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A Lactobacillus buchneriGBS3 strain isolated from the traditional Chinese pickles was used for the production of 3-phenyllactic acid (PLA), an important compound with antimicrobial activities against a wide species of gram-positive and gram-negative bacteria and some fungi. The growth performance of this strain in the de Man, Rogosa and Sharpe (MRS) medium, the production of metabolites of valuable organic acids, and the biosynthesis of PLA using this strain as the whole-cell biocatalyst and phenylpyruvic acid (PPA) as the precursor, were investigated experimentally. The uniform design method with overlay sampling was developed for the optimization of the biotransformation conditions. The results showed that although it produced naturally lactic acid with the maximum concentration of 1.84 g·L−1and PLA with the concentration of 0.015 g·L−1after 66 to 72 h cultivation in MRS broth by fermentation, the present strain displayed an effective utilization ability by transforming PPA to PLA. By the uniform design method with overlay sampling for the design and optimization of transformation conditions, a maximum yield of 10.93 g·L−1PLA with the mole conversion ratio of 83.07% from PPA to PLA was achieved under the optimized condition, i.e., 20 g·L−1glucose, 270 g·L−1cells, 13 g·L−1PPA, pH 8.0 and the reaction time of 15 h, indicating that Lactobacillus buchneriGBS3 was an interesting strain for the biosynthesis of PLA viathe microbial transformation. The prediction of PLA yield under different conditions was achieved successfully based on the limited information of only a small number of experiments by the uniform design with overlay sampling. Therefore, the present methodology is effective and helpful for the optimization of the biosynthesis processes of PLA.