Secretome analysis of Ashbya gossypii

To explore the potential Ashbya gossypii extracellular secretome two computational protocols were used. The 4726 protein-encoding genes predicted in the genome of A. gossypii strain ATCC 10895 were the data source for the analysis. Depending on the computational methods used, 171 to 185 proteins were predicted to be secreted proteins. Based on the results of the present study, A. gossypii is more similar to the closely related yeast Saccharomyces cerevisiae than to other filamentous fungi in its secretion ability

The N-glycome of Ashbya gossypii´s native secreted proteins

Within the framework of a project aimed at analysing the basis of Ashbya gossypii protein secretory capabilities, we focused our attention on the processing of glycoproteins. Complex carbohydrate structures, glycans, are essential components of glycoproteins, contributing to the functional conformation and to the selection of the final destination of these proteins. Presently, the glycosylation pattern and degree of glycosylation performed by A. gossypii remains unexplored. In this study, using MALDI-TOF mass spectrometric profiling, we have examined the N-glycans present in A. gossypii‘s native glycoproteins secreted under different culture conditions. N-glycan profiling revealed that the major glycan species derived from A. gossypii‘s secreted proteins are small glycans containing core-type structures with eight to eleven hexoses (H8±11N2). Growth in defined minimal medium also resulted in charged glycan structures that were slightly bigger and were either phosphorylated or sulphated (H13±15N2P/S). In contrast, no charged glycans could be detected when complex rich medium was used. Some signals detected in the spectra may correspond to more complex N-glycans structures containing fucose, phosphate and pentose residues. Understanding A. gossypii’s glycobiology offers a basis for future studies of its protein secretion processes and may possibly be of practical impact in the production of heterologous proteins

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

Identification in the mould Hypocrea jecorina of a gene encoding an NADP+: d-xylose dehydrogenase

A gene coding for an NADP+-dependent d-xylose dehydrogenase was identified in the mould Hypocrea jecorina (Trichoderma reesei). It was cloned from cDNA, the active enzyme was expressed in yeast and a histidine-tagged enzyme was purified and characterized. The enzyme had highest activity with d-xylose and significantly smaller activities with other aldose sugars. The enzyme is specific for NADP+. The Km values for d-xylose and NADP+ are 43 mM and 250 μM, respectively. The role of this enzyme in H. jecorina is unclear because in this organism d-xylose is predominantly catabolized through a path with xylitol and d-xylulose as intermediates and the mould is unable to grow on d-xylonic acid

Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi

<p>Abstract</p> <p>Background</p> <p>The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-<it>threo</it>-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion.</p> <p>Results</p> <p>Keto-deoxy-L-galactonate (3-deoxy-L-<it>threo</it>-hex-2-ulosonate) accumulated in the culture supernatant when <it>Trichoderma reesei </it>Δ<it>lga1 </it>and <it>Aspergillus niger </it>Δ<it>gaaC </it>were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate l^-1was produced from 20 g D-galacturonate l^-1and <it>A. niger </it>Δ<it>gaaC </it>produced 15.0 g keto-deoxy-L-galactonate l^-1from 20 g polygalacturonate l^-1, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate [g D-galacturonate consumed]^-1. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g l^-1intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in <it>A. niger </it>Δ<it>gaaC</it>, but decreased in <it>T. reesei</it>.</p> <p>Conclusions</p> <p>Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both <it>A. niger </it>Δ<it>gaaC </it>and <it>T. reesei </it>Δ<it>lga1</it>, although production (titre, volumetric and specific rates) was better with <it>A. niger </it>than <it>T. reesei</it>. <it>A. niger </it>was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g l^-1were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from <it>T. reesei </it>mycelia.</p

Biotechnological versatility of riboflavin producer Ashbya gossypii Expression of Trichoderma reesei cellulases CBHI and EGI

The filamentous fungus Ashbya gossypii shows the potential for the production of, yet unexploited, valuable compounds other than riboflavin. To explore the ability of A. gossypii as a host for the expression of recombinant proteins, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were expressed in A. gossypii under Saccharomyces cerevisiae PGK1 promoter. The proteins were secreted into the culture medium, but there were differences in the amount or activity of the protein being produced. In one hand, CBHI activity was not detected using 4-methylumbelliferyl--Dlactoside as substrate, being only detected by Western blot. On the other hand, EGI activity was detectable, the level of activity being comparable to that produced by a S. cerevisiae strain containing the same plasmid. Thus more EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared to the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

BACKGROUND: Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. RESULTS: We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1(+)Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1(+)Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. CONCLUSIONS: The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1

Production of d-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae

d-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of d-glucose to d-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of d-glucose to biomass, and to increase d-glucaric acid yield, the four step d-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High d-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of d-glucose to d-glucaric acid. In batch bioreactor cultures with pulsed d-fructose and ethanol provision 1.3 g d-glucaric acid L−1 was produced. The d-glucaric acid titer (0.71 g d-glucaric acid L−1) was lower in nitrogen limited conditions, but the yield, 0.23 g d-glucaric acid [g d-glucose consumed]−1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield d-glucaric acid yeast strains.</p