基于小RNA调控工具在蓝藻合成生物学中的应用<sup>*</sup>

doi:10.13523/j.cb.2307017

中国生物工程杂志

2024, Vol. 44

Issue (2/3): 176-189 DOI: 10.13523/j.cb.2307017

综述

基于小RNA调控工具在蓝藻合成生物学中的应用^*

王瑞彬,董世瑞^**(

)

天津商业大学生物技术与食品科学学院天津市食品生物技术重点实验室天津 300134

Application of Small RNA Regulatory Tools in Cyanobacteria Synthetic Biology

WANG Ruibin,DONG Shirui^**(

)

Tianjin Key Laboratory of Food Biotechnology, School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China

全文: PDF(1286 KB) HTML

摘要：

基因调控工具在蓝藻合成生物学领域的应用尤为重要,可实现基因表达调控、工程株开发以及附加经济产物的生产。小RNA调控工具是基于小RNA的靶向特异性调控原理,并与合成生物学相结合,进行靶基因特异性调控的定量、全局调控工具。它将小RNA与其靶标作为互作反应模块,并以诱导表达开关、支架序列与伴侣蛋白等作为辅助模块。这类工具已被应用于高附加经济产物合成与藻株对燃料与化学品的耐受性修饰。根据小RNA工具的不同调控特性,对近年来小RNA调控工具的种类、调控原理、辅助模块的选择与改造,以及在合成附加经济产物、提高蓝藻耐受性实验中获得的研究进展进行综述,探讨小RNA工具在应用中可能存在的问题及未来发展前景,为设计高效、精确的分子编辑工具提供参考。

关键词： 合成生物学; 调控基因表达; 小RNA工具

Abstract:

The application of gene regulatory tools in the field of cyanobacterial synthetic biology is particularly important for controlling gene expression, engineering strain development, and the production of additional economic products. Small RNA regulatory tools, which are based on the principle of targeted specificity regulation of small RNAs, are quantitative and global regulatory tools that are combined with synthetic biology for specific regulation of target genes. These tools use small RNAs and their targets as interaction modules and use inducible expression switches, scaffold sequences, and partner proteins as ancillary modules. Such tools have been applied to the synthesis of high-value economic products and the modification of algal strains for fuel and chemical tolerance. This paper provides an overview of different types of small RNA regulatory tools, their regulatory principles, the selection and modification of ancillary modules, as well as research progress in the synthesis of additional economic products and improvement of cyanobacterial tolerance. It also discusses potential problems and future development prospects of small RNA tools in applications, providing a reference for the design of efficient and precise molecular editing tools.

Key words: Synthetic biology Regulates gene expression Small RNA tools

收稿日期: 2023-07-13 出版日期: 2024-04-03

ZTFLH:

Q819

基金资助: *天津市研究生科研创新(2022SKYZ134)

通讯作者: **电子信箱:dongshirui@tjcu.edu.cn

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引用本文:

王瑞彬, 董世瑞. 基于小RNA调控工具在蓝藻合成生物学中的应用^*[J]. 中国生物工程杂志, 2024, 44(2/3): 176-189.

WANG Ruibin, DONG Shirui. Application of Small RNA Regulatory Tools in Cyanobacteria Synthetic Biology. China Biotechnology, 2024, 44(2/3): 176-189.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307017 或 https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/176

图1 核糖体调节系统的调控原理

表1 核糖体调控系统在蓝藻合成生物学中的应用

图2 CRISPR/Cas系统的调控机制

图3 CRISPRi系统的调控机制

表2 CRISPR系统在蓝藻合成生物学中的应用

图4 Hfq结构与sRNA支架-Hfq调控机制 A: 球棒结构表示酸性氨基酸,即天冬氨酸和谷氨酸。蓝色表示二级结构β折叠, 橙色区域表示Hfq蛋白核心的卷曲结构 B:质粒元件结构组成和sRNA支架-Hfq复合物 C、D:展示sRNA支架序列-Hfq的调控机制

表3 sRNA支架-Hfq在蓝藻合成生物学中的应用

图5 核糖开关调控机制以及质粒构建

表4 核糖开关在蓝藻合成生物学中的应用

表5 小 RNA调控工具的特点

类型	辅助元件	藻株	工程系统		靶点	结果	参考文献
CRISPRi	核糖开关	聚球藻PCC 7002 Synechococcus sp. PCC 7002	无水四环素系统融合启动子区调控dCas9表达		ypet报告基因	外源加入1 μg/mL aTc,Ypet荧光强度受到抑制	[6]
CRISPRi	核糖开关	鱼腥藻PCC 7120 Anabaena sp. PCC 7120	P_petE-tetR-P_L03-茶碱核糖开关E-dCas9		谷氨酰胺合成酶 (glnA)	3.3倍抑制glnA表达,胞内产铵,并能够控制光合产铵的时间,诱导后分泌铵至胞外	[51]
核糖体调控系统	核糖开关	鱼腥藻PCC 7120 Anabaena sp. PCC 7120	P_petE-tetR-P_L03-taRNA/P_nifB-crRNA-lacZ		lacZ报告基因	实现对lacZ的二元调控	[73]
核糖体调控系统	支架序列- Hfq	集胞藻PCC 6803 Synechocystis sp. PCC 6803	P_nrsB-taR^2-MicF M7.4 /P_trcΔlacO-crR^2-gfp		gfp报告基因	taR^2-MicF M7.4的表达量最高,比taR^2-MicF高约1.5倍,比taR^2高2.5倍,有效提高集胞藻中taR^2的基因调控能力	[59]
反义sRNA	支架序列- Hfq,核糖开关	聚球藻PCC 7942 Synechococcus elongatus PCC 7942、聚球藻UTEX 2973 (Synechococcus sp. UTEX 2973	P_tho-lacZ/cm^R-P_cpc560-nanT/P_N-sRNA micC- P_cpc560-hfq-P_trc-nanR		lacZ报告基因	实现对靶基因的二元调控	[77]
反义sRNA	核糖开关	鱼腥藻PCC 7120 Anabaena sp. PCC 7120	P_petE-tetR/P_L03-gfp/P_L03-asRNA		gfp报告基因	通过添加无水四环素,增加GFP荧光蛋白表达量	[76]
反义sRNA	支架序列- Hfq,核糖开关	集胞藻PCC 6803 Synechocystis sp. PCC 6803	P_psbA2M-as(zwf、pgi、 pfkA)-micC-T_rbcL-P_trc-核糖开关(茶碱)-hfq-T_rbcL		葡萄糖-6-磷酸脱氢酶基因(zwf)、葡萄糖-6-磷酸异构酶(pgi)基因、磷酸果糖激酶基因(pfkA)	提高MI产量至7.93 mg/L,增加对asRNA表达的控制	[75]
反义sRNA	支架序列- Hfq,核糖开关	集胞藻PCC 6803 Synechocystis sp. PCC 6803	Spe^R-P_psbA2M-asRNA-micC-T_rbcL-P_trc-核糖开关(茶碱)-hfq-T_rbcL	FabD(slr2023)、FabH(slr1511)、FabF (sll1069、slr1332)		sRNA受到严格调控	[60]

表6 小RNA调控工具之间的偶联

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