Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.     

Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris

Economical and thermostable phytase enzymes are needed to release phytate-phosphorus in plant foods for human and animal nutrition and to reduce phosphorus pollution of animal waste. Our objectives were to determine if a methylotrophic yeast, Pichia pastoris, was able to express a phytase gene (phyA) from Aspergillus niger efficiently and if suppression of glycosylation by tunicamycin affected its functional expression. The gene (1.4 kb) was inserted into an expression vector pPICZalphaA with a signal peptide alpha-factor, under the control of AOX1 promoter. The resulting plasmid was transformed into two P. pastoris strains: KM71 (methanol utilization slow) and X33 (wild-type). Both host strains produced high levels of active phytase (25-65 units/ml of medium) that were largely secreted into the medium. The expressed enzyme was cross-reacted with the polyclonal antibody raised against the wild-type enzyme and showed two pH optima, 2.5 and 5.5, and an optimal temperature at 60 degrees C. Compared with the phyA phytase overexpressed by A. niger, this phytase had identical capacity in hydrolyzing phytate-phosphorus from soybean meal and slightly better thermostability. Deglycosylation of the secreted phytase resulted in reduction in the size from 95 to 55 kDa and in thermostability by 34%. Tunicamycin (20 microg/ml of medium) resulted in significant reductions of both intracellular and extracellular phytase activity expression. Because there was no accumulation of intracellular phytase protein, the impairment did not seem to occur at the level of translocation of phytase. In conclusion, glycosylation was vital to the biosynthesis of the phyA phytase in P. pastoris and the thermostability of the expressed enzyme.     

Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.     

Properties of beta-propeller phytase expressed in transgenic tobacco

Phytases are enzymes that liberate inorganic phosphates from phytate. In a previous study, a beta-propeller phytase (168phyA) from Bacillus subtilis was introduced into transgenic tobacco, which resulted in certain phenotypic changes. In the study described herein, the recombinant phytase (t168phyA) was purified from transgenic tobacco to near homogeneity by a three-step purification scheme. The biochemical properties and kinetic parameters of t168phyA were compared with those of its counterpart from B. subtilis. t168phyA was glycosylated, and it showed a 4 kDa increase in molecular size in SDS-PAGE (44 kDa vs. 40 kDa). Although its thermostability remained unchanged, its temperature optimum shifted from 60 degrees C to 45-50 degrees C and its pH optimum shifted from pH 5.5 to 6.0. Kinetic data showed that the t168phyA had a lower Kcat, but a higher Km than the native enzyme. Despite these changes, t168phyA remained catalytically active and has a specific activity of 2.3 U/mg protein. These results verify the activity of recombinant Bacillus phytase that is expressed in plants.     

Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics

AIMS: Using gene cloning and overexpression to obtain a potential industrial phytase as a feed additive to upgrade the nutritional quality of phytate-rich seed-based animal feed. METHODS AND RESULTS: A phyA gene from a high extracellular phytase-producing Aspergillus niger sp. was cloned and overexpressed in Pichia pastoris GS115 using the secretive expression vector pPICZalphaA. After cultivation for 4 days in buffered methanol complex medium (BMMY) containing methanol for induction, catalytically active phytase was secreted as a predominantly extracellular protein. The activity of the expressed phytase in fermented broth was 30 000-fold higher than that of native phytase with a specific activity of 503 U mg(-1). The Lineweaver-Burk plot indicated K(m) values of 0.196 mmol l(-1) for sodium phytate and 18.16 mmol l(-1) for p-nitrophenylphosphate (pNPP). Thermostability studies showed that recombinant phytase retained 70% activity after exposure to 90 degrees C for 5 min and 65% activity after 30 min, much higher than for commercial phytase. CONCLUSIONS: The higher activity and high thermostability of recombinant phytase enable it to withstand the temperatures of the feed pelleting process. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of this recombinant phytase, especially the good thermostability, are likely to render it of potential industrial importance.     

Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme

Aspergillus fumigatus phytase is a heat-stable enzyme of great potential. Our objective was to determine if a high level of functional expression of the A. fumigatus phytase gene could be produced in Pichia pastoris and how the recombinant phytase reacted to different substrates, heating conditions, and proteases. A 1.4-kb DNA fragment containing the coding region of the gene was inserted into the expression vector pPICZalphaA and expressed in P. pastoris as an active, extracellular phytase (r-Afp). The yield was 729 mg of purified protein per liter of culture, with a specific activity of 43 units/mg of protein. The enzyme r-Afp shared similar pH and temperature optima, molecular size, glycosylation extent, and specificity for p-nitrophenyl phosphate and sodium phytate to those of the same enzyme expressed in A. niger. Given 20 min of exposure to 65 to 90 degrees C, the enzyme retained 20 to 39% higher residual activity in 10 and 200 mM sodium acetate than that in sodium citrate. The enzyme seemed to be resistant to pepsin digestion, but was degraded by high levels of trypsin. In conclusion, P. pastoris is a potential host to express high levels of A. fumigatus phytase and the thermostability of the recombinant enzyme is modulated by the specificity of buffer used in the heat treatment.     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils

Transgenic Nicotiana tabacum plants expressing a chimeric phytase gene (ex::phyA) from the soil fungus Aspergillus niger were generated. Three independently transformed lines showed increased extracellular phytase activity compared with a vector control and wild-type plants, both of which had no detectable extracellular phytase. Transgenic N. tabacum plants grown in sterile agar supplied with phosphorus (P) as phytate accumulated 3.7-fold more P than vector control plants. Despite this, the expression of ex::phyA in plants did not lead to an improved accumulation of P from two unamended P-deficient soils. However, when soils were amended with either phytate or phosphate and lime, transgenic plants accumulated up to 52% more P than controls. Positive responses by transgenic plants were, in some instances, coincident with a putative increase in soil phytate. We conclude that the development of plants that exude phytase to the soil may not ensure improved plant P nutrition, as the availability of phytate in the soil also appears to be critical. Nevertheless, if plants that express ex::phyA are combined with soil amendments that promote the availability of phytate, there is the potential to enhance the P nutrition of crop plants and to improve the efficiency of P fertilizer use in agricultural systems.     

Fungal phyA gene expressed in potato leaves produces active and stable phytase

Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90% of the activity at 20 microM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60 nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. 'Biofarming,' therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum (niger) phyA gene in potato leaf.     

Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris

The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed andsynthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering theprotein sequence.The expression vector,pP9K-phyCs,was linearized and transformed in P.pastoris.Theyield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium(BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale,respectively,improving over 90 folds compared with the wild-type isolate.Purified enzyme showed temperature optimumof 70℃ and pH optimum of 7.5.The enzyme activity retained 97% of the relative activity afterincubation at 80℃ for 5 min.Because of the heavy glycosylation the expressed phytase had a molecularsize of approximately 51 kDa.After deglycosylation by endoglycosylase H (EndoH_f),the enzyme had anapparent molecular size of 42 kDa.Its property and thermostability was affected by the glycosylation.     

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris . The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL⁻¹, respectively. Both recombinant phytases exhibited high affinity for phytate but not for p -nitrophenyl phosphate. Optimal phytase activity was observed at 50 °C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 °C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.     

Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves.

Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.     

植酸酶酵母工程菌的构建*

从无花果曲霉(Aspergillus ficuum)3．4322中用RT-PCR方法扩增出一条约1．4kb的特异性条带,DNA序列测定表明,目的片段为不含信号肽的植酸酶编码序列,全长1347bp。无花果曲霉(Aspergillus ficuum)3．4322phyA基因序列已在GenBank注册(注册号为：AF537344)。将该基因克隆到酵母表达载体pYES2中,构建成不带信号肽phyA基因的重组表达载体pYPA2。用醋酸锂法将pYPA2转进urd缺陷型的酿酒酵母(s．oeraisiae INVSc1),筛选获得含植酸酶基因的酵母转化子。经半乳糖诱导表达后,用磷钼蓝显色(AMES)法对酵母菌体进行酶活测定,测出了明显的植酸酶活性,pYPA2胞内植酸酶活性约11．55IU／mL,表明无花果曲霉(Aspergillus ficuum)3．4322phyA基因能在酿酒酵母中表达。     

植酸酶phyAm基因结构延伸突变改善酶的热稳定性

将来源于黑曲霉N25的植酸酶基因phyA^m重组于大肠杆菌表达载体pET-30b（＋）,以重组表达载体pET30b-FphyA^e为模板经PCR扩增获得结构延伸突变植酸酶基因phyA^m（在植酸酶基因C端增加了来源于pET-30b-FphyA^m载体上13氨基酸残基）。含突变基因的重组表达载体pPIC9k-phyA^e在GS115酵母中表达。纯化的突变酶pp-NP^e与野生型酶PP-NP^m-8相比：PP-NPA^e的最适反应温度上升了3气,75℃处理10min,热稳定性提高21％,比活力略有提高。最适反应pH为5．6,有效pH范围pH4,6到pH6．6。比未突变酶扩大了0.4单位。     

Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris

Escherichia coli pH 2.5 acid phosphatase gene (appA) and three mutants were expressed in Pichia pastoris to assess the effect of strategic mutations or deletion on the enzyme (EcAP) biochemical properties. Mutants A131N/ V134N/D207N/S211N, C200N/D207N/S211N, and A131N/ V134N/C200N/D207N/S211N had four, two, and four additional potential N-glycosylation sites, respectively. Extracellular phytase and acid phosphatase activities were produced by these mutants and the intact enzyme r-AppA. The N-glycosylation level was higher in mutants A131N/V134N/D207N/S211N (48%) and A131N/V134N/ C200N/D207N/S211N (89%) than that in r-AppA (14%). Despite no enhancement of glycosylation, mutant C200N/ D207N/S211N was different from r-AppA in the following properties. First, it was more active at pH 3.5-5.5. Second, it retained more (P < 0.01) phytase activity than that of r-AppA. Third, its specific activity of phytase was 54% higher. Lastly, its apparent catalytic efficiency kcat/Km for either p-nitrophenyl phosphate (5.8 x 10(5) vs 2.0 x 10(5) min(-1) M(-1)) or sodium phytate (6.9 x 10(6) vs 1.1 x 10(6) min(-1) M(-1)) was improved by factors of 1.9- and 5.3-fold, respectively. Based on the recently published E. coli phytase crystal structure, substitution of C200N in mutant C200N/D207N/S211N seems to eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein. This change may modulate the domain flexibility and thereby the catalytic efficiency and thermostability of the enzyme.     

植酸酶和甘露聚糖酶双功能毕赤酵母工程菌的构建和产酶分析

根据已发表的植酸酶基因和甘露聚糖酶基因序列设计并合成引物,应用PCR技术,分别以土曲霉总DNA和质粒pHBM1201为模板,扩增出均不含假定信号肽序列的植酸酶基因phyA和甘露聚糖酶基因man,将它们各自克隆到毕赤酵母表达载体pHBM907C上,分别得到重组质粒pHBM907C-phyA和pHBM907C-man。将质粒pHBM907C-phyA上由乙醇氧化酶(AOX1)启动子和终止子引导表达、酿酒酵母α信号肽序列引导分泌的phyA表达盒式结构插入到质粒pHBM907C-man中,构成双基因表达分泌质粒pHBM907C-phyA-man。pHBM907C-phyA-man经SalⅠ酶切线性后转化毕赤酵母(Pichiapastoris)GS115,获得了同时分泌表达植酸酶和甘露聚糖酶的双功能酵母工程菌。研究了该酵母工程菌所分泌表达的重组植酸酶和甘露聚糖酶的相关酶学性质,并进行了双功能酵母工程菌的稳定性测试。     

Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA)

The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically. Although A. fumigatus phytase shows 66.2% sequence homology with A. ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase. The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A. ficuum phytase. Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase. The cloned A. fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identically affected by cyclohexanedione. Both the thermal characterization data and kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequence difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases.     

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.     

Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.