Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus

fmicb-14-1108975 March 28, 2023 Time: 16:58 # 1 TYPE Original Research PUBLISHED 03 April 2023 DOI 10.3389/fmicb.2023.1108975 OPEN ACCESS EDITED BY Yijuan Xu, South China Agricultural University, China REVIEWED BY Ari Mikko Hietala, Norwegian Institute of Bioeconomy Research (NIBIO), Norway Eliane Ferreira Noronha, University of Brasília, Brazil *CORRESPONDENCE Miroslav Kolaˇ rík [email protected] SPECIALTY SECTION This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology RECEIVED 28 November 2022 ACCEPTED 13 March 2023 PUBLISHED 03 April 2023 CITATION Cheng T, Veselská T, Kˇ rížková B, Švec K, Havlíˇ cek V, Stadler M and Kolaˇ rík M (2023) Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus. Front. Microbiol. 14:1108975. doi: 10.3389/fmicb.2023.1108975 COPYRIGHT © 2023 Cheng, Veselská, Kˇ rížková, Švec, Havlíˇ cek, Stadler and Kolaˇ rík. 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Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus Tian Cheng1,2, Tereza Veselská1, Barbora Kˇ rížková1, Karel Švec1, Václav Havlíˇ cek1, Marc Stadler2and Miroslav Kolaˇ rík1* 1Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology, Czech Academy of Sciences, Praha, Czechia, 2Department of Microbial Drugs, Helmholtz Centre for Infection Research, Braunschweig, Germany Spruce bark beetle Ips typographus can trigger outbreaks on spruce that results in significant losses in the forest industry. It has been suggested that symbiotic microorganisms inhabiting the gut of bark beetles facilitate the colonization of plant tissues as they play a role in the detoxification of plant secondary metabolites, degrade plant cell wall and ameliorate beetle’s nutrition. In this study, we sequenced and functionally annotated the genomes of five yeasts Kuraishia molischiana,Cryptococcus sp., Nakazawaea ambrosiae,Ogataea ramenticola, and Wickerhamomyces bisporus isolated from the gut of Ips typographus. Genome analysis identified 5314, 7050, 5722, 5502, and 5784 protein coding genes from K. molischiana,Cryptococcus sp., N. ambrosiae, O. ramenticola, and W. bisporus, respectively. Protein-coding sequences were classified into biological processes, cellular and molecular function based on gene ontology terms enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was used to predict gene functions. All analyzed yeast genomes contain full pathways for the synthesis of essential amino acids and vitamin B6, which have nutritional importance to beetle. Furthermore, their genomes contain diverse gene families related to the detoxification processes. The prevalent superfamilies are aldo-keto reductase, ATP-binding cassette and the major facilitator transporters. The phylogenetic relationships of detoxification-related enzymes aldo-keto reductase, and cytochrome P450 monooxygenase, and ATP-binding cassette are presented. Genome annotations also revealed presence of genes active in lignocellulose degradation. In vitro analyses did not confirm enzymatic endolytic degradation of lignocellulose; however, all species can utilize and pectin and produce a large spectrum of exolytic enzymes attacking cellulose, chitin, and lipids. KEYWORDS Scolytinae, gut microbiome, symbiosis, spruce, plant cell wall, detoxification, yeast, nutrition Frontiers in Microbiology 01 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 2 Cheng et al. 10.3389/fmicb.2023.1108975 1. Introduction The European spruce bark beetle, or Eight-toothed spruce bark beetle, Ips typographus (Coleoptera: Scolytinae), infecting Norway Spruce (Picea abies) is one of the most destructive forest pests in Europe (Biedermann et al.,2019). Bark beetles are known to vector microorganisms that assist them during plant phloem colonization (Six,2013;Hofstetter et al.,2015). They are sources of nutrients, i.e., sterols, vitamins, essential amino acids, and other nitrogenous compounds (Ayres et al.,2000;Bentz and Six,2006;Veselská et al., 2019;Ibarra-Juarez et al.,2020), facilitate plant tissue colonization by detoxification of tree defense compounds (e.g., Zhao et al.,2019) or necrotize healthy plant tissue by which they also can cause tree mortality (Hofstetter et al.,2015;Li et al.,2022). It is assumed that microorganisms introduced by beetles under the bark of a tree have a greater effect on the wood modifications than beetle themselves (Hýsek et al.,2021). Bark beetles’ habitat is a hot spot of microbial diversity and this is also true for I. typographus. Despite long-term investigation of its fungal assemblage, novel fungal associates of this beetle are continuously being discovered (Linnakoski et al., 2016a;Jankowiak et al.,2017). Recent studies reported spatial and temporal differences in fungal assemblages (Linnakoski et al., 2016b;Netherer et al.,2021) and showed that volatile compounds influence the beetle-fungus interactions (Kandasamy et al.,2019). Traditionally the most studied are ectosymbiotic filamentous fungi as they massively grow around and inside beetle’s galleries, often causing coloration of surrounding plant tissues or having plant pathogenic potential. Endoconidiophora polonica and Ophiostoma bicolor, together with Grosmannia penicillata and G. europhioides, are well-known filamentous fungal symbionts of I. typographus, having ability to detoxify host tree defense system (Zhao et al.,2019; Netherer et al.,2021). Although yeasts have been recognized as constant components in the guts of bark beetles (Rivera et al.,2009; Davis,2015) including I. typographus (Chakraborty et al.,2020; Veselská et al.,2023), they were mostly overlooked in majority of the previous studies, mainly because of the difficulties in their taxonomic identification and absence of phytopathogenicity. Recent studies (Barcoto et al.,2020;Ibarra-Juarez et al.,2020) emphasize the importance of whole microbial community, called holobiont (Six,2013), including endosymbiotic gut inhabiting yeasts and bacteria. It was shown that gut of bark beetles provides an environment for microbial inhabitation (Engel and Moran, 2013), where plant allelochemical digestion, detoxification, and nutritional exchange take place (Linser and Dinglasan,2014; Stefanini,2018). Thus, gut-associated fungi can facilitate those important intestinal processes (Itoh et al.,2018). Despite the functional importance of gut inhabiting fungi, factors that maintain these complex interactions are still poorly understood (Biedermann and Vega,2020) and there are very few studies that have comprehensively investigated bark beetles’ microbial intestinal community, particularly in terms of genomics. These studies show that yeasts together with bacteria belong to the first organisms that inhabit bark beetle galleries and synergically prepare galleries for growth of filamentous fungi (Barcoto et al.,2020;Ibarra-Juarez et al.,2020). In the prior study (Veselská et al.,2023), we described I. typographus gut core microbiome based on the metatranscriptomic and DNA metabarcode analysis. We found that it is largely dominated by ascomycetous yeasts (Saccharomycetales), especially by Kuraishia molischiana, which took 25.6% of total fungal reads, Nakazawaea ambrosiae, 20.7% of the reads, Wickerhamomyces bisporus, 16.8% of the reads and Ogataea ramenticola, 2% of the reads. One unidentified species of the genus Cryptococcus (Basidiomycota: Tremellomycetes) with 0.1% of the reads, was selected as representative of basidiomycetous yeasts, which represent another important component of intestinal yeast diversity. On NCBI database, assembled genomes of K. molischiana (PPKW02; 10.36 Mb, 50.7% GC) and O. ramenticola (PPKK02; 12.99 Mb, 32.3% GC) are available; however, they are not annotated. Genomes of the other above-mentioned yeast species have not been sequenced yet. In the present study, we attempt to study dominant gutassociated yeasts with an insight in their genomes. Yeast species remain an untapped source of carbohydrate-active enzymes (CAZymes) since their contribution to biomass degradation has been largely overlooked (Sun and Cheng,2002;Zhao et al.,2014; Despres et al.,2016). In the present study, we focus on the analysis of yeasts’ CAZymes targeting plant cell wall, which is comprised mainly by pectin, cellulose, hemicellulose, other polysaccharides, lignin, lipids and proteins (Glass et al.,2013). We also searched for genes related to detoxification processes as the detoxification of plant secondary metabolites is a complex process for the bark beetles and their associated symbionts. It is performed in different physiological phases involving a variety of genes and proteins. At present, it is known that in microorganisms, enzymes such as cytochrome P450 (CYP) monooxygenases, glutathione-Stransferases (GST), multicopper oxidases, carboxylesterases, flavincontaining monooxygenases (FMO), and aldo-keto reductases (AKR) participate in xenobiotics detoxifications (Sheehan et al., 2001;Barski et al.,2008;Sehlmeyer et al.,2010;Lah et al.,2013; Ramya et al.,2016). ATP-binding cassette (ABC) transporters, major facilitator superfamily (MFS), and multidrug and toxic compound extrusion (MATE) transporters are involved in drug and stress resistance (Lah et al.,2013;Eisinger et al.,2018). Finally, we were looking for pathways related to synthesis of nutrients essential for beetle development. Such information fills the existing knowledge gaps and provides a scientific basis for future studies in the functional analysis of gut associated fungi. 2. Materials and methods 2.1. Collection, isolation, and identification of yeast strains Strains were isolated from the gut of I. typographus larvae collected within a study of its intestinal microbial diversity (Veselská et al.,2023). In brief, larvae were collected during September and October 2020 in the surroundings of forest area in Nižbor (Czechia, 49◦59009.900N 13◦56047.500E, 390 m.a.s.l.). Larvae were taken out of the galleries and surface sterilized by subsequent washing with 70% ethanol, 2% Tween 80 (Avantor, USA) and sterile distilled water. The larvae were dissected straight after collection, their guts were homogenized and plated in various dilutions (diluted with saline) on Petri dishes with YES (5 g of Frontiers in Microbiology 02 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 3 Cheng et al. 10.3389/fmicb.2023.1108975 yeast extract, 30 g of glucose, 20 g agar, 1 l of distilled water) and antibiotics (streptomycine and chloramphenicol, 60 mg/l). Cultivation took place for 5–7 days at 24◦C in dark. DNA was extracted by Nucleo Spin kit (Machery Nagel), the ITS sequence was used for determination based on the BlastN similarity search in NCBI Genbank database. Cultures were deposited in Culture Collection of Fungi (CCF, Department of Botany, Faculty of Sciences, Charles University, Prague) as Cryptococcus sp. CCF 6641, Kuraishia molischiana CCF 6642, Nakazawaea ambrosiae CCF 6643, Ogataea ramenticola CCF 6644, and Wickerhamomyces bisporus CCF 6645. 2.2. Genome sequencing, assembly, and evaluation Genomic DNA (gDNA) of yeasts was obtained with Nucleo Spin kit (Machery Nagel) kit. DNA yield was quantified on Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using Qubit dsDNA BR Assay Kit and DNA quality was checked on Nanodrop (NanoDropTM 2000 c, ThermoFisher scientific). gDNA samples were sent to Macrogen NGS sequencing service center (Amsterdam, Netherlands) for library preparation and sequencing. A TruSeq DNA PCR free (350) library was prepared and genomes were sequenced on Illumina NextSeq 500 system with 2 ×151 nt PE, resulting in a total of 38.96 Gb and roughly 6.49 Gb read bases per sample. The quality of raw data was analyzed with FASTQC v 0.11.9 (Andrews,2010). Sequencing adapters and lowquality reads were trimmed with Trimmomatic v 0.39 (Bolger et al., 2014), k-mer = 5 was used to remove leading and trailing bases with Phred scores lower than 33, as well as reads shorter than 150 bp. De novo assembly was performed using SPAdes v 3.13.1 (Bankevich et al.,2012) in Python v 3.8.10 environment with – careful setting, k-mer = 121 was selected. The completeness of assemblies was evaluated with Benchmarking Universal SingleCopy Orthologs [BUSCO v5.2.1 (Seppey et al.,2019)] to search against the saccharomycetes_odb10 database for Kuraishia molischiana,Nakazawaea ambrosiae,Ogataea ramenticola, and Wickerhamomyces bisporus. The assembly of Cryptococcus sp. was searched against the tremellomycetes_odb10 database. The assessment of those assemblies was performed with Quast v 5.0.2 (Gurevich et al.,2013). 2.3. Gene prediction and genome annotation Gene prediction was performed using AUGUSTUS v 3.1.0 (Stanke et al.,2006) with Saccharomyces cerevisiae S288C as reference species. Gene Ontology (GO) annotation was carried out in eggnog-mapper v 2 (Cantalapiedra et al.,2021) with an e-value of 10−3. The GO terms were distributed by R package GO.db (Carlson et al.,2019) and plotted by ggplot (Wickham,2006). Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was carried out with BlastKOALA (Kanehisa et al.,2016). KEGG pathways were performed with KEGG mapper (Kanehisa and Sato, 2020). 2.4. Identification of carbohydrate-related proteins Carbohydrate active enzymes (CAZymes) were identified in the predicted proteins of each genome with hmmsearch in HMMER 3.3.2 (Mistry et al.,2013) against the CAZy database dbCAN2 (Zhang et al.,2018)1version 8. The e-value was set as 1e–5. 2.5. Annotation and phylogenetic analysis of detoxification-related enzymes The predicted enzymes associated with the detoxification process were identified with HMMER E-value 1e–5 (Mistry et al.,2013) against specific domain from Pfam database (Mistry et al.,2021): cytochrome P450 monooxygenase (PF00067), aldo-keto reductases (PF00248), carboxylesterase (PF00135), flavin-containing monooxygenase (PF00743), glutathione S-transferase (PF13409, PF14497, PF02798, PF13417, PF00043, and PF13410), multicopper-containing oxidases (PF07732, PF00394, and PF07731), ATP-binding cassette (ABC) transporter (PF00664, PF00005, PF06472, and PF00004), multidrug and toxic compound extrusion (MATE) (PF01554) and major Facilitator Superfamily (MFS) (PF07690, PF16983, PF05631, PF00083, PF12832, PF06779, PF13347, and PF05977). The results were verified and filtered with BlastP (e-value 1e-5) using SEG, a word-size six letters and BLOSUM62 matrix. Multiple sequence alignment was carried out with MAFFT v 7.453 (Rozewicki et al.,2019) using maxiterate 1000. The phylogenetic trees were performed with PhyML v 3.0 (Guindon et al.,2010). The best evolutionary model for protein sequences was calculated with Akaike method and likelihood-based method was selected as aLRT SH-like and bootstrap analysis repeated for 100 times. According to the BlastP results and phylogenetic analysis, enzymes of aldo-keto reductases, cytochrome P450, and ABC transporter family were classified into specific groups. Phylogenetic trees were visualized and edited with iTOL (Letunic and Bork, 2021). 2.6. Prediction of localization of detoxification-related enzymes The subcellular localization of detoxification related enzymes was predicted with Protcomp-AN v 6.0 (Klee and Ellis,2005), molecular mass isoelectric point (pI) was performed with Protein isoelectric point calculator (Kozlowski,2016). 2.7. Extracellular enzyme activities As our functional analysis predicts presence of pathways for the degradation of plant cell wall components, in the next step we 1https://bcb.unl.edu/dbCAN2/download/Databases Frontiers in Microbiology 03 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 4 Cheng et al. 10.3389/fmicb.2023.1108975 verified them by in vitro degradation tests. The analyzed enzymes are listed in Table 1. Activity of endolytic enzymes was assessed using various chromogenic substrates (Megazyme, Bray, Ireland), whereas activity of exolytic enzymes was assessed using fluorogenic substrates (Sigma-Aldrich). For endolytic enzyme analyses, yeasts were cultivated in submerged media in 250 ml Erlenmeyer flasks on rotary shaker (200 rpm) at 24◦C. Basic medium (BM) consisted of 1 g pepton, 1 g yeast extract, 1 g (NH4)2HPO4, 0.5 g K2PO4, 0.5 g MgSO4.7H2O and substrates specific to the enzyme tested [3 g of cellulose, 6 g xylan birchwood, 15 g galactomannan (Locust beam gum), 6 g of starch, 1.5 g of amylopectin and 13.5 g of dextran] in 1 l of McIlvaine buffer, pH 4.5. The cultivation time was determined using the glass tube test. Glass tubes containing water agar (20 g agar in 1 L distilled water) were overlaid with a thin layer of a specific chromogenic substrate dissolved in water agar and the agar surface was inoculated with the tested fungi and cultured at 24◦C. Enzymatic degradation was manifested by diffusion of the chromogenic substrate into the water agar. At this point, the submerged cultivation in the Erlenmeyer flask was stopped. If no staining of the water agar was evident after 14 days of cultivation in the glass tubes, submerged cultivation was also stopped and enzyme activities were evaluated. Enzymes were extracted into extraction buffers as it is recommended by manufacturer for 2 h at 4◦C. After extraction, the mixture was centrifuged for 10 min at 3,000 rpm to exclude the medium and fungal cells. The supernatant containing extracellular enzymes was further filtered through filter paper to remove remaining solid parts. We used manufacturer’s protocols for measurement and calculation of enzymatic activities. Trichoderma viride CCF 4516 was used as a positive control for enzymatic degradation of cellulose, xylan and casein as it is known to degrade these substrates (Gomes et al.,1992;Kredics et al.,2005). Sterile media without inoculated fungi served as blanks. To measure exolytic activity, yeast strains were also cultured in submerged medium in 250 ml Erlenmeyer flasks on a rotary shaker (200 rpm) at 24◦C. The medium consisted of yeast extract (5 g/l), peptone (2 g/l), glucose (2 g/l), casein (2 g/l), cellulose (3 g/l), lignin (3 g/l), starch (3 g/l), lime phloem (63 g/l). The yeast grew for 14 days. Sodium acetate, pH 4.5, was used for enzyme extraction. The fluorescence of the released reaction products was measured as previously described (Baldrian,2009) using a method adapted from Vepsäläainen et al. (2001). The fluorescence value of each MUF fluorogenic substrate was corrected by subtracting the background fluorescence of the growth medium. Laccase activity (EC 1.10.3.2) was measured by monitoring the oxidation of ABTS [2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (Bourbonnais and Paice,1990) in citrate-phosphate buffer (100 mM citrate, 200 mM phosphate, pH 5.0)]. The formation of the resulting green dye was evaluated spectrophotometrically at 420 nm. 3. Results 3.1. Genome sequencing and assembly General genome features, e.g., genome size, scaffolds number, N50, L50, number of predicted genes, GC content, completeness of assembly and NCBI accession numbers are summarized in Table 2. The number of reads generated by Illumina sequencing ranged from 39,806,602 to 47,849,884 reads with an average count of 43,006,421. Established genomes range in size from 9.86 to 18.3 Mb. Genomes assembled with Spades resulted in 315–799 scaffolds and were predicted to contain 5,314–7,050 genes, and GC contents from 34.5 to 59.3%. The N50 of five genomes range from 197,085 to 1,183,430, while L50 align from 5 to 26. BUSCO assessment results showed that these assembles are in high quality with around 96% of genes were covered for all five species. 3.2. GO enrichment and KEGG pathway annotation Gene Ontology enrichment analysis indicated that about 63.06, 32.96, 59.14, 63.54, 70.76, and 70.65% genes from K. molischiana, Cryptococcus sp., N. ambrosiae,O. ramenticola, and W. bisporus were mapped to GO terms (Figure 1 and Supplementary Table 1). They were matched to three ontologies: molecular function, cellular component, and biological process. They were highly enriched in cellular process, metabolic process, intracellular anatomical structure, organelle, cytoplasm, binding and catalytic activity. Cryptococcus sp. differed from the other considered species in several functions, such as antioxidant activity, biological adhesion, multicellular organismal process, carbon utilization, immune system process, biological phase and rhythmic process. TABLE 1 Extracellular enzyme analyzed in our study. Target Enzyme (endo or exolytic) Substrate Cellulose endo-1,4-β-D-glucanase (endo) Azo-CM-Cellulose Cellobiohydrolase (exo) MUF-β-Dcellobioside β-glucosidase (exo) MUF-β-D-glucopyranoside Hemicellulose endo-1,4-β-D-xylanase (endo) Azo-Xylan (Birchwood) β-xylosidase (exo) MUF-β-D-xyloside Pectin endo-1,4-β-D-mannanase (endo) Azo-Carob Galactomannan endo-1,5-α-L-arabinanase (endo) Red Debranched Arabinan (Sugar Beet) β-galactosidase (exo) MUF-α-D-galactoside β-mannosidase (exo) MUF-β-D-mannoside α-glucuronidase (exo) MUF-α-D-glucuronide hydrate Starch α-amylase (endo) Red Starch Pullulanase (endo) Red Pullulan Lignin Laccase (endo) ABTS Proteins endo-protease (endo) Azocasein Chitin Chitinase (exo) MUF-β-D-N.N-diacetylchitobiose hydrate Phosphate monoester Acid phosphomonoesterase (exo) MUF-phosphate Triglycerides Lipase (exo) MUF-oleate MUF, 4-Methylumbelliferone; ABTS, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). All MUF based substrates and ABTS were purchased from Sigma-Aldrich, other substrates were purchased from Megazyme. Frontiers in Microbiology 04 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 5 Cheng et al. 10.3389/fmicb.2023.1108975 TABLE 2 Genome assembly statistics of yeasts isolated from the gut of Ips typographus. Species Assembly size (Mb) Number of scaffolds N50 (bp) L50 Predicted genes GC (%) BUSCO (%) NCBI WGS project K. molischiana CCF 6642 9.86 354 475.982 8 5.314 54.8 98.4 JANBXH01 Cryptococcus sp. CCF 6641 18.30 629 235.228 26 7.050 59.3 95.9 JANBXI01 N. ambrosiae CCF 6643 13.20 315 546.084 5 5.722 42.3 98.6 JANBXG01 O. ramenticola CCF 6644 12.20 269 1.183.430 4 5.502 34.5 98.4 JANBXF01 W. bisporus CCF 6645 11.70 628 300.060 13 5.784 35.9 97.5 JANBXE01 FIGURE 1 Gene Ontology (GO) terms enrichment in five genomes. The GO annotation is presented in Y-axis, while species names were labeled in X-axis. Frontiers in Microbiology 05 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 6 Cheng et al. 10.3389/fmicb.2023.1108975 FIGURE 2 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. FIGURE 3 Carbohydrate active enzymes (CAZymes) present in the yeasts’ genomes. (A) Comparison of CAZymes classes in five species. (B) Comparison of gene counts related to celluloses, hemicelluloses and pectin in six species. GT, glycosyl transferase; GH, glycoside hydrolase; CE, carbohydrate esterase; CBM, carbohydrate-binding module; PL, polysaccharide lyase; AA, auxiliary activity. Frontiers in Microbiology 06 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 7 Cheng et al. 10.3389/fmicb.2023.1108975 TABLE 3 Number of genes involved in the detoxification process. K. molischiana CCF 6642 Cryptococcus sp. CCF 6641 N. ambrosiae CCF 6643 O. ramenticola CCF 6644 W. bisporus CCF 6645 Aldo-keto reductases 17 8 21 19 25 Carboxylesterase 6 2 6 1 3 Cytochrome P450 monooxygenase 4 6 5 4 5 Flavin-containing monooxygenase 3 5 5 3 15 Glutathione S-transferase 6 4 9 8 11 Multicopper oxidases 2 3 4 2 5 Transporter ABC 35 37 30 29 31 Multidrug and toxic compound extrusion (MATE) 5 0 5 5 6 Major facilitator Superfamily (MFS) 132 41 127 113 154 FIGURE 4 Maximum-likelihood tree of aldo-keto reductases predicted from all five yeast genomes. To investigate the function of proteins in those five species, the annotation based on BlastKOALA (Kanehisa et al.,2016) against the KEGG database was performed. It was possible to annotate 58.2, 57.4, 60.1, 56.3, and 35.6% of conserved sequences from K. molischiana,N. ambrosiae,O. ramenticola,W. bisporus, and Cryptococcus sp., respectively, Figure 2. As a result, the highest enriched system was “Genetic information processing,” followed by “Carbohydrate metabolism,” and “Cellular processes.” Vitamin B6 compounds, are synthetized and metabolized in all five species (Supplementary Figure 1). Additionally, essential amino acids such as arginine, lysine, phenylalanine, tyrosine, valine, leucine, and isoleucine are biosynthesized in these yeast strains as well (Supplementary Figure 2). 3.3. Identification of carbohydrate-active enzyme (CAZyme) The carbohydrate-active enzyme (CAZyme) gene contents were predicted for all five genomes (Figure 3A,Supplementary Figure 3 and Supplementary Table 2) in this study. These CAZymes are classified into six classes: auxiliary activity (AA), Frontiers in Microbiology 07 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 8 Cheng et al. 10.3389/fmicb.2023.1108975 carbohydrate-binding module (CBM), carbohydrate esterase (CE), glycoside hydrolase (GH), glycosyltransferase (GT), and polysaccharide lyases (PL). Nakazawaea ambrosiae has more CAZymes (221) than the other four studied species, while the Cryptococcus sp. has the smallest CAZymes count (157). GH accounted for almost half of the identified enzymes (83–98) in those species except for Cryptococcus sp. (41), followed by GH (63– 79), AA (13–28), CE (14–24), and CBM (4–9). PL (0–5) comprised only a few genes and is absent in Nakazawaea ambrosiae. The number of identified genes regulating plant cell walldegrading enzymes varies in five yeast species (Figure 3B). Wickerhamomyces bisporus has the highest (71) and Cryptococcus sp. has the least number of genes (35). The number of genes related to cellulose degradation is lower than those related to hemicelluloses and pectin degradation in all genomes. 3.4. Analysis of genes related to detoxification More than 200 detoxification related genes were identified from each species, K. molischiana (225), N. ambrosiae (235), O. ramenticola (200), and W. bisporus (276), except for Cryptococcus sp. (110) (Table 3 and Supplementary Table 3). We predicted presence of 115 aldo-keto reductase enzymes, with lengths from 107 to 599 amino acids, pIs from 4.62 to 9.00, and predicted molecular weights between 12.02 and 65.57 kDa. They were predicted to be localized in the cytoplasm, plasma membrane, nucleus, mitochondria, vacuoles, and the bonding membranes of the Golgi apparatus. They can be classified into 9 subfamilies (Figure 4), aryl-alcohol dehydrogenases, D-arabinose 1-dehydrogenases, oxidoreductases, and NAD(P)Hdependent D-xylose reductases. They were found in all five genomes with 22, 7, 24, and 10 members, respectively. There are 9, 14, 10, and 16 genes identified in subgroups pyridoxal reductases, NAD(P)H-dependent reductases, D-arabinose 1-dehydrogenases (NAD(P)+) heavy chain, and glycerol 2dehydrogenases (NAD(P)+), respectively. These subgroups were absent in Cryptococcus sp. Three genes putatively encoding for aldehyde reductase I were found in Cryptococcus sp. and N. ambrosiae. We identified 29 cytochrome P450 (CYP) monooxygenases with amino acids lengths from 126 to 591, molecular weights from 13.78 to 66.48 kDa, and pIs from 4.9 to 8.51. They were predicted to localize in cytoplasm, plasma membrane, mitochondria, bounding membrane of mitochondria and endoplasmic reticulum. The topology tree of CYP monooxygenases showed that they can be classified into six subfamilies (Figure 5), with 7, 6, 5, 2, 4, and 5 members for CYP51, CYP61, CYP501, CYP56, CYP52, and CYP5252, respectively. Sequences in the CYP51 and CYP61 subfamilies were observed in all five species. CYP501 and CYP5252 were absent in Cryptococcus sp. CYP52 only existed in Cryptococcus sp., while CYP56 was present in W. bisporus genome. ATP binding cassette (ABC) transporters were predicted from those five genomes, they can be classified into seven subfamilies (Figure 6): 35 pleiotropic drug resistance, 10 eye pigment precursor transporter, 62 drug conjugate transporter, 7 heavy metal transporter, 13 α-factor-pheromone transporter, 43 mitochondrial peptide exporter, and 11 peroxisomal fatty acyl CoA transporter. Their amino acid lengths varied from 120 to 2058 aa, molecular weights from 13.14 to 230.256 kDa, pIs from 4.23 to 9.06. Their predicted subcellular localizations are cytoplasmic, mitochondrial, plasma and endoplasmic reticulum membranes, peroxisomal and vacuolar. We predicted the presence of 21 carboxylesterases with 109 to 566 amino acids and molecular weight of 12.10 to 62.49 kDa. Then we predicted 46 flavin-containing monooxygenases with 137–647 amino acids, and molecular weights 15.86–72.63 kDa. Glutathione S-transferases were predicted with number of amino acids varying from 111 to 1006 and molecular weights 12.21– 110.52 kDa. We also predicted 21 multicopper oxidases with 152–722 amino acids and 17.48–76.62 kDa, 38.10% of them were predicted as extracellular enzymes. The number of amino acids of multidrug and toxic compound extrusion (MATE) ranged from 361–1018 with molecular weights 39.43–113.68 kDa. Major Facilitator Superfamily (MFS), which contains 720 sequences, has 92–1863 amino acids with molecular weights 10–207.86 kDa (Supplementary Table 3). 3.5. Extracellular enzyme activities We measured the activities of endoand exolytic enzymes degrading complex components of plant tissues (see Table 4 for results) as functional analysis predicted the presence of these genes in yeast genomes. We found that the yeast species studied do not produce endolytic enzymes, with the exception of endo-1,4-β-Dmannanase, which cleaves pectin. However, all yeasts were strong producers of exolytic enzymes. Kuraishia molischiana was the only species producing all the enzymes tested, especially β-glucosidase, cellobiohydrolase, β-xyloside and β-mannosidase. Nakazawaea ambrosiae was the strongest producer of acid monophosphatase, lipase and β-mannosidase. Wickerhamomyces bisporus mainly secreted β-glucosidase and lipase, and O. ramenticola β-glucosidase and β-mannosidase. Cryptococcus sp. was the weakest of the yeasts in enzyme activities and produced mainly β-glucosidase and lipase. We successfully measured the enzyme activities of the positive control Trichoderma viride, suggesting that the absence of endolytic enzymes in the yeast species was not a methodological error. 4. Discussion Central Europe is currently facing outbreaks of spruce bark beetles, of which Ips typographus is the most common and widespread species (Biedermann et al.,2019). In the previous study (Veselská et al.,2023) we found that ascomycetous yeasts, namely, K. molischiana,N. ambrosiae,O. ramenticola, and W. bisporus dominate the gut fungal microbiome of I. typographus. The set was supplemented by a representative of basidiomycetous yeasts, Cryptococcus (Tremellales). In the present study we sequenced and annotated genomes of these yeasts to infer their possible roles in the bark beetle habitat. Bark beetles face harsh conditions when they colonize rigid plant tissues which are full of toxic secondary metabolites Frontiers in Microbiology 08 frontiersin.org fmicb-14-1108975 March 28, 2023 Time: 16:58 # 9 Cheng et al. 10.3389/fmicb.2023.1108975 FIGURE 5 Maximum-likelihood tree of P450 predicted from all five yeast genomes. FIGURE 6 Maximum-likelihood tree of ABC transporter predicted from all five yeast genomes. related to tree defense against intruders (e.g., Netherer et al., 2021). The bark beetles associated filamentous fungi facilitate colonization as these fungi are often involved in detoxification and/or cause necrosis of the plant tissues (Hofstetter et al., 2015;Zhao et al.,2019;Li et al.,2022). Carbohydrate-active enzymes (CAZymes) play a vital role in the depolymerization Frontiers in Microbiology 09 frontiersin.org