Analysis of the functions of recombination-related genes in the generation of large chromosomal deletions by loop-out recombination in Aspergillus oryzae

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure.     

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70–ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.     

Nonhomologous end-joining deficiency allows large chromosomal deletions to be produced by replacement-type recombination in Aspergillus oryzae

Gene targeting is a technique of introducing a genetic trait at a predetermined site within a genome; it is also used to eliminate undesirable chromosomal regions from the relevant genome. Thus far, replacement-type recombination between two homologous regions separated by a large nonhomologous sequence has been hardly achieved probably due to the low frequency of homologous recombination in filamentous fungi. In this study, we report the successful and highly efficient deletion by replacement-type recombination of up to 470-kb regions of chromosome 8 and 200-kb region in chromosome 3, which includes a homologue of aflatoxin gene cluster, by nonhomologous end-joining deficient strains of Aspergillus oryzae. Our study results indicate that the deficiency of nonhomologous end-joining increases the distance of nonhomologous regions in replacement-type recombination, i.e., the possible deletion range in generation of large chromosomal deletion by one cycle of replacement-type recombination is increased in nonhomologous end-joining deficient strains.     

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.     

Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger

In this study, the ku70 and ku80 homologs from the Aspergillus niger genome were identified and their function was analyzed using targeted mutagenesis. The role of the ku80 gene in non-homologous end-joining (NHEJ) was investigated by calculating the frequency of homologous recombination. The transformation test verified that the frequency of homologous recombination significantly increased, from 1.78 to 65.6% in ku80 single deletion strains and to 100% in ku70/ku80 double deletion strains. These results suggest that the ku80 gene is important for non-homologous end-joining. Although the morphology of the ku deletion strains colonies was similar to that of the wildtype strain, mutants were more sensitive to the mutagen phleomycin. Furthermore, the purified ku80 deletion strain produced some sectored colonies on hygromycin B-containing plates. This result suggests that the ku80 gene deletion leads to genomic instability in A. niger.     

Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries.     

A highly efficient gene-targeting system for Aspergillus parasiticus

Aims: To establish a system that greatly increases the gene‐targeting frequency in Aspergillus parasiticus. Methods and Results: The ku70 gene, a gene of the nonhomologous end‐joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O‐methylsterigmatocystin (OMST). The NHEJ‐deficient strain, RHΔku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA‐topoisomerase I complex inhibitor, camptothecin, and the DNA‐damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA‐targeting frequency in RHΔku70 reached 96%. Conclusions: The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ‐deficient RHΔku70, easy creation of homologous ends for integration, and the ptr‐based selection form a highly efficient gene‐targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study: The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.     

Homologs of Aflatoxin Biosynthesis Genes and Sequence of aflR in Aspergillus oryzae and Aspergillus sojae

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis in Aspergillus parasiticus and A. flavus was shown for A. oryzae and A. sojae. Homologs of the regulatory gene aflR were cloned and sequenced from A. oryzae and A. sojae.     

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein^1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale^1-4.Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).     

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.     

Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.     

The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome

Background

Chromosomal deletions are used extensively in Drosophila melanogaster genetics research. Deletion mapping is the primary method used for fine-scale gene localization. Effective and efficient deletion mapping requires both extensive genomic coverage and a high density of molecularly defined breakpoints across the genome.

Results

A large-scale resource development project at the Bloomington Drosophila Stock Center has improved the choice of deletions beyond that provided by previous projects. FLP-mediated recombination between FRT-bearing transposon insertions was used to generate deletions, because it is efficient and provides single-nucleotide resolution in planning deletion screens. The 793 deletions generated pushed coverage of the euchromatic genome to 98.4%. Gaps in coverage contain haplolethal and haplosterile genes, but the sizes of these gaps were minimized by flanking these genes as closely as possible with deletions. In improving coverage, a complete inventory of haplolethal and haplosterile genes was generated and extensive information on other haploinsufficient genes was compiled. To aid mapping experiments, a subset of deletions was organized into a Deficiency Kit to provide maximal coverage efficiently. To improve the resolution of deletion mapping, screens were planned to distribute deletion breakpoints evenly across the genome. The median chromosomal interval between breakpoints now contains only nine genes and 377 intervals contain only single genes.

Conclusions

Drosophila melanogaster now has the most extensive genomic deletion coverage and breakpoint subdivision as well as the most comprehensive inventory of haploinsufficient genes of any multicellular organism. The improved selection of chromosomal deletion strains will be useful to nearly all Drosophila researchers.     

Multiple large segment deletion method for Corynebacterium glutamicum

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.     

A trial of minimization of chromosome 7 in Aspergillus oryzae by multiple chromosomal deletions

We aim to create an Aspergillus oryzae mutant with a highly reduced chromosome, but better growth, by eliminating the nonessential regions coding various dispensable functions for its better industrial use. In our previous study, we successfully determined the outline of essential and nonessential regions by constructing a series of large chromosomal deletions in A. oryzae chromosome 7. Based on these results, we here constructed two mutants, designated RkuAF7A and RkuAF7B, lacking 24.7 and 24% (725 and 705 kb) of wild type chromosome 7, respectively, using multiple large-scale chromosomal deletions in a recursive pyrG-mediated transformation system. Both showed higher amylase activity in DPY liquid medium and faster growth rate on malt agar medium relative to the parent strain. The two mutants also displayed soft fluffy hyphal morphology when grown in DPY liquid media. In addition, the gene expression profile obtained by DNA microarray indicated that although the deletion regions were fewer than 2% of the whole genome, the effect on whole gene expression exceeded 20%. Among these, the genes involved in secondary metabolism showed a relatively large change in their gene expression levels. Together, the constructed mutants showing better growth and potential usefulness is possibly suitable for further industrial use.     

Fathoming Aspergillus oryzae metabolomes in formulated growth matrices

The stochasticity of Aspergillus oryzae (Trivially: the koji mold) pan-metabolomes commensurate with its ubiquitously distributed landscapes, i.e. growth matrices have been seemed uncharted since its food fermentative systems are mostly being investigated. In this review, we explicitly have discussed the likely tendencies of A. oryzae metabolomes pertaining to its growth milieu formulated with substrate matrices of varying nature, composition, texture, and associated physicochemical parameters. We envisaged typical food matrices, namely, meju, koji, and moromi as the semi-natural cultivation models toward delineating the metabolomic patterns of the koji mold, which synergistically influences the organoleptic and functional properties of the end products. Further, we highlighted how tailored conditions in sub-natural growth matrices, i.e. synthetic cultivation media blends, inducers, and growth surfaces, may influence A. oryzae metabolomes and targeted phenotypes. In general, the sequential or synchronous growth of A. oryzae on formulated matrices results in a number of metabolic tradeoffs with its immediate microenvironment influencing its adaptive and regulatory metabolomes. In broader context, evaluating the metabolic plasticity of A. oryzae relative to the tractable variables in formulated growth matrices might help approximate its growth and metabolism in the more complex natural matrices and environs. These approaches may considerably help in the design and manipulation of hybrid cultivation systems towards the efficient harnessing of commercial molds.     

A further study on chromosome minimization by protoplast fusion in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1–12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 μg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.     

Non-recurrent 17p11.2 deletions are generated by homologous and non-homologous mechanisms

Several recurrent common chromosomal deletion and duplication breakpoints have been localized to large, highly homologous, low-copy repeats (LCRs). The mechanism responsible for these rearrangements, viz., non-allelic homologous recombination between LCR copies, has been well established. However, fewer studies have examined the mechanisms responsible for non-recurrent rearrangements with non-homologous breakpoint regions. Here, we have analyzed four uncommon deletions of 17p11.2, involving the Smith–Magenis syndrome region. Using somatic cell hybrid lines created from patient lymphoblasts, we have utilized a strategy based on the polymerase chain reaction to refine the deletion breakpoints and to obtain sequence data at the deletion junction. Our analyses have revealed that two of the four deletions are a product of Alu/Alu recombination, whereas the remaining two deletions result from a non-homologous end-joining mechanism. Of the breakpoints studied, three of eight are located in LCRs, and five of eight are within repetitive elements, including Alu and MER5B sequences. These findings suggest that higher-order genomic architecture, such as LCRs, and smaller repetitive sequences, such as Alu elements, can mediate chromosomal deletions via homologous and non-homologous mechanisms. These data further implicate homologous recombination as the predominant mechanism of deletion formation in this genomic interval.