blood bread

A shortage in the supply of essential cancer drugs vinblastine and vincristine, compounded by the emergence of the COVID-19 pandemic, has inspired researchers at the Technical University of Denmark (DTU) to work on a more sustainable alternative. Their response ? Genetically modified yeast. Here we chat with Jie Zhang, Principal Investigator at DTU Biosustain, to learn more.

I joined the Novo Nordisk Foundation Center for Biosustainability (Biosustain) at DTU in 2014 as a researcher. My interests focus on the application of synthetic biology to manufacture valuable plant products in microbial cell factories. We are designing plant biosynthetic pathways in baker’s yeast (Saccharomyces cerevisiae) to synthesize natural plant products with high added value by yeast fermentation. We are currently working on monoterpene indole alkaloids (MIA), which are a class of structurally and functionally diverse natural products found in the order Gentianales. So far, over 3,000 AIMs have been reported, including the cancer therapeutics vinblastine and vincristine, among many others.

I’ve always been fascinated by nature and what biology can do, so bioengineering and applying biotechnology to do something useful is very interesting to me. At the Center for Biosustainability, our goal is to develop enabling technologies for the transition to a more sustainable economy with a reduced environmental footprint. In short, we need technologies to replace petroleum-based chemical production and a more sustainable supply chain for natural plant-based products, including therapeutics.

Synthetic biology has shown great potential here; modified microorganisms, such as S.cerevisiae can produce complex molecules faster, using fewer natural resources.

We chose to work on the MIA family because of its diverse bioactivity, which indicates the opportunity to develop new therapies. These molecules have very complex structures that are too difficult to synthesize chemically, so their supply depends exclusively on plant extracts, resulting in a slow manufacturing process that can be affected by plant diseases, natural disasters, etc

We had no way of predicting the 2019-2021 shortage. We chose vinblastine as a test bed because, to our knowledge, it is the longest and most complex plant biosynthetic pathway. The entire pathway – which was only completed after the last two missing enzymes were identified in 2018 – contains 31 enzymatic steps. To meet the challenge, we have divided the entire course into three modules based on the commercial availability of precursors and standards. The first module converts the native yeast metabolite geranyl pyrophosphate (GPP) to strictosidine – the common precursor of all natural MIAs. The second module converts strictosidine to catharanthine and tabersonine, and the third module further converts tabersonine to vindoline. Each module was expressed in yeast and tested individually to confirm functional expression of that part of the pathway.

We then merged the three modules to construct a strain that produces vindoline and catharanthine from a raw material of glucose and amino acids. This final strain contains 56 genetic modifications, including overexpression of 34 genes from plants and other sources, as well as deletions, knockdowns (reduced expression level) and overexpression of ten yeast genes to improve generation of key precursors in the biosynthetic pathway.

Next, we grew the modified yeast strain in lab-scale bioreactors, and from the fermentation broth we purified vindoline and catharanthine, which were chemically coupled to produce vinblastine.

This project would not have been a success without continued funding from the Novo Nordisk Foundation, the BioInnovation Institute Foundation and the European Union’s Horizon 2020 program.

We can use the yeast strain from the first module, which produces strictosidine, as a platform to produce any of the other 3,000 natural MIAs. One molecule we are working on is ibogaine, an anti-addiction agent, a bioactive ingredient from an African evergreen shrub: Tabernanthe iboga.

We will then try to increase the fermentation process and develop a downstream processing method for purification.

The pharmaceutical industry is extremely complex and its manufacturing processes are highly regulated. I can’t say how they can better prepare for future crises caused by a shortage of raw materials or interrupted logistics, but I think we need to develop alternative processes to manufacture APIs – especially those needed for essential medicines. Here, I believe synthetic biology has great potential in terms of establishing a cost-effective and reliable manufacturing process, as well as expanding the chemical space to synthesize many more valuable unnatural products.