CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of Corynebacterium glutamicum.,ACS Synthetic Biology

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CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of Corynebacterium glutamicum.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2019-12-11 , DOI: 10.1021/acssynbio.9b00361
Karin Krumbach ₁ , Christiane Katharina Sonntag ₁ , Lothar Eggeling ₁ , Jan Marienhagen _{1,

2}

Affiliation

Against the background of a growing demand for the implementation of environmentally friendly production processes, microorganisms are engineered for the large-scale biosynthesis of chemicals, fuels, or food and feed additives from sustainable resources. Since strain development is expensive and time-consuming, continuous improvement of molecular tools for the genetic modification of the microbial production hosts is absolutely vital. Recently, the CRISPR/Cas12a technology for the engineering of Corynebacterium glutamicum as an important platform organism for industrial amino acid production has been introduced. Here, this system was advanced by designing an easy-to-construct crRNA delivery vector using simple oligonucleotides. In combination with a C. glutamicum strain engineered for the chromosomal expression of the β-galactosidase-encoding lacZ gene, this new plasmid was used to investigate CRISPR/Cas12a targeting and editing at various positions relative to the PAM site. Finally, we used this system to perform codon saturation mutagenesis at critical positions in the mechanosensitive channel MscCG to identify new gain-of-function mutations for increased l-glutamate export. The mutations obtained can be explained by particular demands of the channel on its immediate lipid environment to allow l-glutamate efflux.

中文翻译：

CRISPR / Cas12a介导的基因组编辑可将氨基酸取代引入谷氨酸棒状杆菌的机械敏感通道MscCG中。

在对实施环境友好的生产过程的需求不断增长的背景下，对微生物进行了改造，以利用可持续资源大规模合成化学物质，燃料或食品和饲料添加剂。由于菌株开发昂贵且耗时，因此对微生物生产宿主进行遗传修饰的分子工具的持续改进绝对至关重要。最近，已经引入了用于谷氨酸棒杆菌工程改造的CRISPR / Cas12a技术，作为用于工业氨基酸生产的重要平台生物。在此，通过使用简单的寡核苷酸设计易于构建的crRNA传递载体，对该系统进行了改进。与C结合使用。谷氨酸菌株是为编码β-半乳糖苷酶的lacZ基因的染色体表达而设计的，该新质粒用于研究CRISPR / Cas12a在相对于PAM位点的不同位置上的靶向和编辑。最后，我们使用该系统在机械敏感通道MscCG的关键位置进行密码子饱和诱变，以识别新的功能增益突变，以增加l-谷氨酸的输出。可以通过通道在其直接脂质环境上的特殊要求来解释所获得的突变，以允许I-谷氨酸外排。我们使用该系统在机械敏感通道MscCG的关键位置进行密码子饱和诱变，以识别新的功能增益突变，以增加l-谷氨酸的输出。可以通过通道在其直接脂质环境上的特殊要求来解释所获得的突变，以允许I-谷氨酸外排。我们使用该系统在机械敏感通道MscCG的关键位置进行密码子饱和诱变，以识别新的功能增益突变，以增加l-谷氨酸的输出。可以通过通道在其直接脂质环境上的特殊要求来解释所获得的突变，以允许I-谷氨酸外排。

更新日期：2019-12-13

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