Development of an itraconazole resistance gene as a dominant selectable marker for transformation in Aspergillus oryzae and Aspergillus luchuensis

Abstract

There are only a few combinations of antifungal drugs with known resistance marker genes in the Aspergillus species; therefore, the transformation of their wild-type strains is limited. In this study, to develop the novel dominant selectable marker for itraconazole, a fungal cell membrane synthesis inhibitor, we focused on Aspergillus luchuensis cyp51A (Alcyp51A), which encodes a 14-α-sterol demethylase related to the steroid synthesis pathway. We found that the G52R mutation in AlCyp51A and the replacement of the native promoter with a high-expression promoter contributed to itraconazole resistance in Aspergillus oryzae, designated as itraconazole resistant gene (itrA). The random integration in the A. luchuensis genome of the itrA marker cassette gene also allowed for transformation using itraconazole. Therefore, we succeed in developing a novel itraconazole resistance marker as a dominant selectable marker for transformation in A. oryzae and A. luchuensis.

Biosynthesis of Naringenin from p-Coumaric Acid in Engineering Oleaginous Filamentous Fungus Mucor circinelloides

“…The results indicated that a higher electric field intensity contributed to the uptake rate of these plasmids with relatively large sizes in M. circinelloides, consistent with the results reported in Aspergillus fumigatus . In addition, the transformation efficiency at resistance 200 Ω was 14–50 transformants per 10 7 spores, which was 2–8-folds higher than that of resistance 400 Ω (7–30 transformants per 10 7 spores).…”

Molecular Mechanisms of Antifungal Resistance in Mucormycosis

“…In-depth investigations portrayed a higher microsomal P450 levels leading to increased ergosterol synthesis in the resistance strain and was responsible for both azole and amphotericin B resistance [ 44 ]. These findings indicate that the cross-resistance to such two triazoles was caused by elevated P450 levels [ 45 ]. In the small number of clinical isolates with 14 α -demethylase overexpression, this phenomenon was only seen in C. glabrata, and the possibility that other resistance mechanisms are active in the same strain all suggests that increased expression of the target enzyme ends up playing just a minor role in clinical azole resistance [ 46 ].…”