Construction and characterization of the GFAT gene as a novel selection marker in Aspergillus nidulans.

Abstract

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.