It is well-know that A. oryzae is an excellent host used to express homologous and heterologous proteins. In recent years, it has been widely used in the production of heterologous proteins and has received increasing attention. A. oryzae has many advantages as an expression host, such as a strong ability to produce and secrete proteins, and strong post-translational modification[30, 31]. Therefore, it is important to develop an effective A. oryzae transformation system.
There are three kinds of selective markers commonly used in filamentous fungi transformation: auxotrophic complementary genes, drug resistance genes, and genes that can make the host use some unusual carbon or nitrogen sources. The transformation systems, which were based on drug resistance genes as main selectable markers have the advantage of the availability of a wild type strain as a host and easy operation. During the transformation of filamentous fungi, most choose to use some genes encoding resistance as selection markers, such as hygromycin and phleomycin[33, 34]. However, studies have shown that A. oryzae is insensitive to most the common antibiotics such as hygromycin B, geneticin(G418) and bleomycin[9, 10]. Moreover, in this study, we found that hygromycin B cannot inhibit A. oryzae RIB 40 ΔpyrG growth. Compared with the other systems, the auxotrophic complementary genetic transformation system has proved to be more efficient.
As a result, transformation systems for A. oryzae have been developed mainly based on nutritional markers. The pyrG gene, ending orotidine-5’-monophosphate (OMP) decarboxylase participates in uridine biosynthesis but is also a target for the antimetabolite 5-fluoroorotic acid (5- FOA). Thus, pyrG genes in filamentous fungi have been widely used as nutritional/auxotrophic markers for fungal transformation. In the present study, the pyrG deletion mutants were successfully obtained by UV mutagenesis. Mutants were selected with 5-FOA, which selectively allows the growth of pyrG deletion strains. As expected, these strains exhibited uridine/uracil auxotrophy and resistance to 5-FOA. Meanwhile, the deletion of the pyrG gene was further confirmed by genome PCR and DNA sequencing. UV mutagenesis is a simple method, we successfully obtained A. oryzae RIB 40 ΔpyrG through this method. However, mutations induced by UV mutagenesis also have disadvantages such as unstable and uncertain direction of mutation.
With the widespread use of A. oryzae in the expression of heterologous proteins, it is particularly important to construct a safe and efficient expression vector for A.oryzae. In this study, a novel expression-stable vectors pBC-hygro.4 was constructed and stably expressed in A. oryzae RIB 40 ΔpyrG strain. These vectors possess the changeable components including the pyrG cassette gene, His-Tag, amyB promoter, and terminator amyB. The pyrG transformation system has a great advantage of a lower false-positive background in transformation experiments (Hao et al. 2008). Promoters and terminators are important expression elements that play a key role in the efficient expression of genes. Studies have shown that different promoters have different efficiency, and strong promoters can effectively improve the stability and transcription level of mRNA. There are some strong promoters for gene expression in A. oryzae, such as amyB, melO, glaA, gpdA and tef1[38–41]. Based on previous reports, the amyB promoter is much better than the gpdA promoter in the regulation of gene expression in A. oryzae. 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. GFP is the most common fluorescent proteins used for tagging filamentous fungi, which can as a reporter to test the stability of pBC-Hygro.4 in A. oryzae RIB 40 ΔpyrG. In the present study, we could directly detect the expression of the GFP reporter gene by recombinant A. oryzae RIB 40 ΔpyrG cultures presented remarkable green fluorescence in the mycelia. These results suggest that the gfp gene was successfully expressed in A. oryzae RIB 40 ΔpyrG, which demonstrated that vector pBC-Hygro.4 can be used for the expression of foreign genes in A. oryzae.
In addition, A. oryzae has the ability to express large amounts of various enzymes, such as α-amylase, glucoamylase[45, 46], and α-glucosidase. Compared with eukaryotic expression systems based on Pichia pastoris and Saccharomyces cerevisiae, A. oryzae expression has higher safety, so it can be widely used in food industry. Therefore, the A. oryzae expression system constructed in this study provides a prerequisite for the expression of more foreign genes in the future. In future studies we can use this system to express more heterologous and homologous proteins, such as xylanase, cellulose Enzymes, proteases, etc.