The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway.
The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway.
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DOI：
10.1016/j.ymben.2017.08.003
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发表时间：
2017-09
影响因子：
8.4
通讯作者：
Xinna Zhu;Dongdong Zhao;Huanna Qiu;Feiyu Fan;S. Man;C. Bi;Xueli Zhang
中科院分区：
文献类型：
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作者：
Xinna Zhu;Dongdong Zhao;Huanna Qiu;Feiyu Fan;S. Man;C. Bi;Xueli Zhang
代谢工程最重要的研究课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，在原核生物（包括工业主力大肠杆菌）中，同时调节染色体上的多个基因仍然具有挑战性。在这项工作中，开发了CRISPR/Cas9促进的多重途径优化（CFPO）技术来同时调节染色体上多个基因的表达。为了实现这一目标，使用两个质粒将 Cas9 靶向途径基因的调控序列，并构建了一个包含调节器池的供体 DNA 质粒库来调节这些基因的表达。使用模块化质粒构建策略来组装复杂的供体 DNA 质粒库。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，CFPO 技术应用于大肠杆菌中的木糖代谢途径基因。 colito 提高木糖利用率。总共含有四个基因的三个转录单位以70％的效率同时调节，并通过生长富集从所得的组合文库中选择改良菌株。最好的菌株 HQ304 的木糖利用率提高了 3 倍。最后，对HQ304的木糖利用途径进行酶学分析，以确定酶活性的最佳组合。
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 inE. colito 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.