细胞动力学工程增强了毕赤酵母中重组人溶菌酶的分泌生产。
Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii.
机构信息
School of Pharmaceutical Sciences, Taizhou University, Jiaojiang, Zhejiang Province, 318000, China.
Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation, Taizhou University, Taizhou, 318000, China.
出版信息
Microb Cell Fact. 2024 Jun 18;23(1):179. doi: 10.1186/s12934-024-02434-w.
BACKGROUND
Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast.
RESULTS
Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ.
CONCLUSIONS
Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.
背景
人溶菌酶（hLYZ）是一种天然抗菌蛋白，在食品和制药行业有广泛的应用。在毕赤酵母（K. phaffii）中重组生产 hLYZ 引起了相当大的关注，但在酵母中实现其超生产的策略非常有限。
结果
通过基于大气压室温等离子体（ARTP）的诱变和转录组分析，鉴定出编码细胞分裂核心蛋白的两个基因 MYO1 和 IQG1 的表达随 hLYZ 产量的增加而下调。敲除任一基因都会导致严重的胞质分裂缺陷，但会显著提高 hLYZ 的产量。在高密度分批补料发酵中，Δmyo1 突变体的 hLYZ 产量最高，生物活性为 1,052,444±23,667 U/mL，总蛋白浓度为 4.12±0.11 g/L，代表了酵母中 hLYZ 的最佳产量。此外，还对这种重组 hLYZ 的 O-连接甘露糖糖基化进行了表征。
结论
我们的工作表明，基于胞质分裂的形态工程是提高 K. phaffii 中 hLYZ 产量的有效方法。
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