Researchers discover a unique fungal enzyme

Environment and sustainability 26. okt 2023 3 min Professor, Deputy Head of Centre Jesper Velgaard Olsen Written by Kristian Sjøgren

Many species of fungi can turn biomass from plants and trees into bioethanol. Researchers and industry have hoped to imitate this process for many years, but some very special enzymes have been lacking. After several decades of searching, researchers have now identified these enzymes. This discovery could be an important step towards the large-scale production of bioethanol.

For decades, researchers have been looking for a very specific enzyme in a very specific fungus.

The enzyme is a key component of an enzyme cocktail that can produce bioethanol from lignocellulose, which is the most available biological material in nature.

Researchers have now finally identified the enzyme in the filamentous fungus Aspergillus nidulans, and this opens up the potential for more efficient large-scale industrial production of bioethanol from virtually all plant material.

“This discovery has several interesting perspectives. We have the technology that can investigate these unique enzymes for which we have been searching for many years. Second, this opens the possibility of producing enzymes useful to industry on a large scale.,” explains a researcher behind the discovery, Jesper Velgaard Olsen, Professor and Vice Director, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.

The study, which is the result of collaboration between researchers at the University of Copenhagen and the Technical University of Denmark, has been published in Nature Communications.

Fungus with unique attributes

The overall aim is to break down lignocellulose, the substance of which plant walls are made. Lignocellulose is extremely abundant globally but, unfortunately, very difficult to break down.

However, some enzymes present in A. nidulans can break down lignocellulose by oxidation, such that the building blocks in lignocellulose become available for producing many other things, including bioethanol.

The enzymes that A. nidulans uses for this purpose are called lytic polysaccharide monooxygenases (LPMOs), and these could be very useful for industry.

LPMOs achieve their completely unique degradative properties through a special modification of one end of the enzyme (the N-terminal), at which a methyl group is added.

This methyl group means that the enzyme is not degraded during the oxidation of lignocellulose, and the researchers have identified the enzyme that causes the methylation of the LPMOs.

“For decades, scientists have tried to express the LPMOs in bacteria or other fungi to produce large amounts of these interesting enzymes, but since we have not had the enzyme that methylates the LPMOs after they have been made, the enzymes remain vulnerable to oxidation. The search for the enzyme that methylates the LPMOs has been underway for many years but now we have finally discovered and characterised it,” says Jesper Velgaard Olsen.

Detective work in the laboratory

Jesper Velgaard Olsen and colleagues carried out many experiments as part of a major detective investigation to find the enzyme.

First, the researchers cultured A. nidulans on two growth media containing cellulose versus only glucose or potato dextrose.

This means that the fungi that grew on cellulose would need to express the relevant enzymes to break down this stubborn substance, whereas the fungi that grew on glucose would not.

Then the researchers thoroughly analysed all the enzymes and other proteins in the fungi in the two media.

“Thousands of proteins and enzymes were differentially expressed in the two fungi, but using bioinformatics and other tools, we verified whether some have potential to methylate and shortlisted 24 candidates,” explains Jesper Velgaard Olsen.

Systematic knockout of 24 enzyme candidates to find “the one”

In experiments that followed, the researchers used CRISPR-Cas9 to knock out the genes for each of the 24 identified candidates to determine how this changed the fungus’s ability to attach a methyl group to the LPMOs.

The researchers investigated this using mass spectrometry, which enabled them to determine whether LPMOs were not methylated when they knocked out the genes for one of the identified enzymes.

The researchers followed up with a rescue experiment showing that adding back the methyltransferase to the fungi that it had knocked out restored the methylation of the LPMOs.

In the final experiment, the researchers expressed both the gene for an LPMO and the gene for the identified enzyme in yeast – which is precisely what is desirable in industry – and the yeast began to produce methylated LPMOs.

“In the past, the problem was that we could not get them to methylate, but now we can,” says Jesper Velgaard Olsen.

The identified enzyme has been named N-terminal histidine methyltransferase (NHMT) and is encoded by the gene AN4663.

Characterising the structure of the special enzyme

In addition, the researchers characterised the structure of NHMT, showing that it contains a structure that enables it to weave itself into a cell membrane with seven-transmembrane segments.

These transmembrane segments use the enzyme to anchor themselves in the part of the cells called the endoplasmic reticulum, close to the cell nucleus, where the LPMOs are first produced.

On their way from the cell nucleus to the cell surface, where the LPMOs must be released to break down lignocellulose, the enzymes pass the endoplasmic reticulum location of NHMT, and one end of the enzyme is methylated so that it is not broken down by oxidation as soon as it attacks the lignocellulose.

“The possibility of exploiting this discovery in industry is the reason for submitting a patent application,” concludes Jesper Velgaard Olsen.

Jesper Olsen studied analytical chemistry in the group of Roman Zubarev at the University of Southern Denmark in Odense, and obtained his PhD in bioch...

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