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.

Halomonas bluephagenesis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”的基础。非模型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H. bluephagenesis中的高效基因表达调节系统 (PrrF1-2-HfqPa)、来自铜绿假单胞菌的小调节 RNA (sRNA) PrrF1 支架和下调基因表达的靶结合序列，及其同源P. aeruginosa Hfq (HfqPa)，由支架募集以促进 sRNA 与靶 mRNA 的杂交。针对prpC的PrrF1-2-HfqPa 系统与对照的 3.1 mol% 相比，H. bluephagenesis有助于将聚 (3-羟基丁酸酯-co-3-羟基戊酸酯) 中的 3-羟基戊酸酯部分增加到21 mol%。这种 sRNA 系统同时抑制phaP1和minD，产生大的聚羟基丁酸酯颗粒。此外，针对 30 个基因的 sRNA 文库被用于大规模目标识别，以增加甲羟戊酸的产量。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，为利用基于其他 sRNA 的新的强大基因组工程工具提供了一个框架。