摘要
链霉菌139产生一种新型微生物胞外多糖依博素,具有明显抗类风湿性关节炎的作用,已申报临床研究,有望发展成为新药。
我们已确定依博素生物合成基因簇,其包含27个开放阅读框(stel-ste27)。以往已对ste6,ste7,ste10,ste15,ste17,ste19和ste22基因在依博素生物合成中的作用进行了研究。
Ste11基因大小为2100bp。经同源性分析,ste11基因编码的蛋白与不同微生物来源的苏氨酸脱氢酶(TDH)具有较高同源性。
为确定ste11编码蛋白的性质,将该基因克隆至大肠杆菌表达载体pET-30a,转化至E.coli BL21,SDS-PAGE分析表明,在预计的分子量大小处出现了一条新的蛋白条带。重组蛋白采用组氨酸亲合层析柱进行纯化。纯化的重组蛋白酶促反应表明在NAD~+存在的条件下该酶可催化苏氨酸生成2-氨基-3酮基丁酸(AKB)和NADH,表明其为苏氨酸脱氢酶,Km值为0.2mM,最适温度为37℃,最适pH为7.5。
为了解ste11在依博素生物合成中的功能,通过同源重组双交换对ste11进行了基因阻断研究,获得基因阻断变株Streptomyces sp.139(ste11~-),Southern杂交证明ste11基因已被成功阻断。对突变株进行了基因回复实验,得到基因互补株。单糖分析表明Streptomyces sp.139(ste11~-)产生的胞外多糖EPS-m单糖比例与依博素相似。基因互补株Streptomyces sp.139(pKC11C)产生的胞外多糖EPS-c单糖比例与EPS-m相似。IL-1R的拮抗活性显示,EPS-m的拮抗活性较依博素明显降低,但EPS-c的拮抗活性有所恢复。此外EPS-m的分子量明显低于依博素,上述结果表明ste11对依博素的生物活性及单糖重复单元的聚合度有显著影响,据此推测苏氨酸可能存在于依博素分子侧链,ste11在依博素的生物合成中可能起修饰基因作用。
糖基转移酶在微生物胞外多糖的生物合成过程中起重要作用,在多糖生物合成基因簇中引入异源糖基转移酶基因可改变多糖结构获得新衍生物,并可验证被置换基因功能及多糖构效关系。本研究从产生胞外多糖的乳酸菌基因组DNA,PCR扩增获得其多糖生物合成簇的葡萄糖糖基转移酶基因。分别以缺失sre22(编码鼠李糖糖基转移酶)、ste7(编码岩藻糖糖基转移酶)的依博素产生菌突变株为受体菌,通过同源重组双交换将该糖基转移酶基因置换至依博素生物合成基因簇中。Southern杂交验证获得了基因置换菌株Streptomyces sp.139(ste7HC)和Streptomyces sp.139(ste22HC),分离纯化了两菌株产生的胞外多糖EPS-HC22、EPS-HC7,结果表明其单糖组分与依博素相比,葡萄糖均有显著提高;但对白介素Ⅰ受体拮抗活性显著降低;分子量低于依博素。这说明鼠李糖和岩藻糖在依博素生物活性中有重要作用,此外乳酸菌葡萄糖糖基转移酶基因与依博素的葡萄糖糖基转移酶基因同源率仅25%,因此异源表达的葡萄糖可能在糖键类型上与依博素不同,从而影响生物活性及单糖重复单元聚合度,相关研究尚需进一步深入研究。
综上所述,本研究通过对ste11基因的表达、敲除及异源置换ste22、ste 7基因等策略,有效的确定了上述基因在依博素生物合成中的作用,并获得相应的新的依博素衍生物。为通过组合生物学途径研究多糖结构与生物活性关系奠定了较好的基础。苏氨酸脱氢酶在链霉菌中的研究尚属首次报道。
Ebosin produced by Streptomyces sp.139 is a novel exopolysaccharide(EPS) with remarkable anti-rheumatic arthritis activity in vivo. It may be developed to a new drug in the future.
A biosynthesis gene cluster consisting of 27 ORFs (ste1-ste27) for Ebosin was identified in our lab. Among them, ste 6, ste7, ste10 ste15 and ste22 were confirmed to play important roles in the biosynthesis of Ebosin before.
Ste11 (2100bp) was predicted to specify a protein with homology to known threonine dehydrogenase from different sources of microbes. For characterization of the protein encoding by ste11, the gene was cloned and expressed in E. coli BL21. The recombinant Ste11 was purified with the affinity chromatography and found capable of catalyzing NAD~+ and L-threonine to NADH and 2-amino-3-ketobutyrate, hence identified as a threonine dehydrogenase with optimum temperature 37℃, pH 7.5 and Km 0.2mmol/L.
To investigate its function in the biosynthesis of Ebosin , the ste11 gene was knocked out with a double crossover via homologous recombination to produce Streptomyces sp. 139 (ste11~-),which was identified by Southern Blot. Gene complementation of the knock-out mutant was achieved producing the ste11 -complemented mutant strain Streptomyces sp. 139 (pKC11). GC analysis of monosacharride composition in EPS-m produced by Streptomyces sp. 139 (ste11~-) showed that it was composed of the same monosaccharides found in Ebosin but some proportional changes occurred in comparison with Ebosin. Analysis of EPS-c produced by the ste11-complemented mutant strain with GC showed no significant changes compared with EPS-m for most of the monosaccharides. The antagonist activities of EPS-m for IL-1R were much lower than those of Ebosin but the activities of EPS-c recovered partly compared from EPS-m. The Mw of EPS-m and EPS-c are remarkably smaller than Ebosin. With its function verified, we assume that threonine presents in the molecule of Ebosin, although the direct evidence has yet to be provided. Ste11 affects not only the bioactivity significantly but also the polymerization of the repeating units consisting of monosaccharides in Ebosin. The gene may function as a modificator gene of Ebosin during its biosynthesis.
Glycosyltransferases (GTs) that sequentially link sugars from intracellular nucleotide sugar to a lipid carrier are key enzymes in EPSs biosynthesis. Replacing the GTs gene in the biosynthesis of Ebosin with the heterologous GTs gene will be benefit to produce Ebosin derivatives and study the relationship between structure and activity of Ebosin. The gene encoding glucosyltransferase originating from Streptococcus thermophilus was amplified with PCR and cloned into Streptomyces sp.139(ste 7~-) and Streptomyces sp.139 (ste22~-) respectively using a double crossover via homologous recombination. The mutants Streptomyces sp.139 (ste22HC) and Streptomyces sp.139( ste7 HC) were identified by Southern Blot.
GC analysis of monosaccharide composition in EPS-HC22 and EPS-HC7 produced by Streptomyces sp. 139 (ste22 HC) and Streptomyces sp. 139 (ste7 HC) separately showed that glucose increased remarkably , at meant wile rhamnose and fucose decreased tremendously in two Ebosin derivatives in comparison with Ebosin. The antagonist activities of IL-1R for two Ebosin derivatives lost and their Mw were significantly lower than Ebosin. Such results demonstrated that rhamnose, fucose and gluocse are essential in biosynthesis of Ebosin.
In briefly, these studies have laid the ground works for producing more new derivatives of Ebosin with various bioactivities. As our knowledge, the threonine dehydrogenase was reported in Streptomyces at first time.
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