In silico proteomic analysis provides insights into phylogenomics and plant biomass deconstruction potentials of the Tremelalles

Basidiomycetes populate a wide range of ecological niches but unlike ascomycetes, their capabilities to decay plant polymers and their potential for biotechnological approaches receive less attention. Particularly, identification and isolation of CAZymes is of biotechnological relevance and has the potential to improve the cache of currently available commercial enzyme cocktails toward enhanced plant biomass utilization. The order Tremellales comprises phylogenetically diverse fungi living as human pathogens, mycoparasites, saprophytes or associated with insects. Here, we have employed comparative genomics approaches to highlight the phylogenomic relationships among thirty-five Tremellales and to identify putative enzymes of biotechnological interest encoded on their genomes. Evaluation of the predicted proteomes of the thirty-five Tremellales revealed 6,918 putative carbohydrate-active enzymes (CAZYmes) and 7,066 peptidases. Two soil isolates, Saitozyma podzolica DSM 27192 and Cryptococcus sp. JCM 24511, show higher numbers harboring an average of 317 compared to a range of 267–121 CAZYmes for the rest of the strains. Similarly, the proteomes of the two soil isolates along with two plant associated strains contain higher number of peptidases sharing an average of 234 peptidases compared to a range of 226–167 for the rest of the strains. Despite these huge differences and the apparent enrichment of these enzymes among the soil isolates, the data revealed a diversity of the various enzyme families that does not reflect specific habitat type. Growth experiment on various carbohydrates to validate the predictions provides support for this view. Overall, the data indicates that the Tremellales could serve as a rich source of both CAZYmes and peptidases with wide range of potential biotechnological relevance

Optimization of glycolipid synthesis in hydrophilic deep eutectic solvents

Glycolipids are considered an alternative to petrochemically based surfactants because they are non-toxic, biodegradable, and less harmful to the environment while having comparable surface-active properties. They can be produced chemically or enzymatically in organic solvents or in deep eutectic solvents (DES) from renewable resources. DES are non-flammable, non-volatile, biodegradable, and almost non-toxic. Unlike organic solvents, sugars are easily soluble in hydrophilic DES. However, DES are highly viscous systems and restricted mass transfer is likely to be a major limiting factor for their application. Limiting factors for glycolipid synthesis in DES are not generally well understood. Therefore, the influence of external mass transfer, fatty acid concentration, and distribution on initial reaction velocity in two hydrophilic DES (choline:urea and choline:glucose) was investigated. At agitation speeds of and higher than 60 rpm, the viscosity of both DES did not limit external mass transfer. Fatty acid concentration of 0.5 M resulted in highest initial reaction velocity while higher concentrations had negative effects. Fatty acid accessibility was identified as a limiting factor for glycolipid synthesis in hydrophilic DES. Mean droplet sizes of fatty acid-DES emulsions can be significantly decreased by ultrasonic pretreatment resulting in significantly increased initial reaction velocity and yield (from 0.15 ± 0.03 μmol glucose monodecanoate/g DES to 0.57 ± 0.03 μmol/g) in the choline: urea DES. The study clearly indicates that fatty acid accessibility is a limiting factor in enzymatic glycolipid synthesis in DES. Furthermore, it was shown that physical pretreatment of fatty acid-DES emulsions is mandatory to improve the availability of fatty acids

Draft Genome Sequence of the Oleaginous Yeast Saitozyma podzolica (syn. Cryptococcus podzolicus) DSM 27192

We report here the draft genome of Saitozyma podzolica DSM 27192 sequenced based on PacBio chemistry. This yeast isolate produces large amounts of single-cell oil (SCO) and gluconic acid (GA). Information from the genome sequence will provide additional insight into the genetic mechanism of SCO and GA metabolism in this organism

Direct transesterification of microalgae after Pulsed Electric Field ( PEF ) treatment

Background Lipid extraction is a major bottleneck for the commercialization of microalgae due to energy costs involved during solvent recycling. Direct transesterification offers the possibility to bypass the extraction step by immediately converting the lipids to fatty acids methyl esters (FAMEs). In this study, the efficiency of direct transesterification after pulsed electric field (PEF) was evaluated. Freshly harvested Auxenochlorella protothecoides (A. protothecoides), cultivated either autotrophically or mixotrophically, was subjected to PEF. Two treatment energies were tested, 0.25 MJ/kgdw and 1.5 MJ/kgdw and results were compared with conventional two-step transesterification. Results For autotrophically grown A. protothecoides, the percentage of the total FAMEs recovered from untreated biomass and microalgae treated with 0.25 MJ/kgdw was 30% for both cases while for 1.5 MJ/kgdw it was 65%. A 24-h incubation step between PEF-treatment and direct transesterification significantly improved the results. Untreated biomass remained stable with 30% of FAMEs, while with both treatment energies a 97% FAME recovery was achieved. However, for mixotrophic A. protothecoides the process was not as effective. Approximately 30% of FAMEs were recovered for all three conditions immediately after PEF with only a marginal increase after incubation. The reason for this different behavior of the two cultivation modes is unknown and under investigation. Conclusions Overall, the synergy between PEF and direct transesterification was proven to have potential, in particular for autotrophic microalgae. Its implementation and further optimization in a biorefinery therefore merits further attention

Direct transesterification of microalgae after Pulsed Electric Field ( PEF ) treatment

Background Lipid extraction is a major bottleneck for the commercialization of microalgae due to energy costs involved during solvent recycling. Direct transesterification offers the possibility to bypass the extraction step by immediately converting the lipids to fatty acids methyl esters (FAMEs). In this study, the efficiency of direct transesterification after pulsed electric field (PEF) was evaluated. Freshly harvested Auxenochlorella protothecoides (A. protothecoides), cultivated either autotrophically or mixotrophically, was subjected to PEF. Two treatment energies were tested, 0.25 MJ/kgdw and 1.5 MJ/kgdw and results were compared with conventional two-step transesterification. Results For autotrophically grown A. protothecoides, the percentage of the total FAMEs recovered from untreated biomass and microalgae treated with 0.25 MJ/kgdw was 30% for both cases while for 1.5 MJ/kgdw it was 65%. A 24-h incubation step between PEF-treatment and direct transesterification significantly improved the results. Untreated biomass remained stable with 30% of FAMEs, while with both treatment energies a 97% FAME recovery was achieved. However, for mixotrophic A. protothecoides the process was not as effective. Approximately 30% of FAMEs were recovered for all three conditions immediately after PEF with only a marginal increase after incubation. The reason for this different behavior of the two cultivation modes is unknown and under investigation. Conclusions Overall, the synergy between PEF and direct transesterification was proven to have potential, in particular for autotrophic microalgae. Its implementation and further optimization in a biorefinery therefore merits further attention

Co-production of single cell oil and gluconic acid using oleaginous Cryptococcus podzolicus DSM 27192

Background: The co-production of single cell oil (SCO) with value-added products could improve the economic viability of industrial SCO production. The newly isolated oleaginous yeast Cryptococcus podzolicus DSM 27192 was able to co-produce SCO intracellularly and gluconic acid (GA) extracellularly. In this study, the metabolic regulation of carbon distribution between SCO and GA through process optimization was comprehensively investigated. Results: The carbon flow distribution between SCO and GA was significantly influenced by the cultivation conditions, such as nitrogen sources, glucose concentration and dissolved oxygen concentration. It was found that organic nitrogen sources were beneficial for SCO accumulation, while GA production was decreased. Dissolved oxygen concentration (DOC) was found to enhance SCO accumulation, while high glucose concentration was more favorable for GA accumulation. Hence, a two-stage DOC or glucose concentration-controlled strategy was designed to improve cell growth and direct carbon distribution between SCO and GA. Moreover, C. podzolicus DSM 27192 could degrade its stored lipids to synthesize GA in the late stationary phase, although considerable amounts of glucose remained unconsumed in the culture medium, indicating the importance of fermentation time control in co-production systems. All these observations provide opportunity to favor either the production of SCO or GA or rather their simultaneous production. Conclusions: Co-production of SCO and GA by C. podzolicus DSM 27192 can improve the economical value for microbial lipid-derived biodiesel production. Moreover, the results of the proposed co-production strategy might give guidance for other co-production systems

Characterization of newly isolated oleaginous yeasts - Cryptococcus podzolicus, Trichosporon porosum and pichia segobiensis

The yeast strains Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis were isolated from soil samples and identified as oleaginous yeast strains beneficial for the establishment of microbial production processes for sustainable lipid production suitable for several industrial applications. When cultured in bioreactors with glucose as the sole carbon source C. podzolicus yielded 31.8% lipid per dry biomass at 20°C, while T. porosum yielded 34.1% at 25°C and P. segobiensis 24.6% at 25°C. These amounts correspond to lipid concentrations of 17.97 g/L, 17.02 g/L and 12.7 g/L and volumetric productivities of 0.09 g/Lh, 0.1 g/Lh and 0.07 g/Lh, respectively. During the culture of C. podzolicus 30 g/l gluconic acid was detected as by-product in the culture broth and 12 g/L gluconic acid in T. porosum culture. The production of gluconic acid was eliminated for both strains when glucose was substituted by xylose as the carbon source. Using xylose lipid yields were 11.1 g/L and 13.9 g/L, corresponding to 26.8% and 33.4% lipid per dry biomass and a volumetric productivity of 0.07 g/Lh and 0.09 g/Lh, for C. podzolicus and T. porosum respectively. The fatty acid profile analysis showed that oleic acid was the main component (39.6 to 59.4%) in all three strains and could be applicable for biodiesel production. Palmitic acid (18.4 to 21.1%) and linolenic acid (7.5 to 18.7%) are valuable for cosmetic applications. P. segobiensis had a considerable amount of palmitoleic acid (16% content) and may be suitable for medical applications

the gatto study a phase i of the anti egfr tomuzotuximab to in combination with the anti muc1 gatipotuzumab gat in patients with egfr positive solid tumors

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Mutation or loss of Wilms' tumor gene 1 (WT1) are not major reasons for immune escape in patients with AML receiving WT1 peptide vaccination

<p>Abstract</p> <p>Background</p> <p>Efficacy of cancer vaccines may be limited due to immune escape mechanisms like loss or mutation of target antigens. Here, we analyzed 10 HLA-A2 positive patients with acute myeloid leukemia (AML) for loss or mutations of the WT1 epitope or epitope flanking sequences that may abolish proper T cell recognition or epitope presentation.</p> <p>Methods</p> <p>All patients had been enrolled in a WT1 peptide phase II vaccination trial (NCT00153582) and ultimately progressed despite induction of a WT1 specific T cell response. Blood and bone marrow samples prior to vaccination and during progression were analyzed for mRNA expression level of WT1. Base exchanges within the epitope sequence or flanking regions (10 amino acids N- and C-terminal of the epitope) were assessed with melting point analysis and sequencing. HLA class I expression and WT1 protein expression was analyzed by flow cytometry.</p> <p>Results</p> <p>Only in one patient, downregulation of WT1 mRNA by 1 log and loss of WT1 detection on protein level at time of disease progression was observed. No mutation leading to a base exchange within the epitope sequence or epitope flanking sequences could be detected in any patient. Further, no loss of HLA class I expression on leukemic blasts was observed.</p> <p>Conclusion</p> <p>Defects in antigen presentation caused by loss or mutation of WT1 or downregulation of HLA molecules are not the major basis for escape from the immune response induced by WT1 peptide vaccination.</p

Analogue peptides for the immunotherapy of human acute myeloid leukemia

Accepted manuscript. The final publication is available at: http://link.springer.com/article/10.1007%2Fs00262-015-1762-9The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies