NZ17074, a member of the arenicin family from Arenicola marina, exhibited potent antimicrobial activity against bacteria and fungi, but had some toxicity to human erythrocytes and porcine intestinal epithelial cells. To resolve this problem, one novel peptide N2 was designed based on NZ17074 by replacing Gly in position 1,12 with Ala. N2 exhibited a typical beta-sheet conformation in aqueous and almost had no hemolysis. N2 was expressed in Pichia pastoris using the small ubiquitin-like modifier fusion technology. The yield of N2 was approximately 6.5 mg/l, and its molecular weight was 2665.0 Da. The minimum inhibitory concentrations of N2 against Gram-negative bacteria ranged from 0.125 to 4 mu g/ml. The antibacterial activity of N2 against Escherichia coli CVCC 195 and Salmonella enteritidis CVCC 3377 was enhanced up to 1-3-fold over parent NZ17074. This result provides some information that assists in the rational design of toxic antimicrobial peptides. (C) 2016 Elsevier Ltd. All rights reserved.