College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China; Shandong Provincial Research Center for Bioinformatic Engineering and Technology, School of Life Sciences, Shandong University of Technology, Zibo, 255049, China.
MOE Key Lab of Bioinformatics, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China.
Metab Eng. 2022 Sep;73:58-69. doi: 10.1016/j.ymben.2022.06.005. Epub 2022 Jun 20.
Halomonas bluephagenesis, a robust and contamination-resistant microorganism has been developed as a chassis for "Next Generation Industrial Biotechnology". The non-model H. bluephagenesis requires efficient tools to fine-tune its metabolic fluxes for enhanced production phenotypes. Here we report a highly efficient gene expression regulation system (PrrF1-2-HfqPa) in H. bluephagenesis, small regulatory RNA (sRNA) PrrF1 scaffold from Pseudomonas aeruginosa and a target-binding sequence that downregulate gene expression, and its cognate P. aeruginosa Hfq (HfqPa), recruited by the scaffold to facilitate the hybridization of sRNA and the target mRNA. The PrrF1-2-HfqPa system targeting prpC in H. bluephagenesis helps increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to 21 mol% compared to 3.1 mol% of the control. This sRNA system repressed phaP1 and minD simultaneously, resulting in large polyhydroxybutyrate granules. Further, an sRNA library targeting 30 genes was employed for large-scale target identification to increase mevalonate production. This work expands the study on using an sRNA system not based on Escherichia coli MicC/SgrS-Hfq to repress gene expression, providing a framework to exploit new powerful genome engineering tools based on other sRNAs.
蓝盐单胞菌是一种强大且抗污染的微生物，已被开发为“下一代工业生物技术”的底盘。非模式生物 H. bluephagenesis 需要有效的工具来微调其代谢通量，以增强生产表型。在这里，我们报告了在 H. bluephagenesis 中一种高效的基因表达调控系统（PrrF1-2-HfqPa），该系统来自铜绿假单胞菌的小调控 RNA（sRNA）PrrF1 支架和一个靶标结合序列，可下调基因表达，以及其同源的铜绿假单胞菌 Hfq（HfqPa），由支架招募来促进 sRNA 和靶标 mRNA 的杂交。针对 H. bluephagenesis 中 prpC 的 PrrF1-2-HfqPa 系统有助于将聚（3-羟基丁酸酯-co-3-羟基戊酸酯）中的 3-羟基戊酸分数从 3.1mol%增加到 21mol%，而对照物的 3.1mol%。该 sRNA 系统同时抑制了 phaP1 和 minD，导致聚羟基丁酸酯颗粒增大。此外，还使用了一个靶向 30 个基因的 sRNA 文库进行大规模靶标鉴定，以提高甲羟戊酸产量。这项工作扩展了使用不基于大肠杆菌 MicC/SgrS-Hfq 的 sRNA 系统来抑制基因表达的研究，为利用其他 sRNA 提供了一个新的强大基因组工程工具的框架。
