Investigation of protein secretion and secretion stress in Ashbya gossypii

Background: Ashbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress. Results: A computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitol Conclusions: This study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.This work was financially supported by Fundacao para a Ciencia e a Tecnologia, Portugal, through: PhD grant SFRH/BD/30229/2006 to OR, MIT-Portugal Program PhD grant SFRH/BD/39112/2007 to TQA, Project AshByofactory (PTDC/EBB-EBI/101985/2008 - FCOMP-01-0124-FEDER-009701), Project RECI/BBB-EBI/0179/2012 - FCOMP-01-0124-FEDER-027462, Strategic Project PEst-OE/EQB/LA0023/2013 and Project BioInd (NORTE-07-0124-FEDER000028) co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. We thank Dominik Mojzita and Mari Hakkinen from VTT Finland for their assistance with the microarray sample preparation, hybridization and data acquisition

Comparison of protein coding gene contents of the fungal phyla Pezizomycotina and Saccharomycotina

BACKGROUND: Several dozen fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been used to analyse specific sub groups of fungi. Notably, analysis of the phylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively. RESULTS: Our analysis shows that based on genome redundancy, the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism in N. crassa. Interestingly in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The genes specific to Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have diverged from those of Pezizomycotina. In addition, we highlight several individual gene families with interesting phylogenetic distributions. CONCLUSION: Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the responsible gene families are likely to evolve fast. Both types of fungal products can be of commercial value, or on the other hand cause harm to humans. In addition, a great number of novel predicted and known enzymes are found from all fungi except Saccharomycotina. Therefore further studies and exploitation of fungal metabolism appears very promising

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion

Errata: 10.1016/j.jcyt.2017.06.003Background. Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Methods. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. Results. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T-SCM, CD95(+)CD45RO(-)CD45RA(+)CD27(+)) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CART cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CART cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10 fold cell expansion and the cells were functionally potent. Discussion. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost:effective T-cell manufacturing.Peer reviewe

Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties

<p>Abstract</p> <p>Background</p> <p><it>Trichoderma reesei </it>is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of <it>T. reesei </it>by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general.</p> <p>Results</p> <p>We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production.</p> <p>Conclusions</p> <p>aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.</p

Prediction and impact of personalized donation intervals

Publisher Copyright: © 2021 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.Background and Objectives: Deferral of blood donors due to low haemoglobin (Hb) is demotivating to donors, can be a sign for developing anaemia and incurs costs for blood establishments. The prediction of Hb deferral has been shown to be feasible in a number of studies based on demographic, Hb measurement and donation history data. The aim of this paper is to evaluate how state-of-the-art computational prediction tools can facilitate nationwide personalized donation intervals. Materials and Methods: Using donation history data from the last 20 years in Finland, FinDonor blood donor cohort data and blood service Biobank genotyping data, we built linear and non-linear predictors of Hb deferral. Based on financial data from the Finnish Red Cross Blood Service, we then estimated the economic impacts of deploying such predictors. Results: We discovered that while linear predictors generally predict Hb relatively well, they have difficulties in predicting low Hb values. Overall, we found that non-linear or linear predictors with or without genetic data performed only slightly better than a simple cutoff based on previous Hb. However, if any of our deferral prediction methods are used to assign temporary prolongations of donation intervals for females, then our calculations indicate cost savings while maintaining the blood supply. Conclusion: We find that even though the prediction accuracy is not very high, the actual use of any of our predictors in blood collection is still likely to bring benefits to blood donors and blood establishments alike.Peer reviewe

Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae

BACKGROUND: Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR) pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. RESULTS: Chemostat cultures of T. reesei expressing human tissue plasminogen activator (tPA) and batch bioreactor cultures treated with dithiothreitol (DTT) to prevent correct protein folding were analysed with cDNA subtraction and cDNA-amplified fragment length polymorphism (AFLP) experiments. ESTs corresponding to 457 unique genes putatively induced under secretion stress were isolated and the expression pattern of 60 genes was confirmed by Northern analysis. Expression of these genes was also studied in a strain over-expressing inositol-requiring enzyme 1 (IREI) protein, a sensor for the UPR pathway. To compare the data with that of S. cerevisiae, published transcriptome profiling data on various stress responses in S. cerevisiae was reanalysed. The genes up-regulated in response to secretion stress included a large number of secretion related genes in both organisms. In addition, analysis of T. reesei revealed up regulation of the cpc1 transcription factor gene and nucleosomal genes. The induction of the cpcA and histone gene H4 were shown to be induced also in cultures of Aspergillus nidulans treated with DTT. CONCLUSION: Analysis of the genes induced under secretion stress has revealed novel features in the stress response in T. reesei and in filamentous fungi. We have demonstrated that in addition to the previously rather well characterised induction of genes for many ER proteins or secretion related proteins also other types of responses exist

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