Engineering T7 RNA polymerase-cascaded systems controlled by nisin and theophylline for protein overexpression and targeted gene mutagenesis in Lactococcus lactis

IF 4.4 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Ying Huang , Kang Ma , Yan Li , Qingyan Li , Fuping Lu , Xueli Zhang , Zhe Sun
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Abstract

Lactococcus lactis serves as an important platform for heterologous protein production, with the nisin-controlled gene expression (NICE) system being widely employed for regulated protein overexpression. However, the NICE system relies on the native RNA polymerase, which limits transcriptional efficiency, and there remains a lack of tools enabling continuous target gene mutagenesis in L. lactis. In this study, we enhanced the NICE system by integrating the highly processive T7 RNA polymerase (T7RNAP) to boost protein expression. A theophylline-dependent riboswitch, RbxE, was incorporated into the nisin-induced promoter to mitigate the toxicity caused by basal T7RNAP expression in Escherichia coli. Directed mutagenesis of the riboswitch region between the stem-loop and the ribosome binding site optimized T7RNAP expression, leading to a 2.4-fold increase upon nisin and theophylline induction in L. lactis. The resulting NICE-T7 system achieved a 2.8-fold increase in GFP compared to the original NICE system. Furthermore, adenosine deaminase TadA8e was fused to T7RNAP to generate the MutaT7LL system, facilitating targeted A-to-G mutagenesis and successfully reactivated an erythromycin resistance gene with a mutation efficiency of 1.33 × 10−6. Overall, this study presents an upgraded NICE system that enhances protein production and enables continuous in vivo mutagenesis of target genes in L. lactis.
乳酸菌蛋白和茶碱控制的T7 RNA聚合酶级联系统在乳酸乳球菌中的蛋白过表达和靶向基因诱变
乳酸乳球菌是异源蛋白生产的重要平台,nisin-controlled gene expression (NICE)系统被广泛用于调节蛋白过表达。然而,NICE系统依赖于天然RNA聚合酶,这限制了转录效率,并且仍然缺乏能够在乳杆菌中连续诱变靶基因的工具。在这项研究中,我们通过整合高加工性T7RNA聚合酶(T7RNAP)来增强NICE系统,以提高蛋白质表达。一种依赖茶碱的核糖开关RbxE被加入到nisin诱导的启动子中,以减轻大肠杆菌中T7RNAP基础表达引起的毒性。在乳酸菌茎环与核糖体结合位点之间的核糖体开关区进行定向诱变,优化了T7RNAP的表达,在乳酸菌素和茶碱诱导下,T7RNAP的表达量增加了2.4倍。由此产生的NICE- t7系统与原始NICE系统相比,GFP增加了2.8倍。此外,将腺苷脱氨酶TadA8e与T7RNAP融合生成MutaT7LL系统,实现了a -to- g的靶向诱变,成功激活了一个红霉素耐药基因,突变效率为1.33 × 10−6。总的来说,本研究提出了一个升级的NICE系统,可以提高乳杆菌的蛋白质产量,并使靶基因在体内连续突变。
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来源期刊
Synthetic and Systems Biotechnology
Synthetic and Systems Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
6.90
自引率
12.50%
发文量
90
审稿时长
67 days
期刊介绍: Synthetic and Systems Biotechnology aims to promote the communication of original research in synthetic and systems biology, with strong emphasis on applications towards biotechnology. This journal is a quarterly peer-reviewed journal led by Editor-in-Chief Lixin Zhang. The journal publishes high-quality research; focusing on integrative approaches to enable the understanding and design of biological systems, and research to develop the application of systems and synthetic biology to natural systems. This journal will publish Articles, Short notes, Methods, Mini Reviews, Commentary and Conference reviews.
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