Cloning, expression and characterisation of two tyrosinase cDNAs from Agaricus bisporus.

Using primers designed on the basis of sequence homologies in the copper-binding domains for a number of plant and fungal tyrosinases, two tyrosinase encoding cDNAs were cloned from an Agaricus bisporus U1 cDNA-library. The sequences AbPPO1 and AbPPO2 were, respectively, 1.9 and 1.8 kb in size and encoded proteins of approximately 64 kDa. The cDNAs represent different loci. Both AbPPO1 and AbPPO2 occur as single copies on the genomes of the U1 parental strains H39 and H97. The genomic size of AbPPO1 and AbPPO2 is minimally 2.3 and 2.2 kb, respectively. Alignment and phylogenetic analysis of 35 tyrosinase and polyphenol oxidase sequences of animal, plant, fungal, and bacterial origin indicated conserved copper-binding domains, and stronger conservation within genera than between them. The translation products of AbPPO1 and AbPPO2 possess putative N-glycosylation and phosphorylation sites and are recognised by antibodies directed against a 43-kDa tyrosinase. The observations are consistent with previously proposed maturation and activation models for plant and fungal tyrosinases.

A system of categorizing enzyme-cell wall associations in Agaricus bisporus, using operational criteria.

Enzymes were investigated for their occurrence in the cell wall fraction (4,000 g sediment of the homogenate) of Agaricus bisporus sporocarps. Besides the markers malate dehydrogenase (MalDH), hexokinase (HK) and ATPase, the range of entities studied included gamma-glutamyl transferase (gamma-GT), mannitol dehydrogenase (MDH), phenoloxidase, chitin and beta-1,3-glucan synthases (ChS, beta-GS), chitinase, beta-N-acetylhexosaminidase (HexNAc'ase) and beta-glucanase. Using the extractability in dilute buffer, digitonin and NaCl at high ionic strength as the operational criteria, four categories (I-IV) of enzyme-wall associations could be discerned: category I encompasses enzymes which are artefactually present (i.e. contaminants); category II, enzymes that are hydrophobically bound (which may or may not be genuinely wall-associated), III includes enzymes that are ionically bound and IV, enzymes whose bonding to the wall is in all probability covalent. The same enzyme entity may have representatives in more than one category, e.g. ChS and beta-GS (I, II, IV), phenolase (I, II, III, IV), beta-glucanase, chitinase and HexNAc'ase (I, IV). It is thought that the categorization presented could be of general applicability in fungi as well as in higher plants to specify enzyme-wall associations in a straightforward, comparable manner, thus avoiding some of the ambiguous terms prevailing in the literature, such as "weakly", "strongly" or "tightly" wall bound. The results are discussed in more detail for several of the more economically important enzymes studied.

Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced lichenan utilization and solvent production.

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.

The crystallographic structure of the mannitol 2-dehydrogenase NADP+ binary complex from Agaricus bisporus.

Mannitol, an acyclic six-carbon polyol, is one of the most abundant sugar alcohols occurring in nature. In the button mushroom, Agaricus bisporus, it is synthesized from fructose by the enzyme mannitol 2-dehydrogenase (MtDH; EC ) using NADPH as a cofactor. Mannitol serves as the main storage carbon (up to 50% of the fruit body dry weight) and plays a critical role in growth, fruit body development, osmoregulation, and salt tolerance. Furthermore, mannitol dehydrogenases are being evaluated for commercial mannitol production as alternatives to the less efficient chemical reduction of fructose. Given the importance of mannitol metabolism and mannitol dehydrogenases, MtDH was cloned into the pET28 expression system and overexpressed in Escherichia coli. Kinetic and physicochemical properties of the recombinant enzyme are indistinguishable from the natural enzyme. The crystal structure of its binary complex with NADP was solved at 1.5-A resolution and refined to an R value of 19.3%. It shows MtDH to be a tetramer and a member of the short chain dehydrogenase/reductase family of enzymes. The catalytic residues forming the so-called catalytic triad can be assigned to Ser(149), Tyr(169), and Lys(173).

Crystallization and preliminary crystallographic analysis of mannitol dehydrogenase (MtDH) from the common mushroom Agaricus bisporus.

Mannitol dehydrogenase (MtDH) is a key enzyme controlling the reductive synthesis of mannitol from fructose in the common mushroom Agaricus bisporus. A better understanding of the control of mannitol metabolism can be obtained by studying the structure of this enzyme. Here, the purification and crystallization of recombinant MtDH are reported. Crystals generally belonged to the space group C2, with unit-cell parameters a = 227, b = 125, c = 133 A, beta = 118 degrees, and diffracted to at least 1.8 A resolution, although a tantalum derivative belonged to the space group P2(1) and diffracted to the lower resolution of 2.9 A.

Utilisation of saccharides in extruded domestic organic waste by Clostridium acetobutylicum ATCC 824 for production of acetone, butanol and ethanol.

Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production. Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively. The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824. This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement. This strain produced 4 g ABE/100 g extruded DOW. When C. acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and beta-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW. The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria. These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium.

Alternative sources of natural rubber.

Rubber (cis-1,4-polyisoprene) is one of the most important polymers naturally produced by plants because it is a strategic raw material used in more than 40,000 products, including more than 400 medical devices. The sole commercial source, at present, is natural rubber harvested from the Brazilian rubber tree, Hevea brasiliensis. Primarily due to its molecular structure and high molecular weight (> 1 million daltons) this rubber has high performance properties that cannot easily be mimicked by artificially produced polymers, such as those derived from, e.g., bacterial poly-hydroxyalkanoates (PHAs). These high performance properties include resilience, elasticity, abrasion resistance, efficient heat dispersion (minimizing heat build-up under friction), and impact resistance. Medical rubber gloves need to fit well, be break-resistant, allow the wearer to retain fine tactile sensation, and provide an effective barrier against pathogens. The sum of all these characteristics cannot yet be achieved using synthetic gloves. The lack of biodiversity in natural rubber production renders continuity of supply insecure, because of the risk of crop failure, diminishing acreage, and other disadvantages outlined below. A search for alternative sources of natural rubber production has already resulted in a large number of interesting plants and prospects for immediate industrial exploitation of guayule (Parthenium argentatum) as a source of high quality latex. Metabolic engineering will permit the production of new crops designed to accumulate new types of valued isoprenoid metabolites, such as rubber and carotenoids, and new combinations extractable from the same crop. Currently, experiments are underway to genetically improve guayule rubber production strains in both quantitative and qualitative respects. Since the choice for gene activities to be introduced or changed is under debate, we have set up a complementary approach to guayule with yeast species, which may more quickly show the applicability and relevance of genes selected. Although economic considerations may prevent commercial exploitation of new rubber-producing microorganisms, transgenic yeasts and bacteria may yield intermediate or alternative (poly-)isoprenes suitable for specific applications.

Cloning and characterization of the Yarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of the Saccharomyces cerevisiae erg9 mutation.

The squalene synthase (SQS) gene encodes a key regulatory enzyme, farnesyl-diphosphate farnesyltransferase (EC 2.5.1.21), in sterol biosynthesis. The SQS1 gene was isolated from a subgenomic library of the industrially important yeast Yarrowia lipolytica, using PCR-generated probes. Probes were based on conserved regions of homologues from different organisms. The complete nucleotide sequence of the coding region and the corresponding amino acid sequence were determined. The sequences showed extensive homologies with squalene synthase genes and enzymes from a number of other organisms and extreme amino acid conservation within the binding and catalytic domains. Direct cloning of a 4.3 kb genomic Y. lipolytica fragment, also comprising its own promoter and terminator sequences, into autonomously replicating plasmid YEp352 and subsequent transformation of a Saccharomyces cerevisiae mutant strain with relevant erg9: ura3-1 markers, resulted in functional complementation of these deficiencies, although Northern blot analyses did not reveal a unique full-length messenger. The availability of the Y. lipolytica SQS1 gene (GenBank Accession No. AF092497) offers prospects for metabolic engineering of the isoprenoid and sterol biosynthetic pathways.

High-yield secretion of recombinant gelatins by Pichia pastoris.

Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.

Cloning and characterization of NADP-mannitol dehydrogenase cDNA from the button mushroom, Agaricus bisporus, and its expression in response to NaCl stress.

Mannitol, a six-carbon sugar alcohol, is the main storage carbon in the button mushroom, Agaricus bisporus. Given the physiological importance of mannitol metabolism in growth, fruit body development, and salt tolerance of A. bisporus, the enzyme responsible for mannitol biosynthesis, NADP-dependent mannitol dehydrogenase (MtDH) (EC 1.1.1.138), was purified to homogeneity, and MtDH cDNA was cloned, sequenced, and characterized. To our knowledge, this represents the first report on the isolation of a cDNA encoding an NADP-dependent mannitol dehydrogenase. The MtDH cDNA contains an open reading frame of 789 bp encoding a protein of approximately 28 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified MtDH subunit, whereas the amino acid composition of the deduced protein was nearly identical to that of the purified MtDH. The MtDH cDNA showed high homology with a plant-induced short-chain dehydrogenase from Uromyces fabae. Phylogenetic analysis based on amino acid sequences from mannitol(-1-phosphate) dehydrogenases indicated a close relationship between the substrate specificity of the enzymes and phylogenetic differentiation. Salt-stressed fruit bodies showed an overall increase in mannitol biosynthesis, as was evident from the increase in MtDH activity, MtDH abundance, and MtDH RNA accumulation. Furthermore, the MtDH transcript level seems to be under developmental control, as MtDH RNA accumulated during maturation of the fruit body.

Highly efficient homologous integration via tandem exo-beta-1, 3-glucanase genes in the common mushroom, Agaricus bisporus.

Homologous integration was studied in the common mushroom, Agaricus bisporus, using a plasmid (pHAG3-1) carrying the hygromycin-resistance gene and a 3.2-kb genomic fragment from A. bisporus. Homologous integration was found in 30-60% of the transformants obtained with pHAG3-1 linearized at three different positions within the homologous sequence, generating either blunt, 5'- or 3'-protruding ends. The genomic fragment was found to contain two homologous open reading frames in tandem, which showed 60% similarity to exo-beta-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The level of the corresponding mRNA is low in the vegetative mycelium and relatively high in fruiting bodies. In the vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position, exoglucanase mRNA was strongly increased without any apparent effect on growth rate or morphology.

Cloning, sequence and expression in Escherichia coli of the gene encoding phosphofructokinase from Bacillus macquariensis.

A chromosomal DNA fragment containing the Bacillus macquariensis (Bm) ATP-dependent phosphofructokinase-encoding gene (pfk) was cloned from a subgenomic library in pUC19 using a PCR-derived probe. The region containing pfk, including flanking sequences, was sequenced and the deduced amino acid sequence (aa) was found to be homologous to other PFK, but it contained two single-aa changes conserved in a range of other organisms from pro- and eukaryotic origins. Enzymatic studies with PFK purified from overproducing Escherichia coli (Ec) host cells showed that the Bm enzyme is similar to B. stearothermophilus (Bs) PFK in many respects and that it is relatively cold stable.

Transformation of the cultivated mushroom, Agaricus bisporus, to hygromycin B resistance.

Application of biotechnology to the cultivated mushroom, Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain of A. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into the A. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomycete Aspergillus nidulans and from A. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from the A. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.

Introduction of hygromycin B resistance into Schizophyllum commune: preferential methylation of donor DNA.

Cotransformation of a trp1 strain of Schizophyllum commune with the homologous TRP gene and the Escherichia coli HPT gene was used to study the feasibility of transformation of S. commune to hygromycin B resistance. Southern blot analysis showed that 75% of the TRP transformants contained multiple integrated copies of the HPT gene. However only 7% of the transformants were resistant to 25 micrograms/ml hygromycin B and direct selection for hygromycin B resistance was hampered by the high incidence of spontaneously arising resistant colonies. Rescue of the HPT gene was possible with E. coli JA221 (mcr-) but not with JM83, suggesting methylation of the integrated donor DNA. Isoschizomer analyses confirmed heavy methylation in the HPT gene and flanking vector sequences but not in the homologous donor TRP gene and its flanking vector sequences. Also cotransforming S. commune Sc4 gene and flanking vector sequences were not methylated. A fusion between the S. commune TRP1 and the E. coli HPT genes resulted in only slight or no methylation of both vector and HPT sequences and in a higher hygromycin B resistance level. This suggests that transformation with DNA exclusively containing foreign sequences results in integration into regions where methylation occurs, possibly entailing poor transcription. Methylation of the HPT gene was also indicated by the stimulation of growth by 5-azacytidine of transformants on hygromycin B containing medium.

Loss of heterozygosity for a chromosome 3 sequence presumably at 3p21 in small cell lung cancer.

A recombinant DNA fragment detecting a chromosome #3 restriction fragment length polymorphism presumably at p21 was hybridized to HindIII-digested DNA isolated from the leukocytes of 12 patients of small cell lung cancer. Four of them appeared to be heterozygous. Analysis of tumor material from these four patients revealed homozygosity for either one or the other restriction fragment in every case. Our findings suggest the presence on the short arm of chromosome #3 of a recessive mutant cancer gene contributing to the development of small cell lung cancer.

Genome analysis of small cell lung cancer (SCLC) and clinical significance.

A chromosome analysis of three cell lines derived from SCLC showed deletions of the short arm of chromosome 3 with bands p21-p23 as the shortest region of overlap. Hybridization of a polymorphic 3p21 probe to DNA from leukocytes of seven SCLC patients revealed heterozygosity for two of them. In the tumours of both these patients the probe detected homozygosity. This suggests the presence of a mutant cancer gene in the short arm of chromosome 3 which might express itself and/or activate some oncogene(s) after deletion of a suppressing normal allele. Amplification of the oncogene C-MYC was found in four cell lines including the ones cytogenetically analyzed. Amplification of C-MYC, though to a lesser degree, was also found in an available pleural effusate from which one of these lines had been established. As shown by in situ hybridization, the amplified oncogene was present in double minutes in three of the cell lines. In the remaining line it was in a homogeneously staining chromosome region. All patients from whom cell lines with C-MYC amplification were obtained had a negative response to chemotherapy. The observed correlation between amplification of C-MYC, occurrence of so-called variant type SCLC-derived cell lines, and negative response to chemotherapy indicates that a genome analysis of SCLC might provide further criteria for the characterization and subdivision of this highly malignant cancer and thereby a base for an optimal selection of therapy for distinct cases of SCLC.

Effect of concentration on the subsequent fate of plasmid DNA in human fibroblasts.

The physical fate of plasmid DNA after entry into human fibroblasts was studied using Southern hybridisation and electron microscopy. Exposure of the cells (5 X 10(5) per well) to pC194 DNA-CaPi, containing 50 micrograms plasmid DNA, resulted in the occasional formation of interlocked molecules. Exposure to a co-precipitate containing 100 micrograms pC194 plasmid DNA per well resulted in an increase of interlocked molecules by a factor of 10-20 relative to the number of monomers. In addition, new classes of molecules were observed. After prolonged incubation of the cells exposed to the higher DNA concentration, the plasmid DNA was partly contained in structures with a very low electrophoretic mobility. Upon restriction endonuclease digestion of the re-extracted DNA, a pattern of bands was observed, suggesting the involvement of illegitimate recombination between non-random plasmid DNA sequences in the formation of the new classes of molecules.

Repair of UV damage in plasmid DNA by human fibroblasts.

Plasmid DNA from Bacillus subtilis was introduced into monolayers of human fibroblasts by means of a modification of the calcium phosphate coprecipitation technique, comprising centrifugation of the coprecipitate onto the cells and treatment with polyethyleneglycol. The amount of DNA resistant to removal from the monolayers ranged from 10% to 15% of the input DNA. By determination of the biological activity of the plasmid DNA, re-extracted after various periods following entry into the fibroblasts and subsequently used as donor for B. subtilis protoplasts, it was shown that the activity of the plasmid DNA was gradually lost. When ultraviolet light-inactivated plasmid DNA was used as donor, reactivation of the plasmid was observed, which was completed within 2 h. The dose-dependent incorporation of [14C]-thymidine suggests that DNA repair processes were involved in reactivation of the plasmid DNA.

Effect of 4,5',8-trimethylpsoralen interstrand cross-links present in recipient Bacillus subtilis on the integration of transforming DNA.

When recipient Bacillus subtilis carrying chromosomal trimethylpsoralen cross-links were transformed, the donor marker activity decreased with the extent of cross-linking. Additional donor marker activity was lost upon incubation of the reextracted DNA with nuclease S1, particularly at higher levels of cross-linking. Physical analysis of the reextracted DNA showed that the donor DNA was progressively excluded from heteroduplex formation as the frequency of cross-links in the recipient DNA increased. In the donor-recipient complexes still being formed, increasing amounts of donor DNA became susceptible to nuclease S1 digestion under these conditions. These results suggest that resident interstrand cross-links interfere both with initiation of recombination and with the completion of heteroduplex formation.

The effect of chlorpromazine on transformation in Bacillus subtilis.

In the presence of the widely used tranquilizer, chlorpromazine, transforming DNA of Bacillus subtilis is photoinactivated by long-wave ultraviolet light. The loss of biological activity is predominantly caused by lack of binding of the DNA to recipient cells and the introduction of single-strand breaks in the treated DNA.