Improved degradation of organophosphate dichlorvos by Trichoderma atroviride transformants generated by restriction enzyme-mediated integration (REMI)

A simple technique, REMI (restriction enzyme-mediated integration), was used to construct transformants of Trichoderma atroviride with improved capability of degrading organophosphate pesticide dichlorvos. Linearized DNA of plasmid pV2 bearing the hygromycin B phosphotransferase (hph) gene was inserted into chromosomes of wild strain T23 and transformation was confirmed by PCR and Southern blot analysis, respectively. Of 247 transformants, 76% showed improved dichlorvos degradation ability as compared to the parent strain T23 based on the least significant difference (LSD) test at p=0.01. Among them, 8 transformants exhibited 30% higher in degradation rate than the parent isolate. The highest dichlorvos degradation rate of the transformants was up to 96%. This study provided an effective approach for improving organophosphate pesticide-degrading capability of T. atroviride.     

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI)

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.     

Tagging pathogenicity genes inUstilago maydis by restriction enzyme-mediated integration (REMI)

In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.     

Tagging pathogenicity genes inUstilago maydis by restriction enzyme-mediated integration (REMI)

In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.     

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ～3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A.?nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.     

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ∼3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi. Received: 22 August 1997 / Accepted: 20 November 1997     

Transformation of the medicinal basidiomycete Trametes versicolor to hygromycin B resistance by restriction enzyme mediated integration

Trametes versicolor, a white-rot basidiomycete, degrades cellulose and lignin as well as many recalcitrant chemicals. There have been many reports about the cloning of laccase and peroxidase genes of T. versicolor which are involved in lignin degradation. In order to analyze a gene function and introduce foreign genes into an organism, genetic transformation is required. Here we have successfully transformed T. versicolor to hygromycin B resistance using pAN 7-1 plasmid by restriction enzyme mediated integration and have obtained many mutants in peroxidase activity and growing patterns. The transformation frequency was 25-50 transformants (microg plasmid DNA)(-1). The transformants were quite stable after 10 consecutive transfers in non-selectable medium.     

Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis.

Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiquitin-conjugating (E2) enzyme is required for cellular processes as diverse as DNA repair, selective proteolysis, and normal growth. For most RAD6-dependent functions, the relevant in vivo targets, as well as the mechanisms and cofactors that govern RAD6 substrate selectivity, are unknown. We have explored the utility of "charge-to-alanine" scanning mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that are nevertheless severely handicapped with respect to several in vivo functions. Five of the nine mutants we generated show defects in their in vivo functions, but almost all of the most severely affected mutants displayed unfacilitated ubiquitin-conjugating activity in vitro. We suggest that E2 mutants obtained by this approach are likely to be defective with respect to interaction with other, trans-acting factors required for their intracellular activity or substrate selectivity and therefore will be useful for further genetic and biochemical studies of ubiquitin-conjugating enzyme function.     

Metalloporphyrin mediated DNA cleavage by a low concentration of HaeIII restriction enzyme

The plasmid DNA scission by the restriction enzyme HaeIII was investigated in the presence of tetrakis(1-methylpyridinium-4-yl)porphyrin (H2TMPyP) and its manganese(III), iron(III), nickel(II), cobalt(III) and zinc(II) derivatives. The effect of metalloporphyrins on plasmid DNA cleavage was ascertained by gel electrophoresis. UV-Vis absorption spectroscopy and circular dichroism (CD) spectroscopy. In the absence of the metalloporphyrins, plasmid DNA scission did not occur in the presence of a low concentration of HaeIII (0.2 units microL(-1)) at 37 degrees C after 1 h incubation. However, DNA cleavage occurred in the presence of the metalloporphyrins and HaeIII (0.2 units microL(-1)) at 37 degrees C after 1 h incubation. Gel electrophoresis results indicate the catalytic effect of metalloporphyrins (Mn(III)-, Fe(III)-, Co(III)- and Zn(II)TMPyP) by binding to both DNA and the enzyme through electrostatic interaction, which was confirmed by the change in UV-Vis and CD spectra. A mechanism for the enhanced DNA cleavage is proposed.     

Degradation of ferrous(II) cyanide complex ions by Pseudomonas fluorescens

Degradation of ferrous(II) cyanide complex (ferrocyanide) ions by free cells of P. fluorescens in the presence of glucose and dissolved oxygen was investigated as a function of initial pH, initial ferrocyanide and glucose concentrations and aeration rate in a batch fermenter. The microorganism used the ferrocyanide ions as the sole source of nitrogen. The ferrocyanide biodegradation rate was 30.7 mg g⁻¹ h⁻¹ under the conditions of initial pH: 5, stirring rate: 150 rpm, aeration rate: 0.15 vvm, initial ferrous(II) cyanide complex ion and glucose concentrations: 100 mg l⁻¹ and 0.465 g l⁻¹, respectively. The culture utilized glucose as the main substrate following the non-competitive toxic component inhibition model in the presence of 100 mg l⁻¹ initial ferrous(II) cyanide complex ion concentration. The inhibition of ferrous(II) cyanide complex ions as a secondary substrate began at very low concentrations. A mathematical model, based on non-competitive substrate inhibition was used to describe the inhibitory effect of ferrous(II) cyanide complex ions on the growth of microorganism and the best fitted model parameters were determined by non-linear regression techniques.     

Screening and identification of insertion mutants from Bipolaris eleusines by mutagenesis based on restriction enzyme-mediated integration

Ophiobolin A is sesterterpenoid-type phytotoxin and may be an important candidate for development of new crop protection and pharmaceutical products. The restriction enzyme-mediated integration (REMI) method was used to introduce the plasmid pSH75 into the ophiobolin A-producing filamentous fungus Bipolaris eleusines. A total of 323 stable transformants were obtained, all of which were capable of growing on potato-dextrose agar medium containing 200?μg?mL(-1) hygromycin B. The transformation frequency was about 4-5 transformants?μg(-1) plasmid DNA. An ophibolin A-deficient transformant (B014) was assessed and the presence of the hph gene in this transformant was confirmed by PCR. The cell-free cultural filtrates of this transformant showed significantly less inhibition on mycelial growth of the fungal pathogen Rhizoctoni solani but little effect on barnyard grass as opposed to that of the wild-type B.?eleusines. There was no detectable amount of ophiobolin A in B014 samples measured with HPLC. This research suggests REMI as a potential approach for improving the production of ophiobolin A by B.?eleusines via genetic engineering to upregulate certain genes responsible for desired biosynthetic pathways.     

Degradation of Cellulosic Materials by Trichoderma viride Cellulase

The extra-cellular filtrates of Trichoderma viride ITCC–1433 showed considerable cellulolytic activity against native celluloses, cellulose derivatives and raw materials. Newspaper-yellow and the rice straw were the prominent waste materials which were preferentially attacked by the enzyme. The alkali treatment of the latter doubled the sugar formation from it. As a result of cellulase action 80.4 per cent of the MN-Cellulose and 60.4 per cent of the alkali treated rice straw lost weight in 96 and 48 hr respectively. The weight loss was more or less equivalent to the reducing sugars formed.     

Kinetic study of partially purified cellulase enzyme produced by Trichoderma viride FCBP-142 and its hyperactive mutants

Cellulases are the enzymes that cleave beta-1,4 linkages of cellulose, and carbohydrate that is main part of plants' cell walls. Presently, cellulase isolation and partial purification was executed through ammonium sulfate precipitation. The isolated protein of parental and derived mutants conferred molecular weights of 30, 45 and 55 kDa. The optimum temperature for maximal cellulase activity was 50 degrees C with Ea for substrate hydrolysis of 77.73, 83.97 and 83.14 kJ mol(-1) and temperature quotient of 1.0020, 1.0022 and 1.0022 by Trichoderma viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3, respectively. The enzyme was stable at 50 degrees C for about 60 min but rapid denaturation occurred above 55 degrees C. The enzyme showed optimum activity at pH 4.0 and involved two types of acidic and basic limbs with pKa1 and pKa2. The pKa1 of active site presented a significant shift from 2.55 to 2.9 and 3.1 by Tv-UV-5.6 and Tv-Ch-4.3, respectively in comparison to parental strain. Likewise, pKa2 moved from 6.05 to 6.5 and 6.4. Enzyme kinetics displayed Michaelis-Menten constant Km 0.6, 0.5 and 0.28 mg mL(-1) and Vmax value of 8.33, 10 and 9.09 Units mL(-1) for parental, Tv-UV-5.6 and Tv-Ch-4.3, respectively.     

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

Summary Three strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Bürrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a K_s value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.     

Degradation of amylin by insulin-degrading enzyme

A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.     

Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis

Abstract Methylosinus trichosporium OB3b synthesizes a soluble cytoplasmic methane monooxygenase when grown in copper-depleted medium and a membrane-bound particulate methane monooxygenase under copper-replete conditions. The genes encoding the hydroxylase component of soluble methane monooxygenase, carried on a plasmid in Escherichia coli , were insertionally inactivated using a kanamycin cassette and transferred back into M. trichosporium by conjugation. Marker-exchange mutagenesis, via a double homologous recombination event, yielded a soluble methane monooxygenase-negative mutant which grew only on methane using the particulate methane monooxygenase during copper-replete growth conditions, thus proving that the two methane oxidation systems in this methanotroph are genetically distinct.     

Degradation of low rank coal by Trichoderma atroviride ES11

A new isolate of Trichoderma atroviride has been shown to grow on low rank coal as the sole carbon source. T. atroviride ES11 degrades approximately 82% of particulate coal (10 g l(-1)) over a period of 21 days with 50% reduction in 6 days. Glucose (5 g l(-1)) as a supplemented carbon source enhanced the coal solubilisation efficiency of T. atroviride ES11, while 10 and 20 g l(-1) glucose decrease coal solubilisation efficiency. Addition of nitrogen [1 g l(-1) (NH(4))(2)SO(4)] to the medium also increased the coal solubilisation efficiency of T. atroviride ES11. Assay results from coal-free and coal-supplemented cultures suggested that several intracellular enzymes are possibly involved in coal depolymerisation processes some of which are constitutive (phenol hydroxylase) and others that were activated or induced in the presence of coal (2,3-dihydrobiphenyl-2,3-diol dehydrogenase, 3,4-dihydro phenanthrene-3,4-diol dehydrogenase, 1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase, 1,2-dihydro-1,2-dihydroxyanthracene dehydrogenase). GC-MS analysis of chloroform extracts obtained from coal degrading T. atroviride ES11 cultures showed the formation of only a limited number of specific compounds (4-hydroxyphenylethanol, 1,2-benzenediol, 2-octenoic acid), strongly suggesting that the intimate association between coal particles and fungal mycelia results in rapid and near-quantitative transfer of coal depolymerisation products into the cell.