Isolation and Charactization of Novel Terpene Synthases from Diverse Metagenomic Libraries | AIChE

Isolation and Charactization of Novel Terpene Synthases from Diverse Metagenomic Libraries


Crook, N. - Presenter, The University of Texas at Austin
Yoneda, A., Washington University in Saint Louis
Dantas, G., Washington University in Saint Louis

Nature produces a stunning diversity of molecular functionality, of which humanity has only exploited a small fraction for industrial and energy applications.  To date, chemical space has been confined to that enabled by petroleum feedstocks or abundant natural products, in turn putting strong constraints on achievable material properties.  Terpenoids are one of the most abundant and diverse classes of biomolecules produced in nature, collectively enabling a variety of therapeutic, energy, and cosmetic applications.  However, this class of molecules remains recalcitrant to synthetic chemistry approaches, necessitating the use living systems.  Although there has been recent outstanding progress in the high-level microbial production of several key terpenoids, much of the remaining molecular diversity is locked within production hosts which produce small product titers and are expensive to cultivate.  This limitation is primarily due to a lack of diverse biological parts, as current databases of protein function are currently populated by proteins which conform to well-understood sequence-function relationships.  Interestingly, despite this known diversity, recent genomics investigations have shown a vast untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivable organisms living in the soil, indicating a vast array of terpenoids waiting to be discovered and offering a wider palette of biological parts for the production of these compounds in robust microbial hosts.  However, in order to validate these predictions and expand our catalogs of useful biocatalysts, techniques for high-throughput enzyme discovery, characterization, and annotation are urgently needed.  In this work, we exploited the toxicity of a key node in the terpenoid biosynthesis pathway in order to develop a high-throughput approach for discovery of terpene synthase activity.  We first develop optimized culture conditions and screening protocols in order to maximize enrichment of terpene synthases.  Then, we experimentally mine hundreds of gigabases of genetic material isolated from diverse habitats in order to discover novel biocatalysts with the ability to produce new classes of terpenoid molecules in E. coli.  After biochemical characterization of terpenoid products generated by these novel enzymes, we then perform bioinformatics analysis to generate improved predictors of terpene synthase function and use these predictors in a focused approach to experimentally investigate terpene synthase activity in sequenced microbial genomes.