The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae

Abstract

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

Development of an Efficient and Stable Transformation System for Aspergillus Oryzae Based on the pyrG Gene

“…There are some strong promoters for gene expression in A. oryzae, such as amyB, melO, glaA, gpdA and tef1 [38][39][40][41] . Based on previous reports, the amyB promoter is much better than the gpdA promoter in the regulation of gene expression in A. oryzae [42] . Therefore, in this study, the amyB promoter and terminator have been considered as the strongest elements in the construction of expression vectors for A. oryzae to produce homologous and heterologous proteins.…”

A simple, efficient and universal method for the extraction of genomic DNA from bacteria, yeasts, molds and microalgae suitable for PCR-based applications

“…The genomic DNA extraction method was adapted from some previously published protocols for fungi [7,10,14] with suitable modifications for each microorganism employed in this study.…”

“…The accurate identification of these microorganisms for specific purposes is usually performed on the basis of barcode ribosomal DNA sequences that include bacterial 16S rRNA, fungal ITS (internal transcribed spacer) region, and microalgal 18S rRNA [3][4][5][6]. Furthermore, the selected useful microorganisms can be subjected to further genetic improvement to enhance beneficial traits [1,2,7]. Consequently, the development of efficient genomic DNA extraction methods with respect to different microbial species is always considered a central step in PCR-based molecular biology applications that include molecular taxonomy, molecular diagnosis, recombinant DNA cloning studies, etc.…”

A Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042

“…establish an ATMT system in A. oryzae were unsuccessful until 2016, when Nguyen et al (2016) established ATMT in A. oryzae using uracil auxotrophy as a selectable marker [17]. Herein, we constructed a dual selection marker transformation system using the ATMT method in an industrial A. oryzae 3.042 strain.…”

“…S1A). Therefore, we replaced the pyrG cassette of pEX1 (the plasmid used for ATMT in A. oryzae RIB40 [17]) with a ptrA cassette to construct the pEX1-ptrA vector (Fig. S1B).…”