Construction of a CRISPR-Cas9-Based Genetic Editing Tool for Serratia marcescens Using a Stationary Phase Promoter and Its Application in Putrescine Production

IF 3.5 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology and Bioengineering Pub Date : 2025-02-05 DOI:10.1002/bit.28949

Linbo Gou, Di Liu, Tai-Ping Fan, Huaxiang Deng, Yujie Cai

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Abstract

Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in Serratia marcescens. S. marcescens has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in S. marcescens. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from Streptococcus pyogenes protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various S. marcescens strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in S. marcescens HBQA7, optimizing the expression of ornithine decarboxylase from Clostridium aceticum DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances S. marcescens gene editing and metabolic engineering.

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基于固定相启动子的粘质沙雷菌crispr - cas9基因编辑工具的构建及其在腐胺生产中的应用

腐胺通过促进植物生长和提高作物品质，在绿色食品生产和农业中发挥着重要作用。它的应用减少了对化肥和农药的依赖，从而支持了可持续农业实践的发展。本研究在粘质沙雷菌中实现了腐胺的高效生产。粘质葡萄球菌已被广泛用于合成抗菌物质和表达蛋白质，但由于缺乏高效的基因组编辑工具，其应用受到限制。本研究提出了一种基于crispr - cas9的粘质葡萄球菌基因编辑工具。构建双质粒系统，将编辑模板与靶特异性sgRNA结合到质粒pEdit中。利用固定相启动子从化脓性链球菌蛋白中表达Cas9，避免了对额外诱导剂的需要，并确保了高效的一步基因敲除和整合。该工具在各种粘质葡萄球菌菌株中显示了超过80%的编辑效率，并实现了成功的单碱基突变。利用该工具，我们提高了S. marcescens HBQA7的腐胺产量，优化了来自Clostridium aceticum DSM1496的带P2启动子的鸟氨酸脱羧酶的表达，并确定了最佳整合位点。48 h内腐胺产量达到8.46 g/L。该研究对粘质葡萄球菌基因编辑和代谢工程具有重要意义。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biotechnology and Bioengineering 工程技术-生物工程与应用微生物

CiteScore

7.90

自引率

5.30%

发文量

280

审稿时长

2.1 months

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