<P><B>Abstract</B></P> <P>Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The <SMALL>L</SMALL>-valine producer <I>Corynebacterium glutamicum</I> Δ<I>aceE</I> was equipped with an <SMALL>L</SMALL>-valine-responsive sensor based on the transcriptional regulator Lrp of <I>C. glutamicum.</I> Evolved strains featured a significantly higher growth rate, increased <SMALL>L</SMALL>-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene <I>ureD</I> (urease accessory protein), which was shown to increase <SMALL>L</SMALL>-valine production by up to 100%. Furthermore, decreased <SMALL>L</SMALL>-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We report biosensor-driven evolution as a new metabolic engineering approach. </LI> <LI> This approach improved growth and <SMALL>L</SMALL>-valine production of <I>C. glutamicum</I> Δ<I>aceE</I>. </LI> <LI> <I>C. glutamicum</I> Δ<I>aceE</I> sensor cells were iteratively sorted by FACS and cultivated. </LI> <LI> Evolved strains show increased valine levels and decreased by-product levels. </LI> <LI> Sequencing of single strains revealed seven non-intuitive SNPs. </LI> </UL> </P>