核酸研究2013-09-05 2:58 AM

Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation

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 prophage 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-constructed libraries to directly isolate L-lysine-producing cells. A mixture of 19 different oligonucleotides 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 different amino acid exchanges in the targeted murE codon, which caused different L-lysine production titers. Apart from introducing a rapid genome construction 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.

KEYWORDS

SHARE & LIKE

COMMENTS

ABOUT THE AUTHOR

核酸研究

0 Following 0 Fans 0 Projects 44 Articles

SIMILAR ARTICLES

Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative

Read More

Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative

Read More

The continuously increasing amount of RNA sequence and experimentally determined 3D structure data drives the development of computational methods supp

Read More

The continuously increasing amount of RNA sequence and experimentally determined 3D structure data drives the development of computational methods supp

Read More

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain cau

Read More

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain cau

Read More

Increased synthesis of endothelin-1 (ET-1) by human vascular endothelial cells (HVECs) in response to hypoxia underscores persistent vasoconstriction o

Read More

Increased synthesis of endothelin-1 (ET-1) by human vascular endothelial cells (HVECs) in response to hypoxia underscores persistent vasoconstriction o

Read More

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface

Read More

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface

Read More