【作者】 李慧；

【导师】 赵圣印； 唐功利；

【作者基本信息】 东华大学 ， 有机化学， 2013， 硕士

【摘要】 近年来,一种全新的从基因到产物的天然产物发现方法——基因组挖掘,得到广泛应用,它利用生物基因信息学为导向发现新的天然产物。这种从源头上寻找新化合物的理念和方法均具有很大的挑战性,而且对于新药的发现也具有十分重要的意义。Micromonospora. sp. A1304是2002年由日本科学家五十岚康弘(Tamotsu Furumai)丛富士山湾深海中分离得到的一株小单孢菌,并在其中发现了具有良好抗肿瘤活性的葸环类抗生素越野他汀(Kosinostatin)。通过生物信息学分析,我们发现在小单孢菌的基因组中存在与安莎霉素类化合物生物合成中的关键起始单元——3-氨基-5-羟基苯甲酸(AHBA)相关的三个连锁的保守基因：AHBA合成酶、氧化还原酶和磷酸酶基因,进而我们推测该菌中存在某种安莎霉素类化合物的生物合成基因簇,因此该产生菌可能可以产生一种安莎霉素类化合物。我们首先利用pCC1FOS为载体,构建了高质量的基因组文库,并己通过多种策略克隆获得部分生物合成基因簇。同时对发酵产物HPLC分析条件进行了摸索,并构建了基因置换和缺失突变株对其生物合成途径进行初步研究。并且我们尝试对该未知化合物进行分离,并取得初步成果。另外,在对越野他汀的生物合成途径的研究中,我们从基因中断突变株mKOSD3中分离得到一种新的葸环类天然产物——脱氧异醌环素B。该化合物是异醌环素B的类似物,并且是越野他汀生物合成途径中的一种副产物。该化合物的抗肿瘤活性相比于异醌环素B有明显下降。同时我们发现KstD5是一个对糖基底物识别不专一的糖基转移酶,这为进一步利用该酶的特性获得更多结构类似物奠定了基础。通过以上实验,我们利用基因组挖掘的方法,以生物信息学为导向,在海洋小单孢菌Micromonospora. sp. A1304中进行新的天然产物的发掘。后续工作中,我们希望得到完整的生物合成基因簇,并通过基因信息与化合物表征相结合的手段,最终确定目标化合物的分子结构。更多还原

【Abstract】 In recent years, a new method for natural products discovery, named "genome mining", has been widely used. Discovery of new natural products using this method is oriented by bioinformatics analysis. This method of discovery of new chemical entities from the source is much more challenging, and may form the basis for new drug leads.Micromonospora sp. A1304was discovered by the Japanese scientist Tamotsu Furumai in2002. And Kosinostatin, an anthracycline antibiotics with anti-tumor activity, was isolated from it. Recently, we have found the key genes, AHBA synthase, oxidoreductase and phosphatase, involved in ansamycin biosynthesis in the genome of Micromonospora sp. A1304based on bioinformatics analysis. These genes are always conversed and play an important role in the biosynthesis of3-amino-5-hydroxy benzoic acid (AHBA), the key start unit of ansamycin biosynthesis. Thus, we speculated that the bacteria may be able to produce ansamycin.To explore the new ansamycin, the fosmid genomic library of Micromonospora sp. A1304with high efficiency was constructed with pCC1FOS as the vector. And part of the biosynthetic gene cluster has been cloned through a variety of strategies. The HPLC analysis conditions of the fermentation product were explored at the same time. Gene replacement and in frame deletion mutants was constructed for preliminary study of its biosynthetic pathway. Also, we tried to isolate the target compounds, and initial results have been achieved.In addition, we constructed the gene disruption mutant strain mKOSD3to investigate the function of kstD3gene, encoding a sugar3,5-epimerase involved in the KST biosynthetic gene cluster. A novel compound, deoxy-isoquinocycline B was discovered and isolated from this mutant strain and its structure was characterized. This compound could be considered as an analogue of isoquinocycline B and a byproduct of the KST biosynthetic pathway. Compared with isoquinocycline B, its anti-tumor activity decreased significantly. We also found that the glycosyltransferase KstD5is more substrate-flexible than anticipated, which lay the foundation for further using the characteristics of this enzyme to obtain more analogues.In conclusion, we have used the method of geneome mining to explore new natural products from Micromonospora sp. A1304. In the follow-up work, we hope to clone the complete biosynthetic gene cluster, and determine the molecular structure of the target compounds by the means of combination of genetic information analysis and compounds characterization.更多还原