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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引用次数: 0
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.
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.