Abstract

In microbial strain development for industrial production of small molecules, the screening for cells with enhanced production properties is a major limitation. Most small molecules of interest have an inconspicuous phenotype, and screening is therefore a costly venture. We present here a novel system to visualize the cytosolic concentration of small molecules at the single-cell level of E. coli or C. glutamicum. The system is based on transcriptional regulators which sense the intracellular concentration of the molecule of interest, and then drive transcription of an autofluorescent protein. 

We developed individual metabolite sensors to monitor the cytosolic concentrations of L-lysine, L-histidine, L-arginine, O-acetyl-serine, L-serine, and L-leucine in C. glutamicum or E. coli. This methodology enables a number of novel FACS-based applications, including high-throughput isolation of individual producers from large mutant populations, or the screening of cellular plasmid libraries coding for modified variants of enzymes.

In one application, we applied our L-lysine sensor to identify new chromosomal mutations leading to L-lysine overproduction. After treatment with mutagen, 200 L-lysine accumulating cells were selected via FACS out of a population of 6.5x106 cells. Targeted sequencing identified 13 new chromosomal mutations in known target genes. Whole genome sequencing unraveled a so far unknown mutation (murE), that improves L-lysine titers significantly even in existing L-lysine producers.

Because transcriptional regulators exist for sugars, sugar phosphates, vitamins, oxoacids, mevalonate, antibiotics, and other small molecules, the presented technology is expected to substantially boost metabolic engineering.