Studies On Cloning And Expression Of Aspergillus Phytase Genes

Phytase, a feed additive for mono-gastric animals is widely acknowleged for its effect. However, there are two major drawbacks that constrain the wide use of phytase: the limited activity of wild-type strain producing phytase and the thermostability constrain of current commercial phytase. It seems to be a good strategy to circumvent the aforementioned problems by screening new phytase-producing strain, cloning phytase gene using molecular biology method and over-expressing the gene in bioreactor.In this study, two phytase-producing strains, an Aspergillus niger strain and an Aspergillus fumigatus strain were screened from soil, the phytase genes from the selected strains were cloned and over-expressed in Pichia pastoris GS 115. In addition, the A.fumigatus phytase was expressed in tobacco. Plant was used as bioreactor to produce the thermostable enzyme for the first time. Meanwhile, in view of great potential of plant gene engineering in improving crop phosphorous (P) nutrition, the growth performance of transgenic tobaccos under phosphpate-starvig conditions was also investigated. The research contents and results in detail were stated as follows:1. Two phytase-producing strains, WY-6 and WY-2 were screened from soil samples at 30℃and 45℃by using calcium phytate screening plate and evaluating phytase activity, respectively, and their enzyme activities were all 0.14U/mL. The phytase produced by strain WY-6 showed the optimal pH and temperature at pH3.0 and 55℃. The phytase produced by strain WY-2 showed the optimal pH and temperature at pH5.5 and 55℃. The two strains were identified as A. niger and A.fumigatus, respectively, and their accession no. in China Center of Industrial Culture Collection were CICC 40700 and CICC 40699.2. The phytase genes from A.niger WY-6 and A.fumigatus WY-2 were obtained by PCR amplification and were named as phyA (GenBank accession no. AY745739) and fphyA (GenBank accession no. AY745738). phyA was 1506bp in size and encoded a polypeptide of 467 amino acids, fphyA was 1409bp in size and encoded a polypeptide of 465 amino acids. The amino acid sequence deduced from phyA showed 97％homology wtih A.ficuum NRRL3135 phytase. The amnio acid sequence deduced from fphyA showed 91％and 66％homology wtih A.fumigatus ATCC34625 phytase and A.ficuum NRRL3135 phytase, respectively.3. The phyA was cloned into expression vector pPIC9K and over-expressed in P.pastoris GS115. After 60h of induction by methanol, the recombinant GS115/phyA produced the highest phytase activity of medium at 28.2U/mL, which was 200 fold of that of A. niger WY-6 strain. The recombinant phytase was purified to homogeneity by using a combination of ammonium sulfate fractionation, ultrafiltration and anion exchange chromatography. Its specific activity was 147U/mg and the apparent molecular mass was 67kDa. A high activity was displayed in the pH range of 2.5-6.5. The two optimal pH were 3.0 and 5.5, respectively, and the optimum temperature was 55℃. The K_m for sodium phytate was 0.112mmol/L and the K_cat was 243/s. Incubated with pepsin for 2h at the ratio (pepsin/phytase, wt/wt) of 0.01, the enzyme still retained 70.9％of the initial phytase activity.4. The fphyA was cloned into expression vector pPIC9K and over-expressed in P.pastoris GS115. After 60h of induction by methanol, the recombinant GS115/fphyA produced the highest phytase activity of medium at 16.2U/mL, which was 114 fold of that of A.fumigatus WY-2 strain. The recombinant phytase was purified to homogeneity by using a combination of ammonium sulfate fractionation, ultrafiltration, anion exchange chromatography and gel filtration chromatography. The purified enzyme showed a specific activity of 51U/mg with an approximate molecular mass of 88kDa. The optimal pH and temperature for activity were pH5.5 and 55℃, respectively. After incubation at 90℃for 15min, it still remained 43.7％of the initial activity. The enzyme showed higher affinity for sodium phytate than other phosphate conjugates, and the K_m for sodium phytate was 0.114mmol/L and the K_cat was 102/s. Incubated with pepsin for 2h at the ratio (pepsin/phytase, wt/wt) of 0.1, it kept 90.1％residual activity. Conformational changes in secondary structure of the enzyme depended on the pH value of buffer solution, and the highestα-helix content was showed at pH6.0 (35.6％).5. The A.fumigatus WY-2 phytase gene fusing with the signal sequence from tobacco calreticulin was constitutively expressed in tobacco. PCR Screening and Southern blotting analysis demonstrated that the phytase gene had been integrated into tobacco genomic DNA, and the results of RT-PCR and Western blotting revealed that the phytase gene had been transcripted and expressed in protein levels. After 4 weeks of growth, the transgenic tobaccos produced the highest phytase expression level, the phytase was found to accumulate in leaves up to 2.3％of total soluble protein and the phytase activity was up to 13 fold of that of the wild-type tobocoos. The recombinant phytase showed an apparent molecular mass of 63kDa and the maximum phytase activity at pH5.5 and 55℃. After 15min of exposure to 90℃, it kept 28.7％of the initial enzyme activity.6. The recombinant enzyme was secreted from plant roots into the rhizosphere under direction of the tobacco calreticulin signal peptide. After 40 days of plant growth, the phytase was found to accumulate in transgenic tobacco media up to 2.012％of total root secreted protein and the phytase activity was 15.3 fold of that of wild-type plants. Significant imorivements in plant growth and P utilization were observed in the transgenics. When phytate was supplied as the sole P source, 45-day-old transgenic tobaccos accumulated 2.0-fold shoot biomass, 1.5-fold root biomass and 2.7-fold total P concentration of those of the wild-type tobcoos. The expreriment of quantifying phytate consumpsion in the growth media showed that the improvements in plant P nutrition were connected with the phytase gene expression.