Biosensor Directed Protein Engineering

Microbial species can be used to convert renewable feed-stocks into valuable chemical compounds in a cost-effective and environmentally friendly manner. Through the expression of a heterologous enzymatic pathway in Saccharomyces cerevisiae, the polymer precursor muconic acid was synthesized drawing off flux from the Shikimate pathway. With the application of metabolic engineering of the host yeast strain, production of muconic acid was improved 40-fold. To facilitate protein engineering of pathway elements using high throughput screening, a biosensor targeting the downstream products of the Shikimate pathway was developed utilizing an inducible hybrid-promoter based on the ARO9 promoter. First, the limits of rational engineering were reached on the target strain. Once this point was reached, the biosensor was integrated into the engineered strain. Utilizing this biosensor, an initial round of mutagenesis and selection was performed on the TKL1 gene, mutant proteins isolated and their improvements quantified. Protein engineering of TKL1 and ARO1 will allow the production of a yeast strain which functions as a strong host for production of muconic acid and other Shikimate pathway products. The process provides insight into the regulatory landscape of amino acid biosynthesis and principles of enzymatic catalysis.