Fig. 1: Development of eLACCO2.1 and R-iLACCO1.

a Schematic representation of TTHA0766-based eLACCO and its mechanism of response to l-lactate. b Schematic representation of LldR-based R-iLACCO and its mechanism of response to l-lactate. c Schematic of directed evolution workflow. Specific sites (i.e., the linkers) or the entire gene of template l-lactate biosensor were randomly mutated and the resulting mutant library was used to transform E. coli. Bright colonies were picked and cultured, and then proteins were extracted to examine ΔF/F upon addition of 10 mM l-lactate. A mixture of the variants with the highest ΔF/F was used as the template for the next round. d ΔF/F rank plot representing all crude proteins tested during the directed evolution of eLACCO. For each round, tested variants are ranked from lowest to highest ΔF/F value from left to right. e Excitation and emission spectra of purified eLACCO2.1 in the presence (10 mM) and absence of l-lactate. f Overall representation of the eLACCO1 crystal structure (PDB ID: 7E9Y [https://www.rcsb.org/structure/7E9Y]) with the position of mutations indicated. l-Lactate and Ca²⁺ (black) are shown in a sphere representation. In the primary structure of eLACCO2.1 (bottom), linker regions are shown in black and the two “gate post” residues³¹ in cpGFP are highlighted in dark orange (His195) and purple (Phe437). g ΔF/F rank plot representing all crude proteins tested during the directed evolution of R-iLACCO. h Excitation and emission spectra of purified R-iLACCO1 in the presence (10 mM) and absence of l-lactate. i Overall representation of the R-iLACCO1 model structure⁴¹ with the position of mutations indicated. In the primary structure of R-iLACCO1 (bottom), linker regions are shown in black and the two “gate post” residues³¹ in cpmApple are highlighted in dark orange (Val112) and purple (Trp351). Source data are provided as a Source Data file.