【作者】 张天宇；

【导师】 李元；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2005， 博士

【摘要】 链霉菌属于革兰氏阳性细菌，是一种重要的以产生抗生素著称的重要工业微生物。尽管在其它菌种中(如乳酸杆菌)胞外多糖(EPS)生物合成的分子生物学研究已经快速发展，但是链霉菌EPS的生物合成研究尚未见报道。本研究室自行构建了以基因重组白细胞介素1可溶性受体为靶位的受体拮抗剂筛选模型，获得了链霉菌139。该菌产生一种新型胞外多糖依博素，依博素是一种杂多糖，由葡萄糖，半乳糖，甘露糖，鼠李糖，阿拉伯糖，木糖，岩藻糖和半乳糖醛酸八种单糖组成。该多糖对类风湿性关节炎有显著疗效且毒性较低，目前正在申报临床研究，有可能发展成为新型药物。 我室王玲燕博士首次从链霉菌139中克隆了长度为34kb，包含24个开放阅读框(opening reading frame，ORF)的依博素生物合成基因簇。为了研究这些ORFs在依博素生物合成中的功能，从而确定该多糖的生物合成途径，为通过组合生物学定向改造代谢途获得新型依博素衍生物奠定基础，需对上述ORF进行深入研究。本文选择了ste19，ste22和ste23基因为研究对象。 依博素生物合成基因簇(ste)中ste19基因经过同源性比较编码一个新的UDP-葡萄糖-4-差向异构酶，该酶催化UDP-葡萄糖和UDP-半乳糖间的转化。该基因大小为1026bp，编码一个341-aa的蛋白，分子量为36.5kD。我们将ste19基更多还原

【Abstract】 Streptomyces, a gram-positive bacterium, is well known as an important industrial microorganism for its production of antibiotics. However, exopolysaccharide (EPS) production in Streptomyces was not reported before despite the fast progress at molecular level in other bacterial species (e.g. in lactic acid bacteria). A screening model for IL-1R antagonist was constructed in our lab, through which Streptomyces sp. 139 was obtained. Ebosin produced by S. sp. 139 is a new heteroexopolysaccharide with repeating unit consisting of galactose, mannose, glucose, arabinose, fucose, xylose, rhamnose and galacturonic acid. Ebosin has obvious anti-rheumatic arthritis activity in vivo and very low toxity. Now Ebosin is being applied for clinic study and may be a promising new drug.Ebosin biosynthesis gene cluster (ste) with the length of 34kb containing 24 ORFs was first cloned from 5. sp. 139 by Dr. Wang Lingyan. Functions of these ORFs should be characterized and identified profoundly to elucidate Ebosin biosynthesis pathway and to lay a foundation for obtaining new derivatives of Ebosin through modify the biosynthesis paths consciously by recombinative biology. We selected ste\9, ste22 and ste23 as research objects in this paper.The stel9 gene in ste gene cluster was identified to encode a new UDP-Glucose-4-epimerase by data base comparison and experimental validation. The enzyme catalyzes the interconversion of UDP-Glucose and UDP-galactose. The gene had a length of 1026 bp and encoded for a protein with 341 amino acids with deduced molecular weight of 36.5kD. To identify the function of ste 19, ste19 was cloned into E. coli expression plasmid pET-30a. SDS-PAGE of expressed product showed there was a new band in site of 37KD as expected. Recombiant protein was purified by His-Bind Resin with the His-tag and the purity was 92.9%. Activity analysis showed the expressed product had the activity of UDP-Glucose 4-epimerase. The characterizations of the enzyme such as Km, opium temperature and opium pH were investigated.The gene ste22 had a length of 948bp and encoded for a protein with 315 amino acids with deduced molecular weight of 35.5kD. The product encoded by ste22更多还原