【作者】 邓名荣；

【导师】 郭俊；

【作者基本信息】 华南理工大学 ， 微生物学， 2011， 博士

【摘要】 粤蓝链霉菌(Streptomyces vietnamensis)GIMV4.0001是本研究室近年从热带原始森林土壤中分离获得的一株链霉菌新种。前期研究发现该菌株能产生色调优美的紫罗兰蓝色素。本研究对该菌的产色发酵条件进行了优化,发现高氏合成一号培养基是适合的产色培养基,该培养基原料价格低廉,可作为工业化生产的基础培养基;优化的培养参数组合为:接种量5%,培养温度30℃,180rpm振动培养7d。对该菌产生的次级代谢产物进行了系统的研究,发现其发酵产物对革兰氏阳性细菌有较强的广谱抗菌活性,对部分革兰氏阴性菌也有不同程度的抑制作用,同时对HeLa肿瘤细胞也显现出很强的抑制作用;薄层层析–生物显影发现2个蓝色组分B1、B2是主要的抗菌活性成分,通过质谱、核磁共振等波谱分析,两个蓝色化合物分别被鉴定为榴菌素和榴菌素B。从发酵产物中还分离、鉴定了其它5个相关化合物,分别为醌茜兰类色素Zg、Zgg、二氢Zg、MM44785和去鼠李糖基MM44785,其中二氢Zg和去鼠李糖基MM44785是两个新的榴菌素相关化合物注1。对粤蓝链霉菌次级代谢产物生物合成途径的多样性进行了分析,发现该菌除榴菌素生物合成途径外,还可能存在合成大环内酯类、角环类化合物以及孢子色素等多条PKS途径,为深入挖掘该菌可能存在的其它生物活性物质提供了指导。采用分段、分步的策略,克隆测序了包含粤蓝链霉菌榴菌素完整生物合成基因簇的序列37480bp;发现粤蓝链霉菌榴菌素生物合成基因数目、基因排列等与紫红链霉菌(S. violaceoruber)Tü22的榴菌素生物合成基因簇完全相同,但序列长度与要短1260bp,整个基因簇中存在大约100个小片段的插入或缺失,提示插入或缺失小片段可能是链霉菌基因组进化的基本机制之一。粤蓝链霉菌与紫红链霉菌的榴菌素生物合成基因簇总体上同源率较高,提示二者具有较近的共同祖先;然而基于16S rDNA序列的链霉菌属系统进化发育树分析发现,粤蓝链霉菌与紫红链霉菌存在相对较远的亲缘关系,已知的榴菌素产生菌在链霉菌属中也呈离散分布,因此,榴菌素生物合成基因簇在链霉菌属内可能发生了水平转移,为抗生素生物合成基因簇存在水平转移的观点提供了有力的证据;侧翼基因orf35在粤蓝链霉菌中的缺失和orf35基因3个碎片的发现,呈现出了榴菌素生物合成基因簇在水平转移之后进一步进化的一个情景,目前存在于粤蓝链霉菌基因组中的榴菌素生物合成基因簇可被诠释为基因水平转移和垂直变异的共同结果。随后分析了粤蓝链霉菌对常用抗生素的天然抗性水平,发现粤蓝链霉菌对阿普拉霉素、卡那霉素、链霉素、壮观霉素、硫链丝菌素等均可用作粤蓝链霉菌遗传操的抗性标记,并通过大肠杆菌–粤蓝链霉菌属间接合转移的方法成功建立了粤蓝链霉菌的遗传转化体系。以衍自pCR2.1的重组质粒替代基因组文库cosmid作为突变模板质粒,尝试将PCR targeting系统应用于粤蓝链霉菌,以阿普拉霉素抗性基因片段置换榴菌素生物合成的关键基因orf1、orf2、orf3,成功构建了不产榴菌素的突变株,这是首次成功建立榴菌素产生菌的有效遗传改造体系,为深入了解榴菌素生物合成及其调控机制奠定了基础。通过体外重组表达和体内基因敲除等方法对榴菌素生物合成基因orf20进行了功能研究,发现携带表达orf20重组质粒的大肠杆菌对百草枯的抗性显著提高,而敲除orf20基因后的粤蓝链霉菌突变株对百草枯的抗性水平没有发生显著变化,但其色素产量(榴菌素)大幅提高,是野生株的3.3倍。这说明ORF20能够替代大肠杆菌SoxR参与抗氧化胁迫的调控,但在原宿主粤蓝链霉菌中并不参与抗氧化胁迫调控,而是对榴菌素的生物合成存在负调控效应。对榴菌素外排基因orf15在粤蓝链霉菌中的转录表达规律进行了分析,发现在野生株开始产色前orf15转录表达水平突然升高,产色后迅速降低;在敲除orf20基因后的突变株中,orf15的转录表达水平在产色前也突然升高,但与野生株相比,提高的幅度要大、高水平转录持续的时间也要长。这是orf20突变株榴菌素的产量要比野生株高的原因。更多还原

【Abstract】 Streptomyces vietnamensis GIMV4.0001 is a newly streptomycete species isolated from tropical forest soil designated by our laboratory. Previous study revealed that this strain posseses the ability to produce a diffusible violet-blue pigment. In this study, an optimization for pigment production was carried out. The Gauze’s synthetic No.1 medium was found to be the suitable medium for pigment production. This medium is also suitable for industrial production since its materials are relatively cheep. The optimized fermentation conditions are as follows: inoculation quantity 5%, culture temperature 30°C, rotation speed 180 r/min and culture time 7days. A further study was carried out on its secondary metabolites. The ethyl acetate extracts of the fermentation broth exhibited broad antibacterial activity against Gram-positive bacteria as well as some strains of Gram-negative bacteria to different extents. The crude extracts also showed excellent anticancer activity. Two blue pigments (B1, B2) in the extract content are the main active compounds as revealed by TLC-bioautography. By a combination of physical and chemical characterization, nuclear magnetic resonance and mass spectrometry, these two blue pigments were identified as granticin and granaticin B, respectively. Five more related compounds were also isolated and identified as quinizarin Zg, Zgg, dihydro-Zg, MM44785 and L-rhodinoside-deglycosylated MM44785, among which dihydro-Zg and L-rhodinoside-deglycosylated possess novel structures.The diversity of secondary metabolite pathways of S. vietnamensis was investigated. Except polyketide synthase (PKS) for granaticin, there are still at least three more PKS pathways possibly responsible for macrolides, angucyclines or spore pigments in S. vietnamensis. This provided guidance for deeper mining the bioactive secondary metabolites potentially produced by this strain.By a sequential cloning strategy, a 37480-long fragment containing the whole granaticin biosynthetic gene cluster (gra) was sequenced. All granaticin biosynthetic genes (orf9~orf34) were found as expected, and the gene arrangement was identical with that of S. violaceoruber Tü22 (AJ011500). When aligning the two gra cluster, about one hundred short insertions or deletions (hereinafter referred as indels) were observed throughout the whole cluster. The full gra cluster from S. vietnamensis was 1260 bp shorter than that of S. violaceoruber. This result suggested that insertion and deletion of tiny fragment might be one of the basic evolution mechanisms for streptomycete genomes.High overall homology of the two gra clusters clearly shows that they share a very recent ancestor, whereas phylogenetic analysis of the genus Streptomyces based on 16S rDNA, revealed a distant evolutionary relationship between S. vietnamensis and S. violaceoruber, and a scattered distribution of the granaticin producers within the genus. This provided compelling evidence that antibiotic biosynthetic gene cluster can be acquired horizontally. The remnants of the disabled flanking gene orf35 found in the gra cluster from S. vietnamensis not only indicated the full orf35 gene once resided in this host genome, making the current case a more convincing example of horizontal gene transfer, but also present a scenario how the antibiotic gene clusters evolved after horizontal gene transfer. Then the contemporary gra cluster held by S. vietnamensis can be interpreted as a combination of horizontal gene transfer and variable vertical transmission.The assay of resisitance level of S. vietnamensis to common antibiotics showed that apramycin, kanamycin, streptomycin, spectromycin and thiostrepton can be used as selection markers in genetic manipulation experiments.An efficient genetic manipulation system was established by intergeneric conjugation between E. coli and S. vietnamensis. Plasmids derived from the TA cloning vector pCR2.1, substituted for genomic library cosmid, were applied to S. vietnamensi for gene disruption. A granaticin-deficient mutant was constructed by replacement of the biosynthetic genes orf1, orf2, orf3 with the aac(3)-IV cassette, demonstrating the modified PCR targeting system was successfully applied to S. vietnamensis. To our knowledge, this is the first report of efficient genetic manipulation on the granaticin-producing strain and can pave a way to study the mechanisms of regulations and biosynthesis of granaticin in vivo.Functional studies were carried out on the biosynthetic gene orf20 by in vivo disruption and in vitro expression. E. coli carrying the recombinant plasmid pET28b-orf20 received an elevated resistance to paraquat, whereas S. vietnamensis orf20 disrupant showed no visible changes of resistance to paraquat. Unexpectedly, the granaticin production of the disrupant was improved for more than three folds. These data suggested that the orf20 gene can complement the soxR gene in E. coli, but is not involved in the regulation of anti-oxidative stress response in S. vietnamensis. Instead, it imposes a negative effect on granaticin production.Transcription of the granaticin-export gene orf15 was analysed by real time PCR. The transcription level of orf15 was abruptly elevated just before the granaticin production and then dropped down with speed in S. vietnamensis wild type, whereas the transcription level of orf15 was much more elevated at that stage and longer duration time of high transcription level was observed in the orf20 disrupant. This accounted for the promotion of granaticin production in the orf20 disrupant.更多还原