Mao Ya-Hui, Li Feng, Luo Li-Zhen, Yin Yu, Ma Jin-Cheng, Zhang Wen-Bin, Wang Hai-Hong, Zhang Changyi, Hu Zhe
Guangdong Provincial Key Laboratory for the Development Biology and Environmental Adaptation of Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, 510642, China.
Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables, College of Life Science and Technology, Hubei Engineering University, Xiaogan, Hubei, 432000, China.
BMC Microbiol. 2024 Dec 4;24(1):517. doi: 10.1186/s12866-024-03645-2.
Sinorhizobium meliloti is noted for its exceptional capacity to produce unsaturated fatty acids (UFAs). Earlier studies have indicated that S. meliloti primarily employs the FabA-FabB pathway for UFA synthesis, however, the mechanisms remain elusive. This study was conducted to elucidate these mechanisms responsible for the significant UFA production in S. meliloti.
The genes encoding enoyl-acyl carrier protein (ACP) reductase (ENR) were disrupted using the suicide plasmid pK18mobsacB, followed by the creation of single-crossover and double-crossover mutants. The ENR proteins were expressed in Escherichia coli BL21(DE3) strains and subsequently purified. Their enzymatic activities were assessed through gel electrophoresis and NADH oxidation assays. Additionally, the fatty acid composition was determined using gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography.
Our findings demonstrate that the heterologous expression of fabI2 in a temperature-sensitive E. coli fabI mutant results in a significant enhancement of UFA production. Genetic analyses confirmed that fabI2 is an indispensable gene in the S. meliloti genome, as it cannot be disrupted. Interestingly, we observed that fabI2 could only be functionally replaced by the Enterococcus faecalis fabI gene and not by the homologous fabI1 from S. meliloti, E. coli fabI, or Pseudomonas aeruginosa fabV. Furthermore, we validated that the deletion of fabI1 in S. meliloti triggered an increase in UFA production compared to the wild-type strain Rm1021.
In this study, we identified the ENR, encoded by the S. meliloti SMc00326 gene (fabI2), as playing a pivotal role in the biosynthesis of UFAs. Additionally, the FabI1 enzyme, encoded by SMc00005, was found to modulate the fatty acid composition within S. meliloti. Together, these discoveries establish a foundation for the development of a model that explains the significant contribution of FabI2 to the robust synthesis of UFAs in S. meliloti.
苜蓿中华根瘤菌以其产生不饱和脂肪酸（UFA）的特殊能力而闻名。早期研究表明，苜蓿中华根瘤菌主要利用FabA-FabB途径进行UFA合成，然而，其机制仍不清楚。本研究旨在阐明苜蓿中华根瘤菌中大量产生UFA的这些机制。
使用自杀质粒pK18mobsacB破坏编码烯酰-酰基载体蛋白（ACP）还原酶（ENR）的基因，随后创建单交换和双交换突变体。将ENR蛋白在大肠杆菌BL21(DE3)菌株中表达，随后进行纯化。通过凝胶电泳和NADH氧化测定评估其酶活性。此外，使用气相色谱-质谱联用仪（GC-MS）和薄层色谱法测定脂肪酸组成。
我们的研究结果表明，fabI2在温度敏感的大肠杆菌fabI突变体中的异源表达导致UFA产量显著提高。遗传分析证实，fabI2是苜蓿中华根瘤菌基因组中不可或缺的基因，因为它无法被破坏。有趣的是，我们观察到fabI2只能被粪肠球菌的fabI基因功能性取代，而不能被苜蓿中华根瘤菌的同源fabI1、大肠杆菌fabI或铜绿假单胞菌fabV取代。此外，我们验证了苜蓿中华根瘤菌中fabI1的缺失与野生型菌株Rm1021相比引发了UFA产量的增加。
在本研究中，我们确定由苜蓿中华根瘤菌SMc00326基因（fabI2）编码的ENR在UFA生物合成中起关键作用。此外，发现由SMc00005编码的FabI1酶可调节苜蓿中华根瘤菌内的脂肪酸组成。总之，这些发现为建立一个解释FabI2对苜蓿中华根瘤菌中UFA强大合成的重要贡献的模型奠定了基础。
