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Aims: The present study aimed to specifically transform stevioside (ST) into rubusoside (RS) through bioconversion with high efficiency, seeking to endow steviol glycosides (SGs) with subtle flavours for commercial acceptability., Methods and Results: An endophytic bacterium named Lelliottia LST-1 was screened and confirmed to specifically convert ST into RS, reaching a conversion rate of 75.4% after response surface optimization. Phylogenetic analysis combined with complete genome sequencing demonstrated that LST-1 was also presumed to be a new species. To further explore the principle and process of biological transformation, the potential beta-glucosidases GH3-1, GH3-2, GH3-3 and GH3-4 were expressed, purified and reacted with SGs. High-performance liquid chromatography revealed that all enzymes hydrolysed ST and generated RS, but substrate specificity analysis indicated that GH3-2 had the highest substrate specificity towards STs and the highest enzyme activity., Conclusion: The potential β-glucosidase GH3-2 in Lelliottia sp. LST-1 was found to specifically and efficiently convert ST to RS., Significance and Impact of Study: The efficient biotransformation of ST into RS will be beneficial to its large-scale production and extensive application in the food and pharmaceutical industries., (© 2021 The Society for Applied Microbiology.)