Applied evolution: Dual dynamic regulations-based approaches in engineering intracellular malonyl-CoA availability.
Applied evolution: Dual dynamic regulations-based approaches in engineering intracellular malonyl-CoA availability.
复制标题
应用进化：基于双动态调节的方法设计细胞内丙二酰辅酶A的可用性。
DOI：
10.1016/j.ymben.2021.08.004
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发表时间：
2021
影响因子：
8.4
通讯作者：
Jingwen Zhou
中科院分区：
文献类型：
--
作者：
Junjun Wu;Lin Zhou;X. Duan;Hu Peng;Shike Liu;Q. Zhuang;Cruz Pablo;Xia Fan;Shijie Ding;Mingsheng Dong;Jingwen Zhou
丙二酰辅酶A是许多具有药用价值或脂肪酸衍生化合物的微生物合成的重要组成部分，包括聚酮、类黄酮、苯丙烷和脂肪酸。然而，严格控制的细胞内丙二酰辅酶A的可获得性往往阻碍整体产品的形成。在这里，为了释放这种紧密的细胞行为，我们提出了进化：基于双重动态规则的方法，将人工健壮和动态功能写入复杂的细胞背景。首先，在大多数生物中，丙二酰辅酶A仅通过乙酰辅酶A的羧化反应产生丙二酰辅酶A，因此，基于保守核心结构域的进化原理被引入到基因组挖掘中，以探索离散乙酰辅酶A(ACC)家族的生物合成多样性。对代表种的50个菌株的基因组进行了全面的系统基因组学分析和进一步的实验分析，总结了进化的历史，发现来自肠沙门氏菌的ACC家族在所有候选菌株中表现出最高的活性。进一步设计了一套基于群体感应(QS)的正交双功能调控工具，并与T7 RNA聚合酶相连接作为遗传放大器，实现了高动态范围内的双重动态控制，使我们能够高效地动态和独立地激活和抑制不同的基因集合。然后将这些遗传电路与ACC OFS相结合。ENTERICA和CRISPRi系统重新编程中央代谢，将严格调控的丙二酰辅酶A途径重新连接到强健和自主的行为，导致丙二酸辅酶A的可用性增加29倍。我们应用这种双重调控工具成功地合成了丙二酰辅酶A衍生化合物(2S)-柚皮苷元，并获得了迄今报道的最高产率(1073.8 mg/L)，同时副产物生成量大幅减少。值得注意的是，整个发酵过程呈现为一种自主行为，完全消除了人类的监督和诱导剂的补充。因此，构建的进化：基于双重动态调节的方法为开发经济上可行和可扩展的丙二酰辅酶A衍生化合物的微生物生产程序铺平了道路。
Malonyl-CoA is an important building block for microbial synthesis of numerous pharmaceutically interesting or fatty acid-derived compounds including polyketides, flavonoids, phenylpropanoids and fatty acids. However, the tightly regulated intracellular malonyl-CoA availability often impedes overall product formation. Here, in order to unleash this tightly cellular behavior, we present evolution: dual dynamic regulations-based approaches to write artificial robust and dynamic function into intricate cellular background. Firstly, a conserved core domain based evolutionary principles were incorporated into genome mining to explore the biosynthetic diversities of discrete acetyl-CoA carboxylase (ACC) families, as malonyl-CoA is solely derived from carboxylation of acetyl-CoA by ACC in most organisms. A comprehensive phylogenomic and further experimental analysis, which included genomes of 50 strains throughout representative species, was performed to recapitulate the evolutionary history and reveal that previously unnoticed ACC families from Salmonella enterica exhibited the highest activities among all the candidates. A set of orthogonal and bi-functional quorum-sensing (QS)-based regulation tools were further designed and connected with T7 RNA polymerase as genetic amplifier to achieve dual dynamic control in a high dynamic range, which allowed us to efficiently activate and repress different sets of genes dynamically and independently. These genetic circuits were then combined with ACC ofS. entericaand CRISPRi system to reprogram central metabolism that rewired the tightly regulated malonyl-CoA pathway to a robust and autonomous behavior, leading to a 29-fold increase of malony-CoA availability. We applied this dual regulation tool to successfully synthesizing malonyl-CoA-derived compound (2S)-naringenin, and achieved the highest production (1073.8 mg/L) reported to date associate with dramatic decreases of by-product formation. Notably, the whole fermentation presents as an autonomous behavior, totally eliminating human supervision and inducer supplementation. Hence, the constructed evolution: dual dynamic regulations-based approaches pave the way to develop an economically viable and scalable procedure for microbial production of malonyl-CoA derived compounds.