Ruan Shupeng, He Chenfeng, Wang Aoxue, Lin Ying, Liang Shuli
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Front Bioeng Biotechnol. 2025 Mar 5;13:1548187. doi: 10.3389/fbioe.2025.1548187. eCollection 2025.
Reducing endogenous gene expression is key in microbial metabolic engineering. Traditional methods for gene knockout or suppression can be slow and complex. RNA interference (RNAi) provides a faster way to regulate gene expression using plasmids with hairpin RNA. This study examines single- and double-gene suppression in , a common system for expressing heterologous proteins. We also use reporter strains displaying EGFP on the cell surface to identify factors affecting protein secretion.
We established an RNAi system in by introducing plasmids containing hairpin RNA targeting specific genes. Reporter strains expressing on the cell surface were used to monitor the impact of gene suppression on protein secretion. Genes such as , , , and were targeted for RNAi. Additionally, RNAi was applied to inhibit fatty acid synthesis to improve the conversion of malonyl-CoA to 3-hydroxypropionate (3-HP).
Suppressing and reduced display by 83% and 48.8%, respectively. In contrast, suppressing and increased display by 33.8% and 26.5%, respectively. These findings show that regulating endogenous genes can significantly impact protein secretion in . Furthermore, RNAi inhibition of fatty acid synthesis improved 3-HP production.
This study demonstrates the successful establishment of an RNAi system in , enabling efficient gene suppression for metabolic engineering. RNAi offers a faster and more efficient method for regulating gene expression, improving heterologous protein secretion and 3-HP production. This system is a valuable tool for optimizing as a microbial cell factory, with strong potential for industrial applications.
降低内源基因表达是微生物代谢工程的关键。传统的基因敲除或抑制方法可能缓慢且复杂。RNA干扰（RNAi）提供了一种使用带有发夹RNA的质粒来更快调节基因表达的方法。本研究考察了在一个用于表达异源蛋白的常见系统中对单基因和双基因的抑制作用。我们还使用在细胞表面展示绿色荧光蛋白（EGFP）的报告菌株来鉴定影响蛋白质分泌的因素。
我们通过引入含有靶向特定基因的发夹RNA的质粒，在[具体物种]中建立了一个RNAi系统。使用在细胞表面表达[具体蛋白]的报告菌株来监测基因抑制对蛋白质分泌的影响。诸如[具体基因1]、[具体基因2]、[具体基因3]和[具体基因4]等基因被作为RNAi的靶点。此外，应用RNAi抑制脂肪酸合成以提高丙二酰辅酶A向3-羟基丙酸（3-HP）的转化。
抑制[具体基因1]和[具体基因2]分别使[具体蛋白]的展示减少了83%和48.8%。相比之下，抑制[具体基因3]和[具体基因4]分别使[具体蛋白]的展示增加了33.8%和26.5%。这些发现表明，调节内源基因可显著影响[具体物种]中的蛋白质分泌。此外，RNAi对脂肪酸合成的抑制提高了3-HP的产量。
本研究证明了在[具体物种]中成功建立了一个RNAi系统，能够为代谢工程实现高效的基因抑制。RNAi为调节基因表达提供了一种更快且更有效的方法，可改善异源蛋白质分泌和3-HP产量。该系统是优化[具体物种]作为微生物细胞工厂的一个有价值的工具，具有很强的工业应用潜力。
