CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of .

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 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 strain engineered for the chromosomal expression of the β-galactosidase-encoding 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.