Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae.

OBJECTIVES: To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins. RESULTS: To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations. CONCLUSIONS: We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

Magnetic separation of human podocalyxin-like protein 1 (hPCLP1)-positive cells from peripheral blood and umbilical cord blood using anti-hPCLP1 monoclonal antibody and protein A expressed on bacterial magnetic particles.

Hemangioblasts are common progenitors of hematopoietic and angiogenic cells, which have been demonstrated in the mouse to possess a unique cell surface marker, podocalyxin-like protein 1 (PCLP1) (Hara, T. et al., Immunity, 11: 567-578. 1999). In this study, we prepared a novel monoclonal antibody against human PCLP1 (hPCLP1) and attempted to isolate human hematopoietic progenitor cells from umbilical cord blood and peripheral blood using nano-sized bacterial magnetic particles (BacMPs) coupled with the anti-hPCLP1 antibody. Flow cytometric analysis demonstrated that the purity of separated hPCLP1-positive cells from peripheral blood was approximately 95% whereas peripheral blood mononuclear cells contained only 0.1% PCLP1+ cells. Umbilical cord blood was demonstrated to be a better source for PCLP1+ cells than peripheral blood. These results suggest that the separation of human PCLP1+ cells using BacMPs with anti-hPCLP1 were extremely effective and may be useful as a means to prepare human hematopoietic progenitor cells.

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

Highly efficient gene targeting in Aspergillus oryzae industrial strains under ligD mutation introduced by genome editing: Strain-specific differences in the effects of deleting EcdR, the negative regulator of sclerotia formation.

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

Leukocyte cell-derived chemotaxin 2 is a zinc-binding protein.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secreted hepatic protein that has been associated with several physiological activities. LECT2 belongs to the peptidase M23 family, suggesting that it is a zinc-binding protein. To test this possibility, electrospray ionization mass spectrometry and X-ray absorption fine-structure analysis were performed. Results of these experiments indicated that recombinant mouse LECT2 produced by an animal cell line contains a zinc atom. Furthermore, the recombinant LECT2 was found to be self-oligomerized by disulfide bonds in vitro, but this was suppressed by addition of zinc. These results indicated that zinc stabilizes the LECT2 structure.

Identification and assignment of three disulfide bonds in mammalian leukocyte cell-derived chemotaxin 2 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Mammalian leukocyte cell-derived chemotaxin 2 (LECT2) contains six evolutionarily conserved cysteine residues. To date, however, the presence of disulfide linkages between these residues has not been determined. To search for disulfide bonds, the protein was proteolitically digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization?time of flight mass spectrometry. The analysis showed that murine and human LECT2 have three intramolecular disulfide bonds (Cys25-Cys60; Cys36-Cys41; Cys99-Cys142) and no free cysteine residues.