Light-guided in vivo evolution of dynamic, multi-state and computational protein functionalities

The present invention relates to a method for the evolution of a protein of interest by applying light-based selection pressure to a yeast cell, (i) wherein in the yeast cell the expression of one or more gene(s) that encode a protein controlling the progression of the yeast cell through the cell cycle is/are optogenetically controlled by an optogenetic gene expression system that is activated by light pulses of a specific wavelength, and wherein the protein of interest is the protein or one of the proteins constituting the optogenetic gene expression system; or (ii) wherein in the yeast cell the expression of one or more gene(s) that encode a protein controlling the progression of the yeast cell through the cell cycle is/are controlled by the protein of interest or a signal transduction cascade that comprises the protein of interest, and wherein the expression of the protein of interest is optogenetically controlled by an optogenetic gene expression system that is activated by light pulses of a specific wavelength, wherein the method comprises (a) exposing the yeast cell to light pulses that differ from the light pulses that optimally activate gene expression but that still in part activate gene expression; (b) culturing the yeast cell contemporaneously under conditions where it divides and wherein random mutations are introduced for a time whereby a light-based selection pressure towards yeast cells being capable of optimally dividing under the light of (a) occurs, and (c) optionally isolating one or more yeast cells being capable of optimally dividing under the light pulses of (a) and preferably identifying the one or more mutation(s) in the gene encoding the protein of interest.