Yu Wei-Sheng, Chen Ming-Hung, Ting Chia-Li, Tsung Wen-Hsuan, Sun Pei-Yu, Liu Po-Yu, Li Si-Yu
Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.
Division of Infectious Diseases, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan.
Probiotics Antimicrob Proteins. 2025 Mar 18. doi: 10.1007/s12602-025-10517-2.
Antimicrobial peptides (AMPs) are a class of naturally occurring molecules found in animals, plants, and microorganisms. Ω76 is an AMP specifically designed for treating Carbapenem-resistant Enterobacteriaceae (CRE). Escherichia coli, engineered to produce Ω76, may serve as a potential whole-cell drug delivery system for treating CRE, yet Ω76 exhibited potent antimicrobial activity against the production host. In this study, P, a tightly controlled, high-level, modulable, and stationary-phase-specific promoter, was employed for the functional expression of Ω76 in E. coli. P facilitates the decoupling of bacterial growth from Ω76 production, enabling efficient Ω76 expression. The results show that P-governed Ω76 expression exhibited a relatively high OD and biomass concentration. In contrast, P-goverend Ω76 expression, which occurred during the log phase, showed a lower OD and dormancy with cell size reducing to less than 1 μm. The expression of Ω76 was confirmed in SDS-PAGE. The extraction of Ω76 was demonstrated using methanol and ethanol as solvents, while acetate and acetonitrile were found to be ineffective. The bacteria extract containing Ω76 exhibited antimicrobial activity against both Gram-negative E. coli and Gram-positive B. subtilis. Moreover, pTOL03F-Ω76 had a Ω76 concentration of 25 ± 3 mg/L compared to 21 ± 3 mg/L for pT7-Ω76. The solid antibacterial testing suggests the formation of Ω76 tertiary structures, indicating that conventional assays designed for small-molecule antibiotics may not be suitable for AMP assays. This study first reveals the phenotypic differences between log-phase and stationary-phase Ω76 expression, suggesting two potential applications for the stationary-phase promoter: (1) the development of a whole-cell drug delivery system, where the recombinant E. coli could grow along with CRE and produce Ω76 during the stationary phase to suppress CRE, and (2) its use in the production of secondary metabolites.
抗菌肽（AMPs）是一类在动物、植物和微生物中天然存在的分子。Ω76是一种专门设计用于治疗耐碳青霉烯类肠杆菌科细菌（CRE）的抗菌肽。经过基因工程改造以产生Ω76的大肠杆菌可能作为治疗CRE的潜在全细胞药物递送系统，但Ω76对生产宿主表现出强大的抗菌活性。在本研究中，使用了P，一个严格控制、高水平、可调节且特定于稳定期的启动子，用于在大肠杆菌中功能性表达Ω76。P促进细菌生长与Ω76产生的解耦，实现高效的Ω76表达。结果表明，由P调控的Ω76表达表现出相对较高的光密度（OD）和生物量浓度。相比之下，在对数期发生的由P调控的Ω76表达显示出较低的OD和细胞休眠，细胞大小减小至小于1μm。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中证实了Ω76的表达。以甲醇和乙醇作为溶剂证明了Ω76的提取，而发现乙酸盐和乙腈无效。含有Ω76的细菌提取物对革兰氏阴性大肠杆菌和革兰氏阳性枯草芽孢杆菌均表现出抗菌活性。此外，pTOL03F-Ω76的Ω76浓度为25±3mg/L，而pT7-Ω76为21±3mg/L。固体抗菌测试表明形成了Ω76三级结构，这表明为小分子抗生素设计的传统检测方法可能不适用于抗菌肽检测。本研究首次揭示了对数期和稳定期Ω76表达之间的表型差异，表明稳定期启动子的两个潜在应用：（1）开发全细胞药物递送系统，其中重组大肠杆菌可与CRE一起生长并在稳定期产生Ω76以抑制CRE，以及（2）其在次级代谢产物生产中的应用。
