pyrG Locus Research Articles

NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger

Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3’ end and by HDR at the 5’ end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.

Polycistronic gene expression in Aspergillus niger

BackgroundGenome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger.ResultsIn this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three.ConclusionsThe P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.

A new vector for efficient gene targeting to the pyrG locus in Aspergillus niger.

BackgroundThe possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics.FindingsIn this study, we report a new targeting vector based on the pyrG marker in Aspergillus niger. The DNA sequence to be targeted is surrounded by two fragments of the pyrG gene to allow homologous recombination of the recombinant DNA at the pyrG locus. The 5’ end of the targeting cassette contains a non-functional truncated pyrG open reading frame (first 112 bases deleted) and the 3’ untranslated region (3’ UTR). At the 3’ end, the targeting cassette consists of the 3’ flanking region of the pyrG gene. A unique NotI site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the A. niger pyrG mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene (mluc) as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 (ku70 −) transformants contained the reporter gene at the pyrG locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the mluc reporter was integrated at the pyrG locus, showed comparable and reproducible lux activities.ConclusionThe new pyrG targeting vector is an important improvement to the existing method for gene targeting in A. niger. Although the vector is specific for A. niger, the presented design and approach is easily applicable for constructing integration vectors for other fungi.

Repetitive genomic sequences as a substrate for homologous integration in the Rhizopus oryzae genome

The vast number of repetitive genomic elements was identified in the genome of Rhizopus oryzae. Such genomic repeats can be used as homologous regions for integration of plasmids. Here, we evaluated the use of two different repeats: the short (575 bp) rptZ, widely distributed (about 34 copies per genome) and the long (2053 bp) rptH, less prevalent (about 15 copies). The plasmid carrying rptZ integrated, but did so through a 2256-bp region of homology to the pyrG locus, a unique genomic sequence. Thus, the length of rptZ was below the minimal requirements for homologous strand exchange in this fungus. In contrast, rptH was used efficiently for homologous integration. The plasmid bearing this repeat integrated in multicopy fashion, with up to 25 copies arranged in tandem. The latter vector, pPyrG-H, could be a valuable tool for integration at homologous sequences, for such purposes as high-level expression of proteins.

Efficient and versatile transformation systems in entomopathogenic fungus Lecanicillium species

Lack of genetic tools in entomopathogenic fungi, especially those for targeted homologous recombination, hindered the advance in this field. To facilitate the genetic study, we constructed a transformation system in entomopathogenic fungus Lecanicillium sp. strain HF627 using the uridine auxotrophic pyrG mutant strain as host and endogenous pyrG as marker. pUC19 harboring endogenous pyrG successfully restored the uridine auxotrophy of the host strain, and the integration of the vector DNA was confirmed by Southern hybridization. An autonomously replicating vector harboring an AMA1 sequence was constructed and applied to the constructed transformation system, which improved the transformation efficiency 16.7-fold. Southern hybridization revealed replication of the AMA1-harboring vector with an average copy number of 2.4. A ku80 knock-out strain was created to improve the efficiency of gene targeting. Deletion of the pyrG locus, which is homologous to the marker gene, from the ku80 knock-out strain achieved a targeting efficiency of 62.5% against both trp1 and his3; the levels of these genes were 3.2- and 5-fold higher, respectively, than the ku80-intact strain. With the pyrG-deleted and ku80-inactivated strain constructed in this study, transformation and targeted homologous recombination were highly enhanced, by which genetic analysis in Lecanicillium spp. will be performed quickly and efficiently.

Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus

An efficient gene-targeting system based on impairment of the nonhomologous end-joining pathway and the orotidine monophosphate decarboxylase gene (pyrG) in Aspergillus flavus was established. It was achieved by replacing the ku70 gene with the Aspergillus oryzae pyrithiamine resistance (ptr) gene and by inserting the Aspergillus parasiticus cypA gene into the pyrG locus. The utility of this system was demonstrated by disruption of nine candidate genes for conidial pigment biosynthesis. The gene-targeting frequencies ranged from 80 to 100%. Two linked genes on chromosome 4, wA and olgA, were confirmed to be involved in pigment formation. In contrast to the parental strain which produced yellowish-green conidia, the knockout mutants produced white and olive-green conidia, respectively. The system was further refined by restoring the pyrithiamine sensitivity and uracil auxotrophy in the A. flavus transformation recipient with an engineered pyrG marker. The improvement allowed gene manipulation using the reusable pyrG marker as shown by the restoration of laeA-mediated aflatoxin production in an A. flavus laeA-deleted mutant.

Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae.

Genetic transformation of the Mucorales fungi has been problematic, since DNA transformed into the host rarely integrates and usually is mitotically unstable in the absence of selective pressure. In this study, transformation of Rhizopus oryzae was investigated to determine if the fate of introduced DNA could be predicted based on double-strand break repair and recombination mechanisms found in other fungi. A transformation system was developed with uracil auxotrophs of Rhizopus oryzae that could be complemented with the pyrG gene isolated in this work. DNA transformed as circular plasmids was maintained extrachromosomally in high-molecular-weight (>23 kb) concatenated arrangement. Type-I crossover integration into the pyrG locus and type-III pyrG gene replacement events occurred in approximately 1-5% of transformants. Linearization of the plasmid pPyr225 with a single restriction enzyme that cleaves within the vector sequence almost always resulted in isolates with replicating concatenated plasmids that had been repaired by end-joining recombination that restored the restriction site. The addition of a 40-bp direct repeat on either side of this cleavage site led to repair by homologous recombination between the repeated sequences on the plasmid, resulting in loss of the restriction site. When plasmid pPyr225 was digested with two different enzymes that cleave within the vector sequence to release the pyrG containing fragment, only pyrG gene replacement recombination occurred in transformants. Linearization of plasmid pPyr225 within the pyrG gene itself gave the highest percentage (20%) of type-I integration at the pyrG locus. However, end-joining repair and gene replacement events were still the predominant types of recombination found in transformations with this plasmid topology.

Intrachromosomal recombination after targeted monocopy integration in Penicillium chrysogenum: stabilization of the direct repeats to prevent loss of the inserted gene.

Monocopy systems obtained by targeted integration at the pyrG locus of P. chrysogenum led to the formation of unstable direct repeats in the genome. A previously isolated pyrG mutant was sequenced and the mutation was found to be located at nucleotide position 665 of the pyrG gene. A different pyrG mutation was introduced in vitro at the BamHI site of this gene. Recombination products arising from monocopy systems using the bleomycin/phleomycin resistance gene (ble) as a model were studied to elucidate the intrachromosomal recombination mechanisms. Experimental results showed that both gene conversion and deletion events occurred spontaneously at the integration site. Gene conversion products were obtained at a frequency of one in 3.4x10(4) viable transformant spores. When gene conversion occurred, the inserted exogenous gene was retained and was flanked by rearranged direct repeats of the pyrG gene, each containing at least one pyrG mutation. Deletion events resulted in the loss at high frequency of the inserted exogenous gene. Genetic stabilization of a monocopy system was obtained when both pyrG repeats (formed at the targeted integration site) contained at least one identical mutation, since in this case further recombinations can be easily counter-selected.

A Novel Heptameric Sequence (TTAGTAA) Is the Binding Site for a Protein Required for High Level Expression of pcbAB, the First Gene of the Penicillin Biosynthesis in Penicillium chrysogenum

The first two genes pcbAB and pcbC of the penicillin biosynthesis pathway are expressed from a 1.01-kilobase bidirectional promoter region. A series of sequential deletions were made in the pcbAB promoter region, and the constructions with the modified promoters coupled to the lacZ reporter gene were introduced as single copies at the pyrG locus in Penicillium chrysogenum npe10. Three regions, boxes A, B, and C, produced a significant decrease in expression of the reporter gene when deleted. Protein-DNA complexes were observed by using the electrophoretic mobility shift assay with boxes A and B (complexes AG1, BG1, BG2, and BL1) but not with box C. Uracil interference assay showed that a protein in P. chrysogenum cell extracts interacts with the thymines in a palindromic heptanucleotide TTAGTAA. Point mutations and deletion of the entire TTAGTAA sequence supported the involvement of this sequence in the binding of a transcriptional activator named penicillin transcriptional activator 1 (PTA1). In vivo studies using constructions carrying point mutations in the TTAGTAA sequence (or a deletion of the complete heptanucleotide) confirmed that this intact sequence is required for high level expression of the pcbAB gene. The TTAGTAA sequence resembles the target sequence of BAS2 (PHO2), a factor required for expression of several genes in yeasts.

Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination.

Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A. tumefaciens-mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA. Recombinant A. awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.

Transcription of the pcbAB, pcbC and penDE genes of Penicillium chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs.

Glucose repressed transcription of the penicillin biosynthesis genes pcbAB, pcbC and penDE when added at inoculation time to cultures of Penicillium chrysogenum AS-P-78 but it had little repressive effect when added at 12 h and no effect when added at 24 or 36 h. A slight increase in the expression of pcbC and penDE (and to a smaller extent of pcbAB) was observed in glucose-grown cultures at pH 6.8, 7.4 and 8.0 as compared with pH 6.2, but alkaline pHs did not override the strong repression exerted by glucose. Transcription of the actin gene used as control was not significantly affected by glucose or alkaline pHs. Repression by glucose of the three penicillin biosynthetic genes was also observed using the lacZ reporter gene coupled to each of the three promoters in monocopy transformants with the constructions integrated at the pyrG locus. Glucose repression of the three genes encoding enzymes of penicillin biosynthesis therefore appears to be exerted by a regulatory mechanism independent from pH regulation.

Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase.

A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function.

The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori.

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, inframe fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-pro-sequence. The multicopy strains showed a 6-to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion.

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori.

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5' end of the pyrG gene with vectors containing a mutation near the 3' end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae.

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr+ transformants at a frequency of up to 20 per micrograms of DNA, whereas with linear pAO4-2 up to 200 transformants per micrograms DNA were obtained. In 75% of the Pyr+ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding beta-galactosidase, and uidA, encoding beta-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans

We have modified the transformation procedures of Ballance et al. [Biochem. Biophys. Res. Commun. 112 (1983) 284-289] to give increased rates of transformation in Aspergillus nidulans. With the modified procedures we have been able to complement pyrG89, a mutation in the orotidine-5'-phosphate decarboxylase gene of A. nidulans, by transformation with a library of wild-type (wt) sequences in pBR329. We have recovered, by marker rescue from one such transformant, a plasmid (pJR15) that carries an A. nidulans sequence that complements pyrG89 efficiently. In three experiments, this plasmid gave an average of 1985 stable transformants/μg of transforming DNA. We have analyzed ten of these genetically and by Southern hybridization. In five transformants a single copy of the transforming plasmid had integrated at the pyrG locus, in one transformant several copies of pJR15 had integrated at this locus, in one transformant several copies of the plasmid had integrated into other sites, and in three transformants, the wt allele had apparently replaced the mutant allele with no integration of pBR329 sequences. Sequence and S1 nuclease protection analysis revealed that pJR15 contains a gene that predicts an amino acid sequence with regions of strong homology to the orotidine-5'-phosphate decarboxylases of Neurospora crassa and Saccharomyces cerevisiae. We conclude that this gene is the wt pyrG allele. Finally, we have compared the 5'- and 3'-noncoding sequences and intron splice sequences to other genes of A. nidulans and have mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I.

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene.

The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5'-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr+ transformants at a frequency of 40 transformants per microgram of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per microgram of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr+. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50–100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ansl. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 × 10 3 stable transformants per μg of plasmid DNA.