Fig. 1: Development of a genetically encoded l-lactate biosensor, eLACCO1.

a Schematic of the directed evolution workflow. Specific sites (i.e., the linkers) or the entire gene of template l-lactate biosensor genes 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 and ΔF/F upon addition of 10 mM l-lactate was determined. The variant with the highest ΔF/F was used as the template for the next round. b ΔF/F rank plot representing all proteins tested during the directed evolution. For each round, tested variants are ranked from lowest (negative responses indicate inverse response) to highest ΔF/F value from left to right. The first round of evolution used a library in which residues of the C-terminal linker were randomized. Screening of this library led to the discovery of eLACCO0.2 with a direct response (fluorescence increase upon binding) to l-lactate (ΔF/F = 0.3) while the template eLACCO0.1 decreased the fluorescence intensity (inverse response) in response to l-lactate. Nine rounds of the evolution led to eLACCO1 indicated with a magenta circle. c Excitation and emission spectra of eLACCO1 in the presence and absence of 10 mM l-lactate. Excitation and emission peak is at 494 and 512 nm, respectively. d Schematic representation of eLACCO1 and its mechanism of response to l-lactate. Linker regions are shown in black and the two “gate post” residues⁵ in cpGFP are highlighted in dark orange (His195) and purple (Phe437). e Crystal structure of eLACCO1. Right panel represents a zoom-in view around the chromophore. Source data of b, c are provided as a Source Data file.