Light-mediated gene expression enables the noninvasive regulation of cellular functions. Apart from their classical application of regulating single cells with high spatiotemporal resolution, we highlight the potential of light-mediated gene expression for biotechnological issues. Here, we demonstrate the first light-mediated gene regulation in Saccharomyces cerevisiae using the repressible pMET17 promoter and the photolabile NVOC methionine that releases methionine upon irradiation with UVA light. In this system, the expression can be repressed upon irradiation and is reactivated due to consumption of methionine. The photolytic release allows precise control over the methionine concentration and therefore over the repression duration. Using this light regulation mechanism, we were able to apply an in-house constructed 48-well cultivation system which allows parallelized and automated irradiation programs as well as online detection of fluorescence and growth. This system enables screening of multiple combinations of several repression/derepression intervals to realize complex expression programs (e.g., a stepwise increase of temporally constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction, although we used a repressible promoter.) Thus, we were able to control all general parameters of a gene expression experiment precisely, namely start, pause, and stop at desired time points, as well as the ongoing expression rate. Furthermore, we gained detailed insights into single-cell expression dynamics with spatiotemporal resolution by applying microfluidics cultivation technology combined with fluorescence time-lapse microscopy.