The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study， we constructed a 2 kb-triple-acting fusion gene () encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase)， a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The gene was expressed in plants using the non-replicating (CPMV)-based vector pEAQ-HT and the replicating AltMV)-based pGD5TGB123-MCS-CP3 vector， and in using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a gene codon-optimized for expression in tobacco plants， resulting in an improved codon adaptation index (CAI) from 0.79 ( gene) to 0.93 ( gene). Incorporation of the nt gene into the pEAQ-HT vector， followed by transient expression in ， led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against 305， showing 14% of growth inhibition， while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains， showing 68 (IC 25 µg/ml) and 60% (IC 71 µg/ml) growth inhibition against 305 and Newman， respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants， the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.