Phosphatidylserine synthase plays a critical role in the utilization of n-alkanes in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica can assimilate n-alkane as a carbon and energy source. To elucidate the significance of phosphatidylserine (PS) in the utilization of n-alkane in Y. lipolytica, we investigated the role of the Y. lipolytica ortholog (PSS1) of Saccharomyces cerevisiae PSS1/CHO1, which encodes a PS synthase. The PSS1 deletion mutant (pss1Δ) of Y. lipolytica could not grow on minimal medium in the absence of ethanolamine and choline but grew when either ethanolamine or choline was supplied to synthesize phosphatidylethanolamine and phosphatidylcholine. The pss1Δ strain exhibited severe growth defects on media containing n-alkanes even in the presence of ethanolamine and choline. In the pss1Δ strain, the transcription of ALK1, which encodes a primary cytochrome P450 that catalyses the hydroxylation of n-alkanes in the endoplasmic reticulum, was upregulated by n-alkane as in the wild-type strain. However, the production of functional P450 was not detected, as indicated by the absence of reduced CO-difference spectra in the pss1Δ strain. PS was undetectable in the lipid extracts of the pss1Δ strain. These results underscore the critical role of PSS1 in the biosynthesis of PS, which is essential for the production of functional P450 enzymes involved in n-alkane hydroxylation in Y. lipolytica.

Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

SNF1 plays a crucial role in the utilization of n-alkane and transcriptional regulation of the genes involved in it in the yeast Yarrowia lipolytica.

Yarrowia lipolytica is an ascomycetous yeast that can assimilate hydrophobic carbon sources including oil and n-alkane. The sucrose non-fermenting 1/AMP-activated protein kinase (Snf1/AMPK) complex is involved in the assimilation of non-fermentable carbon sources in various yeasts. However, the role of the Snf1/AMPK complex in n-alkane assimilation in Y. lipolytica has not yet been elucidated. This study aimed to clarify the role of Y. lipolytica SNF1 (YlSNF1) in the utilization of n-alkane. The deletion mutant of YlSNF1 (ΔYlsnf1) exhibited substantial growth defects on n-alkanes of various lengths (C10, C12, C14, and C16), and its growth was restored through the introduction of YlSNF1. Microscopic observations revealed that YlSnf1 tagged with enhanced green fluorescence protein showed dot-like distribution patterns in some cells cultured in the medium containing n-decane, which were not observed in cells cultured in the medium containing glucose or glycerol. The RNA sequencing analysis of ΔYlsnf1 cultured in the medium containing n-decane exhibited 302 downregulated and 131 upregulated genes compared with the wild-type strain cultured in the same medium. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses suggested that a significant fraction of the downregulated genes functioned in peroxisomes or were involved in the metabolism of n-alkane and fatty acids. Quantitative real-time PCR analysis confirmed the downregulation of 12 genes involved in the metabolism of n-alkane and fatty acid, ALK1-ALK3, ALK5, ADH7, PAT1, POT1, POX2, PEX3, PEX11, YAS1, and HFD3. Furthermore, ΔYlsnf1 exhibited growth defects on the medium containing the metabolites of n-alkane (fatty alcohol and fatty aldehyde). These findings suggest that YlSNF1 plays a crucial role in the utilization of n-alkane in Y. lipolytica. This study provides important insights into the advanced biotechnological applications of this yeast, including the bioconversion of n-alkane to useful chemicals and the bioremediation of petroleum-contaminated environments.

Phosphatidylcholine levels regulate hyphal elongation and differentiation in the filamentous fungus Aspergillus oryzae.

Filamentous fungi are eukaryotic microorganisms that differentiate into diverse cellular forms. Recent research demonstrated that phospholipid homeostasis is crucial for the morphogenesis of filamentous fungi. However, phospholipids involved in the morphological regulation are yet to be systematically analyzed. In this study, we artificially controlled the amount of phosphatidylcholine (PC), a primary membrane lipid in many eukaryotes, in a filamentous fungus Aspergillus oryzae, by deleting the genes involved in PC synthesis or by repressing their expression. Under the condition where only a small amount of PC was synthesized, A. oryzae hardly formed aerial hyphae, the basic structures for asexual development. In contrast, hyphae were formed on the surface or in the interior of agar media (we collectively called substrate hyphae) under the same conditions. Furthermore, we demonstrated that supplying sufficient choline to the media led to the formation of aerial hyphae from the substrate hyphae. We suggested that acyl chains in PC were shorter in the substrate hyphae than in the aerial hyphae by utilizing the strain in which intracellular PC levels were controlled. Our findings suggested that the PC levels regulate hyphal elongation and differentiation processes in A. oryzae and that phospholipid composition varied depending on the hyphal types.

Detection limitations of prion seeding activities in blood samples from patients with sporadic prion disease.

BACKGROUND: Human prion diseases (HPDs) are fatal neurodegenerative disorders characterized by abnormal prion proteins (PrPSc). However, the detection of prion seeding activity in patients with high sensitivity remains challenging. Even though real-time quaking-induced conversion (RT-QuIC) assay is suitable for detecting prion seeding activity in a variety of specimens, it shows lower accuracy when whole blood, blood plasma, and blood-contaminated tissue samples are used. In this study, we developed a novel technology for the in vitro amplification of abnormal prion proteins in HPD to the end of enabling their detection with high sensitivity known as the enhanced quaking-induced conversion (eQuIC) assay. METHODS: Three antibodies were used to develop the novel eQUIC method. Thereafter, SD50 seed activity was analyzed using brain tissue samples from patients with prion disease using the conventional RT-QUIC assay and the novel eQUIC assay. In addition, blood samples from six patients with solitary prion disease were analyzed using the novel eQuIC assay. RESULTS: The eQuIC assay, involving the use of three types of human monoclonal antibodies, showed approximately 1000-fold higher sensitivity than the original RT-QuIC assay. However, when this assay was used to analyze blood samples from six patients with sporadic human prion disease, no prion activity was detected. CONCLUSION: The detection of prion seeding activity in blood samples from patients with sporadic prion disease remains challenging. Thus, the development of alternative methods other than RT-QuIC and eQuIC will be necessary for future research.

Chicken Interleukin-5 is Expressed in Splenic Lymphocytes and Affects Antigen-Specific Antibody Production.

Vaccination is important for reducing disease incidence in the poultry industry. To enhance immunity and vaccine efficacy, chicken cytokines associated with antibody production must be identified. In this study, we focused on interleukin-5 (IL-5), involved in antibody production in mice, measuring its expression and effects on antibody production. Concanavalin A-stimulated splenocytes were used for RT-PCR to clone IL5 cDNAs. Recombinant IL-5 was prepared from the clone and administered to chickens with antigen via the ocular-topical route twice every alternate week. IL-5 enhanced antigen-specific IgY and inhibited antigen-specific serum IgA production in serum. Our findings suggest that IL-5 plays an important role in chicken antibody production, with possible unique functions.

Evaluation of expression systems for recombinant protein production in chicken egg bioreactors.

Chicken eggs have gained attention as excellent bioreactors because of their genetic modifications. However, the development of chicken egg bioreactors requires a long time from the construction of the production system to the evaluation of the products. Therefore, in this study, a chicken cell line producing ovalbumin (OVA) was established and constructed a system for the rapid evaluation of the production system. First, the EF1α promoter was knocked in upstream of the OVA locus in chicken DF-1 cells for continuous OVA expression. Furthermore, an ideal position at the OVA locus for the insertion of useful protein genes to maximize recombinant protein yield was analyzed and identified. The knocking in the EF1α promoter upstream of exon1 yielded the maximum production of OVA protein was achieved. In addition, Linking a recombinant hFGF2 cDNA to the 5' side of the OVA was found to increase production efficiency. Therefore, an OVA-expressing cell line and an evaluation system for proteins in chicken egg bioreactors was established. The findings may improve the efficiency of chicken expression systems and expand their applications in protein production.

Wnt signaling blockade is essential for maintaining the pluripotency of chicken embryonic stem cells.

Activation of Wnt/ß-catenin signaling supports the self-renewal of mouse embryonic stem cells. We aimed to understand the effects of Wnt signaling activation or inhibition on chicken embryonic stem cells (chESCs), as these effects are largely unknown. When the glycogen synthase kinase-3 ß inhibitor CHIR99021-which activates Wnt signaling-was added to chESC cultures, the colony shape flattened, and the expression levels of pluripotency-related (NANOG, SOX2, SOX3, OCT4, LIN28A, DNMT3B, and PRDM14) and germ cell (CVH and DAZL) markers showed a decreasing trend, and the growth of chESCs was inhibited after approximately 7 d. By contrast, when the Wnt signaling inhibitor XAV939 was added to the culture, dense and compact multipotent colonies (morphologically similar to mouse embryonic stem cell colonies) showing stable expression of pluripotency-related and germline markers were formed. The addition of XAV939 stabilized the proliferation of chESCs in the early stages of culture and promoted their establishment. Furthermore, these chESCs formed chimeras. In conclusion, functional chESCs can be stably cultured using Wnt signaling inhibitors. These findings suggest the importance of Wnt/ß-catenin signaling in avian stem cells, offering valuable insights for applied research using chESCs.

[A Long-Term Survival Case of Gastric Cancer with Pyloric Stenosis and Peritoneum Dissemination That Received Intravenous and Intraperitoneal Paclitaxel Combined with S-1 Therapy after Bypass Surgery].

Although a 74-year-old man with gastric cancer with pyloric stenosis(cT4aN[+]M0, Stage Ⅲ)had undergone surgery, he was diagnosed with peritoneum dissemination. He received bypass surgery, and an intraperitoneal access port was implanted in his subcutaneous space. Postoperatively, he received 4 courses of SOX therapy. In treatment effect, the primary tumor showed no change, and ascites developed. Therefore, we changed the chemotherapy regimen in intravenous and intraperitoneal paclitaxel combined with S-1 therapy. After starting this regimen, the primary tumor decreased in size, and the pyloric stenosis improved. Currently, the patient is alive without recurrence for 5 years and 8 months after intravenous and intraperitoneal paclitaxel combined with S-1 therapy and receiving this treatment regularly.

Comprehensive analysis of the composition of the major phospholipids during the asexual life cycle of the filamentous fungus Aspergillus nidulans.

Filamentous fungi undergo significant cellular morphological changes during their life cycle. It has recently been reported that deletions of genes that are involved in phospholipid synthesis led to abnormal hyphal morphology and differentiation in filamentous fungi. Although these results suggest the importance of phospholipid balance in their life cycle, comprehensive analyses of cellular phospholipids are limited. Here, we performed lipidomic analysis of A. nidulans during morphological changes in a liquid medium and of colonies on a solid medium. We observed that the phospholipid composition and transcription of the genes involved in phospholipid synthesis changed dynamically during the life cycle. Specifically, the levels of phosphatidylethanolamine, and highly unsaturated phospholipids increased during the establishment of polarity. Furthermore, we demonstrated that the phospholipid composition in the hyphae at colony margins is similar to that during conidial germination. Furthermore, we demonstrated that common and characteristic phospholipid changes occurred during germination in A. nidulans and A. oryzae, and that species-specific changes also occurred. These results suggest that the exquisite regulation of phospholipid composition is crucial for the growth and differentiation of filamentous fungi.

Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.

Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p < 0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine 2000 and our PGC medium were the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them.

The N-terminal disordered region of ChsB regulates its efficient transport to the hyphal apical surface in Aspergillus nidulans.

In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region.

Transcription activator-like effector nuclease-mediated deletion safely eliminates the major egg allergen ovomucoid in chickens.

Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate allergens. However, recent genome editing technology has made it possible to generate OVM-knockout chicken eggs. To use this OVM-knockout chicken egg as food, it is important to evaluate its safety as food. Therefore, in this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum TALENs. The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities, and immunoblotting showed that the albumen contained neither the mature OVM nor the OVM truncated variant. Whole genome sequencing (WGS) revealed that the potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. The WGS information confirmed that plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluation and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.

Fate Decisions of Chicken Primordial Germ Cells (PGCs): Development, Integrity, Sex Determination, and Self-Renewal Mechanisms.

Primordial germ cells (PGCs) are precursor cells of sperm and eggs. The fate decisions of chicken PGCs in terms of their development, integrity, and sex determination have unique features, thereby providing insights into evolutionary developmental biology. Additionally, fate decisions in the context of a self-renewal mechanism have been applied to establish culture protocols for chicken PGCs, enabling the production of genome-edited chickens and the conservation of genetic resources. Thus, studies on the fate decisions of chicken PGCs have significantly contributed to both academic and industrial development. Furthermore, studies on fate decisions have rapidly advanced owing to the recent development of essential research technologies, such as genome editing and RNA sequencing. Here, we reviewed the status of fate decisions of chicken PGCs and provided insight into other important research issues that require attention.

srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans.

Conidiation is an important reproductive process in Aspergillus. We previously reported, in A. nidulans, that the deletion of a putative glycosyltransferase gene, rseA/cpsA, causes an increase in the production of extracellular hydrolases and a severe reduction in conidiation. The aim of this study was to obtain novel genetic factors involved in the repression of conidiation in the rseA deletion mutant. We isolated mutants in which the rseA deletion mutant conidiation defect is suppressed and performed a comparative genomic analysis of these mutants. A gene encoding a putative transcription factor was identified as the associated candidate causative gene. The candidate gene was designated as srdA (suppressor gene for the conidiation defect of the rseA deletion mutant). The conidiation efficiency of the rseAsrdA double-deletion mutant was increased. Introduction of wild-type srdA into the suppressor mutants caused a conidiation defect similar to that of the rseA deletion mutant. Notably, the conidiation efficiencies of the rseAsrdA double-deletion and srdA single-deletion mutants were higher than that of the wild-type strain. These results indicate that srdA is a novel genetic factor that strongly represses conidiation of the rseA deletion mutant, and a putative transcriptional regulator, SrdA is a negative regulator of conidiation in A. nidulans.

Screening of Optimal CpG-Oligodeoxynucleotide for Anti-Inflammatory Responses in the Avian Macrophage Cell Line HD11.

CpG-oligodeoxynucleotides (. CpG-ODNs: ) have been shown to possess immunostimulatory features in both mammals and birds. However, compared to their proinflammatory effects, little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, the anti-inflammatory response in the chicken macrophage cell line HD11 was characterized under stimulation with five types of CpG-ODNs: CpG-A1585, CpG-AD35, CpG-B1555, CpG-BK3, and CpG-C2395. Single-stimulus of CpG-B1555, CpG-BK3, or CpG-C2395 induced interleukin (IL)-10 expression without causing cell injury. The effects of pretreatment with CpG-ODNs before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, pretreatment with only CpG-C2395 resulted in high expression levels of IL-10 mRNA in the presence of lipopolysaccharide. Finally, gene expression analysis of inflammation-related cytokines and receptors revealed that pre-treatment with CpG-C2395 significantly reduced the mRNA expression of tumor necrosis factor-α, IL-1ß, IL-6, and Toll-like receptor 4. Overall, these results shed light on the anti-inflammatory responses triggered by CpG-C2395 stimulation through a comparative analysis of five types of CpG-ODNs in chicken macrophages. These results also offer insights into the use of CpG-ODNs to suppress the expression of proinflammatory cytokines, which may be valuable in the prevention of avian infectious diseases in the poultry industry.

Prediction of sex-determination mechanisms in avian primordial germ cells using RNA-seq analysis.

In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism underlying sex determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method.

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.

Orthologs of Saccharomyces cerevisiae SFH2, genes encoding Sec14 family proteins, implicated in utilization of n-alkanes and filamentous growth in response to n-alkanes in Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has an ability to assimilate n-alkanes as carbon and energy sources. In this study, the roles of orthologs of Saccharomyces cerevisiae SEC14 family gene SFH2, which we named SFH21, SFH22, SFH23 and SFH24, of Y. lipolytica were investigated. The transcript levels of SFH21, SFH22 and SFH23, determined by RNA-seq analysis, qRT-PCR analysis and northern blot analysis, were found to increase in the presence of n-alkanes. The deletion mutant of SFH21, but not that of SFH22, SFH23 or SFH24, showed defects in growth in the media containing n-alkanes and in filamentous growth on the solid media containing n-alkanes. Additional deletions of SFH22 and SFH23 significantly exaggerated the defect in filamentous growth of the deletion mutant of SFH21, and expression of SFH22 or SFH24 using the SFH21 promoter partially suppressed the growth defect of the deletion mutant of SFH21 on n-alkanes. These results suggest that SFH2 orthologs are involved in the utilization of n-alkanes and filamentous growth in response to n-alkanes in Y. lipolytica.

Gene manipulation in the Mucorales fungus Rhizopus oryzae using TALENs with exonuclease overexpression.

The Mucorales fungal genus Rhizopus is used for the industrial production of organic acids, enzymes and fermented foods. The metabolic engineering efficiency of Rhizopus could be improved using gene manipulation; however, exogenous DNA rarely integrates into the host genome. Consequently, a genetic tool for Mucorales fungi needs to be developed. Recently, programmable nucleases that generate DNA double-strand breaks (DSBs) at specific genomic loci have been used for genome editing in various organisms. In this study, we examined gene disruption in Rhizopus oryzae using transcription activator-like effector nucleases (TALENs), with and without exonuclease overexpression. TALENs with an overexpressing exonuclease induced DSBs, followed by target site deletions. Although DSBs are repaired mainly by nonhomologous end joining in most organisms, our results suggested that in R. oryzae microhomology-mediated end joining was the major DSB repair system. Our gene manipulation method using TALENs coupled with exonuclease overexpression contributes to basic scientific knowledge and the metabolic engineering of Rhizopus.