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

Stephan Binder,Solvej Siedler,Lothar Eggeling,Jan Marienhagen,Michael Bott

doi:10.1093/nar/gkt312

Abstract

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.

Highlights

For the development of microbial producer strains, fast methodologies are required for introducing genomic mutations
For our initial studies to assay on the functionality of recombinases in C. glutamicum, we focused on RecT and cauri_1962
We chose the well-studied red gene bet, as well as the SSAPs of the mycobacteriophage Che9c and Halo. The reason for this is that both Mycobacterium and Corynebacterium belong to the order Corynebacteriales, and genes of M. tuberculosis show functionality in C. glutamicum [27,28]

Summary

Introduction

For the development of microbial producer strains, fast methodologies are required for introducing genomic mutations. The problem of ultrahigh-throughput detection and isolation of productive recombinants has recently been solved by the development of optical nanosensors based on transcription factors They allow the detection of intracellularly synthesized small molecules at the single-cell level [6,7,8], and, together with fluorescence-activated cell sorting (FACS), they enable the ultrahigh-throughput screening of large libraries. We have developed an L-lysine sensor for Corynebacterium glutamicum, which transmits the cytosolic L-lysine concentration of a single cell into a graded optical output Using this sensor, we isolated new L-lysine producers via FACS from a library of 107 randomly mutagenized wild-type cells and identified novel relevant mutations causing increased L-lysine synthesis by whole-genome sequencing [6]

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