The microbial production of D-lysine to replace chemical approach has gained great interest with the rising concerns over the environment. Here, we employed recombinant E. colt 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 optical purity of D-lysine, the optimal reaction condition for resolution of DL-lysine after the racemization was investigated. 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 racemization and asymmetric degradation process. The final o-lysine enantiomeric excess was over 99%. Meanwhile, a valuable compound 5-aminovaleric add was synthesized with the production of D-lysine, indicating the economic feasibility of the two-strain coupling system. (C) 2020 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd. All rights reserved.