Abstract

       Directed evolution is a powerful synthetic biology approach used to optimize proteins especially for enzyme. It is commonly employed to enhance the catalytic efficiency of target enzymes to meet production demands. EvolvR systems including enCas9 and error-prone DNA polymerase have been developed to enable continuous directed evolution in Escherichia coli and Saccharomyces cerevisiae, yielding promising results. However, sensitive and high-throughput substrate/product detection technologies are urgently needed for the directed evolution of certain synthetic enzymes. Many substances, such as cyclic monoterpenes and monosaccharides, are difficult to detect endogenously in real-time. Here, we aim to expand the application of EvolvR to nonconventional yeast species and establish synthetic platforms for two difficult-to-detect compounds, borneol and tagatose. Additionally, we have developed biosensors capable of endogenous detection, in order to facilitate directed evolution pathways for difficult-to-detect substances and provide a paradigm for similar molecules.