By Laszlo Kozak, Chemical Researcher, Biotechnological, Teva api’s R&D & Professor István Pócsi, Department of Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, Hungary, Professor István Molnár, Natural Products Center, University of Arizona, Tucson, USA
“The story of the Claviceps Paspali, Genetic transformation of the ergot producer” is a real-life story about a research done by Teva api’s experts.
This story involves a fungus from the Middle Ages (that was responsible for the outbreak of poisoning epidemics called “St. Antony’s fire”), ergots genetic transformation and a group of Molecular Biology experts.
This story describes a research that will later enable superior productivity and better safety profile for fermentation and downstream processing.
Claviceps paspali is a parasitic fungus that infects wild grasses. Claviceps species are notorious toxin producers that have caused several poisoning epidemics over the history of mankind by infecting various crops and grains. The most well-known member of the Claviceps genus is Claviceps purpurea. During the Middle Ages, this fungus was responsible for the outbreak of poisoning epidemics called St. Antony’s fire (now referred to as “ergotism”).
Ingestion of rye infected with C. purpurea causes a variety of symptoms, including horrible visions, burning pain, and gangrene of limbs. These symptoms are caused by a variety of mycotoxins produced by the fungus. These mycotoxins can be divided into two major classes.
Chemical modification of ergot alkaloids – yielding excellent starting materials for drug discovery.
The first class encompasses the ergot alkaloids. These compounds are bioactive molecules which show structural similarities to neurotransmitters such as adrenalin, nor-adrenalin and dopamine. Neurotransmitters are endogenous chemicals which transfer the signal from one neuron to another.
Because of this structural similarity, ergot alkaloids are able to stimulate certain receptors of the central nervous system, thus triggering an array of symptoms. Beyond their harmful effects, these chemicals also show various advantageous properties. Documents from approximately 1100 BC describe that ergot-infected crops were used in obstetric practice in ancient China because of their stimulatory effects on uterine contraction.
In modern times, chemical modification of ergot alkaloids was used to modulate the bioactivities of these molecules, yielding excellent starting materials for drug discovery and manufacture. Nowadays, ergot alkaloids are widely used to prepare drugs for the treatment of several diseases, such as Parkinson’s disease, migraine and hypertension, and are produced on a multi-ton scale in the pharmaceutical industries.
The classical method for ergot manufacture is field production, when fungal conidia are sprayed on field-cultivated rye. In the infected rye, the fungus forms a hard and compact mass (sclerotium) which contains high concentrations of ergot alkaloids. Another way to produce ergot alkaloids is the cultivation of Claviceps strains (mainly Claviceps paspali or Claviceps purpurea) in bioreactors. This process is called fermentation. By optimizing all fermentation parameters, the fungus can be “forced” to produce large amounts of the desired alkaloids, which are then purified from the fermentation broth.
Utilizing additional toxins
The other important class of toxins produced by Claviceps species is the indole diterpenes. Some members of this class are referred to as tremorgenic mycotoxins, because the ingestion of these toxins causes involuntary shaking and trembling, and can also lead to seizures and even death. Grazing on Claviceps paspali-infected grass by large animals such as horses and cattle causes millions of dollars in losses to the livestock industry. On the other hand, some indole diterpene molecules have beneficial properties which may be exploited in the future, for example as pesticides or potential drugs for the treatment of lung cancer.
Using genetic transformation
Despite of the pharmaceutical and ecological importance of Claviceps paspali, there have not been any publications describing practical methods for the genetic manipulation of this fungus up till now. Genetic modification, most importantly genetic transformation (the introduction of DNA into the fungal cells) is a critical tool to study the biochemical and physiological processes of a microorganism. Using genetic transformation, we are able to reduce or strengthen the effects of individual genes, thereby revealing their function. Even more importantly, we can create modified microorganisms with better productivity or increased safety.
Microorganism to the rescue
We at the Molecular Biology group at the Debrecen, Hungary facility of TEVA, in collaboration with the University of Debrecen and the University of Arizona (USA), recently reported in the journal Applied Microbiology and Biotechnology (vol. 102, pp. 3255-3266, 2018) that we successfully established an efficient and practical genetic transformation method for Claviceps paspali.
Such method will help us understand the individual biochemical steps that lead to the production of ergot alkaloids and indole diterpenes in this organism. To devise such a genetic transformation system, we turned for help to another microorganism, Agrobacterium tumefaciens. Agrobacterium tumefaciens is a plant-pathogenic bacterium which is able to transfer specific regions of its DNA (the so-called Ti DNA) into the cells of the infected plant.
By modifying the Ti DNA and determining the optimal conditions for its transfer, we were able to adapt this natural phenomenon to introduce any selected DNA segment into the cells of Claviceps paspali. In order to demonstrate the usefulness of this method, we inactivated four of the genes proposed to be involved in the biosynthesis of indole diterpenes in Claviceps paspali. As expected, “knocking out” these four genes eliminated the production of the complete spectrum of indole diterpene metabolites in Claviceps paspali.
In addition to demonstrating the practicality of this transformation method and validating the proposed function of the genes, fermentations with the Claviceps paspali mutant that we created are now devoid of indole diterpene mycotoxins, thus display an improved safety during downstream processing of the fermentation broth. Our work also paves the way for the further genetic characterization of Claviceps paspali and other Claviceps species, and will contribute to the creation of new, industrially useful strains with superior productivity and better safety profile.