Identification of traits to improve co-assimilation of glucose and xylose by adaptive evolution of Spathaspora passalidarum and Scheffersomyces stipitis yeasts.

Lignocellulosic biomass is a renewable raw material for producing several high-value-added chemicals and fuels. In general, xylose and glucose are the major sugars in biomass hydrolysates, and their efficient utilization by microorganisms is critical for an economical production process. Yeasts capable of co-consuming mixed sugars might lead to higher yields and productivities in industrial fermentation processes. Herein, we performed adaptive evolution assays with two xylose-fermenting yeasts, Spathaspora passalidarum and Scheffersomyces stipitis, to obtain derived clones with improved capabilities of glucose and xylose co-consumption. Adapted strains were obtained after successive growth selection using xylose and the non-metabolized glucose analog 2-deoxy-D-glucose as a selective pressure. The co-fermentation capacity of evolved and parental strains was evaluated on xylose-glucose mixtures. Our results revealed an improved co-assimilation capability by the evolved strains; however, xylose and glucose consumption were observed at slower rates than the parental yeasts. Genome resequencing of the evolved strains revealed genes affected by non-synonymous variants that might be involved with the co-consumption phenotype, including the HXT2.4 gene that encodes a putative glucose transporter in Sp. passalidarum. Expression of this mutant HXT2.4 in Saccharomyces cerevisiae improved the cells' co-assimilation of glucose and xylose. Therefore, our results demonstrated the successful improvement of co-fermentation through evolutionary engineering and the identification of potential targets for further genetic engineering of different yeast strains. KEY POINTS: • Laboratory evolution assay was used to obtain improved sugar co-consumption of non-Saccharomyces strains. • Evolved Sp. passalidarum and Sc. stipitis were able to more efficiently co-ferment glucose and xylose. • A mutant Hxt2.4 permease, which co-transports xylose and glucose, was identified.

Diversity of genomic adaptations to the post-fire environment in Pezizales fungi points to crosstalk between charcoal tolerance and sexual development.

Wildfires drastically impact the soil environment, altering the soil organic matter, forming pyrolyzed compounds, and markedly reducing the diversity of microorganisms. Pyrophilous fungi, especially the species from the orders Pezizales and Agaricales, are fire-responsive fungal colonizers of post-fire soil that have historically been found fruiting on burned soil and thus may encode mechanisms of processing these compounds in their genomes. Pyrophilous fungi are diverse. In this work, we explored this diversity and sequenced six new genomes of pyrophilous Pezizales fungi isolated after the 2013 Rim Fire near Yosemite Park in California, USA: Pyronema domesticum, Pyronema omphalodes, Tricharina praecox, Geopyxis carbonaria, Morchella snyderi, and Peziza echinospora. A comparative genomics analysis revealed the enrichment of gene families involved in responses to stress and the degradation of pyrolyzed organic matter. In addition, we found that both protein sequence lengths and G + C content in the third base of codons (GC3) in pyrophilous fungi fall between those in mesophilic/nonpyrophilous and thermophilic fungi. A comparative transcriptome analysis of P. domesticum under two conditions - growing on charcoal, and during sexual development - identified modules of genes that are co-expressed in the charcoal and light-induced sexual development conditions. In addition, environmental sensors such as transcription factors STE12, LreA, LreB, VosA, and EsdC were upregulated in the charcoal condition. Taken together, these results highlight genomic adaptations of pyrophilous fungi and indicate a potential connection between charcoal tolerance and fruiting body formation in P. domesticum.

Comparative genomics of pyrophilous fungi reveals a link between fire events and developmental genes.

Forest fires generate a large amount of carbon that remains resident on the site as dead and partially 'pyrolysed' (i.e. burnt) material that has long residency times and constitutes a significant pool in fire-prone ecosystems. In addition, fire-induced hydrophobic soil layers, caused by condensation of pyrolysed waxes and lipids, increase post-fire erosion and can lead to long-term productivity losses. A small set of pyrophilous fungi dominate post-fire soils and are likely to be involved with the degradation of all these compounds, yet almost nothing is currently known about what these fungi do or the metabolic processes they employ. In this study, we sequenced and analysed genomes from fungi isolated after Rim fire near Yosemite National Park in 2013 and showed the enrichment/expansion of CAZymes and families known to be involved in fruiting body initiation when compared to other basidiomycete fungi. We found gene families potentially involved in the degradation of the hydrophobic layer and pyrolysed organic matter, such as hydrophobic surface binding proteins, laccases (AA1_1), xylanases (GH10, GH11), fatty acid desaturases and tannases. Thus, pyrophilous fungi are important actors to restate the soil's functional capabilities.

Evolution and comparative genomics of the most common Trichoderma species.

BACKGROUND: The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. RESULTS: We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. CONCLUSIONS: Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Physiological and comparative genomic analysis of new isolated yeasts Spathaspora sp. JA1 and Meyerozyma caribbica JA9 reveal insights into xylitol production.

Xylitol is a five-carbon polyol of economic interest that can be produced by microbial xylose reduction from renewable resources. The current study sought to investigate the potential of two yeast strains, isolated from Brazilian Cerrado biome, in the production of xylitol as well as the genomic characteristics that may impact this process. Xylose conversion capacity by the new isolates Spathaspora sp. JA1 and Meyerozyma caribbica JA9 was evaluated and compared with control strains on xylose and sugarcane biomass hydrolysate. Among the evaluated strains, Spathaspora sp. JA1 was the strongest xylitol producer, reaching product yield and productivity as high as 0.74 g/g and 0.20 g/(L.h) on xylose, and 0.58 g/g and 0.44 g/(L.h) on non-detoxified hydrolysate. Genome sequences of Spathaspora sp. JA1 and M. caribbica JA9 were obtained and annotated. Comparative genomic analysis revealed that the predicted xylose metabolic pathway is conserved among the xylitol-producing yeasts Spathaspora sp. JA1, M. caribbica JA9 and Meyerozyma guilliermondii, but not in Spathaspora passalidarum, an efficient ethanol-producing yeast. Xylitol-producing yeasts showed strictly NADPH-dependent xylose reductase and NAD+-dependent xylitol-dehydrogenase activities. This imbalance of cofactors favors the high xylitol yield shown by Spathaspora sp. JA1, which is similar to the most efficient xylitol producers described so far.

Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Sequencing and Analysis of the Entire Genome of the Mycoparasitic Bioeffector Fungus Trichoderma asperelloides Strain T 203 (Hypocreales).

The filamentous mycoparasitic fungus Trichoderma asperelloides (Hypocreales, Ascomycota, Dikarya) strain T 203 was isolated from soil in Israel by the Ilan Chet group in the 1980s. As it has been the subject of laboratory, greenhouse, and field experiments and has been incorporated into commercial agricultural preparations, its genome has been sequenced and analyzed.

Wildfire-dependent changes in soil microbiome diversity and function.

Forest soil microbiomes have crucial roles in carbon storage, biogeochemical cycling and rhizosphere processes. Wildfire season length, and the frequency and size of severe fires have increased owing to climate change. Fires affect ecosystem recovery and modify soil microbiomes and microbially mediated biogeochemical processes. To study wildfire-dependent changes in soil microbiomes, we characterized functional shifts in the soil microbiota (bacteria, fungi and viruses) across burn severity gradients (low, moderate and high severity) 1 yr post fire in coniferous forests in Colorado and Wyoming, USA. We found severity-dependent increases of Actinobacteria encoding genes for heat resistance, fast growth, and pyrogenic carbon utilization that might enhance post-fire survival. We report that increased burn severity led to the loss of ectomycorrhizal fungi and less tolerant microbial taxa. Viruses remained active in post-fire soils and probably influenced carbon cycling and biogeochemistry via turnover of biomass and ecosystem-relevant auxiliary metabolic genes. Our genome-resolved analyses link post-fire soil microbial taxonomy to functions and reveal the complexity of post-fire soil microbiome activity.

Draft Genome Sequence of Muricauda sp. Strain K001 Isolated from a Marine Cyanobacterial Culture.

We report the whole-genome sequence of Muricauda sp. strain K001 isolated from a marine cyanobacterial culture. This genome sequence will improve our understanding of the influence of heterotrophic bacteria on the physiology of cyanobacteria and may contribute to the development of new natural products.

Genome-wide identification, characterization and expression profile analysis of expansins gene family in sugarcane (Saccharum spp.).

Expansins refer to a family of closely related non-enzymatic proteins found in the plant cell wall that are involved in the cell wall loosening. In addition, expansins appear to be involved in different physiological and environmental responses in plants such as leaf and stem initiation and growth, stomata opening and closing, reproduction, ripening and stress tolerance. Sugarcane (Saccharum spp.) is one of the main crops grown worldwide. Lignocellulosic biomass from sugarcane is one of the most promising raw materials for the ethanol industry. However, the efficient use of lignocellulosic biomass requires the optimization of several steps, including the access of some enzymes to the hemicellulosic matrix. The addition of expansins in an enzymatic cocktail or their genetic manipulation could drastically improve the saccharification process of feedstock biomass by weakening the hydrogen bonds between polysaccharides present in plant cell walls. In this study, the expansin gene family in sugarcane was identified and characterized by in silico analysis. Ninety two putative expansins in sugarcane (SacEXPs) were categorized in three subfamilies after phylogenetic analysis. The expression profile of some expansin genes in leaves of sugarcane in different developmental stages was also investigated. This study intended to provide suitable expansin targets for genetic manipulation of sugarcane aiming at biomass and yield improvement.

Correction: Genome-wide identification, characterization and expression profile analysis of expansins gene family in sugarcane (Saccharum spp.).

[This corrects the article DOI: 10.1371/journal.pone.0191081.].