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ORIGINAL RESEARCH ARTICLE

Engineering the chimeric lysin Art-15 via NZ2114–PlySs2 fusion enhances strain-specific antibacterial activity

Meng-Lin Lee1† ,  Zhitao Wu1† ,  Xingyu Zhou1 ,  Yishu Xiao1 ,  Ruilin Lai1 ,  Shuang Song1 ,  Bingjie Yan1 ,  Jinmei Huang1*
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1 Department of Research and Development, Green Long Biotech Co., Ltd., Wuhan, Hubei , China
†These authors contributed equally to this work.
MI, 026300088 https://doi.org/10.36922/MI026300088
Received: 23 July 2026 | Revised: 21 August 2026 | Accepted: 1 September 2026 | Published online: 14 September 2026
© 2026 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

The global rise of multidrug-resistant Gram-positive pathogens demands new antimicrobial strategies. Phage-derived endolysins such as PlySs2 show promising cell-wall targeting activity but may be limited by strain-dependent cell-envelope accessibility. We engineered Art-15, a chimeric lysin that fuses the lipid II-targeting peptide NZ2114 to PlySs2, to test whether enhanced targeting improves antibacterial activity. Art-15 was constructed using a (GGGGS)3 linker, expressed in E. coli, and purified by nickel affinity chromatography. Structural features were analyzed using AlphaFold2 and a transmembrane hidden Markov model. Antibacterial activity was evaluated through spot-on-lawn assays, minimum inhibitory concentration (MIC) determination, and five-hour OD600 turbidity assays. Art-15 was successfully expressed, with a recoverable soluble fraction corresponding to a ~36 kDa recombinant protein. Structural prediction indicated a compact fold without transmembrane helices. In spot assays, Art-15 showed a narrower activity spectrum than PlySs2 (12/51 vs. 27/51 isolates). However, against strain STA2505001, Art-15 exhibited a lower MIC (64 µg/mL) than PlySs2 or NZ2114 alone (128 µg/mL). In OD600 assays, Art-15 suppressed growth more effectively over five hours than either parental component.  Art-15 appears to trade broader-spectrum activity for increased strain-specific potency. The findings suggest NZ2114 may contribute to altered cell-envelope targeting and local exposure of the PlySs2 catalytic domain to peptidoglycan, although host-range narrowing indicates target accessibility remains strain-dependent.

Graphical abstract
Keywords
Endolysin
Antimicrobial peptide
PlySs2
NZ2114
Art-15
Bactericidal kinetics
Enzyme engineering
Funding
None.
Conflict of interest
Meng-Lin Lee, Zhitao Wu, Xingyu Zhou, Yishu Xiao, Ruilin Lai, Shuang Song, Bingjie Yan, and Jinmei Huang are employees of Green Long Biotech Co., Ltd. The company has filed a patent application related to the technology described in this manuscript. These interests have been disclosed and did not influence the design, execution, or interpretation of the study.
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Microbes & Immunity, Electronic ISSN: 3029-2883 Print ISSN: 3041-0886, Published by AccScience Publishing
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