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The microbial production of D-lysine to replace chemical approach has gained great interest with the rising con-cerns over the environment. Here, we employed recombinant E. coli strain BL21-LYR with lysine racemase and strain BL-22A-RB-YB with L-lysine monooxygenase and 5-aminovaleramide amidohydrolase to establish a two-strain coupling whole-cell bioconversion system for D-lysine production from L-lysine. To improve the opti-cal purity of D-lysine, the optimal reaction condition for resolution of DL-lysine after the racemization was inves-tigated. The specificity of BL-22A-RB-YB for L-lysine and the effects of reaction condition on bioconversion efficiency of whole-cell were accordingly determined. Under the optimal condition, a maximum 53.5 g?L-1 D-lysine and 48.2 g ? L-1 5-AVA were obtained with yield of 47.4％ and 42.3％, respectively, by the microbial racemi-zation and asymmetric degradation process. The final D-lysine enantiomeric excess was over 99％. Meanwhile, a valuable compound 5-aminovaleric acid was synthesized with the production of D-lysine, indicating the eco-nomic feasibility of the two-strain coupling system.