嗜热链球菌IMAU20246胞外多糖基因簇及其表达分析

Identification and expression profiling of exopolysaccharide gene cluster in Streptococcus thermophilus IMAU20246

摘要:

【目的】嗜热链球菌IMAU20246是一株具有良好发酵特性且高产胞外多糖(exopolysaccharides,EPS)的菌株，但其EPS基因簇及合成途径尚不清晰。因此可通过全基因组测序及生物信息学分析菌株基因组序列，探究EPS合成及调控机制。【方法】本实验对嗜热链球菌IMAU20246进行全基因组测序并进行生物信息学分析，解析EPS生物合成相关基因簇及EPS合成途径，同时采用实时荧光定量PCR技术(quantitative real-time PCR,qRT-PCR)对其不同时间点EPS基因簇的表达进行定量分析。【结果】嗜热链球菌IMAU20246基因组中有一个18.1kb的EPS生物合成基因簇，编码15个与EPS生物合成相关的基因。嗜热链球菌IMAU20246通过转运葡萄糖、甘露糖、果糖、半乳糖、乳糖、海藻糖、纤维二糖及蔗糖合成UDP-葡萄糖、dTDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖、UDP-呋喃半乳糖、UDP-N-乙酰葡萄糖胺和UDP-N-乙酰半乳糖胺等7种糖核苷酸。qRT-PCR的结果表明，EPS基因簇中的基因在细胞生长阶段均能表达，特别是糖基转移酶基因epsE、epsF、epsH和epsJ在培养6h时表达量最高，此时EPS产量达到最高。【结论】本研究从基因组解析了嗜热链球菌IMAU20246 EPS基因簇及其合成途径，为菌株的进一步开发提供了理论依据。

关键词:嗜热链球菌;胞外多糖;EPS基因簇;生物合成;生物信息学分析

Abstract:

[Objective] Streptococcus thermophilus IMAU20246 is a strain with good fermentation performance and high production of exopolysaccharides (EPS), while its gene cluster and pathway for EPS synthesis remain unclear. Therefore, whole-genome sequencing and bioinformatics tools can be employed to identify the gene cluster and decipher the mechanism of EPS synthesis. [Methods] The whole genome of S. thermophilus IMAU20246 was sequenced and bioinformatic analysis was performed to analyze the EPS biosynthesis-related gene clusters and pathway. Further, quantitative real-time PCR (qRT-PCR) was carried out to determine the expression levels of the EPS gene cluster at different time points. [Results] An EPS gene cluster of 18.1 kb was identified in the genome of S. thermophilus IMAU20246, including 15 genes. The strain synthesized 7 sugar nucleotides including UDP-glucose, dTDP-glucose, dTDP-rhamnose, UDP-galactose, UDP-galactofuranose, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine by transporting glucose, mannose, fructose and galactose, lactose, trehalose, cellobiose, and sucrose, respectively. The results of qRT-PCR showed that the genes in the EPS gene cluster were expressed during the cell growth. In particular, the glycosyltransferase genes epsE, epsF, epsH, and epsJ reached the highest expression levels at the time point of 6 h, when the EPS yield peaked. [Conclusion] In this study, the gene cluster and pathway for EPS synthesis in S. thermophilus IMAU20246 were analyzed, which will provide a theoretical basis for further utilization of the strain.

Key words:Streptococcus thermophilus;exopolysaccharides (EPS);EPS gene clusters;biosynthesis;bioinformatics analysis