A preliminary study on Agrobacterium tumefaciens-mediated transformation of the food filamentous fungus Aspergillus luchuensis AL1

Abstract

Aspergillus luchuensis is a filamentous fungus renowned in the traditional alcohol production industry in Japan. This species has been proven to be safe and does not produce fungal toxins. The A. luchuensis fungus is capable of utilising various inexpensive substrates and synthesising many valuable enzymes such as amylase, protease, and xylanase. However, research on genetic modification in A. luchuensis remains relatively limited, especially in Vietnam. This study introduced an A. luchuensis AL1 strain derived from food, which was classified based on morphological characteristics and sequence analysis of three independent gene regions, including the ITS of rDNA, β-tubulin (benA) gene, and calmodulin (CaM) gene. Initial research on gene transfer into A. luchuensis AL1 using Agrobacterium tumefaciens and the hygromycin B resistance gene has been successful. The efficiency of genetic transformation reached 40 transformants per 10⁶ fungal spores, and no false-positive colonies were observed. With this gene transfer method, the gene encoding green fluorescent protein (GFP), regulated by the gpdA promoter from Aspergillus nidulans, was successfully transferred into the A. luchuensis AL1 strain. Analysis of the GFP-tagged transformants under a fluorescence microscope showed strong expression of the GFP protein in the fungal mycelia.