Expression Of Optimized Designed Phytase PhyC And PhyA～m-phyCs Fusion Gene In Pichia Pastoris

Phytase(myo-inositol hexaphosphate 3- or 6-phosphohydrolase, EC3. 1. 3. 8 or EC3. 1. 3. 26) is a kind of safe feed additive widely used in feedstuff for poultry and aquatic anmimals. Many phytases, including fungal phytase from Aspergillus spp. (phyA), have been cloned, characterized, expesssed and fermented. This kind of phytases are histidine acid phosphatases which exhibit an optimum pH at 2.5 to 6, have a pH activity profile ideally suited for maximal activity in the digestive tract of either wines or poultry. The phytases from Bacillus spp. (phyC) are Beta-propeller phytases which exhibit an optimum pH around 6 to 9 are suitable for neutral animal tracts and more thermostable. Due to less studies on neutral phytase and low level of production of neutal phytase, it was not sufficient for commercial prodution for feed additive applicaitons. In addition, the studies on fusion phytase has not been reported yet.To obtain high production of neutral phytase, we optimized the phyC gene and synthesized the netural phytase gene phCs without altering the protein sequence according to the P. pastoris codon usage bias. As a result, the phyCs gene was expressed successfully in P. pastoris. To obtain a new phytase revealed broad pH optima and reduce the production price, the phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were amplified, ligated, inserted into expression vector pPIC9K in correct orientation and transformed into P. pastosis. Furthermore, the recombinant yeasts producing phytase were cultivated in the fermentor. And the properties of the phytase were measured.The paper carried out the following studies:1. The biology informations of phyC gene ( GenBank No: AF AY220075, successfully coined in our previous study were analized. The result showed that the phyC gene was 1152bp in length and encoded 383-amino acid long polypeptide. And the mautre peptide gene was 1074bp in length and encoded 358-amino acid long polypeptide. The (G+C)% content was 46.2% in the phyC gene. And the (G+C)% content of the third site of codon was 56.7%. The (G+C)% content altered to 44.3% and 43.6%respectively when theα-factor singal peptide sequence was added in the phyC gene. According to P. pastoris preferred codons, the analysis ofphyC gene showed that the number of codons whose relative synonymous codon usage (RSCU) value less than 0.3 was 118, accounted for 33% of the whole codons. And AG of the folding mRNA was -437.30 kcal/mol of the original mRNA. The start codon AUG was in the loop of the mRNA secondary structure.2. The phyCs gene was optimized designed and synthesized fistly after the optimized scheme was established. The nucleic acid sequence of synthetic gene was designed from the amino acid sequence of neutral phytase based on the P. pastoris preferred codons. The synthesized 1074-bp phyCs gene showed 73.5% homology with the wild type phyC gene. All codons in phyCs were designed to be preferential for methylotrophic yeast P. pastoris. The (G+C)% content was 49.1% in the phyCs gene. And the (G+C)% content of the third site of codon was 60.1%. The (G+C)% content altered to 46.2% and 52% respectively when theα-factor singal peptide sequence was added in the phyC gene. AT-rich stretches were eliminated to avoid premauture termination. The mRNA secondary structure around the AUG start codon were adjusted, so that AUG is relatively free of secondary structure to ensure efficient translation of mRNA as predicated by the RNA fold software analysis. And AG of the folding mRNA was increased to -434.90kcal/mol higher than -437.40kcal/mol of the original mRNA. The synthetic gene was designed with SnaBâ… and Notâ… restriction enzyme sites at the 5' and 3' terminal, respectively.3. The neutral phytase gene expression vector, pPICgK-phyCs, was constructed successfully after sequencing. And the neutral phytase was expressed successfully. The expression vector pPICgK-phyCs used for transformation, was linearized by Sacâ… . P. pastoris GS115 strains were made competent and transformed with Sacâ… -linearized pPgK-phyCs by electroporation. After selection by MM plates, MD plates, colony-PCR and phyase activity measurement, one colony, which exhibited the highest phytase activity among the clonies examined was selected for shake-flask expression. After 120 h of methanol induction, the yield of total extracellular phytase activity was 18.5U/ml in WBEI medium at the flask scale, increased over 9-fold than phyC phytase expressed in P. pastoris. After 96 h of methanol induction, the yield of total extracellular neutral phytase activity was 54U/ml for high cell-density fermentation induced in WBSG medium. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 51kDa. After deglycosylation by EndoH_f, the enzyme had an apparent molecular size of 42kDa.4. To widen the range of pH effect and reduce the product price, the fusion phytase gene expression vector, pPIC9K-A^mCs, was constructed successfully. The phyA^m and phyCs gene were amplified by PCR using phyA^m and phyCs containing vetcror as the template. To amplify phyA^m gene, the upstream primer and the downstream primer were designed with SnaBâ… and Notâ… restriction enzyme sites at 5' and 3' terminal, respectively. To amlify phyCs gene, an upstream primer was synthesized with Avrâ…¡restriction enzyme sites and linker (G₄S)₃ at the 5' terminal, and a downstream primer was designed with Notâ… restriction enzyme sites at 3'terminal. The two PCR fragments were cloned into pUCm-T vector, named pUCm-phyA^m and pUCm-phyCs, respectively. The phyCs gene was prepared by digestion of pUCm-phyCs with restriction endonucleases Notâ… and Avrâ…¡. The expression vector was prepared by analogous procedures. The purified phyCs fragment was ligated to the purified Avrâ…¡-Notâ… double-digested secretory expression vector pPIC9K with the correct orientation. E. coli strain JM109 was transformed with the ligation products. The bacterial transformants were screened by colony-PCR amplification. The recombinant vector, pPIC9K-phyCs, was prepared using a plasmid miniprep kit (Ω), indentified by Avrâ…¡-Notâ… double-digested restriction endonucleases analysis and sequenced by TakaRa. The phyA^m gene, digested of pUCm-phyA^m with restriction endonucleases SnaBâ… and Avrâ…¡, was inserted into pPIC9K-phyCs by analogous procedures as above. The phytase expression plasmids, pPIC9K-phyA^mCs, was also purified, digested and sequenced as above.5. The fusion phytase was expressed successfully in Ppastoris. The expression vector pPIC9K-phyA^mCs used for transformation, was lineafized by BspEI. P. pastoris GS115 strains were made competent and transformed with BspEI-linearized pPIC9K-phyA^mCs by electroporation. After selection by MM plates, MD plates, colony-PCR and phyase activity measurement, one colony, which exhibited the highest phytase activity among the clonies examined was selected for shake-flask expression. After 120 h of methanol induction, the yield of total extracellular phytase activity was 20.5U/ml in WBEI medium at the flask scale. After 84 h of methanol induction, the yield of total extracellular fusion phytase activity was 176U/ml for high cell-density fermentation induced in WBSG medium. Due to the heavy glycosylation, the expressed phytase had a broad and diffuse band in SDS-PAGE. After deglycosylation by EndoHf, the fusion enzyme had an apparent molecular size of 94.6kDa. And the phyA^m phytase had an apparent molecular size of 52.6kDa after deglycosylation by EndoH_f.6. The recombinant neutral phytase and fusion phytase was purified and characterized. After purified by centrifugation, cold ethanol and aceton precipitation, Q-Sepharose FF anion-exchange chromatography and dialyzed methods, the thermosbility, pH optima and temperture optima were measured.Before and after deglycosylation the enzymatic activity of the expressed neutral phytase both showed temperature optima at 70℃. And they both showed high relative activity at pH between 6 to 9 and optima pH at 7.5. But less phytase acitivity was detected at pH below 4 and above 11. The glycosylated and deglycosylated enzyme activity retains 97% and 77% of the relative activity after incubation at 80℃for 5 min. At 80℃for 10 min the phytase lost 17% and 31% of its activity. Compared with the phyC gene expressed in P. pastris, the phyCs phytase had a broad pH optima, an improve temperture optima and the same thermosbility. The result also showed that the its thermostability was affected by the glycosylation.Purified fusion phytase had temperature optima at 55℃and three pH optima at 2.5, 5.5-6.0 and 7.0, but the enzyme was found to be most acitve in the range of pH5.5 to 6.0. Relative activity of fusion phtase at pH2.5 and 5.5 was 82% and 84%. To test the thermostability, the activity of fusion phytase was measured after incubaiton at varous temperatures for 5min and 10min. Enzyme activity remained around 63% of the initial activity after incubation at 80℃for 5rain and 10min. At 85℃, 90℃, 95℃and 100℃for 5min and 10 min treatment, the fusion phytase also can remain 50% to 60% of its activity. Therefore, the fusion phytase had a broad pH optima ranged from 2.5 to 7.0. Compared with the acid phytase, the thermosbility was also improved.This is the first report that optimizing the phyC gene, successful expression of optimized neutral phyCs gene and fusion phytase phyA^m-phyCs gene in P. pastoris. The neutral phytase activity improved over 9-fold compared with the recombinant phyC phytase. The fusion phytase had a broad pH optima ranged fi'om 2.5 to 7.0 and a good thermosbility. The yield of total extracellular neutral phytase and fusion phytase activity was 58U/ml and 176U/ml for high cell-density fermentation induced in WBSG medium.