Hungateiclostridium thermocellum DSM 1313 has biotechnological potential as a whole‐cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been hampered due to the lack of simple and high‐throughput genome engineering tools. Recently in our research group, a thermophilic bacterial CRISPR–Cas9‐based system has been developed as a transcriptional suppression tool for regulation of gene expression. We applied ThermoCas9‐based CRISPR interference (CRISPRi) to repress the H. thermocellum central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes. The effects of repression on target genes were studied based on transcriptional expression and product formation. Single‐guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62% respectively. This resulted in 24% and 17% decrease in lactate and acetate production, correspondingly. Hence, the CRISPRi approach for H. thermocellum to downregulate metabolic genes can be used for remodelling of metabolic pathways without the requisite for genome engineering. These data established for the first time the feasibility of employing CRISPRi‐mediated gene repression of metabolic genes in H. thermocellum DSM 1313.

中文翻译：

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CRISPR干扰（CRISPRi）作为Hungateiclostridium thermocellum DSM 1313的转录抑制工具。

Hungateiclostridium thermocellum DSM 1313作为使用木质纤维素可再生资源生产乙醇的全细胞生物催化剂具有生物技术潜力。由于缺乏简单和高通量的基因组工程工具，H.thermocellum的充分利用受到了阻碍。最近，在我们的研究小组中，基于嗜热细菌CRISPR–Cas9的系统已被开发为调节基因表达的转录抑制工具。我们应用了基于ThermoCas9的CRISPR干扰（CRISPRi）来抑制嗜热纤维菌中央代谢乳酸脱氢酶（ldh）和磷酸转乙酰酶（pta）基因。基于转录表达和产物形成，研究了抑制对靶基因的影响。在来自嗜热链球菌的天然基因间16S / 23S rRNA启动子的控制下，将ThermodCas9引导至pta和ldh沉默转化子的启动子区域的单向导RNA（sgRNA）分别将表达降低了67％和62％。相应地，乳酸和乙酸盐的产量分别下降了24％和17％。因此，对于CRISPRi方法H.热纤下调代谢基因可以用于代谢途径的重塑，而无需基因组工程。这些首次采用代谢基因的CRISPRi介导的基因抑制的可行性建立的数据H.热纤DSM 1313。