Characterization of lysine sensor and lysine-producing recombinant cells. (a) Cytosolic L-lysine concentration in C. glutamicum WT and five defined lysine producer strains, all carrying pSenLys, and specific fluorescence (Sp. fluorescence) of the cultures. Error bars give the means of three independent cultures for each strain. Fitting the data to the Hill equation describes the signal transfer function by an napp of 3.19 ± 1.45 and this is shown as the red curve. (b) Extracellular accumulation of lysine after 48 h by the strains used in (a). (c) Lysine excretion rates of the same strains showing that strains with increased final lysine accumulation have increased excretion rates. Strain DM1920 has two copies of the lysine exporter gene lysE present in its chromosome and shows the highest excretion rate, but an intermediate cytosolic lysine concentration (color code as in (b)). (d) Differentiation of an equal mixture of cells of ATCC13032, DM1728 and DM1919 each carrying pSenLys by flow cytometry. The success of strain-specific sorting using gates P1 to P3 was over 90%. (e) Influence of dipeptide addition on specific fluorescence of C. glutamicum WT carrying pSenLys. The peptides Lys-Ala (circle), Arg-Ala (triangle) and His-Ala (square) were added to the cultures at the indicated concentrations. Additionally, Ala-Ala was added to give a total dipeptide concentration of 3 mM. The specific fluorescence was measured 1.5 h after dipeptide addition. FSC, forward scatter; RU, relative units.