Functional analysis of OAT gene in Aspergillus Niger *

Mitochondrial oxaloacetate transporter protein encoded by OAT gene transports oxaloacetate from cytoplasm into mitochondria. To investigate the primary effects of OAT gene on relative metabolism in Aspergillus niger, a oat-deleted mutant was derived from wild-type A. niger ATCC1015 using the double-crossover chromosome replacement technique. Then batch fermentation was performed to evaluate the mutant. Compared with the wild type, the mutant showed lower organic acids production, with the experimental data that the production of pyruvate and 2-ketoglutarate decreased by 31.6% and 35.7%, respectively, and by contrast, the mutant showed higher glycerol formation. The results suggest that OAT gene plays significant roles on metabolism in A. niger.


Introduction
The filamentous fungus Aspergillus niger was widely applied to industrial enzymes and organic acids production [1].Despite decades of study, the production process is not well understood in details yet [2].The release of the genomic sequences of Aspergillus niger ATCC1015 in 2006 [3][4] provided a very good opportunity to characterize the function of genes in A. niger.Additionally, the deletion of non-homologous end-joining gene Ku80 showed high recombination frequency, which provided a useful tool for gene targeting in A. niger [5][6][7].
In the fugus strain A. niger, citrate can be synthesed from oxaloacetate and acetyl-CoA by citrate synthase.The progress of citrate synthesis constantly accompanied with oxaloacetate consumption, so it is important to ensure adequate oxaloacetate for the production of citric acid [8].Formers' researches verified that oxaloacetate transporter delivers oxaloacetate from the cytoplasm to the mitochondria in A. niger [3].In this study, oxaloacetate transporter OAT null mutant was generated, and the effect of deletion of OAT on the metabolites of A. niger was investigated.

Strains
Aspergillus niger strain ATCC 1015 used in this study was kindly provided by Dr Sun Ji Bin, Tianjin Institute of Industrial Biotechnology, CAS.
Escherichia coli DH5α was used for routine cloning experiments.
Agrobacterium tumefaciens AGL1 was used for the transformation of A. niger.

Strain Growth
A. niger strains were grown at 30 ℃ on potato dextrose agar (PDA) slant for sporulation and stock [9].
E. coli DH5α was grown at 37 °C on LB medium.A. tumefaciens AGL1 was grown at 28°C on LB medium.

OAT Gene Deletion
E-mail address: liuhao@tust.edu.cnTherelative sequences of OAT gene were obtained from the genome sequences database DOE Joint Genome Institute (http://genome.jgi.doe.gov/Aspni5/Aspni5.home.html).The plasmid p44 containing hph gene was derived from pCAMBIA1300.The oatΔ strain was constructed using the standard one-step gene replacement strategy in which hph gene was used as selective marker [11].Briefly, about 1 kb of 5' UTR and 3' UTR fragments were amplified by PCR using the primer pairs oat-upF/oat-upR and oat-downF/oat-downR, respectively.The two fragments Were ligated to flank the hygromycin resistance cassette in p44, sequentially.The gene replacement construct was introduced into A.niger ATCC 1015 via Agrobacterium tumefaciens-mediated transformation.CM containing 200 μg/mL hygromycin was used for selecting transformants.Correct gene replacement event was confirmed by PCR using primer pairs oat-upF1/oat-downR1.The sequences of the pimers are presented in Table 1.

Cultivation Conditions
For inoculation of batch cultivations, spores of A. niger incubated at 30 °C on PDA solid medium for 5-7 days were harvested with 0.05% (w/w) Tween-80.All strains were cultivated in 5L-Braun bioreactors with a working volume of 4 L. The bioreactors were equipped with two Rushton four-blade disc turbines used for culture broth mixing.No baffles were incorporated in the bioreactors thereby reducing the surface area available for wall growth.Subsequently, Spores were added to the bioreactors to an initial concentration of 5×10 8 /L.The temperature was maintained at 30 °C.The initial aeration rate, rotate speed and pH were 0.3 vvm, 200 rpm and 2.5, respectively.After germination (approximately 16 h), the culture condition described above were gradually adjusted to 1 vvm, 600 rpm and pH 4.5 and kept constantly throughout the rest of the fermentation time.The pH was controlled by automatic addition of 1 M NaHCO 3 or 2 M HCl.

Biomass Determination
The biomass was determined by the use of 0.45 μm nitrocellulose filter.Initially, the filters were pre-dried in a microwave oven at 150 W for 10 min, and then weighed.A known weight of cell culture was filtered, and the cells were washed with distilled water.Finally, the filter was dried in the micro-wave at 150 W for 15 min and the dry weight was determined.

Extracellular Metabolites Quantification
To determine the extracellular metabolites, a culture sample was taken and immediately filtered through a 0.45 μm nitrocellulose filter.The filtrate was frozen and kept at -20 °C until analysis.Glucose, pyruvate, citrate, succinate, oxalate and glycerol concentrations were determined using an cationic-exchange column (Aminex HPX-87H, BioRad, USA) eluted with 5mM H 2 SO 4 with the flow rate of 0.6 mL min -1 at 65 °C.Metabolites were detected by refractive index and UV.

The Construct of p 44-oat::hph Gene Deletion Cassette
The 3' UTR fragment (966 bp) was amplified and inserted into BamH I and Kpn I sites of p44 to construct the plasmid p44-oat-dw.The 5' UTR fragment (855 bp) was amplified and inserted into Hind III and Pst I sites of p44-oat-dw to obtain the plasmid p44-oat::hph (Figure 1).The result of double digestion of p44-oat::hph to confirm the successful cloning being constructed (Figure 2).

The Generation of the OAT Gene Deletion in A. Niger
T-DNA fragment containing the 5' UTR (855 bp) and 3' UTR (966 bp) fragments of the OAT gene was transferred into the strain A. niger ATCC 1015 and the transformants were selected with hygromycin.
The resistant colonies grown in the plates were picked out and total DNA were extracted from the transformants, and then used as the template for PCR amplification and examination.More than 30 transformants were examined, and two gene deletion candidates were obtained.When the OAT gene was replaced by hph gene, the length of the DNA fragment amplified by PCR using primer pairs oat-upF1/ oat-downR1 is about 3.5 kb (Figure 3, Lane 3) compared to 3.0 kb obtained from the wild type strain(Figure 3, Lane 1).The length of the PCR product obtained from transformants1 (Figure 3, Lane 2) is the same as that obtained from the wild type strain, indicating that the transformants1 was not the oatΔ strain.All the data suggested that the transformants2 was an OAT gene deletion strain.

Characterization of OAT Deletion Strain
The biomass of the oatΔ strain and the wild type strain were almost the same along with the culture time (Figure 4).Moreover, the glucose consumptions of the two strains were also similar, indicating that the deletion of the OAT gene has little influence on the growth of A. niger (Figure 4).Organic acids production of the oatΔ strain and the wild type strain were tested and compared with each other.The results are shown in Table 2. Generally, oatΔ strain produced less quantity of organic acids than the wild type strain.Oxalate and citrate were the most abundant acids produced at pH 4.5.It was observed that the productions of oxalate and citrate in the oatΔ strain were reduced compared to the wild type strain.When the OAT gene was deleted, pyuvate production decreased, less pyuvate was transferred into the mitochondria which caused the synthese of citrate blocked (The data was shown in Table 2).pyruvate and α-ketoglutarate of the oatΔ strain was reduced by 31.6% and 35.7% respectively compared to the wild type strain.In contrast, the glycerol production in the oatΔ strain was increased than the wild type strain.

Conclusion
It is concluded that deletion of OAT has significant effect on the metabolic flux of oxaloacetate.For the reason that oxaloacetate in mitochondria can be supplemented by muliple pathways [12], so it is necessary to further study the details of oxaloacetate metabolic processes in A.niger in organic acids metabolism.