One of the most important research subjects of metabolic engineering is the pursuit of balanced metabolic pathways, which requires the modulation of expression of many genes. However, simultaneously modulating multiple genes on the chromosome remains challenging in prokaryotic organisms, including the industrial workhorse - Escherichia coli. In this work, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique was developed to simultaneously modulate the expression of multiple genes on the chromosome. To implement it, two plasmids were employed to target Cas9 to regulatory sequences of pathway genes, and a donor DNA plasmid library was constructed containing a regulator pool to modulate the expression of these genes. A modularized plasmid construction strategy was used to enable the assembly of a complex donor DNA plasmid library. After genome editing using this technique, a combinatorial library was obtained with variably expressed pathway genes. As a demonstration, the CFPO technique was applied to the xylose metabolic pathway genes in E. coli to improve xylose utilization. Three transcriptional units containing a total of four genes were modulated simultaneously with 70% efficiency, and improved strains were selected from the resulting combinatorial library by growth enrichment. The best strain, HQ304, displayed a 3-fold increase of the xylose-utilization rate. Finally, the xylose-utilization pathway of HQ304 was analyzed enzymologically to determine the optimal combination of enzyme activities.

中文翻译：

---

CRISPR / Cas9促进的多重途径优化（CFPO）技术及其在改善大肠杆菌木糖利用途径中的应用。

代谢工程最重要的研究主题之一是追求平衡的代谢途径，这需要调节许多基因的表达。然而，同时调节染色体上的多个基因在原核生物中仍然具有挑战性，包括工业主力大肠杆菌。在这项工作中，开发了CRISPR / Cas9促进的多重途径优化（CFPO）技术，以同时调节染色体上多个基因的表达。为了实现这一点，采用了两个质粒将Cas9靶向途径基因的调控序列，并构建了一个供体DNA质粒文库，其中包含一个调控池来调节这些基因的表达。使用模块化的质粒构建策略来组装复杂的供体DNA质粒文库。使用该技术进行基因组编辑后，获得了具有可变表达途径基因的组合文库。作为演示，将CFPO技术应用于大肠杆菌中的木糖代谢途径基因，以提高木糖利用率。同时以70％的效率同时调节总共包含四个基因的三个转录单位，并通过生长富集从所得组合文库中选择改良的菌株。最好的菌株HQ304显示木糖利用率提高了3倍。最后，通过酶学分析HQ304的木糖利用途径，以确定酶活性的最佳组合。获得具有可变表达的途径基因的组合文库。作为演示，将CFPO技术应用于大肠杆菌中的木糖代谢途径基因，以提高木糖利用率。同时以70％的效率同时调节总共包含四个基因的三个转录单位，并通过生长富集从所得组合文库中选择改良的菌株。最好的菌株HQ304显示木糖利用率提高了3倍。最后，通过酶学分析HQ304的木糖利用途径，以确定酶活性的最佳组合。获得具有可变表达的途径基因的组合文库。作为演示，将CFPO技术应用于大肠杆菌中的木糖代谢途径基因，以提高木糖利用率。同时以70％的效率同时调节总共包含四个基因的三个转录单位，并通过生长富集从所得组合文库中选择改良的菌株。最好的菌株HQ304显示木糖利用率提高了3倍。最后，通过酶学分析HQ304的木糖利用途径，以确定酶活性的最佳组合。同时以70％的效率同时调节总共包含四个基因的三个转录单位，并通过生长富集从所得组合文库中选择改良的菌株。最好的菌株HQ304显示木糖利用率提高了3倍。最后，通过酶学分析HQ304的木糖利用途径，以确定酶活性的最佳组合。同时以70％的效率同时调节总共包含四个基因的三个转录单位，并通过生长富集从所得组合文库中选择改良的菌株。最好的菌株HQ304显示木糖利用率提高了3倍。最后，通过酶学分析HQ304的木糖利用途径，以确定酶活性的最佳组合。