Overexpression of phytase genes in yeast systems

IS 2 255 281 T3

DESCRIPTION

Overexpression of phytase genes in yeast systems.

Field of the invention

The present invention relates to a method of producing phytase in yeast, to yeast strains expressing heterologous phytase and to heterologous phytase produced by yeast.

Background of the invention

Phytases, a specific group of monoester phosphatases, are necessary to initiate the release of phosphate ("P") from phytate (myoinositol hexaphosphate), the main storage form of P in cereal foods or feeds (Reddy, NR et al., "Phytates in Legumes and Cereals", Advances in Food Research, 28: 1 (1982)). Because single-stomach animals such as pigs and poultry, as well as humans, have little phytase activity in their gastrointestinal tracts, almost all of the P ingested phytate is indigestible. This results in the need to supplement with inorganic P, an expensive and non-renewable nutrient, the diets for these animals. More undesirably, the unused phytate P excreted through compost from these animals becomes P contamination of the environment (Cromwell, GL et al., “P - A Key Essential Nutrient, Yet a Possible Major Pollutant- Its Central Role in Animal Nutrition ”, Biotechnology in the Feed Industry; Proceedings Alltech 7th Annual Symposium, p. 133 (1991)). In addition, phytate contains essential trace elements such as zinc and produces nutrient deficiencies such as growth retardation and mental retardation in children who eat mainly plant-based foods without elimination of phytate.

Two phytases, phyA and phyB, have been cloned and sequenced from Aspergillus niger NRRL3135 (Ehrlich, KC et al., "Identification and Cloning of a Second Phytase Gene (phys) from Aspergillus niger (ficuum)", Biochem. Biophys Res. Commun., 195: 53-57 (1993), Piddington, CS et al., "The Cloning and Sequencing of the Genes Encoding Phytase (phy) and pH 2.5-optimum Acid Phosphatase (aph) from Aspergillus niger var. awamori ”, Gene, 133: 56-62 (1993)). Recently, new phytase genes have been isolated from Aspergillus terreus and Myceliophthora thermophila (Mitchell et al., "The Phytase Subfamily of Histidine Acid Phosphatases: Isolation of Genes for Two Novel Phytases From the Fungi Aspergillus terreus and Myceliophilamobiology thermobiology" , 143: 245-252, (1997)), Aspergillus fumicatus (Pasamontes et al., "Gene Cloning, Purification, and Characterization of a Heat-Stable Phytase from the Fungus Aspergillus fumigatus", Appl. Environ. Microbiol., 63: 1696-1700 (1997)), Emericella nidulans and Talaromyces thermophilus (Pasamontes et al., "Cloning of the Phytase from Emericella nidulans and the Thermophilic Fungus Talaromyces thermophilus", Biochim. Biophys. Acta., 1353: 217 -223 (1997)), and corn (Maugenest et al., "Cloning and Characterization of a cDNA Encoding a Maize Seedling Phytase", Biochem. J., 322: 511-517 (1997)).

Various types of phytase enzymes have been isolated and / or purified from Enterobacter sp. 4 (Yoon et al., "Isolation and Identification of Phytase-Producing Bacterium, Enterobacter sp. 4, and Enzymatic Properties of Phytase Enzyme", Enzyme and Microbial Technology 18: 449-454 (1996)), Klebsiella terrigena (Greiner et al. , "Purification and Characterization of a Phytase from Klebsiella terrigena", Arch. Biochem. Biophys. 341: 201-206 (1997)), and Bacillus sp. DS11 (Kim et al., "Purification and Properties of a Thermostable Phytase from Bacillus sp. DS11", Enzyme and Microbial Technology 22: 2-7 (1998)). The properties of these enzymes have been studied. Furthermore, the crystal structure of Aspergillus ficuum phyA has been reported (Kostrewa et al., "Crystal Structure of Phytase from Aspergillus ficuum at 2.5 A Resolution", Nature Structure Biology 4: 185-190 (1997)).

Hartingsveldt et al. introduced the phyA gene into A. niger and obtained a ten-fold increase in phytase activity, compared to the wild type ("Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger", Gene 127: 87-94 (1993)). Supplemental microbial phytase from this source in pig and poultry diets has been shown to be effective in enhancing zinc and phytate P utilization (Simons et al., "Improvement of Phosphorous Availability By Microbial Phytase in Broilers and Pigs ", Br. J. Nutr., 64: 525 (1990); Lei, XG et al.," Supplementing Corn-Soybean Meal Diets With Microbial Phytase Linearly Improves Phytate P Utilization by Weaning Pigs ", J. Anim. Sci ., 71: 3359 (1993); Lei, XG et al., "Supplementing Corn-Soybean Meal Diets With Microbial Phytase Maximizes Phytate P Utilization by Weaning Pigs", J. Anim. Sci., 71: 3368 (1993); Cromwell, GL, et al., “P- A Key Essential Nutrient, Yet a Possible Major Pollutant- Its Central Role In Animal Nutrition”, Biotechnology in the Feed Industry “, Proceedings Alltech 7^th Annual Symposium ”, p. 133 (1991)). However, the expense of the limited supply of commercially available phytase and the instability of the enzyme's activity under the heat of pelletization of feed precludes its practical use in the animal industry (Jongbloed, AW et al., “Effect of Pelleting Mixed Feeds on Phytase Activity and Apparent Absorbability of Phosphorous and Calcium in Pigs ”, Animal Feed Science and Technology, 28: 233-242 (1990)). Furthermore, the phytase produced from A. Niger is not allegedly the safest source for the manufacture of human food.

Yeast can be used to efficiently produce enzymes while growing in simple and inexpensive media. With an appropriate signal sequence, the enzyme can be secreted into the media for convenient harvesting. Some yeast expression systems have the added benefit of being well accepted in the food industry and are safe and efficient producers of food products.

IS 2 255 281 T3

Pichia pastoris is a methylotrophic yeast capable of metabolizing methanol as its only carbon source. This system is well known for its ability to express high levels of heterologous proteins. Because it is a eukaryote, Pichia has many of the advantages of higher eukaryotic expression systems such as protein processing, folding, and post-transcriptional modification.

Therefore, there is a need to develop an efficient and simple system for economically producing phytase for application to the food and feed industry.

Summary of the invention

The present invention relates to a method for the production of phytase in yeast that comprises providing an appA gene isolated from bacterial cells, said appA gene encoding a protein or polypeptide with phytase activity, expressing said appA gene in a yeast strain and isolating the expressed protein or polypeptide, wherein said protein or polypeptide has increased thermostability compared to said protein or polypeptide expressed in a non-yeast host cell.

The present invention also relates to a protein or polypeptide obtainable by said method and having phytase activity, with an optimal activity in the temperature range of 57-65 ° C at a pH of 2.5 to 3.5 or 5 ,5. The optimum pH at 2.5 to 3.5 is particularly important for phytase because that is the pH of the stomach of animals.

The invention further provides a yeast strain comprising an appA gene isolated from bacterial cells, said appA gene encoding a protein or polypeptide with phytase activity and which is functionally linked to a promoter capable of expressing phytase in yeast, wherein said protein or polypeptide has increased thermostability when expressed in a yeast host cell compared to said protein or polypeptide expressed in a non-yeast host cell.

Still another aspect of the invention is a vector comprising an appA gene isolated from bacterial cells, said gene encoding a protein or polypeptide with phytase activity; a promoter functionally linked to the appA gene, said promoter being capable of initiating transcription in yeast; and an origin of replication capable of maintaining the vector in yeast, wherein said protein or polypeptide has increased thermostability when expressed in a yeast host cell compared to said protein or polypeptide expressed in a non-yeast host cell. .

The invention also provides a method of producing a protein or polypeptide having phytase activity, which comprises providing an appA gene isolated from bacterial cells, said gene encoding a protein or polypeptide with phytase activity, expressing said gene in a host cell, in that said host cell is a yeast strain; and isolating the expressed protein or polypeptide.

The invention also includes a method of converting phytate to inositol and inorganic phosphate. The appA gene isolated from bacterial cells expresses a protein or polypeptide with phytase activity in a host cell. The protein or polypeptide is then contacted with phytate to catalyze the conversion of phytate to inositol and inorganic phosphate.

Brief description of the drawings

Figure 1 shows a PAGE-SDS analysis of soluble protein prepared from E. coli transformed with the IPTG-induced phytase gene. Cells were grown 4 hours before harvesting. Lane 1: marker; Lanes 2 and 3: pEP1 transformants (protein expressed was approximately 55 kDa); lane 4: transformant with only the expression vector pET25b (+).

Figure 2 shows Western blot analysis of phytase protein expressed in E. coli. The antibody was raised against purified native phytase from A. niger. Each lane contained 50 pg of total intracellular protein. Lanes 1 and 2: recombinant after and before induction; lanes 3 and 4: control (expression vector only) after and before induction.

Figure 3 is a Northern blot analysis scan image of PhyA mRNA in E. coli. A 1.4 kb PhyA probe was used. Each lane contained 20 pg of total RNA. Lanes 1 and 2: RNA isolated from control cells (expression vector only) before and after induction; Lanes 3 and 4: RNA isolated from the PhyA-containing recombinants before and after induction.

Figure 4 is a time course of induced expression of phytase (pEP1) in E. coli BL21 (DE3). Cells were induced when DO₆₀₀ reached 0.5. The soluble protein, prepared at each moment, was quantified by SDS-PAGE analysis.

Figure 5 shows a PAGE-SDS analysis of extracellular phytase protein expressed by S. lividans transformed with phytase after growing for 72 hours. Cells were centrifuged for 15 minutes at 8,000 xg, and the supernatant was subjected to gel electrophoresis. Lane 1: marker; lane 2: control with only the expression vector; Lane 3: the positive colony expressed phytase and the size was approximately 57 kDa.

IS 2 255 281 T3

Figure 6 shows Western blot analysis of S. lividans expressed phytase using a phytase antibody raised against purified native A. niger phytase. Each lane was loaded with 20 pg of protein medium (supernatant). Lane 1: vector transformed control cell culture supernatant; lane 2: culture supernatant inoculated with the positive colony.

Figure 7 represents a PAGE-SDS analysis of the extracellular phytase expressed by S. cerevisiae. Each lane was loaded with 50 pg of protein medium (supernatant). Lanes 1 to 3: culture supernatant inoculated with the positive colony collected at 5, 10 and 25 hours after induction, respectively; lanes 4 to 6: vector-transformed control cell culture supernatant collected at 5, 10, and 25 hours after induction, respectively; lane 7: marker (kDa). The expressed phytase was approximately 110 kDa (confirmed by Western blotting).

Figure 8 is a time course of extracellular phytase activity expressed by S. cerevisiae transformed with the pYPP1 construct after induction by galactose. The collected medium supernatant was analyzed for activity.

Figure 9 shows Western blot analysis of extracellular phytase expressed by S. cerevisiae before and after deglycosylation (Endo H), using a phytase antibody raised against purified native A. niger phytase. Lane 1: Prestained SDS-PAGE standards (kDa) from Bio-Rad; lanes 2 and 3: 10 and 20 pg deglycosylated phytase protein, respectively; lane 4: glycosylated phytase (20 pg protein).

Figure 10 is a Northern blot analysis scan image for total RNA isolated from transformed S. cerevisiae cells. Lane 1: control (with only the expression vector pYES2); lanes 2 and 3: transformants of pYPP1.

Figure 11 is a chronological course of extracellular phyA phytase activity produced by Pichia pastoris Mut transformants.^s (KM71) and Mut + (X33) after induction.

Figure 12 represents a PAGE-SDS analysis of the overexpressed phytase in Pichia with the pPICZaAPhyA construct in KM71 (MUT^s). Lane 1: protein scale. Lane 2: 40 µl of AK1 supernatant (one colony showed 21,700 mU / ml extracellular phytase), collected 108 hours after induction. Lane 3: 40 µl of the supernatant from a control strain overexpressing human serum albumin (HAS, 6.7 kDa) at a level of 1 g / l. Lane 4: 40 µl of the KM71 control supernatant.

Figure 13 depicts the effects of Endo H deglycosylation on the thermostability of phytase expressed in Pichia. Phytase activity was measured after heating the enzymes for 15 minutes at 37 ° C or 80 ° C in 0.2M citrate buffer, pH 5.5.

Figure 14 is a Northern scan scan image of the phyA mRNA expressed by the transformed strains of Pichia pastoris (KM71 and X33). A 1.3 kb phyA probe was used for blotting. Lanes 1 and 2: the KM71 transformant before and after induction; Lanes 3 and 4: the X33 transformant after and before induction.

Figure 15 shows the optimum pH of the extracellular phytase expressed by Pichia (X33). 0.2 M glycine-HCl buffers were used for pH 1.5, 2.5, 3.5; 0.2 M sodium citrate for pH 4.5, 5.5, 6.5 and 0.2 M Tris-HCl for pH 7.5 and 8.5.

Figure 16 shows the optimum temperature of the extracellular phytase expressed by Pichia (X33). The assays were performed in 0.2M citrate buffer, pH 5.5.

Figure 17 represents the release of free phosphorus from soybeans by phytase expressed in Pichia (X33). Five grams of soybeans were suspended in 25 ml of 0.2M citrate, pH 5.5, with different amounts of the enzyme. Incubation was carried out for 4 hours at 37 ° C and the phosphorus released in the supernatant was determined.

Figure 18 shows a time course of the expression of extracellular phytase activity of five Pichia pastoris transformants containing the E. coli appA gene.

Figure 19 graphically shows the relationship between the pH of the medium and the expression of phytase activity by Pichia pastoris.

Figure 20 is a PAGE-SDS analysis of E. coli phytase overexpressed in Pichia pastoris. Lane 1: protein scale; Lanes 2 to 4: supernatants collected from the cultures of the positive colonies 23, 22 and 11, respectively, 118 hours after induction.

Figure 21 graphically shows the optimum pH of the E. coli phytase overexpressed by Pichia pastoris.

Figure 22 graphically shows the optimum temperature of the E. coli phytase overexpressed by Pichia pastoris.

IS 2 255 281 T3

Figure 23 shows the amount of free phosphorus released from soybean meal by E. coli phytase overexpressed by Picha pastoris.

Detailed description of the invention

The present invention provides a process for the production of phytase in yeast. According to this method, an appA gene isolated from bacterial cells is expressed in a yeast strain, said appA gene encoding a protein or polypeptide with phytase activity, and the expressed protein or polypeptide is isolated, wherein said protein or polypeptide has improved thermostability compared to that of said protein or polypeptide expressed in a non-yeast host cell.

The enzymes that catalyze the conversion of phytate to inositol and inorganic phosphorus are widely known as phytases. Phytase-producing microorganisms include bacteria such as Bacillus subtilis (Paver et al., J. Bacteriol. 151, 1102 (1982), hereby incorporated by reference) and Pseudomonas (Cosgrove, Austral. J. Biol. Sci. 23: 1207 (1970), hereby incorporated by reference); yeasts, such as Saccharomyces cerevisiae (Nayini et al., Lebensmitttel Wissenschaft und Technologie 17:24 (1984), hereby incorporated by reference) and fungi such as Aspergillus terreus (Yamada et al., Agric. Biol. Chem., 32: 1275 (1986), which is hereby incorporated by reference) and Aspergillus ficuum (van Gorcom et al., European Patent Application 89 / 202,436, which is hereby incorporated by reference).

Phytases are also present endogenously in many plant species. Loewus, in "Plant Biology" vol. 9, "Inositol Metabolism in Plants" (eds. DJ Morre, WF Boss, FA Loewus) 13 (1990); and Gellatly et al., Plant Physiology (supplement) 93: 562 (1990), which are hereby incorporated by reference; cite the isolation and characterization of a phytase cDNA clone obtained from potato tubers. Gibson et al., J. Cell. Biochem., 12C: L407 (1988) and Christen et al., J. Cell Biochem., 12C: L402 (1988), which are incorporated herein by reference, cite endogenous phytase synthesis during soybean germination.

Preferably, the protein or polypeptide with phytase activity is secreted from the cell in the growth media. This allows for higher levels of expression and easier isolation of the product. The protein or polypeptide with phytase activity is coupled to a signal sequence capable of directing the protein outside the cell. Preferably, the signal sequence is excised from the protein.

The appA gene can be spliced ​​in phase with a transcriptional enhancer element.

Proteins encoding the appA gene with phytase activity are isolated from a bacterial cell.

A preferred appA gene is the appA gene isolated from E. coli. The gene, originally defined as the E. coli periplasmic phosphoanhydride phosphohydrolase gene (appA), contains 1,298 nucleotides (GeneBank accession number M58708). The gene was first found to encode a pH optimum 2.5 acid phosphatase protein (EcAP) in E. coli. Acid phosphatase is a monomer with a molecular mass of 44,644 Da. Mature EcAP contains 410 amino acids (Dassa, J. et al., "The Complete Nucleotide Sequence of the Escherichia Coli Gene AppA Reveals Significant Homology Between pH 2.5 Acid Phosphatase and Glucose-1 Phosphatase", J. Bacteriology, 172: 54975500 (1990), which is hereby incorporated by reference) . Ostanin et al. overexpressed appA in E. coli BL21 using a pT7 vector, and increased its acid phosphatase activity by approximately 400 times (440 mU / mg protein) (Ostanin, K. et al., "Overexpression, Site-Directed Mutagenesis, and Mechanism of Escherichia Coli Acid Phosphatase", J. Biol. Chem., 267: 22830-22836 (1992), hereby incorporated by reference). The appA gene product was not previously known to have phytase activity.

The appA gene can be expressed in any prokaryotic or eukaryotic expression system. A variety of host-vector systems can be used to express the protein coding sequence (s). Preferred vectors include a viral vector, plasmid, cosmid, or oligonucleotide. Above all, the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (eg, baculovirus); and plant cells infected with bacteria. The expression elements of these vectors vary in their potency and specificities. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements can be used.

Hosts for expressing appA include yeast species, which can be used as host cells in accordance with the present invention. Preferred yeast host cells include different strains of Saccharomyces cerevisiae. Other yeasts such as Kluyveromyces, Torulaspora and Schizosaccharomyces can also be used. In a preferred embodiment, the yeast strain used to overexpress the protein is Saccharomyces cerevisiae.

In another preferred embodiment of the present invention, the yeast strain is a methylotrophic yeast strain. Methylotrophic yeasts are those yeast genera capable of using methanol as a carbon source for the production of the energy resources necessary to maintain cell function and that contain a gene

ES 2 255 281 T3 for the expression of alcohol oxidase. Typical methylotrophic yeasts include members of the genera Pichia, Hansenula, Torulopsis, Candida, and Karwinskia. These yeast genera can use methanol as the sole carbon source. In a more preferred embodiment, the methylotrophic yeast strain is Pichia pastoris.

The present invention also provides a protein or polypeptide with phytase activity. AppA is expressed in Pichia and Saccharomyces cerevisiae, with the resulting protein having a much higher extracellular activity and a much more preferred optimum pH of 2.5 to 3.5.

A preferred embodiment of the invention is a protein or polypeptide having phytase activity with optimal activity in a temperature range of 57 to 65 ° C. A more preferred embodiment is a protein or polypeptide having phytase activity in which its temperature range for optimal activity is 58 to 62 ° C.

Still another preferred embodiment is a protein or polypeptide having phytase activity in which the protein retains at least 40% of its activity after heating the protein for 15 minutes at 80 ° C. More preferred is a protein or polypeptide having phytase activity in which the protein retains at least 60% of its activity after heating the protein for 15 minutes at 60 ° C.

The purified protein can be obtained by various methods. The protein or polypeptide of the present invention is preferably produced in purified form (preferably at least about 80%, more preferably 90% pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is secreted into the growth medium of recombinant host cells. Alternatively, the protein or polypeptide of the present invention is produced, but not secreted into the growth medium. In such cases, to isolate the protein, the host cell carrying a recombinant plasmid is propagated, lysed by sonication, heat, or heat treatment, and the homogenate is centrifuged to remove cellular debris. The supernatant is then subjected to sequential precipitation with ammonium sulfate. The fraction containing the polypeptide or protein of the present invention is subjected to gel filtration on a dextran or polyacrylamide column of appropriate size to separate the proteins. If necessary, the protein fraction can be further purified by HPLC.

The present invention also provides a yeast strain comprising an appA gene isolated from bacterial cells, said appA gene encoding a protein or polypeptide with phytase activity and being functionally linked to a promoter capable of expressing phytase in yeast, wherein said protein or polypeptide has increased thermostability when expressed in a yeast host cell compared to said protein or polypeptide expressed in a non-yeast host cell.

Still another aspect of the invention is a vector for expressing phytase in yeast. The vector carries an appA gene isolated from bacterial cells, said gene encoding a protein or polypeptide with phytase activity. The appA gene can be cloned into any vector that autonomously replicates or integrates into the yeast genome. The copy number of autonomous replicating plasmids, for example YEp plasmids, may be high, but their mitotic stability may be insufficient (Bitter et al., "Expression and Secretion Vectors for Yeast", Meth. Enzymol., 153: 516-544 (1987), which is hereby incorporated by reference). They may contain the 2 mu plasmid sequence responsible for autonomous replication, and an E. coli sequence responsible for replication in E. coli. The vectors preferably contain a genetic marker for selection of yeast transformants, and an antibiotic resistance gene for selection in E. coli. Episomal vectors containing the ARS and CEN sequences appear as a single copy per cell, and are more stable than YEp vectors. Integrative vectors are used when a DNA fragment is integrated as one or multiple copies into the yeast genome. In this case, the recombinant DNA is stable and does not need selection (Struhl et al., "High-Frequency Transformation of Yeast: Autonomous Replication of Hybrid DNA Molecules", Proc. Natl. Acad. Sci. USA 76: 1035-1039 (1979); Powels et al., "Cloning Vectors I-IV" and following, Elsevier (1985); Sakai et al., "Enhanced Secretion of Human Nerve Growth Factor from Saccharomyces cerevisiae Using an Advanced δ-Integration System", Biotechnology 9: 1382-1385 (1991), which are hereby incorporated by reference). Some vectors have an origin of replication that functions in the selected host cell. Suitable origins of replication include 2 µ, ARS1, and 2 5 µΜ. Vectors have restriction endonuclease sites for insertion of the fusion gene and promoter sequences, and selection markers. Vectors can be modified by removal or addition of restriction sites, or removal of other unwanted nucleotides.

The appA gene can be arranged under the control of any promoter (Stetler et al., "Secretion of Active, Fulland Half-Length Human Secretory Leukocyte Protease Inhibitor by Saccharomyces cerevisiae", Biotechnology 7: 5560, (1989), which is incorporated by the present for reference). A constitutive or regulated yeast promoter can be chosen. Suitable promoter sequences for yeast vectors include, but are not limited to, metallothionein, 3-phosphoglycerate kinase promoters (Hitzeman et al., J. Biol. Chem. 255: 2073 (1980), hereby incorporated by reference) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149 (1968), and Holland et al., Biochem. 17: 4900 (1978), hereby incorporated by reference) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructolanase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate phosphoglucose isomerase and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in EP-A73,657 to Hitzeman, which is hereby incorporated by reference. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al., J. Biol. Chem. 258: 2674 (1982) and Beier et al., Nature 300: 724 (1982), which are hereby incorporated by reference. .

IS 2 255 281 T3

A constitutive or regulated yeast promoter can be chosen. Strong promoters, for example, from the phosphoglycerate kinase (PGK) gene, from other genes encoding glycolytic enzymes, and from the alpha factor gene, are constitutive. When a constitutive promoter is used, the product is synthesized during cell growth. The ADH2 promoter is regulated with ethanol and glucose, the GAL-1-10 and GAL7 promoters with galactose and glucose, the PHO5 promoter with phosphate, and the metallothionin promoter with copper. Heat shock promoters, to which the HSP150 promoter belongs, are regulated by temperature. Hybrid promoters can also be used. A regulated promoter is used when continuous expression of the desired product is harmful to host cells. Instead of yeast promoters, a strong prokaryotic promoter such as the T7 promoter can be used, but in this case the yeast strain has to be transformed with a gene encoding the respective polymerase. For termination of transcription, the HSP150 terminator, or any other functional terminator, is used. Here, promoters and terminators are called control elements. The present invention is not limited to any specific vector, promoter, or terminator.

The vector can also carry a detectable marker. Detectable markers are often antibiotic resistance genes or genes capable of complementing yeast strains that have well characterized metabolic deficiencies such as tryptophan or histidine deficient mutants. Preferred detectable markers include URA3, LEU2, HIS3, TRP1, HIS4, ARG4, or antibiotic resistance genes.

The vector may also have an origin of replication capable of replication in a bacterial cell. Manipulation of vectors is more efficient in bacterial strains. Preferred bacterial origins of replication are ColE1, Ori, or oriT.

A leader sequence from yeast or phytase genes or other sources can be used to support the secretion of phytase enzyme expressed in the medium. The present invention is not limited to any specific type of leader sequence or signal peptide.

Suitable leader sequences include the yeast factor alpha leader sequence, which can be used to direct the secretion of phytase. The alpha factor leader sequence is often inserted between the promoter sequence and the structural gene sequence (Kurjan et al., Cell 30: 933 (1982); Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330 (1984); US Patent No. 4,546,082 and European Published Patent Application No. 324,274, which are hereby incorporated by reference). Another suitable leader sequence is S. cerevisiae MF alpha 1 (alpha factor), synthesized as a 165 amino acid preproform, comprising a 19 amino acid signal peptide or prepeptide followed by a 64 amino acid "leader" peptide or propeptide, comprising three N-linked glycosylation sites followed by (LysArg (Asp / Glu, Ala) 2-3 alpha factor) 4 (Kurjan et al., Cell 30: 933-943 (1982), hereby incorporated by reference) . The signal-leader part of preproMF alpha 1 has been used extensively to obtain the synthesis and secretion of heterologous proteins in S. cerevisiae. The use of homologous yeast signal / leader peptides is known from US Patent No. 4,546,082, European Patent Application Nos. 116,201, 123,294, 123,544, 163,529 and 123,289, and DK Patent Application No. 3614/83, which are hereby incorporated by reference. In European Patent Application No. 123,289, which is hereby incorporated by reference, the use of the factor a precursor of S. cerevisiae, while WO 84/01153, which is hereby incorporated by reference, indicates the use of invertase signal peptide from Saccharomyces cerevisiae, and German patent application DK 3614/83, which is hereby incorporated as Reference indicates the use of the signal peptide PH05 from Saccharomyces cerevisiae for the secretion of foreign proteins.

Saccharomyces cerevisiae alpha factor leader signal (MF alpha 1 or MF alpha 2) can also be used in a secretion process of heterologous proteins expressed in yeast (US Patent No. 4,546,082, European Patent Applications Nos. 16,201, 123,294, 123,544 and 163,529, which are hereby incorporated by reference). By condensing a DNA sequence encoding the MF alpha 1 signal / leader sequence of S. cerevisiae at the 5 'end of the gene, secretion of the desired protein and processing of the desired protein were demonstrated. The use of mouse salivary amylase signal peptide (or a mutant thereof) to provide secretion of heterologous proteins expressed in yeast has been described in published PCT application Nos. WO 89/02463 and WO 90/10075, which are incorporated hereby for reference.

US Patent No. 5,726,038 describes the use of yeast aspartic protease 3 signal peptide, which is capable of providing enhanced secretion of yeast expressed proteins. Other suitable leader sequences are known to those of skill in the art to facilitate secretion of recombinant polypeptides from yeast hosts. A leader sequence near its 3 'end can be modified to contain one or more restriction sites. This will facilitate the fusion of the leader sequences with the structural gene.

Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929 (1978), which is hereby incorporated by reference. The Hinnen et al. selects Trp transformants on a selective medium, in which the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 jug / ml adenine, and 20 jug / ml uracil .

The appA gene can be maintained in a stable expression vector, an artificial chromosome, or by integration into the yeast host cell chromosome. Integration into the chromosome can be achieved by cloning the appA gene into a vector that will recombine into a yeast chromosome. The right vectors

ES 2 255 281 T3 can include nucleotide sequences that are homologous to nucleotide sequences on the yeast chromosome. Alternatively, the appA gene can be located between recombination sites such as transposable elements, which can mobilize the gene on the chromosome.

The present invention also provides a method of phytase production by providing an appA gene isolated from bacterial cells that encodes a protein or polypeptide with phytase activity, and by expressing the gene in a host cell that is a yeast strain. Preferably, the appA gene is isolated from Escherichia coli. Preferred yeast strains are Saccharomyces, Klyuveromyces, Torulaspora and Schizosaccharomyces, in particular the yeast strain Saccharomyces cerevisiae.

A method of converting phytate to inositol and inorganic phosphorus is also provided. An appA gene is isolated from bacterial cells using techniques well known in the art. A protein or polypeptide with phytase activity is then expressed from the gene in a host cell. The resulting protein or polypeptide is mixed with or contacted with phytate. This technique is especially useful for treating phytate in food or animal feed.

The preferred appA gene is isolated from Escherichia coli.

Although the phytase enzyme produced in a yeast system released P from phytate from corn and soybeans as efficiently as currently commercial phytase, it appeared to be more heat stable. This yeast phytase overexpression system can be used to provide thermostable phytase for use in the food and feed industries.

Examples

Example 1

Materials and methods for the overexpression of PhyA in E. coli, S. lividans and a Saccharomyces system. Phytase gene, host strains, and expression plasmids

The phytase gene, phyA, was kindly provided by USDA's Dr. EJ Mullaney. The gene (GenBank accession number M94550) was contained in plasmid pMD4.21 in E. coli strain HB101. A 2.7 kb SphI fragment of A. niger DNA contained the deglycosylated phytase coding region and its 5 'and 3' flanking sequences. The plasmid containing the signal peptide sequence, Spxy, of the Aureobasidium pullulans active xylanase gene (GenBank accession number U10298) was kindly provided by Dr. XL Li of the University of Georgia. E. coli strain DH5a was used as the initial host for all recombinant plasmids. To express phyA in E. coli, the expression vector pET25b (+) (Novagen, Madison, WI) and the expression host BL21 (DE3) pLysS were used. To express phyA in TK24 from S. lividans, the plasmid pSES1 (Jung, ED, et al., "DNA Sequences and Expression in Streptomyces Lividans glucanase Gene and an Endoglucanase Gene from Thermomonospora Fusca", Appl. Environ. Microbiol. 59 : 3032-3043 (1993), hereby incorporated by reference) to construct the shuttle plasmid (from Dr. DB Wilson of Cornell University, and obtained from Dr. DA Hopwood, John Innes Institute, Norwich; England). To express phyA in yeast, the expression vector pYES2 and the host strain INVScl S. cerevisiae (Invitrogen, San Diego, CA) were used.

Plasmid module constructions and transformations. All constructed plasmids and corresponding hosts are listed in Table 1. A 1.4 kb PCR fragment of the phyA gene was amplified from pMD4.21 using two primers: upstream 5'-CGG AAT TCG TCA CCT CCG GAC T-3 '(SEQ ID NO.1) and downstream 5 '-CCC AAG CTT CTA AGC AAA ACA CTC-3' (SEQ ID NO 2). The resulting fragment contained the sequence encoding the deglycosylated phytase of A. niger, PhyA, and the EcoRI and HindIII restriction site upstream and downstream, respectively. After purification with the Geneclean II kit (Biol01, Inc., La Jolla, CA), the fragment was inserted into pET25b (+) and the resulting construct pEP1 (6893 bp) was transformed into BL21 (DE3) pLysS after initial confirmation in DH5a cells. Expression was under the control of the T7 promoter, followed by the leader sequence (pel B) encoding 21 amino acids, and phyA. The host transformed only with the vector pET25 (+) was used as a control.

IS 2 255 281 T3

TABLE 1

Expression vectors, constructs and their host strains used in the study

<td>Plasmid</td><td>Host</td><td>Description¹</td><td>Reference²</td>

<td>pET25b (+)</td><td>DH5a and BL21 (DE3) pLysS from E. coli</td><td>Expression vector</td><td>Novagen</td>

<td>pEP1</td><td>BL21 (DE3) pLysS de E. coli</td><td>pET25ba (+) + phyA gene</td><td>This article</td>

<td>pSES2</td><td>DH5a from E. coli and TK24 from S. lividans</td><td>Expression vector</td><td>Jung et al.², 1993</td>

<td>pSPP1</td><td>DH5a from E. coli and TK24 from S. lividans</td><td>PSES2 + Spe2 + phyA</td><td>This article</td>

<td>pYES2</td><td>DH5a from E. coli e INVScl of S. cerevisiae</td><td>Expression vector</td><td>Invitrogen</td>

<td>pYEP1</td><td>DH5a from E. coli e INVScl of S. cerevisiae</td><td>pYES2 + Spe2 + phyA</td><td>This article</td>

<td>pYXP1</td><td>DH5a from E. coli e INVScl of S. cerevisiae</td><td>pYES2 + Spxy + phyA</td><td>This article</td>

<td>pYPP1</td><td>DH5a from E. coli e</td><td>pYES2 + phyA + Sphy</td><td>This article</td>

INVScl of S. cerevisiae ¹ Spe2 is the endoglucanase E2 signal peptide from T. fusca (Wilson, DB "Biochemistry and Genetics of Actinomycete Cellulases", Crit. Rev. Biotechnol., 12: 45-63 (1992), which is hereby incorporated by reference ); Spxy is the xylanase signal peptide from A. pullulans (Li and Ljungdahl, "Cloning, Sequencing and Regulation of a Xylanase Gene from the Fungus Aureobasidium pullulans Y-2331-1", Appl. Environ. Microbiol., 60: 3160-3166 (1994); Li and Ljungdahl, "Expression of Aureobasidium pullulans xynA in, and Secretion of the Xylanase from, Saccharomyces cerevisiae", Appl. Environ. Microbiol., 62: 209-213 (1996), hereby incorporated by reference); and Sphy is the phyA signal peptide from A. niger (Hartingsveldt et al., "Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger", Gene 127: 87-94 (1993), hereby incorporated by reference).

² Jung, ED et al., "DNA Sequences and Expression in Streptomyces Lividans glucanase Gene and an Endoglucanase Gene from Thermonospora Fusca", Appl. Environ. Microbiol., 59: 3032-3043 (1993), hereby incorporated by reference).

The construction of the plasmid for the expression of phyA in S. lividans began with the synthesis of a fragment containing the promoter pLT1 and the signal peptide Spe2 (Lao, G. et al., "DNA Sequences of Three Beta-1,4 -endoglucanase Genes from Thermomonospora Fusca ", J. Bacteriol., 173: 3397-3407 (1991), hereby incorporated by reference) by PCR. One upstream primer, 5'-CAG CTA TGA CCA TGA TTA CGC C-3 '(SEQ ID NO.3) and one downstream primer 5'-CCT AGA ACG GGA ATT CAT TGG CCG CC-3' (SEQ ID NO. ID 4) contained PstI and EcoRI restriction sites, respectively. The fragment was amplified from pBW2 (Jung. ED et al., "DNA Sequences and Expression in Streptomyces lividans of an Exoglucanase Gene and an Endoglucanase Gene From Thermomonospora Fusca", Appl. Environ. Microbiol., 59: 3032-3042 (1993), hereby incorporated by reference), and then digested with PstI and EcoRI, while the pEP1 construct and the pBluescript SK + plasmid (Stratagene, La Jolla, CA) were digested with EcoRI and HindIII, and PstI and HindIII, respectively. The three digested fragments were subsequently purified using the Geneclean II kit and ligated into a single recombinant construct containing the desired PstI and KpnI restriction sites (from pBluescript SK +), the pLT1 promoter, and the endoglucanase E2 Spe2 leader peptide ( 551 bp, Lao, G. et al., "DNA Sequences of Three Beta-1,4-endoglucanase Genes from Thermomonospora Fusca", J. Bacteriol., 173: 3397-3407 (1991), hereby incorporated by reference) and the phyA gene (1365 bp). After digesting the construct with PstI and KpnI, the resulting fragment was inserted into the expression vector pSES1, and the formed shuttle plasmid (pSPP1, 9131 bp) was transformed into protoplasts of the host S. lividans according to Hopwood et al. (Hopwood. DA et al., "Genetic Manipulation of Streptomyces-A Laboratory Manual", John Innes Foundation, Norwich, England (1985), hereby incorporated by reference). Likewise, a control was prepared by transforming S. lividans with the expression vector pSES2.

IS 2 255 281 T3

Three shuttle plasmids with three different signal peptide sequences were constructed to express phyA in the yeast system (see Table 2). The first plasmid originated from a HindIII-digested fragment of pSPp1, including the pLT1 promoter, the Spe2 leader sequence, and the phyA coding region sequence. The fragment was ligated to the HindIII site of pYES2 treated with calf intestinal alkaline phosphatase, and the plasmid was named pYEP1 (7783 bp) after confirming its correct orientation. The second plasmid contained Spxy, a signal peptide sequence of the A. pullulans xylanase gene (Li, XL et al., "Cloning, Sequencing and Regulation of a Xylanase Gene From the Fungus Aureobasidium pullulans) Y-2311-1", Appl. Environ. Microbiol., 60: 31603166 (1994); Li, XL et al., "Expression of Aureobasidium pullulans XynA in, and Secretion of the Xylanase From, Saccharomyces cerevisiae", Appl. Environ. Microbiol., 62: 209-213 (1996), hereby incorporated by reference), and the phyA gene. Spxy was spxy with phyA by overlap extension (Horton, RM, "In vitro Recombination and Mutagenesis of DNA: SOEing Together Tailor-Made Genes", PCR Protocols: Currents Methods and Applications, 251-261 (1993), which is incorporated by present for reference) with two successive PCR steps. One was to amplify the Spxy sequence from pCE4 (Li, XL et al., "Expression of Aureobasidium Pullulans XynA in, and Secretion of the Xylanase From, Saccharomyces cerevisiae", Appl. Environ. Microbiol. 62: 209-213 (1996), which is hereby incorporated by reference) using the upstream primer (5'-CCC AAG CTT Gat CAC ATC CAT TCA -3 ') (SEQ ID NO: 5) with a HindIII restriction (primer 1) and an overlapping downstream primer (5'-CGG GGA CTG CTA GCG CAC GTT CGA T-3 ', primer 2) (SEQ ID NO: 6). The other PCR was to amplify the phyA coding region of pEP1 using the overlapping upstream primer (5'- ATC GAA CGT GCG CTA GCA GCA GTC CCC G -3 ', primer 3) (SEQ ID NO 7) and the downstream primer (5'- GCT CTA GAC TAA GCA AAA CAC TCC-3 ', primer 4) (SEQ ID NO: 8) with an XbaI restriction site. The second PCR step was performed to fuse the two fragments generated by the two previous PCRs using the two purified fragments as templates and primers 1 and 4. The resulting fragment contained the HindIII and XbaI restriction sites and was cloned into pSES2. This plasmid was named pYXP1 (7219 bp). The third plasmid contained the signal peptide (Sphy) sequence of phyA and the coding region of phyA, excluding the intron between them (Hartingsveldt, W. van., et al., "Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger", Gene 127: 87-94 (1993), which is hereby incorporated by reference). Two primers were used, including 70 bp of the upstream primer containing the signal peptide with an engineered KpnI restriction site and a downstream primer that was the same used for the construction of pYXP1 (primer 4) to amplify the fragment. desired from pEP1. The PCR product was digested with KpnI and XbaI and cloned into pSES2, resulting in a plasmid named pYPP1 (7176 bp). The three above constructs were transformed into S. cerevisiae by the method of Ito et al., "Transformation of Intact Yeast Cells Treated with Alkali Cations", J. Bacteriol., 153: 163-168 (1983), which is incorporated by the present for reference.

TABLE 2

Signal peptides used for the expression of phyA in S. cerevisiae

<td>Construct size (bp)</td><td>Peptide</td><td>Gen</td><td>Organism</td><td>Phytase activity¹(mUF / ml)</td>

<td>pYEP1 7783</td><td>Spe2 (93 bp)</td><td>Cellulase E2</td><td>T. fusca</td><td> 0,80</td>

<td>pYXP1 7219</td><td>Spxy (102 bp)</td><td>Xylanase A</td><td>A. pullulans</td><td>It did not detect.</td>

<td>pYPP1 7176</td><td>Sphy (57 bp)</td><td>PhyA of phytase</td><td>A. niger</td><td> 146</td>

<td>pSES1² 7224</td><td></td><td></td><td>S. cerevisiae</td><td>It did not detect.</td>

¹ Phytase activity was detected in the cell culture supernatant of Sabouraud-raffinose medium 15 hours after induction by adding galactose. See text for definition of phytase units.

² Expression vector for S. cerevisiae, used as a control.

Growth medium and induction of gene expression. Transformants were grown in the E. coli system in 50 ml of LB medium containing 50 µng / ml of ampicillin at 30 ° C. After the DO value₆₀₀ of the medium reached 0.5 to 0.6, the phytase gene expression was induced by adding IPTG (isopropyl bD-thiogalactopyranoside) to the medium at a final concentration of 1 mM. Three hours after induction, cells were harvested by centrifugation at 8,000 xg for 15 minutes, washed with 1 x PBS, and lysozyme lysed. Soluble and insoluble cell fractions were prepared and a sample containing 500 pg of total protein (Lowry, OH et al, "Protein Measurement With the Folin Phenol Reagent", J. Biol. Chem., 193: 265-275 (1951), hereby incorporated by reference) in the same volume of 2 x SDS buffer, and analyzed by SDS-PAGE (Laemmli, UK, "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4", Nature (London), 227: 680-685 (1970), hereby incorporated by reference).

Recombinant S. lividans was grown in TSB broth with 5 ng / ml of thiostrepton at 30 ° C (Jung, ED et al., "DNA Sequences and Expression in Streptomyces lividans of an Exoglucanase Gene and an Endoglucanase Gene from

IS 2 255 281 T3

Thermomonospora Fusca ”, Appl. Environ. Microbiol., 59: 3032-3043 (1993), hereby incorporated by reference). After 72 hours of incubation, cells and medium were harvested and prepared for SDS-PAGE (Wilson, DB "Biochemistry and Genetics of Actynomycete Cellulases", Crit. Rev. Biotechnol., 12: 45-63 (1992), which is hereby incorporated by reference).

The S. cerevisiae transformants were initially grown in Sabouraud-raffinose medium (4%) (100 ml) without uracil for 48 hours, then sterile galactose was added to the medium (2%) to induce phytase expression. Cell and medium samples were collected at various time points, and extracellular and intracellular samples were prepared as described by Li and Ljungdahl, "Expression of Aureobasidim pullulans xynA in, and Secretion of the Xylanase from, Saccharomyces cerevisiae", Appl. Environ. Microbiol., 62: 209-213 (1996), which is hereby incorporated by reference. When necessary, the supernatant from the cell culture fractions expressed with stirred cells from Amicon (Beverly, MA) was concentrated using YM10 membranes (MW cutoff 10,000). Other means were tried accordingly.

Enzyme protein and activity assay. The amounts of phytase protein expressed under various conditions were quantified by relative densitometry of specific bands on SDS-PAGE, using the IS-1000 Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA). Phytase activity in media and cell samples was determined as described above (Piddington, CS et al., “The Cloning and Sequencing of the Genes Encoding Phytase (phy) and pH 2.5-optimum Acid Phosphatase (aph) from Aspergillus niger var. awamori ", Gene, 133: 56-62 (1993), hereby incorporated by reference) and released inorganic phosphate was assayed by the method of Chen, PS et al.," Microdetermination of P ", Anal. Chem., 28: 1756-1758 (1956), which is hereby incorporated by reference. One phytase unit (UF) was defined as the amount of enzyme that liberates 1 // mole of inorganic phosphate from sodium phytate per minute at 37 ° C.

Western blot analysis (immunoblot). The soluble fraction of the phytase transformed E. coli cell mass and the supernatant from the S. lividans and S. cerevisiae transformant medium were collected as for SDS-PAGE. After electrophoresis, the proteins were transferred to Protran® nitrocellulose membrane (Schleicher &amp; Schuell, Keene, NH, USA) in 20 mM Tris-HCl (pH 8.3), 20% methanol and 0.1 % SDS, using a Mini Trans-Blot cell (Bio-Rad Laboratories). The transfer was carried out overnight at constant 50 V, and the initial buffer temperature was 4 ° C. The membranes were then subjected to Western blot analysis. Rabbit polyclonal IgG (kindly provided by USDA Dr. AHJ Ullah. Dilution: 1: 5,000) against purified native A. niger phytase was used as the first antibody. The blot was completed using the Immuno-Blot Assay kit (Bio-Rad Laboratories) containing a second antibody conjugated to horseradish peroxidase.

Isolation and analysis of total RNA. Total RNA was isolated with TRIzol ™ reagent (GIBCO BRL, Gaithersburg, MD) from E. coli and S. cerevisiae transformants 3 and 15 hours after induction, respectively. RNA samples (10 pg per lane) were then separated by formaldehyde-agarose gel electrophoresis (1.5% w / v) and transferred to Hyblot membranes (National Labnet, Woodbridge, NJ) (Davis et al. , "Basic Methods in Molecular Biology", 2^to ed., Ed. Appleton and Lange, Norwalle, CT. (1994), which is hereby incorporated by reference). A 1.4 kb EcoRI-HindIII fragment was prepared in plasmid pEP1 and randomized primer labeled with³²P using a DNA labeling kit followed by G-50 column purification (Pharmacia Biotech., Piscataway, NJ), and then hybridized to the blotted RNA membranes in a hybridization oven (Hybaid, Middlesex, UK). Hybridized membranes were exposed for examination on Fuji Imaging Plate and analyzed by Bio-Imaging Analyzer (Kohshin Graphic Systems, Fuji, Japan).

Example 2

PhyA expression in E. coli

Four hours after induction, a specific band (~ 55 kDa) was observed in SDS-PAGE (12.5%) of the soluble cell fraction, compared to the control transformed with expression vector only (see Figures 1 and 2). This band represented 3.8% of the total soluble protein in this fraction. Correspondingly, Northern analysis showed phyA mRNA overexpression in these phytase gene transformants and no signal was observed in control cells (see Figure 3).

To optimize protein phytase expression, the time course and the effects of a number of factors on expression were studied. These factors included incubation temperature (30 and 37 ° C), pH of the medium (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0), anaerobiosis (add oil sterile mineral on growing cells), the level of inorganic phosphate in the medium (Dassa, E. et al., “The Acid Phosphatases with Optimum pH of 2.5 of Escherichia coli.” J. Bio. Chem., 257: 6669-6676 (1982), which is hereby incorporated by reference) and sodium phytate (0, 0.1, 0.2.0.3, 0.4 and 0.5 mM) . The results indicated that phytase protein expression accumulated linearly with time during the first six hours after induction (see Figure 4). Thereafter, the expression remained relatively unchanged although the bacterial cells continued to grow. Only the pH of the medium and the concentration of sodium phytate significantly affected the expression of protein phytase. Maximum protein was shown at pH 6.0 and 0.3 mM sodium phytate, where the phytase protein increased from 3.8 to 9.6% of the total soluble protein.

IS 2 255 281 T3

No extracellular or intracellular phytase activity was observed. This may not be completely unexpected because native A. niger phytase is a glycoprotein with a size of 70-80 kDa (Hartingsveldt et al., "Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger" , Gene 127: 87-94 (1993), which is hereby incorporated by reference). The protein expressed in the E. coli system of this study was approximately 55 kDa in size. Presumably, the lack of glycosylation of the protein and any other necessary post-translational modification during secretion would preclude phytase activity.

Example 3

PhyA expression in S. lividans

Heterologous genes have been expressed in S. lividans, and the resulting products have been secreted into the medium with enzymatic activity (Ghangas, GS et al., "Cloning of the Thermomonospora Fusca Endoglucanase E2 Gene in Streptomyces lividans: Affinity Purification and Functional Domains of the Cloned Gene Product ", Appl. Environ. Microbiol., 54: 2521-2526 (1988); Wilson, DB," Biochemistry and Genetics of Actinomycete Cellulases ", Crit. Rev. Biotechnol. 12: 45-63 (1992); Jung, ED et al., "DNA Sequences and Expression in Streptomyces lividans of an Exoglucanase Gene and an Endoglucanase Gene from Thermomonospora fusca", Environ. Microbiol., 59: 3032-3043 (1993), which are hereby incorporated by reference). Similarly, the phyA gene was expressed in S. lividans and the protein was introduced into the medium, as shown by a specific band in the medium samples analyzed by PAGeSDS (see Figures 5 and 6). This suggested that the signal peptide of the T. fusca endoglucanase E2 gene was capable of driving the phytase protein out of the cell. This protein was 57 kDa and represented 16.2% of the total protein in the medium. Changing the pH of the medium to 6.0 and adding 0.3 mM sodium phytate to the medium improved the protein yield to 20.3% of the total protein. Because the phytase protein was secreted into the medium at such a high level, it should be easy to purify and effectively use for a variety of purposes such as producing phytase antibody. Again, no increased phytase activity was found either in the medium or in the lysed cells. Although the protein size increased a little (2-3%), compared to that expressed in E. coli, presumably due to glycosylation of the phytase protein in this expression system, there was still no phytase activity.

Example 4

PhyA expression in S. cerevisiae

Three different signal peptides were used to compare the efficiency in driving the expressed protein out of cells (see Table 2). Phytase activity increased substantially in the Sabouraud-raffinose medium in which the transformants of pYEP1 and pYPP1 grow, but not pYXP1. Visible phytase protein was shown by PAGESDS 20 hours after induction (Figure 7).

The expression of transformants of pYEP1 and pYPP1 was determined in three different types of medium: Sabouraudraffinose (Li, XL, et al., "Expression of Aureobasidium pullulans XynA in, and Secretion of the Xylanase From, Saccharomyces cerevisiae", Appl. Environ. Microbiol., 62: 209-213 (1996), hereby incorporated by reference), Sabouraud-glycerol, and a modified general purpose YPED medium. As for the transformants of pYEP1, a similar phytase activity was expressed in Sabouraud-raffinose and Sabouraud medium, but there was no activity detected in YPED medium. In contrast, the phytase activity in the medium grown with pYPP1 transformants varied greatly with the different types of medium. The activity was enhanced to 375 mU / ml when Sabouraud-glycerol medium was used. The activity increased further to 1675 mU / ml when the medium was changed to YEPD (see Table 3). Although YEPD medium was much cheaper than Sabouroaud-raffinose medium, the phytase yield increased more than tenfold. Thus, the putative signal peptide of the fungal phytase gene achieved the most efficient expression of extracellular phytase activity. Almost all of the protein produced was secreted in YEPD medium, because very little activity was detected in yeast cells. The chronological course of phytase expression in this system was shown in Figure 8.

TABLE 3

Phytase activity expressed from transformant with pYPP1 in different media Hours after induction (mUF / mlfi

<td>Half</td><td></td><td> 0</td><td> 10</td><td> 15</td>

<td></td><td>Sabouraud-raffinose</td><td> 22</td><td> 136</td><td> 146</td>

<td></td><td>Sabouraud-glycerol</td><td> 6</td><td> 174</td><td> 375</td>

<td></td><td>YEPD</td><td> 18</td><td> 1238</td><td> 1675</td>

¹ Phytase activity was detected in the cell culture supernatant of the three media 0, 10 and 15 hours after induction by adding galactose. See text for definition of phytase units

IS 2 255 281 T3

A variety of microorganisms, including bacilli, yeast, and filamentous fungi, have phytase activity, while A. niger strain NRRL3135 produces the highest activity (340 mU / ml, Shieh, TR et al., “Survey of Microorganisms for the Production of Extracellular Phytase ", Appl. Environ. Microbiol., 16: 1348-1351 (1968), which is hereby incorporated by reference). CAS 2863 from Schwanniomyces castellii has the highest phytase activity among 21 yeast strains (140 mU / ml, Lambrechts, C. et al., "Utilization of Phytate by Some Yeasts", Biotechnology Letters, 14: 61-66 (1992), which is hereby incorporated by reference). Clearly, the recombinant yeast strain transformed with pYPP1 in the present study produced much higher phytase activity (1,675 mU / ml) than A. niger (4 times) and S. castellii CBS 2683 (11 times). Maximum phytase production in the system can be obtained by optimizing incubation conditions and modifying plasmid modules (Demolder, JW et al., "Efficient Synthesis of Secreted Murine Interleukin-2 by Saccharomyces cerevisiae: Influence of 3'-Untranslated Regions and Codon Usage ", Gene, 111: 207-213 (1992), which is hereby incorporated by reference).

The high level of expression of phytase activity in S. cerevisiae was most likely due to sufficient glycosylation of the phytase protein and other post-translational modifications by yeast. After concentrating the supernatant medium and subject to SDS-PAGE analysis, there was a band of approximately 110 kDa (see Figures 7 and 9), which was larger than the size of the native A. niger protein (Hartingsveldt, W. go. et al., "Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger", Gene 127: 87-94 (1993), hereby incorporated by reference). Northern analysis confirmed the specific overexpression of phyA mRNA (see Figure 10). These results indicated that the yeast system was effective in actively overexpressing the extracellular phytase enzyme. The yeast system has several advantages over bacteria or other systems such as A. niger (Hartingsveldt, W. van. Et al., "Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger", Gene 127: 87-94 (1993), which is hereby incorporated by reference). It carries out post-translational modifications, including proper folding, glycosylation, disulfide bonding, and proteolysis, during the translocation of proteins through the endoplasmic reticulum and cell membrane. Protein secretion is facilitated by short hydrophobic signal peptides in the N-terminal regions of protein precursors (Li, XL et al., "Expression of Aureobasidium pullulans XynA in, and Secretion of the Xylanase From, Saccharomyces cerevisiae", Appl. Environ. Microbiol., 62: 209-213 (1996), hereby incorporated by reference). Proteins secreted by yeast cells are protected from aggregation and degradation by protease. Most importantly, the enzyme proteins produced by S. cerevisiae are easily purified because it secretes only a few proteins. Considering the well-known safety of yeast products for both humans and animals, the system has great potential for the human food and animal feed industry.

Example 5

Properties of PhyA phytase overexpressed in Saccharomyces cerevisiae

Overexpressed phytase from plasmid pYPP1 transformants was concentrated and used to study their properties (see Table 4). The enzyme showed two optimal pH ranges: 2 to 2.5 and 5.0 to 5.5. However, the apH 2 to 2.5 enzyme activity was only 60% of the apH 5 to 5.5 activity. No activity was detected at pH 1 or 8. The optimum pH was virtually the same as for A. phytase. niger (Simons et al., "Improvement of Phosphorous Availability By Microbial Phytase in Broilers and Pigs", Br. J. Nutr., 64: 525 (1990), hereby incorporated by reference), therefore, it would be expected the active role in hydrolysis of P from phytate in the gastrointestinal tracts. The optimum temperature of the enzyme was 60 ° C, while the current on the market produced by Gist-Brocades is 55 ° C (BASF, 1996). More than 80% of the activity remained at 50 to 55 ° C, but little activity was detected at 75 or 80 ° C. When the enzyme was heated for 15 min at 37 ° C and 80 ° C, the remaining activity of the expressed yeast phytase of the present invention was 100 and 63%, respectively, and for the Gist-Brocades phytase it was 100 and 52%, respectively. The differences between the two enzyme sources at any given temperature were significant (see Table 5). Thus, yeast phytase appears to be more heat stable than the current commercial phytase product.

IS 2 255 281 T3

TABLE 4

Characteristics of yeast overexpressed phytase¹ optimum pH²

<td>pH</td><td> 1,0</td><td> 2,0</td><td> 2,5</td><td> 3,0</td><td> 4,0</td><td> 5,0</td><td> 5,5</td><td> 6,0</td><td> 8,0</td>

<td>Exercise</td><td> 0,5^and</td><td> 59,7^c</td><td> 64,8^c</td><td> 48,1^d</td><td> 81,0^b</td><td> 100,0^to</td><td> 95,0^to</td><td> 66,3^c</td><td> 0,8^and</td>

<td>relative (%)</td><td> ±0,2</td><td> ±3</td><td> ±6</td><td> ±4</td><td> ±5</td><td> ±1</td><td> ±6</td><td> ±1</td><td> ±0,4</td>

<td>Optimal temperature³° C</td><td> 25</td><td> 37</td><td> 45</td><td> 50</td><td> 55</td><td> 60</td><td> 75</td><td> 80</td><td></td>

<td>Exercise</td><td> 24,2^and</td><td> 44,6^d</td><td> 63,9^c</td><td> 83,6^b</td><td> 89,8^b</td><td> 100,0^to</td><td> 0,6^F</td><td> 0,9^F</td><td></td>

<td>relative (%)</td><td> ±0,8</td><td> ±3</td><td> ±8</td><td> ±2</td><td> ±4</td><td> ±4</td><td> ±0,1</td><td> ±0,2</td><td></td>

¹ Data are means of relative activity ± standard deviation (n = 4). The means in a row with different superscript letters differ (p &lt;0.05). The general linear model of the statistical analysis system (1988) was used to analyze the effects of the main treatment, while the complete randomized designs and Bonferroni t test were used for the comparison of means of multiple treatments. The level of significance was set at p &lt;0.05.

² Activity was tested at 37^or C (see context for definition of phytase unit). Different buffers were used: 0.2 mM glycine-HCl buffer for pH 1.0 to 3.5; 0.2 mM sodium citrate buffer for pH 4.0 to 6.5; and 0.2 mM Tris-HCl buffer for pH greater than 7.

³ Optimum temperature was determined at pH 5.5 (0.2 mM sodium citrate buffer)

TABLE 5

Comparison of the thermostability of yeast overexpressed phytase and Gist-Brocades phytase produced by A. niger¹·²

<td>Relative activity (%)</td><td></td><td>37 ° C</td><td>80 ° C</td>

<td></td><td>Yeast phytase</td><td> 100^to±1</td><td> 63^b ±1</td>

<td></td><td>Phytase from A. niger</td><td> 100^x±3</td><td> 52^v ±2</td>

<td></td><td>P³&lt;</td><td></td><td> 0,03</td>

¹ Data are means of relative activity ± standard deviation (n = 3). The means in a row with different superscript letters differ (p &lt;0.05). The general linear model of the statistical analysis system (1988) was used to analyze the effects of the main treatment, while the complete randomized designs and Bonferroni's t test were used for the comparison of means of multiple treatments. The level of significance was set at p &lt;0.05.

² The enzyme was heated for 15 minutes at different temperatures before reacting at 37^or C and pH 5.5 ³ Significance (P values) of the t test between the activity of the two phytases at each temperature condition

Although it is not clear how said improvement in thermostability is related to different post-translational modifications (folding, cleavage, glycosylation, etc.). (Li, XL et al., "Expression of AureobasidiumpullulansXynA in, and Secretion of the Xylanase From, Saccharomyces cerevisiae", Appl. Environ. Microbiol., 62: 209-213 (1996), which is hereby incorporated by reference), it is certainly advantageous to have a more thermostable phytase enzyme that can be hopefully resistant to heat during feed pelletization, which is a problem with Current Gist-Brocades phytase.

Example 6

In vitro hydrolysis of P from corn, soybean, and ground wheat phytate by yeast-expressed phytase

Yeast expressed phytase released phytate P from corn and soybean meal as efficiently as Gist-Brocades phytase based on activity per unit (see Table 6). As expected, the hydrolysis of P from phytate was a function of time and activity rate. Yeast expressed phytase was also effective in liberating P from phytate from ground wheat, indicating its great potential in bread fermentation. Because the ground wheat used in this study contained much higher intrinsic phytase activity than the wheat flour commonly used, a much greater effect of yeast-expressed phytase on improving hydrolysis would be expected.

ES 2 255 281 T3 of flour phytate P and in the release of trace elements, when used in a bakery (Hall, MN et al., "The Early Days of Yeast Genetics", Cold Spring Harbor Laboratory Press (1993), which is hereby incorporated by reference).

TABLE 6

Free phosphorus released from corn, soybean (SMB) and ground wheat flour by yeast overexpressed phytase and fungal phytase from A. niger in vitro¹

<td>Yeast phytase (UF / kg)</td><td>of 0</td><td> 100</td><td> 250</td><td> 500</td><td> 1000</td><td>250 (fungal phytase)</td>

<td>Corn: 1 hour</td><td> 0,23^d±</td><td> 0,64^c±</td><td> 1,14^b±</td><td> 1,46^to±</td><td> 1,54^to±</td><td> 1,16^b± 0,15</td>

<td></td><td> 0,03</td><td> 0,08</td><td> 0,18</td><td> 0,04</td><td> 0,04</td><td></td>

<td>4 hours</td><td> 0,36^c ±</td><td> 1,26^b±</td><td> 1,60^to±</td><td> 1,66^to±</td><td> 1,72^to±</td><td>1.68a ± 0.04</td>

<td></td><td> 0,02</td><td> 0,04</td><td> 0,03</td><td> 0,06</td><td> 0,04</td><td></td>

<td>SBM: 1 hour</td><td> 0,68^d±</td><td> 1,18^CD±</td><td> 1,62^c±</td><td> 2,48^b±</td><td> 3,13^to±</td><td> 1,68^c± 0,2</td>

<td></td><td> 0,01</td><td> 0,02</td><td> 0,18</td><td> 0,32</td><td> 0,19</td><td></td>

<td>4 hours</td><td> 0,73^d</td><td> 1,67^c</td><td> 2,69^b</td><td> 3,41^to</td><td> 3,71^to</td><td> 2,78^b</td>

<td>Shredded Wheat: 1 hour</td><td> 3,56±</td><td></td><td> 4,11±</td><td> 4,67²±</td><td></td><td></td>

<td></td><td> 0,39</td><td></td><td> 0,64</td><td> 0,05</td><td></td><td></td>

<td>4 hours</td><td> 5,63±</td><td></td><td> 6,02±</td><td> 6,38²±</td><td></td><td></td>

<td></td><td> 0,5</td><td></td><td> 0,48</td><td> 0,07</td><td></td><td></td>

¹ Each 5 g sample was shaken in 20 ml of 0.2 mM sodium citrate buffer at 37^or C for 1 or 4 hours. The supernatant was obtained by centrifuging for 15 minutes at 8,000 g. After passing through Whatman 541 filter paper, the sample was tested for free P by the method of Chen, PS et al. , "Microdetermination of P", Anal. Chem., 28: 1756-1758 (1956), which is hereby incorporated by reference. The data in the table are means of relative activity ± standard deviation (n = 4). The General Linear Model of the Statistical Analysis System (1988) was used to analyze the effects of the main treatment, while the complete randomized designs and Bonferroni t test were used for the comparison of means of multiple treatments. The level of significance was set at p &lt;0.05. There was a significant difference between 1 and 4 hours for each feed at each dose of enzyme analyzed by t-test. The means in a row with different superscript letters differ (p &lt;0.05).

² n = 2

Aspergillus niger (phyA) phytase overexpression in Escherichia coli, Streptomyces lividans, and Saccharomyces cerevisiae was compared to develop a simple and efficient system for economically producing phytase. A soluble 55 kDa intracellular protein, representing 9.6% of the total soluble protein, was expressed in E. coli using the pET25b (+) system. It was expressed in S. lividans a 57 kDa extracellular protein, representing 20.3% of the total protein in the medium, using a shuttle plasmid containing the pLT1 promoter and the endoglucanase E2 leader peptide SpelI. No increase in phytase activity was shown in any expression system, presumably due to lack of glycosylation and other necessary post-translational modifications. In contrast, high extracellular phytase activity occurred in S. cerevisiae transformed with the phyA gene. Three different signal peptides and three different types of medium were compared to identify the best expression vector and condition. The use of the Sphy signal peptide from the phyA gene and YEPD medium produced the highest extracellular phytase activity. The yeast overexpressed phytase was approximately 110 kDa, had two pH optics: 2.0 to 2.5 and 5.5 to 6.0, and the optimum temperature was 60 ° C.

Example 7

Methods and materials for the expression of phyA in Pichia

Host and vector. A Pichia EasySelect ™ expression kit was purchased from Invitrogen (San Diego, CA). The kit provides hosts and vectors to express the gene intracellularly or extracellularly in Mut + or Mut strains.^s(from use of normal or slow methanol). X33 was used as the Mut + strain and KM71 as the Mut strain.^s. Two vectors were used, pPICZ B (3.3 kb) and pPICZaA (3.6 kb), both use AOX1 as promoter.

Construction of expression vectors. To compare the effect of different signal peptides on PhyA expression in the Pichia system, two constructs were prepared. First, a 1.4 kb EcoRI-KpnI fragment, containing the PhyA sequence encoding the mature phytase protein, was ligated into pPICZaA. In this plasmid

ES 2 255 281 T3 (pPICZa-phyA), PhyA was driven by an alpha factor, a very generally used signal peptide from Saccharomyces cerevisiae. Second, a 1.4 kb KpnI-XbaI fragment of pYPP1 was ligated into the vector (the coding region of phyA was driven by its own signal peptide, which was very efficient in secreting the phytase expressed in Saccharomyces cerevisiae).

Transformation and expression. Constructs confirmed by PmeI were linearized and transformed into GS115 and KM71 using the EasyComp ™ provided by the kit. Neocin ™ was used to select for positive colonies. After inoculating a single colony in 10 ml of MGY medium and growing to OD₆₀₀ at 2-6 at 30 ° C, cells were harvested by centrifugation and resuspended in 10 ml of MMY medium (containing 0.5% methanol). Samples were collected every 12 or 24 h after induction. Cells were separated from the supernatant and lysed with glass beads in disruption buffer. The supernatant and cells were assayed for phytase activity as described above. SDS-PAGE and Western blotting were performed to determine the size and relative amount of the protein expressed.

Example 8

PhyA phytase activity in Pichia

The expression construct was transformed using alpha factor as a signal peptide for phyA in two Pichia strains. KM71 is a slow-utilization strain of methanol, while X33 is a wild-type Pichia that utilizes methanol efficiently. Examination and incubation were performed in 10 ml shake flasks at 29-30 ° C. For the KM71 transformants, 19 out of 20 harvested colonies had extracellular phytase activity greater than 6 units / ml culture supernatant after induction for 24 hours. Colony # 13 showed the highest activity, 26 units / ml after incubation for 108 hours. For the X33 transformants, all colonies (20/20) were greater than 10 units / ml after induction for 24 hours. One of the colonies (# 101) produced a phytase activity of 65 units / ml of supernatant. A study of the time course of phytase expression in KM71 and X33 was summarized in Figure 11. Despite the difference of these two strains in the use of methanol and, therefore, the ability to express phytase, it was found that the alpha factor was processed correctly by yeast cells. Furthermore, almost all of the expressed protein was secreted into the medium, since no more than 5% of the total activity expressed intracellularly was found.

The effects of inorganic phosphorus and media pH on phytase expression in media (BMGY and BMMY) were studied using a recombinant X33 phyA (# 101). Medium containing 50 mM phosphate produced the highest phytase activity, 66 units / ml at 168 hours after induction. By including 50 mM phosphate in the media, the effect of different pH's of this buffer (3, 4, 5, 6, 7 and 8) on expression was also studied. When the pH was 6, this X33 transformant produced 75 phytase units / ml supernatant. Based on the protein concentration and PAGE-SDS analysis, the protein phytase expression yield was estimated to be between 3 and 4 mg / ml. Example 9

Properties of PhyA phytase expressed in Pichia

Molecular size and deglycosylation of the expressed phytase. After subjecting the medium supernatant inoculated with the phyA transformant to SDS-PAGE, a strong band was observed at approximately 95 kDa (Figure 12). This was almost the only protein observed in the supernatant. The expressed phytase reacted efficiently with the rabbit polyclonal antibody raised against native phytase purified from A. niger. This indicated that the immunoreactivity of the expressed phytase was essentially the same as that of native A. niger phytase. The size was reduced to 50 kDa by deglycosylation using Endo H. The phyA antibody also reacted with deglycosylated phytase. Furthermore, deglycosylation, performed under native conditions, reduced phytase activity by approximately 15%, indicating that glycosylation was important for phytase activity. Furthermore, glycosylation affected the thermostability of the enzymes (Figure 13).

Northern analysis. As shown in Figure 14, a 1.3 kb phyA DNA probe hybridized to mRNA from both KM71 (# 13) and X33 (# 101) induced transformants. Transformants' response was also observed before induction. PhyA expression in this system was probably not strictly controlled at the transcriptional level.

Optimum pH and temperature and hydrolysis of phosphorus defitate. Similar to the A. niger phytase, the expressed phytase had two optimal pH's, 2.5 and 5.5 (Figure 15). The optimum temperature of the expressed phytase was 60 ° C (Figure 16). When the expressed phytase was incubated with soybean samples at 100, 200,400, 800 mU / g of sample at 37 ° C, phosphorus was released in a linear fashion with the dose of phytase (Figure 17).

Example 10

Methods and materials for the overexpression of the appA gene of E. coli in Saccharomyces cerevisiae

Gene and protein. This gene, originally defined as the E. coli periplasmic phosphoanhydride phosphohydrolase gene (appA), contains 1,298 nucleotides (GenBank accession number: M58708). It was found first that the gene

ES 2 255 281 T3 encoded a pH optimum 2.5 acid phosphatase protein (EcAP) in E. coli. Acid phosphatase is a monomer with a molecular mass of 44,644 Da. Mature EcAP contains 410 amino acids (Dassa, J. et al., "The Complete Nucleotide Sequence of the Escherichia Coli Gene appA Reveals Significant Homology Between pH 2.5 Acid Phosphatase and Glucose-1 Phosphatase", J. Bacteriology, 172: 5497-5500 (1990), which is hereby incorporated by reference). Ostanin, K. et al., "Overexpression, Site-Directed Mutagenesis, and Mechanism of Escherichia Coli Acid Phosphatase", J. Biol. Chem., 267: 22830-22836 (1992), hereby incorporated by reference), overexpressed appA in E. coli BL21 using a pT7 vector, and increased their acid phosphatase activity by approximately 400 times (440 mU / mg protein).

The gene and a host E. coli strain CU 1869 (# 47092) were purchased from the ATCC. The gene, a 1.3 kb insert, was transformed into E. coli strain BL21 (# 87441) using an expression vector pAPPA1 (Ostanin, K. et al., "Overexpression, Site-Directed Mutagenesis, and Mechanism of Escherichia Coli Acid Phosphatase ", J. Biol. Chem., 267: 22830-22836 (1992), hereby incorporated by reference).

Host and vector. The vector to overexpress the appA gene in Saccharomyces cerevisiae was pYES2 and the host was INVScl (Invitrogen, San Diego, CA).

Construction of the expression vector. Initially, a 1.3 kb XbaI fragment was isolated from pAPPA1. The fragment contained the appA gene with its own signal peptide. After binding to the XbaI site of pYES2, the construct (pYES2-appA) was transformed into Saccharomyces cerevisiae. But the phytase activity in the extra or intracellular parts did not increase, compared to the controls, and pAPPA and pYPP1 (PhyA and its signal peptides in pYES2) were co-transformed in the yeast strain. Again, no increase in phytase activity due to pAPPA1 was detected in yeast cells or media.

Two primers were synthesized to construct the signal peptide of the PhyA gene with the coding region of the appA gene. One was 80 bp in length and contained the PhyA signal peptide and a KpnI site at the 5 'end: GGG GTA CCA TGG GCG TCT CTG CTG TTC TAC TTC CTT TGT ATC TCC TGT CTG GAG TCA CCT CCG GAC AGA GTG AGC CGG AG (SEQ ID NO 9). The other primer was 24 bp in length, with an EcoRI site at its 3 'end: GGG AAT TCA TTA CAA ACT GCA GGC (SEQ ID NO: 10). PCR was performed for 25 cycles, with 1 min of denaturation at 95 ° C, 1 min of annealing at 58 ° C and 1 min of chain extension at 72 ° C. The 1.3 kb fragment was amplified, digested and ligated into pYES2. After confirming the insert by restriction mapping, the construct (pYES2-SphyA-appA) was transformed into INVScl by the lithium acetate method.

Expression. The selected transformants were inoculated in YEPD medium. Expression was induced by adding galactose to the culture after OD₆₀₀ will reach 2, as described above. Cells were harvested 15 or 20 h after induction.

Activity test. Acid phosphatase activity was assayed at 37 ° C in 25 mM HCl-glycine buffer (pH 2.5) using p-nitrophenyl phosphate as substrate (250 mM stock solution). 1.7 ml of reaction buffer was added to 0.1 ml samples. After incubating for 5 min in a 37 ° C water bath, 0.2 ml of the prewarmed substrate was added and mixed. The reaction solution was transferred to a prewarmed cuvette and incubated for 2 min in a spectrophotometric compartment at 37 ° C. Released p-nitrophenol was read continuously for 5 min at 405 nm for the calculation of enzyme activity.

In vitro study. Soy flour (5.0 g) was suspended in 20 ml of 20 mM citrate buffer, pH 5.5, mixed with 200 mU of phytase and incubated at 37 ° C for 4 h with continuous shaking. After cooling on ice for 10 min, the suspension was transferred to a centrifuge tube and centrifuged for 15 min at 15,000 x g. The supernatant was used to determine free phosphorus.

Example 11

Quantification of phytase activity from the overexpression of the appA gene of E. coli in Saccharomyces cerevisiae

The intracellular acid phosphatase activity in appA overexpressing E. coli (pAPPA1) was 440 mU / mg protein. An unprecedented intracellular phytase activity greater than 4,900 mU / mg protein was found in the transformed strain. However, there was only minimal phytase activity in the control (BL21). Thus, this acid phosphatase gene also encodes a phytase. The sequence of the appA gene was aligned with that of PhyA and these two genes were found to share 23% identity.

Transformation of INVScl with the pYES2-Sphy-appA construct (driven by the PhyA signal peptide) produced extracellular phytase activity in the supernatant that was 2,000 times greater than that of the wild type or the transformant containing the appA gene plus its own signal peptide (see Table 7).

IS 2 255 281 T3

TABLE 7

Extracellular phytase activity in transformants of the appA gene with different signal peptides

<td>Construct</td><td>Sign</td><td>Activity (U / ml)</td><td>Activity (mU / mg protein)</td>

<td>pYES-appA</td><td>appA</td><td>Indetect.</td><td>Indetect.</td>

<td>pYES2-SphyA-appA</td><td>PhyA</td><td> 1.158</td><td> 445</td>

The effects of the medium (YEPD), inorganic phosphorus, phytate, pH and temperature on the expression of phytase activity by pYES2-SPhyA-AppA are presented in Table 8. The highest activity was 2,286 mU / ml (633 mU / mg protein) under optimal conditions.

TABLE 8

Effect of the different conditions in the YEPD medium on the expression of phytase activity of pYES2-SphyA-appA in yeast

Medium conditions

Phosphorus, mg / 100 ml 0 1 5

Sodium phytate, g / 100 ml 0

0,1

1.0 pH

5,0

7,0

8,0

Temperature ° C

Activity (mU / ml)

1402

714

722

456

870

1019

1748

892

996

2286

312

1036

996

The thermostability of the overexpressed extracellular phytase activity produced by the yeast transformant was higher than that of the intracellular phytase produced by E. coli transformed with pAPPA1 (see Table 9). Heating the extracellular phytase for 15 min at 80 ° C resulted in a 30% loss of its phytase activity, while almost all of the phytase activity of E. coli was lost under the same conditions.

TABLE 9

Effect of heating different sources of phytase at 80 ° C for 15 min on their activities

<td>Phytase</td><td></td><td>Relative activity after heating,%</td>

<td></td><td>appA in E. coli</td><td> 0,1</td>

<td></td><td>appA in S. cerevisiae</td><td> 69</td>

<td></td><td>PhyA in S. cerevisiae</td><td> 66</td>

<td></td><td>BASF phytase</td><td> 50</td>

IS 2 255 281 T3

Comparisons of the effect on phosphorus release from soybean meal by phytases (200 mU) of E. coli, AppA overexpressed in yeast and BASF are presented in Table 10. The results indicate that all three sources of phytases released phosphorus from phytase efficiently from soybean meal.

TABLE 10

Free phosphorus released from soybean meal by different sources of phytases

<td>Phytase</td><td></td><td>Phosphorus (mg / g)</td>

<td></td><td>appA in E. coli</td><td> 1,11</td>

<td></td><td>appA in S. cerevisiae</td><td> 0,69</td>

<td></td><td>BASF</td><td> 0,87</td>

The E. coli appA gene (acid phosphatase), when expressed in Saccharomyces cerevisiae, produced extracellular phytase activity in the media that was more than 2,000 times greater than the control. Overexpressed phytase efficiently releases phytate phosphorus from soybean meal, and appears to be more heat stable than currently available commercial phytase or intracellular phytase produced in E. coli by the same gene (appA).

Example 12

Methods and materials to overexpress the E. coli appA gene encoding an acid phosphatase / phytase in Pichia pastoris

Gene and protein. The appA gene and the host E. coli strain CU 1867 (# 47092) were obtained from ATCC. The gene, a 1.3 kb insert, was transformed into E. coli strain BL21 (# 87441) using an expression vector pAPPAI (Ostanin, K. et al., "Overexpression, Site-Directed Mutagenesis, and Mechanism of Escherichia Coli Acid Phosphatase ", J. Biol. Chem., 267: 22830-22836 (1992), hereby incorporated by reference).

Host and vector. A Pichia EasySelect ™ expression kit was obtained from Invitrogen (San Diego, CA). The kit provides hosts and vectors to express the gene intracellularly or extracellularly in a wild-type strain (X-33). Two vectors were used, pPICZ B (3.3 kb) and pPICZaA (3.6 kb), both use AOX1 as promoter.

Construction of the expression vector. Two primers were used to amplify the pAPPA1 appA gene and two EcoRI and KpnI restriction sites were produced at the 5 'and 3' ends, respectively.

Upstream primer: GGA ATT CCA GAG TGA GCC GGA (SEQ ID NO: 11)

Downstream primer: GGG GTA CCT TAC AAA CTG CAC G (SEQ ID NO: 12)

Template: pAPPA1. DNA isolated from ATCC 87441.

PCR was performed for 30 cycles, with 1 min of denaturation at 94 ° C, 1 min of re-association at 55 ° C, and 1 ml of chain extension at 72 ° C. A 1,245 base pair fragment was amplified, digested with EcoRI and KpnI, and ligated (at 16 ° C overnight) into pPICZ B (3.3 kb) and pPICZaA (3.6 kb). Ligation was confirmed by restriction mapping after transforming the constructs into DH5a.

Transformation of the construct in Pichia (X33). For each transformation, 100 pg of plasmid DNA was prepared and linearized by digestion with PmeI. After linearization, DNA was purified and resuspended in 10 µl of sterile deionized water. Half of the DNA was actually used for each transformation. Both electroporation and the EasyComp chemistry kit (Invitrogen) were used to transform the DNA into X33. In the case of electroporation, an Electro Cell Manipulator (ECM 600, Gentromics, BTX Instrument Division, San Diego, CA 92121) and 2 mm cuvettes were used. The resistance was 186 ohms, the charging voltage was 1.5 kilovolts, and the actual charging duration was approximately 7 milliseconds. Electroporated cells were incubated on YPD agar plates containing 100 mg zeocin / ml at 30 ° C for 2-4 days for colony growth. In the case of chemical transformation, cells were grown on YPDS agar plates containing 100 mg of zeocin / ml. Compared to electroporation, the chemical method had lower transformation efficiency.

Expression. Single colonies were inoculated into 10 ml of MGY medium (30 ml tube) and grown (16-18 h) until OD₆₀₀ 5-6 to 28-30 ° C in a shaking incubator (200 rpm). Cells were harvested by centrifugation (2,000 rpm) and resuspended in 10 ml of BMMY medium (containing 0.5% methanol) to induce expression. Samples (200 µl) were collected every 12 or 24 hours after induction. Methanol (100%) was added to 100 µl every 24 hours to maintain a concentration of 0.5-1% in the media.

IS 2 255 281 T3

Essays. Cells were separated from the media (supernatant) and lysed with glass beads in disruption buffer. Extracellular phytase activity in the supernatant and intracellular phytase activity in lysed cells were assayed as described above (0.2M citrate buffer, pH 5.5 at 37 ° C, using 10mM sodium phytate). Acid phosphatase activity was assayed at 37 ° C in 25 mM HCl-glycine buffer (pH 2.5) using p-nitrophenyl phosphate as substrate (250 mM stock solution). 1.7 ml of reaction buffer was added to 0.1 ml samples. Released p-nitrophenol was read continuously for 5 min at 405 nm for the calculation of enzyme activity. SDS-PAGE (12%) was performed to determine the size and relative amount of the protein expressed. The optimal pH and temperature of the expressed phytase were determined as described in the results.

In vitro study. Soy flour (5.0 g) was suspended in 20 ml of 20 mM citrate buffer, pH 5.5, mixed with different levels of phytase and incubated at 37 ° C for 4 h with continuous shaking. After cooling on ice for 10 min, the suspension was transferred to a centrifuge tube and centrifuged for 15 min at 15,000 x g. The supernatant was used to determine free phosphorus.

Example 13

Colony phytase activity test for Pichia pastoris overexpressing the appA gene of E. coli

Wild-type Pichia X33 produces minimal intracellular (&lt;0.03 U / mg protein) or extracellular (&lt;0.05 U / ml) phytase activity. The X33 cells transformed with the appA gene inserted into pPICZB (without factor a and presumably produces intracellular phytase) did not show any increase in phytase activity (extracellular 0.2 U / ml and intracellular 0.05 U / mg protein).

Transforming Pichia X33 cells with the pPIZaA-appA construct (driven by the α-factor signal peptide) produced extracellular phytase activity in the media. Initially, 72 colonies were examined. Only two colonies had activity &lt;1 U / ml 40 hours after induction. Most of the colonies had activity in the range of 10-20 U / ml 40 hours after induction. All 70 colonies had phytase activity&gt; 80 U / ml 118 hours after induction. The highest phytase activity detected so far was 215 U / ml 192 hours after induction (see Table 11).

TABLE 11

Range of extracellular phytase activity in X33 colonies transformed with pPIZaA-appA 40 and 118 hours after induction

<td>Number of colonies</td><td>40 hours after induction</td><td>118 hours after induction</td>

<td> 2</td><td>&lt;1 U / ml</td><td></td>

<td> 6</td><td>1 to 10 U / ml</td><td></td>

<td> 36</td><td>11 to 20 U / ml</td><td></td>

<td> 28</td><td>&gt; 20 U / ml</td><td></td>

<td> 70</td><td></td><td>&gt; 80 U / ml</td>

The phytase and acid phosphatase activities in the transformant expressing 215 U phytase activity / ml were compared with those of the wild type X33 (192 hours after induction) (see Table 12). Almost all of the expressed phytase protein was secreted from the cells, indicating that α-factor was a very efficient signal peptide for phytase secretion.

TABLE 12

Phytase and acid phosphatase activities in transformant pPIZaA-appA and wild-type X33 192 hours after induction

<td></td><td></td><td>Wild-type X33</td><td colspan="2">PP1ZaA-appA transformant</td>

<td></td><td colspan="2">Extracellular Intracellular U / mg</td><td>Extracellular</td><td>Intracellular U / mg</td>

<td></td><td>U / ml</td><td>protein</td><td>U / ml</td><td>protein</td>

<td>Phytase</td><td> 0,05</td><td> 0,03</td><td> 215</td><td> 0,5</td>

<td>Acid phosphatase</td><td> 0,01</td><td> 0,002</td><td> 5,88</td><td> 0,9</td>

IS 2 255 281 T3

E. coli transformants with the same acid phosphatase appA gene had an intracellular phytase activity of 5 U / mg protein (Ostanin, K. et al., "Overexpression, Site-Directed Mutagenesis, and Mechanism of Escherichia Coli Acid Phosphatase ", J. Biol. Chem., 267: 22830-22836 (1992), which is hereby incorporated by reference). The transforming PhyA gene in A. niger produced an extracellular activity of 7.6 U / ml (Hartingsveldt et al., "Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus niger", Gene 127: 8794 (1993), which is incorporated by this for reference). Compared to these results, the phytase expression system in Pichia is a very efficient expression system.

Example 14

Chronological course of phytase expression

There was a linear increase in extracellular phytase activity in the media in almost all the selected colonies up to 192 hours after induction. Figure 18 summarizes the activity changes of five selected colonies from 24 to 163 hours after induction.

Example 15

Effects of the pH of the medium on the expression of phytase (colony n ° 23, activity 136 U / ml at 186 h)

Using 0.1 M phosphate buffered media, the effects of different pH's on extracellular phytase production in transformants were studied against a control medium without buffer (pH 7.0). The pH 6 buffered medium produced the highest phytase activity (see Figure 19).

Example 16

Size of the extracellular phytase expressed

Using SDS-PAGE analysis (12% gel), a clear band was observed in the culture medium supernatant inoculated with three different colonies (see Figure 20). The size was approximately 55 kDa, probably partially glycosylated. Since the expressed protein represented almost the only visible band in the supernatant, it would be convenient to collect the enzyme product without the need for tedious purification. Example 17 Optimum pH and temperature of the expressed extracellular phytase (colony # 23)

The optimum pH of the expressed phytase was 2.5 to 3.5 (see Figure 21). This is significantly different from A. niger phyA phyAse (BASF) or our other expression systems. It is ideal for the function of phytase at the pH of the stomach.

The optimum temperature of the expressed enzyme was 60 ° C (see Figure 22).

Example 18

Effect of expressed phytase on the hydrolysis of phosphorus defitate from soybean meal

This overexpressed E. coli phytase (colony # 23) efficiently hydrolyzed phytate phosphorus from soybean meal (see Figure 23). The release of free phosphorus in the mixture was linear from 0 to 800 mU of phytase / g of feed. Example 19

Effects of E. coli phytase AppA expressed by Pichia pastoris on the bioavailability of phosphorus defitate in weaned pigs

To determine the nutritional values ​​of E. coli phytase expressed by Pichia in pig diets, the efficacy of this new phytase was compared with that of inorganic phosphorus or commercially available microbial phytase (Natuphos ™, BASF Corp., Mt. Olive, NJ). 48 weaned pigs were selected from multiparous sows at the Cornell Swine Research Farm. Pigs were weaned at 21 days of age and fed a commercial fluid feed until day 28. Two were then arranged per sty, with six pens randomly assigned for treatment. Pigs were given two weeks to adjust to a basal cornmeal-soybean diet (Table 13).

IS 2 255 281 T3

TABLE 13

Formulating experimental diets for pigs

<td>Ingredient</td><td>% diet + C</td><td>% diet -C</td><td>% FL diet</td><td>% FM diet</td>

<td>Corn</td><td> 60,50</td><td> 61,57</td><td> 61,07</td><td> 61,07</td>

<td>Protein concentrate</td><td></td><td></td><td></td><td></td>

<td>serum</td><td> 3,00</td><td> 3,00</td><td> 3,00</td><td> 3,00</td>

<td>SBM at 44%</td><td> 30,00</td><td> 30,00</td><td> 30,00</td><td> 30,00</td>

<td>Corn oil</td><td> 3,00</td><td> 3,00</td><td> 3,00</td><td> 3,00</td>

<td>Lime</td><td> 0,80</td><td> 0,93</td><td> 0,93</td><td> 0,93</td>

<td>Dicalcium phosphate</td><td> 1,20</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>

<td>Vitamin and premix</td><td></td><td></td><td></td><td></td>

<td>mineral</td><td> 0,50</td><td> 0,50</td><td> 0,50</td><td> 0,50</td>

<td>ECAP Premix</td><td> 0,00</td><td> 0,00</td><td> 0,50</td><td> 0,00</td>

<td>FM premix</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,50</td>

<td>Salt</td><td> 0,50</td><td> 0,50</td><td> 0,50</td><td> 0,50</td>

<td>CSP 250</td><td> 0,50</td><td> 0,50</td><td> 0,50</td><td> 0,50</td>

<td>Total</td><td> 100,00</td><td> 100,00</td><td> 100,00</td><td> 100,00</td>

<td>CP</td><td> 20,60</td><td> 20,60</td><td> 20,60</td><td> 20,60</td>

<td>AC</td><td> 0,73</td><td> 0,47</td><td> 0,47</td><td> 0,47</td>

<td>^Ptotal</td><td> 0,60</td><td> 0,39</td><td> 0,39</td><td> 0,39</td>

Note: All premixes use corn as the vehicle

Vitamin and mineral premix supplies: 2,540 IU of vit A, 660 IU of vit. D, 15 IU of vit. E, 2.2 mg vit K, 3.3 mg riboflavin, 13.2 mg pantothenic acid, 17.6 mg niacin, 110.1 mg choline, 1.98 μg B-12, 37 , 4 mg of Mn, 0.6 mg of I, 10 mg of Cu, 0.3 mg of Se, 100 mg of Zn and 100 mg of Fe per kg of diet

Each sty then received one of four treatment diets. The positive control group (+ C) received the basal diet supplemented with dicalcium phosphate. The negative control group (-C) received only the basal diet. The yeast phytase (FL) group received the basal diet supplemented with E. coli phytase expressed at 1,200 U / kg of feed. The microbial phytase (FM) group received the basal diet supplemented with BASF phytase at 1,200 U / kg of feed. The pigs were given free access to feed and water. The body weight gain of the individual pigs was recorded weekly. Daily feed intake from individual pigsty was recorded daily. Blood samples were taken weekly from each of the individual pigs to test plasma inorganic phosphorus concentrations. The results of body weight (BW), mean daily gain (GDM), mean feed intake (TPM), feed / gain ratio (P: G) and plasma inorganic phosphorus (PP) are presented in Table 14.

TABLE 14

Summary of PP, PC, GDM, TPM and P: G from pigs affected by dietary phytase

<td></td><td>+ C</td><td>-C</td><td>FL</td><td>FM</td>

<td colspan="5">Initial</td>

<td>PP</td><td> 12,99</td><td> 13,02</td><td> 13,07</td><td> 13,54</td>

<td>Pc</td><td> 11,54</td><td> 11,63</td><td> 12</td><td> 11,5</td>

<td colspan="5">Week 1</td>

<td>PP</td><td> 10,83^to</td><td> 6,48^C</td><td> 8,59^B</td><td> 8,35^B</td>

<td>Pc</td><td> 14</td><td> 13,83</td><td> 14,29</td><td> 13,92</td>

<td>GDM</td><td> 0,351</td><td> 0,316</td><td> 0,327</td><td> 0,345</td>

<td>TPM</td><td> 0,700</td><td> 0,684</td><td> 0,697</td><td> 0,697</td>

<td>P: G</td><td> 2,04</td><td> 2,20</td><td> 2,18</td><td> 2,13</td>

IS 2 255 281 T3

TABLE 14 (continued)

<td></td><td>+ C</td><td>-C</td><td>FL</td><td>FM</td>

<td colspan="5">Week 2</td>

<td>PP</td><td> 9,76^to</td><td> 5,64^D</td><td> 8,72^B</td><td> 7,84^D</td>

<td>Pc</td><td> 18,04</td><td> 17,42</td><td> 17,83</td><td> 17,71</td>

<td>GDM</td><td> 0,578</td><td> 0,512</td><td> 0,506</td><td> 0,542</td>

<td>TPM</td><td> 0,833</td><td> 0,855</td><td> 0,784</td><td> 0,837</td>

<td>P: G</td><td> 1,46^B</td><td> 1,67^to</td><td> 1,56^AB</td><td> 1,55^AB</td>

<td colspan="5">Week 3</td>

<td>PP</td><td> 11^to</td><td> 6,26^D</td><td> 8,64^B</td><td> 8,13^B</td>

<td>Pc</td><td> 22,58</td><td> 21,17</td><td> 22</td><td> 22,21</td>

<td>GDM</td><td> 0,649^to</td><td> 0,536^B</td><td> 0,595^AB</td><td> 0, 643^ab</td>

<td>TPM</td><td> 1,166</td><td> 1,02</td><td> 1,001</td><td> 1,003</td>

<td>P: G</td><td> 1,8</td><td> 1,92</td><td> 1,71</td><td> 1,36</td>

<td colspan="5">Week 4</td>

<td>PP</td><td> 10,94^to</td><td> 6,31^C</td><td> 9,65^B</td><td> 9,2^B</td>

<td>Pc</td><td> 27,54</td><td> 25,29</td><td> 27,79</td><td> 27,38</td>

<td>GDM</td><td> 0,708^AB</td><td> 0,589^B</td><td> 0,827^to</td><td> 0,738^AB</td>

<td>TPM</td><td> 1,395^to</td><td> 1,049^B</td><td> 1,309^to</td><td> 1,273^AB</td>

<td>P: G</td><td> 1,98</td><td> 1,87</td><td> 1,59</td><td> 1,73</td>

<td colspan="5">¹ Numbers in the same row without sharing a common letter are significantly different. The analysis of the difference was performed with the Bonferroni t test (Dunn) with alpha = 0.05 and df = 20</td>

In addition, there were several severe phosphorus deficiencies in the negative control group at the end of the four-week experiment. However, there was no sign of phosphorus deficiency in the other three groups. Clearly, the E. coli phytase expressed by Pichia was at least as effective, if not more, than commercial microbial phytase in improving the bioavailability of phytate phosphorus from corn-soybean meal diets for weaned pigs. . It can be used to replace inorganic phosphorus supplement for weaned pigs.

Example 20

Effects of E. coli phytase AppA expressed by Pichia pastoris on the bioavailability of iron (Fe) and phosphorus defitate in weaned pigs

To determine the effect of Pichia overexpressed E. coli phytase on the bioavailability of Fe bound to dietary phytate for weaned pigs, 20 anemic pigs (21 days old and 7.3 g of hemoglobin (Hb) / dl of blood). Pigs were fed Fe-deficient fluid chow for 7 days and housed in metabolic cages at the age of 28 days. Pigs were then fed the experimental diets at the age of 35 days for 5 weeks. The treatment diets were as follows: basal iron-deficient diet (-C, with added inorganic phosphorus), iron-supplemented diet (+ C), iron-phosphorus-deficient diet supplemented with expressed E. coli phytase (FL) or commercial microbial phytase (BASF, FM) at 1,200 U / kg of feed. Body weight (PC), packed cell volume (ECV), Hb and plasma inorganic phosphorus (PP) were determined weekly. The results are presented in Table 15.

IS 2 255 281 T3

TABLE 15

Summary of VCE, Hb, PC and PP from pigs affected by dietary phytase¹

<td></td><td>+ C</td><td>-C</td><td>FL</td><td>FM</td>

<td colspan="5">Initial</td>

<td>VCE</td><td> 25</td><td> 25</td><td> 26</td><td> 24</td>

<td>Hb</td><td> 7,73</td><td> 7,22</td><td> 7,85</td><td> 7,08</td>

<td>Pc</td><td> 8,14</td><td> 8,27</td><td> 8,17</td><td> 7,45</td>

<td>PP</td><td> 7,92</td><td> 7,76</td><td> 7,21</td><td> 7,36</td>

<td colspan="5">Week 1</td>

<td>VCE</td><td> 25</td><td> 26</td><td> 29</td><td> 27</td>

<td>Hb</td><td> 7,62</td><td> 8,3</td><td> 8,77</td><td> 7,88</td>

<td>Pc</td><td> 9,44</td><td> 8,84</td><td> 9,63</td><td> 8,57</td>

<td>PP</td><td> 8,41</td><td> 8,45</td><td> 8,48</td><td> 8,22</td>

<td colspan="5">Week 2</td>

<td>VCE</td><td> 29</td><td> 26</td><td> 30</td><td> 28</td>

<td>Hb</td><td> 8,6</td><td> 7,34</td><td> 8,93</td><td> 8,27</td>

<td>Pc</td><td> 12,32</td><td> 10,13</td><td> 11,91</td><td> 10,84</td>

<td>PP</td><td> 10,28^to</td><td> 9,05^ab</td><td> 8,89^ab</td><td> 8,22^to</td>

<td colspan="5">Week 3</td>

<td>VCE</td><td> 36^to</td><td> 29^b</td><td> 34^to</td><td> 33^to</td>

<td>Hb</td><td> 11,55^to</td><td> 8,2^b</td><td> 10,84^to</td><td> 9,96^ab</td>

<td>Pc</td><td> 16,77^to</td><td> 13^b</td><td> 15,62^ab</td><td> 14,62^ab</td>

<td>PP</td><td> 12,14^to</td><td> 11,37 ^ab</td><td> 10,25^bc</td><td> 9,71^c</td>

<td colspan="5">Week 4</td>

<td>VCE</td><td> 39</td><td> 34</td><td> 38</td><td> 36</td>

<td>Hb</td><td> 12,99^to</td><td> 10,11^b</td><td> 12,27^to</td><td> 11,35 ^ab</td>

<td>Pc</td><td> 21,36^to</td><td> 17,37^b</td><td> 19,44^ab</td><td> 18,56 ^ab</td>

<td>PP</td><td> 10,19^to</td><td> 9,34^ab</td><td> 9,49^ab</td><td> 8,8^b</td>

<td colspan="5">Week 5</td>

<td>VCE</td><td> 40</td><td> 38</td><td> 40</td><td> 39</td>

<td>Hb</td><td> 13,52^to</td><td> 12,24^b</td><td> 13,64^to</td><td> 13,13 ^ab</td>

<td>Pc</td><td> 26,53</td><td> 22,59</td><td> 24,27</td><td> 23,43</td>

<td>PP</td><td> 9,27^to</td><td> 8,95^ab</td><td> 8,79^ab</td><td> 8,02ⁿ</td>

Values ​​are means (n ​​= 5). The means in the same row without sharing a common superscript letter are significantly different (p &lt;0.10)

In conclusion, the E. coli phytase overexpressed by Pichia was at least as effective as the BASF phytase in enhancing the utilization of Phytate phosphorus and Fe in corn-soybean diets for weaned pigs.

Although the preferred embodiments have been represented and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention, and these are considered within the scope of the invention. scope of the invention as defined in the following claims.