Sun Tao, Li Shubin, Song Xinyu, Pei Guangsheng, Diao Jinjin, Cui Jinyu, Shi Mengliang, Chen Lei, Zhang Weiwen
1Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin, 300072 People's Republic of China.
2Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072 People's Republic of China.
Biotechnol Biofuels. 2018 Feb 5;11:26. doi: 10.1186/s13068-018-1032-0. eCollection 2018.
Photosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts.
Aiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in . Both regulatory tools functioned well in regulating exogenous reporter gene and endogenous gene in sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch.
In this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.
光合蓝细菌因其利用太阳能和一氧化碳的能力，作为生产可再生燃料和化学品的有前景的底盘而备受关注。值得注意的是，增加丙二酰辅酶A等关键前体的供应将有利于许多生物化合物的生产。然而，蓝细菌中缺乏遗传工具，尤其是必需途径的敲低策略，严重限制了将碳通量从中心碳水化合物代谢重新导向生物产品合成的尝试。
为了开发新的遗传工具，报道了两种基于配对末端RNA以及先前在[具体文献]中开发的外源Hfq伴侣蛋白和MicC支架（Hfq-MicC）的小RNA调控工具，用于模式蓝细菌集胞藻PCC6803。这两种调控工具在调控集胞藻PCC6803中的外源报告基因和内源基因方面都发挥了良好作用，与野生型相比，基因表达下调高达90%。此外，开发了Hfq-MicC工具来同时调控与必需脂肪酸生物合成相关的多个基因，这导致脂肪酸含量降低了11%。此外，为了重新导向碳通量，利用Hfq-MicC工具干扰丙二酰辅酶A的竞争途径，与野生型相比，细胞内丙二酰辅酶A丰度增加高达41%（约698.3 pg/mL/OD）。最后，基于茶碱诱导型核糖开关，将Hfq-MicC系统进一步改造为诱导型系统。
在本研究中，报道了两种用于操纵集胞藻PCC6803中必需代谢途径和重新导向碳通量的小RNA调控工具。这项工作为光合蓝细菌引入了高效且有价值的代谢调控策略。
