Productive against all expectations

| by Charlotte Fuchs

Fungal cultures on agar and soil. Structural formula of lindoline. Comparison of the number of natural substances from basic and higher fungi. Demonstration of anti-oomycetic effect of Lindolin.
Lindolin is formed by EDF via a previously unknown synthetic pathway. The joint JMRC strain collection of Leibniz-HKI and FSU Jena contains thousands of basal fungi collected over several decades. "This means we have a treasure chest right on our doorstep that could still contain many unknown natural products," says Greßler, looking to the future. Source: Robin Sonnabend/Leibniz-HKI

Contrary to previous theories, Early diverging fungi (EDF) could also be producers of important natural products. One division of EDF contradicts this common dogma by possessing a secondary metabolism. Via a two-step biosynthesis, the fungi produce lindolin, a natural substance that is effective against pathogens of fruit rot. This was discovered by researchers of the Leibniz-HKI and the Friedrich-Schiller-University during the investigation of a representative of the Zoopagomycota.

Early diverging fungi arose early in the course of evolution and branched off in the phylogenetic tree. While the higher fungi are already well studied and about 40,000 natural compounds are known, only up to 500 substances have been discovered so far in EDF. The preliminary work on which the research team led by Markus Greßler of the Pharmaceutical Microbiology Research Group is based dates back a good 50 years. One reason for the stepmotherly treatment of these fungi is the assumption, still valid today, that they do not possess their own secondary metabolism that produces interesting substances.

A neglect unjustly as it seems. While studying Linderina pennispora, a fungus from the Zoopagomycota division, the researchers discovered that it independently produces certain indole alkaloids. "We named the substance Lindolin, combining the name of the fungus and the basic structure of the active ingredient," Greßler explains. The independent production is significant because it is often not the EDF themselves that produce a natural substance, but bacterial endosymbionts living within them.

Fungi as plant protectors

Lindolin acts selectively against oomycetes - fungus-like algae that can cause dreaded plant diseases. One of the most popular cases was the famine in Ireland in the mid-19th century. As a result, about one million people died and two million left the country looking for a better life in America. The reason for the misery was potato blight, caused by the oomycete Phytophthora infestans.

Phytophthora species are fruit rot pathogens that, in addition to potatoes, primarily attack tomatoes, but also soybeans and stone fruits. "What is exciting is that Linderina pennispora produces Lindolin not just in vitro in the laboratory," Greßler explains, "In fact, Lindolin is actively released into the soil by the fungi and, as cultivation in garden soil shows, could protect plants from infestation by oomycetes." The potential of the compound for biological plant protection will be further investigated via a collaboration with an industrial partner.

Lindolines are not only formed by this one fungal species, but also by other representatives of the order Kickxellales, as a collaboration with the Jena Microbial Resource Collection (JMRC) showed. Further derivatives of Lindolin are now to be produced and analyzed.

New biosynthesis

The researchers investigated the biosynthesis of the natural compound and came to a surprising conclusion. Usually, peptide synthesis in fungi takes place via megaenzymes, so-called non-ribosomal peptide synthetases. But the synthesis of Lindolin is more similar to a plant pattern with two small enzymes working in sequence instead of one megaenzyme.

The gene analysis of Linderina pennispora revealed another surprise. The genes for the synthesis enzymes are not located in clusters next to each other on one DNA strand, as is common in higher fungi and bacteria, but are scattered throughout the genome. The researchers thus refuted the so-called gene cluster hypothesis. Based on this, EDF were often excluded as producers of natural products in previous genome analyses because no clusters for synthesis enzymes were found. Thus, EDF may be able to produce many more natural products than previously thought. Identifying them, exploring their role in nature and deriving possible applications from them is what Greßler with his team sees as a great potential for the future and at the same time the motivation for his research work.

Original publication

Rassbach J, Hilsberg N, Haensch VG, Dörner S, Gressler J, Sonnabend R, Semm C, Voigt K, Hertweck C, Gressler M. (2023) Non-canonical two-step biosynthesis of anti-oomycete indole alkaloids in Kickxellales. Fungal Biol Biotechnol, doi: 10.1186/s40694-023-00166-x.


Sebastian Dörner
Julia Greßler
Markus Greßler
Veit Hänsch
Christian Hertweck
Johannes Raßbach
Caroline Semm
Robin Sonnabend
Kerstin Voigt