Sense and Screen-ability: Development of tuneable, biosensor-based screening platforms for abscisic acid

The activities of heterologous enzymes often limit the production titers, rates and yields of cell factories. With the help of biosensors, large random mutagenesis libraries can be screened for improved enzyme variants in a high-throughput manner, even if the enzyme-of-interest is poorly characterised. We previously constructed a Saccharomyces cerevisiae cell factory for the heterologous production of abscisic acid (ABA), a high-value product with a broad range of applications in medicine, agriculture and nutrition. In the current study, we developed high-throughput screening platform strains for two rate-limiting cytochrome P450 monooxygenases, BcABA1 and BcABA2, in the ABA biosynthetic pathway. The screening platforms are designed to minimize the occurrence of false positives during screening experiments. We thoroughly characterised two plant protein-based ABA biosensor candidates. Furthermore, we designed a simple genetic switch, based on the thiamine-repressible promoter p THI4 , to regulate the expression level of enzyme variants. We demonstrated that ABA production can be fine-tuned by varying thiamine concentration in the media. In-depth analysis of the platform strains revealed that screening conditions can be optimized by varying thiamine concentration and cultivation time, making it possible to utilize the full dynamic and operational range of the biosensor. In the future, the constructed strains can be used to screen for improved BcABA1 and BcABA2 variants.