Ectopic expression of a soybean phytase in developing seeds of <Emphasis Type="Italic">Glycine max</Emphasis> to improve phosphorus availability

A transgenic approach was used to alter soybean seed phytate content by expressing a soybean phytase gene (GmPhy) during seed development to degrade accumulating phytic acid (IP₆). An expression vector containing the soybean phytase cDNA controlled by the seed-specific -conglycinin promoter (-subunit) was used to transform embryogenic soybean cultures. Plants from four independent transgenic lines were analyzed for transgene integration and seed IP₆ levels. The reduction in IP₆ levels in transgenic seeds compared to control Jack soybeans ranged from 12.6 to 24.8 as determined by HPLC. A low copy transformant was propagated to the T₄ generation and examined in more detail for phytase expression and enzyme activity during seed development. Expression of phytase mRNA and phytase activity increased during seed development, consistent with the use of an embryo-specific promoter. Ectopic phytase expression during seed development offers potential as an effective strategy for reducing phytate content in soybean seed.     

Phytase enzymology, applications, and biotechnology

Phytases are phosphohydrolases that initiate the step-wise removal of phosphate from phytate. These enzymes have been widely used in animal feeding to improve phosphorus nutrition and to reduce phosphorus pollution of animal waste. The potential of phytases in improving human nutrition of essential trace minerals in plant-derived foods is being explored. This review covers the basic biochemistry and application of phytases, and emphasizes the emerging biotechnology used for developing new effective phytases with improved properties.     

Microbial phytase addition resulted in a greater increase in phosphorus digestibility in dry-fed compared with liquid-fed non-heat-treated wheat–barley–maize diets for pigs

The objective was to evaluate the effect of microbial phytase (1250 FTU/kg diet with 88% dry matter (DM)) on apparent total tract digestibility (ATTD) of phosphorus (P) in pigs fed a dry or soaked diet. Twenty-four pigs (65±3 kg) from six litters were used. Pigs were housed in metabolism crates and fed one of four diets for 12 days; 5 days for adaptation and 7 days for total, but separate collection of feces and urine. The basal diet was composed of wheat, barley, maize, soybean meal and no mineral phosphate. Dietary treatments were: basal dry-fed diet (BDD), BDD with microbial phytase (BDD+phy), BDD soaked for 24 h at 20°C before feeding (BDS) and BDS with microbial phytase (BDS+phy). Supplementation of microbial phytase increased ATTD of DM and crude protein (N×6.25) by 2 and 3 percentage units (P<0.0001; P<0.001), respectively. The ATTD of P was affected by the interaction between microbial phytase and soaking (P=0.02). This was due to a greater increase in ATTD of P by soaking of the diet containing solely plant phytase compared with the diet supplemented with microbial phytase: 35%, 65%, 44% and 68% for BDD, BDD+phy, BSD and BSD+phy, respectively. As such, supplementation of microbial phytase increased ATTD of P in the dry-fed diet, but not in the soaked diet. The higher ATTD of P for BDS compared with BDD resulted from the degradation of 54% of the phytate in BDS by wheat and barley phytases during soaking. On the other hand, soaking of BDS+phy did not increase ATTD of P significantly compared with BDD+phy despite that 76% of the phytate in BDS+phy was degraded before feeding. In conclusion, soaking of BDS containing solely plant phytase provided a great potential for increasing ATTD of P. However, this potential was not present when microbial phytase (1250 FTU/kg diet) was supplemented, most likely because soaking of BDS+phy for 24 h at 20°C did not result in a complete degradation of phytate before feeding.     

Effects of phytase and 25-hydroxyvitamin D3 inclusions on the performance, mineral balance and bone parameters of grower-finisher pigs fed low-phosphorus diets

Two experiments, a performance experiment and a mineral balance study, were conducted on grower-finisher pigs (42 to 101 kg live weight) to investigate the effects of Peniophora lycii phytase enzyme and 25-hydroxyvitamin D3 (25-OHD3) on growth performance, carcass characteristics, nutrient retention and excretion, and bone and blood parameters. The two experiments were designed as a 2 × 2 factorial (two levels of phytase and two levels of 25-OHD3). The four diets were T1, low-phosphorous diet; T2, T1 + phytase; T3, T1 + 25-OHD3 and T4, T1 + phytase + 25-OHD3 diet. In all, 25 μg of 25-OHD3 was used to replace 1000 IU of vitamin D3 in diets T3 and T4. Diets were pelleted (70°C) and formulated to contain similar concentrations of energy (13.8 MJ DE/kg), lysine (9.5 g/kg) and digestible phosphorus (P; 1.8 g/kg). Neither the inclusion of phytase nor 25-OHD3 in the diet had any effect on pig performance. There was an interaction between phytase and 25-OHD3 on calcium (Ca) and P retention (P < 0.01) and on the apparent digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.001). Pigs offered phytase diets only, had a higher retention of Ca and P and digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.01) compared with pigs offered unsupplemented diets. However, when the combination of phytase and 25-OHD3 were offered, no effects were detected compared with 25-OHD3 diets only. Pigs fed phytase diets had higher bone ash (P < 0.01), bone P (P < 0.01) and bone Ca (P < 0.05) concentrations compared with pigs offered non-phytase diets. In conclusion, pigs offered phytase diets had a significantly increased bone ash, Ca and P than pigs offered unsupplemented phytase diets. However, there was no advantage to offering a combination of phytase and 25-OHD3 on either bone strength or mineral status compared to offering these feed additives separately.     

Ability of <Emphasis Type="Italic">Emericella rugulosa</Emphasis> to mobilize unavailable P compounds during Pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] crop under arid condition

Phosphate solubilizing microorganisms are ubiquitous in soils and could play an important role in supplying P to plants where plant unavailable P content in soil was more. A phosphatase and phytase producing fungus Emericella rugulosa was isolated and tested under field condition (Pearl millet as a test crop) in a loamy sand soil. In the experimental soil 68% organic phosphorous was present as phytin; less than 1% of phosphorous was present in a plant available form. The maximum effect of inoculation on different enzyme activities (acid phosphatase, alkaline phosphatase, phytase, and dehydrogenase) was observed between 5 and 8 weeks of plant age. The depletion of organic P was much higher than mineral and phytin P. The microbial contribution was significantly higher than the plant contribution to the hydrolysis of the different P fractions. A significant improvement in plant biomass, root length, seed and straw yield and P concentration of root and shoot resulted from inoculation. The results suggest that Emericella rugulosa produces phosphatases and phytase, which mobilize P and enhance the production of pearl millet.     

Production and purification of a novel thermostable phytase by Pichia pastoris FPHY34

A genetically engineered Pichia pastoris FPHY34 strain containing a 1.3 kb thermostable phytase gene (fphy) evolved by DNA shuffling was constructed and screened. Expression and purification conditions for the recombinant phytase were developed in this study. The effect of Pi on recombinant phytase expression and cell growth of P. pastoris FPHY34 was tested in shake flask culture. Optimization of carbon sources for cell growth and methanol feeding strategies for phytase expression in P. pastoris FPHY34 was carried out in a 50-L fermenter by fed-batch fermentation. The purification of phytase was investigated by micro-filtration and ultra-filtration followed by desalting, ion-exchange chromatography, and gel filtration in the ÄKTA system. It showed that the optimum inorganic phosphorus is 13.6 g L⁻¹ and that glucose can be used as a substrate for P. pastoris cell growth instead of glycerol; the biomass yield of glycerol (Y_X/S) is slightly higher than that of glucose. Different profiles of lag phase and respiratory quotient (RQ) displayed between glucose and glycerol as the sole carbon source. The maximum phytase activity in per millimetre reached 2508 U mL⁻¹ at a methanol feed rate of 3.0 mL L⁻¹ h⁻¹ after 80 h period of induction. A purification factor of 41.1 with a 32% yield was achieved after chromatographic purification. The specific enzyme activity was 80 U mg⁻¹ and 3281 U mg⁻¹ in that supernatant fraction and after gel filtration purification, respectively. The strain P. pastoris FPHY34 showed a promising application in phytase industrial production.     

Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.     

Nutrient availability and management in the rhizosphere: exploiting genotypic differences

Crop nutrition is frequently inadequate as a result of the expansion of cropping into marginal lands, elevated crop yields placing increasing demands on soil nutrient reserves, and environmental and economic concerns about applying fertilizers. Plants exposed to nutrient deficiency activate a range of mechanisms that result in increased nutrient availability in the rhizosphere compared with the bulk soil. Plants may change their root morphology, increase the affinity of nutrient transporters in the plasma membrane and exude organic compounds (carboxylates, phenolics, carbohydrates, enzymes, etc.) and protons. Chemical changes in the rhizosphere result in altered abundance and composition of microbial communities. Nutrient-efficient genotypes are adapted to environments with low nutrient availability. Nutrient efficiency can be enhanced by targeted breeding through pyramiding efficiency mechanisms in a desirable genotype as well as by gene transfer and manipulation. Rhizosphere microorganisms influence nutrient availability; adding beneficial microorganisms may result in enhanced availability of nutrients to crops. Understanding the role of plant-microbe-soil interactions in governing nutrient availability in the rhizosphere will enhance the economic and environmental sustainability of crop production.     

High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.