Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res 41: 6360–6369

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of pro-phage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-con-structed libraries to directly isolate L-lysine-producing cells. A mixture of 19 different oligo-nucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause L-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 dif-ferent amino acid exchanges in the targeted murE codon, which caused different L-lysine production titers. Apart from introducing a rapid genome con-struction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology