Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.

The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A. ficuum phytase. Although the characteristic bi-hump pH optima were retained in the cloned phytase, the optimal pH shifted downward from 5.5 to 5.0. Also, the recombinant phytase was inhibited by the pseudo-substrate myo-inositol hexasulfate and also by antibody raised against a 20-mer peptide belonging to fungal phytase. The expressed phytase in alfalfa could also be modified by phenylglyoxal. Taken together, the results indicate that fungal phytase when cloned and expressed in alfalfa leaves produces stable and catalytically active phytase while retaining all the properties of the benchmark phytase. This affirms our view that "molecular biofarming" could be an alternative means of producing stable hydrolytic enzymes such as phytase.     

Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves.

The phyA gene from Aspergillus ficuum coding for a 441-amino-acid full-length phytase was expressed in Nicotiana tabacum (tobacco) leaves. The expressed phytase was purified to homogeneity using ion-exchange column chromatography. The purified phytase was characterized biochemically and its kinetic parameters were determined. When the recombinant phytase was compared with its counterpart from Aspergillus ficuum for physical and enzymatic properties, it was found that catalytically the recombinant protein was indistinguishable from the native phytase. Except for a decrease in molecular mass, the overexpressed recombinant phytase was virtually the same as the native fungal phytase. While the temperature optima of the recombinant protein remain unchanged, the pH optima shifted from pH 5 to 4. The results are encouraging enough to open the possibility of overexpressing phyA gene from Aspergillus ficuum in other crop plants as an alternative means of commercial production of this important enzyme.     

植酸酶phyA基因在解脂耶氏酵母po1h中的表达

本实验通过PCR方法从毕赤酵母GS115-phyA中扩增出不含有信号肽及内含子的黑曲霉NRRL3135植酸酶phyA基因,并将其克隆到表达载体pINA1297中,得到表达载体pINA1297-phyA,利用醋酸锂转化法将线性化载体转化到解脂耶氏酵母po1h中,通过YNBcasa和PPB平板筛选出阳性表达菌株,阳性菌株在YM培养基中28℃培养6d后酶活达到最大为636.23U/mL。表达上清经SDS-PAGE分析得到表达植酸酶分子量约为130kDa,但通过去糖基化处理后其分子量变为51kDa,与理论值相符。经过酶学性质分析表明重组植酸酶最适pH为5.5,最适温度为55℃,该酶在pH2.0~8.0处理1h后仍有较高酶活,并且90℃处理10min后还有86.08%的残留酶活,其抵抗胃蛋白酶和胰蛋白酶能力也较强。     

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.     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

黑曲霉N-2植酸酶phy4基因的克隆及其重组酵母表达载体的构建

根据已发表的植酸酶phyA基因序列设计并合成1对引物,应用PCR技术,以黑曲霉N-2总DNA为模板,扩增出不包含假定信号肽序列的phyA基因,将其克隆到pMD18-T载体中,测定其核苷酸序列,并推导其氨基酸序列。该基因全长为1350bp,与已发表的黑曲霉NRRL3135的phyA基因的同源性为92．4％(不计内含子),编码1个含449个氨基酸残基的蛋白质,推导的氨基酸序列同源性为95．1％。将该基因与分泌型载体pPIC9K连接,构建了植酸酶基因的重组酵母表达载体pPIC9K／phyA。     

Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis

Functional expression of a thermostable phytase from A. niger was achieved in Kluyveromyces lactis GG799 cells. Effective secretion of recombinant enzyme (198 U ml⁻¹) in the fermentation broth at 72 h incubation at 22 °C was obtained. Purified enzyme showed a specific activity of 72 U mg⁻¹) and was detected on SDS-PAGE as a heavily glycosylated protein with a molecular weight of ≥140 kDa. Optimum temperature of the enzyme was at 55 °C and it showed a characteristic bi-hump pH profile with two pH optima (at pH 2.5 and 5.5). Enzyme showed considerable pepsin resistance with 60% activity retention after incubation with pepsin at the ratio of 1:1000. Enzyme was thermostable retaining 69 and 37% activity at 90 and 100 °C for 10 min respectively and remained active at these temperatures till 1 h. Deglycosylation studies demonstrated negligible effect of N-linked glycans on thermal properties. Multiple sequence alignment data revealed a conserved Asn at position 345 of this phytase which might contribute to its thermal properties. This thermostable phytase coupled with its noticeable protease resistance could be a better alternative to current commercial phytases.     

Characterisation of a gene that is expressed in leaves at higher levels upon tuberisation in potato and upon flowering in tobacco

A partial cDNA clone has been isolated which represents a gene that is induced in potato (Solanum tuberosum L.) leaves upon tuber initiation. The gene is also present in tobacco (Nicotiana tobacum L.) where it is induced upon flowering, and in tomato (Lycopericon esculentum, L.) where it is induced when the plant starts to form fruit. This provides further evidence for the similarity of the floral-induction and tuber-induction pathways, and raises the possibility that this gene may be involved in a common step in both of these pathways. Alternatively, it could be involved in a secondary process that is induced during tuberisation, flowering and fruit development.     

Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering

Role of disulfide bridges in phytase's unfolding-refolding was probed using dynamic light scattering. Phytase was unfolded by guanidinium chloride and then refolded by removing the denaturant by dialysis. Thiol reagents prevented refolding; thus, disulfide bridge formation is an integral step in phytase folding. Catalytic demise of phytase after unfolding and refolding in presence of Tris(2-carboxyethyl)phosphine (TCEP) indicates that disulfide bridges are necessary for refolding. The hydrodynamic radius (rh) of active and unfolded phytase is 4 and 14 nm, respectively. Removal of denaturant through dialysis refolds phytase; its rh shifts back to 4 nm. When TCEP remains in the refolding media, the rh remains high. The unfolded phytase when diluted in assay medium refolds as a function of time at 25 and 37 degrees C, but not at higher temperature. Monitoring rh under denaturing and renaturing condition gives an accurate measure of the folding status of phytase.     

Potato virus X-induced gene silencing in leaves and tubers of potato

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.     

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.     

Factors affecting gene expression of patatin and proteinase-inhibitor-II gene families in detached potato leaves:

In whole intact potato (Solanum tuberosum L.) plants, the gene families of class-I patatin and proteinase inhibitor II (Pin 2) are constitutively expressed in the tubers. However, they are also induced in detached potato leaves in the presence of light. To further characterize this light action, the detached leaves were subjected to monochromatic light of different wavelengths and to darkness in the presence of metabolites and inhibitors. Patatin genes could be induced by the simultaneous application of sucrose (sugars) and glutamine in darkness. Neither of these metabolites was active when supplied alone. When photosynthesis was blocked by 3-(3,4-Di-chlorophenyl)-1, 1-dimethylurea (DCMU) in the light, patatin genes were not expressed; however, the inhibition was overcome in the presence of sucrose. This indicates that besides its role in photosynthetic carbohydrate production, light may be essential for the supply of amino acids (or reduced nitrogen). Unlike patatin, Pin 2 genes were, to a small extent, also active in darkness, and sucrose weakly enhanced this expression. However, DCMU did not affect Pin 2 expression in the light. Both abscisic acid and methyl jasmonate strongly promoted the accumulation of Pin 2 mRNA independent of the light conditions, indicating that the gene family is probably under hormonal control. The phytohormones did not affect patatin gene expression. Inhibitors of cytosolic (cycloheximide) and organellar (chloramphenicol) translation had opposite effects on the two gene families. Careful evaluation of the inhibitors' action indicates that protein synthesis (cytosol) is required for the expression of Pin 2 genes but not for the patatin genes. These results clearly demonstrate that, although in situ both gene families are constitutively expressed in the same plant organ (tuber) in intact plants, their expression is mediated by different factors.Abbreviations ABA cis-abscisic acid - DCMU 3-(3,4-dichlorphenyl)-1,1-dimethylurea - GUS -glucuronidase activity - MeJA methyl jasmonate - Pin 2 proteinase inhibitor II We thank Beate Küsgen and Regina Breitfeld for the greenhouse work. This work was supported by a grant from the Bundesministerium für Forschung und Technologie.     

Freezing injury in potato leaves

Time-temperature profiles of freezing leaves from frost-resistant (Solanum acaule Bitt.) and frost-susceptible (Solanum tuberosum L. subsp. tuberosum Hawkes) types of potatoes did not reveal any major differences. The pattern of change in resistance of leaves to low voltage, low frequency current during freezing was different in the frost-resistant and susceptible leaves. In tissue sections from both types of leaves, cells freeze extracellularly at cooling velocities lower than 5 C per minute. Cells from leaves of resistant plants showed a higher osmotic pressure but not a higher water permeability than those from susceptible plants. The extent of injury caused by even very slow freezing was greater than that caused by equivalent isopiestic desiccation, particularly in susceptible leaves. The higher osmotic pressure in cells of leaves from resistant plants can account for the greater desiccation resistance but not for the frost resistance observed.     

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.     

Selection of Lactobacillus sp. PSC101 that produces active dietary enzymes such as amylase, lipase, phytase and protease in pigs

This study was carried out to screen lactic acid bacteria that produce active dietary enzymes, such as amylase, lipase, phytase, and protease, using a two-step process in pigs. We isolated a total of 210 and 132 strains of bacteria, grown under aerobic and anaerobic conditions, respectively, in Man Rogosa Sharpe agar containing 0.13% bile after treatment of intestinal samples at pH 3 for 30 min. From fecal samples, a total of 134 aerobic and 111 anaerobic strains were isolated in the same manner. In the second screening test, we selected four strains that produced four dietary enzymes from isolates obtained in the first screening test. Each strain was characterized as lactobacilli based on the following criteria: rod shape, negative for catalase, Gram positive, and lack of acute oral toxicity in mice. Of these four strains, we finally selected Lactobacillus spp. PSC101, which was resistant to pH 3 for 8 h and grew in the presence of 1% bile. In summary, Lactobacillus sp. PSC101 may be a strong probiotic candidate in swine due to its resistance to both acid and bile, its production of dietary enzymes that promote animal growth, and its non-toxic nature in mice.     

Gene note. An alcohol dehydrogenase-like gene is specifically expressed in potato pistils

The reproductive function of the pistil requires the production of compounds essential for pollen tube growth. A cold-plaque screening of a pollinated pistil cDNA library of Solanum tuberosum resulted in the isolation of cDNA clone cp67. Northern blot analyses revealed that cp67 is specifically expressed in potato pistils and in a limited number of plant species. The deduced CP67 protein displays similarity to long-chain zinc-containing alcohol dehydrogenases (ADHs), although at a similarity level much lower than between other plant ADHs.     

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.     

Oxylipin profiling in pathogen-infected potato leaves

Plants respond to pathogen attack with a multicomponent defense response. Synthesis of oxylipins via the lipoxygenase (LOX) pathway appears to be an important factor for establishment of resistance in a number of pathosystems. In potato cells, pathogen-derived elicitors preferentially stimulate the 9-LOX-dependent metabolism of polyunsaturated fatty acids (PUFAs). Here we show by oxylipin profiling that potato plants react to pathogen infection with increases in the amounts of the 9-LOX-derived 9,10,11- and 9,12,13-trihydroxy derivatives of linolenic acid (LnA), the divinyl ethers colnelenic acid (CnA) and colneleic acid (CA) as well as 9-hydroxy linolenic acid. Accumulation of these compounds is faster and more pronounced during the interaction of potato with the phytopathogenic bacterium Pseudomonas syringae pv. maculicola, which does not lead to disease, compared to the infection of potato with Phytophthora infestans, the causal agent of late blight disease. Jasmonic acid (JA), a 13-LOX-derived oxylipin, accumulates in potato leaves after infiltration with P. syringae pv. maculicola, but not after infection with P. infestans.