Genomic analysis of the xylanolytic yeast Pseudozyma brasiliensis GHG001

Orientadores: Gustavo Henrique Goldman, Diego Mauricio Riaño PachónDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os fungos do Filo Basidiomycota são os organismos mais relevantes na decomposição de matéria orgânica no planeta, sendo que os membros do subfilo Agaricomycotina são as principais espécies envolvidas na desconstrução de biomassa lignocelulósica. A família Ustilaginaceae pertence ao subfilo Ustilaginomycotina, grupo-irmão de Agaricomycotina, e inclui importantes fitopatógenos de gramíneas e leveduras não patogênicas que têm se destacado pelo potencial biotecnológico, incluindo a produção de enzimas de degradação de polímeros da parede celular de plantas. O xilano é o principal componente da porção hemicelulósica da parede celular de plantas e o entendimento de sua degradação por fungos, bem como do transporte de seu monômero, a xilose, para o interior celular e seu metabolismo interno, são essenciais para a aplicação industrial e o melhoramento genético destes organismos, incluindo a produção de bioetanol de segunda geração. A levedura xilanolítica Kalmanozyma brasiliensis GHG001 (previamente Pseudozyma brasiliensis) é um membro de Ustilaginaceae isolada do trato intestinal de uma larva de Chrysomelidae que parasitava a cana-de-açúcar, durante o screening de leveduras capazes de fermentar xilose, tendo se destacado pela produção de uma endoxilanase da família GH11. As abordagens ômicas são valiosas para a exploração do potencial de degradação de biomassa lignocelulósica. Trabalhos previamente realizados incluem genômica comparativa de diversos grupos de fungos ascomicetos e basidiomicetos com foco na degradação de lignocelulose e a transcriptômica com RNA-seq em basidiomicetos já foi estudada para entendimento da degradação de lignocelulose limitando-se, no entanto, aos membros de Agaricomycotina. Apesar do seu potencial biotecnológico e da disponibilidade das sequências genômicas de diversos membros, a família Ustilaginaceae permanece subexplorada. Neste trabalho, abordagens ômicas são usadas para a exploração do potencial biotecnológico desta família de fungos com foco na degradação de xilano e no metabolismo de xilose. Inicialmente, a análise de transcriptômica com RNA-seq é usada para melhorar a compreensão do metabolismo de xilano e xilose em K. brasiliensis GHG001, incluindo a expressão de genes que codificam as enzimas de degradação de xilano, os transportadores putativos de xilose e possíveis proteínas reguladoras da transcrição. A análise de RNA-seq foi usada também para o melhoramento da anotação estrutural desta levedura. Em seguida, uma abordagem de genômica comparativa foi aplicada para a exploração de outros membros da família com genomas sequenciados, incluindo leveduras isoladas de flores de Heliconia psittacorum, Moesziomyces spp. e Pseudozyma sp. pro tempore, potencialmente novas espécies. As análises de RNA-seq melhoraram significativamente a anotação estrutural do genoma de K. brasiliensis GHG001 e contribuíram para o melhor entendimento do metabolismo de pentoses e da regulação da expressão gênica. As análises filogenéticas de Ustilaginaceae contribuíram para a Sistemática da família de fungos e permitiram não apenas alocar as novas leveduras sequenciadas, mas também possibilitaram o estudo da genômica comparativa. Por fim, a prevalência de Enzimas Ativas em Carboidratos (CAZymes) e a evolução (expansões e contrações) de famílias CAZymes e de Proteínas Associadas à Transcrição (TAPs) destacaram aspectos importantes para o levantamento de hipóteses sobre a família de fungosAbstract: Fungi in phylum Basidiomycota comprise the most relevant in decomposition of organic matter in our planet, and members of the Agaricomycotina sub-phylum are the main involved in deconstruction of lignocellulosic biomass. The family Ustilaginaceae belongs to sub-phylum Ustilaginomycotina, a sister clade of Agaricomycotina, and includes important grass phytopathogens and non-pathogenic yeasts, both have been highlighted because of their biotechnological potential, including production of enzymes for deconstruction of plant cell wall polymers. Xylan is the main component of the hemicellulosic portion of plant cell wall and the comprehension of its degradation by fungi, as well as the transport of its monomer, xylose, and its internal metabolism are essential for industrial application and genetic improvement of these organisms, including production of Second Generation Bioethanol. The xylanolytic yeast Kalmanozyma brasiliensis GHG001 (previously Pseudozyma brasiliensis) is a member of Ustilaginaceae isolated from the gut of a Chrysomelidae larvae that parasitized sugarcane, during a screening of yeasts able to ferment xylose, and was outstanding in overproduction of an endoxylanase of family GH11. Omics approaches are valuable for exploitation of the potential of lignocellulosic biomass deconstruction. Previous works have been carried out that included comparative genomics of several fungal groups within ascomycetes and basidiomycetes with focus on lignocellulose degradation and transcriptomics employing RNA-seq in basidiomycetes has already been carried out for understanding of lignocellulose degradation, but limited to Agaricomycotina. Despite its biotechnological importance and availability of genome sequences for several of its members, the family Ustilaginaceae remains underexplored. In this work, omics approaches have been applied to exploit the biotechnological potential of this fungal family with focus on xylan degradation and xylose metabolism. First, a transcriptomic analysis with RNA-seq was employed to improve our comprehension of xylan and xylose metabolism in K. brasiliensis GHG001, including the expression of genes coding for xylan degrading enzymes, putative xylose transporters, and possible transcriptional regulatory proteins. RNA-seq analysis was also used to improve the genome structural annotation of the yeast. Next, a comparative genomics approach was used to exploit other members of the fungal family with sequenced genomes, including three novel ustilaginaceous yeasts, possibly new species, isolated from flowers of Heliconia psittacorum, Moesziomyces spp. F16C1 and F8B2, and Pseudozyma sp. F5C1 pro tempore, potentially novel species. RNA-seq analyses improved the structural annotation of the K. brasiliensis GHG001 genome and allowed a better understanding of the pentose metabolism and regulation of gene expression. Phylogenetic analyses of the family Ustilaginaceae contributed to its Systematics, allowing the placement of sequenced yeasts and providing an accurate phylogeny for the comparative genomic analysis. Lastly, the prevalence of CAZYmes and the evolution (expansions and contractions) of CAZYme and TAP families highlighted important aspects for raising hypotheses about the fungal familyMestradoBioinformaticaMestre em Genética e Biologia Molecular2014/15799-7, 2016/13968-1FAPES

Examining Signatures of Natural Selection in Antifungal Resistance Genes Across Aspergillus Fungi

Certain Aspergillus fungi cause aspergillosis, a set of diseases that typically affect immunocompromised individuals. Most cases of aspergillosis are caused by Aspergillus fumigatus, which infects millions of people annually. Some closely related so-called cryptic species, such as Aspergillus lentulus, can also cause aspergillosis, albeit at lower frequencies, and they are also clinically relevant. Few antifungal drugs are currently available for treating aspergillosis and there is increasing worldwide concern about the presence of antifungal drug resistance in Aspergillus species. Furthermore, isolates from both A. fumigatus and other Aspergillus pathogens exhibit substantial heterogeneity in their antifungal drug resistance profiles. To gain insights into the evolution of antifungal drug resistance genes in Aspergillus, we investigated signatures of positive selection in 41 genes known to be involved in drug resistance across 42 susceptible and resistant isolates from 12 Aspergillus section Fumigati species. Using codon-based site models of sequence evolution, we identified ten genes that contain 43 sites with signatures of ancient positive selection across our set of species. None of the sites that have experienced positive selection overlap with sites previously reported to be involved in drug resistance. These results identify sites that likely experienced ancient positive selection in Aspergillus genes involved in resistance to antifungal drugs and suggest that historical selective pressures on these genes likely differ from any current selective pressures imposed by antifungal drugs.RS was supported by the Brazilian São Paulo Research Foundation (FAPESP) grant numbers 2017/21983-3 and 2019/07526-4. JS and AR are supported by the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study Program. AR’s laboratory received additional support from a Discovery grant from Vanderbilt University, the Burroughs Wellcome Fund, the National Science Foundation (DEB-1442113), and the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R56AI146096). GHG was supported by FAPESP (2016/07870-9) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq).S

RNAseq reveals hydrophobins that are involved in the adaptation of aspergillus nidulans to lignocellulose

Background Sugarcane is one of the world’s most profitable crops. Waste steam-exploded sugarcane bagasse (SEB) is a cheap, abundant, and renewable lignocellulosic feedstock for the next-generation biofuels. In nature, fungi seldom exist as planktonic cells, similar to those found in the nutrient-rich environment created within an industrial fermenter. Instead, fungi predominantly form biofilms that allow them to thrive in hostile environments. Results In turn, we adopted an RNA-sequencing approach to interrogate how the model fungus, Aspergillus nidulans, adapts to SEB, revealing the induction of carbon starvation responses and the lignocellulolytic machinery, in addition to morphological adaptations. Genetic analyses showed the importance of hydrophobins for growth on SEB. The major hydrophobin, RodA, was retained within the fungal biofilm on SEB fibres. The StuA transcription factor that regulates fungal morphology was up-regulated during growth on SEB and controlled hydrophobin gene induction. The absence of the RodA or DewC hydrophobins reduced biofilm formation. The loss of a RodA or a functional StuA reduced the retention of the hydrolytic enzymes within the vicinity of the fungus. Hence, hydrophobins promote biofilm formation on SEB, and may enhance lignocellulose utilisation via promoting a compact substrate-enzyme-fungus structure. Conclusion This novel study highlights the importance of hydrophobins to the formation of biofilms and the efficient deconstruction of lignocellulose

Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30

The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ~70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, β-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol

Os principais tipos e manifestações da Cirrose Hepática: uma atualização clínica

Introdução: A cirrose hepática é um processo patológica crônico, considerado a hepatopatia mais comum, definido como a conversão difusa morfoestrutural por nódulos de arquitetura anômalo envoltos por fibrose. Objetivou-se descrever os tipos mais relevantes de cirrose e suas devidas manifestações. Metodologia: Trata-se de uma revisão bibliográfica, fundamentada nas plataformas do SciELO, PubMed, Scopus, utilizando os termos “hepatical cirrhosis”, “liver disease” e “hepatocellular insufficiency” a qual através da revisão narrativa, abordou amplamente a respeito da contextualização da cirrose e as principais etiologias. Resultados e Discussão: Foi analisado que tal condição afeta qualquer faixa etária, sexo, etnia e independe da classe socioeconômica, mas as diversas etiologias impõem um perfil epidemiológico específico conforme a aparição. As principais origens abordam o tipo alcoólico, hepatite, aplicação crônica de alguns fármacos e esteatose gordurosa ou não. Ademais, estima-se que estas afetam a anatomofuncionalidade do órgão responsável por grande parte da homeostase, culminando em diversas manifestações clínicas.  Conclusão: A cirrose é uma consequência grave de fatores de base em estágio avançado, a qual devido ao seu curso geralmente silencioso culmina no desenvolvimento e progressão clínica. Neste contexto, a atenção aos fatores predisponentes como alimentação rica em lipídios, estilismo, negligência a exames de rotina, sedentarismo e obesidade contribuem constituem medidas eficazes de prevenção primária.&nbsp

Carvão (Sporisorium scitamineum) e podridão-abacaxi (Ceratocystis paradoxa) em cana-de-açúcar (Saccharum spp.): uma revisão

Sugarcane (Saccharum sp.) is an important grass cultivated in tropical and subtropical regions of the world, such as in Br, Ind, and Ch, and has its biomass being raw material for production of sugar, fuel ethanol, and some other derivatives. Fungal diseases infect sugarcane fields worldwide, damaging crops and thus, causing great economic losses. Fungal specialized structures act during all Pathogen-Host Relationship Cycle (PHRC) (survival, dissemination, infection, colonization, and reproduction of pathogen), maintaining fungal populations in cultivation soil, infecting plants in following crops and vegetative propagation of sugarcane by infected seeds may allow fungal transportation into regions where diseases haven’t occured before. Biotechnological methods and approaches have significantly contributed to understanding of the relationship among parasite and host, as to diseases management (control, detection, and prevention). Some techniques have daily applications in Agriculture, while others are only used in research and to breeding of host resistant varieties. Among notable diseases, smut (Sporisorium scitamineum) and pineapple disease (Ceratocystis paradoxa) are important because they cause damage and losses in sugarcane regions, although there are different periods for each one to occur. This work aims to review the PHRC for each patosystem, the biotechnological methods and approaches and its perspectives in the study and management of these diseases. As environment is an important factor to the effectiveness of PHRC, one chapter is dedicated to Global Climate Change (GCC) and its possible influences over these diseases in a longterm periodCana-de-açúcar (Saccharum sp.) é uma gramínea de grande importância, cultivada nas regiões tropicais e subtropicais do planeta, como no Brasil, na China e na Índia, sendo sua biomassa matéria-prima para a produção de açúcar, etanol combustível e diversos outros produtos. As doenças fúngicas frequentemente acarretam prejuízos à cultura em todo o planeta, gerando danos ao campo e, consequentemente, grandes perdas econômicas. Estruturas fúngicas especializadas atuam durante todo o Ciclo de Relação Patógeno-Hospedeiro (CRPH) (sobrevivência, disseminação, infecção, colonização e reprodução do patógeno), podendo as populações do patógeno permanecer no solo de cultivo, infectando plantas em safras subsequentes e a propagação vegetativa utilizando toletes infectados pode permitir, ainda, o transporte para regiões onde não havia incidência da doença. Métodos e abordagens da Biotecnologia têm contribuído significativamente para o entendimento da relação entre o parasita e o hospedeiro, assim como para o manejo das doenças (controle, detecção e prevenção). Muitas de suas técnicas têm aplicabilidade no cotidiano do agricultor, enquanto outras são utilizadas na pesquisa e no desenvolvimento de variedades resistentes do hospedeiro. Entre as doenças de destaque, carvão (Sporisorium scitamineum) e podridão-abacaxi (Ceratocystis paradoxa) são importantes e causam prejuízos já registrados em todas as regiões onde há o cultivo de cana-de-açúcar, embora os períodos de maior incidência sejam distintos. Neste trabalho, são revisados os CRPHs para estes patossistemas, os métodos e perspectivas da Biotecnologia aplicados no estudo e manejo das doenças. Considerando a importância do fator ambiente para a efetivação do CRPH, um capítulo trata as Mudanças Climáticas Globais (MCG) e as possíveis influências sobre os patossistemas, a longo praz

Standardization of Comparative Transcriptomic in Analyses in Plants Using Experimental Metadata andOntology Terms

<p>Analyzing gene expression levels in plants with stress condition contexts can discern nuances in gene regulation that provide deep insights into different biological conditions. However, to extract precise meaning from the complex RNA-Seq data sets, ontology assignment is essential to standardize comparisons, enabling more robust and integrated analyses. Thus the goals of this work are: to assign ontology terms to RNA-Seq data based on different tissues and experimental conditions; to develop Python modules for the CoNekT Grasses platform (<a href="http://conekt.cena.usp.br:82/conekt/">http://conekt.cena.usp.br</a>) using ontology terms; and to identify gene expression markers associated with stress in <i>Setaria viridis</i>. We downloaded RNA-Seq metadata/datasets for <i>S. viridis</i> from NCBI and stored the results in a SQLite database (<a href="https://gitlab.com/SantosRAC/cena_usp_racs/-/blob/8c33e6bd640c5ca241d18bab7e08cee24948f1cc/scripts/get_store_metadata/get_metadata.py">get_metadata.py</a>). Read mapping rates and strand-specific libraries were assessed using Salmon v1.8.0. To assign Plant Ontology (PO) and Plant Experimental Condition Ontology (PECO) terms from NCBI metadata, we developed a Python script (<a href="https://gitlab.com/SantosRAC/cena_usp_racs/-/blob/main/scripts/metadata_to_ontology/scripts/lemmatization.py">lemmatization.py</a>) using library 'spaCy'. We filtered part of speech from the lemmatized words to identify plant tissues/organs and stress conditions. Another script (<a href="https://gitlab.com/SantosRAC/cena_usp_racs/-/tree/main">bioportal.py</a>) was created to retrieve terms from ontology libraries on the BioPortal website (PO and PECO). Integration of ontology annotations into the CoNekT Grasses platform involved back-end and front-end modifications. We expanded the platform's capabilities for sample annotation input. To identify gene expression markers under stress conditions, we analyzed an expression matrix involving abiotic stress studies. This matrix was normalized by <i>Remove Unwanted Variation</i> (RUVg). RUVg was used for batch effect correction, library preparation, and other nuisance effects; specificity calculations were performed using the TSPEX package. Genes with TAU > 0.85 were considered highly specific, and we compared results with and without RUVg correction. We obtained SQLite databases for <i>S. viridis </i>containing 51 datasets associated with a publication involving abiotic stress of luminosity, heat and caffeic acid exposure. Annotation of public RNA-Seq samples was implemented in the CoNekT Grasses platform. An exploratory analysis of stress datasets revealed sample heterogeneity and batch effects, with RUVg correction improving sample grouping. Comparing specificity results between matrices with and without RUVg correction showed differences in 8110 genes, while 1834 genes were consistent in both matrices. Using TSPEX, we identified highly specific genes associated with different stress conditions, including 65 light stress, 18 heat stress, and 206 caffeic acid stress. In conclusion, our specificity calculations yielded potential gene markers of stress-associated datasets. Future work should focus on functional annotation and validation of these genes for filtering stress-related datasets in CoNekTGrasses platform and building co-expression networks.</p&gt

Draft Genome Sequences of Four Aspergillus Section Fumigati Clinical Strains.

Aspergillus fungi in section Fumigati include important human pathogens. Here, we sequenced the genomes of two strains of Aspergillus hiratsukae and two strains of Aspergillus felis The average genome sizes are 29.5 Mb for A. hiratsukae and 31.8 Mb for A. felis.This research was funded by Brazilian São Paulo Research Foundation (FAPESP) grant 2016/07870-9 (to G.H.G.) and scholarships 2017/21983-3 and 2019/07526-4 (to R.A.C.D.S.) and by research projects from the Fondo de Investigación Sanitaria (PI13/02145 and PI16CIII/00035) of the Instituto de Salud Carlos III. J.L.S. and A.R. were funded by the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program. A.R. also received funding from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (1R56AI146096-01A1).S