枯草芽孢杆菌中人乳铁蛋白的表达与分泌
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国家自然科学基金(32270096, 32021005)


The expression and secretion of human lactoferrin in Bacillus subtilis
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    摘要:

    人源乳铁蛋白(human lactoferrin, HLF)是母乳中重要的营养成分,具有抗菌、消炎、提高机体免疫力等功能。本研究以枯草芽孢杆菌(Bacillus subtilis) G601为宿主,对比了3种组成型(P21、P43和Pveg)和3种诱导型启动子(Pgrac100、PxylA和Ptet*)对HLF表达的影响,摇瓶发酵结果显示,启动子Ptet*驱动的HLF的表达量最高,为651.57μg/L;进一步对核糖体结合位点(ribosome binding site, RBS)和信号肽进行组合筛选,获得的RBS-信号肽组合RBS6-SPyycP促使HLF的总表达量提升至 1 099.87 μg/L,其中分泌至胞外的蛋白量为498.68 μg/L;为了提高蛋白的胞外分泌量,敲除细胞壁表面离子相关基因dltD,菌株HLF分泌产量达到637.28μg/L。本研究通过表达元件筛选与优化等策略成功实现了HLF在B. subtilis的分泌表达,为构建B. subtilis细胞工厂高效合成乳蛋白奠定了基础。

    Abstract:

    Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 μg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 μg/L, with 498.68 μg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 μg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

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张予婷,李洋,武耀康,刘延峰,李江华,堵国成,吕雪芹,刘龙. 枯草芽孢杆菌中人乳铁蛋白的表达与分泌[J]. 生物工程学报, 2024, 40(6): 1895-1908

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历史
  • 收稿日期:2023-11-15
  • 最后修改日期:
  • 录用日期:2024-01-17
  • 在线发布日期: 2024-06-06
  • 出版日期: 2024-06-25
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