The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain. The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for producing other important products that require malonyl-CoA as a precursor.

中文翻译：

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通过基于CRISPR干扰系统的大肠杆菌系统地调节中央代谢途径来增强类黄酮的产生。

大肠杆菌中细胞内丙二酰辅酶A的有限供应阻碍了聚酮化合物，类黄酮和生物燃料的生物合成。在这里，建立了一个有规律的，相互间隔的短回文重复序列（CRISPR）干扰系统，用于微调中心代谢途径，以有效地将碳通量引导至丙二酰辅酶A。使用合成的sgRNA沉默候选基因，可以将细胞内丙二酰辅酶A水平提高223％以上的基因用作目标基因。调节这些基因的抑制效率以达到合适的水平，从而在不显着改变最终生物量积累的情况下提高细胞内丙二酰辅酶A水平（最终OD600降低不到10％）。根据结果​​，多基因抑制成功地达到了生产植物特异性次级代谢产物（2S）-柚皮素所需的丙二酰辅酶A量的极限。通过将遗传修饰与细胞生长耦合，这些遗传扰动的综合作用使最终（2S）柚皮素效价提高至421.6 mg / L，比对照菌株高7.4倍。此处描述的策略可用于表征细胞生长必不可少的基因，并使大肠杆菌成为组织完善的细胞工厂，以生产需要丙二酰辅酶A作为前体的其他重要产品。6 mg / L，比对照菌株高7.4倍。此处描述的策略可用于表征细胞生长必不可少的基因，并使大肠杆菌成为组织完善的细胞工厂，以生产需要丙二酰辅酶A作为前体的其他重要产品。6 mg / L，比对照菌株高7.4倍。此处描述的策略可用于表征细胞生长必不可少的基因，并使大肠杆菌成为组织完善的细胞工厂，以生产需要丙二酰辅酶A作为前体的其他重要产品。