The phenomenon of degeneration of industrial Trichoderma reesei strains.

BACKGROUND: Even if the loss of production capacity of a microorganism is said to be a serious problem in various biotechnology industries, reports in literature are rather rare. Strains of the genera Trichoderma reesei are used for large-scale production of cellulases, which are needed in food and feed, textile, paper industries and biofuel production. RESULTS: Here, we describe the phenomenon of spontaneous degeneration of T. reesei strains during large-scale cultivation. The phenotype of the degenerated population is characterized most importantly by a loss of any cellulase formation. Interestingly, promoter regions of relevant genes had a more compact chromatin in the (cel -) strains compared to productive strains. For a systematic investigation of the phenomenon a protocol for artificially induced and lab-scaled strain degeneration was developed. This workflow allows to determine the degeneration rate and thus, to compare the occurrence of a degenerated population in differently productive strains on the one hand, and to monitor the success of any strategies to prevent or decrease the degeneration on the other hand. While highly productive strains have higher degeneration rates compared to moderate producers, the degeneration can hardly be triggered in moderate producers. The observed (cel -) phenotype is not caused by a mutation in the gene encoding the essential transactivator Xyr1. The development of a non-producing population is also not triggered by any compounds released by either producing or non-producing cells. CONCLUSIONS: The extent of the occurrence of a degenerated strain population relates to the production capacity of the strain and goes along with chromatin condensation in relevant promoter regions.

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae.

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Fungal Genetics and Biology 29, 28-37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzae pyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.

Targeted mutations in a Trametes villosa laccase. Axial perturbations of the T1 copper.

Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.

Polygalacturonase gene expression in ripe melon fruit supports a role for polygalacturonase in ripening-associated pectin disassembly.

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.

Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 A resolution.

Laccase catalyses the oxidation of a variety of organic substrates coupled to the reduction of oxygen to water. It is widely believed to be the simplest representative of the ubiquitous blue multi-copper oxidase family. Laccase is implicated in a wide spectrum of biological activities and, in particular, plays a key role in morphogenesis, development and lignin metabolism in fungi and plants. The structure of laccase from the fungus Coprinus cinereus has been determined by X-ray crystallography at a resolution of 2.2 A. Laccase is a monomer composed of three cupredoxin-like beta-sandwich domains, similar to that found in ascorbate oxidase. In contrast to ascorbate oxidase, however, the mononuclear type-1 Cu site lacks the axial methionine ligand and so exhibits trigonal planar coordination, consistent with its elevated redox potential. Crucially, the structure is trapped in a Cu depleted form in which the putative type-2 Cu atom is completely absent, but in which the remaining type-1 and type-3 Cu sites display full occupancy. Type-2 Cu depletion has unexpected consequences for the coordination of the remaining type-3 Cu atoms.

Crystallization and preliminary X-ray analysis of the laccase from Coprinus cinereus.

The laccase from the fungus Coprinus cinereus has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small plate-like crystals of an enzymatically deglycosylated form of the enzyme have been grown by the hanging-drop method using polyethylene glycol as precipitant. These crystals diffract to at least 2.2 A. They belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.4, b = 85.7, c = 143.1 A with a single molecule of laccase in the asymmetric unit.

Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family.

Three laccase genes were isolated from the white-rot basidiomycete Trametes villosa (Tv). The predicted protein products have 63-71% identity to the previously cloned Tv laccase genes lcc1 and lcc2. The genes lcc3, lcc4 and lcc5 contain 12, 10 and 11 introns, respectively. The position of several of the introns is conserved among all 5 genes. The 5 genes appear to be differentially regulated, and message has only been detected for lcc1 and lcc2. The karyotype of Tv was determined by CHEF, and 8 bands ranging in size from approximately 5.7 to 2.2 Mb were resolved of which 2 appear to be doublets. The 5 laccase genes have been mapped to specific bands resolved by CHEF. The lcc1 and lcc2 genes hybridize to a band of approximately 5.7 Mb. The lcc4 and lcc5 genes are on a chromosome of approximately 3.7 Mb, and lcc3 is on a chromosome of approximately 2.8 Mb.

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

Nucleotide sequence of the Aspergillus niger srpA gene.

The Aspergillus niger (An) gene srpA, encoding a protein with homology to the signal recognition particle (SRP) 54-kDa protein from Saccharomyces cerevisiae (Sc), has been isolated and the nucleotide sequence determined. The putative An srpA gene is comprised of two exons of 78 and 1527 bp separated by a 49-bp intron, and encodes a protein of 534 amino acids that is 53% identical to Sc SRP54.

Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica.

Two sequences (ARS18 and ARS68) displaying autonomous replication activity were previously cloned in the yeast Yarrowia lipolytica. The smallest fragment (1-1.3 kb) required for extrachromosomal replication of a plasmid is significantly larger in Y. lipolytica than in Saccharomyces cerevisiae. Neither autonomously replicating sequence (ARS) is homologous with known ARS or centromere (CEN) consensus sequences. They share short regions of sequence similarity with each other. These ARS fragments also contain Y. lipolytica centromeres: (i) integration of marker genes at the ARS loci results in a CEN-linked segregation of the markers, (ii) an ARS on a plasmid largely maintains sister chromatid attachment in meiosis I, and (iii) integration of these sequences at the LEU2 locus leads to chromosome breakage. Deletions performed on ARS18 show that CEN and ARS functions can be physically separated, but both are needed to establish a replicating plasmid.

Vacuolar ATPase mutants accumulate precursor proteins in a pre-vacuolar compartment.

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates an electrochemical potential across the vacuole membrane. Strains with chromosomal disruptions of the genes encoding the A, B, and c subunits of the vacuolar ATPase accumulate precursor forms of the vacuolar membrane protein alkaline phosphatase, and the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. We have found that the intracellular precursors in delta vat strains accumulate within the secretory pathway at some point before delivery to the vacuole but after transit to the Golgi complex. Purified vacuoles from delta vat cells do not contain the precursor forms of carboxypeptidase Y or alkaline phosphatase. In addition, vacuolar hydrolase-invertase hybrid proteins are inefficiently delivered to the vacuole in delta vat strains as demonstrated by vacuole isolation. Further subcellular fractionation to separate organelles indicate that significant amounts of the carboxypeptidase Y-invertase and alkaline phosphatase-invertase hybrid proteins are located in the late Golgi complex and/or post Golgi compartments.

Mutations in the yeast vacuolar ATPase result in the mislocalization of vacuolar proteins.

The vacuolar ATPase of the yeast Saccharomyces cerevisiae acidifies the vacuolar lumen and generates an electrochemical gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of genes (delta vat) encoding the A (69 x 10(3) M(r)), B (57 x 10(3) M(r)) or c (16 x 10(3) M(r)) subunits of the vacuolar ATPase accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. A kinetic analysis suggests that these precursor proteins accumulate in, and are secreted from, the Golgi complex or post-Golgi vesicles. In addition, subcellular fractionation shows that vacuolar hydrolase-invertase hybrid proteins are inefficiently localized to the vacuole in delta vat strains. This result suggests that the vat mutations cause a steady-state defect in vacuolar protein sorting. The vat mutations also affect the sorting of vacuolar membrane proteins. Precursor forms of alkaline phosphatase are accumulated in vat mutant cells, but to a lesser extent than is seen for the soluble vacuolar hydrolases. This finding, coupled with the insensitivity of alkaline phosphatase to the ATPase inhibitor bafilomycin A1, suggests that vacuolar membrane protein sorting is less sensitive to changes in lumenal pH when compared with the targeting of soluble vacuolar proteins. These results indicate that acidification of the vacuolar system is important for efficient sorting of soluble proteins to the vacuole.

Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway.

The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.

A mutation in the signal recognition particle 7S RNA of the yeast Yarrowia lipolytica preferentially affects synthesis of the alkaline extracellular protease: in vivo evidence for translational arrest.

Replacement of the signal recognition particle (SRP) 7S gene (SCR1) on a replicating plasmid with scr1-1 (G to A at 129 and A to T at 131 in the consensus sequence -GNAR- in the loop of domain III) resulted in temperature sensitivity for growth of cells in which both chromosomal SRP 7S RNA genes were deleted. Pulse-chase immunoprecipitation experiments were done after a shift to non-permissive temperature using the major secreted protein the alkaline extracellular protease (AEP) as a reporter molecule. No untranslocated AEP precursor was detected in a strain with scr1-1 on a plasmid, but the amount of the largest AEP precursor (55 kD) immunoprecipitated as a percentage of total protein synthesized was reduced 68% compared to an isogenic strain with SCR1 on the plasmid. The possibility that an untranslocated precursor was synthesized but not detected because of instability was largely eliminated by detection of a 53-kD untranslocated precursor of a mutated AEP (P17M; methionine replaced proline in the second position of the pro-peptide) which chased to the 55-kD translocated AEP precursor. Thus, SRP has a role in the biosynthesis of AEP. Possibly, the scr1-1 mutation does not affect signal recognition or translational arrest but instead results in maintenance of translational arrest of AEP synthesis. The results also suggest that AEP can be translocated in vivo either co-translationally in which SRP is at least involved in biosynthesis or posttranslationally without SRP involvement.

The yeast Yarrowia lipolytica has two, functional, signal recognition particle 7S RNA genes.

Cells containing a deletion of either the SCR1 or SCR2 genes, which code for the 7SL RNA component of the signal recognition particle (SRP) homologue, were found to be viable. Two independent approaches demonstrated that cells containing deletions of both genes were inviable. Therefore, Yarrowia lipolytica contains two (and only two) functional 7SL RNA genes.