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Phosphatases with improved phytase activity Number:6,974,690 from the United States Patent and Trademark Office (PTO) owispatent

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Title: Phosphatases with improved phytase activity

Abstract: The present invention provides phosphatases with improved phytase activity. The invention provides proteolytic fragments of phosphatase having improved phytase activity. A recombinant gene encoding a phosphatase fragment having improved phytase activity is also provided. The invention also includes a method of increasing the phytase activity of phosphatase by treating the phosphatase with a protease. In addition, the invention provides a new phosphatase, AppA2, having improved properties.

Patent Number: 6,974,690 Issued on 12/13/2005 to Lei

Inventors: Lei; Xingen (Ithaca, NY)
Assignee: Cornell Research Foundation, Inc. (Ithaca, NY)
Appl. No.: 266041
Filed: October 7, 2002

Current U.S. Class: 435/252.3; 435/320.1; 435/325; 435/6; 435/196; 536/23.2
Intern'l Class: C12N 001/20; C12N 015/00; C12N 005/00; C12Q 001/68; C07H 021/04
Field of Search: 536/232 435/196,6,252.3,320.1,325,255.1,255.5

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Primary Examiner: Monshipouri; Maryam
Attorney, Agent or Firm: Nixon Peabody LLP
Parent Case Text


This application is a continuation of U.S. patent application Ser. No. 09/540/149 filed Mar. 31, 2000, now U.S. Pat. No. 6,511,699, issued Jan. 28, 2003, which claims the benefit of U.S. Provisional patent application Ser. No. 60/127,032, filed Mar. 31, 1999, the disclosures of each of which are hereby incorporated by reference in their entirety.
Claims


1. A recombinant gene encoding a phosphatase having improved phytase activity, comprising:

a promoter;

a coding region encoding the phosphatase comprising a polypeptide having an amino acid sequence of SEQ ID NO:1; and

a terminator.

2. A vector carrying the gene according to claim 1.

3. A host cell transformed with the vector according to claim 2.

4. The host cell according to claim 3, wherein the host cell is yeast.

5. The host cell according to claim 4, wherein the yeast is Pichia pastoris.

6. The host cell according to claim 4, wherein the yeast is Saccharomyces cerevisiae.

7. An isolated nucleic acid molecule encoding a phosphatase comprising a polypeptide having an amino acid sequence of SEQ ID NO:1.

8. A vector carrying the nucleic acid molecule according to claim 7.

9. A host cell transformed with the vector according to claim 8.

10. The host cell according to claim 9, wherein the host cell is yeast.

11. The host cell according to claim 10, wherein the yeast is Pichia pastoris.

12. The host cell according to claim 11, wherein the yeast is Saccharomyces cerevisiae.
Description


BACKGROUND OF THE INVENTION

Phytases are myo-inositol hexakisphosphate phosphohydrolases that catalyze the stepwise removal of inorganic orthophosphate from phytate (myo-inositol hexakisphosphate) (1). There are two types of phytases. One is called 3-phytase (EC.3.1.3.8) which initiates the removal of phosphate groups at the positions 1 and 3 of the myo-inositol ring. The other is called 6-phytase (EC.3.1.3.26) which first frees the phosphate at the position 6 of the ring. While no phytase has been identified from animal tissues, plants usually contain 6-phytases and a broad range of microorganisms, including bacteria, filamentous fungi, and yeast, produce 3-phytases (2-9). Because over 70% of the total phosphorus in foods or feeds of plant origin is in the form of phytate that is poorly available to simple-stomached animals and humans, phytases are of great uses in improving mineral nutrition of these species (10-16). Supplemental microbial phytases in diets for swine and poultry effectively enhance bioavailability of phytate phosphorus and reduce the need for inorganic phosphorus supplementation (11-15), resulting less phosphorus pollution in areas of intensive animal production (8-15). However, a relatively high level of phytase supplementation is necessary in animal diets (10-16), because a considerable amount of the enzyme is degraded in stomach and small intestine (13), probably by proteolysis of pepsin and trypsin. Meanwhile, the proteolytic profiles of various phytases were not studied. Clearly, a better understanding of their sensitivities to trypsin and pepsin hydrolysis could be helpful for improving the nutritional value of phytases. Aspergillus niger phytase gene (phyA) has been overexpressed in its original host (17) and the recombinant enzyme (r-PhyA, EC 3.1.3.8) has been used in animal diets as a commercial phytase (13, 14). This enzyme is a glycoprotein of approximately 80 kDa. Escherichia coli pH 2.5 acid phosphatase gene (appA) has also been characterized (18, 19). Animal experiments have demonstrated that the recombinant enzyme (r-AppA, EC: 3.1.3.2) is as effective as r-PhyA in releasing phytate phosphorus in animal diets (14).

But, expenses of the limited available commercial phytase supply and the activity instability of the enzyme to heat of feed pelleting preclude its practical use in animal industry. Therefore, there is a need for enzymes which have a high level of phytase activity and a high level of stability for use in animal feed.

SUMMARY OF THE INVENTION

The present invention provides a phosphatase fragment having improved phytase activity. A fragment of a phosphatase having increased phytase activity is produced by treating the phosphatase with a protease.

The invention further provides a recombinant gene encoding a phosphatase fragment having improved phytase activity. The vector consists of a promoter, a coding region encoding the phosphatase fragment, and a terminator.

In another embodiment, the invention provides a method of increasing the phytase activity of phosphatase by treating the phosphatase with a protease.

The invention also provides a phosphatase having improved phytase activity, which has an amino acid sequence as shown in SEQ. ID No. 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the change in phytase activity after protease digestion. FIG. 1A shows phytase activity changes of r-PhyA and r-AppA incubated with different ratios of trypsin/protein (w/w) (r=0.001, 0.005, 0.01, and 0.025). Symbols: r-PhyA (▪) and r-AppA (●). The results are the mean±SEM from five independent experiments. *indicates statistical significance (P<0.01) versus untreated r-PhyA or r-AppA control. FIG. 1B shows phytase activity changes of r-PhyA and r-AppA incubated with different ratios of pepsin/protein (w/w) (r=0.001, 0.002, 0.005, and 0.01. Symbols: r-PhyA (□) and r-AppA (custom character). The results are the mean±SEM from seven independent experiments. *indicates statistical significance (P<0.01) versus untreated r-PhyA or r-AppA control.

FIG. 2 shows residual phytase activity of r-PhyA and r-AppA after trypsin or pepsin hydrolysis during a time course (0, 1, 51 30, and 120 min). Symbols: trypsin-digested r-PhyA (▪) or r-AppA (●); pepsin-digested r-PhyA (□) and r-AppA (custom character). The ratio of trypsin/phytase (w/w) used was: r=0.01 (w/w). The ratio of pepsin/phytase used was: r=0.005. The results are the mean±SEM from six independent experiments. *indicates statistical significance (P<0.01) versus untreated r-PhyA or r-AppA control.

FIG. 3 shows the results of SDS-polyacrylamide gel electrophoresis of r-AppA (12%, Panel A) or r-PhyA (20%, Panel B) digested products by different amounts of trypsin (r=0.001, 0.005, 0.01, and 0.025, (w/w). Proteins were stained using Coomasie blue. T: trypsin control, C: purified r-AppA (FIG. 3A) or r-PhyA (FIG. 3B). The protein marker (M) is a 10 kDa ladder [10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, and 200 kDa) (Gibco). The results are representative from four independent experiments.

FIG. 4 shows the results from SDS-polyacrylamide gel (20%) electrophoresis of r-AppA (FIG. 4A) or r-PhyA (FIG. 4B) digested products by different amounts of pepsin (r=0.001, 0.002, 0.005, and 0.01, (w/w)). Proteins were stained using Coomasie blue. T: trypsin control, C: purified r-AppA (FIG. 4A) or r-PhyA (FIG. 4B). The protein marker (M) is a 10 kDa ladder [10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, and 200 kDa) (Gibco). The results are representative from six independent experiments.

FIG. 5 shows the amounts of inorganic phosphorus (iP) released from soybean meal by r-PhyA and r-AppA incubated with different concentrations of trypsin (r=0.001, 0.005, 0.01, and 0.025, w/w) (FIG. 5A), or pepsin (r=0.001, 0.002, 0.005, and 0.01) (FIG. 5B). Symbols: r-AppA (▪), r-PhyA (□). The results are the mean SEM from three independent experiments. *indicates statistical significance (P<0.01) versus untreated r-AppA or r-PhyA control.

FIG. 6 shows the nucleotide sequence of the appA2 gene and its deduced amino acid sequence. The untranslated region is indicated by lowercase letters. The underlined sequences are the primers used to amplify appA2 (Pf1: 1-22, and K2: 1468-1490), appA2 (E2: 243-252, and K2: 1468-1490). Potential N-glycosylation sites are boxed. The sequence of appA2 has been transmitted to Genebank data library with accession number 250016.

FIG. 7 is a time course of extracellular phytase (□) and acid phosphatase (custom character) activities, and CIPPA2 mRNA expression (▴) in Pichia pastoris transformed with appA2 after induction. Results are expressed as the mean±SEM from three experiments.

FIG. 8 shows a northern blot analysis of appA2 mRNA expression in Pichia pastoris transformed with appA2 after induction (FIG. 8A). Hybridization was realized using [α-32P] labeled appA2 as a probe. Twenty μg of total RNA was loaded per lane. FIG. 8B represents the equal RNA loading visualized by the yeast rRNA under UV.

FIG. 9 shows the pH dependence of the enzymatic activity at 37° C. of the purified r-appA2 (●), r-appA (▴), and r-phyA (□) with sodium phytate as the substrate. Buffers: pH 1.5-4.5, 0.2M glycine-HCl; pH 5.5-7.5, 0.2 M citrate; pH 8.5-11, 0.2 M Tris-HCl. Results are expressed as the mean SEM from three experiments.

FIG. 10 shows a non-denaturing gel (15%) electrophoresis analysis of the remaining acid phosphatase activity of r-appA2 after incubated at different temperatures for 20 min. After the heat treatment, the samples were put on ice for 5 min before being loaded onto the gel (200 μg protein/lane).

FIG. 11 shows the hydrolysis of phytate phosphorus in soybean meal by different amounts (100, 300, 600, and 900 PU) of purified r-appA2 (●), r-appA (▴), and r-phyA (□) enzymes. * indicates significant differences (P<0.05) between r-appA2 and other two enzymes. Results are expressed as the mean±SEM from three experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides phosphatases having improved phytase activity.

One embodiment of the invention provides a phosphatase fragment having improved phytase activity. The phosphatase is treated with a protease and fragments having phosphatase activity are selected. As discussed in further detail below, these fragments, have improved phytase activity compared to the full length peptide.

In a preferred embodiment, the protease is pepsin.

In addition to producing the active fragment by proteolysis of the full length peptide, the present invention also provides a recombinant gene having a promoter, a coding region encoding the phosphatase fragment according to claim 1, and a terminator. The recombinant gene can be used to express the truncated product directly.

The improved phosphatases can by used in animal feed to improve the accessibility of phosphate to the animal.

In addition to the phosphatase, the invention provides a method of increasing the phytase activity of phosphatase by treating the phosphatase with a protease.

In another embodiment, the invention provides a phosphatase having improved phytase activity, which has an amino acid sequence as shown in SEQ. ID No. 1 as shown in FIG. 6.

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

In a preferred embodiment, the heterologous gene, which encodes a protein or polypeptide with phytase activity, is spliced in frame with a transcriptional enhancer element.

A preferred phosphatase is encoded by the appA gene of E. coli. The gene, originally defined as E. coli periplasmic phosphoanhydride phosphohydrolase (appA) gene, contains 1,298 nucleotides (GeneBank accession number: M58708). The gene was first found to code for an acid phosphatase protein of optimal pH of 2.5 (EcAP) in E. coli. The acid phosphatase is a monomer with a molecular mass of 44,644 daltons. Mature EcAP contains 410 amino acids (18). Ostanin, et al. overexpressed appA in E. coli BL21 using a pT7 vector and increased its acid phosphatase activity by approximately 400-folds (440 mU/mg protein) (20). The product of the appA gene was not previously known to have phytase activity.

The phosphatase can be expressed in any prokaryotic or eukaryotic expression system. A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Preferred vectors include a viral vector, plasmid, cosmid or an oligonucleotide. Primarily, 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 virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Preferred hosts for expressing phosphatase include fungal cells, including species of yeast or filamentous fungi, may be used as host cells in accordance with the present invention. Preferred yeast host cells include different strains of Saccharomyces cerevisiae. Other yeasts like Kluyveromyces, Torulaspora, and Schizosaccharomyces can also be used. In a preferred embodiment, the yeast strain used to overexpress the protein is Saccharomyces cerevisiae. Filamentous fungi host cells include Aspergillus and Neurospora.

In another embodiment of the present invention, the yeast strain is a methylotrophic yeast strain. Methylotrophic yeast are those yeast genera capable of utilizing methanol as a carbon source for the production of the energy resources necessary to maintain cellular function and containing a gene 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 a sole carbon source. In a preferred embodiment, the methylotrophic yeast strain is Pichia pastoris.

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

Yet another preferred embodiment is a protein or polypeptide having phytase activity where 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 where the protein retains at least 60% of its activity after heating the protein for 15 minutes at 60° C.

Purified protein may be obtained by several 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 growth medium. In such cases, to isolate the protein, the host cell carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove cell debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the polypeptide or protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.

The present invention also provides a yeast strain having a heterologous gene which encodes a protein or polypeptide with phytase activity. The heterologous gene should be functionally linked to a promoter capable of expressing phytase in yeast and followed by a transcriptional terminator.

Yet another aspect of the invention is a vector for expressing phytase in a host. The vector carries a phosphatase gene which encodes a protein or polypeptide with phytase activity.

For cloning into yeast, the gene can be cloned into any vector which replicates autonomously or integrates into the genome of yeast. The copy number of autonomously replicating plasmids, e.g. YEp plasmids may be high, but their mitotic stability may be insufficient (48). 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. The episomal vectors containing the ARS and CEN sequences occur as a single copy per cell, and they are more stable than the 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 no selection is needed (49-51). Some vectors have an origin of replication, which functions in the selected host cell. Suitable origins of replication include 2μ, ARS1, and 25 μM. The vectors have restriction endonuclease sites for insertion of the fusion gene and promoter sequences, and selection markers. The vectors may be modified by removal or addition of restriction sites, or removal of other unwanted nucleotides.

The phytase gene can be placed under the control of any promoter (52). One can choose a constitutive or regulated yeast promoter. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (53) or other glycolytic enzymes (54), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in EP A-73,657 to Hitzeman, which is hereby incorporated by reference. Another alternative is the glucose-repressible ADH2 promoter (56, 57), which are hereby incorporated by reference.

One can choose a constitutive or regulated yeast promoter. The strong promoters of e.g., phosphoglycerate kinase (PGK) gene, other genes encoding glycolytic enzymes, and 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 metallothionine promoter with copper. The 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 for the 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 transcription termination, the HSP150 terminator, or any other functional terminator is used. Here, promoters and terminators are called control elements. The present invention is not restricted to any specific vector, promoter, or terminator.

The vector may also carry a selectable marker. Selectable markers are often antibiotic resistance genes or genes capable of complementing strains of yeast having well characterized metabolic deficiencies, such as tryptophan or histidine deficient mutants. Preferred selectable 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 origin of replications are ColE1, Ori, or oriT.

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

Suitable leader sequences include the yeast alpha factor leader sequence, which may be employed to direct secretion of the phytase. The alpha factor leader sequence is often inserted between the promoter sequence and the structural gene sequence (58; U.S. Pat. No. 4,546,082; and European published patent application No. 324,274, which are hereby incorporated by reference). Another suitable leader sequence is the S. cerevisiae MF alpha 1 (alpha-factor) is synthesized as a prepro form of 165 amino acids comprising signal or prepeptide of 19 amino acids followed by a "leader" or propeptide of 64 amino acids, encompassing three N-linked glycosylation sites followed by (LysArg(Asp/Glu, Ala)2-3 alpha-factor)4 (58). The signal-leader part of the preproMF alpha 1 has been widely employed to obtain synthesis and secretion of heterologous proteins in S. cerivisiae. Use of signal/leader peptides homologous to yeast is known from U.S. Pat. No. 4,546,082, European Patent Applications 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, utilization of the S. cerevisiae a-factor precursor is described whereas WO 84/01153, which is hereby incorporated by reference, indicates utilization of the Saccharomyces cerevisiae invertase signal peptide, and German Patent Application DK 3614/83, which is hereby incorporated by reference, indicates utilization of the Saccharomyces cerevisiae PH05 signal peptide for secretion of foreign proteins.

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

U.S. Pat. No. 5,726,038 describes the use of t


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