Transcriptomics and proteomics analysis to elucidate the exopolysaccharide (EPS) production pathway and structure of EPS produced by streptococcus thermophilus ASCC 1275

Abstract

Exopolysaccharides (EPS) are biopolymers of saccharides or derivatives of saccharides synthesized by microbes and plants. EPS from lactic acid bacteria (LAB) is of huge demand in food industry due generally regarded as safe (GRAS) status of LAB. Incorporation of EPS producing LAB such Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus was found to enhance the rheological properties of fermented dairy foods. Moreover, certain LAB-EPS exhibited health promoting benefits like antioxidant activity, antitumor activity, immunomodulatory effects and cholesterol lowering effects. However, the low-yield and high purification cost of LAB-EPS restrict its commercialisation. Also, the knowledge about the global changes occurring in gene- and protein-level in LAB during EPS production is not known. To overcome these issues, studies have been performed in this project to understand the underlying mechanism of EPS production in high EPS-producing dairy starter bacterium Streptococcus thermophilus ASCC 1275 using transcriptomics and proteomics approaches. Sugars are one of the major determinants of EPS production. Therefore, three sugars, glucose, sucrose and lactose were selected based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of S. thermophilus 1275 and grown in M17- broth supplemented with each sugar (1%), separately, from 0 h to 24 h. EPS was extracted, purified by dialysis and estimated using phenol sulphuric acid method while residual sugar analysis was performed using high performance liquid chromatography (HPLC). Samples for transcriptomics and proteomics analysis were collected at log phase (5 h) and stationary phase (10 h). IIIumina based complete RNA-seq analysis was performed to understand the differentially expressed genes (DEGs) responsible for EPS biosynthesis in S. thermophilus 1275. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis was done to understand the differentially expressed proteins (DEPs) involved in EPS production. KEGG and Clusters of Orthologous Group (COG) functional enrichment analysis were also performed to validate the transcriptomics and proteomics results. Finally, the EPS structure was characterized by glycosyl composition analysis, glycosyl linkage analysis and nuclear magnetic resonance (NMR) spectroscopy. S. thermophilus 1275 synthesized high amount of EPS (~430 mg/L) when M17 broth was supplemented with sucrose (1%) when compared to glucose (1%: ~276 mg/L) and lactose (1%: ~163 mg/L) at 12 h. Analysis of DEGs and DEPs showed that phosphoenolpyruvate phosphotransferase system (PEP-PTS) was responsible for sucrose and glucose uptake and lacS was responsible for lactose uptake. Biosynthesis pathways for UDP-glucose and UDP- galactose were upregulated in presence of all the three sugars. The glycosyl transferase gene epsG was highly expressed in sucrose medium in both transcriptomics and proteomics study. Wzx/Wxy-dependent pathway was found to be responsible for transport of EPS to the external environment. Furthermore, the upregulation of genes involved in amino acid metabolism especially arginine metabolism was observed. Structural analysis of EPS revealed a branched heteropolymer of glucose and galactose. Overall, this study provided insights into the EPS production mechanism of S. thermophilus 1275 and the global

transcriptional and translational level changes occurring in this bacterium in the presence of three sugars at two growth conditions along with the elucidation of the EPS structure.