摘要
链霉菌 139能够产生一种新的胞外多糖 139A ,该多糖具有抗类风湿性关节炎的活性。为研究多糖 139A的生物合成基因簇 ,首要策略是克隆到在多糖 139A的生物合成中起关键作用的引导糖基转移酶基因。根据其他几个种属的糖基转移酶氨基酸序列的两个保守区域设计简并引物 ,通过PCR方法扩增出相应的DNA片段作为探针 ,从链霉菌 139基因组文库中分离到引导糖基转移酶基因ste5 ,并定位于约 32kb的基因簇上。序列分析发现其蛋白序列与引导糖基转移酶具有较高的同源性 ,其C 端含有A ,B和C 3个保守区 ,N 端具有 5个跨膜区。引导糖基转移酶基因阻断突变株不能够产生多糖 139A表明其参与多糖 139A的生物合成。
Recently in our laboratory, Streptomyces sp.139 has been identified to produce a new exopolysaccharide designated EPS 139A that shows anti-rheumatic arthritis activity.The strategy of studying EPS 139A biosynthesis is to clone the key gene in the EPS biosynthesis pathway,i.e.the priming glycosyltransferase gene catalyzing the first step of nucleotide sugar transfer.Degenerate primers-based PCR approach was adopted to isolate the putative priming glycosyltransferase gene in Streptomyces sp.139.According to the genes encoding the priming glycosyltransferases that have been identified in several microorganisms,a multiple alignment of the amino acid sequences of these genes was used to identify regions conserved between all genes.To clone the priming glycosyltransferase gene in Streptomyces sp.139,degenerate primers were designed from these conserved regions taking into account information on Streptomyces codon usage to amplify an internal DNA fragment of this gene.A distinctive PCR product with the expected size of 0.3 kb was amplified from Streptomyces sp.139 total genomic DNA.Sequence analysis showed that it is part of a putative priming glycosyltransferase gene and contains the predicted conserved domain B.To isolate the complete priming glycosyltransferase gene,a Streptomyces sp.139 genomic library was constructed in the E.coli-Streptomyces shuttle vector pOJ446.Using the 0.3 kb PCR product of priming glycosyltransferase gene as a probe,17 positive colonies were isolated by colony hybridization.A 4.0 kb Bam HI fragment from all positive cosmids that hybridized to this probe was sequenced,which revealed the complete priming glycosyltransferase gene.The priming glycosyltransferase gene ste5 (GenBank under accession number AY131229) most likely begins with GTG,preceded by a probable ribosome binding site (RBS),GGGGA.It encodes a 492-amino-acid protein with molecular weight of 54 kDa and isoelectric point of 10.6.The G+C content of ste5 is 73%,close to the average of G+C content (74%) for Streptomyces .Moreover,the preference usage of G or C as third base of codons are found in the ste5 ,which is in accordance with the Streptomyces codon usage.A BlastP search showed that the C-terminal region of Ste5 shows highly homology with a number of priming glycosyltransferases from many different organisms.Ste5 contains two putative catalytic residues,Glu and Asp (residues 423 and 474) with a spacing of approximately 50 amino acids that conserved in various β- glycosyltransferases.Moreover,the C-terminal one third of Ste5 contains three domains,A,B and C that is reported to be common to glycosyltransferases.By hydrophilicity plot prediction,the N-terminal two thirds of Ste5 exhibits 5 putative transmembrane domains.To investigate the involvement of the identified polysaccharide gene cluster in EPS 139A biosynthesis,the gene ste5 encoding priming glycosyltransferase was insertionally disrupted by a single-crossover homologous recombination event.A 0.85 kb internal fragment of ste5 was cloned into vector pKC1139 to yield pLY5015 that was transduced into Streptomyces sp.139.Correct integration in Streptomyces LY1001 ste5 - mutant strain was confirmed by Southern hybridization.After fermentation,no EPS 139A could be detected in the cultures of ste5 - mutant strain Streptomyces LY1001.Therefore,the gene ste5 identified in this work is involved in the synthesis of the Streptomyces sp.139 EPS.
基金
国家 8 63计划 (2 0 0 1AA2 14 0 91)~~
