct cloning and Redαβrecombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression[J]. Nature Protocols,2016,11(7):1175-1190.
[17] ZHANG Y,MUYRERS J P,TESTA G,et al. DNA cloning by homologous recombination in Escherichia coli[J]. Nature Biotechnology,2000,18(12):1314-1317.
[18] ZHANG Y,BUCHHOLZ F,MUYRERS J P,et al. A new logic for DNA engineering using recombination in Escherichia coli[J]. Nature Genetics,1998,20(2):123-128.
[19] WANG J,SAROV M,RIENTJES J,et al. An improved recombineering approach by adding RecA to lambda red recombination[J]. Journal of Microbiology and Biotechnology,2006,32(1):43-53.
[20] FU J,BIAN X,HU S,et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting[J]. Nature Biotechnology,2012,30(5):440-446.
[21]郑文韬,张友明,卞小莹. Red/ET同源重组技术及其在微生物基因组挖掘中的应用进展[J].微生物学报,2017,57(11):1735-1746.ZHENG Wentao, ZHANG Youming, BIAN Xiaoying.Advances in Red/ET recombineering and its application for microbial genome mining[J]. Acta Microbiologica Sinica,2017,57(11):1735-1746.
[22] TU Q,YIN J,FU J,et al. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency[J]. Scientific Reports,2016,20(6):246-248.
[23]邬敏辰,孙崇荣,邬显章.平板扩散法粗略确定碱性脂肪酶的活性[J].无锡轻工大学学报,2000,19(2):168-172.WU Minchen,SUN Congrong,WU Xianzhang. Diffusion plate assay for quick and rough estimation of alkaline lipase activity[J]. Journal of Wuxi University of Light Industry,2000,19(2):168-172.
[24] GAELLE P,JACQUES C B. Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase:a simple test for the determination of lipase activity in organic media[J]. Enzyme and Microbial Technology,1996,18:417-422.
[25]滕昆,张吉福,牛福星,等.脂肪酶活力测定中终止反应方法的比较分析[J].广西科学,2014,21(2):115-118.TENG Kun,ZHANG Jifu,NIU Fuxing,et al. Comparative analysis of termination reaction methods in lipase activity assay[J]. Guangxi Science,2014,21(2):115-118.