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

Background Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. An ecient and stable transformation system is the key to the successful expression of the gene of interest in A. oryzae. Results To improve the expression eciency of the gene of interest in A. oryzae, we constructed the uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG by Ultraviolet (UV) mutagenesis of pyrG gene deletion which would be used as a host for further transformation. In addition, a novel and ecient expression vector pBC-hygro.4 was constructed, including the pyrG cassette gene, His-Tag, amyB promoter and terminator,and green uorescent protein GFP marker. pBC-hygro.4 transformed A. oryzae RIB40ΔpyrG eciently via the PEG-CaCl 2 -mediated transformation method, and the stability of pBC-hygro.4 was tested by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verication, we successfully obtained a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG. At the same time, the developed vectors are fully functional for heterologous expression of the GFP uorescent proteins in the A. oryzae RIB40ΔpyrG. Conclusion Our work provides a new method that can be applied to other lamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.

Currently the most commonly used methods for lamentous fungi transformation including PEG (polyethylene glycol)-mediated protoplast transformation(PMPT) and Agrobacterium tumefaciensmediated transformation (ATMT) [18,19] . In comparison with the ATMT method, PEG-CaCl 2 -mediated transformation has some disadvantages, such as low transformation e ciency, di culty in obtaining high concentrations of viable protoplasts, high percentages of transient transformants [20,21] . However, due to its simplicity in technical operation and equipment required, the PEG-CaCl 2 -mediated transformation remains the most commonly used method to conduct transformation in lamentous fungi [22] .
In recent years, A. oryzae as an expression host for heterologous gene expression has attracted a lot of interest, so the construction of highly e cient A. oryzae expression vectors is particularly important. Many previous studies have shown that vectors constructed with plasmid pBC-hygro as the backbone can be effectively transformed in various lamentous fungi, such as Aspergillus fumigates [23] , Aspergillus sydowii [24] , Thermomyces lanuginosus [25] , and Podospora anserine [26] . Taka-amylase (amyB) promoters and terminators were widely used for the e cient expression of many genes. In this study, a novel vectors pBC-hygro.4 carrying A. oryzae pyrG marker was constructed for the transformation of uridine/uracil auxotrophic A. oryzae via an adapted PEG-CaCl 2 -mediated transformation method. The construction of the A. oryzae expression system will lay the foundation for the successful expression of foreign genes in A. oryzae and provide a new method that can be applied to other lamentous fungi to develop similar fungal transformation systems based on auxotrophic / nutrition markers.

Determination of 5-FOA minimum use concentration
The resistance of wild-type A. oryzae RIB40 to different concentrations 5-FOA was analyzed. To isolate a selection agent suitable for pyrG deletion mutants screening, the growth of wild-type A. oryzae RIB40 on CD plates containing 5-FOA at different concentrations (0.5, 0.75, 1.0, 1.25, 1.5 mg/mL) was observed.
The result shown that 1.0 mg/mL 5-FOA inhibited the growth of wild-type A. oryzae RIB40 for the longest time ( Fig. 1). Therefore, 1.0 mg/mL 5-FOA was chosen as the selection agent for pyrG deletion mutants screening.

Screening and characterization of pyrG deletion mutants
We obtained many mutant strains by UV mutagenesis. After preliminary screening through 5-FOA plates, we succeeded to gain ve resistant strains that can grow on 5-FOA plates, named P1-P5. Then, mutant strains to resistant to 5-FOA were transferred simultaneously to the CD, CD + Uri/Ura and CD + Uri/Ura + 5-FOA (1 mg/mL) to examine their growth (Fig. 2). The result showed that the wild type was unable to grow on CD + Uri/Ura + 5-FOA plates and grew well on CD, CD + Uri/Ura. However, the pyrG deletion mutants P1-P5 were unable to grow on CD plates without uridine and uracil and grew well on CD + Uri/Ura, CD + Uri/Ura + 5-FOA plates (Fig. 2), indicating that strains P1-P5 were uridine auxotrophs. These uridine auxotrophs were detected by further PCR to identify pyrG deletion.
For the ve uridine auxotrophs, the predicted 1.8 kb pyrG cassette fragments (Fig. 3) were ampli ed from the genomic DNA of the mutants using the primer pair pyrGF/pyrGR. The P1-P5 strain ampli ed a speci c band of about 1.8 kb, which was consistent with the wild-type A. oryzae RIB40 pyrG cassette sequence size. We selected strain P1 and puri ed its PCR product by gel recovery kit and sent it to invitrogen for sequencing. The sequencing results were compared with the wild type A. oryzae RIB40 pyrG cassette sequence, nucleotide sequencing revealed that strain containing pyrG mutations were successfully obtained. Strain P1 contain 1 bp deletion and 1 bp insertion at the target sequence of the pyrG gene (Fig. 4). These mutations all cause frameshifts, which suggests that the pgrG gene does not function in the strains.

Dna Manipulation And Plasmid Construction
First, we veri ed the resistance of A. oryzae RIB40 ΔpyrG to hygromycin B. The spore suspension was cultivated in CD medium containing 0, 0.5, 1.0, 1.5, 2.0 mg/mL for 4 days at 28 °C. We found that hygromycin B has no inhibitory effect on A. oryzae RIB40 ΔpyrG, so we need to delete the hygromycin B gene sequence. Analysis of the plasmid pBC-Hygro's sequence revealed that it contained three Nde I restriction sites, which were located at sequences 3861 bp, 4539 bp, and 4991 bp, and the sites at 4539 bp and 4991 bp were located in hygromycin B gene sequence. Therefore, we can choose to delete the Nde I restriction sites at 3861 bp and use Nde I digestion to destroy the hygromycin B resistance gene so that it loses its activity. We use site-directed mutagenesis to mutate base C at 3861 bp to base T. For speci c methods, refer to the site-directed mutation kit. Design mutant primers TbndeF/TbndeR and sequencing primers TucxF/TucxR. The primer sequences are shown in Table 2. sec, 60ºC for 10 sec, and 68ºC for 210 sec, then a nal incubation of 68ºC for 5 min. Reactions were cooled on ice and digested with 5 units of Dpn I for 1 h at 37ºC to cleave methylated and hemimethylated parental DNA, but not the newly synthesized mutant DNA molecules. The mutant plasmid was veri ed by DNA-sequencing using the sequencing primers TucxF/TucxR (Table 2), Name the correct mutant plasmid pBC-Hygro.1.
The primers used in this study are listed in Table 2. The novel protein expression vector pBC-Hygro.4 consisted of There fragments (One, Two, Three). Fragment One, the pyrG cassette as the auxotrophic marker was ampli ed from the genome of the wild-type A. oryzae RIB40 strain using primers pyrgbdhF/pyrgbdhR. Fragment Two includes the promoter amyB ampli ed from wild-type A. oryzae RIB40 strain with primers amybF/amybR, and a green uorescent protein GFP marker ampli ed from plasmid pHBT-GFP-NOS using primers GFPf/GFPr, which is fused by SOE-PCR. The fused fragment amgfp was inserted into pET30b at the restriction enzyme sites Sal I/Xho I, resulting in the pET-amgfp expression vector. Meanwhile, the amgfp + His-Tag ampli ed from plasmid pET-amgfp with primers amygfpHTf/amygfpHTr. Fragment Three includes terminator amyB ampli ed from the genome of the

Discussion
It is well-know that A. oryzae is an excellent host used to express homologous and heterologous proteins [29] . In recent years, it has been widely used in the production of heterologous proteins and has received increasing attention [8] . A. oryzae has many advantages as an expression host, such as a strong ability to produce and secrete proteins, and strong post-translational modi cation [30,31] . Therefore, it is important to develop an effective A. oryzae transformation system.
There are three kinds of selective markers commonly used in lamentous fungi transformation: auxotrophic complementary genes, drug resistance genes, and genes that can make the host use some unusual carbon or nitrogen sources [22] . 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 [32] . During the transformation of lamentous 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 e cient.
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-uoroorotic acid (5-FOA). Thus, pyrG genes in lamentous fungi have been widely used as nutritional/auxotrophic markers for fungal transformation [35] . 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 con rmed 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 [36] .
With the widespread use of A. oryzae in the expression of heterologous proteins, it is particularly important to construct a safe and e cient 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 e cient expression of genes. Studies have shown that different promoters have different e ciency, and strong promoters can effectively improve the stability and transcription level of mRNA [37] . 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. GFP is the most common uorescent proteins used for tagging lamentous fungi, which can as a reporter to test the stability of pBC-Hygro.4 in A. oryzae RIB 40 ΔpyrG [43] . 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 uorescence 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 [44] , glucoamylase [45,46] , and α-glucosidase [47] . 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.

Conclusions
In summary, in the present study, we successfully obtained A. oryzae RIB 40 ΔpyrG strain by UV mutagenesis which would be used as a host for further transformation. In addition, we have constructed a new type of safe and e cient A. oryzae expression vector, which can stably express in A. oryzae RIB 40 ΔpyrG. This will be a new method that can be applied to other lamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.

Bacterial strains and plasmids
All bacterial strains and plasmids used in this work are listed in Table 1. A. oryzae RIB40, a wild-type strain, was stored previously in our laboratory. Escherichia coli DH5α was used for routine plasmid construction and maintenance, which grown in Luria-Bertani (LB) medium. 0.05% MgSO 4 •7H 2 O, 1% glucose, 0.122% uridine 2.0% agar, 0.5-1.5 mg/mL 5-FOA. CD + Uri/Ura medium containing 0.5% uridine and 0.2% uracil was used for the selection of pyrG deletion mutants. CD + 5-FOA + Uri/Ura medium containing 1 mg/mL 5-FOA, 0.5% uridine and 0.2% uracil was used for the growth of pyrG mutants. E. coli DH5a was used for the construction, propagation, and ampli cation of hybrid plasmids.

Spore Preparation And Determination Of 5-foa Minimum Use Concentration
The wild-type A. oryzae RIB40 was grown on (CD) medium for 3-5 days until the spores are mature. Sterile distilled water was added to the culture plate and fungal spores were released from the mycelium by scraping the agar surface with a sterile glass spreader under a laminar ow hood. The resulting mixture was collected with a micropipette and ltered through 500 mesh nylon cloth before centrifugation at 4000 rpm for 10 min. The spore pellet was washed twice with sterile distilled water and resuspended in sterile distilled water to obtain the spore suspension. The spore concentration was examined using a thoma counting chamber and adjusted to 1.0 × 10 7 spores/ml for fungal transformation. The prepared spore suspension was pipetted into 200 µL of 5-FOA medium, which is containing 5-FOA at different concentrations, and allowed to stand for 4-6 days at 28 °C to observe the growth of A. oryzae.
Screening and characterization of pyrG deletion mutants Pipette 3 mL of the 1.0 × 10 7 spores/ml spore suspension prepared above into a Petri dish, place it under UV light (30 W) and irradiated for 5 minutes. The prepared spore suspension was pipetted into 500 µL of 5-FOA medium, the above plate was wrapped with tin foil and allowed to stand for 3 to 4 days at 28 °C. Mutant strains to resistant to 5-FOA were transferred simultaneously to the CD, CD + Uri/Ura and CD + Uri/Ura + 5-FOA (1 mg/mL) to examine their growth. The mutants, which were unable to grow on the CD minimal medium, but could grow normally on CD + Uri/Ura as well as on CD + Uri/Ura + 5-FOA, were selected for single spore isolation. These mutant strains were tested for mitotic stability for ve successive generations on CD + Uri/Ura + 5-FOA before re-growing on CD medium.
The wild-type strains and the uridine/uracil auxotrophic mutants were cultivated in CD medium or CD + Uri/Ura for 4 days, at 28 °C. The obtained mycelia were used for genomic DNA extraction, and the primer pyrGF/pyrGR were used for PCR screening of pyrG deletion mutants.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional les.
Ethics approval and consent to participation Not applicable.

Consent for publication
Not applicable.