Data_Sheet_4_Transcriptomic Insights Into the Growth Phase- and Sugar-Associated Changes in the Exopolysaccharide Production of a High EPS-Producing Streptococcus thermophilus ASCC 1275.XLSX

In a previous study, incorporation of high exopolysaccharide (EPS) producing dairy starter bacterium Streptococcus thermophilus ASCC 1275 was found to improve functionality of low fat mozzarella cheese and yogurt. This bacterium in its eps gene cluster has a unique pair of chain length determining genes, epsC- epsD, when compared to other sequenced S. thermophilus strains. Hence, the aim of this study was to understand the regulatory mechanism of EPS production in this bacterium using transcriptomic analysis to provide opportunities to improve the yield of EPS. As sugars are considered as one of the major determinants of EPS production, after preliminary screening, we selected three sugars, glucose, sucrose and lactose to identify the EPS producing mechanism of this bacterium in M17 medium. Complete RNA-seq analysis was performed using Illumina HiSeq 2000 sequencing system on S. thermophilus 1275 grown in three different sugars at two-time points, 5 h (log phase) and 10 h (stationary phase) to recognize the genes involved in sugar uptake, UDP-sugar formation, EPS assembly and export of EPS outside the bacterial cell. S. thermophilus 1275 was found to produce high amount of EPS (∼430 mg/L) in sucrose (1%) supplemented M17 medium when compared to other two sugars. Differential gene expression analysis revealed the involvement of phosphoenolpyruvate phosphotransferase system (PEP-PTS) for glucose and sucrose uptake, and lacS gene for lactose uptake. The pathways for the formation of UDP-glucose and UDP-galactose were highly upregulated in all the three sugars. In the presence of sucrose, eps1C1D2C2D were found to be highly expressed which refers to high EPS production. Protein homology study suggested the presence of Wzx/Wzy-dependent EPS synthesis and transport pathway in this bacterium. KEGG pathway and COG functional enrichment analysis were also performed to support the result. This is the first report providing the transcriptomic insights into the EPS production mechanism of a common dairy bacterium, S. thermophilus.

既往研究显示，将高产胞外多糖（exopolysaccharide, EPS）的乳品发酵剂菌株嗜热链球菌（Streptococcus thermophilus）ASCC 1275应用于低脂马苏里拉奶酪与酸奶的制作，可改善其产品功能特性。与其他已完成全基因组测序的嗜热链球菌菌株相比，该菌株的eps基因簇中存在一对独特的链长决定基因epsC与epsD。因此，本研究旨在通过转录组学分析解析该菌株的EPS合成调控机制，为提升EPS产量提供理论依据。由于糖类是影响EPS合成的核心调控因素之一，本研究在初步筛选后，选取葡萄糖、蔗糖与乳糖三种碳源，探究该菌株在M17培养基中的EPS合成机制。本研究依托Illumina HiSeq 2000测序平台，对分别以三种糖类为碳源培养的嗜热链球菌1275进行全转录组测序，采样时点分别为培养5小时（对数生长期）与10小时（稳定生长期），以识别参与糖类摄取、UDP-糖合成、EPS组装以及EPS胞外分泌的相关基因。实验结果显示，与另外两种碳源相比，在添加1%蔗糖的M17培养基中，嗜热链球菌1275的EPS产量最高，可达约430 mg/L。差异基因表达分析结果表明，该菌株通过磷酸烯醇式丙酮酸磷酸转移酶系统（phosphoenolpyruvate phosphotransferase system, PEP-PTS）摄取葡萄糖与蔗糖，而通过lacS基因完成乳糖的转运。在三种碳源培养条件下，UDP-葡萄糖与UDP-半乳糖的合成通路均呈现显著上调表达。在蔗糖培养条件下，eps1C1D2C2D基因簇呈现高表达，这与该条件下EPS高产的表型一致。蛋白质同源性分析结果显示，该菌株存在依赖Wzx/Wzy的EPS合成与转运通路。本研究同时通过KEGG通路富集分析与COG功能富集分析验证了上述实验结果。本研究首次针对常见乳品发酵菌株嗜热链球菌的EPS合成机制提供了转录组层面的解析与见解。