RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

Background  

Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA ^- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions.     

Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions

Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the -glucuronidase (gusA) and -galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Cloning, characterization of the acyl-CoA : 6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene

Summary The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5 region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39 240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.     

Transformation of Penicillium chrysogenum by a vector containing a mitochondrial origin of replication

Summary In order to establish a transformation system for P. chrysogenum autonomously replicating vectors were constructed using mitochondrial DNA sequences from the fungus. A physical map of the mt DNA of a production strain was established using ten different restriction enzymes. Unexpectedly, the mt DNA of this strain proved to be significantly smaller than that of a second strain from a culture collection (27 kb versus 49 kb). Various fragments representing about 71% of the 27 kb mt DNA were cloned and, at first, preselected for replicating activity in an intermediate host (Saccharomyces cerevisiae). Two of these fragments also promoted autonomous replication in P. chrysogenum, which was confirmed by isolation of bulk DNA and transfer into E. coli. For selection of transformants in P. chrysogenum the prokaryotic kanamycin resistance gene was used which increased about twofold the resistance against G418. Present address: Institut für Biotechnologie, Fachgebiet Mikrobiologie, Techn. Universität Berlin, Seestr. 13, D-1000 Berlin 65     

Identification and physical characterization of yeast maltase structural genes

Summary Each of at least five unlinked MAL loci (MAL1 through MAL4 and MAL6) on the yeast genome controls the ability to synthesize an inducible -D-glucosidase (maltase). A subcloned fragment of the coding sequence of the MAL6 maltase structural gene was used as a hybridization probe to investigate the physical structure of the family of MAL structural genes in the genomes of different Saccharomyces strains. Mal⁺ strains, each carrying a genetically defined MAL locus, were crossed with a Mal^- strain and the segregation behavior of the functional locus and of sequences complementary to the maltase structural gene at that locus analyzed. The maltase structural gene sequences of each MAL locus were detected by Southern blot hybridization using BamH1 digests of genomic DNA of the meiotic products. This restriction enzyme was previously shown to cleave outside the confines of the MAL6 locus.The results of such experiments indicate that each MAL locus encompasses at least one maltase structural gene sequence homologous to that of MAL6, that yeast strains that lack functional MAL loci may or may not contain the corresponding maltase structural gene sequence, that the MAL1 maltase structural gene sequence or one of its alleles can be detected in all laboratory yeast strains examined and that each MAL locus can be identified as a characteristic BamH1 fragment of genomic DNA which includes a maltase structural gene.Yeast strains vary in the number of maltase structural gene sequences that they carry. By using the approach described in this report, the ones corresponding to the different functional MAL loci and residing within a BamH1 generated restriction fragment can be identified.     

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9′

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9 was found to be the best lipase producer. The maximum lipase activity was 68 units mL^–1 culture medium on the fifth day of incubation at pH 6.0 and 20°C. Therefore, P. chrysogenum 9 was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30°C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The K_m and V_max were calculated to be 2.33 mM and 22.1 units mL^–1, respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca²⁺. The P. chrysogenum 9 lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.     

Resolution of four large chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome 1 (10.4 Mb) in Penicillium chrysogenum

Four chromosomes were resolved by pulsed field gel electrophoresis in Penicillium notatum (10.8, 9.6, 6.3 and 5.4 Mb in size) and in five different strains of Penicillium chrysogenum (10.4, 9.6, 7.3 and 6.8 Mb in the wild type). Small differences in size were found between the four chromosomes of the five P. chrysogenum strains. The penicillin gene cluster was localized by hybridization with a pcbAB probe to chromosome II of P. notatum and to chromosome I of all P. chrysogenum strains except the deletion mutant P. chrysogenum npe10, which lacks this DNA region. The pyrG gene was localized to chromosome I in P. notatum and to chromosome II in all P. chrysogenum strains except in the mutant AS-P-78 where the probe hybridized to chromosome 111. A major chromosomal rearrangement seems to have occurred in this high penicillin producing strain. A fast moving DNA band observed in all gels corresponds to mitochondrial DNA. The total genome size has been calculated as 32.1 Mb in P. notatum and 34.1 Mb for the P. chrysogenum strains.     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5 flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5 regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Improvement of Cephalosporin C Production by Recombinant DNA Integration in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Cephalosporins are widely used as anti-infectious β-lactam antibiotics in clinic. For the purpose of increasing the yield of cephalosporin C (CPC) fermentation, especially in an industrial strain, A. chrysogenum genes cefEF and cefG, which encode the ultimate and penultimate steps in CPC biosynthesis, cefT, which encodes a CPC efflux pump, and vgb, which encodes a bacterial hemoglobin gene were transformed in various combinations into an industrial strain of A. chrysogenum. Both PCR and Southern blotting indicated that the introduced genes were integrated into the chromosome of A. chrysogenum. Carbon monoxide difference spectrum absorbance assay was performed and the result showed that Vitreoscilla hemoglobin was successfully expressed in A. chrysogenum and had biological activity. HPLC analysis of fermentation broth of recombinant A. chrysogenum showed that most transformants had a higher CPC production level than the parental strain. Multiple transformants containing an additional copy of cefG showed a significant increase in CPC production. However, cefT showed little effect on CPC production in this high producer. The highest improvement of CPC titer was observed in the mutant with an extra copy of cefG + cefEF + vgb whose CPC production was increased by 116.3%. This was the first report that three or more genes were introduced simultaneously into A. chrysogenum. Our results also demonstrated that the combination of these genes had a synergy effect in a CPC high producer.     

Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease

Summary A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO₄ after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 × 10³ transformants/g DNA per 10⁷ protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.     

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

Summary A plant gene vector cassette to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.Abbreviations OCS octopine synthase (gene) - NOS nopaline synthase (gene) - NPT-II neomycin phosphotransferase (gene) of transposon Tn5 - vir Ti-plasmid region encoding virulence functions - Cb carbenicillin - Gm gentamycin - Km kanamycin - Cm chloramphenicol - Sm streptomycin - Sp spectinomycin - Rif rifampicin - Ery erythromycin - bom basis of mobilization - ori _r origin of conjugational plasmid transfer - Tra, Mob functions required for conjugational transfer of plasmids - BAP N⁶-benzylaminopurine - NAA -naphthaleneacetic acid - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide     

Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a self-cloning sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5 upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.     

Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.