Development and application of a single cell biosensor for the intracellular detection of L-methionine and branched-chain amino acids

Since the early 20$^{th}$ century, microbes have written a success story as hosts for the production of various small molecules. Traditional approaches for strain development or analysis are, however, typically based on bulk measurements, which do not interface with high-throughput technologies and provide average data for the whole population. Current efforts in the field of biotechnology aim at the development of novel techniques enabling the detection and quantification of metabolites in single microbial cells. In the present work, a genetically-encoded single cell biosensor was developed, which enables the detection of L-methionine and the branched-chain amino acids in the industrial amino acid producer $\textit{Corynebacterium glutamicum}$. The principal design of the biosensor is based on the native Lrp-BrnFE module of $\textit{C. glutamicum}$. In response to cytosolic accumulation of L-methionine and the branched-chain amino acids, the transcriptional regulator Lrp was shown to activate expression of the $\textit{brnFE}$ operon, encoding the transport system for these amino acids. For the construction of the biosensor, a sensor module including $\textit{lrp}$, the intergenic region of $\textit{lrp}$ and $\textit{brnF}$, and a transcriptional fusion of $\textit{brnF}$ to the reporter gene $\textit{eyfp}$ was designed. Characterization of the biosensor performance features revealed the highest sensitivity of the Lrp-sensor towards L-methionine followed by L-leucine, Lisoleucine,and L-valine. In the case of L-methionine, the minimal linear range of detection extended from 78-fold dynamic range) and, thus, covered a range, which is relevant to the development of production strains. In the following, the biosensor was implemented in FACS-(fluorescence-activated cell sorting) based high-throughput screenings of$\textit{ C. glutamicum}$ mutant libraries for the isolation of amino acid producing strains. A secondary uHPLC screen for the measurement of amino acid levels in the supernatant of isolated strains revealed about 20% positive clones producing branched-chain amino acids. Further screening attempts, aiming in particular at the isolation of L-methionine producing mutants, emphasized the strong impact of the strain background, medium composition, and FACS gating strategy on the screening outcome. For example, screening of mutagenized $\textit{C. glutamicum ΔmcbR}$, lacking the master regulator of L-methionine and L-cysteine synthesis, resulted in about 50% positive clones, which exhibited at least a 100% improvement in L-methionine production in comparison to the parental strain (up to 8 mM). Whole genome sequencing of selected mutants revealed mutations in genes contributing to L-methionine biosynthesis as well as in pathways supplying building blocks, precursors (C$_{1}$- and sulfur metabolism), redox power (pentose phosphate pathway), and transcriptional regulators involved in the control of sulfur utilization (SsuR and CysR). In further studies the Lrp-sensor was successfully applied for online monitoring of L-valine production strains based on pyruvate dehydrogenase-deficient $\textit{C. glutamicum}$ strain Δ$\textit{aceE}$ and was shown to provide information with respect to production start and course of metabolite production over time. Furthermore, the sensor was suitable to reveal different levels of productivity in basic as well as in high yield production strains. In order to investigate the phenotypic structure of L-valine production strains, isogenic microcolonies of $\textit{C. glutamicum}$ strains were grown under constant environmental cultivation conditions in microfluidic chip devices. The studies displayed cell-to-cell variation with respect to cell size, doubling time, and metabolite production, which significantly depends on the particular growth conditions. Altogether, the obtained results emphasize such like sensor systems as convenient and valuable tool for strain development and single cell analysis and reveal versatile applications for future studies