New scalable strategy for challenging plant-based medicines A European consortium working on the MIAMi project is using yeast to support sustainable, scalable production of complex chemical compounds in rare plants, to harness their valuable healthcare and pharmaceutical properties. We speak to Dr. Michael Krogh Jensen, Group Leader & Senior Researcher from the Technical University of Denmark. About half of modern medicines are plant-based and plants provide the ingredients of highly effective medicines for several debilitating and life-threatening conditions. For just a couple of examples to show the usefulness of plant-derived medicines, take the anti-malarial drug Artemisinin, from the sweet wormwood plant – it damages the malaria parasite in red blood cells, or the opium poppy, harbouring opiates for pain medicine and used in morphine. Plants are invaluable and can provide us with an enormous range of uses for healthcare. The MIAMi project focuses specifically on tropical plants possessing the molecules known as MIAs (monoterpenoid indole alkaloids), which are plant secondary metabolites. There are more than 2000 MIAs in nature, but they can be rare, so studying them is not always possible. This rarity also means it is near impossible to harvest them and furthermore, exploiting them could lead to their extinction. However, it is known they can be used for anti-cancer therapeutics, anti-psychotic drugs and anaesthetics so finding better methods to utilise them for treatments is an important goal in healthcare. The challenges of extracting MIAs “We have millions of species of plant on this planet, most of them full of interesting chemicals and apart from problems like habitat destruction, and the extinction of plants, even if we find interesting substances in a plant this does not mean we can directly use them, because if you use a substance you have to have it in sufficient amounts, you need to produce them cheaply and sustainably and that’s where we often find problems,” said Linus Naumann, from the Max-Planck Institute, who is working on MIAMi. “Some plants require you to harvest tonnes of them for milligrams of the substance. Some may grow extremely slowly. There may be some that you cannot cultivate at all because they need symbiotic interactions that only occur in their natural habitat. Many interesting plants are also very rare so they would become extinct if exploited. You can see the dilemma.”
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By machine learning-guided elucidation of biosynthetic pathways for monoterpenoid indole alkaloids found in plants, the EU-funded MIAMi consortium are refactoring alkaloid biosynthesis in yeast cells using computer-aided design tools, advanced genome engineering, mass-spectrometry-assisted imaging, and enzyme characterization to enable fermentation-based manufacturing of natural and new-to-nature alkaloids from engineered yeast cells. Lærke M.M. Lassen
As if this wasn’t enough to dissuade the exploitation of the plants, the process for extracting them can involve solvents and large amounts of energy which shows it’s not kind to the environment. Production of pharmaceutical ingredients can also be hindered when there are gaps in knowledge about biosynthetic pathways. “Sometimes, these molecules quickly become so complex and intricate that even our biochemists cannot efficiently reproduce them in the lab. Even in the year 2020, we rely on growing these plants and extracting the molecules.”
The chemical mastery of plants It may seem strange that modern science cannot keep pace with this natural manufacturing process but there are good reasons. Plants rely on chemicals for nearly
all of their interaction with their environment because plants cannot move from where their seed falls. They face threats such as being attacked by insects and eaten by larger grazing animals, they have to deal with extremes in weather and they must be attractive to pollinators to reproduce. Therefore, they rely on a specific, targeting, chemical production for each job, for defence, for attraction, even for signalling and conveying warnings of attack, to other plants. They have developed chemical mastery in nature and this specialism, honed over evolutionary time frames is beyond our abilities to mimic and reproduce them. The impracticalities and inefficiencies in the process from extraction to synthesis, mean that whilst the range of plant-derived pharmaceuticals is potentially vast, only a fraction can be used for medical treatments.
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New solutions and strategies
Who’s involved?
The secret to these substances is encoded in the organism’s DNA. The best hope is in genetic engineering, finding a way to trick the natural production of the valuable chemical in a more accessible, quick growing, cheap and abundant organism, compared to the rare plant. That ideal organism would take on the task of ramping up chemical production. To accomplish this feat, you first need to extract the genetic sequence from the plant, then analyse it and create a new DNA construct, and finally insert it into the alternative organism. Each of these processes is incredibly complex and there are many unknowns, so trial and error and going back to the start are part of the painstaking process. The MIAMi project is made up of four university groups and three industry partners collaborating to address the challenge to develop a sustainable bioproduction route based on yeast, which can ferment simple and cheap feedstocks such as sugar into bioactive MIAs. It essentially means taking the DNA from the plants and placing it into yeast to grow it. “This organism, yeast, that has been used for baking bread and brewing beer for thousands of years, grows quickly and on cheap media, and it possesses advanced cellular machinery. This organism is already part of many great achievements in biotechnology. It provides a means of cheaper, more sustainable production,” says Dr. Michael Krogh Jensen.
The Dutch company, Future Genomics Technologies take on the task of sequencing the plant. The company showcases one of the most advanced DNA sequencing technologies available, called Nanopore sequencing. Nanopore sequencing relies on a thin electrically insulating membrane in a small liquid-filled well. In the membrane is one tiny hole called a pore protein which allows
measuring these changes in the electrical current, it is possible to calculate each and every base that passes through the hole. The sequence of changes in electrical current can be matched with correlating DNA bases. This is the method used to sequence genomes for the MIAMi project. For reading and deciphering the sequence, Universite de Tours, Max Planck Institute for Chemical Ecology and University of
Doulix has created a tool for biologists to upload the DNA sequences, which returns a recommendation on how to assemble them to fit the new target organism. Building a library of well characterised and standardised biological parts means it will become possible for plug-and-play designs of new genetic circuits, over time. This kind of innovation is
key to accelerating biosynthetic solutions.
DNA to pass through it. The scientists put the DNA that needs to be sequenced on one side of the membrane. DNA is a negatively charged molecule which means when an electrical voltage is fed into the membrane the DNA gets sucked to the positive pole, through the one single pore. When it passes through the pore, every base of this DNA changes the electrical current that flows through it for a short amount of time. By
Copenhagen step in with their expertise. After finding out which part of the genome produces which compound, Explorer Biotech and their business unit, Doulix, attempt to streamline and automate DNA construct design. The next step is the production of these sought-out substances, when they are inserted into yeast cells, which act as busy high-throughput biological factories for the desired molecular compounds.
There will need to be ‘bug-fixes’ in the genetic pathways but using yeast allows scientists to refine the process, testing variable genetic constructs until an adequate ‘pilot’ design is reached. Optimisation will also need consideration, determining the best conditions to grow the compounds on media, taking account of criteria such as Ph value, nutrients, oxygen levels and other environmental variables. One of the partners in the programme is the French company, Axythtis, which has experience in pharmaceutical compounds and can medically test new substances in pre-clinical studies like animal testing, for example measuring the bio-activity of compounds introduced in mice. When scaleup is approved, large, industrial bio-reactors can be used for production.
Saving lives with MIAs In summary, the project discovers genes in the target plants that need to be assembled in a new way, to be suitable to genetically engineer in yeast cells. Doulix has created a tool for biologists to upload the DNA sequences, which returns a recommendation on how to assemble them to fit the new target organism. Building a library of well characterised and standardised biological parts means it will become possible for plug-and-play designs of new genetic circuits, over time. This
kind of innovation is key to accelerating biosynthetic solutions. The project intends to deliver at least three lead MIA chemicals, and 15 MIA analogues. An important milestone has already been accomplished when researchers from the University of Tours, France, working on the MIAMi project, assembled a complex seven-step biosynthetic pathway of the MIA, vindoline, the precursor of the cancer medications vinblastine and vincristine. Furthermore, the DTU team has recently showcased the biosynthesis of vindoline and catharanthine in yeast, and from those compounds enabled synthesis of vinblastine, purely from fermentation of sugar and amino acid feedstocks by yeast (ref: Zhang et al., Nature, 2022). Currently, cancer patients face shortages in the supply chain, so the achievement is a positive step toward a more reliable, sustainable production of the drugs. The wider implications of this process, mean taking the emphasis off discovering the plants and manufacturing drugs directly from the harvest. It would be game-changing for increasing production without destroying the source. It would save on resources like land and water and it would also save time. Most of all, it makes possible the creation and mass production of new drugs with these key pharmaceutical ingredients, that could change healthcare outcomes and save lives, potentially for millions of people.
MIAMI Refactoring monoterpenoid indole alkaloid production in microbial cell factories
Project Objectives
Our mission is to develop new tools and methodologies to discover complex biosynthetic pathways of bioactive natural products from plants, and to optimize their production in yeast cell factories in order to establish robust fermentation-based manufacturing of essential plant-derived medicines facing supply chain challenges and environmental concerns related to their sourcing from plants.
Project Funding
This project has received funding from the EU Horizon 2020 research and innovation programme. Grant agreement No 814645.
Project Partners
https://www.miami-project.eu/about-us/ meet_the_team/
Contact Details
Dr. Michael Krogh Jensen Group leader & Senior researcher Technical University of Denmark DTU Biosustain Kemitorvet 220 DK-2800 Kgs. Lyngby Denmark T: +45 61284850 E: mije@biosustain.dtu.dk W: http://www.miami-project.eu animation video: https://www.youtube.com/ watch?v=PSRY0oGu4x0 https://www.youtube.com/watch?v=plwcUWIKWts https://www.youtube.com/ watch?v=eS9cFjagaY0&t=984s Newsletter: https://www.miami-project.eu/ newsletter-2021/ https://pubmed.ncbi.nlm.nih.gov/36045295/
Dr. Michael Krogh Jensen
Dr. Michael Krogh Jensen is Principal Investigator at the Center for Biosustainability at the Technical University of Denmark, and coordinator of the Horizon 2020 consortium MIAMi focused on developing new supply chains of human therapies using yeast cell factories. He is a molecular biologist by training, and conducted his post-doctoral research at Max-Planck Institute in Germany, University of Copenhagen, and Stanford University in the US.
MIAMi Team Photo
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