美达霉素生物合成途径中的新型转录调控因子Med-ORF10的作用机制初步研究
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摘要
美达霉素是由包括链霉菌AM-7161(Streptomyces sp. AM-7161)在内的多个链霉菌积累的抗生素,具有抗菌和抗癌活性,可以作为多种肿瘤细胞的信号转导抑制剂。美达霉素生物合成基因簇中的med-ORF10是美达霉素生物合成途径中的一个功能未知基因。因为其同源基因在多个抗生素基因簇中都存在,所以推测med-ORF10及同源基因在相应抗生素合成途径中都是必须基因,可能调控抗生素的生物合成。
本研究以美达霉素生物合成基因簇中的新型转录调节基因(med-ORF10)为研究对象,综合运用遗传学、分子生物学和分析化学等相关研究方法,通过体外和体内实验确定med-ORF10的功能,并对其作用机制展开初步研究,具体内容如下：
1.体内遗传学实验证明med-ORF10具有调控功能
将含有基因med-ORF10的链霉菌自主复制型质粒pHSL98经原生质体转化导入美达霉素产生菌-链霉菌AM-7161中进行超量表达(Overexpression);对超量表达菌株AM-7161/pHSL98进行发酵液及R4平板菌落产素(暗示美达霉素的积累)颜色观察,初步推断wed-ORF10的超量表达可以促进产素；然后通过对AM-7161/pHSL98发酵液进行LC/MS检测,发现基因med-ORF10的导入和过量表达使发酵上清中美达霉素产量增加大约2.3倍(并且一个中间产物的量也增加了大约6倍)。这表明med-ORF10可能是正效调控基因。
2.通过RT-PCR初步确定在美达霉素基因簇中受med-ORF10调控的靶基因
提取美达霉素“异源野生菌株”CH999/pIK340和“异源突变菌株”CH999/pAYT64的总RNA,以看家基因23 S rRNA基因为内参,利用半定量RT-PCR检测两个菌株中美达霉素基因簇中med-ORF12、med-ORF1、med-ORF11等基因的转录水平,发现在“异源野生菌株”和“异源突变菌株”两菌株中,这些基因转录水平存在差异,说明这几个基因的转录分别在不同程度上受到med-ORF10的调控；而且“异源突变菌株”中med-ORF10的缺失使这些靶基因的转录水平不同程度的提高。
本实验初步确定在异源表达体系中受到med-ORF10调控的靶基因。
3.med-ORF10与靶基因启动子的关系初步研究
首先构建了在美达霉素产生菌-野生型链霉菌AM-7161-中进行启动子活性检测的阳性对照系统：将已知功能的链霉菌启动子PactIII克隆到启动子探针载体pIJ8660的EGFP基因上游；通过原生质体转化导入野生型链霉菌AM-7161；荧光显微镜检测到EGFP绿色荧光,说明这套启动子检测系统在AM-7161菌株中是适用的。
利用生物信息学软件分析预测了美达霉素基因簇中几个靶基因上游的启动子；对主要的靶基因med-ORF12上游的可能的启动子进行了克隆和测序,并进一步插入到启动子探针载体pIJ8660的EGFP基因上游,将获得的质粒pHSL33导入美达霉素产生菌AM-7161(野生菌株)中。
以上研究结果初步确定了med-ORF10的调控功能以及在美达霉素基因簇中的可能的靶基因,为抗生素高产菌遗传育种积累优势调控基因。
Medermycin is an antibiotic produced by many streptomycetes, including Streptomyces sp AM-7161. Medermycin possesses antitumor and antibacterial activity as a strong inhibitor against signal transduction pathways in many types of tumor cells. In medermycin biosynthetic gene cluster, med-ORF10 was a gene with undesignated function. Because its homologies are found in many other antibiotic biosynthetic gene clusters, we speculated that med-ORF10 and its homologous genes are essential in these antibiotics biosynthetic pathways, and they may regulate the biosynthesis of these antibiotics.
In order to investigate the function and mechanism of a proposed transcription regulator gene (med-ORF10) in the medermycin biosynthetic gene cluster, we utilized genetics, molecular biology, analytical chemistry methods to determine med-ORF10 function in vivo, and performed a preliminary study on its action mode:
1. Genetic experiments show that med-ORF10 possesses a regulatory function
Firstly, we introduced a med-ORF10-containing plasmid pHSL98, derived from a streptomyces auto-replicating plasmid pWHM4*, into a medermycin-producing strain, Streptomyces sp. AM-7161, by protoplast transformation for overexpression of med-ORF10; Secondly, we determined the pigmentation level of over-expression strain AM-7161/pHSL98. Our data showed that the over-expression strain could accumulate obviouly stronger pigmentation than the wild type strain AM-7161 both on solid and liquid media, implying the over-expression of med-ORF10 can promote the production of medermycin; Thirdly, we measured the fermentation broth of AM-7161/pHSL98 by LC/MS, and found that the production of medermycin in the over-expression strain AM-7161/pHSL98 was promoted by about 2.3-fold as well as the production of an intermediate increased by about 6-fold. These data show that med-ORF10 may be a regulatory gene as an activator.
2. Target genes located in the medermycin gene cluster and regulated by med-ORF10 were determined using RT-PCR
Firstly, we extracted total RNAs respectively from two strains ("heterologous-expression mutant strain Streptomyces coelicolor CH999/pAYT64" and "heterologous-expression wild-type strain Streptomyces coelicolor CH999/pIK340); Secondly, we performed half-quantiative RT-PCR using these total RNAs as templates and 23 S rRNA as control. Our data showed that the transcription of some genes including med-ORF12, med-ORF1 and med-ORF11 in the mutant CH999/pAYT64 was promoted at different levels, comparing to that in CH999/pIK340, suggesting that the expression of these genes might be regulated by med-ORF10 in a direct or indirect way.
3. Preliminary study on the mechanism of med-ORF10 regulating target genes
Firstly, we constructed a positive control system for promoter-detection in the wild-type Streptomyces sp AM-7161 using a proved constitutive promoter PactⅢwhich was cloned onto a streptomyces-promoter-probe vector pIJ8660 (the resultant plasmid was named as pHSL32). We introduced pHSL32 into AM-7161 by protoplast transformation, and could detect green fluorescence from GFP expression in AM-7161/pHSL32 under the fluorescence microscopy. Secondly, we analyzed the proposed promoter regions on the medermycin gene cluster and cloned the proposed promoter (Pmed-ORF12) of a main target gene med-ORF12 onto the same promoter-probe vector pIJ8660, and obtained a new plasmid pHSL33. The transformation of pHSL33 into AM-7161 was under the way.
本研究以美达霉素生物合成基因簇中的新型转录调节基因(med-ORF10)为研究对象,综合运用遗传学、分子生物学和分析化学等相关研究方法,通过体外和体内实验确定med-ORF10的功能,并对其作用机制展开初步研究,具体内容如下：
1.体内遗传学实验证明med-ORF10具有调控功能
将含有基因med-ORF10的链霉菌自主复制型质粒pHSL98经原生质体转化导入美达霉素产生菌-链霉菌AM-7161中进行超量表达(Overexpression);对超量表达菌株AM-7161/pHSL98进行发酵液及R4平板菌落产素(暗示美达霉素的积累)颜色观察,初步推断wed-ORF10的超量表达可以促进产素；然后通过对AM-7161/pHSL98发酵液进行LC/MS检测,发现基因med-ORF10的导入和过量表达使发酵上清中美达霉素产量增加大约2.3倍(并且一个中间产物的量也增加了大约6倍)。这表明med-ORF10可能是正效调控基因。
2.通过RT-PCR初步确定在美达霉素基因簇中受med-ORF10调控的靶基因
提取美达霉素“异源野生菌株”CH999/pIK340和“异源突变菌株”CH999/pAYT64的总RNA,以看家基因23 S rRNA基因为内参,利用半定量RT-PCR检测两个菌株中美达霉素基因簇中med-ORF12、med-ORF1、med-ORF11等基因的转录水平,发现在“异源野生菌株”和“异源突变菌株”两菌株中,这些基因转录水平存在差异,说明这几个基因的转录分别在不同程度上受到med-ORF10的调控；而且“异源突变菌株”中med-ORF10的缺失使这些靶基因的转录水平不同程度的提高。
本实验初步确定在异源表达体系中受到med-ORF10调控的靶基因。
3.med-ORF10与靶基因启动子的关系初步研究
首先构建了在美达霉素产生菌-野生型链霉菌AM-7161-中进行启动子活性检测的阳性对照系统：将已知功能的链霉菌启动子PactIII克隆到启动子探针载体pIJ8660的EGFP基因上游；通过原生质体转化导入野生型链霉菌AM-7161；荧光显微镜检测到EGFP绿色荧光,说明这套启动子检测系统在AM-7161菌株中是适用的。
利用生物信息学软件分析预测了美达霉素基因簇中几个靶基因上游的启动子；对主要的靶基因med-ORF12上游的可能的启动子进行了克隆和测序,并进一步插入到启动子探针载体pIJ8660的EGFP基因上游,将获得的质粒pHSL33导入美达霉素产生菌AM-7161(野生菌株)中。
以上研究结果初步确定了med-ORF10的调控功能以及在美达霉素基因簇中的可能的靶基因,为抗生素高产菌遗传育种积累优势调控基因。
Medermycin is an antibiotic produced by many streptomycetes, including Streptomyces sp AM-7161. Medermycin possesses antitumor and antibacterial activity as a strong inhibitor against signal transduction pathways in many types of tumor cells. In medermycin biosynthetic gene cluster, med-ORF10 was a gene with undesignated function. Because its homologies are found in many other antibiotic biosynthetic gene clusters, we speculated that med-ORF10 and its homologous genes are essential in these antibiotics biosynthetic pathways, and they may regulate the biosynthesis of these antibiotics.
In order to investigate the function and mechanism of a proposed transcription regulator gene (med-ORF10) in the medermycin biosynthetic gene cluster, we utilized genetics, molecular biology, analytical chemistry methods to determine med-ORF10 function in vivo, and performed a preliminary study on its action mode:
1. Genetic experiments show that med-ORF10 possesses a regulatory function
Firstly, we introduced a med-ORF10-containing plasmid pHSL98, derived from a streptomyces auto-replicating plasmid pWHM4*, into a medermycin-producing strain, Streptomyces sp. AM-7161, by protoplast transformation for overexpression of med-ORF10; Secondly, we determined the pigmentation level of over-expression strain AM-7161/pHSL98. Our data showed that the over-expression strain could accumulate obviouly stronger pigmentation than the wild type strain AM-7161 both on solid and liquid media, implying the over-expression of med-ORF10 can promote the production of medermycin; Thirdly, we measured the fermentation broth of AM-7161/pHSL98 by LC/MS, and found that the production of medermycin in the over-expression strain AM-7161/pHSL98 was promoted by about 2.3-fold as well as the production of an intermediate increased by about 6-fold. These data show that med-ORF10 may be a regulatory gene as an activator.
2. Target genes located in the medermycin gene cluster and regulated by med-ORF10 were determined using RT-PCR
Firstly, we extracted total RNAs respectively from two strains ("heterologous-expression mutant strain Streptomyces coelicolor CH999/pAYT64" and "heterologous-expression wild-type strain Streptomyces coelicolor CH999/pIK340); Secondly, we performed half-quantiative RT-PCR using these total RNAs as templates and 23 S rRNA as control. Our data showed that the transcription of some genes including med-ORF12, med-ORF1 and med-ORF11 in the mutant CH999/pAYT64 was promoted at different levels, comparing to that in CH999/pIK340, suggesting that the expression of these genes might be regulated by med-ORF10 in a direct or indirect way.
3. Preliminary study on the mechanism of med-ORF10 regulating target genes
Firstly, we constructed a positive control system for promoter-detection in the wild-type Streptomyces sp AM-7161 using a proved constitutive promoter PactⅢwhich was cloned onto a streptomyces-promoter-probe vector pIJ8660 (the resultant plasmid was named as pHSL32). We introduced pHSL32 into AM-7161 by protoplast transformation, and could detect green fluorescence from GFP expression in AM-7161/pHSL32 under the fluorescence microscopy. Secondly, we analyzed the proposed promoter regions on the medermycin gene cluster and cloned the proposed promoter (Pmed-ORF12) of a main target gene med-ORF12 onto the same promoter-probe vector pIJ8660, and obtained a new plasmid pHSL33. The transformation of pHSL33 into AM-7161 was under the way.
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