A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background

Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on d-glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background

Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation

The aim of this study was to apply a generated Δtku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δtku70Δlcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1

Bragg coherent x-ray diffractive imaging of a single indium phosphide nanowire

Three-dimensional (3D) Bragg coherent x-ray diffractive imaging (CXDI) with a nanofocused beam was applied to quantitatively map the internal strain field of a single indium phosphide nanowire. The quantitative values of the strain were obtained by pre-characterization of the beam profile with transmission ptychography on a test sample. Our measurements revealed the 3D strain distribution in a region of 150 nm below the catalyst Au particle. We observed a slight gradient of the strain in the range of ±0.6% along the [111] growth direction of the nanowire. We also determined the spatial resolution in our measurements to be about 10 nm in the direction perpendicular to the facets of the nanowire. The CXDI measurements were compared with the finite element method simulations and show a good agreement with our experimental results. The proposed approach can become an effective tool for in operando studies of the nanowires

X-ray induced grain structure dynamics in Bi2Se3

Grain rotation in crystals often results in coarsening or refinement of the grains that modify the mechanical and thermal properties of materials. While many studies have explored how externally applied stress and temperature drive grain structure dynamics in nano-polycrystalline materials, the analogous studies on colossal grains have been limited, especially in the absence of external force. In this work, we used X-ray free electron laser pulses to irradiate single-crystalline bismuth selenide (Bi2Se3) and observed grain boundary formation and subsequent grain rotation in response to the X-ray radiation. Our observations with simultaneous X-ray diffraction and transmission X-ray microscopy demonstrate how intense X-ray radiation can rapidly change grain morphologies of initially single-crystalline material.Comment: 20 pages, 8 figures including 3 supplemental figure

Correlation of gene expression and protein production rate - a system wide study

<p>Abstract</p> <p>Background</p> <p>Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on <it>Saccharomyces cerevisiae </it>chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.</p> <p>Results</p> <p>We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of <it>T. reesei</it>. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.</p> <p>Conclusions</p> <p>Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).</p

Direct imaging of ultrafast lattice dynamics

Under rapid high-temperature, high-pressure loading, lattices exhibit complex elastic-inelastic responses. The dynamics of these responses are challenging to measure experimentally because of high sample density and extremely small relevant spatial and temporal scales. Here, we use an x-ray free-electron laser providing simultaneous in situ direct imaging and x-ray diffraction to spatially resolve lattice dynamics of silicon under high–strain rate conditions.We present the first imaging of a new intermediate elastic feature modulating compression along the axis of applied stress, and we identify the structure, compression, and density behind each observed wave. The ultrafast probe x-rays enabled time-resolved characterization of the intermediate elastic feature, which is leveraged to constrain kinetic inhibition of the phase transformation between 2 and 4 ns. These results not only address long-standing questions about the response of silicon under extreme environments but also demonstrate the potential for ultrafast direct measurements to illuminate new lattice dynamics

Horizontal Transfer of a Nitrate Assimilation Gene Cluster and Ecological Transitions in Fungi: A Phylogenetic Study

High affinity nitrate assimilation genes in fungi occur in a cluster (fHANT-AC) that can be coordinately regulated. The clustered genes include nrt2, which codes for a high affinity nitrate transporter; euknr, which codes for nitrate reductase; and NAD(P)H-nir, which codes for nitrite reductase. Homologs of genes in the fHANT-AC occur in other eukaryotes and prokaryotes, but they have only been found clustered in the oomycete Phytophthora (heterokonts). We performed independent and concatenated phylogenetic analyses of homologs of all three genes in the fHANT-AC. Phylogenetic analyses limited to fungal sequences suggest that the fHANT-AC has been transferred horizontally from a basidiomycete (mushrooms and smuts) to an ancestor of the ascomycetous mold Trichoderma reesei. Phylogenetic analyses of sequences from diverse eukaryotes and eubacteria, and cluster structure, are consistent with a hypothesis that the fHANT-AC was assembled in a lineage leading to the oomycetes and was subsequently transferred to the Dikarya (Ascomycota+Basidiomycota), which is a derived fungal clade that includes the vast majority of terrestrial fungi. We propose that the acquisition of high affinity nitrate assimilation contributed to the success of Dikarya on land by allowing exploitation of nitrate in aerobic soils, and the subsequent transfer of a complete assimilation cluster improved the fitness of T. reesei in a new niche. Horizontal transmission of this cluster of functionally integrated genes supports the “selfish operon” hypothesis for maintenance of gene clusters

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens