Abstract
Almost all iron on earth can be found in its ferric (Fe3+) state which is insoluble in water. This property diminishes the bioavailability of iron for organisms greatly, which is especially problematic due to it playing an essential role in vital metabolic pathways. To address this, organisms have developed iron scavenging systems for which they secrete secondary metabolites that can chelate ferric iron, resulting in a water soluble complex which can be taken up into the cell. These properties make siderophores attractive candidates for a diverse range of biotechnological applications.
Currently, desferrioxamine is the most economically relevant siderophore and finds application in life-saving chelation therapy for patients suffering from thalassemia and other iron overload disorders. It is also being utilized in novel technologies such as gallium recovery from industrial wastewater. However, desferrioxamine is very expensive to produce, limiting its economic viability.
The high production cost partly stems from the specialized fermentation processes that relies on Streptomyces pilosus. Streptomyces suffer from major limitations such as slow growth, low product yields, and complex lifecycles that complicate maintaining consistent fermentation conditions at industrial scale making it an unsuitable production platform. Because of this our team aimed to move to a more suitable production platform by isolating the desferrioxamine biosynthetic gene cluster and introducing it into Rhodococcus which does not produce desferrioxamine endogenously.
We chose Rhodococcus because it is well established in industry for its production of complex secondary metabolites, fast turnover rates and its scalability for industrial fermentation. Both species belong to the Actinobacteria phylum, therefore they share a similar genetic architecture, codon usage, and metabolic framework necessary for expressing the pathway for desferrioxamine biosynthesis.
To monitor transcriptional activity of the biosynthesis gene cluster during transfer, we designed a split-GFP system that confirms successful pathway integration and functionality.
With our project, we aim to reduce desferrioxamine production costs and increase its accessibility for medical applications and industrial processes worldwide.
Desferrioxamine in medicine
It is estimated that 400,000 children are born worldwide annually suffering from either sickle cell disease or thalassemia.[1] Those are genetic diseases that result in the body of the affected patients not being capable of producing sufficient amounts of functioning red blood cells as a result of which they have lower counts of red blood cells.
A healthy adult has a hemoglobin concentration of ~15 g/dL, while someone suffering from a hemoglobin disorder like thalassemia major only has a hemoglobin concentration of ~6 g/dL, so less than half. Consequently their body is not capable of supplying its tissue with enough oxygen which results in a multitude of health complications. Treatment consists of regular blood transfusion which is essential for thalassemia patients and greatly reduces risk of stroke in sickle cell patients.[2]
But this comes at a cost, with every blood transfusion the patients absorb up to 350 mg of iron into their body which accumulates in organs like the heart, liver, pancreas and kidneys. Left untreated this results in oxidative stress and organ failure which can culminate in the death of the patient. The total mortality burden of sickle cell diseases alone was estimated to be 370,000 deaths in 2021.[3]
With the majority of sickle cell cases occurring on the African continent while thalassemia is more common in central to south eastern asia.[4] Both Regions are projected to have a growing population until well into the 21st century, with every second birth on earth occurring in Africa by 2050.[5] As a result, it is expected that the health burden of these diseases will only increase in the future.
Established treatments consist of chelation therapy and has been proven to be an efficacious method to remove excess iron before any damage can occur. During treatment chelators like desferrioxamine can be administered, travel through the blood stream to the iron deposits where they form a strong inert complex which is consequently excreted through the urine or fecal matter.
But chelation treatment is expensive, making it inaccessible in the regions where the disease is most common. By moving production to Rhodococcus, we aim to reduce costs through faster growth rates, higher product yields and more scalable fermentation processes. This would make desferrioxamine more affordable and accessible to patients worldwide who desperately need this life-saving treatment.
Desferrioxamine in Gallium and Germanium recovery
Germanium and gallium are essential elements in modern industry and are required for a wide range of applications, particularly in the technology sector. Germanium is used in diodes, camera lenses, microscopes, fiber optic cables, and solar cells, among other things. Gallium, on the other hand, is mainly found in everyday devices such as LEDs, smartphones, computer chips, and laser pointers, but also in certain solar cell technologies. Both elements are classified as critical raw materials, often referred to as “rare earth metals,” although chemically they do not belong to the rare earth metals. Their extraction is increasingly problematic because global reserves are limited and mining is often associated with high environmental and political risks. For this reason, the recovery of these metals from industrial waste and wastewater is becoming increasingly important.
A promising recovery method is the use of biological chelators, in particular desferrioxamine B (DFOB), a natural siderophore that is originally used for iron binding by microorganisms.[6],[7] were able to show that DFOB could be used to recover more than 80% of the germanium contained in wastewater from the glass fiber industry, with a purity of 84.5%. [8] also reported promising results in gallium recovery. With the help of DFOB and desferrioxamine E (DFOE), 100% of the gallium from industrial wastewater could be bound. In the subsequent processing and recovery from the DFO gallium complexes, a total of 70% (DFOB) and 93% (DFOE) could be successfully purified.
These results clearly demonstrate the potential of desferrioxamines for the sustainable recovery of critical metals from industrial waste. Researchers who are already working with desferrioxamine B see great potential in this method. At the same time, they point out that the production of DFOB is currently one of the limiting factors. In order to make recovery economically attractive and scalable, the industrial production of DFOB must be significantly expanded.
Engineering
Our primary goal is to develop a biosensor that enables the successful detection of successful translation within a gene cascade. We have used the DesA to DesD genes, which are responsible for the production of desferrioxamine B, as the gene cascade. The split-GFP system will be used as the sensor system. The 1–10ß of GFP is integrated either on a plasmid or in the genome, while the 11ß-fold of GFP is located on a separate plasmid. Our gene cascade is designed to be located upstream of the 11ß-fold GFP and is therefore transcribed and translated first, before the 11ß-fold GFP is expressed. If the gene cascade is successfully translated, the 11ß-fold GFP is then also produced. Upon contact with the 1–10ß GFP, fluorescence occurs under UV light (black light), which generates a visible signal and confirms the successful completion of translation.
One advantage of this system is that 11ß-GFP is not fused to the target protein and therefore does not interfere with its structure or function. If, on the other hand, you want to investigate a protein-protein interaction, 11ß GFP can be fused to the target protein in a similar way to a His tag, while 1–10ß GFP is coupled to the potential interaction partner. If interaction occurs, a fluorescence signal is also generated under black light, confirming the interaction of the two components.
We would like to establish this system and the gene cascade in Rhodococcus. The reason for this is that Rhodococcus is becoming increasingly important in industry, particularly due to its high adaptability and ability to metabolize a wide range of substrates. In addition, Rhodococcus, like Gordonia, from which we took the gene cascade, belongs to the group of actinobacteria. Due to this phylogenetic proximity, the probability of successful expression of the target genes in Rhodococcus is higher than in E. coli, for example.
In addition to establishing the gene cascade in Rhodococcus, we are pursuing the goal of expanding the molecular biology toolkit for this organism with the help of the split-GFP system. This should facilitate work with Rhodococcus and contribute to the fundamental elucidation of molecular interactions in this organism.
Education
Through our educational programs, we aim to build public trust in science by helping people better understand our research. To reach audiences of all ages, our activities are organized into three main areas:
- Children and young people: We engaged with them during the summer festival of the Botanical Garden at Ruhr-University Bochum, where we offered fun, hands-on ways to explore biology.
- Teenagers and young adults: We connected with them through high school visits and at the RUB open house day, where we talk with them about biotechnology, our project and life as a biology student in general.
- Older generations: We involve them at the summer festival of the Botanical Garden. While their children were busy with the experiments, we spoke with parents about topics more suited to an adult audience. To learn more about our educational events please take a look at our education section.
Sources
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