Exploring Lysine Riboswitch for Metabolic Flux Control and Improvement of l‑Lysine Synthesis in <i>Corynebacterium glutamicum</i>

Riboswitch, a regulatory part of an mRNA molecule that can specifically bind a metabolite and regulate gene expression, is attractive for engineering biological systems, especially for the control of metabolic fluxes in industrial microorganisms. Here, we demonstrate the use of lysine riboswitch and intracellular l-lysine as a signal to control the competing but essential metabolic by-pathways of lysine biosynthesis. To this end, we first examined the natural lysine riboswitches of <i>Eschericia coli</i> (ECRS) and <i>Bacillus subtilis</i> (BSRS) to control the expression of citrate synthase (<i>gltA</i>) and thus the metabolic flux in the tricarboxylic acid (TCA) cycle in <i>E. coli</i>. ECRS and BSRS were then successfully used to control the <i>gltA</i> gene and TCA cycle activity in a lysine producing strain <i>Corynebacterium glutamicum</i> LP917, respectively. Compared with the strain LP917, the growth of both lysine riboswitch-<i>gltA</i> mutants was slower, suggesting a reduced TCA cycle activity. The lysine production was 63% higher in the mutant ECRS-<i>gltA</i> and 38% higher in the mutant BSRS-<i>gltA</i>, indicating a higher metabolic flux into the lysine synthesis pathway. This is the first report on using an amino acid riboswitch for improvement of lysine biosynthesis. The lysine riboswitches can be easily adapted to dynamically control other essential but competing metabolic pathways or even be engineered as an “on-switch” to enhance the metabolic fluxes of desired metabolic pathways