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NIH grant 1F31AI200331-01: Improving Access to Malaria Treatment and Prevention Measures via Biosensor-Guided Evolution
Institute: {'code': 'AI', 'abbreviation': 'NIAID', 'name': 'National Institute of Allergy and Infectious Diseases', 'admin_org_id': '12500003', 'admin_funding_url': ''}. PI: Cassidy Oliverio. Award: $41953.
Start: 2026-07-25T00:00:00.
Project Summary
Terpenes, otherwise known as isoprenoids, are a large and diverse class of natural products that
display a variety of biological activities. Two such molecules, nootkatone and artemisinin, play critical roles in
both the prevention and treatment of one of the most devastating infectious diseases: malaria. Nootkatone has
been proven to be a potent insect repellent, rivaling the properties of harsh synthetic insecticides such as
DEET. Artemisinin has been used for years in the treatment of malaria as a part of artemisinin combination
therapies, saving billions of lives. However, despite the importance of these molecules, the main source of
production remains plant extractions. The natural abundance of terpenes is extremely low and often results in
poor extraction efficiency. This, coupled with any variations in growth conditions, crop disease, and the
geopolitical climate, can impact the availability of these invaluable products. To circumvent these issues, the
use of microbes as high-yielding production facilities should be established, thus ensuring the accessibility of
both nootkatone and artemisinin. However, various bottlenecks in these microbial biosynthetic pathways have
resulted in low yields or high production costs, preventing this method from being used on a large scale. The
process of directed evolution, where random mutations are introduced to the rate-limiting enzyme in the
biosynthetic pathway, can be used to mimic how nature optimizes biosynthesis. However, this method
generates thousands of samples to screen. The lack of robust, high-throughput methods for screening
terpenes has severely hindered the ability to apply directed evolution to enhance the microbial production of
terpenes such as nootkatone and artemisinin. To address this, the objective of this proposal is to develop
genetically encoded biosensors that detect nootkatone and artemisinin and to leverage them for high-
throughput screening of enzyme/pathway libraries to overcome complex bottlenecks and identify
improved production strains.
In this proposal, the macrolide-sensing transcription factor MphR will serve as a template for the
development of a terpene biosensor, based on preliminary results indicating that MphR variants can detect
several sesquiterpenes. The goal of the first aim is to enhance the in vivo production of nootkatone by directed
evolution of cytochrome P450 enzymes in E. coli, utilizing a genetically encoded biosensor to report the P450-
catalyzed hydroxylation of valencene. The goal of the second aim is to transfer the E. coli-based biosensor
capabilities into yeast and develop a yeast-based artemisinin biosensor. This tool can then be used to evolve
the first-ever in vivo production of artemisinin. The impacts of the work described here not only contribute
substantially towards the fight against malaria but can also impact the development of other pharmaceutically
relevant terpenes, such as the anti-cancer therapy Taxol.document fetched 2026-08-02 08:24