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Development of a Fungal Synthetic Biology Platform

Abstract

Despite recent advances in medical science, humans are still facing some global health challenges, including emerging infectious diseases and debilitating diseases such as cancer. Hence, there is an unmet clinical need for new and effective drugs. However, a long-existing problem in drug discovery is the cost-effectiveness issue in finding effective drugs for clinical use. To address this, 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 consists of the genetically modified 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. We have successfully obtained three novel variecolin analogues as confirmed by LC-MS and NMR analyses, and experiments are currently underway to characterize their biological activities.

Background

The Importance of Health
Figure 1. Maslow's hierarchy of needs, Simply psychology. According to Maslow’s Hierarchy of Needs theory, health is the foundation of life needs. In Maslow’s pyramid, health is among the safety needs at the bottom layer (McLeod, 2007). Only by satisfying the needs at the bottom can a person pursue the needs at a higher level, to reach the spiritual levels of culture, morality, and ideals. Thus, if we cannot fulfill the requirement of health, we cannot enjoy a high-quality life; being free of illness or injury directly affects our capacity to enjoy life.

A population’s health can be measured via life expectancy, which measures how many years, on average, a person is expected to live based on the current age- and sex-specific death rate.
Figure 2. Life Expectancy, Our World in Data. This study indicated that the world life expectancy was about 70 years in 2019, compared to only 30 in 1900 (Roser, Ortiz-Ospina & Ritchie, 2019). Global life expectancy has increased dramatically during the most recent centuries, with the advent of modern medicine and science.

Figure 3. Life Expectancy, Our World in Data. Healthcare expenditure is among the most important inputs to health; it improves and leads to increased life expectancy. This visualization shows the worldwide relationship between life expectancy and healthcare expenditure per capita (Roser, Ortiz-Ospina & Ritchie, 2019). As the figure shows, countries with higher healthcare expenditure tend to have a higher life expectancy. The changes over time indicate that the greater the expenditure on health, the higher the population’s life expectancy.

Problems

1. Unavailability of medicines in developing countries due to low affordability
Figure 4. Life Expectancy, Our World in Data

Research has revealed that vast differences remain between developed and developing countries, despite the increased worldwide life expectancy (Roser, Ortiz-Ospina & Ritchie, 2019). As the graph illustrates, people in developing countries like South Africa only have a life expectancy of about 60 years, compared to about 80 years in developed countries like Japan; this 20 year-gap is significantly wide. The difference can be extremely large because the medicine availability in developing countries remains a barrier to affordable treatment access. Thus, there is a need for access to less expensive, affordable medicine.

2. Number and types of diseases increasing
Human diseases have been on the rise around the world in both number and type, despite major advances in medical science and the apparent global increase in average life expectancy. Due to global warming, rising temperatures may increase the reproductive rate of the insects that are infectious disease vectors (Kurane, 2010). These insects can carry bacteria and viruses to the human body, leading to disease outbreaks.

3. Higher demands in the market for effective antimicrobial and anticancer drugs
To treat infectious diseases which are continuously evolving, we need more effective antimicrobial and anticancer drugs. However, because drug discovery is expensive, complicated, and time-consuming regarding medicines being made industry-ready, producing drugs for the world population is highly challenging.


Inspiration

In order to solve these problems, our team at VarieCure decided to find a more cost-effective, easier, and faster drug discovery process that would align with our modern society’s needs.
To solve the challenge of low-yield drug discovery, we seek to utilize natural products. Natural products are organic molecules derived from microorganisms, plants, and animals. Fungi represent a major source of natural products.
Figure 5. European Biopharmaceutical Review, Samedan Limited Pharmaceutical Publishers. According to the study of Samedan Limited Pharmaceutical Publishers (2011), natural products and their derivatives play a significant role in drug development. Currently, approximately 30% of all small-molecule drugs are either natural products or derived from natural products, which accounts for a large proportion.

Advantages of Natural Products

One of the benefits is great molecular rigidity, because natural products contain more carbon and oxygen atoms than nitrogen and halogen atoms. Moreover, natural products have high molecular mass, which is valuable in tackling protein-protein interactions. In addition, the high hydrophilicity enables easy delivery of drugs to the body. Furthermore, the complex structures provide interaction with different organisms, which is highly relevant for infectious diseases and the ability to treat both diseases and cancer. Thus, natural products can be used in the pharmaceutical field, as antibacterial drugs, anticancer agents, opioid analgesic drugs, cholesterol-lowering statins, and anti-Alzheimer’s drugs.

Current Procedure in Drug Discovery

The first phase is extraction of natural products from organisms, such as bacteria. The next step is identification of a crude extract with promising pharmacological activity. The last phase comprises consecutive bioactivity-guided fractionation.


Our Project: Generating novel bioactive variecolin analogues using synthetic biology

What is variecolin?

Variecolin is a fungal sesterterpenoid, originally isolated from Aspergillus variecolor; it reportedly possesses diverse therapeutic activities. Variecolin is a natural product with anticancer and antimicrobial activities. Although researchers have worked intensively on the chemical synthesis of variecolin, the total synthesis of the molecule has never been achieved. Our team will use a synthetic biology approach to produce variecolin and variecolin analogues.

We aim to synthesize variecolin and to create novel variecolin analogues in an Aspergillus oryzae fungal host, using P450 genes derived from several different fungi. In addition, we will evaluate the antimicrobial and anticancer properties of variecolin and its analogues.


References

1. European Biopharmaceutical Review. (2011). Retrieved from http://www.samedanltd.com/magazine/12/issue/146/article/2839
2. Kurane, I. (2010). The effect of global warming on infectious diseases. Osong public health and research perspectives, 1(1), 4-9.
3. McLeod, S. (2007). Maslow's hierarchy of needs. Simply psychology, 1(1-18).
4. Roser, M., Ortiz-Ospina, E., & Ritchie, H. (2013). Life expectancy. Our World in Data.