International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology, Central South University of Forestry and Technology, Changsha, China.
Hunan Provincial Key Laboratory of Forestry Biotechnology, Central South University of Forestry and Technology, Changsha, China.
Appl Environ Microbiol. 2024 Aug 21;90(8):e0007524. doi: 10.1128/aem.00075-24. Epub 2024 Jul 12.
Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of , which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for expression in 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of gene significantly decreased the transcription level of in 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in , which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in , which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.
甘油脱水酶是 1,3-丙二醇合成途径中的关键和限速酶，它决定了 1,3-丙二醇的产率和产量。然而，甘油脱水酶基因的表达调控机制仍知之甚少。在本研究中，鉴定并表征了一种组蛋白样核小体结构（H-NS）蛋白，作为先前研究中我们发现的对 2e 中 表达具有高耐受性的正向转录调节剂。在 2e 中删除 基因显著降低了 基因的转录水平，导致菌株生长、甘油脱水酶活性和甘油发酵过程中 3-羟基丙醛生成显著缺陷。与纯甘油相比，在粗甘油中 基因的转录水平显著上调，而 H-NS 的失活导致前者中 基因的转录水平产生更负的影响。尽管两种底物中 H-NS 的表达水平几乎相当，但与纯甘油相比，其多聚体状态在粗甘油中减少，表明 H-NS 的寡聚状态可能有助于甘油脱水酶表达的正向调节。此外，电泳迁移率变动和 DNase I足迹实验表明，H-NS 可以通过识别富含 AT 的区域直接结合到 的上游启动子区域。这些发现为 H-NS 对 的甘油脱水酶表达的转录调控机制提供了新的见解，这可能为工程菌工业生产 1,3-丙二醇提供新的目标。
重要性
微生物发酵从甘油生产 1,3-丙二醇的生物生产在工业应用上具有广阔的前景。甘油脱水酶催化甘油代谢的倒数第二步，被认为是 1,3-丙二醇生产的关键和限速酶之一。在大多数研究中，H-NS 被报道为一种多效调节剂，对基因表达有负作用。在这里，我们首次报道 H-NS 正向调节甘油脱水酶基因的表达。该结果提供了一个关于 H-NS 正向调节 中甘油脱水酶基因表达的新分子机制的见解，这对于促进高效 1,3-丙二醇生产工程菌株的构建具有很大的潜力。
