【作者】 雷健；

【导师】 阎浩林； 王以光；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2007， 硕士

【摘要】 吸水链霉菌17997（Streptomyces hygroscopicus 17997）是中国医学科学院医药生物技术研究所从中国土壤中分离到的格尔德霉素（geldanamycin，GDM）产生菌。本实验室从S. hygroscopicus 17997中克隆了GDM的部分生物合成基因簇，并在其中发现了两个调节基因gdmRⅠ和gdmRⅡ，经ORF分析和同源比较发现二者与LuxR家族调节基因同源性很高。基因阻断和回复实验证明GdmRⅠ和GdmRⅡ均是GDM生物合成的正调控蛋白。本研究在可溶性培养基上，研究了S.hygroscopicus 17997的生长及发酵周期，表明其生长始于0～12h，对数生长期是24～60h，60h～120h进入稳定期；发酵培养的生长期为12-48h，抗生素合成期为48-96h。采用半定量RT-PCR技术，在转录水平上研究了两个调节基因gdmRⅠ和gdmRⅡ与GDM生物合成相关基因的关系。结果表明gdmRⅠ和gdmRⅡ基因在48-72h开始转录，96h转录活性达到最大，以后逐渐降低，与格尔德霉素生物合成期相符。并证明gdmRⅠ和gdmRⅡ调节基因主要调控参与GDM聚酮体生物合成的聚酮合酶（pks）、酰氨合酶（gdmF）和3—氨基—5—羟基苯甲酸合酶（gdnA）等基因的转录，而对其PKS后修饰基因氨甲酰基转移酶（gdmN）基因不具有调控作用。根据生物信息学分析，从一端缩短gdmRⅠ和gdmRⅡ基因启动区序列(P_gdmRⅠ-Ⅴ：739bp，649bp，451bp，278bp，152bp；P_gdmRⅡ-Ⅴ：331bp，293bp，233bp，186bp，120bp)，将这些启动区序列分别构建到链霉菌启动子探测质粒pIJ486中，并以红霉素启动子PermE为阳性对照，以质粒pIJ486为阴性对照。将P_{gdmRⅠ-Ⅰ-Ⅴ}和P_{gdmRⅡ-Ⅰ-Ⅴ}重组载体分别转化到变铅青链霉菌TK24中，以卡那霉素（Km）抗性为指标。研究发现P_{gdmRⅠ-Ⅰ-Ⅴ}的转化子Km抗性水平分别为250μg／ml、300μg／ml、200μg／ml、50μg／ml、0μg／ml。P_{gdmRⅠ-Ⅰ-Ⅲ}的Km抗性急剧下降至50μg／ml，初步判断gdmRⅠORF上游-649到-278之间可能为其启动子活性区；而P_{gdmRⅡ-Ⅰ-Ⅴ}的表达活性依次为350μg／ml、250μg／ml、200μg／ml、500μg／ml和0μg／ml。P_ermE阳性对照的抗性水平为500μg／ml。提示在gdmRⅡORF起始点上游-331到-233可能为其启动活性区，而在其下游-233到-186或有部分重叠序列可能存在另一方向的启动活性区。这些结果为进一步精密研究gdmRⅠ和gdmRⅡ的启动子的活性区打下了基础。本研究在分子水平上研究了调控基因gdmRⅠ和gdmRⅡ与GDM生物合成的相互关系，并初步定位了两个调节基因的启动区序列，为深入研究LuxR家族调节基因调控GDM生物合成机制打下了基础，并为定向改良GDM生产菌株提供了可能。更多还原

【Abstract】 Streptomyces hygrscopicus 17997 was isolated as a geldanamycin producer from a Chinese soil by the Institute of Medicinal Biotechnology, CAMS. Geldanamycin biosynthetic gene cluster has been cloned from S. hygroscopicus 17997, in which two regulatory genes, gdmRⅠand gdmRⅡ, were found, and their deduced products were identified homologous with LuxR transcriptional regulatory proteins. Disruption of gdmRⅠand gdmRⅡgenes, respectively, resulted in completely loss of the GDM production, which suggested that they all played the positive regulatory role in GDM biosynthesis.The growth pattern studies showed that the exponential phase of S. hygroscopicus 17997 was from 24 to 60h, and the lag phase was from 60 to 120h. The typical trophophase （12-48h） and idiophase （48-96h） could be distinguished in S. hygroscopicus 17997 fermentation course.The results were in agreement with the GDM biosynthesis cycle. Semi-quantitative RT-PCR was carried out to investigate the transcription activities of the GDM biosynthetic genes in S. hygroscopicus 17997 and the two regulatory gene disruptants. The results suggested the transcription of gdmRⅠand gdmRⅡappeared at 72h, reached peak at 96h, and after 96h decreased gradually, which were in agreement with the time of initiation of biosynthesis GDM in S. hygroscopicus 17997. And both of gdmRⅠand gdmRⅡgenes positively controlled the transcription of the genes involved in polyketide formation of GDM biosynthesis, but not the gdmN （carbamyltransferase） gene, a post-PKS modification gene of GDM biosynthesis.The gdmRⅠand gdmRⅡpromoter, P_gdmRⅠ-P_gdmRⅡ regions were deduced according to bio-informatics analysis. The different length fragments of both deduced promoter regions (P_{gdmRⅠ^--1-V}:739bp, 649bp, 451bp, 278bp, 152bp; P_{gdmRⅡ^-Ⅰ-V}: 331bp, 293bp, 233bp, 186bp, 120bp) were generated by PCR and constructed in the streptomyces promoter-probe plasmid pIJ486, separately, taking ermE promoter as positive control and pIJ486 itself as negative control. The resultant plasmids were introduced into the S. lividans TK24 and the kanamycin （Km） resistance level of the transformants was considered to roughly estimate the transcription level of these promoter regions. The results showed that Km resistance level of P_{gdmRⅠ-Ⅰ-V} was 250μg/ml, 300μg/ml, 200μg/ml, 50μg/ml, 0μg/ml, respectively. It is suggested that P_gdmRⅠ might be located at -649 and -278 or -152 of the upstream of the gdmRⅠORF. The Krn resistance level of P_{gdmRⅡ-Ⅰ-V} was apparently350μg/ml, 250μg/ml, 200μg/ml, 500μg/ml, 0μg/ml, respectively. In the same experimental condition the ermE promoter activity was reached to 500μg/ml. Rather high level of Km resistance in the region between -331 and -120 and highest Km resistance in the region between -186 and -120 suggested that possible existence of divergent transcription promoters localized upstream of the gdmRⅡORF. In -331 to -120 region two promoters with same transcription direction were present. And in -186 region might be a promoter with convergent transcription existed upstream of the gdmRⅡORF. Intensive studies will be required to clarify the phenomenon.In this studies the relationships of the regulatory genes gdmRⅠ、gdmRⅡand GDM biosynthesis at the transcriptional level were discussed, and the two regulatory gene promoter regions were roughly localized.. It is of great importance both for a fundamental understanding of the regulatory mechanisms of GDM biosynthesis and for the further rational improvement of GDM producers.更多还原