α-Linolenic acid (ALA) is an important essential omega-3 (ω-3) polyunsaturated fatty acid for the maintenance of human health. Although ALA has traditionally been obtained from plant sources, microbial fermentation has emerged as a promising alternative for its sustainable and cost-effective production. However, most of the present approaches rely on genetically modified organisms, which present regulatory and consumer-acceptance concerns. In this study, we aimed to develop a high-ALA-producing strain of Aspergillus oryzae, a Generally Recognized As Safe (GRAS) microorganism widely used in food production in Japan, through self-cloning, a form of genetic engineering that utilizes only the host’s own DNA. To achieve this, an endogenous ω-3 desaturase gene (fad3), which catalyzes the conversion of linoleic acid to ALA, was identified via BLASTP analysis. Subsequently, a multicopy A. oryzae strain (Aofad3-MC) overexpressing fad3 was constructed. This strain increased ALA production, with ALA comprising 30.7% of the total lipids. Furthermore, k-mer analysis confirmed the absence of foreign vector sequences, verifying that Aofad3-MC was constructed through self-cloning. In addition to the identification of the A. oryzae ω-3 desaturase gene, this study provides a microbial platform for the sustainable production of ALA, with potential applications across the food, feed, and related industries.

Lactate-rich byproducts and fermentation residues are generated during various food and fermentation processes, presenting opportunities for their conversion into value-added products through microbial bioprocesses. Aspergillus oryzae is a promising microbial host for such applications because of its ability to use diverse substrates and produce valuable products. However, the ability of A. oryzae to assimilate lactate and the factors limiting this process remain poorly understood despite its industrial importance. In this study, we investigated the transcriptional responses of A. oryzae during lactate assimilation and evaluated the culture conditions promoting this process. A. oryzae demonstrated only limited growth and lactate consumption when lactate was provided as its only carbon source in the synthetic minimal medium. Transcriptome analysis suggested possible metabolic constraints associated with carbon flux into the tricarboxylic acid (TCA) cycle, limiting lactate assimilation. To alleviate these constraints, the culture conditions were examined. Supplementation with small amounts of sugars, specific amino acids, and α-ketoglutarate promoted lactate assimilation. Moreover, low-pH conditions further improved lactate assimilation, reducing residual lactate concentrations to below the detection limit. These findings indicate that efficient lactate assimilation is promoted by small amounts of metabolic co-substrates, potentially through effects on central carbon metabolism, together with favorable low-pH conditions.