Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.     

The Putative Protein Methyltransferase LAE1 of Trichoderma atroviride Is a Key Regulator of Asexual Development and Mycoparasitism

In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δ lae1 and 30–50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δ lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δ lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.     

Variant RONΔ160 of the RON receptor tyrosine kinase promotes the growth and invasion in vitro and in vivo in gastric cancer cell lines

Background

Recepteur d’origine nantais (RON) is a receptor tyrosine kinase whose overexpression has been observed in human gastric cancers. This study aimed to determine whether overexpression of the variant RONΔ160 could induce tumorigenicity of gastric cancer cells in vitro or in vivo, and whether its specific small molecule inhibitor (Compound I) could inhibit the effect of RONΔ160.

Methods

We constructed human gastric cancer cell line MGC-803 that was stably transfected with a recombinant plasmid expressing RONΔ160, and the effect of RONΔ160 overexpression and macrophage-stimulating protein (MSP) activation on proliferation, migration and invasion abilities of MGC-803 cells were evaluated. Tumor-bearing mice with gastric cancer cells were used to analyze the effects of RONΔ160 overexpression and Compound I on implanted tumor growth.

Results

In vitro, overexpression of RONΔ160 in MGC-803 cells resulted changes to their cell morphology, and promoted cell proliferation, migration and invasion. In addition, overexpression of RONΔ160 increased the proportion of cells in the S phase. The effect of RONΔ160 was significantly enhanced by induction of MSP inducing (p < 0.05). In vivo, RONΔ160 promoted the growth of MGC-803 cells in nude mice, including increased tumor size and weight, and lower tumor incubation period. The Compound I inhibited the tumorigenic abilities of RONΔ160 (p <0.05).

Conclusions

The results indicate that overexpression of the variant RONΔ160 altered the phenotype and tumorigenicity of MGC-803 cells. Its specific small molecule inhibitor could inhibit the effect of RONΔ160. Therefore, the variant RONΔ160 may become a potential therapeutic target for gastric cancer.     

The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex.     

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

Background

Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus.

Methodology/Principal Findings

Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Δ mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Δ cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Δ cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Δ mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p.

Conclusions/Significance

The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Δ cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Δ cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Δ cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target.     

The influences of PRG-1 on the expression of small RNAs and mRNAs

Background

In metazoans, Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that mutation of prg-1 causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.

Results

In this study, we wanted to systematically demonstrate the function of PRG-1 in the regulation of small RNAs and their targets. By analyzing small RNAs and mRNAs with and without a mutation in prg-1 during C. elegans development, we demonstrated that (1) mutation of prg-1 leads to a decrease in the expression of 21U-RNAs, and causes 35 ~ 40% of miRNAs to be down-regulated; (2) in C. elegans, approximately 3% (6% in L4) of protein-coding genes are differentially expressed after mutating prg-1, and 60 ~ 70% of these substantially altered protein-coding genes are up-regulated; (3) the target genes of the down-regulated miRNAs and the candidate target genes of the down-regulated 21U-RNAs are enriched in the up-regulated protein-coding genes; and (4) PRG-1 regulates protein-coding genes by down-regulating small RNAs (miRNAs and 21U-RNAs) that target genes that participate in the development of C. elegans.

Conclusions

In prg-1-mutated C. elegans, the expression of miRNAs and 21U-RNAs was reduced, and the protein-coding targets, which were associated with the development of C. elegans, were up-regulated. This may be the mechanism underlying PRG-1 function.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-321) contains supplementary material, which is available to authorized users.     

Asc1 Supports Cell-Wall Integrity Near Bud Sites by a Pkc1 Independent Mechanism

Background

The yeast ribosomal protein Asc1 is a WD-protein family member. Its mammalian ortholog, RACK1 was initially discovered as a receptor for activated protein C kinase (PKC) that functions to maintain the active conformation of PKC and to support its movement to target sites. In the budding yeast though, a connection between Asc1p and the PKC signaling pathway has never been reported.

Methodology/Principal Findings

In the present study we found that asc1-deletion mutant (asc1Δ) presents some of the hallmarks of PKC signaling mutants. These include an increased sensitivity to staurosporine, a specific Pkc1p inhibitor, and susceptibility to cell-wall perturbing treatments such as hypotonic- and heat shock conditions and zymolase treatment. Microscopic analysis of asc1Δ cells revealed cell-wall invaginations near bud sites after exposure to hypotonic conditions, and the dynamic of cells'' survival after this stress further supports the involvement of Asc1p in maintaining the cell-wall integrity during the mid-to late stages of bud formation. Genetic interactions between asc1 and pkc1 reveal synergistic sensitivities of a double-knock out mutant (asc1Δ/pkc1Δ) to cell-wall stress conditions, and high basal level of PKC signaling in asc1Δ. Furthermore, Asc1p has no effect on the cellular distribution or redistribution of Pkc1p at optimal or at cell-wall stress conditions.

Conclusions/Significance

Taken together, our data support the idea that unlike its mammalian orthologs, Asc1p acts remotely from Pkc1p, to regulate the integrity of the cell-wall. We speculate that its role is exerted through translation regulation of bud-site related mRNAs during cells'' growth.     

Methanol extract from Vietnamese Caesalpinia sappan induces apoptosis in HeLa cells

Background

This study evaluated the cytotoxic activity of extracts from Caesalpinia sappan heartwood against multiple cancer cell lines using an MTT cell viability assay. The cell death though induction of apoptosis was as indicated by DNA fragmentation and caspase-3 enzyme activation.

Results

A methanol extract from C. sappan (MECS) showed cytotoxic activity against several of the cancer cell lines. The most potent activity exhibited by the MECS was against HeLa cells with an IC₅₀ value of 26.5 ± 3.2 μg/mL. Treatment of HeLa cells with various MECS concentrations resulted in growth inhibition and induction of apoptosis, as indicated by DNA fragmentation and caspase-3 enzyme activation.

Conclusion

This study is the first report of the anticancer properties of the heartwood of C. sappan native to Vietnam. Our findings demonstrate that C. sappan heartwood may have beneficial applications in the field of anticancer drug discovery.     

Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus

Background

The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3–16); hence, the mechanisms responsible for changes in copy number at this locus are unclear.

Results

In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure.

Conclusions

Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-614) contains supplementary material, which is available to authorized users.     

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

Background

Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.

Results

Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2^nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.

Conclusion

Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.     

How slug herbivory of juvenile hybrid willows alters chemistry,growth and subsequent susceptibility to diverse plant enemies

Background and Aims

Selective feeding by herbivores, especially at the seedling or juvenile phase, has the potential to change plant traits and ultimately the susceptibility of surviving plants to other enemies. Moreover, since hybridization is important to speciation and can lead to introgression of traits between plant species, differential feeding (herbivore-induced mortality) can influence the expression of resistance traits of hybrids and ultimately determine the consequences of hybridization. While it would be expected that herbivore-induced mortality would lead to greater resistance, there may be trade-offs whereby resistance to one herbivore increases susceptibility to others. The hypothesis was tested that the exotic slug, Arion subfuscus, causes non-random survival of hybrid willows and alters plant: (1) susceptibility to slugs; (2) secondary and nutritional chemistry, and growth; and (3) susceptibility to other phytophages.

Methods

Two populations of plants, control and selected, were created by placing trays of juvenile willows in the field and allowing slugs access to only some. When ≤10 individuals/tray remained (approx. 85 % mortality), ‘selected’ and undamaged ‘control’ trays were returned to a common area. Traits of these populations were then examined in year 1 and in subsequent years.

Key Results

The selected population was less palatable to slugs. Surprisingly, foliar concentrations of putative defence traits (phenolic glycosides and tannins) did not differ between treatments, but the selected population had higher foliar nitrogen and protein, lower carbon to nitrogen ratio and greater above-ground biomass, indicating that vigorously growing plants were inherently more resistant to slugs. Interestingly, selected plants were more susceptible to three phytophages: an indigenous pathogen (Melampsora epitea), a native herbivorous beetle (Chrysomela knabi) and an exotic willow leaf beetle (Plagiodera versicolora).

Conclusions

This exotic slug changed the population structure of F₂ hybrid willows in unanticipated ways. Defence expression remained unchanged, while nutritional and growth traits changed. These changes caused plants to be more susceptible to other plant enemies. Other exotic herbivore species are anticipated to have similar direct and indirect effects on native plant populations.