The role of acid phosphatase activity during enzymic dephosphorylation of phytates byAspergillus niger phytase

Acid phosphatase present in preparations ofAspergillus niger phytase accelerated dephosphorylation of sodium phytate. Its influence on the reaction rate and distribution ofmyo-inositol phosphates was most apparent at low pH value (2.5) and when acid-hydrolysed substrate was de-esterified enzymatically. With partly purified phytase, the predominant inositol form was tetraphosphate but a preparation having acid phosphatase activity caused an even distribution of lower inositol phosphates after a few hours.     

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg     

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping

Summary TheAspergillus nidulans gene coding for acetamidase (amdS) was introduced intoA. niger by transformation. Twelve Amd⁺ transformants were analysed genetically. TheamdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed)A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for theamdS insert have the Amd⁺ phenotype. We exploited the opportunity for two-way selection inA. niger: transformants can be isolated based on the Amd⁺ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologousamdS gene inA. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI betweenpabA1 andcnxA1, providing evidence for a single transformational insert. The results also show that theamdS transformants ofA. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use ofamdS transformants for mitotic gene mapping in other fungi.     

Transformation of Aspergillus oryzae using the A. niger pyrG gene

Summary A transformation system for Aspergillus oryzae based on the orotidine-5-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per g of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Identification, cloning and analysis of theAspergillus niger genepacC, a wide domain regulatory gene responsive to ambient pH

A wide domain regulatory gene implicated in modulating gene expression in response to ambient pH has been cloned and sequenced from the industrially useful filamentous fungusAspergillus niger. This gene,pacC, is able to restore apacC ⁺ phenotype toA. nidulans pacC ^c 11 andpacC ^c 14 mutants with respect to extent of conidiation, conidial pigment intensity and acid phosphatase regulation. ThepacC gene ofA. niger comprises three exons, encodes a three-zinc-finger protein of 677 amino acids, and shows pH-dependent regulation of expression: mRNA levels are elevated under alkaline conditions and considerably reduced under acidic conditions. The occurrence of PacC consensus binding targets within the sequences upstream ofpacC may indicate autoregulation.     

Expression of fungal phytase on the cell surface ofSaccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast,Saccharomyces cerevisiae by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) ofAspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast α-agglutinin, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced intoS. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.     

Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppA_Ec and rAppA_Sc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppA_Sc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppA_Ec.     

Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.     

Strain improvement of Aspergillus niger for phytase production

A strain improvement program was developed to increase extracellular phytase (E.C. 3.1.3.8.) production by Aspergillus niger (syn. A. ficuum) NRRL 3135. Ultraviolet (UV) radiation was used as the mutagen and resistance to 50 g/ml of hygromycin B as the selection method. Mutant 2DE, the product of two UV treatments, had phytase (PhytA) activity at pH 5.0 in the extracellular filtrate that was 3.3-fold higher than the wild-type activity. The activity of the non-specific acid phosphatase with a pH optimum of 6.0 (Pase) was one-fifth the activity of the wild type and the non-specific acid phosphatase with a pH optimum of 2.5 (PhytB) was not significantly different from the wild type. The mutant and wild-type PhytA, PhytB and Pase responsed similarly in inhibition studies. However, the wild-type enzymes were inhibited more by 1 mm sodium fluoride and 1 mm phosphomycin. PhytA production by the mutant was repressed 60% by inorganic phosphate concentrations of 0.006% (wt/vol) or above. The mutant had an extracellular protein concentration 3.2-fold higher than the wild type, which correlated with its higher phytase activity at pH 5.0, but not with phytase activity at pH 2.5 and acid phosphatase activities. The isolate may be a phytase catalytic mutant, as well as, on overproducer of phytase. In addition, a mutant with an apparent lack of activity of all three acid phosphatases was isolated.Correspondence to: R. J. Wodzinski     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus niger niaD gene encoding nitrate reductase

Summary A transformation system for Gibberella fujikuroi based on the Aspergillus niger nitrate reductase gene (niaD) was developed. A strain (designated SG140) carrying a non-reverting niaD mutation (niaD11) was generated by screening mutagenised cells for non-growth on nitrate as sole nitrogen source. Transformation frequencies of 1–2 transformants per g DNA were observed when strain SG140 was transformed to nitrate utilisation. Southern blot analyses of niaD⁺ transformants showed that the vector DNA sequences were integrated into the chromosomal DNA. The results demonstrate that the A. niger niaD gene is expressed in G. fujikuroi.     

A single amino acid substitution in soybean VSPα increases its acid phosphatase activity nearly 20-fold

Soybean [Glycine max (L.) Merr.] contains two proteins called vegetative storage proteins (VSPs) that function as temporary storage reserves, but are also closely related to plant acid phosphatases of the haloacid dehalogenase (HAD) superfamily. This study examined the biochemical basis for the relatively low catalytic activity previously reported for these VSPs. The specific activity of purified recombinant VSP on GMP was about 40-fold lower than for a related soybean root nodule acid phosphatase (APase), which had a specific activity of 845 U mg^–1 protein. Conversion of Ser¹⁰⁶ to Asp increased VSP activity about 20-fold. This Asp residue is present in nodule APase and is a highly conserved nucleophile in the HAD superfamily. Related VSPs from cultivated soybean and from three wild perennial soybeans, as well as a pod storage protein (PSP) from Phaseolus vulgaris L. all lack the catalytic Asp, suggesting they too are catalytically inefficient. Phylogenetic analysis showed the VSPs and PSP are more closely related to each other than to 21 other VSP-like proteins from several plant species, all of which have the nucleophilic Asp. This study suggests that loss of catalytic activity may be a requirement for the VSPs and PSP to function as storage proteins in legumes.Abbreviations APase Acid phosphatase - GST Glutathione S-transferase - HAD Haloacid dehalogenase - pNPP Para-nitrophenol phosphate - PSP Pod storage protein - RIP Ribosome inactivating protein - VSP Vegetative storage protein Accession numbers for the VSP sequences reported in this paper are from G. falcata, AY523602; G. tomentella, AY523603; G. curvata, AY523604     

Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5 promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5 up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.     

Introduction and maintenance of prokaryotic DNA inUstilago violacea

Summary A strain of the basidiomycete,Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin.U. violacea transformants were selected at a frequency of 5 per g pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipientU. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction ofU. violacea transformants failed to produceE. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction inU. violacea transformants producedE. coli transformants resistant to neomycin. TheE. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harborU. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryoteU. violacea and acquire endogenous sequences from this organism.     

Expression of <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA2 phytase in four yeast systems

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ A), and one constitutive system: P. pastoris (pGAPZA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml^–1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml^–1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower K_mfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.Revisions requested 18 November 2004; Revisions received 7 January 2005     

Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatus Phytase, A. niger Phytase, and A. niger pH 2.5 Acid Phosphatase

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.