Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-ß-glucanases that efficiently hydrolyse cellulosic substrates.

The hydrolysis of cellulose into fermentable sugars is a costly and rate-limiting step in the production of biofuels from renewable feedstocks. Developing new cellulase systems capable of increased cellulose hydrolysis rates would reduce biofuel production costs. With this in mind, we screened 55 fungal endoglucanases for their abilities to be expressed at high levels by Aspergillus niger and to hydrolyze amorphous cellulose at rates significantly greater than that obtained with TrCel5A, one of the major endoglucanases in the Trichoderma reesei cellulase system. This screen identified three endoglucanases, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A and Sporotrichum thermophile StCel5A. We determined that A. niger expressed the three endoglucanases at relatively high levels (≥0.3 g/l) and that the hydrolysis rate of ApCel5A and StCel5A with carboxymethylcellulose 4M as substrate was five and two times greater than the T. reesei Cel5A. The ApCel5A, GtCel12A and StCel5A enzymes also demonstrated significant synergy with Cel7A/CbhI, the major exoglucanase in the T. reesei cellulase system. The three endoglucanases characterized in this study are, therefore, promising candidate endoglucanases for developing new cellulase systems with increased rates of cellulose saccharification.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

CDC42 is required for polarized growth in human pathogen Candida albicans.

Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42p(G12V) or CaCdc42p(D118A) ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p(G12V) was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.

Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes.

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP-mediated gene deletion. Both wild-type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site-specific recombinase FLP. Expression of a C. albicans-adapted FLP gene under the control of an inducible promoter generated cell populations consisting of > or = 99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by aggregating and expressing hypha-specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP-mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.