期刊
ACS SYNTHETIC BIOLOGY
卷 8, 期 12, 页码 2726-2734出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.9b00361
关键词
CRISPR/Cas; recombineering; L-glutamate; Corynebacterium glutamicum; metabolic engineering; genome editing
资金
- European Research Council (ERC) under the European Union [638718]
- European Research Council (ERC) [638718] Funding Source: European Research Council (ERC)
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 beta-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.
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