Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (cat1, sucD, 4hbd, cat2, bld, bdh) in the 1,4-BDO biosynthesis pathway. The successive E. coli engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (gabD, ybgC and tesB) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as E. coli and production of chemicals (e.g., 1,4-BDO).

中文翻译：

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结合CRISPR和CRISPRi系统进行大肠杆菌和1,4-BDO生物合成的代谢工程

在大肠杆菌中生物合成1,4-丁二醇（1,4-BDO）需要一个人工途径，该途径涉及六个基因和耗时的迭代基因组工程。CRISPR是一种有效的基因编辑工具，而CRISPR干扰（CRISPRi）则被重新用于可编程的基因抑制。这项研究旨在将CRISPR和CRISPRi结合起来用于大肠杆菌的代谢工程和1,4-BDO生产。我们首先利用CRISPR进行glt A的点突变，用异源lpdA替代天然lpdA，敲除悲伤并敲入两个大的基因盒（长度分别为6.0和6.3 kb），编码六个基因（cat 1，suc D ，41,4-BDO生物合成途径中的hbd，cat 2，bld和bdh）。连续的大肠杆菌工程使48小时内1,4-BDO的滴定度达到0.9 g / L。通过组合CRISPRi系统以同时抑制竞争基因，使竞争基因从1,4-BDO生物合成途径（gabD，ybgC和tesB）转移，> 85％，我们将1,4-BDO滴度进一步提高了100％至1.8克/升，同时降低副产物γ-丁内酯和琥珀酸酯的滴度分别为55％和83％。这些数据证明了结合CRISPR和CRISPRi进行微生物等基因组工程和代谢通量调节的潜力。以及化学品的生产（例如1,4-BDO）。