Recombinant dna in Filamentous Fungi: Progress and Prospects

Rambosek, J A; Leach, Jeanette; Kinsey, John A.

doi:10.3109/07388558709089387

Cited by 150 publications

(40 citation statements)

References 143 publications

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“…USA 85 (1988) washed twice with 10 ml of buffer 11 (25 mM 20-fold. This is in marked contrast to results obtained with other filamentous fungi, where linearization of the plasmid usually has little or no effect on transformation frequency (17). In Saccharomyces cerevisiae improved transformation has also been achieved with linear DNA and has been attributed to the recombinogenic nature of free ends of DNA (18).…”

contrasting

confidence: 52%

Gene transfer system for the phytopathogenic fungus Ustilago maydis.

Wang

Holden

Leong

1988

Proc. Natl. Acad. Sci. U.S.A.

211

121

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A selectable marker for transformation was constructed by transcriptional fusion of a Ustiago maydis heat shock gene promoter with the hygromycin B phosphotransferase gene of Escherichia coli. U. maydis was transformed to hygromycin B resistance by polyethylene glycol-induced fusion of spheroplasts following exposure to plasmid DNA that carried the marker gene. Transformation frequencies of 50 and 1000 transformants per ,ug of DNA per 2 x 107 spheroplasts were obtained for circular and linear vector DNA, respectively. In the majority of transformants, the vector was integrated at a single chromosomal site, in either single copy or tandem duplication, as determined by Southern hybridization analysis of electrophoretically separated chromosomes and of restriction-endonuclease-cleaved DNA. The predominant form (82%) of vector integration was by nonhomologous recombination; the remainder carried the plasmid at the homologous heat shock gene locus. No evidence for gene conversion or gene replacement was obtained in 28 transformants. Hygromycin B phosphotransferase activity and resistance to hygromycin B were roughly correlated with the copy number of the integrated vector at the homologous location. Transforming DNA was stably maintained during mitosis and meiosis. This transformation procedure and associated vector should permit the cloning of genes by direct complementation in U. maydis.Research on the basidiomycete Ustilago maydis, a pathogen of corn, has deepened our understanding of the genetics and biochemistry of DNA replication, repair, and recombination (1,2). This fungus can be cultured with ease on simple, chemically defined medium, and the yeast-like, uninucleate haploid stage of growth facilitates genetic analysis. U. maydis infects corn and forms smut galls, plant neoplastic structures. Due to the rapid expansion of knowledge of the molecular biology and genetics of corn, U. maydis should prove to be valuable for molecular studies of host-parasite interactions. Studies on this fungus may also provide models for research on other basidiomycete phytopathogens including the cereal smuts and rusts. These fungi are causative agents of many severe plant diseases that can result in significant crop losses (3).The long-term goal of our work is to elucidate the structure, regulation, and role of genes involved in phytopathogenic and saprophytic growth of U. maydis. Moleculargenetic analysis of these genes and others requires the development of a gene-transfer system. Genetic transformation in U. maydis based on kanamycin resistance has been reported by Banks (4); however, only low-frequency, unstable transformants were obtained. Here we report the establishment of an efficient and reproducible DNA transformation system for U. maydis based on resistance to hygromycin B (HygB), an antibiotic that has been successfully utilized as a selective agent in a variety of organisms ranging from yeast to mouse cells (5-9).MATERIALS AND METHODS Strains and Media. Escherichia coli HB101 (F-hsdS20-(rB ,mB ) recA13 ara-...

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contrasting

confidence: 52%

Gene transfer system for the phytopathogenic fungus Ustilago maydis.

Wang

Holden

Leong

1988

Proc. Natl. Acad. Sci. U.S.A.

211

121

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“…Seven introns are present in the gene, which were all confirmed by cDNA sequencing. The introns are of 50-58 nucleotides, have fungal consensus splice sequences (Rambosek and Leach, 1987) and all but one (intron IV) have in-frame stop codons. None of the introns are in an identical position to the intron in the A. tubingensis pgaII gene or to any of the introns of the A. niger endopolygalacturonase genes pgal, pgall and pguC (Bussink et al, 1991(Bussink et al, a,b, 1992.…”

Section: Resultsmentioning

confidence: 99%

Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis

Kester

Someren

Muller

et al. 1996

European Journal of Biochemistry

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From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly( 1,4-a-~-ga~acturonide)galacturonohydrolasel that cleaves monomer units from the non-reducing end of the substrate molecule.K,,, and V,,,, for polygalacturonic acid hydrolysis were 3.2 mg mlV' and 3.1 mg ml-' and 255 U mg I and 262U mg-' for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2 -7), the Michaelis constant (K,,,) decreased with increasing chain length (n). The V,;,, value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid ( K , = 0.3 mM) as well as by reduced digalacturonate (K, = 0.4 mM).The exopolygalacturonase gene (pgax) was cloned by reverse genetics and shows only 13 % overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pguX-coding region was used to achieve high level production of exopol ygalacturonase under conditions where no endopolygalacturonases were produced.

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“…The amino acid sequence of the 29-and 11-kDa proteins from P. chrysogenum crossed the intron-exon boundaries (3, 25a). Intron lengths fell within expected limits for fungi, and splice junction sites in both genes conformed to the consensus sequences for fungal genes (20). Possible branch sites were less similar.…”

Section: Resultsmentioning

confidence: 90%

“…4. The protein-coding sequences were colinear with a predicted size of 357 amino acids, and they were 76.5% identical at the (20). A possible polyadenylation signal 3' to the open reading frame is also overlined.…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli

et al. 1990

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The final step in the biosynthesis of ,B-lactam antibiotics in Penicilium chrysogenum and AspergiUus nidulans involves removal of the L-a-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids Over the last two decades, workers in several laboratories (1,9,10,13,14,23) have investigated the production of semisynthetic penicillins in Penicillium chrysogenum by exchange of the a-aminoadipyl side chain of isopenicillin N (IPN), an obligate intermediate (18), with one of many acyl-coenzyme A (acylCoA) substituents derived from exogenously added monosubstituted acetic acids. In the absence of these derivatives, 6-aminopenicillanic acid (6-APA) production increases as well as penicillins with side chains derived from the endogenous monosubstituted acetic acids (e.g., penicillin G [PenG] from phenylacetic acid). Queener and Neuss (18) proposed a model (Fig. 1) for the conversion of IPN to penicillins and suggested the existence of a two-step enzymatic process. However, it has been unclear whether a single enzyme (acylCoA:6-APA acyltransferase [AT]) can directly accept IPN as a substrate and exchange side chains (i.e., it contains IPN amidolyase activity and is multifunctional).Alvarez et al. (2) and Alonso et al.(1) have independently purified AT to apparent homogeneity as a monomeric protein with an Mr of approximately 29,000 as judged by gel chromatography. However, the latter authors did not report activity with IPN, and the former found that the specific activity with IPN was nearly sevenfold lower than with 6-APA, even in the crude extract. Luengo et al. (13) substrate specificity. Therefore, the reaction to produce nonpolar penicillins and the enzyme(s) necessary for this activity remained unclear.Recently, Veenstra et al. (24) and Barredo et al. (3), using the N-terminal amino acid sequence of the putative AT protein (29 kilodaltons [kDa]) from P. chrysogenum, generated a mixed oligonucleotide probe and cloned DNA that complemented mutants of P. chrysogenum Wis54-1255 (2). These mutants were reported to have little or no AT activity. By further molecular characterization, the code for the 29-kDa protein was demonstrated in the DNA sequence. The N-terminal sequence of an 11-kDa protein that was present after a several-hundred-fold purification of the 29-kDa protein was also encoded in the cloned DNA sequence (24, 25a).Moreover, the gene coding for IPN synthetase (pcbC [5]) in P. chrysogenum is also closely linked to penDE (8,24). Sequence and preliminary transcription analysis indicated that penDE contains introns and c...

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Recombinant dna in Filamentous Fungi: Progress and Prospects

Cited by 150 publications

References 143 publications

Gene transfer system for the phytopathogenic fungus Ustilago maydis.

Gene transfer system for the phytopathogenic fungus Ustilago maydis.

Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli

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