<br>应用于 Caco-2 细胞的双 sgRNA CRISPRa 系统可增强纳豆枯草芽孢杆菌中 MK-7 的产生并减轻沙门氏菌感染,Journal of Agricultural and Food Chemistry

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应用于 Caco-2 细胞的双 sgRNA CRISPRa 系统可增强纳豆枯草芽孢杆菌中 MK-7 的产生并减轻沙门氏菌感染
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-02-12 , DOI: 10.1021/acs.jafc.3c08866
Chaoyong Liao ₁ , Jian Cui ₁ , Mingkun Gao ₁ , Bo Wang ₁ , Koichi Ito ₂ , Yuming Guo ₁ , Bingkun Zhang ₁

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本研究优化了源自纳豆的枯草芽孢杆菌 ( B. subtilis ) 纳豆 NB205 中的甲基萘醌 7 (MK-7) 合成途径，以提高其 MK-7 的产量。利用突变育种，我们开发了 NBMK308，这是一种突变菌株，其 MK-7 产量显着增加了 117.23%。全面的转录组分析确定了两个关键基因ispA和ispE在 MK-7 合成中至关重要。利用双sgRNA CRISPRa系统在新工程菌株A3E3中实现了ispA和ispE的精确调控。这种策略性调控导致MK-7产量显着增强，与传统过表达系统和原始菌株NB205相比，分别增加了20.02%和201.41%。此外，A3E3 的发酵上清液显着抑制沙门氏菌侵入 Caco-2 细胞，显示出其对抗此类感染的潜力。双sgRNA CRISPRa系统的安全性通过细胞测定得到证实。本研究中双 sgRNA CRISPRa 系统的利用对于 MK-7 合成中关键基因的精确调控至关重要，从而导致产量显着增加，并展示了抑制病原体感染的额外治疗潜力。这种方法有效地结合了微生物发酵和生物技术的优势，解决了健康和营养挑战。

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Dual-sgRNA CRISPRa System for Enhanced MK-7 Production and Salmonella Infection Mitigation in Bacillus subtilis natto Applied to Caco-2 Cells

This study optimized the menaquinone-7 (MK-7) synthetic pathways in Bacillus subtilis (B. subtilis) natto NB205, a strain that originated from natto, to enhance its MK-7 production. Utilizing mutation breeding, we developed NBMK308, a mutant strain that demonstrated a significant 117.23% increase in MK-7 production. A comprehensive transcriptome analysis identified two key genes, ispA and ispE, as being critical in MK-7 synthesis. The dual-sgRNA CRISPRa system was utilized to achieve precise regulation of ispA and ispE in the newly engineered strain, A3E3. This strategic modulation resulted in a significant enhancement of MK-7 production, achieving increases of 20.02% and 201.41% compared to traditional overexpression systems and the original strain NB205, respectively. Furthermore, the fermentation supernatant from A3E3 notably inhibited Salmonella invasion in Caco-2 cells, showcasing its potential for combating such infections. The safety of the dual-sgRNA CRISPRa system was confirmed through cell assays. The utilization of the dual-sgRNA CRISPRa system in this study was crucial for the precise regulation of key genes in MK-7 synthesis, leading to a remarkable increase in production and demonstrating additional therapeutic potential in inhibiting pathogenic infections. This approach effectively combined the advantages of microbial fermentation and biotechnology, addressing health and nutritional challenges.

更新日期：2024-02-12

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