The demand for robust microbial cell factories that can produce valuable biomaterials while being resistant to stresses imposed by current bioprocesses is rapidly growing. R. toruloides is an emerging host that presents desirable features for bioproduction, since it can grow in a wide range of substrates and tolerate a variety of toxic compounds. In order to explore R. toruloides suitability for application as a cell factory in biorefineries, we sought to understand the transcriptional responses of this yeast when growing under experimental settings that simulated those used in biofuels-related industries. Thus, we performed RNA sequencing of the oleaginous, carotenogenic yeast in different contexts. The first ones were stress-related: two conditions of high temperature (37 ˚C and 42 ˚C) and two ethanol concentrations (2% and 4%), while the other was using the inexpensive and abundant sugarcane juice as substrate. Using transcriptomic data, differential expression and functional analysis were implemented to select differentially expressed genes and enriched pathways from each set-up. A reproducible bioinformatics workflow was developed for mining new regulatory elements. We then predicted, for the first time in this yeast, binding motifs for several transcription factors, including HAC1, ARG80, RPN4, ADR1, and DAL81. Most of the putative transcription factors uncovered here were involved in stress responses and found in the yeast genome. Our method for motif discovery provides a new realm of possibilities in the study of gene regulatory networks, not only for the emerging host R. toruloides, but for other organisms of biotechnological importance.
RESUMO.A haptoglobina é a principal proteína de fase aguda em ruminantes, sendo o seu uso amplamente pesquisado na clínica médica animal como biomarcador precoce de processos inflamatórios, infecciosos, danos teciduais e condições de estresse. Isto se deve ao fato de seus valores sanguíneos se apresentarem próximos de 0,05 mg/ml em bovinos saudáveis, e se elevarem em mais de 100 vezes durante a ocorrência das condições patológicas, antes mesmo que surjam sinais clínicos e ocorram alterações no leucograma. Com estas características, a haptoglobina apresenta um alto potencial para uso em diferentes segmentos da cadeia produtiva pecuária brasileira, sendo o objetivo desta revisão de literatura compilar os trabalhos científicos que utilizaram a esta proteína como bioindicador de saúde animal na bovinocultura de corte e leite. Verificou-se que, na pecuária de corte, a quantificação da haptoglobina pode contribuir com a triagem dos animais na inspeção ante morten, permitindo a identificação dos animais com doença subclínica que não foram diagnosticados pela inspeção visual; pode também contribuir para a verificação do bem-estar animal nos momentos após o transporte e pré-abate, evitandose o abate de animais com possíveis quadros de estresse que possam alterar a qualidade dos produtos cárneos. Na pecuária leiteira, a haptoglobina pode contribuir diretamente com a identificação de vacas com diferentes níveis de mastite clínica e subclínica, além de permitir a identificação de lotes de leite com alta concentração de células somáticas ou oriundos de rebanhos acometidos por agentes infecciosos. Para que seja fomentada a sua inclusão como ferramenta de monitoramento dentro cadeia produtiva pecuária brasileira, para uso por ambos médicos veterinários e estabelecimentos industriais de produtos de origem animal, recomenda-se a realização de estudos de validação do uso da haptoglobina como bioindicador de saúde animal em parceria com empresas do setor, de forma que a sua utilidade prática seja verificada diretamente pelos profissionais envolvidos.Palavras chave: diagnóstico, pecuária, proteína de fase aguda, sanidade animal Applicability of haptoglobin as a health bioindicator in beef and dairy cattle ABSTRACT. Haptoglobin is the main acute phase protein in ruminants, and its use is being widely investigated in animal clinics as an early biomarker of inflammation, infection, tissue damage and stress conditions. This occurs due to the fact that this protein presents blood values close to 0.05 mg / ml in healthy cattle, but it increases more than 100 times during the pathological conditions, even before clinical signs appear and changes in
The demand for robust microbial cell factories that produce valuable biomaterials while resisting stresses imposed by current bioprocesses is rapidly growing. Rhodosporidium toruloides is an emerging host that presents desirable features for bioproduction, since it can grow in a wide range of substrates and tolerate a variety of toxic compounds. To explore R. toruloides suitability for application as a cell factory in biorefineries, we sought to understand the transcriptional responses of this yeast when growing under experimental settings that simulated those used in biofuels-related industries. Thus, we performed RNA sequencing of the oleaginous, carotenogenic yeast in different contexts. The first ones were stress-related: two conditions of high temperature (37 and 42°C) and two ethanol concentrations (2 and 4%), while the other used the inexpensive and abundant sugarcane juice as substrate. Differential expression and functional analysis were implemented using transcriptomic data to select differentially expressed genes and enriched pathways from each set-up. A reproducible bioinformatics workflow was developed for mining new regulatory elements. We then predicted, for the first time in this yeast, binding motifs for several transcription factors, including HAC1, ARG80, RPN4, ADR1, and DAL81. Most putative transcription factors uncovered here were involved in stress responses and found in the yeast genome. Our method for motif discovery provides a new realm of possibilities in studying gene regulatory networks, not only for the emerging host R. toruloides, but for other organisms of biotechnological importance.
INTEGRATIVE ANALYSIS OF GENE EXPRESSION DATA OF RHODOSPORIDIUM TORULOIDES UNDER RELEVANT CONDITIONS TO THE BIOTECHNOLOGICAL INDUSTRY"Rhodosporidium toruloides (R. toruloides) has been highlighted as an alternative to model organisms already used to conduct industrial bioprocesses due to some characteristics such as: (i) efficient processing of complex carbon sources, (ii) high storage and production of lipids, (iii) carotenogenesis, (iv) innate resistance to some chemical inhibitors present in lignocellulosic hydrolisates, and (v) absence of pathogenicity. However, to better exploit the potentials of this yeast and improve the efficiency of these bioprocesses, it is needed to know how it biologically deals with different growth conditions. Thus, our work aimed to understand the landscape of the transcriptional response of R. toruloides under different culture conditions through the integration of gene expression data.For this purpose, 54 RNA-Seq data of R. toruloides IFO0880 ∆ku70 strain were analysed. The quality control of raw reads was performed by FastQC and Trimmomatic softwares, as well as read alignment was done by Hisat2, transcripts assembly was done by Stringtie, differential expression analysis was performed by DESeq2, transcripts functional annotation and functional enrichment analysis was performed by Blast2GO. The results of the growth profile analysis indicate that the yeast is able to grow even in the presence of stresses induced by 5% ethanol, 0.15% acetic acid and 40º C. By analyzing the total number of differentially expressed genes (DEGs) of each condition we can infer that the number of DEGs changes in function of the culture conditions as a result of the level of system disturbance caused by each condition. Despite this difference in total DEGs number between conditions, the number of up-regulated and down-regulated DEGs inside each condition is really near to the balance, supposedly as a result of a fight to achieve a system homeostasis. Furthermore, our enrichment analysis results of the stress conditions provide some evidences that the major players in the response to stress are placed at the cell membrane and the main molecular functions associated to this response are the oxidoreductase activity and transmembrane transporter activity. Considering the carbon source conditions, the response is associated with carbohydrate metabolism, ATP production, ribosome biogenesis, translation and cell division. Thus, this work provides a more integrated biological knowledge of the transcriptional response of R. toruloides under conditions relevant to the biotechnology industry.
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