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 of S. enterica and 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.

丙二酰辅酶 A 是微生物合成许多药学上有趣的或脂肪酸衍生化合物的重要组成部分，包括聚酮化合物、类黄酮、苯丙烷和脂肪酸。然而，受到严格调控的细胞内丙二酰辅酶A 可用性通常会阻碍整体产物的形成。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，将人工鲁棒和动态功能写入复杂的细胞背景。首先，将基于保守核心域的进化原理纳入基因组挖掘，以探索离散乙酰辅酶A羧化酶 (ACC) 家族的生物合成多样性，因为丙二酰辅酶 A 仅来源于大多数生物体中 ACC 对乙酰辅酶 A 的羧化。肠沙门氏菌在所有候选人中表现出最高的活性。进一步设计了一套基于正交和双功能群体感应（QS）的调控工具，并与 T7 RNA 聚合酶作为遗传放大器连接，以实现高动态范围内的双重动态控制，从而使我们能够有效地激活和抑制不同的动态和独立的基因组。然后将这些遗传回路与S. enterica的 ACC 相结合和 CRISPRi 系统对中枢代谢进行重新编程，将严格调节的丙二酰辅酶 A 途径重新连接为稳健和自主的行为，从而使丙二酰辅酶 A 的可用性增加 29 倍。我们应用这种双重调控工具成功合成了丙二酰辅酶A衍生的化合物（2S）-柚皮素，并实现了迄今为止报道的最高产量（1073.8 mg/L），这与副产物形成的显着减少有关。值得注意的是，整个发酵呈现为一种自主行为，完全消除了人为监督和诱导剂的补充。因此，构建的进化：基于双重动态法规的方法为开发用于微生物生产丙二酰辅酶A衍生化合物的经济可行且可扩展的程序铺平了道路。