High-Throughput Screening for Terpene-Synthase-Cyclization Activity- and Directed Evolution of a Terpene Synthase

摘要

The development of high-throughput assays can be extremely challenging, yet is essential for many applications in drug discovery and enzyme engineering. Directed evolution has proven to be a reliable method for optimizing the performance of enzymes in a variety, of applications but it requires an appropriate high-throughput assay for screening mutant libraries. Terpene synthases catalyze the key cyclization step in the biosynthesis of terpenoids, which are by far the largest class of natural compound and are highly valued as medicines, materials, fuels, and chemicals. Although metabolic engineering efforts have improved access to some terpenoids by production in microbial hosts, the terpene synthase enzymes responsible for the cyclization of linear isoprenoid diphosphates into cyclic terpenoids have proven difficult to engineer. The cyclizations proceed through complex carbocation bond-forming reactions and migrations, and are terminated by either elimination or aqueous quenching of carbocationic intermediates. The complexity of these processes renders rational engineering of terpene synthases nearly impossible. Furthermore, mutagenic studies are reliant upon tedious GC-MS analysis of products, and thus, such studies have explored only small selections of amino acid mutations. The use of directed evolution, therefore, has been difficult because no easy high-throughput assay for productive cyclization activity has been described.