Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker

Summary The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency. Several transformants were tested and shown to be mitotically stable. Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites.     

Development of a gene transfer system for Penicillium chrysogenum

Summary We report here the development of an endogenous gene transfer system for the industrially-important Deuteromycete Penicillium chrysogenum, utilising a recombinant plasmid designated pPC-31 to complement a tryptophan — auxotrophic strain. Transformation frequencies in the order of 300–1800 transformants per g DNA have been obtained, and Southern hybridisation analysis has demonstrated that in the majority of cases, integration is mediated by homologous recombination between pPC-31 and the host genome at the site of the resident mutant allele.     

Transformation of Aspergillus flavus: construction of urate oxidase-deficient mutants by gene disruption

Summary A transformation procedure based on the complementation of a genetic defect was developed using a nitrate reductase-deficient mutant of Aspergillus flavus. The initial transformation efficiency was improved 40-fold by combining factors in a planned experimental program. Although low, this transformation rate was sufficient to obtain transformants in which the urate oxidase-encoding gene (uaZ) was disrupted in a gene replacement experiment. These new uaZ^- strains were unable to utilize uric acid as the unique nitrogen source and could be reversed directly to the wild-type phenotype in second order transformation experiments using a urate oxidase-expressing vector.     

Electrophoretic karyotypes and gene mapping in eight species of the Fusarium sections Arthrosporiella and Sporotrichiella

Pulsed-field gel electrophoresis was used to identify karyotypes for eight species of the Fusarium sections Arthrosporiella and Sporotrichiella. The total number of chromosome-sized DNA molecules varied from six to nine, depending on the species. The sizes of chromosomes ranged from 0.4 to approximately 6.5 Mb which gave estimates of genome size of between 27.0 and 29.9 Mb. When fractionated chromosomes of the eight species were probed with Tox5, a gene coding for the key-enzyme of trichothecene biosynthesis, strong hybridization signals developed in F. poae and F. sporotrichioides, suggesting that of the eight species examined only these two have the genetic potentiality to produce trichothecene mycotoxins. By using heterologous probes from Aspergillus different rRNA loci have also been mapped on Fusarium chromosomes.     

Transformation of Penicillium chrysogenum with a dominant selectable marker

Summary We have cloned a mutant oligomycin resistance allele of the mitochondrial ATP synthase subunit 9 gene from the filamentous fungus Penicillium chrysogenum. The gene was isolated using the equivalent gene from Aspergillus nidulans as a hybridisation probe. Using the cloned gene it is possible to select for oligomycin resistance in P. chrysogenum transformation experiments. This transformation system was used to introduce further copies of the P. chrysogenum isopenicillin N synthetase gene, which were stably maintained without selection. An assessment of the frequency with which homologous integration occurs was also made. With this system, it should prove possible to transform any strain of P. chrysogenum.     

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora     

Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum

Summary A hybrid cefE gene was constructed by juxtaposing promoter sequences from the Penicillium chrysogenum pcbC gene to the open reading frame of the Streptomyces clavuligerus cefE gene. In S. clavuligerus the cefE gene codes for the enzyme penicillin N expandase [also known as deacetoxycephalosporin C synthetase (DAOCS)]. To insert the hybrid cefE gene into P. chrysogenum the vector pPS65 was constructed; pPS65 contains the hybrid cefE gene and the Aspergillus nidulans amdS gene. The amdS gene encodes acetamidase and provides for dominant selection in P. chrysogenum. Protoplasts of P. chrysogenum were transformed with pPS65 and selected for the ability to grow on acetamide medium. Extracts of cells cultivated in penicillin production medium were analyzed for penicillin N expandase activity. Penicillin N expandase activity was detected in approximately one-third of the transformants tested. Transformants WG9-69C-01 and WG9-61L-03 had the highest specific activities of penicillin N expandase: 4.3% and 10.3%, respectively, relative to the amount of penicillin N expandase in S. clavuligerus. Untrasformed P. chrysogenum exhibited no penicillin N expandase activity. Analysis of the penicillin V titer revealed that WG9-61L-03 produced titers equal to that of the recipient strain while the amount of penicillin V produced in WG9-69C-01 was reduced by five fold.     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast

A disruption cassette has been constructed containing the LEU2 gene flanked by directly repeated sitespecific recombination sites of the yeast plasmid, pSB3, which resembles the 2 m DNA of Saccharomyces cerevisiae. A disruption constructed by inserting this DNA fragment acquires a Leu⁺ phenotype, which can be easily removed by expressing the FLP-PSB3 gene encoding the site-specific recombinase of pSB3. A test was made using a Schizosaccharomyces pombe host. The ura4 ⁺ gene of S. pombe was replaced with the ura4::LEU2 gene constructed by inserting the disruption cassette into the ura4 ⁺ gene. Then, the FLP-pSB3 gene driven by the nmt1 ⁺ promoter was introduced into this disruptant. Upon de-repression of the nmt1 promoter by removing thiamine from the medium, the rate of appearance of Leu^- was increased. As expected the ura4 ⁺ locus underwent a structural change. Thus, the FLP-pSB3 protein and its target site can function adequately in S. pombe.     

Linear mitochondrial genome organization in vivo in the genus Pythium

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.     

Chromosome rearrangements in improved cephalosporin C-producing strains of Acremonium chrysogenum

Summary A number of A. chrysogenum strains from a lineage improved in cephalosporin C production were analysed by contoured-clamped homogenous electric field gel electrophoreis (CHEF). Although antibiotic titre was increased across the lineage, chromosome rearrangements were only observed at two points in it. In one member of the lineage the chromosomal changes included those which altered the size of the chromosome on which the isopenicillin N synthetase gene (pcbC) was located. It is proposed that chromosome changes are a chance event in an industrial strain improvement programme.     

Conversion of homothallic yeast to heterothallism through HO gene disruption

A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeast strains to heterothallism through HO gene disruption. An integrative ho::neo disrupted allele was constructed by cloning a dominant selectable marker, the bacterial neo gene, within the HO gene. Transformation of a homothallic diploid S. cerevisiae strain with plasmid DNA containing the ho::neo allele yielded G418-resistant yeast transformants in which one of the HO alleles was replaced by the disrupted ho::neo allele. Meiotic tetrad analysis of four-spored asci from these G418-resistant transformants gave rise to haploid heterothallic and diploid homothallic tetrad progeny. The presence of the ho::neo and HO alleles in the heterothallic and homothallic progeny was confirmed by Souther-blot analysis.     

Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassaa

Summary Pyrimidine auxotrophs of Penicillium chrysogenum have been isolated at a high frequency among mutants resistant to 5-fluoroorotic acid (5.2 mM). Some of the pyrimidine auxotrophs (e.g. strain pyrG1) showed no reversion. A radiometric assay based on the conversion of (6-¹⁴C)orotidine 5-monophosphate (OMP) into (6-¹⁴C)uridine 5-monophosphate (UMP) was developed to determine OMP-decarboxylase activity. One of the pyrimidine auxotrophs (P. chrysogenum pyrGl) was studied in detail. It was deficient in OMP-decarboxylase activity, whereas the parental strain (P. chrysogenum Wis. 54-1255) showed a normal enzyme activity. A five-fold higher OMP-decarboxylase activity was found in a P. chrysogenum pyrGI clone transformed with plasmids containing the Neurospora crassa pyr4 gene (which codes for the same enzyme).Abbreviations OMP orotidine 5-monophosphate - UMP uridine 5-monophosphate     

Genome analysis of imperfect fungi: electrophoretic karyotyping and characterization of the nuclear gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Curvularia lunata

Summary The gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes.     

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Summary Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35–36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.     

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.