Identification of genomic regions related to catabolism of lignocellulosic biomass by the fungus Trichoderma harzianum IOC-3844

Orientadores: Anete Pereira de Souza, Sindélia Freitas AzzoniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O Brasil é hoje referência mundial na produção de etanol produzido a partir da cana-de-açúcar, cujo consumo tem aumentado significativamente nos últimos anos. Entretanto, a produção atual de etanol a partir do suco da cana-de-açúcar é insuficiente para atender a demanda do mercado nacional e internacional. Nesse contexto, a produção de etanol celulósico (de segunda geração) emergiu como uma alternativa promissora ao bioetanol de primeira geração. O fungo filamentoso Trichoderma harzianum IOC-3844 é uma linhagem brasileira que se destaca pela alta capacidade de produção de enzimas do complexo das celulases e hemicelulases, característica de grande interesse em biocatálise para conversão de biomassa em monômeros de açúcar fermentáveis. Apesar de seu comprovado potencial, há poucos dados de literatura disponíveis a respeito de sua capacidade celulolítica. Desta forma, este projeto teve como objetivo principal contribuir para o conhecimento básico sobre regiões do genoma de T. harzianum IOC-3844 envolvidas na via de hidrólise de compostos celulósicos, através da construção de uma biblioteca genômica de BAC (bacterial artificial chromosome). A biblioteca de BACs conta com 5760 clones, com insertos de DNA de tamanho médio de 90 kb, o que dá uma cobertura de aproximadamente 12 vezes o genoma de T. harzianum. Através da seleção de clones contendo genes de interesse, foram identificadas regiões com altas concentrações de genes relacionados à hidrólise de biomassa. Além disso, a combinação de dados genômicos, obtidos através da biblioteca de BACs, juntamente com dados de transcriptoma possibilitou a identificação de novos potenciais genes regulatórios. Os resultados trazem grande contribuição para a pesquisa associada a T. harzianum e à genômica de fungos relacionada à produção de etanol de segunda geraçãoAbstract: Brazil is a world reference in sugarcane ethanol production, whose consumption has increased significantly in recent years. However, the current production of ethanol from sugarcane juice is insufficient to meet the demand of national and international market. In this context, the production of cellulosic ethanol (second generation) has emerged as a promising alternative to first-generation bioethanol. The filamentous fungus Trichoderma harzianum IOC-3844 is a Brazilian strain known for its high ability to produce enzymes of cellulosic and hemicellulosic complex, characteristic of great interest in biocatalysis for conversion of biomass into fermentable sugar monomers. Despite its potential, there are few published data available regarding its cellulolytic ability. Thus, this project aimed to contribute to the basic knowledge about regions of the genome of T. harzianum IOC-3844 involved in the hydrolysis of cellulosic compounds pathway, through the construction of a genomic BAC (bacterial artificial chromosome) library. The BAC library comprises 5,760 clones with an average DNA insert size of 90 kb, which represents about 12-fold coverage of the T. harzianum genome. Through the selection of clones containing genes of interest, regions containing high concentrations of genes related to biomass hydrolysis were identified. Furthermore, the combination of genomic data obtained from BAC library together with transcriptome data allowed the identification of novel potential regulatory genes. The results bring great contribution to studies related to T. harzianum and to fungal genomics regarding second generation bioethanol productionDoutoradoGenetica de MicroorganismosDoutora em Genética e Biologia Molecula

Analysis Of Genomic Regions Of Trichoderma Harzianum Ioc-3844 Related To Biomass Degradation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (similar to 150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes.104Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [proc. 2009/13185-3

Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions

Additional file 3: Table S3. Percent identity matrix between the GH3 β-glucosidase. The multiple sequence alignment was performed using the Clustal Omega server ( http://www.ebi.ac.uk/Tools/msa/clustalo/ )

Transcriptome Profile of Trichoderma harzianum IOC-3844 Induced by Sugarcane Bagasse

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. in the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. the sequencing generated 14.7 Gbp for downstream analyses. de novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. the present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Campinas UNICAMP, CBMEG, Campinas, SP, BrazilBrazilian Ctr Res Energy & Mat CNPEM, Brazilian Bioethanol Sci & Technol Lab CTBE, Campinas, SP, BrazilUniv São Paulo, Phys Inst Sao Carlos, Sao Carlos, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilUniv Campinas UNICAMP, Dept Plant Biol, Inst Biol, Campinas, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilWeb of Scienc

Vapd In Xylella Fastidiosa Is A Thermostable Protein With Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.10e014576

Microbial oil production in sugarcane bagasse hemicellulosic hydrolysate without nutrient supplementation by a Rhodosporidium toruloides adapted strain

Sugarcane bagasse hemicellulosic hydrolysate (SCBH) is a low-cost substrate for single-cell oil (SCO) production. However, this hydrolysate has an undesirable low carbon/nitrogen (C/N) ratio and contains inhibitors. Yeast adaptation is a simple strategy to overcome the presence of inhibitors, while the concentration of the hydrolysate and glycerol supplementation are alternatives to solve the low C/N limitation. This work aimed to investigate the adaptation of the yeast Rhodosporidium toruloides in undetoxified SCBH and its use in SCO production. The adapted strain produced more lipids than the parental strain: the concentration of SCBH or the addition of glycerol increased the lipid content, concentration, and productivity to at least 108%, 175%, and 118%, respectively. Lipid production in SCBH was done without salts or nitrogen supplementation. Transcriptomic study showed that hydrolysate-tolerance- and lipid accumulation-related genes were strongly induced. These genes can be targets for metabolic engineering571625CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação2013/03103-

Transcriptome sequencing reveals genes and adaptation pathways in salmonella typhimurium inoculated in four low water activity foods

Salmonella enterica serotypes have been reported as the agent of various outbreaks occurred after the consumption of low water activity (a(w)) foods. When the pathogen encounters harsh conditions, several regulatory networks are activated through dynamic differential gene expression that lead to cell survival for prolonged periods. In this work, the transcriptome of S. enterica serovar Typhimurium using RNA-Seq, after cells' inoculation in four distinct types of low a(w) foods ( milk chocolate, powdered milk, black pepper, and dried pet food), following storage at 25 degrees C per 24 and 72 h was studied. The findings of this study suggest that gene regulation is influenced by the food composition mainly in the first 24 h post-inoculum, proceeded by the induction of similar genes shared among all samples. It was possible to evaluate the differences on each type of food matrix regarding the bacteria adaptation, as well as the similarities provoked by low a(w). The results reveal genes that may play key roles in response to desiccation in Salmonella, as well as the pathways in which they are involved82426435CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302763/2014-7; 305804/2017-0não tem2014/17387-

Sporeforming bacteria in beer: Occurrence, diversity, presence of hop resistance genes and fate in alcohol-free and lager beers

The aim of this study was to assess the occurrence of sporeforming bacteria in different types of beers (n = 163) and to assess the presence of hor genes in the isolates. Additionally, the study aimed to evaluate the fate of five representative sporeforming bacteria harboring horA and horC genes in alcohol-free and lager beers. Two hundred and sixty (n = 260) sporeforming bacteria belonging to eight different genera were isolated from beers, i.e., Bacillus (n = 118), Paenibacillus (n = 89) and Brevibacillus (n = 41), Lysinbacillus (n = 6), Cohnella (n = 3), Rummeliibacillus (n = 1), Alicyclobacillus (n = 1), and Anoxybacillus (n = 1), respectively. A predominance of members within the Bacillus cereus sensu lato (n = 72; 27.1%), followed by B. megaterium (n = 18; 7%), P. validus (n = 16; 6.1%), P. humicus (n = 13; 5%), P. alginolyticus (n = 13; 5%) and Br. brevis (n = 13; 5%) was observed in beer samples analyzed. Only 5% (n = 14) out of 260 sporeforming bacterial isolates recovered from beers harbored one or both horA and horC genes. Only one (0.3%) isolate, i.e., Bacillus cereus sensu lato (identified as B. thuringiensis LMQA 206) presented both horA and horC genes. None of the five bacterial sporeforming strains harboring horA or horC genes inoculated was able to grow in the beers throughout the storage period studied, and no spoilage was detected. The results of this study indicated a widespread occurrence of sporeforming bacteria in several types of beers from different brands, highlighting that measures should be taken to reduce the occurrence of sporeforming bacteria considering stability and safety concerns.Fil: Munford, Allan R. G.. Universidade Estadual de Campinas; BrasilFil: Alvarenga, Verônica O.. Universidade Estadual de Campinas; BrasilFil: Prado Silva, Leonardo do. Universidade Estadual de Campinas; BrasilFil: Crucello, Aline. Universidade Estadual de Campinas; BrasilFil: Campagnollo, Fernanda B.. Universidade Estadual de Campinas; BrasilFil: Chaves, Rafael D.. Universidade Estadual de Campinas; BrasilFil: Oteiza, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centro de Investigación y Asistencia Técnica a la Industria; ArgentinaFil: Sant'Ana, Anderson de Souza. Universidade Estadual de Campinas; Brasi

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from xylella fastidiosa overexpressed in escherichia coli

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa822284289CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2001/07533-7; 2008/55690-