Maiko FurubayashiView all speakers
Maiko Furubayashi is a PhD student at Chiba University under the supervision of Dr. Daisuke Umeno, and serves as a JSPS Research Fellow. Her research interest is on testing “synthetic” approach to inquire how metabolic pathways evolve new functions and become more specialized. Specifically, she is now working on the construction of long step pathways for non-natural carotenoids and terpenoids.
Synthetic biology aspires to construct novel metabolic pathways to useful, sometimes unnatural compounds. In extending natural pathways in novel directions, pathway engineers usually rely on the promiscuous activities (the ability to accept alternative substrates and/or synthesize alternative products) of recruited or mutated enzymes. However, the promiscuous enzymes that enable molecular discovery can generate hyperbranched pathways that preclude specific production of the target novel product. Here, we assembled a six-enzyme pathway in Escherichia coli for the 15-step synthesis of C50-astaxanthin, a non-natural purple carotenoid, by the judicious pairing of laboratory-engineered promiscuous enzymes. The initial combination of promiscuous enzymes encompassing the six desired biochemical functions resulted only in a mixture of non-target carotenoids. We then evolved variants of the first two pathway enzymes, isoprenyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), with shifted specificity ranges. The combinatorial pairing of the selected variants resulted in specific production of C50 carotenoid backbones. The specific C50 backbone pathway enabled the directed evolution of the third enzyme, carotenoid desaturase (CrtI), to synthesize C50-lycopene. Coexpression of three downstream promiscuous enzymes (CrtY, CrtW and CrtZ) enabled the further extension of the C50 carotenoid pathway. The resultant pathway to C50-astaxanthin is both highly efficient and selective, despite containing not a single catalytically specific enzyme. We believe our results and analysis provide a framework for understanding how complex and precise biosynthetic networks can be built out of imperfect biosynthetic parts, either by synthetic biologists or natural evolution.