杀虫肽U10-MYRTX-Mri1a的抗菌活性验证及其酵母表达产物的活性评价

Antibacterial activity verification of insecticidal peptide U10-MYRTX-Mri1a and activity evaluation of its yeast-expressed product

摘要:

我国“饲料禁抗、养殖减抗”政策实施后，新型抗生素替代产品的研发已成为迫切需求。在这种背景下，杀虫肽因具有功能多效性特征，且其杀虫活性与抗菌活性可能共享膜靶向机制，被认为是极具潜力的抗生素替代产品。本研究旨在验证源自欧洲红蚁毒液的杀虫肽U10-MYRTX-Mri1a的抗菌活性，并建立基于毕赤酵母表达系统的该肽生物合成平台，为开发抗生素替代产品提供实验依据与技术支持。基于“结构相似-功能跨界”的思路，本研究验证了U10-MYRTX-Mri1a对大肠杆菌和金黄色葡萄球菌的抗菌活性。通过化学合成获得U10-MYRTX-Mri1a，测定其对2种致病菌的最小抑菌浓度(minimum inhibitory concentration, MIC)分别为16 μmol/L和8 μmol/L，最小杀菌浓度(minimum bactericidal concentration, MBC)均为16 μmol/L。扫描电镜观察到肽处理导致菌体膜结构塌陷和细胞碎裂。同时，本研究采用组成型启动子甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAP)，在毕赤酵母X33中表达融合肽U10-MYRTX-Mri1a-GNA，该融合肽对大肠杆菌和金黄色葡萄球菌的抑菌率分别为(89.37±2.22)%、(88.27±2.81)%，证明酵母表达的融合肽同样具有抗菌活性。上述研究结果初步证明杀虫肽U10-MYRTX-Mri1a是有抗生素替代潜力的天然产物，该结果拓宽了替抗产品来源。同时，成功建立的基于毕赤酵母生物合成肽平台，也为后续替抗研究与开发奠定了基础。

Abstract:

After China implemented the policy of banning the application of antibiotics in feed and reducing the application of antibiotics in animal production, there has been an urgent need to develop new antibiotic alternatives. In this context, animal venom peptides are considered highly promising alternatives to antibiotics because they possess multifunctional properties and appear to employ the same membrane-targeting mechanism for both insecticidal and bactericidal activities. This study examines the antibacterial activity of U10-MYRTX-Mri1a, an insecticidal peptide derived from the venom of European red ants (Manica rubida), and assesses the functionality of its recombinant fusion protein produced in Pichia pastoris, thereby providing experimental support for the development of antibiotic alternatives. According to the concept of structural similarity-functional crossover, we evaluated the antibacterial activities of the insecticidal peptide U10-MYRTX-Mri1a derived from Myrmica rubra against Escherichia coli O157:H7 and Staphylococcus aureus ATCC 25923. U10-MYRTX-Mri1a was chemically synthesized. Its minimum inhibitory concentrations (MIC) were 16 μmol/L for E. coli O157:H7 and 8 μmol/L for S. aureus ATCC 25923, and the minimum bactericidal concentrations for both strains were 16 μmol/L. Scanning electron microscopy showed that the peptide treatment caused collapse of the bacterial membrane structure and cell fragmentation. Meanwhile, the fusion peptide U10-MYRTX-Mri1a-GNA was expressed via the constitutive promoter glyceraldehyde-3-phosphate dehydrogenase (GAP) in Pichia pastoris X33. The fusion peptide inhibited E. coli O157:H7 and S. aureus ATCC 25923 by (89.37±2.22)% and (88.27±2.81)%, respectively. The findings indicate that the insecticidal peptide U10-MYRTX-Mri1a exhibits promising potential as a natural antibiotic substitute, thereby broadening the spectrum of available antibiotic-alternative agents. Furthermore, a reliable biosynthesis platform using Pichia pastoris was successfully developed, which lays a solid groundwork for future research and development of antibiotic alternatives.