地衣芽孢杆菌中木糖操纵子受葡萄糖胁迫的转录调控特性
|
摘要
木糖操纵子是芽孢杆菌中常用的表达元件,但目前对其认识只停留在静态机理层面,关于其在发酵过程中转录调控特性的研究还鲜见报道.利用qPCR技术探究在葡萄糖胁迫下地衣芽孢杆菌木糖诱导的木糖异构酶基因在发酵过程中的转录水平,考察菌体的生长状态,并通过二硝基水杨酸(DNS)法及高效液相色谱(High performance liquid chromatography,HPLC)法测定发酵过程中糖浓度变化.结果显示:在实验所设置的地衣芽孢杆菌代谢相对稳定的条件下,地衣芽孢杆菌木糖启动子转录强度在稳定期以前均呈增加的趋势,在对数生长末期或稳定前期转录强度最高,约是7 h时的14倍,随后呈下降趋势;进一步研究发现20-180 g/L葡萄糖浓度均对其表现为抑制,且抑制程度一致,当葡萄糖含量极少或者没有而木糖存在的情况下,启动子转录强度极高.本研究表明以地衣芽孢杆菌为宿主的木糖诱导系统在菌体对数生长末期诱导效果最佳;当环境中葡萄糖含量极少或者没有而木糖存在的情况下,更有利于启动子表达;结果对利用木糖诱导型重组地衣芽孢杆菌诱导发酵有一定的启示与指导意义.(图5表2参24)
The xylose operon is a commonly used as a expression element in Bacillus spp., but our present understanding of its function remains only at the level of its static mechanism, whereas studies on its transcriptional regulation characteristics in the fermentation process have only been done rarely. Obtaining further understanding of this operon's characteristics during the fermentation process should help to provide a scientific basis for the application of xylose-inducible expression systems in the fermentation process. In this study, the dinitrosalicylic acid(DNS) method was used to control the glucose content during the fermentation process, and then quantitative reverse transcriptase polymerase chain reaction(RT-qPCR) technology and high-performance liquid chromatography(HPLC) were used to accurately analyze the residual sugar content and quantify the transcription levels of the operon gene, respectively, in samples collected during the fermentation process. As a result, the glucose concentration was controlled within a desired range so that the growth and metabolism of Bacillus licheniformis in the samples were stable. Meanwhile, a systematic method was established for detecting the expression levels of the xyloseinducible promoter gene during the fermentation process, which revealed the transcriptional regulation characteristics of the xylose-inducible promoter gene in the fermentation process. The transcription level of the xylose-inducible promoter gene increased until the stationary phase, with the highest transcription level attained at the end of the logarithmic growth or prestabilization phase, which was increased by about 14 times after 7 h, and then after this it began to decline. Further, glucose concentrations of 20-180 g/L inhibited the transcription of this promotor gene, and the degree of inhibition was consistent across this concentration range. When the glucose content was low or zero, and xylose was present, the transcription level of the promoter was extremely high. The results of this study indicated that the xylose-inducible system with Bacillus licheniformis as the host has the best induction effect at the end of the logarithmic growth phase of microbial fermentation, and when there is little or no glucose in the environment and xylose is present conditions are even more favorable for promoter expression. These findings are significant because they could be used to guide the optimization of the application of inducible fermentation by xylose-induced recombinant Bacillus licheniformis.
The xylose operon is a commonly used as a expression element in Bacillus spp., but our present understanding of its function remains only at the level of its static mechanism, whereas studies on its transcriptional regulation characteristics in the fermentation process have only been done rarely. Obtaining further understanding of this operon's characteristics during the fermentation process should help to provide a scientific basis for the application of xylose-inducible expression systems in the fermentation process. In this study, the dinitrosalicylic acid(DNS) method was used to control the glucose content during the fermentation process, and then quantitative reverse transcriptase polymerase chain reaction(RT-qPCR) technology and high-performance liquid chromatography(HPLC) were used to accurately analyze the residual sugar content and quantify the transcription levels of the operon gene, respectively, in samples collected during the fermentation process. As a result, the glucose concentration was controlled within a desired range so that the growth and metabolism of Bacillus licheniformis in the samples were stable. Meanwhile, a systematic method was established for detecting the expression levels of the xyloseinducible promoter gene during the fermentation process, which revealed the transcriptional regulation characteristics of the xylose-inducible promoter gene in the fermentation process. The transcription level of the xylose-inducible promoter gene increased until the stationary phase, with the highest transcription level attained at the end of the logarithmic growth or prestabilization phase, which was increased by about 14 times after 7 h, and then after this it began to decline. Further, glucose concentrations of 20-180 g/L inhibited the transcription of this promotor gene, and the degree of inhibition was consistent across this concentration range. When the glucose content was low or zero, and xylose was present, the transcription level of the promoter was extremely high. The results of this study indicated that the xylose-inducible system with Bacillus licheniformis as the host has the best induction effect at the end of the logarithmic growth phase of microbial fermentation, and when there is little or no glucose in the environment and xylose is present conditions are even more favorable for promoter expression. These findings are significant because they could be used to guide the optimization of the application of inducible fermentation by xylose-induced recombinant Bacillus licheniformis.
引文
1牛丹丹,石贵阳,王正祥.分泌高效蛋白的地衣芽孢杆菌及其工业应用[J].生物技术通报,2009(6):45-50[Niu DD,Shi GY,Wang ZX.Research progress of high protein secretion Bacillus licheniformis and its industrial application[J].Biotechnol Bull,2009(6):45-50]
2 Kim JH,Yang YM,Ji CJ,Ryu SH,Won YB,Ju SY,Kwon Y,Lee YE,Youn H,Lee JW.The inability of Bacillus licheniformis perR mutant to grow is mainly due to the lack of PerR-mediated fur repression[J].JMicrobiol,2017,55(6):457-463
3 Li YR,Gu ZH,Zhang L,Ding ZY,Shi GY.Inducible expression of trehalose synthase in Bacillus licheniformis[J].Protein Expression Purif,2016,130:115-122
4 Ying Q,Zhang C,Guo F,Wang S,Bie X,Lu F,Lu Z.Secreted expression of a hyperthermophilicα-amylase gene from Thermococcus sp.HJ21 in Bacillus subtilis[J].J Mol Microbiol Biotechnol,2012,22(6):392
5杨韵霏,李由然,张梁,李赢,顾振华,丁重阳,石贵阳.细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达[J].微生物学通报,2017,44(2):263-273[Yang YF,Li YR,Zhang L,Li Y,Gu ZH,Ding ZY,Shi GY.Inducible heterogenous expression of bacterial maltogenic amylase in Bacillus subtilis[J].Microbiol Chin,2017,44(2):263-273]
6 Scheler A,Hillen W.Regulation of xylose utilization in Bacillus licheniformis:Xyl repressor-xyl-operator interaction studied by DNAmodification protection and interference[J].Mol Microbiol,2010,13(3):505-512
7 Silvia H,Angelika H,Christopher T,Margot S,Esther E,Reingard G,Stefan H.Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum[J].Microb Cell Fact,2016,15(1):50
8 Derveaux S,Vandesompele J,Hellemans J.How to do successful gene expression analysis using real-time PCR[J].Methods,2010,50(4):227-230
9尹萌萌,贺婷停,王超,宋婷,王海燕.响应面法优化短小芽孢杆菌SCU11发酵产碱性蛋白酶及关键基因转录调控分析[J].应用与环境生物学报,2016,22(3):371-376[Yin MM,He TT,Wang C,Song T,Wang HY.Optimization of fermentation conditions for alkaline proteases production by Bacillus pumilus SCU11 and tanscriptional regulation analysis of critical genes[J].Chin J Appl Environ Biol,2016,22(3):371-376]
10 Wiegand S,Voigt B,Albrecht D,Bongaerts J,Evers S,Hecker M,Daniel R,Liesegang H.Fermentation stage-dependent adaptations of Bacillus licheniformis during enzyme production[J].Microb Cell Fact,2013,12(1):1-18
11谭忠元,张智,付茂红,罗笛,钟娟,周金燕,杨杰,肖亮,谭红.补料对发酵工艺中枯草芽胞杆菌ZK8产伊枯草菌素A调控基因的影响[J].中国农业科技导报,2015,17(3):35-41[Tan ZY,Zhang Z,Fu MH,Luo D,Zhong J,Zhou JY,Yang J,Xiao L,Tan H.Effect of feeding on regulatory genes of Bacillus subtilis ZK8 synthesizing Iturin A in fermentation process[J].J Agric Sci Technol,2015,17(3):35-41]
12王珊瑛.枯草芽孢杆菌木糖诱导型穿梭质粒的构建[D].无锡:江南大学,2016[Wang SY.Construction of shuttle vectors for Bacillus Subtills[D].Wuxi:Jiangnan University,2016]
13范贺超.新型木糖利用酵母的评价及其遗传表达系统构建[D].无锡:江南大学,2016[Fan HC.Evaluation of physiological and metabolic character istics of new xylose-utilizing yeasts and their genetic engineering.[D]:Wuxi:Jiangnan University,2015]
14 Bustin SA,Benes V,Garson JA,.The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments[J].Clin Chem,2009,55(4):611-622
15 Hoffmann K,Wollherr A,Larsen M,Rachinger M,Liesegang H,Ehrenreich A,Meinhardt F.Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence[J].Appl Environ Microbiol,2010,76(15):5046-5057
16 Jakobs M,Hoffmann K,Grabke A,Neuber S,Liesegang H,Volland S,Meinhardt F.Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945a strains[J].Microbiology,2014,160(10):2136-2147
17 Qiu YM,Zhang JY,Li L,Wen ZY,Nomura CT,Wu SL,Chen SW.Engineering Bacillus licheniformis for the production of meso-2,3-butanediol[J].Biotechnol Biofuels,2016,9(1):117-130
18张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125[Zhang YF,Zhao LJ,Zeng YL.Selection and application of reference genes for gene expression studies[J].Plant Physiol J,2014,50(8):1119-1125]
19 Hruz T,Wyss M,Docquier M,Pfaff l MW,Masanetz S,Borghi L,Verbr ugghe P,Kalaydjieva L,Bleuler S,Laule O,Descombes P,Gruissem W,Zimmermann P.RefGenes:Identification of reliable and condition specific reference genes for RT-qPCR data normalization[J].BMC Genomics,2011,12(1):156-170
20 Rachinger M,Volland S,Meinhardt F,Daniel R,Liesegang H.First Insights into the completely annotated genome sequence of Bacillus licheniformis strain 9945a[J].Genome Announce,2013,1(4):1-2
21 Voigt B,Hoi LT,Jürgen B,Albrecht D,Ehrenreich A,Veith B,Evers S,Maurer KH,Hecker M,Schweder T.The glucose and nitrogen starvation response of Bacillus licheniformis[J].Proteomics,2010,7(3):413-423
22范如意.基因工程技术改造地衣芽孢杆菌实现中温α-淀粉酶高效表达[D].无锡:江南大学,2015[Fan RY.Heterologous expression ofα-amylase in Bacillus licheniformis with genetic engineering modification.[D]:Wuxi:Jiangnan University,2015]
23 Bastet L,Turcotte P,Wade JT,Lafontaine DA.Maestro of regulation:riboswitches orchestrate gene expression at the levels of translation,transcription and mRNA decay[J].RNA Biol,2018,15(3):1-4
24 Gu Y,Ding Y,Ren C,Sun Z,Rodionov DA,Zhang WW,Yang S,Yang C,Jiang WH.Reconstruction of xylose utilization pathway and regulons in Firmicutes[J].BMC Genomics,2010,11(1):255-269
2 Kim JH,Yang YM,Ji CJ,Ryu SH,Won YB,Ju SY,Kwon Y,Lee YE,Youn H,Lee JW.The inability of Bacillus licheniformis perR mutant to grow is mainly due to the lack of PerR-mediated fur repression[J].JMicrobiol,2017,55(6):457-463
3 Li YR,Gu ZH,Zhang L,Ding ZY,Shi GY.Inducible expression of trehalose synthase in Bacillus licheniformis[J].Protein Expression Purif,2016,130:115-122
4 Ying Q,Zhang C,Guo F,Wang S,Bie X,Lu F,Lu Z.Secreted expression of a hyperthermophilicα-amylase gene from Thermococcus sp.HJ21 in Bacillus subtilis[J].J Mol Microbiol Biotechnol,2012,22(6):392
5杨韵霏,李由然,张梁,李赢,顾振华,丁重阳,石贵阳.细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达[J].微生物学通报,2017,44(2):263-273[Yang YF,Li YR,Zhang L,Li Y,Gu ZH,Ding ZY,Shi GY.Inducible heterogenous expression of bacterial maltogenic amylase in Bacillus subtilis[J].Microbiol Chin,2017,44(2):263-273]
6 Scheler A,Hillen W.Regulation of xylose utilization in Bacillus licheniformis:Xyl repressor-xyl-operator interaction studied by DNAmodification protection and interference[J].Mol Microbiol,2010,13(3):505-512
7 Silvia H,Angelika H,Christopher T,Margot S,Esther E,Reingard G,Stefan H.Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum[J].Microb Cell Fact,2016,15(1):50
8 Derveaux S,Vandesompele J,Hellemans J.How to do successful gene expression analysis using real-time PCR[J].Methods,2010,50(4):227-230
9尹萌萌,贺婷停,王超,宋婷,王海燕.响应面法优化短小芽孢杆菌SCU11发酵产碱性蛋白酶及关键基因转录调控分析[J].应用与环境生物学报,2016,22(3):371-376[Yin MM,He TT,Wang C,Song T,Wang HY.Optimization of fermentation conditions for alkaline proteases production by Bacillus pumilus SCU11 and tanscriptional regulation analysis of critical genes[J].Chin J Appl Environ Biol,2016,22(3):371-376]
10 Wiegand S,Voigt B,Albrecht D,Bongaerts J,Evers S,Hecker M,Daniel R,Liesegang H.Fermentation stage-dependent adaptations of Bacillus licheniformis during enzyme production[J].Microb Cell Fact,2013,12(1):1-18
11谭忠元,张智,付茂红,罗笛,钟娟,周金燕,杨杰,肖亮,谭红.补料对发酵工艺中枯草芽胞杆菌ZK8产伊枯草菌素A调控基因的影响[J].中国农业科技导报,2015,17(3):35-41[Tan ZY,Zhang Z,Fu MH,Luo D,Zhong J,Zhou JY,Yang J,Xiao L,Tan H.Effect of feeding on regulatory genes of Bacillus subtilis ZK8 synthesizing Iturin A in fermentation process[J].J Agric Sci Technol,2015,17(3):35-41]
12王珊瑛.枯草芽孢杆菌木糖诱导型穿梭质粒的构建[D].无锡:江南大学,2016[Wang SY.Construction of shuttle vectors for Bacillus Subtills[D].Wuxi:Jiangnan University,2016]
13范贺超.新型木糖利用酵母的评价及其遗传表达系统构建[D].无锡:江南大学,2016[Fan HC.Evaluation of physiological and metabolic character istics of new xylose-utilizing yeasts and their genetic engineering.[D]:Wuxi:Jiangnan University,2015]
14 Bustin SA,Benes V,Garson JA,.The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments[J].Clin Chem,2009,55(4):611-622
15 Hoffmann K,Wollherr A,Larsen M,Rachinger M,Liesegang H,Ehrenreich A,Meinhardt F.Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence[J].Appl Environ Microbiol,2010,76(15):5046-5057
16 Jakobs M,Hoffmann K,Grabke A,Neuber S,Liesegang H,Volland S,Meinhardt F.Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945a strains[J].Microbiology,2014,160(10):2136-2147
17 Qiu YM,Zhang JY,Li L,Wen ZY,Nomura CT,Wu SL,Chen SW.Engineering Bacillus licheniformis for the production of meso-2,3-butanediol[J].Biotechnol Biofuels,2016,9(1):117-130
18张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125[Zhang YF,Zhao LJ,Zeng YL.Selection and application of reference genes for gene expression studies[J].Plant Physiol J,2014,50(8):1119-1125]
19 Hruz T,Wyss M,Docquier M,Pfaff l MW,Masanetz S,Borghi L,Verbr ugghe P,Kalaydjieva L,Bleuler S,Laule O,Descombes P,Gruissem W,Zimmermann P.RefGenes:Identification of reliable and condition specific reference genes for RT-qPCR data normalization[J].BMC Genomics,2011,12(1):156-170
20 Rachinger M,Volland S,Meinhardt F,Daniel R,Liesegang H.First Insights into the completely annotated genome sequence of Bacillus licheniformis strain 9945a[J].Genome Announce,2013,1(4):1-2
21 Voigt B,Hoi LT,Jürgen B,Albrecht D,Ehrenreich A,Veith B,Evers S,Maurer KH,Hecker M,Schweder T.The glucose and nitrogen starvation response of Bacillus licheniformis[J].Proteomics,2010,7(3):413-423
22范如意.基因工程技术改造地衣芽孢杆菌实现中温α-淀粉酶高效表达[D].无锡:江南大学,2015[Fan RY.Heterologous expression ofα-amylase in Bacillus licheniformis with genetic engineering modification.[D]:Wuxi:Jiangnan University,2015]
23 Bastet L,Turcotte P,Wade JT,Lafontaine DA.Maestro of regulation:riboswitches orchestrate gene expression at the levels of translation,transcription and mRNA decay[J].RNA Biol,2018,15(3):1-4
24 Gu Y,Ding Y,Ren C,Sun Z,Rodionov DA,Zhang WW,Yang S,Yang C,Jiang WH.Reconstruction of xylose utilization pathway and regulons in Firmicutes[J].BMC Genomics,2010,11(1):255-269