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A genome-based systematic analysis combined with phenotypic experiments was conducted to evaluate the safety properties and metabolic characteristics of Streptococcus thermophilus DMST-H2, isolated from Chinese traditional homemade yogurt in Inner Mongolia. Comparative genomic analysis showed that DMST-H2 had a closer genetic relationship with S. thermophilus KLDS.3.1003 based on the sequence similarities of the 1193 core-genes. No genes related to antibiotic resistance and virulence factor as well as no complete biogenic amine biosynthesis gene clusters were identified in DMST-H2 genome. DMST-H2 was sensitive to various antibiotics and had no hemolytic activity and no biogenic amine production capacity, confirming that DMST-H2 is safe and non-toxic. In addition, multiple genes for carbohydrate metabolism were predicted, including various carbohydrate-active enzymes and sugar transporters that could be involved in the transport of multiple carbon sources. Two exopolysaccharide (EPS) gene clusters (eps-1 and rgp-2) were also detected, indicating that DMST-H2 has the potential to synthesize heteropolysaccharides. Gene functional features revealed a cyclic antimicrobial peptide related gene cluster in DMST-H2, which displayed high identities with the streptide biosynthesis gene cluster as previously reported. Our comprehensive analysis of the whole genome and corresponding phenotypes of S. thermophilus DMST-H2 provides important theoretical basis for its application in food products. • Comparative genomic analysis of S. thermophilus DMST-H2 was displayed. • Systematic analysis of its safety and metabolism characteristics based on phenotype and genotype. • Two exopolysaccharide (EPS) gene clusters were found in the genome of S. thermophilus DMST-H2. • Carbohydrate metabolism pathways were predicted in the genome of S. thermophilus DMST-H2. [ABSTRACT FROM AUTHOR]