Native D-amino acid oxidase (DAAO) that is expressed mostly as inclusion body and its toxicity for E. coli hamper efficient heterologous expression. In this study, the soluble expression of DAAO from Rhodosporidium toruloides (RtDAAO) was improved in E. coli through N-terminal modification, but the cell biomass was decreased. Then a trade-off between DAAO expression and biomass was achieved to obtain the highest volumetric activity of DAAO through regulated the number of N-terminus histidine residues. When variant (2)H(3)G was fused with three N-terminus histidine residues, the volumetric activity was increased by 3.1 times and the biomass was not significant change compared with the wild type. Finally, the N-terminus disordered region of RtDAAO (HSQK) was replaced with HHHG and the variant enzyme activity reached 80.7 U/mL (with a 40 percent of inactive DAAO reduced) in a 7.5 L fermenter in 24 h. (C) 2017 Elsevier Ltd. All rights reserved.