NOXFUN

The Noxfun project was aimed at the in vitro production of new fungal oxidases.
Oxidases are a kind of enzyme that catalyses the incorporation of oxygen atoms into different organic compounds. Those enzymes are very important in many biological processes, including the synthesis of hormones and other bioactive compounds, decomposing toxins and immune responses. In this project, we focused on two kinds of oxidases, lytic polysaccharide monooxigenase (LPMO) and lipoxygenases.

LPMO are a kind of oxidase found in fungus, bacteria and other organisms. Their primary function is to decomposes cellulose and other polysaccharides present in plant cell walls. Because of that capacity, they have been widely studied as an enzymatic tool for producing biofuels and decomposing organic waste. Furthermore, LPMO can also have applications in food production, and in improving the properties of some industrial materials. In this project, a new application in the field of healthcare using those enzymes for decomposing bacterial biofilms made of cellulose has been studied.

For its part, lipoxygenases are another kind of oxidase that is found in many organisms, including humans. These enzymes oxidate polyunsaturated fats to produce different bioactive products, like leukotrienes, lipoxins and hepoxilins, which play an important role in the immune response and inflammation. In addition, some lipoxygenases have also been related with developing cancer and other diseases. In this project, the production of fungal origin lipoxygenases was studied, because fungus is a group of organisms that has been explored very little in the production of these enzymes, whose role in their physiology is not clear.
Obtaining new enzymes from new organisms is of great importance for biotechnology, because the enzymatic diversity of different organisms on the planet can provide a wide variety of options for improving or developing new biotechnological processes that are more efficient, sustainable and environmentally friendly.

Along those lines, at Noxfun we looked for new oxidases produced by fungi different from the ones generally available. In the first place, we determined the characteristics we wanted in the target protein (resistance to high temperatures, salt resistance). Subsequently, we looked for fungi that live in natural environments that selected those characteristics (thermophilic fungi, salt stress resistant fungi). We did the search in the database of fungi whose genome has been sequenced (Mycocosm from the Joint Genome Institute). We thus had access to the gene sequence that codified the relevant proteins.

In this process, our reasoning was the following: Instead of trying to thermo-stabilise a protein produced by a conventional organism, our approach was to purify a protein from a thermophilic organism such that the adaptation of the protein to high temperatures would have occurred during natural evolution, in a more effective way than laboratory guided evolution. The problem of accessing an interesting extremophile was solved by having access to the gene sequence, which avoided the need to cultivate the microorganism that produces it.

After identifying the microorganism and gene, several genes corresponding to interesting LPMOs and lipoxygenases were synthesised in vitro. The synthesis was done by optimising the gene sequence to achieve a greater expression of it in the heterologous plichia pastoris system, which is a methylotrophic yeast used frequently in biotechnology as a recombinant protein expression system, because of its high capacity to produce large quantities of heterologous proteins.

Twenty genes were initially selected to study and start the production process with this approach. Of those genes, the work focused on six that were expressed in the heterologous system and, finally, on two LPMOs that were taken to production in 4 litre fermenters. The glycosylation system was studied in these enzymes, because they are extracellular enzymes, an analysis system for their enzymatic activity was fine-tuned, and their three-dimensional structure was studied using the AlphaFold artificial intelligence platform. Knowing the structure of the protein and determining the function of some of the enzymes in the laboratory, whose function had only been predicted biocomputationally and not experimentally, opens up the possibility of applying this production procedure to other enzymes of industrial or biotechnological interest.

In conclusion, during the Noxfun project several enzymes were selected by identifying the microorganisms that were selected because they live in environments where enzymes with special properties are produced. After the organisms were identified, the codifying genes of the enzymes were identified, so they could be expressed in a laboratory yeast, which made it possible to produce the enzymes without ever having contact with the initial producer microorganisms. In several cases, these enzymes were produced in the laboratory at a sufficient level to determine their enzymatic activity and, complementarily, their structure was established, with which a level was reached at which it is possible to consider transferring these products to higher production levels and apply the technology to searching for other new products.