College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China.
Beijing Enhalor Biotechnology Co., Ltd, Beijing, 100081, China.
Appl Microbiol Biotechnol. 2022 May;106(9-10):3669-3678. doi: 10.1007/s00253-022-11942-x. Epub 2022 May 3.
Plectasin is a promising and potent antimicrobial peptide isolated from the fungus Pseudoplectania nigrella which has been heterologously expressed in various hosts. In this study, a four-copy cassette of plectasin was constructed via 2A peptide assembly to further increase its expression level in recombinant Pichia pastoris. The yeast transformant 4Ple-61 harboring four-copy cassette of plectasin could secrete 183.2 mg/L total protein containing 60.8% of plectasin at the flask level within 120 h, which was 2.3 times higher than that of the yeast transformant Ple-6 carrying one-copy cassette of plectasin. Western blot confirmed the significant peptide expression level in the transformant 4Ple-61. Furthermore, it yielded as high as 426.3 mg/L total protein within 120 h during a 5-L fermentation. The purified plectasin shows superior stability and good antimicrobial activity against conventional Staphylococcus aureus ATCC 26,001 and some food-borne antibiotic-resistant S. aureus strains with the MICs ranging from 8 to 32 μg/mL. Therefore, the strategy based on 2A peptide assembly can enhance the expression of plectasin and further expand its application prospect. KEY POINTS: • A yeast transformant 4Ple-61 with four-copy cassette of plectasin was constructed. • The plectasin level yield by the transformant 4Ple-61 was boosted by 2.3 times. • The plectasin showed good activity against food-borne antibiotic-resistant S. aureus.
佩拉菌素是一种从真菌假黑瘤黑粉菌中分离出来的有前途的、有效的抗菌肽，已在各种宿主中异源表达。在这项研究中，通过 2A 肽组装构建了一个包含四个拷贝的佩拉菌素盒，以进一步提高重组毕赤酵母中的表达水平。含有四个拷贝的佩拉菌素盒的酵母转化子 4Ple-61 在摇瓶水平下可在 120 小时内分泌 183.2mg/L 的总蛋白，其中含有 60.8%的佩拉菌素，比携带一个拷贝的佩拉菌素盒的酵母转化子 Ple-6 高 2.3 倍。Western blot 证实了转化子 4Ple-61 中显著的肽表达水平。此外，在 5L 发酵过程中，在 120 小时内可获得高达 426.3mg/L 的总蛋白。纯化的佩拉菌素表现出优异的稳定性和良好的抗菌活性，对传统的金黄色葡萄球菌 ATCC 26001 和一些食源性病原体抗生素抗性金黄色葡萄球菌菌株具有 8-32μg/mL 的 MIC 值。因此，基于 2A 肽组装的策略可以增强佩拉菌素的表达，并进一步扩大其应用前景。要点：• 构建了一个含有四个拷贝的佩拉菌素盒的酵母转化子 4Ple-61。• 转化子 4Ple-61 的佩拉菌素产量提高了 2.3 倍。• 佩拉菌素对食源性病原体抗生素抗性金黄色葡萄球菌表现出良好的活性。
