Most people want to avoid mould. These filamentous fungi can pose a real health hazard in water-damaged houses but they also produce several substances that can potentially combat diseases. Laboratory tests have shown that one such substance, calbistrin, can kill cancer cells. Now Danish researchers have found a way to get moulds to produce calbistrin in large quantities. This discovery can pave the way for future clinical trials with calbistrin and change how similar drugs are produced in the future.
When humans inherit genes, we get half from each parent. This is not always the case for moulds. Through evolution, different species have shared useful genes with each other so that they can inherit traits that benefit their survival. Danish scientists have now used this fact to determine how the Penicillium decumbens mould produces calbistrin, a potential anticancer drug.
“We searched for the genes the moulds use to produce calbistrin using a technique called retrosynthetic analysis. Based on the chemical structure of calbistrin, we could predict the required enzymes and thereby which genes the moulds must use to produce calbistrin. When we later found these genes in three types of moulds that could produce calbistrin but not in other closely related species of moulds, we knew we were on the right path,” explains a main author, Rasmus J.N. Frandsen, Associate Professor, Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark.
Thirteen relevant genes
Retrosynthetic analysis can be compared to trying to figure out how a LEGO house is built by examining its surface. The bricks can be assembled in many ways and yet still end up with the same surface. The number of possible solutions can be reduced by removing one LEGO brick at a time from the outermost layer and thereby reveal the underlying bricks.
For calbistrin and similar substances, we work backward from the final structure by removing the chemical groups most likely to be attached at the end of the biosynthesis, which results in a simpler substance, and then we can then repeat this process. Each chemical group is attached by one specific type of enzyme, and we can therefore very precisely predict which enzyme and genes are needed to produce calbistrin.
Based on these predictions, the researchers could then search for relevant types of genes in the genome of the moulds known to produce calbistrin. By comparing the genomes – the DNA – of three remotely related fungal species, which surprisingly all produce calbistrin, the researchers found genes that could encode calbistrin’s unique biosynthetic pathway.
“Of these, we found 13 relevant genes next to one another in the genome. We proved that we had found the right genes by cutting the genes out of the mould genome using the CRISPR-Cas9 editing tool. When we removed these genes, the mould no longer produced calbistrin.”
Solution leads to a new mystery
The mechanism by which mould species share genes across the normally impermeable boundaries between species is not known, but the fact that this happens gives the researchers a unique tool for finding the few relevant genes for producing the desired substances among the thousands of candidates in the genome of the moulds.
“Nature helps us. Each mould species can produce many bioactive substances, each formed through a unique biosynthetic pathway comprising many enzyme-catalysed steps. Fortunately, the genes for each biosynthetic pathway are co-located in small discrete clusters in the moulds’ genome.
In practice, finding one gene that is essential for producing a substance means that the neighbouring genes are also very likely to be essential for producing this substance. The researchers therefore also know, based on their initial analysis of the production of calbstrincalbistrin, that they still have to find one additional gene before they have completely mapped the moulds’ production mechanism.
“Fortunately, this step is not limiting foris not a limiting factor for the mouldsmoulds’ production of calbistrin, but we are continuing the search for its genetic basis. We often experience this in science - : when one mystery is solved, this usually leads to several new mysteries that need unravelling.”
Creating the basis for cell factories
The genes identified in the moulds enable the researchers to alter the genetic material of the mould so that it produces calbistrin in sufficient quantities for the researchers to thoroughly examine whether calbistrin is useful for treating people with cancer. The results were created in connection with a project carried out by a large EU consortium (QuantFung) comprising both universities and companies.
These companies are interested in using new species of moulds in their production of substances, including medicine and enzymes. Today, they use very few species of fungi and bacteria to produce useful substances, and this limits what substances can be produced.
The reason for this practice is that companies have invested decades of research into developing the genetic tools required to modify the genome of the fungi and to develop variants that enable growth to be controlled more easily in large cultivation tanks.
“However, most new substances with pharmaceutical potential are usually found in species of fungi that are not considered industrial workhorses, and which do not produce enough of the desired substance when they are cultivated in fermenters -– cultivation tanks. Also, many assume that it is difficult to culturecultivate and control the non-standard fungi in fermenters.”
Long, hard road ahead
The typical solution for the companies today is to move production to one of the standard moulds. However, this requires identifying the genes that encode the production of the medicine. Then the genes have to be transferred into the standard mould a company normally uses.
Unfortunately, the genes often do not function optimally in the new organism, and optimizing production to reach quantities corresponding to those the original moulds can achieve in nature often takes a long time. The research project has aimed to use the original moulds directly to produce the substances in sufficient quantities.
The results show that most of the natural species of moulds thrive in cultivation tanks and can be controlled via standard method,methods but that they do not produce enough of the desired substances under these unnatural growth conditions.
“However, this can be changed, since we have also shown that their genetic material can be modified using CRISPR-Cas9 and production can be upgraded. Overall, in the future, using the natural organisms will often be more profitable, possibly manipulating their genomes to increase production.”
The researchers therefore hope that their study can show companies the potential of using other organisms such as moulds to produce substances including medicine. However, since the pharmaceutical industry is typically highly regulated and requires strict approval, Rasmus J.N. Frandsen is well aware that the shift to new organisms is not imminent.
Changing this will be a long, hard road. This is also why we make a special effort at the Technical University of Denmark to educate our students in all the steps in these processes, so that future generations of researchers in industry have the tools and knowledge to further develop the industrial production of this type of substance using new species of moulds.
“Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin” has been published in Fungal Biology and Biotechnology. In 2015, the Novo Nordisk Foundation awarded Rasmus J.N. Frandsen a grant for the project A Platform for Microbial Production of Aromatic and Cyclic Compounds.