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.     

Characterization of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: growth and P nutrition in laboratory media and soil

Transgenic Trifolium subterraneum expressing a phytase gene (phyA) from Aspergillus niger were generated. Five independently transformed lines showed an average 77‐fold increase in exuded phytase activity in comparison with null segregant and wild‐type controls. Unlike other phosphatases, exuded phytase activity was unaffected by P supply, verifying the constitutive expression of phyA. Transgenic T. subterraneum grown in agar with P supplied as phytate, took up 1.3‐ to 3.6‐fold more P than controls and had equivalent P uptake to plants supplied with orthophosphate. This unique phenotype was compromised when the plants were grown in soil. None of the five lines showed increased shoot biomass or total P uptake in an unfertilized, low‐P soil taken from under permanent pasture. With addition of P, one of the five transgenic lines had consistently greater P nutrition compared with control plants. Despite variable growth and P nutrition responses, P uptake per root length was on average greater for transgenic lines. Exudation of phytase by transgenic T. subterraneum allowed utilization of P from phytate in non‐sorbing, sterile laboratory media, but was less effective when plants were grown in soil. Release of extracellular phytase is therefore not the only requirement for the acquisition of P from endogenous soil phytate by plants.     

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated T_o events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08–1.77, 0.02–0.67 and 0.44–2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4 weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01–1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.     

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 degrees 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.     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

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.     

Survey of Microorganisms for the Production of Extracellular Phytase

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.     

Functional analysis of an Aspergillus ficuum phytase gene in Saccharomyces cerevisiae and its root-specific, secretory expression in transgenic soybean plants

Phytases release inorganic phosphates from phytate in soil. A gene encoding phytase (AfPhyA) was isolated from Aspergillus ficuum and its ability to degrade phytase and release phosphate was demonstrated in Saccharomyces cerevisiae. A promoter from the Arabidopsis Pky10 gene and the carrot extensin signal peptide were used to drive the root-specific and secretory expression of the AfPhyA gene in soybean plants. The phytase activity and inorganic phosphate levels in transgenic soybean root secretions were 4.7 U/mg protein and 439 μM, respectively, compared to 0.8 U/mg protein and 120 μM, respectively, in control soybeans. Our results demonstrated the potential usefulness of the root-specific promoter for the exudation of recombinant phytases and offered a new perspective on the mobilization of phytate in soil to inorganic phosphates for plant uptake. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guilan Li and Shaohui Yang authors contribute equally to the paper.     

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.     

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.     

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.     

转枯草芽孢杆菌植酸酶基因烟草对不同介质中植酸磷的吸收利用

利用转入枯草芽孢杆菌植酸酶基因的不同烟草株系,分别在无菌培养基、砂培和土培试验中研究了转植酸酶基因烟草对植酸磷的吸收和利用.结果表明,在无菌培养基试验中,所有转植酸酶基因烟草对植酸磷的吸收利用能力均显著高于野生型,其生物量比野生型提高了3.6～10.7倍,总磷吸收量提高了2.2～4.6倍;在沙培和土培中,转植酸酶基因烟草对植酸磷的吸收利用与野生型相比,生物量和总磷吸收量差异不显著.这说明转植酸酶基因在无菌条件下可以提高植物吸收利用植酸磷的能力,但是在自然条件下,由于微生物分解或矿物固定等原因,其作用不稳定,需要进一步研究克服土壤中的限制因素,才能使转基因植物充分发挥作用.     

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.     

Assimilation of Phytate-phosphorus by the Extracellular Phytase Activity of Tobacco (Nicotiana tabacum) is Affected by the Availability of Soluble Phytate

Phytate, the major organic phosphorus in soil, is not readily available to plants as a source of phosphorus (P). It is either complexed with cations or adsorbed to various soil components. The present study was carried out to investigate the extracellular phytase activities of tobacco (Nicotiana tabacum variety GeXin No.1) and its ability to assimilate external phytate-P. Whereas phytase activities in roots, shoots and growth media of P_i-fed 14-day-old seedlings were only 1.3–4.9% of total acid phosphatase (APase) activities, P starvation triggered an increase in phytase secretion up to 914.9 mU mg⁻¹ protein, equivalent to 18.2% of total APase activities. Much of the extracellular phytase activities were found to be root-associated than root-released. The plants were not able to utilize phytate adsorbed to sand, except when insoluble phytate salts were preformed with Mg²⁺ and Ca²⁺ ions for supplementation. Tobacco grew better in sand supplemented with Mg-phytate salts (31.9 mg dry weight plant⁻¹; 0.68% w/w P concentration) than that with Ca-phytate salts (9.5 mg plant⁻¹; 0.42%), presumably due to its higher solubility. We conclude that insolubility of soil phytate is the major constrain for its assimilation. Improving solubility of soil phytate, for example, by enhancement of citrate secretion, may be a feasible approach to improve soil phytate assimilation.     

A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5-6.0 (optimum pH 4.5) with an optimum temperature of 55 degrees C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.     

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.     

Limiting nutrients: an old problem with new solutions?

Iron and phosphorus are essential minerals for both humans and plants. Advances in our understanding of the molecular mechanisms involved in the mobilization, transport and storage of these minerals now allow us to engineer plants to improve the yield and mineral nutrition of crops. Strategies range from increasing the expression of endogenous genes, such as that encoding the iron storage protein ferritin, to expressing a phytase gene from the fungus Aspergillus in Arabidopsis, thereby allowing the plants to obtain a previously unusable pool of phosphorus.     

Engineering the root-soil interface via targeted expression of a synthetic phytase gene in trichoblasts

For biochemical modification of the root-soil interface, the engineered secretion of stable enzymes from trichoblasts (= root hair bearing rhizodermal cells) is proposed. As a reporter activity, we chose to express a synthetic gene encoding a secretory phytase (PHY) directed by a trichoblast-specific promoter in root hair cells of the crop plant potato. Transgenic plants produced and secreted phytase in sufficient amounts to release phosphate from phytate in liquid medium. When grown in an unsterile substrate containing phytate, transgenic plants accumulated 40% more P in leaves than wild-type plants. The improved P nutrition driven by trichoblast-targeted expression and subsequent secretion of PHY illustrates the potential of using trichoblast-targeted expression of suitable enzymes for future applications in plant nutrition, phytoremediation and molecular farming.