Implementation in the real world
Despite recent advances in medical science, humans continue to face a variety of global health challenges, including emerging infectious diseases and debilitating diseases such as cancer. Therefore, there is an unmet clinical need for new and effective drugs. However, cost-effectiveness in finding effective drugs for clinical use is a longstanding problem in drug discovery. To address this challenge, our team aims to develop a simple and cost-effective fungal synthetic biology platform to produce various derivatives of variecolin, a fungal natural product that shows potent therapeutic and antimicrobial properties. The platform comprises the genetic modification of an Aspergillus oryzae host with (1) a “terpene synthase” biobrick for the backbone synthesis and (2) a series of different “cytochrome P450 monooxygenase” biobricks to derivatize the variecolin scaffold. It is envisaged that this synthetic biology platform could be used to expand the chemistry of other natural products to produce a broader array of derivatized natural products to facilitate the discovery of new and effective drugs.
Potential stakeholders include pharmaceutical industries involved in drug discovery and production as well as medical institutions. Given the rising demand for new and effective drugs, natural products are important sources for the discovery of new drugs and provide good lead compounds suitable for further modification during drug development.
Even though using natural products in drug discovery can effectively address these issues due to their diverse structure, novelty, and better biological interactions, many industries in drug discovery has been reluctant to use natural products to develop new drugs due to the long-existing problem: cost-effectiveness. However, by genetic modification of a fungal host, we aim to develop a simple and cost-effective method to produce natural product drugs with novel therapeutic properties. We aspire to share our knowledge with pharmaceutical companies, clinical scientists, and medical institutions to help treat various human diseases which are continuously evolving.
Our contribution to future research on natural product drug discovery.
Variecolin is a fungal sesterterpenoid, originally isolated from Aspergillus variecolor; it reportedly possess diverse therapeutic activities. Variecolin is a natural product with antimicrobial (Jouda et al., 2016) and anticancer (Yodsing et al., 2018) activities. Although researchers have worked intensively on the chemical synthesis of variecolin, the total synthesis of the molecule has never been achieved. Our team has used a synthetic biology approach to expand the production of a diversity of variecolin analogues. We have successfully created three novel variecolin analogues (which we named as compound 161, 163, and 313) in an Aspergillus oryzae fungal host, using cytochrome P450 genes derived from several different fungi. We have evaluated the antimicrobial and anticancer properties of variecolin and the newly derivatized variecolin analogues.
Antibacterial activities were tested utilizing several microbial species, including Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, and Escherichia coli. One of the variecolin analogues exhibited a minimum inhibitory concentration (MIC70) of 16 μg/ml on Staphylococcus aureus, compared to 7 μg/ml exhibited by variecolin. Anticancer activities were tested utilizing breast cancer cell MCF-7. The half maximal inhibitory concentration (IC50) of variecolin was 1.4±0.3 μM μM, and the new variecolin analogues exhibited anticancer properties with IC50 values of 8±3 μM, 31±9 μM, and 146±82 μM, respectively.
The present project has successfully identified the biosynthetic pathways for producing bioactive compounds that have a promising application in the future. We have also constructed and tested a number of biobricks, which are responsible for producing novel variecolin analogues, which are available to the iGEM registry. Researchers from both academia and industry may refer to our biobricks to produce these compounds and expand the scope of their bioactivity and usage.
Safety considerations, potential challenges and further studies to implement our project
It is demonstrated that variecolin and its derivatives exhibit antibacterial and anticancer activities. After consulting with iGEMers of a previous CityU team, who have successfully opened a biotechnology startup (Hands Life Science Ltd) that focuses on personal care and cosmetic products, several issues have come to our mind.
The first one is the safety of our derivatized natural products. The pharmacokinetics and toxicity are still yet to be investigated. Although we have examined the drug's safety using in silico modeling approaches, comprehensive clinical trials for risk assessment are still needed before introduction to the drug market. Another safety consideration is the dosage. In most cases, toxicity depends on the concentration of drug administered (Atuah et al., 2004). Although antibacterial and anticancer properties may be more potent at a higher concentration, extensive animal tests should be carried out to determine the safe dosage for our compounds.
The second issue is that we are unsure of the drug targets (in MCF-7) of the compounds, which are related to the observed anticancer and antibacterial activities. For future studies, mechanistic studies could be carried out to better understand the biological properties of these newly synthesized variecolin derivatives.
There are several potential challenges to implement our project. One of the most important ones is the yield of the final product. Generally, the production yield of microbial fermentations is extremely low, even with the help of recombinant DNA technologies. Since we were using a fungal host for the biosynthetic platform, the yield of the final product should be given special attention. An advanced fermentation technique could be developed to alleviate the problem.
1. Atuah, K. N., Hughes, D. & Pirmohamed, M. (2004). Clinical pharmacology: special safety considerations in drug development and pharmacovigilance. Drug Safety, 27(8), 535–554.
2. Yodsing, N., Lekphrom, R., Sangsopha, W., Aimi, T., & Boonlue, S. (2018). Secondary metabolites and their biological activity from Aspergillus aculeatus KKU-CT2. Current Microbiology, 75(5), 513–518. https://doi.org/10.1007/s00284-017-1411-y
3. Jouda, J.-B., Fopossi, J.-L., Mbazoa, C., & Wandji, J. (2016). Antibacterial activity of the major compound of an endophytic fungus isolated from Garcinia preussii. Journal of Applied Pharmaceutical Science, 026–029. https://doi.org/10.7324/japs.2016.60605