【作者】 白利平；

【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， 2007， 博士

【摘要】 链霉菌属于革兰氏阳性细菌,是一种以产生抗生素著称的重要工业微生物。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,获得了一种由链霉菌139产生的新型胞外多糖依博素,该多糖由半乳糖、阿拉伯糖、甘露糖、岩藻糖、木糖、鼠李糖、半乳糖醛酸和葡萄糖以克分子数比19∶16∶5.0∶5.0∶4.0∶3.0∶3.0∶2.0组成,其重复单元的结构已经确定。经药效学研究表明该多糖具有明显的抗类风湿性关节炎的作用且毒性较低,已申报临床研究,有可能发展成为临床治疗类风湿性关节炎的新药。我室王玲燕博士首次从链霉菌139中确定了包含24个开放阅读框(openingreading frame,ORF)的依博素生物合成基因簇(GeneBank Accession Number∶AY131229)。序列分析推测各基因功能如下:转录调控基因(ste1-ste4)、糖基转移基因(ste5,ste7,ste15,ste22)、糖核苷酸前体的合成基因(ste6,ste10,ste11,ste17,ste19)、多糖的聚合与输出基因(ste8,ste9,ste13,ste14,ste21)以及多糖的修饰基因(ste12,ste16,ste18,ste20)。为了阐明依博素生物合成途径,研究依博素结构与生物活性关系并获得新型依博素衍生物,拟对不同开放阅读框(ORF)的功能进行深入研究。本文选择了ste10和ste7基因为研究对象。根据同源性分析发现ste10基因编码的蛋白与不同来源的天冬酰胺合成酶具有很高的同源性。已知天冬酰胺合成酶能够利用谷氨酰胺或氨作为氮源,催化天冬氨酸生成天冬酰胺。该酶广泛分布于原核及真核细胞,但在链霉菌中尚未见报道。为了确定ste10基因表达产物性质,本文将ste10基因克隆至大肠杆菌表达载体pET-30a,经37℃诱导和细胞裂解后,SDS-PAGE分析结果显示在70KD处出现了一条新的蛋白条带,与预计的分子量相符,Ste10蛋白在E.coli中以包涵体形式表达。重组蛋白经复性后,采用镍亲和柱层析进行纯化,HPLC分析纯度达到93%。酶学性质研究证明Ste10能够催化天冬氨酸生成天冬酰胺,该酶的Km值为0.9mM、最适温度为37℃、最适pH为8.0。为了进一步了解ste10在依博素生物合成中的作用,对ste10进行了同源重组双交换基因阻断实验。与依博素相比,基因缺失突变株Streptomyces sp.139(ste10~-)产生的多糖衍生物EPS-10m的单糖组成有一定变化;基因互补株Streptomyces sp.139(ste10c)产生的多糖衍生物EPS-10c,其单糖组成类似于EPS-10m。对IL-1R体外拮抗活性的ELISA分析结果显示,与依博素比较,突变株Streptomyces sp.139(ste10~-)产生的EPS-10m对IL-1R的拮抗活性降低,但这种拮抗活性降低可经基因互补回复。推测天冬酰胺可能存在于依博素的侧链结构,ste10可能作为一个调节基因参与依博素的生物合成。同源性比较结果显示ste7编码的蛋白与不同来源的糖基转移酶有很高的同源性。本研究通过基因同源重组双交换,经Southern杂交和PCR验证,获得了ste7基因缺失突变株。与依博素相比,基因缺失突变株Streptomyces sp.139(ste7~-)产生的多糖衍生物EPS-7m,单糖含量发生了较大的变化。六种单糖含量降低,其中葡萄糖(从3.9%→0.66%)与岩藻糖(从6.8%→1.41%)降低最为显著,约降低80%以上;另外两种单糖,半乳糖和鼠李糖含量增加。基因互补株Streptomyces sp.139(sre7)产生的多糖衍生物EPS-7c,与EPS-7m相比有较大变化,特别是岩藻糖有较明显恢复(从1.41%→2.10%),但葡萄糖基本无变化。与依博素相比,突变株Streptomyces sp.139(ste7~-)产生的EPS-7m对IL-1R的拮抗活性降低;但基因互补株产生的EPS-7c与EPS-7m相比,对IL-1R的拮抗活性显著恢复,在浓度为4.5μg/ml时甚至高于依博素,实验结果显示ste7可能编码岩藻糖糖基转移酶,参与依博素的生物合成。BLAST比较结果显示sre7和ste15编码的蛋白与糖基转移酶家族Ⅰ具有很高的同源性,都含有类似于pfam00534的结构域。以往研究显示ste15基因可能编码葡萄糖糖基转移酶,本研究通过基因同源重组双交换,在ste15基因缺失突变株Streptomyces sp.139(ste15~-)基础上,再进行ste7基因阻断,经Southern杂交和PCR验证,已经得到ste7和ste15双基因缺失突变株Streptomyces sp.139(ste7~- ste15~-),基因互补株筛选及变株单糖组分分析及生物活性测定正在进行中。综上所述,本研究通过对ste10、ste7基因单敲除及ste7-ste15基因双敲除等策略,有效的确定了各基因在依博素生物合成中的作用,为通过组合生物学途径研究多糖结构与生物活性关系,获得具有生物活性的新结构多糖奠定了较好基础。有关链霉菌中天冬酰氨合成酶的性质与功能研究国内外未见报道。更多还原

【Abstract】 Streptomyces,a gram-positive bacterium,is well known as an important industrial microorganism producing antibiotics.A screening model for IL-1R antagonists was constructed in our lab;Ebosin produced by Streptomyces sp.139 was obtained with the model.Ebosin is a new heteroexopolysaccharide with repeating unit consisting of galactose,arabinose,mannose,fucose,xylose,rhamnose,galacturonic acid and glucose with a molar ratio of 19:16:5.0:5.0:4.0:3.0:3.0:2.0.The structure of the repeating units for Ebosin has been identified.The pharmacology studies show that Ebosin has obvious anti-rheumatic arthritis activity in vivo with lower toxicity.It has been applied for clinic trial and may be developed to a new drug treating rheumatic arthritis.Ebosin biosynthesis gene cluster（ste） containing of 24 ORFs was identified by Dr.Wang.The cluster include the functions related to regulation（ste1-ste4）, glycosyltransferation（ste5,ste7,ste15 and ste22）,precursor synthesis of nucleotide sugar（ste6,ste10,ste11,ste17and ste19）,polymerization and export（ste8,ste9,ste13, ste14 and ste21）,and modification（ste12,ste16,ste18 and ste20）.For elucidating the biosynthesis pathway of Ebosin,studying the relationship between structure and bioactivity and obtaining new derivatives,the functions of the ORFs involving in the gene cluster will be studied in more detail.Both ste7 and ste10 were selected for functional study in this thesis.According to the data base analysis,the protein encoded by ste10 showed high homology with asparagine synthetase originated from various microorganisms.It is well known that the asparagine synthetase can catalyze the synthesis of asparagines from aspartate,using either glutamine or ammonia as a nitrogen source.The asparagine synthetase distributes widely in prokaryotes and eukaryotes.As our knowledge,the enzyme has not been reported in Streptomyces before us.To identify the function of ste10,it was expressed in E.coli with plasmid pET-30a as vector.After inducing with IPTG and cultivating at 37℃,SDS-PAGE analysis of and cell lysates showed that there was a new band in site of 70KD as expected and the recombinant Ste10 was expressed in the inclusion body form.After renaturation,the protein was purified with Ni²⁺-NTA affinity chromatogram to 93% purity analyzing with HPLC.Ste 10 was shown to be able of catalyzing the transfer of amide nitrogen of glutamine to the side chain of aspartate to produce asparagine.Its Kin,optimum temperature and pH were determined to be 0.9 mM,37℃and 7.38, respectively.For understanding the function of ste10 in the biosynthesis of Ebosin,the gene was disrupted with a double crossover via homologous recombination.Compared with Ebosin,the monosacchafide composition of EPS-10m produced by the gene deficient strain Streptomyces sp.139（ste10^-） was changeable.EPS-10c produced by the gene complement strain Streptomyces sp.139（ste10c） was similar to EPS-10m. ELISA assay showed that the antagonist activity of EPS-m for IL-1R was much lower than that of Ebosin and this lost in the bioactivity was recovered in EPS-c after the gene complementation.We deduced that asparagine might be in the side chain of Ebosin.Ste10 is probably involved in the biosynthesis of Ebosin as a modificator gene.Base on data base comparison,the protein encoded by ste7 showed high homology with glycosyltransferases originated from different microorganisms.The gene was disrupted with a double crossover via homologous recombination and the mutant Streptomyces sp.139（ste7^-） verified by Southern blot and PCR analyses. Compared with Ebosin,the monosaccharide composition of exopolysaccharide （EPS-7m） produced by the mutant Streptomyces sp.139（ste7^-） was found quite different.Six out of eight sugar components were reduced in proportion but most noticeable were the drops（about 80%） in glucose and fucose contents.The other two, galactose and rhamnose were increased in proportion.Gene complementation largely reversed the composition changes including the decrease of fucose but had little effect on glucose content.The antagonist activity of EPS-m for IL-1R in vitro was remarkably lower than Ebosin at both tested concentrations.Compared with EPS-m this bioactivity of EPS-c produced by the complemented strain was significantly higher and it was even higher than Ebosin at the lower concentration（at 4.5μg/ml）. These results have demonstrated the functional involvement of ste7 gene in the biosynthesis of Ebosin mostly probably by encoding a glycosyltransferse for fucose transfer. According to the data base comparison,proteins encoded by ste7 and ste15 showed high homology with that of the glycosyltransferase groupⅠ（Pfam00534）. Previous studies showed that ste15 gene encodes a glycosyltransferse for glucose transfer.In this study,we disrupted ste7 with the same strategy mentioned above in the mutant Streptomyces sp.139（ste15^-）.The mutant strain Streptomyces sp.139 （ste7^-ste15^-） was identified by Southern blot and PCR.The studies of gene complementation,monosaccharide composition analysis and the IL-1R antagonist bioactivities for mutants have been carried out in the laboratory.In conclusion,the functions of ste7 and ste 10 were identified in the biosynthesis of Ebosin with gene disruptions.Such results demonstrated the relationship of structure and bioactivity of Ebosin and laid on the foundation of studying new derivatives of Ebosin.Studying characterazation and function of asparagine synthetase in streptomyces has not been reported before us.更多还原