CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002.
CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002.
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DOI：
10.1016/j.ymben.2016.07.007
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发表时间：
2016-11
影响因子：
8.4
通讯作者：
Pfleger, Brian F.
中科院分区：
文献类型：
--
作者：
Gordon, Gina C.;Korosh, Travis C.;Cameron, Jeffrey C.;Markley, Andrew L.;Begemann, Matthew B.;Pfleger, Brian F.
反式作用调节因子为研究必需基因和调节代谢途径提供了新的机会。我们已经采用了来自化脓性链球菌的成簇规则散布回文重复序列（CRISPR）系统来抑制蓝细菌聚球藻属菌株PCC 7002（以下称为PCC 7002）中的反式基因。通过这种称为CRISPR干扰（CRISPRi）的方法，特定靶序列的转录被无催化活性的Cas9蛋白抑制，该Cas9蛋白通过与与靶序列互补的单个指导RNA的碱基对相互作用而被募集到靶DNA。我们适应这个系统PCC 7002，并实现了有条件的和可滴定的抑制异源报告基因，黄色荧光蛋白。接下来，我们证明了通过下调藻胆体丰度来微调天然基因表达的实用性。此外，我们创造了一个条件营养缺陷型通过抑制合成的羧基，一个重要组成部分的碳浓缩机制蓝藻用来固定大气中的二氧化碳。最后，我们展示了一种新的策略，通过有条件地下调氮同化的关键节点来增加中心碳通量。所得细胞产生的乳酸比基线工程化细胞系多2倍，代表迄今为止最高的光合产生的生产力。这项工作是使用CRISPRi在蓝藻中进行可滴定抑制的第一个例子，能够通过中心碳代谢动态调节基本过程和操纵通量。该工具通过提供一种直接的方法来重新定向高价值化学品生产中的代谢和碳通量，促进了对未知功能的必需基因的研究，并实现了突破性的代谢工程能力。
Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.
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DOI：
10.1073/pnas.1404097111
发表时间：
2014-06-10
影响因子：
11.1
作者：
Espinosa, Javier;Rodriguez-Mateos, Francisco;Contreras, Asuncion
通讯作者：
Contreras, Asuncion
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
9.9
作者：
Farasat I;Kushwaha M;Collens J;Easterbrook M;Guido M;Salis HM
通讯作者：
Salis HM
影响因子：
6.4
作者：
Hara KY;Araki M;Okai N;Wakai S;Hasunuma T;Kondo A
通讯作者：
Kondo A
影响因子：
2.8
作者：
Gruendel, Marianne;Scheunemann, Ramon;Zilliges, Yvonne
通讯作者：
Zilliges, Yvonne