微生物学通报  2019, Vol. 46 Issue (2): 407−414

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文章信息

倪静姝, 汪焰胜, 吴杭, 张部昌
NI Jing-Shu, WANG Yan-Sheng, WU Hang, ZHANG Bu-Chang
放线菌中与抗生素合成相关TetR家族转录因子的研究进展
Progress in TetR family transcriptional regulator related to antibiotic synthesis in actinomycetes
微生物学通报, 2019, 46(2): 407-414
Microbiology China, 2019, 46(2): 407-414
DOI: 10.13344/j.microbiol.china.180936

文章历史

收稿日期: 2018-11-20
接受日期: 2019-01-22
网络首发日期: 2019-01-25
放线菌中与抗生素合成相关TetR家族转录因子的研究进展
倪静姝1 , 汪焰胜1 , 吴杭2 , 张部昌1     
1. 安徽大学物质科学与信息技术研究院    安徽 合肥    230601;
2. 安徽大学生命科学学院    安徽 合肥    230601
摘要: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。
关键词TetR家族调控因子    放线菌    抗生素    靶基因    配体    
Progress in TetR family transcriptional regulator related to antibiotic synthesis in actinomycetes
NI Jing-Shu1 , WANG Yan-Sheng1 , WU Hang2 , ZHANG Bu-Chang1     
1. Institutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, China;
2. School of Life Sciences, Anhui University, Hefei, Anhui 230601, China
Abstract: Actinomycetes are an important source of natural antibiotics, and have a wide variety of transcription factors that precisely control the biosynthesis of antibiotics as secondary metabolites. As an important family of prokaryotic single-component signaling systems, TetR family transcriptional regulators (TFRs) are involved in the regulation of multiple cellular activities such as antibiotic biosynthesis, drug efflux, primary metabolism. On the basis of the recent studies in our lab and the research advances published, we review here the molecular regulatory mechanism of TFRs involved in the biosynthesis of several important antibiotics from the perspective of regulated target genes, and outlined the ligands of TFRs. Finally, this review summarizes and indicates the application of TFRs in increasing antibiotic production, activating silent cryptic gene clusters and artificially exploiting synthetic biology elements.
Keywords: TetR family transcriptional regulators    Actinomycetes    Antibiotics    Targets    Ligand    

抗生素是微生物通过复杂生物合成途径产生的具有结构多样与生物活性广泛的次级代谢产物[1],例如阿维菌素、红霉素、井冈霉素、林可霉素、多杀菌素等。随着基因组测序技术的发展,许多放线菌的基因组序列被测定完成,发现放线菌基因组含有大量次级代谢物生物合成基因簇,一般包括生物合成的结构基因、抗性基因和调控基因[2]。除了位于抗生素生物合成基因簇内的途径特异性调控基因外,放线菌还编码多效调控或全局性调控等种类繁多的转录因子,形成复杂多样的分子调控模式[3]

近年来,应用基因工程技术与分子生物学等方法[4],对放线菌转录调控基因的功能和作用机制进行了研究,发现TetR家族转录调控因子(TetR family transcriptional regulators,TFRs)不仅可以调控抗生素的生物合成,还可以调节放线菌形态分化、药物外排、初级代谢、群体感应、应对渗透胁迫等生理过程[5-7]。TetR是以该家族第一个成员四环素抗性阻遏蛋白(Tet repressor,TetR)命名的[7],常以二聚体形式发挥作用,二聚体包括两个同型单体结构,每个单体含两个结构域:DNA结合结构域和配体结合结构域,前者一般在N端,序列保守,存在一个螺旋-转角-螺旋基序;后者位于C端,一级序列差异大,表明TFRs配体具有多样性[7]

TFRs广泛分布在细菌或古菌等微生物中,其中在放线菌中该家族成员数量最多。天蓝色链霉菌(Streptomyces coelicolor)编码153个TFRs,阿维链霉菌(Streptomyces avermitilis)编码115个TFRs,灰色链霉菌(Streptomyces griseus)编码104个TFRs,红色糖多孢菌(Saccharopolyspora erythraea)编码101个TFRs等,说明TFRs在放线菌复杂的形态分化和生理代谢途径中发挥着重要作用[8-9]。研究发现,TFRs可作用抗生素生物合成基因簇,还可调控簇外基因的表达,进而负调控或正调控抗生素的生物合成[10-11]。本文从TFRs作用靶基因的角度出发,结合本实验室的研究工作,综述了近几年放线菌中TFRs参与几种重要抗生素生物合成的分子调控机制(表 1),并概述其应答的配体。在此基础上,总结与展望了TFRs在抗生素产量提高、沉默基因簇激活和调控元件设计上的应用。

表 1 放线菌抗生素合成相关TFRs调控的靶基因 Table 1 Target genes regulated by TFRs related to antibiotic biosynthesis in actinomycetes
TFRs 菌株
Strains
调控的靶基因
Target genes
调控抗生素生物合成
Antibiotics
参考文献
References
SACE_7301 Saccharopolyspora erythraea eryA, ermE +: Erythromycin [11]
SACE_3446 Saccharopolyspora erythraea eryA, ermE -: Erythromycin [12]
SACE_3986 Saccharopolyspora erythraea SACE_3985 -: Erythromycin [13]
SACE_5754 Saccharopolyspora erythraea SACE_0388, SACE_6194 -: Erythromycin [14]
SACE_3396 Saccharopolyspora erythraea pccBC, pccA, SACE_3880–3884 -: Erythromycin [15]
SLCG_2919 S. lincolnensis lm cluster, SCGL_2920 -: Lincomycin [10]
AveR1 S. avermitilis aveR, aco, cyp17, avaR1, avaR2, avaR3, aveT, rpsQ, rpmB, sav3560, sig29, pstB, nuoB, leuD, amfC -: Avermectin [16]
AveR2 S. avermitilis aveR, aco, cyp17, avaR1, avaR2, avaR3, aveT, sav1230, sav3560, sig29, pstB, nuoB1, leuD, sav2051, rpsQ, rpmB, folp2, amfC -: Avermectin [17]
SAV7471 S. avermitilis sav1104, sav1258, sav7472-sav7473 -: Avermectin [18]
SAV151 S. avermitilis sav152-sav153-sav154 -: Avermectin [19]
SAV576 S. avermitilis sav575, sav576, sav577 -: Avermectin [20-21]
SAV577 S. avermitilis sav575, sav576 -: Avermectin [21]
AveT S. avermitilis aveT, pepD2, aveM +: Avermectin [22]
XdhR S. coelicolor xdhR, sco1134, actⅡ-4, actⅡ-1, xdhABC -: Actinorhodin [23]
AtrA S. roseosporus dptE, atrA +: Daptomycin [24]
DepR1 S. roseosporus dptE, depR1 +: Daptomycin [25]
JadR* S. venezuelae jadY, jadR1, jadI, jadE -: Jadomycin [26]
JadR2 S. venezuelae jadR1, jml -: Jadomycin [27]
CHlF1 S. antibioticus chlJ, hlF1, chlG, chlK +: Chlorothricin [28]
RifQ Amycolatopsis mediterranei rifP -: Rifamycin [1]
PaaP S. pristinaespiralis paa cluster +: Pristinamycin PI [29]
注:+:促进或激活抗生素生物合成;-:抑制抗生素生物合成.
Note: +: Enhancing or activating antibiotics biosynthesis; -: Repressing antibiotics biosynthesis.
1 TFRs调控簇内基因

TFRs通过其DNA结合结构域与靶基因的启动子区结合,从而调控靶基因的转录。TFRs直接调控抗生素生物合成的方式是与抗生素生物合成基因簇内基因启动子区结合。

红霉素是由红色糖多孢菌产生的一类大环内酯类抗生素,但其合成基因簇中不存在调控基因,推测簇外调控因子在红霉素生物合成中扮演着重要的角色[8, 30]。红色糖多孢菌全基因组中存在大量的转录调控基因,最早报道的多效转录因子BldD可直接调控红霉素生物合成[30]。这些基因编码多种转录调控蛋白家族,其中成员最多的是TFRs。本实验室研究发现两个TFRs (SACE_7301和SACE_3446),直接与红霉素生物合成基因簇中聚酮合成酶基因eryAI和抗性基因ermE启动子区结合,分别正向和负向调控红霉素的生物合成[11-12, 30]。近期,本实验室在林可链霉菌(Streptomyces lincolnensis)中鉴定了第一个TFR (SLCG_2919),可与林可霉素基因簇中所有结构基因、抗性基因与调控基因启动子区结合,通过直接控制这些基因的转录,负调控林可霉素的生物合成[10]

在阿维链霉菌中阿维菌素生物合成基因簇编码一个特异性调控蛋白AveR[31],近年来研究发现的两个TFRs (AveR1和AveR2)都可与aveR启动子区结合,负调控阿维菌素的生物合成[16-17]。在模式放线菌天蓝色链霉菌中,发现一个TFR (XdhR)通过与放线紫红素生物合成基因簇内编码途径特异性调节蛋白的两个基因acIIt-4actII-1启动子区结合,进而负调控放线紫红素的产生[23]。在产达托霉素的玫瑰孢链霉菌(Streptomyces roseosporus)中,目前已报道两个TFRs (AtrA和DepR1)可直接结合达托霉素生物合成基因簇中dptE的启动子,激活达托霉素基因簇的转录表达,正调控达托霉素的生物合成[24-25],其中AtrA同源蛋白在链霉菌中广泛存在(图 1),调控着多种类型抗生素的生物合成,暗示TFRs调控功能的保守性。

图 1 玫瑰孢链霉菌AtrA与林可链霉菌SLCG_2919的系统进化树 Figure 1 Phylogenetic tree of AtrA and SLCG_2919 in Streptomyces 注:A:玫瑰孢链霉菌AtrA的进化树;B:林可链霉菌SLCG_2919的进化树.分支点上数字表示蛋白的相似性. Note: A: Phylogenetic tree of AtrA; B: Phylogenetic tree of SLCG_2919. The number at each knot represents protein identities.
2 TFRs调控簇外基因 2.1 抗生素生物合成前体代谢基因

微生物次级代谢物大多数源自一些关键初级代谢物,如短链脂肪酸、氨基酸、糖与氨基糖等[32],这些前体是决定次级代谢物生物合成产量的关键[33]

红色糖多孢菌中红霉素生物合成的直接前体是丙酰辅酶A和甲基丙二酰辅酶A[34-36],甲基丙二酰辅酶A可通过甲基丙二酰辅酶A变位酶途径和丙酰辅酶A羧化酶途径合成[15],丙酰辅酶A可以通过乙酰辅酶A羧化酶途径和分支氨基酸降解途径生成[37-38]。近期发现了一个TFR (PccD),可通过负调控下游的丙酰辅酶A羧化酶基因簇的转录,影响甲基丙二酰辅酶A的供应;同时下调分支氨基酸降解的bkd操纵子转录水平,减少丙酰辅酶A的供应,进而负调控红霉素的生物合成[15, 37]

阿维菌素的生物合成直接前体是异丁酰基辅酶A、2-甲基丁酰辅酶A、丙二酰辅酶A和甲基丙二酰基辅酶A,合成过程需要大量的辅酶A供应[39]sav7472-sav7473编码的产物可能与泛酸和辅酶A的代谢有关,TFR SAV7471可以直接抑制下游sav7472-sav7473操纵子转录,SAV7471可能是通过抑制CoA浓度影响前体的形成从而负调控阿维菌素的生物合成[18]。另一个TFR (SAV151)是通过直接抑制临近基因簇sav152-sav153-sav154,负调控阿维菌素的生物合成。sav152sav154分别编码的脱氢酶和水解酶可能为阿维菌素生物合成提供能量或前体[19]

始旋链霉菌(Streptomyces pristinaespiralis)中,L-苯基甘氨酸是原始霉素Ⅰ生物合成前体,可由苯乙酰辅酶A生成。TFR PaaR因为抑制paa簇内基因的表达,使得苯乙酰辅酶A更多流向L-苯基甘氨酸生物合成途径,从而促进原始霉素Ⅰ的生物合成[29]

2.2 其他初级代谢基因

TFR除了可以调控上述前体代谢,还可以参与调控脂质代谢、嘌呤代谢等。本实验室发现SACE_3986通过抑制其上游编码短链脱氢酶/还原酶SACE_3985基因的转录,间接负调控红霉素生物合成[13];SACE_3446抑制上游编码长链脂肪酸辅酶A连接酶基因SACE_3447的转录,抑制红霉素的产生[12]。阿维链霉菌中,SAV7471还可以直接结合靶基因sav1104sav1258启动子区,这两个基因都编码乙酰辅酶A合成酶/长链脂肪酸辅酶A连接酶,可能参与脂质代谢[18]xdhABC基因簇编码黄嘌呤脱氢酶复合物,XdhR通过直接抑制靶基因簇xdhABC的转录调控放线紫红素的生物合成[23]

2.3 簇外调控基因与抗性基因

TFRs还可以通过调控簇外调控基因以及自身参与抗生素的生物合成。阿维链霉菌中两个相邻TFRs (SAV576和SAV577)都负调控阿维菌素的生物合成,两者互相抑制对方转录,其中SAV576还抑制自身的转录表达[20-21]

细菌具有不同的抗性机制对抗有毒成分,这些有毒成分包括药物和抗生素等。TFR家族发现的第一个成员TetR,就是调控四环素的转运被命名的[7]。研究发现林可链霉菌SLCG_2919调控的临近靶基因SLCG_2920编码了一个ATP/GTP结合蛋白,推测SLCG_2919与SLCG_2920蛋白之间形成了一个反馈调节回路,SLCG_2920过表达可提高林可链霉菌对林可霉素的抗性[10],SLCG_2919及其临近编码ATP/GTP结合蛋白的基因组合广泛存在于多种链霉菌中(图 1)。AveT作用的靶基因之一aveM编码一个跨膜外排蛋白,该蛋白能够抑制阿维菌素的生物合成,AveT通过抑制aveM的转录表达提高阿维菌素的产量[22]

2.4 γ-丁酸内酯类信号分子的生物合成基因

γ-丁酸内酯等自调控因子可作为信号分子,控制相关次级代谢物的产生与形态分化,如最初发现的灰色链霉菌中的A因子和最近在圈卷产色链霉菌发现的3个丁烯羟酸内酯SAB1、2、3[40-42]。在阿维链霉菌中,AveR1和AveR2编码γ-丁酸内酯受体同系物。Avenolide是阿维链霉菌中的一种新型的丁烯羟酸内酯类小分子,该物质在低浓度下能够诱导阿维菌素的生物合成[43]。AveR1和AveR2则可与Avenolide生物合成基因aco启动子区结合,抑制aco的转录,从而负调控阿维菌素的生物合成[16-17]

3 TFRs的配体

TFRs与其作用靶点的结合受到一些小分子配体的调控,即TFRs与配体结合后改变其构象,增强或抑制其与靶基因的结合力,进而控制其靶基因的转录表达[7]。近年来研究发现,放线菌中与抗生素合成相关的TFRs主要应答γ-丁酸内酯类信号分子、ppGpp、GTP和抗生素及其生物合成中间体或前体(表 2)。阿维链霉菌中AveR1和AveR2可以结合γ-丁酸内酯类信号分子,此外AveR2还能结合外源抗生素如杰多霉素B、安普霉素和潮霉素B[16-17];AveT则是应答阿维菌素B1生物合成的中间体C-5-O-B1[22]。地中海拟无枝酸菌(Amycolatopsis mediterranei)利福霉素生物合成基因簇中TFR RifQ与利福霉素形成了自反馈调控回路,调节胞内利福霉素的浓度[1]。天蓝色链霉菌XdhR结合ppGpp或GTP后减弱了其与靶基因启动子区的结合力[23]。在抗生链霉菌(Streptomyces antibioticus) chl基因簇中CHlF1不仅可以响应终产物氯丝霉素,还可以结合该抗生素生物合成的中间产物去甲基水杨酸-氯丝霉素(Demethylsalicycloyl- chlorothricin)和脱氯-氯丝霉素(Deschloro- chlorothricin),从而调节胞内抗生素的浓度[28]。红色糖多孢菌PccD通过结合甲基丙二酸调控下游靶基因[15]。委内瑞拉链霉菌(Streptomyces venezuelae) JadR*和JadR2是杰多霉素生物合成基因簇中的两个调控因子,其中JadR*与2, 3-脱氢-UWM6 (2, 3-Dehydro-UWM6,DHU)、脱氢腊伯罗霉素(Dehydrorabelomycin,DHR)结合形成前馈调控回路,通过及时供应辅因子FMNH2/FADH2使DHR转化为杰多霉素A,此外JadR*还可以应答杰多霉素B和杰多霉素A[26];而JadR2则是通过结合杰多霉素和氯霉素对这两种抗生素的生物合成进行交叉调控[44-45]

表 2 TFRs的配体与保守的DNA结合序列 Table 2 Ligands and conserved DNA-binding sequences of TFRs
TFRs 配体
Ligand
N端结合的保守序列
Conserved sequence of TFRs (5′→3′)
参考文献
References
RifQ Rifamycin B ACCGACCCTTATATGGTGTATAAGAA [1]
PccD Methylmalonic acid t/aTGACGg/cTGt/cTGt/a [15]
AveR1 Avenolide AWWCCRBBHDDNMSGTWT [16]
AveR2 Avenolide, Apramycin, Jadomycin B, Hygromycin B AWWCCRBBHDDNMSGTWT [17]
AveT C-5-O-B1 CGAAACGKTKYCGTTTCG [22]
XdhR ppGpp, GTP AACGGACAGTTGTCCGCT [23]
JadR* Jadomycin B (JdB), 2, 3-Dehydro-UWM6 (DHU), Dehydrorabelomycin (DHR), Jadomycin A (JdA) [26]
CHlF1 Chlorothricin, Demethylsalicycloyl-chlorothricin, Deschloro-chlorothricin GTAANNATTTAC [28]
PaaP Phenylacetyl coenzyme A (PA-CoA) ACCGA-n4-TCGGT AACGA-n4-TCGGT [29]
JadR2 Jadomycin, Chloramphenicol [44]
4 总结与展望

越来越多的研究表明TFRs作用多样的靶基因,有的甚至作为全局转录因子调控抗生素生物合成,如阿维链霉菌中AveI不仅调控抗生素的生物合成,还参与蛋白质合成和脂质代谢等其他重要生理过程[46]。本文主要从TFRs作用靶基因的角度进行阐述,是为了更好了解TFRs多元化调控的分子机制,以便通过对TFRs及其靶基因的改造提高放线菌抗生素的产量。如阿维链霉菌中aveT基因的过表达及其编码转运蛋白的靶基因aveM的缺失都可提高阿维菌素的产量[22];红色糖多孢菌中缺失SACE_5754后再过表达靶基因SACE_0388SACE_5754可显著提高野生菌与高产菌的红霉素产量[14]。通过基因工程改造TFRs还可以激活沉默的隐性基因簇从而合成新型的天然产物。在天蓝色链霉菌中敲除scbR2激活了cpk基因簇,产生了新的抗生素Coelimycin[47-48]。在委内瑞拉链霉菌中敲除jadR2后,即使没有乙醇胁迫也可以合成杰多霉素B[27, 45]。此外,利用TFRs与靶基因启动子区结合高亲和力的特性,可以设计与开发智能合成生物学元件,如Kouprina等利用TetR与启动子区tetO稳定结合的特性,设计了alphoidtetO-HAC组合元件,用于人类染色体以及基因组和癌症的研究[49]。作为一类别构转录因子(Allosteric transcription factor,aTF),TFRs应答配体后可将信号关联至其调控的DNA分子,Cao等应用TetR开发出可检测环境中四环素的新型生物传感器,具有灵敏度高和实际应用性强等特性[50]

目前寻找转录因子靶基因的方法有:染色质免疫共沉淀(ChIP)[20]、基因组水平的指数富集配体进化系统技术(gSELEX)[51]、转录组技术[14]以及利用相关生物信息学软件预测等方法[18, 52]。近年来,研究者利用上述方法在不同的放线菌中构建了TFRs作用靶基因的调控网络,但目前仍很少涉及到TFRs时序性调控网络的构建,如在抗生素生物合成的不同时期,TFRs作用靶基因是否有差异?又是如何参与调控作用的?调控过程中又有哪些配体参与,其信号如何传导?揭示TFRs控制抗生素生物合成的动态调控网络,并将其应用到合成生物学元件开发及其微生物药物的高效制造是未来的研究热点之一。

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