Bioluminescence underlies a number of key reporter systems used in biology and medicine. The most significant limitation of existing bioluminescent technologies is the need to exogenously add a synthetic substrate to the sample to emit light. This limits the application of existing bioluminescence-based methods to invasive laboratory studies. Recently, our research team made discoveries that led to the emergence of the first technology of eukaryotic autonomous bioluminescence — that is, a glow that does not require the exogenous addition of a synthetic substrate but is based on its in situ biosynthesis. These new technologies open up the possibility of creating organisms that communicate almost any physiological event by emitting light. For example, such organisms can use bioluminescence to visualize a pathological condition, in long-term non-invasive studies, to visualize infections in plants and animals in real time, or to study the physiology of plants and animals in their natural environment, outside the laboratory. However, currently existing autonomous bioluminescence technologies are still far from the level of maturity needed for these new applications. In particular, these technologies are not suitable as quantitative reporters because light emission depends on both gene expression and metabolic activity. The present project aims to overcome these limitations through the systematic molecular engineering of autonomous bioluminescence enzymes. The project will lead to the development of molecular solutions that will solve the problem of dependence of bioluminescence on metabolic activity and allow quantitative measurements of physiological parameters using bioluminescence.