| Penicillium decumbens is a filamentous ascomycete fungus isolated from soil and has capacity to produce large amounts of cellulases and hemicellulases. Due to its high efficiency in enzymatic conversion of renewable cellulosic biomass to fermentable sugars, P. decumbens is becoming particularly attractive for the study of hydrolysis of lignocellulose. To improve the production of biomass-hydrolyzing enzymes, classical mutagenesis strategies and cultivation conditions optimization have been applied for P. decumbens. For example, the strain P. decumbens A10, as a catabolite repression-resistant mutant, was obtained from P. decumbens 114-2 by UV mutagenesis. Recently, genome shuffling was used to further enhance cellulase production by repeating protoplast fusions. Although the strain improvement in this fungus appears to be well established and several genes involved in cellulose and hemicellulose hydrolysis have been cloned (GenBank:EU239661.1; EU239662.1, etc.), the molecular mechanism for the enhancement of cellulase activity is not known. Especially, the P. decumbens genome sequencing project will be completed shortly, thus allowing the development of genome-wide knockouts. Therefore, to develop new molecular tools and novel technologies for functional genomic studies in P. decumbens is becoming increasingly urgent.Gene targeting is one of the most important approaches for comprehensive understanding of gene function. Using this strategy, target gene can be displaced with its deletion cassette, and a preexisting genomic mutation can also be corrected to wild-type with its endogenous allele. Unfortunately, the high frequency of illegitimate integration of transforming DNA has hampered gene targeting approaches in most filamentous fungi. In eukaryotes, the repair of DNA double-strand breaks (DSB) works through two main recombination pathways. One is the homologous recombination (HR) pathway, which involves interactions between homologous DNA sequences. Another is the nonhomologous end-joining (NHEJ) pathway, which involves direct ligation of DNA ends independent of sequence homology. The NHEJ pathway has been described as consisting of several components, including Ku heterodimer (Ku70/Ku80), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Xrcc4-DNA ligase IV complex. The model organism Saccharomyces cerevisiae preferentially utilizes the HR pathway in DSB repair although having NHEJ pathway. In contrast to S. cerevisiae, filamentous fungi mainly utilize the NHEJ pathway of DSB repair and exogenous DNA can be integrated anywhere in the genome. Inhibition of the NHEJ pathway in these fungi is hypothesized to force the transforming DNA to integrate into the genome via HR. Therefore, disruption of Ku70 homologue in P. decumbens is postulated to enhance the gene targeting efficiency and facilitate functional genomic studies.The transcriptional repressor CreA is the main regulatory element that has been proposed to participate in cellulase and xylanase gene expression. CreA is a negatively acting protein that mediates carbon catabolite repression in Aspergilli and Trichoderma. Despite the fact that there is a similar repression mechanism in the Aspergilli and Trichoderma, some distinct modes of CreA regulation exit in cellulose and xylan degradation.In this study, the pku70 gene homologous to A. nidulans ku70 was cloned and identified. The pku70 targeting cassette was obtained by PCR-based fusion technique, and the ability to undergo HR was assessed in pku70-defective strain compared to the wild-type parental strain at two independent loci. Subsequently, Functional analysis of CreA in wild-type strain 114-2 and derepression phenotype in mutant A10 were performed in P. decumbens.1 Isolation and analysis of putative pku 70 geneA 1.36-kb sequence with similarity to A. nidulans ku70 gene was amplified in P. decumbens, on the basis of the three different ortholog sequences of human Ku70 from Aspergillus. Based on the obtained partial sequence, the 3.2-kb and 2.8-kb up- and downstream regions of this sequence were obtained by SEFA PCR. BLAST analysis of these amplified sequences revealed that they contain a full-length open reading frame (ORF) consisting of a 2047-bp. This predicated ku 70 gene, named pku70, has three introns which are located at nucleotides 559 to 611,1459 to 1518 and 1623 to The P. decumbens pku70 gene encodes a protein of 620 amino acids (aa) with a predicated molecular mass of 68.9 kDa. BLASTP analysis showed that the Pku70 protein shares 72,71,70,69,68 and 67% identity with the respective Ku70 proteins of Neosartorya fischeri, A. clavatus, P. chrysogenum, A. sojae, A. terreus and A. nidulans. The predicted Pku70 protein sequence was submitted to the Conserved Domain Search service. The analysis revealed that Pku70 protein containes three distinct domains: Ku70/Ku80 N-terminal (vWA-ku) domain (aa 1 to 193), Ku70-core domain (aa 238 to 516) and SAP-domain (aa 573 to 607). The N-terminal domain is not involved in DNA binding but may very likely mediate Ku heterodimer interactions with other proteins involved in DSB repair process. The Ku70-core domain is constructed from a core of sevenβ-strands arranged in an antiparallel fashion, and it provides an relatively flat surface for DNA binding. The SAP-domain is a putative DNA-binding motif involved in chromosomal organization.2 Development of a highly efficient gene targeting system in Penicillium decumbens To establish highly efficient gene targeting system and confirm the function of pku70 involved in NHEJ pathway in P. decumbens,pku70 targeting cassette was constructed with a pyrithiamine resistance gene ptrA as selectable marker. The 6.4-kb△pku70::ptrA targeting cassette was transformed into P. decumbens wild-type strain 114-2. The coding sequence of the pku 70 locus was removed from the genome of the strain PKU70-42. We therefore concluded that a single gene replacement occurred at the resident pku70 locus, indicating that the pku70 gene had been disrupted in the strain PKU70-42.Since Ku proteins (Ku70/80) are involved in NHEJ repair of DSBs in eukaryotes, the△pku70 strain was examined for phenotypic characterization with respect to vegetative growth, germination rate, sensitivity to mutagen toxicity and osmotic stress, as well as cellulose hydrolysis. Plate assay showed that the growth rate of the△pku70 strain PKU70-42 was identical to that of the wild-type strain on MM. No irregularities in mycelial morphology and vegetative growth were observed in the strain PKU70-42, and the sporulation and spore germination rates of the△pku70 strain were not affected. The sensitivity to mutagen toxicity was determined with plate assay using MM plates independently supplemented with different amounts of EMS, hygromycin B and H2O2. The strain PKU70-42 showed no sensitivity differences in comparison with the wild-type strain. Sensitivity to osmotic stress was examined with spot tests using conidiospore suspension. In all cases, sensitivities to osmotic stress were not significantly different between the wild-type strain and the△pku70 strain PKU70-42. Additionally, in order to effectively utilize△pku70 background for creating deletions in genes regarding cellulose hydrolysis, cellulase activity was assayed and no significant differences were detected between△pku70 and wild-type genetic backgrounds of P. decumbens. These results indicated that the Apku70 strain is a suitable host for gene function analysis.The effect of the disruption of pku70 on homologous gene targeting was evaluated by the deletion of two randomly selected genes (creA and xlnR) in a Apku70 strain. The functions of both genes have not been elucidated in P. decumbens. The homolog of creA gene in A. nidulans encodes a negatively-acting protein, which mediates carbon catabolite repression, and the homolog of xlnR gene in Hypocrea jecorina encodes a transcriptional activator that governs cellulolytic and xylanolytic gene expression. Disruption of creA and xlnR genes by gene targeting may facilitate further study of the important regulatory machinery of carbon metabolism in P. decumbens.The△creA::hph cassette contained 3.5-kb 5'and 3.0-kb 3'of the target creA gene and the△xlnR::hph cassette harbored 1.8-kb and 1.9-kb up- and downstream genomic sequences of xlnR. Deletion of the pku70 gene in P. decumbens clearly increased gene targeting frequencies. The frequency of gene targeting was 100% when flanking regions longer than 1.8-kb were used. In the parental strain, however, gene targeting frequencies were 33% (3/9) and 91% (32/35) at creA and xlnR loci, respectively. These results suggested that gene targeting in△pku 70 was effective at various loci.3 Analysis of integration patterns P. decumbensPrevious studies have shown that ectopic integration events are usually more frequent than homologous integration when foreign DNA is integrated into the fungal genome. In the three independent gene-targeting experiments in P. decumbens wild-type strain, relatively high frequencies of homologous integration were found at xlnR, creA and pku70 loci. Possible factors influencing the homologous integration include the conformation of transforming DNA, length of homology and the recipent strain. Conformation of transforming DNA (circular or linear) can significantly affect gene targeting and transformation frequency. In addition, we discovered that complex integration patterns were existed only in a minority of the transformants, in which homologous integration of targeting cassette was accompanied by additional ectopic integration events. In most transformants foreign DNA was either ectopically or homologously integrated into the genome. Thus it seemed to be a strong incompatibility or competition between two main classes of recombination mechanisms for transforming DNA. On average, approximately 61.5% (16/26) of nontargeted cells incorporated two or multiple copies of targeting cassette integrated randomly into the genome, compared with 9.4% (5/53) of the targeted cells incorporated more than one copy of transforming DNA due to ectopic integration. These results indicated that the nontargeted cells were more likely to incorporate a second fragment by ectopic integration than were targeted cells in P. decumbens.4 Marker exchanging in the△pku70::ptrA strainTo adequately validate the△pku70 genetic background, the P. decumbens△pku70::hph strain was constructed by exchanging selectable markers in Apku70::ptrA strain with hph expression cassettes. The△pku70::hph targeting cassette was created and transformed into the P. decumbens△pku70::ptrA strain PKU70-42 as recipient. Sixteen randomly selected transformants were subjected to PCR analysis and plate assay. All transformants obtained ultimately became resistant to hygromycin B and sensitive to pyrithiamine. These results indicated that allelic exchange between the two pku70 targeting cassettes successfully occurred at the pku70::ptrA locus by homologous integration in strain PKU70-42.5 Analysis of cotransformation in P. decumbensTo determine whether two loci could be independently targeted within a single transformation experiment by using mixtures of two different targeting cassettes, the△pku70::ptrA and△ace1::dsRed2 cassettes were constructed and cotransformed into the wild-type and Apku70::hph strains, respectively. In the first case, cotransformants were found at a frequency of approximately one per thirteen PyrR transformants, and the△ace1::dsRed2 cassette was integrated ectopically at unknown sites in the genome of wild-type strain. The remainder of PyrR transformants showed no cotransforming△ace1::dsRed2 cassette anywhere. In parallel experiments with the same targeting cassettes, thirty-eight PyrR transformants were obtained using the△pku70::hph strain as recipient. However, all of them did not contain cotransforming△ace1::dsRed2 fragment. The analysis confirmed that the hph marker in△pku70::hph strain was replaced by the ptrA expression cassette without additional integration of△pku70::ptrA fragment into genome. Collectively, these data demonstrated that cotransformation with two linear targeting cassettes occurred at a relatively low frequency in P. decumbens, especially in△pku70 strain as recipient. We therefore concluded that simultaneous targeting of independent genes is not a frequent occurrence during cotransformation of two different targeting cassettes.6 Functional analysis of CreA in P. decumbensIn order to analyze the function of the CreA protein, we produced total deletion of creA produced by using in vitro techniques and△creA strain was complemented by another creA allele. In P. decumbens strain 114-2, deletions of the creA gene lead to derepression of cellulase and xylanase production even in the medium containing the D-glucose. The results showed that the regulator CreA, as a carbon catabolite repressor, mediates the repression of genes encoding cellulase and hemicellulase. Our results also indicated that lactose has no obvious effect on induction of cellulase production.7 Analysis of derepression phenotype in P. decumbens A10The known mutation in creA confers a derepressed phenotype in P. decumbens A10. A mutant was constructed in which a total deletion of creA produced by using in vitro techniques in P. decumbens A10. We also produced a strain the creA was corrected by another creA allele cloned from wild-type strain 114-2. In the rectified strain, the phenotype for catabolite repression can be rescued by transformation with the wild-type creA allele. Information from plate analysis of creA mutants, combined with information derived from analysis of rectified strain, allow us to determine that CreA lead to partial derepression and retain some activity in P.decumbens A10, since a total deletion has much more derepression phenotype in medium containing glucose.
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