PHAGE-ENABLED MINING OF GUT METAGENOMES FOR TERPENOID AND CAROTENOID METABOLISM

ABSTRACT Terpenoids are the largest class of naturally occurring small molecules. While the majority of terpenoids originate from plants and fungi, bacteria are also capable of producing them. Terpenoids are generated from sequential additions of isoprene units, followed by derivatization via terpene synthase and cyclase enzymes. We have

analyzed Human Microbiome Project metagenomes using secondary metabolite prediction algorithms and found that half of all human gut microbiomes carry at least one terpene biosynthetic pathway. We also recently leveraged a high-throughput functional metagenomics approach and discovered that a histidine phosphatase

present in the human fecal microbiome without homology to known terpene synthases catalyzed the formation of terpenoids. Carotenoids are mainly tetraterpenoids and are vitamin A precursors with important antioxidant, immunological, and metabolic functions. The overall absorption efficiency of carotenoids can be quite low in the

small intestine; therefore, a significant quantity of carotenoids is transferred to the colon. When carotenoids enter the colon, they can interact with the colonic epithelium by acting on biochemical pathways, as we have previously shown. Despite this knowledge, how the gut microbiota might transform these compounds and how microbially

produced terpenes/carotenoids impact the gut lining are understudied. We have found that 93.3% of subjects from the Human Microbiome Project contain carotenoid-modifying enzymes in their gut microbiota, and over half of these subjects contain a terpene biosynthesis pathway. Other researchers have shown that a reduced

abundance of phytoene dehydrogenase in the gut microbiota is associated with symptomatic atherosclerosis, and that gut bacteria synthesize retinoic acid to modulate host immunity. Carotenoids and their microbial derivatives, therefore, have significant health impacts. In support of this, we have found that carotenoids in

carrots mitigated obesity-associated gut dysbiosis. Considering all this data, we hypothesize that the human gut microbiome possesses yet-undiscovered terpene synthases and carotenoid modifying enzymes, and microbial terpenoids/carotenoids can display immunomodulatory activities in the colon. To test these hypotheses, we first

will use a novel phage-based functional metagenomics approach and high-throughput screens to reveal terpene synthases and carotenoid modifying enzymes. We then will conduct analytical chemistry experiments to characterize the products of these enzymes. Finally, we will illustrate their biological activities in colon cells at

the immunological and proteomic levels. Taken together, this work will reveal new chemistry performed by the gut microbiota and its effects on human health.