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Forests provide invaluable economic, ecological, and social services. At the same time, they are exposed to several threats, such as fragmentation, changing climatic conditions, or increasingly destructive pests and pathogens. Trees, the inherent species of forests, cannot be viewed as isolated organisms. Manifold (micro)organisms are associated with trees playing a pivotal role in forest ecosystems. Of these organisms, fungi may have the greatest impact on the life of trees. A multitude of molecular and genetic methods are now available to investigate tree species and their associated organisms. Due to their smaller genome sizes compared to tree species, whole genomes of different fungi are routinely compared. Such studies have only recently started in forest tree species. Here, we summarize the application of molecular and genetic methods in forest conservation genetics, tree breeding, and association genetics as well as for the investigation of fungal communities and their interrelated ecological functions. These techniques provide valuable insights into the molecular basis of adaptive traits, the impacts of forest management, and changing environmental conditions on tree species and fungal communities and can enhance tree-breeding cycles due to reduced time for field testing. It becomes clear that there are multifaceted interactions among microbial species as well as between these organisms and trees. We demonstrate the versatility of the different approaches based on case studies on trees and fungi. KEY POINTS: • Current knowledge of genetic methods applied to forest trees and associated fungi. • Genomic methods are essential in conservation, breeding, management, and research. • Important role of phytobiomes for trees and their ecosystems., (© 2023. The Author(s).)

To realize biomass refinery without complex downstream processes, we extensively screened for microbial strains that efficiently produce extracellular oil from sugars. Rhodotorula paludigena (formerly Rhodosporidium paludigenum) BS15 was found to efficiently produce polyol esters of fatty acids (PEFAs), which mainly comprised of 3-acetoxypalmitic acid and partially acetylated mannitol/arabinitol. To evaluate the performance of this strain, fed-batch fermentation was demonstrated on a flask scale, and 110 g/L PEFA and 103 g/L dry cells were produced in 12 days. To the best of our knowledge, the strain BS15 exhibited the highest PEFA titer (g/L) ever to be reported so far. Because the PEFA precipitated at the bottom of the culture broth, it could be easily recovered by simply discarding the upper phase. Various carbon sources can be utilized for cell growth and/or PEFA production, which signifies the potential for converting diverse biomass sources. Two different types of next-generation sequencers, Illumina HiSeq and Oxford Nanopore PromethION, were used to analyze the whole-genome sequence of the strain BS15. The integrative data analysis generated a high-quality and reliable reference genome for PEFA-producing R. paludigena. The 22.5-M base genome sequence and the estimated genes were registered in Genbank (accession numbers BQKY01000001-BQKY01000019). KEY POINTS: • R. paludigena BS15 was isolated after an extensive screening of extracellular oil producers from natural sources. • Fed-batch fermentation of R. paludigena BS15 yielded 110 g/L of PEFA, which is the highest titer ever reported to date. • Combined analysis using Illumina and Oxford Nanopore sequencers produced the near-complete genome sequence., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Efficient transporters are necessary for high concentration and purity of desired products during industrial production. In this study, we explored the mechanism of substrate transport and preference of the C4-dicarboxylic acid transporter AoMAE in the fungus Myceliophthora thermophila, and investigated the roles of 18 critical amino acid residues within this process. Among them, the residue Arg78, forming a hydrogen bond network with Arg23, Phe25, Thr74, Leu81, His82, and Glu94 to stabilize the protein conformation, is irreplaceable for the export function of AoMAE. Furthermore, varying the residue at position 100 resulted in changes to the size and shape of the substrate binding pocket, leading to alterations in transport efficiencies of both malic acid and succinic acid. We found that the mutation T100S increased malate production by 68%. Using these insights, we successfully generated an AoMAE variant with mutation T100S and deubiquitination, exhibiting an 81% increase in the selective export activity of malic acid. Simply introducing this version of AoMAE into M. thermophila wild-type strain increased production of malic acid from 1.22 to 54.88 g/L. These findings increase our understanding of the structure-function relationships of organic acid transporters and may accelerate the process of engineering dicarboxylic acid transporters with high efficiency. KEY POINTS: • This is the first systematical analysis of key residues of a malate transporter in fungi. • Protein engineering of AoMAE led to 81% increase of malate export activity. • Arg78 was essential for the normal function of AoMAE in M. thermophila. • Substitution of Thr100 affected export efficiency and substrate selectivity of AoMAE., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels. KEY POINTS: • L. starkeyi metabolism reprograms for consumption of different plant-based sugars. • Non-specific regulation was observed during growth on cellobiose. • L. starkeyi secretes β-glucosidases for extracellular hydrolysis of cellobiose., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Endophytic strains belonging to the Bacillus cereus group were isolated from the halophytes Atriplex halimus L. (Amaranthaceae) and Tamarix aphylla L. (Tamaricaceae) from costal and continental regions in Algeria. Based on their salt tolerance (up to 5%), the strains were tested for their ability to alleviate salt stress in tomato and wheat. Bacillus sp. strain BH32 showed the highest potential to reduce salinity stress (up to + 50% and + 58% of dry weight improvement, in tomato and wheat, respectively, compared to the control). To determine putative mechanisms involved in salt tolerance and plant growth promotion, the whole genome of Bacillus sp. BH32 was sequenced, annotated, and used for comparative genomics against the genomes of closely related strains. The pangenome of Bacillus sp. BH32 and its closest relative was further analyzed. The phylogenomic analyses confirmed its taxonomic position, a member of the Bacillus cereus group, with intergenomic distances (GBDP analysis) pinpointing to a new taxon (digital DNA-DNA hybridization, dDDH < 70%). Genome mining unveiled several genes involved in stress tolerance, production of anti-oxidants and genes involved in plant growth promotion as well as in the production of secondary metabolites. KEY POINTS : • Bacillus sp. BH32 and other bacterial endophytes were isolated from halophytes, to be tested on tomato and wheat and to limit salt stress adverse effects. • The strain with the highest potential was then studied at the genomic level to highlight numerous genes linked to plant growth promotion and stress tolerance. • Pangenome approaches suggest that the strain belongs to a new taxon within the Bacillus cereus group., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A sequential fungal pretreatment of Miscanthus × giganteus was conducted by mixing unsterilized Miscanthus with material previously colonized with the white-rot fungus Ceriporiopsis subvermispora. For three generations, each generation started with inoculation by mixing unsterilized fresh Miscanthus with end material from the previous generation and ended after 28 days of incubation at 28 °C. After the first generation, the cellulose digestibility of the material doubled, compared to that of the unsterilized Miscanthus, but the second and third generations showed no enhancements in cellulose digestibility. Furthermore, high degradation of Miscanthus structural carbohydrates occurred during the first generation. A microbial community study showed that, even though the fungal community of the material previously colonized by C. subvermispora was composed mainly of this fungus (> 99%), by the first generation its relative abundance was down to only 9%, and other microbes had prevailed. Additionally, changes in the bacterial community occurred that might be associated with unwanted cellulose degradation in the system. This reiterates the necessity of feedstock microbial load reduction for the stability and reproducibility of fungal pretreatment of lignocellulosic biomass. KEY POINTS: • Sequential fungal pretreatment of unsterilized Miscanthus was unsuccessful. • Feedstock changes with white-rot fungi favored the growth of other microorganisms. • Feedstock microbial reduction is necessary for pretreatment with C. subvermispora., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mushrooms are high-value products that can be produced from lignocellulosic biomass. Mushrooms are the fruiting body of fungi and are domestically cultivated using lignocellulosic biomass obtained from agricultural byproducts and woody biomass. A handful of edible mushroom species are commercially cultivated at small, medium, and large scales for culinary and medicinal use. Details about different lignocellulosic biomass and their composition that are commonly used to produce mushrooms are outlined in this review. In addition, discussions on four major processing steps (i) producing solid and liquid spawn, (ii) conventional and mechanized processing lignocellulosic biomass substrates to produce mushroom beds, (iii) maintaining growth conditions in climate-controlled rooms, and (iv) energy requirements and managements to produce mushrooms are also provided. The new processing methods and technology outlined in this review may allow mushrooms to be economically and sustainably produced at a small scale to satisfy the growing food needs and create rural jobs. KEY POINTS: • Some of the challenges faced by small-scale mushroom growers are presented. This review is expected to stimulate more research to address the challenges., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rhodosporidium toruloides is an oleaginous yeast capable of producing a variety of biofuels and bioproducts from diverse carbon sources. Despite numerous studies showing its promise as a platform microorganism, little is known about its metabolism and physiology. In this work, we investigated the central carbon metabolism in R. toruloides IFO0880 using transcriptomics and metabolomics during growth on glucose, xylose, acetate, or soybean oil. These substrates were chosen because they can be derived from plants. Significant changes in gene expression and metabolite concentrations were observed during growth on these four substrates. We mapped these changes onto the governing metabolic pathways to better understand how R. toruloides reprograms its metabolism to enable growth on these substrates. One notable finding concerns xylose metabolism, where poor expression of xylulokinase induces a bypass leading to arabitol production. Collectively, these results further our understanding of central carbon metabolism in R. toruloides during growth on different substrates. They may also help guide the metabolic engineering and development of better models of metabolism for R. toruloides.Key points• Gene expression and metabolite concentrations were significantly changed.• Reduced expression of xylulokinase induces a bypass leading to arabitol production.• R. toruloides reprograms its metabolism to allow growth on different substrates., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that Aspergillus homomorphus turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (1) and B (2) by HRMS and NMR. Combined investigations including retro-biosynthesis, genome mining, and gene deletions successfully linked both compounds to their related biosynthetic gene clusters. This demonstrated that homopyrones are biosynthesized by using cinnamoyl-CoA as the starter unit, followed by extension with three malonyl-CoA units, and lactonization to give the core hybrid backbone structure. The polyketide synthase AhpA includes a C-methylation domain, which however seems to be promiscuous since only 2 is C-methylated. Altogether, the homopyrones represent a rare case of hybrid phenylpropanoid- and polyketide-derived natural products in filamentous fungi. KEY POINTS: • Homopyrones represent a rare type of fungal polyketides synthesized from cinnamic-CoA. • CRISPR/Cas9 technology has been firstly applied in Aspergillus homomorphus.

Aryl-alcohol oxidases (AAOs) are FAD-containing enzymes that oxidize a broad range of aromatic as well as aliphatic allylic alcohols to aldehydes. Their broad substrate spectrum accompanied by the only need for molecular oxygen as cosubstrate and production of hydrogen peroxide as sole by-product makes these enzymes very promising biocatalysts. AAOs were used in the synthesis of flavors, fragrances, and other high-value-added compounds and building blocks as well as in dye decolorization and pulp biobleaching. Furthermore, AAOs offer a huge potential as efficient suppliers of hydrogen peroxide for peroxidase- and peroxygenase-catalyzed reactions. A prerequisite for application as biocatalysts at larger scale is the production of AAOs in sufficient amounts. Heterologous expression of these predominantly fungal enzymes is, however, quite challenging. This review summarizes different approaches aiming at enhancing heterologous expression of AAOs and gives an update on substrates accepted by these promising enzymes as well as potential fields of their application. KEY POINTS: • Aryl-alcohol oxidases (AAOs) supply ligninolytic peroxidases with H 2 O 2 . • AAOs accept a broad spectrum of aromatic and aliphatic allylic alcohols. • AAOs are potential biocatalysts for the production of high-value-added bio-based chemicals.

Aspergillus species are closely associated with humanity through fermentation, infectious disease, and mycotoxin contamination of food. Members of this genus produce various enzymes to degrade plant polysaccharides, including starch, cellulose, xylan, and xyloglucan. This review focus on the machinery of the xyloglucan degradation using glycoside hydrolases, such as xyloglucanases, isoprimeverose-producing oligoxyloglucan hydrolases, and α-xylosidases, in Aspergillus species. Some xyloglucan degradation-related glycoside hydrolases are well conserved in this genus; however, other enzymes are not. Cooperative actions of these glycoside hydrolases are crucial for xyloglucan degradation in Aspergillus species. KEY POINTS: •Xyloglucan degradation-related enzymes of Aspergillus species are reviewed. •Each Aspergillus species possesses a different set of glycoside hydrolases. •The machinery of xyloglucan degradation of A. oryzae is overviewed.

Distinct wood degraders occupying their preferred habitats have biased enzyme repertoires that are well fitted to their colonized substrates. Pleurotus ostreatus, commonly found on wood, has evolved its own enzyme-producing traits. In our previous study, transcriptional shifts in several P. ostreatus delignification-defective mutants, including Δhir1 and Δgat1 strains, were analyzed, which revealed the downregulation of ligninolytic genes and the upregulation of cellulolytic and xylanolytic genes when compared to their parental strain 20b on beech wood sawdust medium (BWS). In this study, rice straw (RS) was used as an alternative substrate to examine the transcriptional responses of P. ostreatus to distinct substrates. The vp1 gene and a cupredoxin-encoding gene were significantly upregulated in the 20b strain on RS compared with that on BWS, reflecting their distinct regulation patterns. The overall expression level of genes encoding glucuronidases was also higher on RS than on BWS, showing a good correlation with the substrate composition. Transcriptional alterations in the mutants (Δhir1 or Δgat1 versus 20b strain) on RS were similar to those on BWS, and the extracellular lignocellulose-degrading enzyme activities and lignin-degrading ability of the mutants on RS were consistent with the transcriptional alterations of the corresponding enzyme-encoding genes. However, transcripts of specific genes encoding enzymes belonging to the same CAZyme family exhibited distinct alteration patterns in the mutant strains grown on RS compared to those grown on BWS. These findings provide new insights into the molecular mechanisms underlying the transcriptional regulation of lignocellulolytic genes in P. ostreatus.Key Points• P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates.• A demand to upregulate the cellulolytic genes seems to be present in ligninolysis-deficient mutants.• The regulation of some specific genes probably driven by the demand is dependent on the substrate.

Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl 2 . Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. KEY POINTS: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization.

Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central β-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the β-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.

Cellulose-degrading auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are known to be widely distributed among filamentous fungi and participate in the degradation of lignocellulose via the oxidative cleavage of celluloses, cello-oligosaccharides, or hemicelluloses. AA9 LPMOs have been reported to have extensive interactions with not only cellulases but also oxidases. The addition of AA9 LPMOs can greatly reduce the amount of cellulase needed for saccharification and increase the yield of glucose. The discovery of AA9 LPMOs has greatly changed our understanding of how fungi degrade cellulose. In this review, apart from summarizing the recent discoveries related to their catalytic reaction, functional diversity, and practical applications, the stability, expression system, and protein engineering of AA9 LPMOs are reviewed for the first time. This review may provide a reference value to further broaden the substrate range of AA9 LPMOs, expand the scope of their practical applications, and realize their customization for industrial utilization.Key Points• The stability and expression system of AA9 LPMOs are reviewed for the first time.• The protein engineering of AA9 LPMOs is systematically summarized for the first time.• The latest research results on the catalytic mechanism of AA9 LPMOs are summarized.• The application of AA9 LPMOs and their relationship with other enzymes are reviewed.