Data for a manuscript to be deposited to the FEBS Journal. Abstract as deposited: We present a novel approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated a profound impact of a few nucleotides following the initiator codon (further to as N-terminal sequences) on their expression. Most of these investigations have been limited to selecting from a few rationally designed sequences. In contrast we used directed evolution-based methodology screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increasing expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.