The White-Rot Basidiomycete Dichomitus squalens Shows Highly Specific Transcriptional Response to Lignocellulose-Related Aromatic Compounds

Abstract

Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. Dichomitus squalens is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when D. squalens was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of D. squalens when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of D. squalens is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.

Effect of Lactic Acid Bacteria Strains on the Growth and Aflatoxin Production Potential of Aspergillus parasiticus, and Their Ability to Bind Aflatoxin B1, Ochratoxin A, and Zearalenone in vitro

“…This variability is strain-specific and occurs even when the strain is subjected to similar experimental conditions such as performed here ( Whiting and Golden, 2002 ; Freire et al, 2018 ). Also in agreement with our results, several reports indicate that, once the mycotoxins have reached a maximum production, a decline of its levels occurs, possibly because of the ability of the fungus to degrade or convert the mycotoxins into other metabolites ( Freire et al, 2018 ; Kowalczyk et al, 2019 ). Furthermore, Greeff-Laubscher et al (2020) showed that the level of mycotoxin production by a fungus can vary largely within the replicates of a trial as well as length of time of incubation of the fungus, with episodes of levels increasing and decreasing over the whole period.…”

First Report of Six Macrofungi from Daecheongdo and Socheongdo Islands, Korea

“…Crepidotus praecipuus is recorded as an endemic species of New Zealand, and this is the first time it has been recorded outside of New Zealand (https:// inaturalist.nz). Dichomitus squalens is a white-rot basidiomycete that produces diverse extracellular enzymes for lignocellulose degradation and oxidative enzymes to act on lignin [23][24][25]. The extracts from cultivated Trametes lactinea significantly inhibit the activities of lipoxygenase and hyaluronidase [26].…”

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

“…Due to their broad substrate specificity, laccases also are attractive biocatalysts for a wide range of biotechnology applications. The previous studies have shown that lcc3 is highly expressed by D. squalens in various cultivation conditions [32,34,54], which made it an interesting target to study its role in vivo in more detail. The protoplast mixture was supplied with 7 and 10 µg of ssODNs for the repair of cuts introduced in the sdi1 and lcc3 loci, respectively, while the total amount of Cas9 and gRNA used to assemble RNPs remained unchanged.…”

“…Manganese peroxidases (MnPs) are key enzymes for lignin degradation and uniquely produced by white-rot fungi [31]. As the D. squalens MnP2 encoding gene has been reported to be highly expressed in several wood and other plant biomass containing cultures [32,34,54,55], we were interested to target it to obtain a strain that possibly has an altered ability for lignocellulose conversion. In this case, one out of 10 carboxin-resistant transformants showed the expected EcoRI restriction pattern.…”

“…In this study, we assessed different methods for CRISPR/Cas9-based gene editing in the wild-type Dichomitus squalens strain. D. squalens is a transformable, wood-degrading fungus and a highly suitable reference species to investigate molecular mechanisms behind plant biomass degradation in white-rot basidiomycetes [32][33][34]. Here, we adapted a co-targeting strategy based on preassembled Cas9-sgRNA RNPs for selectable homology and NHEJ-based gene editing in D. squalens.…”