利用CRISPRa技术提高乳酸克鲁维酵母凝乳酶分泌表达
Improvement of the secretary expression of chymosin in Kluyveromyces lactis via CRISPRa
邓宇慧 1王世清 1郑艳丽1
作者信息
- 1. 武汉轻工大学生命科学与技术学院,湖北 武汉 430023
- 折叠
摘要
乳酸克鲁维酵母因其可靠的食品安全性被广泛应用于工业酶制剂生产,利用高通量的基因调控技术,如基于CRISPR-dCas9 的基因表达激活平台(CRISPR activation,CRISPRa)是进一步促进酶制剂产量提高的最有效方法.本研究将激活因子 VPR和 LAC9 分别与 dCas9 融合,针对 PLAC4 启动子上游区域序列设计 gRNA,引导 dCas9-激活因子融合蛋白对其进行特异性结合,促进 RNA聚合酶在该区域的聚集,驱动受 PLAC4 启动子调控的凝乳酶表达.结果显示,dCas9-VPR与 dCas9-LAC9 的融合形式可分别将凝乳酶表达量提高 1.67 倍和 1.65 倍.本研究为利用乳酸克鲁维酵母进行工业酶制剂生产提供了有效的工具.
Abstract
Kluyveromyces lactis is widely used in industrial enzyme production due to its reliable food safety,and utilizing high-throughput gene regulation technologies,such as CRISPR activation(CRISPRa)upon the CRISPR-dCas9 system,is one of the most effective methods to further improve enzyme yields.In this study,activators including VPR and LAC9 were individually fused to dCas9 while gRNAs were designed against sequences in the upstream region of the PLAC4 promoter to direct specific binding of each dCas9-activator fusion,thereby promoting the aggregation of RNA polymerase to drive expression of the chymosin gene controlled by the PLAC4 promoter.The results showed that the fusions of dCas9-VPR and dCas9-LAC9 could increase the chymosin expression yields by the highest 1.67 times and 1.65 times,respectively.This study thus provided an effective tool for industrial enzyme production using K.lactics.
关键词
乳酸克鲁维酵母/CRISPR-dCas9/LAC9/激活因子/VPR激活因子/凝乳酶Key words
Kluyveromyces lactis/CRISPR-dCas9/LAC9/VPR/chymosin引用本文复制引用
出版年
2025
NETL
NSTL
万方数据