Wu Junjun, Du Guocheng, Chen Jian, Zhou Jingwen
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Sci Rep. 2015 Sep 1;5:13477. doi: 10.1038/srep13477.
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.
大肠杆菌细胞内丙二酰辅酶A的供应有限，这阻碍了聚酮化合物、类黄酮和生物燃料的生物合成。在此，构建了一种成簇规律间隔短回文重复序列（CRISPR）干扰系统，用于微调中心代谢途径，以有效地将碳通量导向丙二酰辅酶A。使用合成的sgRNA沉默候选基因，将能够使细胞内丙二酰辅酶A水平提高超过223%的基因用作靶基因。调节这些基因的抑制效率以达到适当水平，从而在不显著改变最终生物量积累（最终OD600降低不到10%）的情况下提高细胞内丙二酰辅酶A水平。基于这些结果，多重基因抑制成功地接近了生产植物特异性次生代谢产物（2S）-柚皮素所需的丙二酰辅酶A量的极限。通过将基因修饰与细胞生长相结合，这些基因扰动的综合作用使最终的（2S）-柚皮素滴度提高到421.6 mg/L，比对照菌株高7.4倍。本文所述策略可用于鉴定对细胞生长至关重要的基因，并将大肠杆菌开发成一个组织良好的细胞工厂，用于生产其他需要丙二酰辅酶A作为前体的重要产品。
