Double knockout of two target genes via genome co-editing using a nitrate transporter gene nrtA and a putative thiamine transporter gene thiI as selectable markers in Aspergillus oryzae.

Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, thiI gene was found as a selectable marker for such genome co-editing in the filamentous fungus Aspergillus oryzae, while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in A. oryzae. In this study, we focused on the membrane transporter gene nrtA (AO090012000623), which promotes uptake of nitrate (NO3-). It is known that, in nrtA knockout strain, chlorate (ClO3-), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene wA was attempted using both nrtA- and wA-specific single-guide RNAs via genome co-editing with KClO3 supplementation in A. oryzae laboratory strain RIB40 and industrial strain KBN616. Resultantly, wA knockout mutant was generated, and nrtA was identified as a selectable marker. Moreover, this genome co-editing system using nrtA was compatible with that using thiI, and thus, a double knockout mutant of two target genes wA and yA was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As nrtA homologs have been found in several industrial Aspergillus species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes.

Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

The beta-1,3-exoglucanase gene exgA (exg1) of Aspergillus oryzae is required to catabolize extracellular glucan, and is induced in growth on a solid surface.

The biological role of ExgA (Exg1), a secretory beta-1,3-exoglucanase of Aspergillus oryzae, and the expression pattern of the exgA (exg1) gene were analyzed. The exgA disruptant and the exgA-overexpressing mutant were constructed, and phenotypes of both mutants were compared. Higher mycelial growth rate and conidiation efficiency were observed for the exgA-overexpressing mutant than for the exgA disruptant when beta-1,3-glucan was supplied as sole carbon source. On the other hand, no difference in phenotype was observed between them in the presence or absence of the inhibitors of cell wall beta-glucan remodeling when grown with glucose. exgA Expression was induced in growth on solid surfaces such as filter membrane and onion inner skin. A combination of poor nutrition and mycelial attachment to a hydrophobic solid surface appears to be an inducing factor for exgA expression. These data suggest that ExgA plays a role in beta-glucan utilization, but is not much involved in cell wall beta-glucan remodeling.