Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.
6-Phytase/genetics* ; Pichia/genetics* ; Plasmids ; Recombinant Proteins/genetics* ; Saccharomycetales

Both phytase and endoglucanase are additives in feed for mono-gastric animal known for their effects. Recombinant vector pPICZalpha-EG was constructed and transformed to GS115-phyA, a Pichia pastoris strain that had integrated with phytase gene, generating GS115-phyA-EG. Both phytase and endoglucanase activities in the supernatant were determined after methanol induction of GS115-phyA-EG. Phytase and endoglucanase activity reached 39.4% and 56.2% activity compared to GS115-phyA and GS115-EG, respectively. Properties of the mixed enzyme suggest that the optimal temperature and pH value be 55 degrees C and 5.5 respectively. Both phytase and endoglucanase showed greater than 80% activity across temperature ranges 45 degrees C to 55 degrees C and pH ranges 4.5 to 5.5. Expressing more than one enzyme in one system could save time and money during induced expression, and the mixed enzyme might apply for treating forge before feeding with poultry.
6-Phytase ; biosynthesis ; genetics ; Cellulase ; biosynthesis ; genetics ; Genetic Vectors ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.
6-Phytase ; genetics ; Fermentation ; Gene Dosage ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombination, Genetic

Using the polymmerse chain reaction (PCR), we amplified the phytase gene phyA from Pichia pastoris GS115-phyA in Aspergillus niger NRRL3135 without the signal peptide sequence and intron sequence,. Then, it was cloned into pINA1297 vector to generate a recombinant vector of pINA1297-phyA. pINA1297-phyA was linearized and transformed into Yarrowia lipolytica po1h by the lithium acetate method. The positive transformants were obtained by YNB(casa) and PPB plates, after induced in YM medium at 28 degrees C for 6 day. The activity of the expressed phytase phyA reached 636.23 U/mL. The molecular weight of the enzyme was 130 kDa measured with SDS-PAGE analysis, whereas its molecular size reduced to 51 kDa after deglycosylation which is correspond with theoretical value. The enzymatic analysis of the recombinant phytase phyA revealed its optimal pH and temperature was 5.5 and 55 degrees C, which had high activity after incubated in pH ranged from 2.0 to 8.0 for 1 h. Moreover, its activity remained 86.08% after exposure to 90 degrees C for 10 min. It also was resistant to pepsin or trypsin treatment.
6-Phytase ; biosynthesis ; genetics ; Aspergillus niger ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Yarrowia ; genetics ; metabolism

High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the Pichia pastoris expression vector pPIC9, then introduced into the host Pichia pastoris by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The result of Southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase proteins with apparent molecular weight in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E. coli. The results of N-terminal sequecing and deglycosylation of the expressed phytase in Pichia pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia pastoris shared similar pH and temperature optima to those of the natural phytase from E. coli and had highly resistant to pepsin digestion. In 5-L fermentor, after induced by 0.5% methanol for 120 h, the expression level of phytase protein was 2.5 mg/mL, and the phytase activity (fermentation potency) exceeded 7.5 x 10(6) IU/mL, which was the highest among those of all kinds of recombinant strains reported now.
6-Phytase ; genetics ; metabolism ; Escherichia coli ; enzymology ; Escherichia coli Proteins ; genetics ; Fermentation ; Pichia ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

Phytase gene of Aspergillus niger 963 was cloned into baculovirus transfer vector. DNA of the recombinant vector was co-transfected with Bm-BacPAK6 DNA into BmN cells, and recombinant virus was selected by plaque assays. The recombinant virus was identified by Dot blot and Southern-blot with the specific probe for phytase gene. Phytase gene was expressed in silkworm larvae and pupae. The expression product was 1.43 g/L haemolymph for silkworm larvae and 1.90 g/L haemolymph for pupae, respectively. The enzymic characteristicses of phytase expressed in baculovirus-expression system were studied in this paper.
6-Phytase ; genetics ; metabolism ; Animals ; Aspergillus niger ; enzymology ; genetics ; Blotting, Southern ; Bombyx ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hydrogen-Ion Concentration

The phytase gene phyA(m) from Aspergillus niger N25 was recombined into E. coli expression vector pET-30b(+). Recombined at expression vectors pET30b-FphyA(m) was served as a template to amplify phytase gene, and the PCR product named elongation mutation gene phyA(e) was expanded with a 13 amino acid sequence from pET-30b-FphyA(m) vector at C-terminal of phytase gene phyA(m). Furthermore, phyA(e) gene was recombined into expression vector pPIC9k and expressed in Pichia pastoris. The comparison experiment of mutant phytase PP-NP0 with wild-type phytase PP-NP(m)-8 showed that: the optimum temperature of PP-NPe was increased by 3 degrees C, and its thermostability was increased by 21% when it was exposed to 10 min at 75 degrees C. Its effective reaction pH range with catalysis efficiency above 70% was pH 4.6 - pH 6.6, and wider 0.4 pH value than that of wild-type phytase.
6-Phytase ; biosynthesis ; genetics ; Amino Acid Sequence ; Aspergillus niger ; enzymology ; genetics ; Enzyme Stability ; genetics ; Escherichia coli ; enzymology ; genetics ; Fungal Proteins ; biosynthesis ; genetics ; Hot Temperature ; Molecular Sequence Data ; Mutation

This research amplified the phyA gene with the designed and synthesized primers specific for the phyA gene full-length coding sequence. The phyA gene was from Aspergillus niger F246 by the polymerase chain reaction(PCR), which is selected and identified in our laboratory. After sequncing the coding sequence, it was confirmed that the construction of cloning vector was succeeded. The phyA gene fragment was recovered from the pMD18T-phyA and ligated with prokaryotic expression vector pET30a+ to construct the recombinant expression plasmid pET30a+ -phyA. It was expressed with IPTG induction in E. coli for high efficiency. A new protein band with apparent molecular weight 50 kDa was detected in the lysate of the transformed cell by using SDS-PAGE. The amount of the soluble fusion protein was about 40% of large intestine bacillus soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation. It's phytase activity was eight times over the natural phyase. So this research provides the basis of the study on obtaining large and high active phytase and developmant of the new microbial ecologicalagent.
6-Phytase ; biosynthesis ; genetics ; Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genes, Fungal ; Genetic Vectors ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Enzymes can degrade the anti-nutrient factors in feedstuff, increase nutrient digestibility, and reduce pollution to environment, and have been widely supplemented in animal feedstuff. However, the use of enzymes is limited because of their undesirable properties, such as thermoliability and susceptibility against protease digestions. And its commercialization is also limited by low production efficiency and high cost. Therefore, the focuses for future enzyme development will be: (1) to obtain novel enzymes with better properties by high-throughput screening of enzyme encoding genes, especially those from extreme and special environments; (2) to improve enzyme properties using directed mutagenesis and protein engineering methods; (3) to achieve high-level fermentation of enzymes by heterogonous expression and optimization of codons, vectors and fermentation conditions; (4) to determine the effect of enzymes to animals and utilize enzymes efficiently.
6-Phytase ; genetics ; metabolism ; pharmacology ; Animal Feed ; Animals ; Dietary Supplements ; Lipase ; genetics ; metabolism ; pharmacology ; Peptide Hydrolases ; genetics ; metabolism ; pharmacology ; Protein Engineering

The phyA(m) gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyA(m)-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4+/-0.53) U/ml at the flask scale and (159.1+/-2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 degrees C and an optimal pH at 5.5~6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 degrees C to 95 degrees C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoH(f)), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyA(m).
6-Phytase ; genetics ; isolation & purification ; metabolism ; Amino Acid Sequence ; Fermentation ; Genetic Vectors ; Molecular Sequence Data ; Pichia ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification