Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker.

The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae.

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.

Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus.

The phyA gene encoding an extracellular phytase from the thermophilic fungus Thermomyces lanuginosus was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. The phyA gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) with Aspergillus niger phytase. The phyA gene was inserted into an expression vector under transcriptional control of the Fusarium oxysporum trypsin gene promoter and used to transform a Fusarium venenatum recipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 micromol of inorganic phosphate released per min per mg of protein at pH 6 and 37 degrees C. The Thermomyces phytase retained activity at assay temperatures up to 75 degrees C and demonstrated superior catalytic efficiency to any known fungal phytase at 65 degrees C (the temperature optimum). Comparison of this new Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range.

Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile.

A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

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.

Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.

A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.

The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter.

Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase-R) of Aspergillus niger. One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR). Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns. The deduced amino acid sequence of catalase-R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group. Increased expression of the catR gene was obtained by transformation of an A. niger host strain with an integrative vector carrying the cloned genomic DNA segment. Several of these transformants produced three- to fivefold higher levels of catalase than the untransformed parent strain. Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome. A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A. niger glucoamylase (glaA) gene. A. niger transformants containing this vector produced from three- to 10-fold higher levels of catalase-R than the untransformed parent strain.

Isolation and characterization of the Aspergillus oryzae gene encoding aspergillopepsin O.

We have cloned and determined the nucleotide sequence of a genomic DNA segment from Aspergillus oryzae which contains pepO, the gene encoding the aspartic proteinase, aspergillopepsin O (PEPO). The organization of pepO is strikingly similar to that of pepA from A. niger var. awamori (previously called A. awamori) in that both are composed of four exons and three introns with virtually identical lengths, and the positions of the introns are exactly conserved. From the deduced amino acid (aa) sequence, it appears that PEPO, like other fungal aspartic proteinases, is synthesized as a zymogen containing a putative N-terminal prepro-region of 77 aa followed by a mature protein of 327 aa. Southern blotting experiments suggest that a single copy of pepO exists in the A. oryzae genome.

Gene expression systems for filamentous fungi.

The extraordinary capacity of filamentous fungi to produce large quantities of extracellular protein, together with the advent of DNA-mediated fungal transformation, has resulted in rapid advances in the development of gene expression systems for filamentous fungi. This review focuses on recent developments in the expression of both fungal and non-fungal genes and improvements to the host.

Commercial levels of chymosin production by Aspergillus.

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates.

We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.

Mutations in the small subunit of cyanobacterial ribulose-bisphosphate carboxylase/oxygenase that modulate interactions with large subunits.

In the cyanobacterium Anacystis nidulans (Synechococcus PCC6301), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) is composed of eight large subunits and eight small subunits. There are three regions of the small subunit that contain amino acids that are conserved throughout evolution, from bacteria to higher plants. Since the function of the small subunit is not fully understood, site-directed mutagenesis was performed on highly conserved residues in the first and second conserved regions. Ser-16, Pro-19, Leu-21, and Tyr-54 were replaced by Asp-16, His-19, Glu-21, and Ser-54, respectively. Crude extracts containing the recombinant His-19 mutant enzyme indicated that there was little effect on either Rbu-P2 carboxylase activity or interactions between large and small subunits. However, the Asp-16, Glu-21, and Ser-54 mutations showed effects on Rbu-P2 carboxylase activity and the interaction between large and small subunits. The large and small subunits of the Asp-16, Glu-21, and Ser-54 enzymes were found to dissociate during nondenaturing gel electrophoresis or sucrose density gradient centrifugation. However, the dissociated small subunits remained functional and were capable of reconstituting Rbu-P2 carboxylase activity when added to large subunits. These results indicated that Ser-16, Leu-21, and Tyr-54 might play an important role in interactions between large and small subunits of the A. nidulans enzyme.

Regulation of the glaA gene of Aspergillus niger.

The glucoamylase gene of Aspergillus niger, glaA, is expressed at high levels in the presence of starch. We have determined the nucleotide sequence of 1966 bp of the 5' flanking region of the glaA gene and have begun to identify sequences important for the control of glaA expression by deletion analysis. Constructs containing deletions extending into the glaA gene promoter were introduced into an A. niger host whose own glaA gene had been disrupted by a gene replacement event. Secreted levels of glucoamylase, expressed from each of the recombinant glaA genes, were measured by enzyme immunoassay. The effect of each deletion on the expression of glaA, when grown on differing carbon sources, was used to determine the limits of sequences upstream of glaA responsible for gene regulation. A region between -562 and -318 appears to direct high-level expression, whereas only 214 bp of 5' flanking sequence is required to initiate the start to transcription.

Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion.

We have extended the work on chymosin production in Aspergillus by constructing an expression vector in which the cDNA encoding bovine prochymosin B was fused in frame immediately following the codon for the last amino acid of the A. awamori glucoamylase (glaA) gene. Transformation of A. awamori with this plasmid led to the secretion of considerably higher amounts of chymosin than obtained with previous chymosin expression vectors. We present evidence that mature chymosin is autocatalytically released from the glucoamylase-chymosin fusion protein after secretion.

The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis.

Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (HyR) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. HyR transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcriptional start points are the same in U. maydis and A. niger.

Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.