目的:研究TetR家族转录调控子(TFR)基因敲除对鲍曼不动杆菌生物被膜(BF)生成能力的影响。方法:通过构建自杀质粒的方法敲除鲍曼不动杆菌SZ042株TFR基因,并设计内部引物和外部引物的方法进行敲除株的验证,采用结晶紫染色法对比观察和定...目的:研究TetR家族转录调控子(TFR)基因敲除对鲍曼不动杆菌生物被膜(BF)生成能力的影响。方法:通过构建自杀质粒的方法敲除鲍曼不动杆菌SZ042株TFR基因,并设计内部引物和外部引物的方法进行敲除株的验证,采用结晶紫染色法对比观察和定量分析基因敲除前后BF生成的情况。结果:成功敲除鲍曼不动杆菌中TFR基因,获得TFR基因敲除株SZ042/ΔtetRt株;与野生株相比,敲除株菌膜形成量明显下降,培养4、8、12、16、24 h BF的生成量差异均具有统计学意义(P<0.05)。结论:TFR基因对鲍曼不动杆菌被膜生成和形态维持有促进作用,可以作为开发针对鲍曼不动杆菌BF生成机制的新型抗菌药物的潜在靶点。展开更多
TetR家族转录调控因子(TetR family transcriptional regulators,TFRs)广泛分布于细菌与古菌中,最早被发现的是可控制大肠杆菌四环素外排泵的TetR。TFRs拥有DNA结合和多样的配体感知能力,其作用机制复杂。配体小分子诱导TFRs产生构象变...TetR家族转录调控因子(TetR family transcriptional regulators,TFRs)广泛分布于细菌与古菌中,最早被发现的是可控制大肠杆菌四环素外排泵的TetR。TFRs拥有DNA结合和多样的配体感知能力,其作用机制复杂。配体小分子诱导TFRs产生构象变化,抑制或促进TFRs对其靶点的控制。目前已知的TFRs配体种类繁多,包括糖类、蛋白质、脂肪酸及其衍生物、金属离子等。配体的多样性使得TFRs调节范围很广,包括从基础碳代谢、氮代谢到群体感应、抗生素生物合成等一系列生理过程。文中主要从介导调控角度介绍TFRs配体的研究进展,并结合笔者实验室的研究工作,重点阐述TFRs配体在基础碳代谢、脂肪酸生物合成与降解等初级和次级代谢产物合成过程中的作用机制,及其在基因线路开发和抗生素合成基因的激活等应用方面的研究进展。展开更多
The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and re...The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock （HS）-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor （TetR） and a HS transcription factor （HSF） controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase （GUS） gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40℃ drives GUS expression to a similar level as the Cauliflower mosaic virus （CaMV） 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.展开更多
链霉菌和其他放线菌是活性天然产物的重要来源。随着基因组测序结果的飞速积累,人们发现链霉菌中蕴含丰富的次级代谢途径,远远超过实验室常规条件下检测到的天然产物数量。链霉菌次级代谢途径的表达受到精密而复杂的代谢调控网络的控制...链霉菌和其他放线菌是活性天然产物的重要来源。随着基因组测序结果的飞速积累,人们发现链霉菌中蕴含丰富的次级代谢途径,远远超过实验室常规条件下检测到的天然产物数量。链霉菌次级代谢途径的表达受到精密而复杂的代谢调控网络的控制。链霉菌中的TetR家族成员数量大、作用机制多样,是调控网络的重要成员。链霉菌TetR家族的成员通过配体结合域结合γ-丁酸内酯(Gamma butyrolactone,GBL)信号分子或其他小分子配体,感受外界环境变化,对下游靶基因进行阻遏或激活,从而对次级代谢进行调控。本文从TetR家族的功能及结合配体的类型对该家族成员进行分类,并总结一些已经被证实在链霉菌次级代谢调控中发挥作用的TFRs(TetR family transcriptional regulators)的作用机制。展开更多
放线菌是天然抗生素的重要来源,放线菌中存在着种类繁多的转录因子,精细控制着作为次级代谢产物的抗生素生物合成。作为原核生物单组分信号传递系统中的一个重要家族,TetR家族转录调控因子(TetR family transcriptional regulators,TFRs...放线菌是天然抗生素的重要来源,放线菌中存在着种类繁多的转录因子,精细控制着作为次级代谢产物的抗生素生物合成。作为原核生物单组分信号传递系统中的一个重要家族,TetR家族转录调控因子(TetR family transcriptional regulators,TFRs)广泛参与调控抗生素生物合成、药物外排、初级代谢等多种生理过程。本文综述了近几年放线菌TFRs的研究进展,并结合本实验室的研究工作,从TFRs作用靶基因的角度着重阐述了放线菌TFRs参与几种重要抗生素生物合成的分子调控机制,概述其应答的配体,并总结与展望了放线菌TFRs在抗生素产量提高、沉默基因簇激活、调控元件设计与开发等方面的应用进展。展开更多
The TetR family of regulators are an important group of transcription regulators that regulate diverse cellular processes in prokaryotes.In this study,we found that XNR_0706,a TetR family regulator,controlled the expr...The TetR family of regulators are an important group of transcription regulators that regulate diverse cellular processes in prokaryotes.In this study,we found that XNR_0706,a TetR family regulator,controlled the expression of XNR_0345,XNR_0454,XNR_0513 and XNR_1438 putatively involved in fatty acidβ-oxidation by interacting with the promoter regions in Streptomyces albus B4.The transcription level of these four genes was downregulated in XNR_0706 deletion strain(ΔXNR_0706)and restored by XNR_0706 complementation inΔ0706/pIB-0706,demonstrating that XNR_0706 was a positive transcriptional regulator of the genes.With toxic long-chain fatty acids addition in TSB media,deletion of XNR_0706 caused significantly poor growth,whereas XNR_0706 complementation increased the utilization of additional fatty acids,resulting in restored growth.Fatty acidβ-oxidation is one source of acetyl-and malonyl-CoA precursors for polyketides biosynthesis in actino-bacteria.Overexpression of XNR_0706 in B4/spnNEW,a spinosad heterologous expression strain derived from S.albus B4,increased spinosad yield by 20.6%.Additionally,supplement of 0.3 g/L fatty acids resulted in a further 42.4%increase in spinosad yield.Our study reveals a regulatory mechanism in long-chain fatty acids metabolism in S.albus and these insights into the molecular regulation ofβ-oxidation by XNR_0706 are instrumental for increasing secondary metabolites in actinobacteria.展开更多
A partition of unity(PU) based four-node tetrahedral element with continuous nodal stress(Tetr4-CNS) was recently proposed for static analysis of three-dimensional solids. By simply using the same mesh as the classica...A partition of unity(PU) based four-node tetrahedral element with continuous nodal stress(Tetr4-CNS) was recently proposed for static analysis of three-dimensional solids. By simply using the same mesh as the classical four-node tetrahedral(Tetr4)element, high order global approximation function in the Tetr4-CNS element can be easily constructed without extra nodes or nodal DOFs. In this paper, the Tetr4-CNS element is further applied in the analysis of three dimensional dynamic problems. A series of free vibration and forced vibration problems are solved using the Tetr4-CNS element. The numerical results show that,for regular meshes, accuracy obtained using the Tetr4-CNS element is superior to that obtained using the Tetr4 and eight-node hexahedral(Hexa8) elements. For distorted meshes, the Tetr4-CNS element has better mesh-distortion tolerance than both the Tetr4 and Hexa8 elements.展开更多
Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Here...Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.展开更多
文摘目的:研究TetR家族转录调控子(TFR)基因敲除对鲍曼不动杆菌生物被膜(BF)生成能力的影响。方法:通过构建自杀质粒的方法敲除鲍曼不动杆菌SZ042株TFR基因,并设计内部引物和外部引物的方法进行敲除株的验证,采用结晶紫染色法对比观察和定量分析基因敲除前后BF生成的情况。结果:成功敲除鲍曼不动杆菌中TFR基因,获得TFR基因敲除株SZ042/ΔtetRt株;与野生株相比,敲除株菌膜形成量明显下降,培养4、8、12、16、24 h BF的生成量差异均具有统计学意义(P<0.05)。结论:TFR基因对鲍曼不动杆菌被膜生成和形态维持有促进作用,可以作为开发针对鲍曼不动杆菌BF生成机制的新型抗菌药物的潜在靶点。
文摘TetR家族转录调控因子(TetR family transcriptional regulators,TFRs)广泛分布于细菌与古菌中,最早被发现的是可控制大肠杆菌四环素外排泵的TetR。TFRs拥有DNA结合和多样的配体感知能力,其作用机制复杂。配体小分子诱导TFRs产生构象变化,抑制或促进TFRs对其靶点的控制。目前已知的TFRs配体种类繁多,包括糖类、蛋白质、脂肪酸及其衍生物、金属离子等。配体的多样性使得TFRs调节范围很广,包括从基础碳代谢、氮代谢到群体感应、抗生素生物合成等一系列生理过程。文中主要从介导调控角度介绍TFRs配体的研究进展,并结合笔者实验室的研究工作,重点阐述TFRs配体在基础碳代谢、脂肪酸生物合成与降解等初级和次级代谢产物合成过程中的作用机制,及其在基因线路开发和抗生素合成基因的激活等应用方面的研究进展。
基金supported by the National High-Tech R&D Program of China(2010AA10060705)the Transgenic Engineering Crops Breeding Special Funds from China’s Ministry of Agriculture(2009ZX08010-005B)
文摘The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock （HS）-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor （TetR） and a HS transcription factor （HSF） controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase （GUS） gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40℃ drives GUS expression to a similar level as the Cauliflower mosaic virus （CaMV） 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.
文摘链霉菌和其他放线菌是活性天然产物的重要来源。随着基因组测序结果的飞速积累,人们发现链霉菌中蕴含丰富的次级代谢途径,远远超过实验室常规条件下检测到的天然产物数量。链霉菌次级代谢途径的表达受到精密而复杂的代谢调控网络的控制。链霉菌中的TetR家族成员数量大、作用机制多样,是调控网络的重要成员。链霉菌TetR家族的成员通过配体结合域结合γ-丁酸内酯(Gamma butyrolactone,GBL)信号分子或其他小分子配体,感受外界环境变化,对下游靶基因进行阻遏或激活,从而对次级代谢进行调控。本文从TetR家族的功能及结合配体的类型对该家族成员进行分类,并总结一些已经被证实在链霉菌次级代谢调控中发挥作用的TFRs(TetR family transcriptional regulators)的作用机制。
文摘放线菌是天然抗生素的重要来源,放线菌中存在着种类繁多的转录因子,精细控制着作为次级代谢产物的抗生素生物合成。作为原核生物单组分信号传递系统中的一个重要家族,TetR家族转录调控因子(TetR family transcriptional regulators,TFRs)广泛参与调控抗生素生物合成、药物外排、初级代谢等多种生理过程。本文综述了近几年放线菌TFRs的研究进展,并结合本实验室的研究工作,从TFRs作用靶基因的角度着重阐述了放线菌TFRs参与几种重要抗生素生物合成的分子调控机制,概述其应答的配体,并总结与展望了放线菌TFRs在抗生素产量提高、沉默基因簇激活、调控元件设计与开发等方面的应用进展。
基金support from the National Key Research and Development Program of China(No.2018YFA0900404).
文摘The TetR family of regulators are an important group of transcription regulators that regulate diverse cellular processes in prokaryotes.In this study,we found that XNR_0706,a TetR family regulator,controlled the expression of XNR_0345,XNR_0454,XNR_0513 and XNR_1438 putatively involved in fatty acidβ-oxidation by interacting with the promoter regions in Streptomyces albus B4.The transcription level of these four genes was downregulated in XNR_0706 deletion strain(ΔXNR_0706)and restored by XNR_0706 complementation inΔ0706/pIB-0706,demonstrating that XNR_0706 was a positive transcriptional regulator of the genes.With toxic long-chain fatty acids addition in TSB media,deletion of XNR_0706 caused significantly poor growth,whereas XNR_0706 complementation increased the utilization of additional fatty acids,resulting in restored growth.Fatty acidβ-oxidation is one source of acetyl-and malonyl-CoA precursors for polyketides biosynthesis in actino-bacteria.Overexpression of XNR_0706 in B4/spnNEW,a spinosad heterologous expression strain derived from S.albus B4,increased spinosad yield by 20.6%.Additionally,supplement of 0.3 g/L fatty acids resulted in a further 42.4%increase in spinosad yield.Our study reveals a regulatory mechanism in long-chain fatty acids metabolism in S.albus and these insights into the molecular regulation ofβ-oxidation by XNR_0706 are instrumental for increasing secondary metabolites in actinobacteria.
基金supported by the National Natural Science Foundation of China(Grant Nos.51609240,11572009,51538001,51579235&41472288)the National Basic Research Program of China(Grant No.2014CB047100)
文摘A partition of unity(PU) based four-node tetrahedral element with continuous nodal stress(Tetr4-CNS) was recently proposed for static analysis of three-dimensional solids. By simply using the same mesh as the classical four-node tetrahedral(Tetr4)element, high order global approximation function in the Tetr4-CNS element can be easily constructed without extra nodes or nodal DOFs. In this paper, the Tetr4-CNS element is further applied in the analysis of three dimensional dynamic problems. A series of free vibration and forced vibration problems are solved using the Tetr4-CNS element. The numerical results show that,for regular meshes, accuracy obtained using the Tetr4-CNS element is superior to that obtained using the Tetr4 and eight-node hexahedral(Hexa8) elements. For distorted meshes, the Tetr4-CNS element has better mesh-distortion tolerance than both the Tetr4 and Hexa8 elements.
基金the National Program on Key Basic Research Project(973 program,2013CB734000)the National Natural Science Foundation of China(Grant Nos.31300081,31570074)the Initial Foundation of Doctoral Scientific Research in Anhui University(01001904,J01001935).
文摘Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.
