(408c) Combinatorial Engineering of Isoprenoid Pathway for Improved Amorpha-4,11-Diene Production in Budding Yeast

Synthetic biology has been emerging to produce value-added compounds with numerous applications in the food, agriculture, chemical, and pharmaceutical industries. In the present study, our group attempted to optimize the metabolic flux of isoprenoid pathway in budding yeast for improved production of terpene derivatives. Specifically, combinatorial engineering of isoprenoid pathway through shuffling pathway gene copy number into the genome was carried out. By exploiting carotenoid biosynthesis as screening module, we successfully created a library through current combinatorial engineering approach, with yeast colonies appeared to have various intensities of orange color. Eight colonies from the library with intense orange color were picked out and characterized by PCR for the validation of genome integration events. As proof of concept that our carotenoid overproducers could serve to boost the titer of other terpenoid production, the engineered strains were subsequently transformed with pRS425-ADS for testing the production of amorpha-4,11-dine. From our preliminary result, the carotenoid overproducers from the library showed significantly high level of amorpha-4,11-diene over the parental strain harboring pRS425ADS. Among them, the best producer obtained at this moment yielded more than 6-fold of final product over the parental strain. Our result clearly suggested that combinatorial engineering approach through tuning the gene copy number into genome for stable expression can be very promising to balance the metabolic flux of isoprenoid pathway and it should be straightforward to exploit the current platform for engineering other metabolic pathways as well. Moreover, anther beauty of our approach is that multiple rounds of combinatorial engineering might be performed to further balance the metabolic flux after marker removal and/or simply through increasing selection pressure to facilitate more genome integration events.