# Patent

## Title
Thermotolerant Non-K12 Escherichia coli phytase and its production

## Abstract

This invention relates to cloning and sequencing of thermotolerant phytase gene from Non-K12 Escherichia coli strain, ATCC 9637, phytase gene expression in Escherichia coli expression system, codon usage optimized and expression in Pichia pastoris, Pichia methanolica and Kluyeromyces lactis. The high level yield and thermotolerant enzyme was produced from fermentation of Pichia pastoris with optimized codon of phytase gene.

## Background
TECHNICAL FIELD
The present invention relates to the fields of molecular biology, biochemistry, fermentation and post-process of phytase. More specifically, the present invention relates to cloning and expression of a novel Non-K12 Escherichia coli gene coding for thermotolerant phytate hydratase enzyme, phytase.
BACKGROUND OF THE INVENTION
Phytases (myo-inositol hexakisphosphate phosphohydrolase: EC 3.1.3.8) are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. The enzymes are known to be valuable feed additives. At the close of the twentieth century, annual sales of phytase as an animal feed additive were estimated to exceed $150 million and were growing.
Poultry and pig diets are currently based primarily on cereals, legumes, and oilseed products. About two-thirds of phosphorus (P) present in these feedstuffs occurs as phytates, the salts of phytic acid. Phytate phosphorus in plants is a mixed calcium-magnesium-potassium salt of phytic acid that is present as chelate and its solubility is very low. Phosphorus in this form is poorly digestible for monogastric animals such as human, swine, and poultry.
For the utilization of phytate phosphorus and minerals and trace elements bound in phytic acid complexes, hydrolysis of the ester-type bonded phosphate groups of phytic acid by phytase is necessary. Phytases belong to a special group of phosphatases which are capable of hydrolyzing phytate to a series of lower phosphate esters of myo-inositol and phosphate. Two types of phytases are known: 3-phytase and 6-phytase, indicating the initial attack of the susceptible phosphate ester bond. Although monogastric animals lack sufficient phytase to effectively utilize phytate phosphorous, many fungi, bacteria and yeasts produce phytase that can be used to supplement animal rations.
The beneficial effects of supplementary phytases on phosphorus digestibility and animal performance have been well documented (Mroz et al., 1994; Kornegay et al., 1996; Rao et al., 1999; Ravindran et al., 1999). The efficacy of enzyme preparation depends not only on the type, inclusion rate and level of activity present, but also on the ability of the enzyme to maintain its activity in the different conditions encountered through the gastrointestinal tract and the conditions used for the pre-treatment of a food or feed formulation.
Although numerous phytases are available for use as a supplement, many of the enzymes have certain disadvantages. For example, many of the currently used phytases lose activity during feed pelleting processes due to heat treatment. Additionally, many of the currently used phytases are not adequate in instability for proteases in animal digestion systems such as pepsins and chymotrypsins.
There is a need for a phytase with improve properties for use in animal feed and food processing.
SUMMARY OF THE INVENTION
Accordingly, in one aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding a polypeptide comprising the amino acid as set forth in SEQ ID NO: 3;
b) a nucleotide sequence hybridizing to a) under a stringent hybridization condition, wherein said nucleotide sequence encoding a polypeptide having the activity of a Non-K12 Escherichia coli phytase; or
c) a nucleotide sequence complementary to either of a) and b).
In one embodiment of the invention, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence consisting of nucleotides 67-1296 as set forth in SEQ ID NO: 1;
b) a nucleotide sequence consisting of nucleotides 1-1230 as set forth in SEQ ID NO: 2;
c) a nucleotide sequence as set forth in SEQ ID NO: 1;
d) a nucleotide sequence hybridizing to any of a) to c) under a stringent condition, and encoding a polypeptide having the activity of a Non-K12 Escherichia coli phytase; or
e) a nucleotide sequence complementary to any of a) to d)
In another aspect, the present invention provides a vector, comprising the isolated nucleic acid molecule of the invention, preferably, said vector is an expression vector. For example, said vector may be selected from the group consisting of pTrcHis2-PhE, pPIC9K-PhE, pMET-PhE and pKLAC-PhE.
In a further aspect, the present invention provides an isolated cell comprising an isolated nucleic acid molecule according to the invention, preferably, said isolated nucleic acid molecule is comprised in an expression vector. Preferably, said cell is a yeast cell. For example, said cell may be selected from the group consisting of Escherichia coli, Pichia pastoris, Pichia methanolica and Kluyeromyces lactis. More specifically, the cell can be derived from a strain selected from the group consisting of E. coli MG1655, P. pastoris GS115, P. methanolica PMAD16 and Kluyeromyces lactis GG799. In one embodiment of the invention, the cell is a strain selected from the group consisting of E. coli MG1655 transformed with pTrcHis2-PhE, P. pastoris SMD1168 transformed by pPIC9K-PhE, P. methanolica PMD16 transformed by pMET-PhE and Kluyeromyces lactis GG799 transformed by pKLAC-PhE.
In a further aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of:
a) an amino acid sequence as set forth in SEQ ID NO: 3; or
b) an amino acid sequence having an identity of at least 99% to SEQ ID NO: 3; wherein said polypeptide has the activity of Non-K12 Escherichia coli phytase.
In a further aspect, the present invention provides a process for production of a Non-K12 Escherichia coil phytase by fermentation, comprising a step of cultivating a cell comprising the isolated nucleic acid molecule of the invention under a condition effective for expression to obtain a polypeptide having the activity of Non-K12 Escherichia coli phytase. Preferably, said cell is a strain selected from the group consisting of E. coli MG1655 transformed with pTrcHis2-PhE, P. pastoris SMD1168 transformed by pPIC9K-PhE, P. methanolica PMD16 transformed by pMET-PhE and Kluyeromyces lactis GG799 transformed by pKLAC-PhE.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B show a nucleic acid sequence (SEQ ID NO: 1) encoding the non-K12 Escherichia coli phytase isolated from the strain Escherichia coli ATCC 9637 and the amino acid sequence of the encoded protein (SEQ ID NO: 4) in an embodiment of the invention. The signal peptide is underlined. The nucleic acid sequence encoding the mature protein is from nucleotide 67 to nucleotide 1296. The stop codon TAA is denoted by “*”.
FIGS. 2A and 2B show a nucleic acid sequence (SEQ ID NO: 2) encoding the non-K12 phytase and the amino acid sequence of the encoded protein (SEQ ID NO: 3) in another embodiment of the invention, wherein the codons are optimized for gene expression in yeast systems. The nucleic acid sequence encoding the mature protein is from nucleotide 1 to nucleotide 1230. The stop codon TAA is denoted by “*”.
FIG. 3 shows the sequences of the primers used for cloning the nucleic acid molecules of the invention in the exemplified embodiments of the invention.
FIG. 4 shows the comparison of amino acid sequence between the Non-K12 phytase of the invention (SEQ ID NO: 4) and two known phytases (SEQ ID NOS: 5 and 6).
FIG. 5 shows the construction maps of the recombinant expression plasmids constructed in several embodiments of the present invention. In the plasmid pTrcHis2-PhE, the insert “Phytase” has the sequence of SEQ ID NO: 1, while the insert “Phytase” in pPIC9k-PhE, pMET-PhE and pKLAC-PhE has the sequence of SEQ ID NO: 2.
FIG. 6 shows the Non-K12 phytase activities at different pH.
FIG. 7 shows the Non-K12 phytase activity at different temperatures.
FIG. 8 shows the Non-K12 phytase activities at different fermentation times.
FIG. 9 shows the Non-K12 phytase product on SDS-PAGE of samples at different time points of a fermentation according to the present invention.
FIG. 10 shows the Non-K12 phytase activity after treatment with the indicated proteases.
FIG. 11 shows the Molecular weight of the phytase secreted by Pichia pastoris in an embodiment of the invention.
FIG. 12 shows the pH-tolerance of the expressed phytase in an embodiment of the invention.
FIG. 13 shows the thermotolerance of the expressed phytase in an embodiment of the invention.
FIG. 14 shows the thermotolerance of a dried formulated phytase of the invention.
FIG. 15: comparison of the thermotolerance between the Non-K12 phytase of the invention and the E. coli K12 phytase and the fungal phytase from Aspergillus niger. 
FIG. 16: comparison of the pH-tolerance between the Non-K12 phytase of the invention and the E. coli K12 phytase and the fungal phytase from Aspergillus niger. 



DETAILED DESCRIPTION OF THE INVENTION
In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.
As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
The term “thermotolerant” characterizes an enzyme that retains activity despite exposure to a given temperature.
As used herein, the term “pTrcHis2-PhE” refers to the plasmid pTrcHis2 (Invitrogen Biotechnology Co., Ltd) containing a nucleotide sequence as set forth in SEQ ID NO: 1 under the control of LacO promoter and a pBR322 origin, and bla(Apm) gene for DNA replication and transformation selection as shown in FIG. 5
As used herein, the term “pPIC9K-PhE” refers to the plasmid pPIC9K (Invitrogen Biotechnology Co., Ltd) containing the nucleotide sequence as set forth in SEQ ID NO: 2 in the orientation as shown in FIG. 5.
As used herein, the term “pMET-PhE” refers to the plasmid pMETalphaA (Novagen, Inc.) containing the nucleotide sequence as set forth in SEQ ID NO: 2 in the orientation as shown in FIG. 5.
As used herein, the term “pKLAC-PhE” refers to the plasmid pKLAC(New England Biolabs, Inc.) containing the nucleotide sequence as set forth in SEQ ID NO: 2 in the orientation as shown in FIG. 5.
As used herein, an “isolated nucleic acid fragment” or “isolated polynucleotide” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the instant microbial polypeptides as set forth in FIGS. 1 and 2. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA), and the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111 20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.). The term “MEME” refers to a software program used to identify conserved diagnostic motifs based on a hidden Markov model (Timothy L. Bailey and Charles Elkan, Fitting a mixture model by expectation maximization to discover motifs in biopolymers, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28 36, AAAI Press, Menlo Park, Calif. (1994)). “MAST” (Timothy L. Bailey and Michael Gribskov, “Combining evidence using p-values: application to sequence homology searches” Bioinformatics, Vol. 14, pp. 48 54 (1998)) is a program that takes the output from the MEME program and searches the identified motifs against the protein databases such as EMBL and SwissProt. Within the context of this application it will be understood that where sequence analysis software is used for analysis, the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters which originally load with the software when first initialized.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described, for example, by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984) (hereinafter “Silhavy”); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987) (hereinafter “Ausubel”).
The present invention provides a polynucleotide isolated from Escherichia coli ATCC 9637 (American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108, USA) and a synthetic nucleic acid sequence that encode the said polypeptide. The isolated polypeptide is a Non-K12 Escherichia coli phytase. When expressed, the phytase hydrates phytates into the corresponding myo-inositol and inorganic phosphate. The invention also provides transformed microbial host cells expressing the polypeptides. The invention further provides a method for producing the polypeptide catalysts using the transformed microbes and a method for using the catalysts for converting phytates to the myo-inositol and inorganic phosphate
In the context of the present invention, the terms “a Non-K12 Escherichia coli phytase”, “a Non-K12 E. coli phytase”, “a Non-K12 phytase” and “a thermotolerant phytase” are used interchangeably as referring to a thermotolerant phytase which retains at least 17% activity as a phytase at a temperature up to 80 for 1 hr, more preferably at least 33% activity at a temperature up to 90 for 1 hr, even more preferably at least 26% activity at a temperature up to 100 for 1 hr, which has gastric stability against acid environment and proteases in animal digestion system.
In one embodiment, the invention provides a method to prepare a thermotolerant phytase. The method comprises expressing in a microbial host cell an expression cassette comprising a promoter linked to a nucleic acid molecule encoding a thermotolerant phytase. The microbial host cell may be a prokaryotic cell, such as a bacterial cell (e.g., Escherichia or Bacillus), yeast (e.g., Saccharomyces, Schizosaccharomyces, Pichia or Kluyeromyces lactis) cell. In one preferred embodiment, the microbial cell which is employed to prepare the recombinant thermotolerant phytase yields a glycosylated form of the recombinant thermotolerant phytase.
The invention provides methods of cloning and sequencing a nucleic acid molecule encoding a thermotolerant phytase which has 1296 nucleic acids coding for 432 amino acids including a signal peptide of 22 amino acids. Sequence comparison (FIG. 4, wherein “W-Phytase” refers to the Non-K12 phytase of the present invention) shows that the present invention provided a novel phytase polypeptide.
It is preferred that the polynucleotide that encodes the thermotolerant phytase (the first polynucleotide) is operably linked to at least one regulatory sequence, such as a promoter, an enhancer, a termination sequence, or any combination thereof, and, optionally, to a second polynucleotide encoding a signal sequence, which directs the phytase encoded by the first polynucleotide to a particular cellular location e.g., an extracellular location. Promoters can be constitutive promoters or inducible (conditional) promoters.
A parent polynucleotide may be obtained from any source including bacterial or fungal nucleic acid, and any method may be employed to prepare a synthetic polynucleotide of the invention from a selected parent polynucleotide, e.g., combinatorial mutagenesis, recursive mutagenesis and/or DNA shuffling.
In one embodiment, the present invention provides a nucleotide sequence hybridizing to the indicated sequences, such as SEQ ID NO: 1 or SEQ ID NO: 2, under a stringent hybridization condition. The “stringent hybridization condition” can be a conventional one as previously described in the relevant articles, e.g., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989), Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). For example, a stringent hybridization condition may be 0.1.times.SSC, 0.1% SDS, 65. And, other exemplary conditions for hybridization include (1) high stringency: 0.1.times.SSPE, 0.1% SDS, 65; (2) medium stringency: 0.2.times.SSPE, 0.1% SDS, 50; and (3) low stringency: 1.0.times. SSPE, 0.1% SDS, 50. Obviously, equivalent stringencies may be achieved using alternative buffers, salts and temperatures.
Thus, in one embodiment of the invention, the thermotolerant phytase has one or more amino acid substitutions relative to a corresponding (reference) phytase, which substitutions are associated with the retention of activity at temperatures equal to or greater than 60. Preferably, the thermotolerant phytase has at least 17% activity remained under 80 for 1 hr, more preferably at least 33% activity at 90 for 1 hr, even more preferably at least 26% activity at 100 for 1 hr. An exemplary thermotolerant phytase of the invention is the Non-K12 Escherichia coli phytase having the amino acid sequence of SEQ ID NO: 3 (also see FIG. 1).
In one embodiment, the present invention also provides a polypeptide comprising an amino acid sequence having a certain identity to SEQ ID NO: 3; wherein said polypeptide has the activity of Non-K12 Escherichia coli phytase. The term “identity” when used in connection with the polypeptide of the present invention, is defined as the percentage of amino acid residues in a candidate sequence that are identical with a subject sequence (such as SEQ ID NO: 3), after aligning the candidate and subject sequences to achieve the maximum percent identity. Amino acid sequence identity can be determined by many known methods, such as the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443-453 (1970)), or using commercially available programs. It is well understood that many levels of sequence identity are useful in identifying related polypeptide sequences. As contemplated by the present invention, the useful identities include but are not limited to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 99% or 100, and also the values between them.
Vectors, which comprise the expression cassette or polynucleotide of the invention and transformed microbial cells comprising the polynucleotide, expression cassette or vector of the invention, are also provided by this invention. A vector of the invention can encode more than one polypeptide including more than one thermotolerant phytase or may encode a fusion polypeptide comprising the thermotolerant phytase of the invention, and a transformed microbial cell may comprise one or more vectors of the invention. The transformed cells of the invention are useful for preparing the recombinant thermotolerant phytase of the invention. Accordingly, the invention provides thermotolerant phytase isolated from the transformed microbial cells of the invention, as well as synthetically prepared phytase(s).
Further, the invention provides the preparation of thermotolerant phytase via fermentation of a yeast strain comprising the Non-K12 Escherichia coli phytase gene. By the provided process of fermentation for production of phytase, the yield of the protein reaches 2.7 g/L. The phytase, which is estimated to be partially glycosylated, showed a molecular weight of 52 kD on SDS-PAGE (example 7). The optimal pH range for the phytase of the invention is pH2-7.5, preferably pH 3-6.
Further, as a phytase of the invention is capable of surviving the heat conditioning step encountered in a commercial pellet mill during feed formulation, the invention provides a method on making animal feed, e.g., hard granular feed pellets comprising the thermotolerant phytase. To make feed, the formulated phytase may be mixed with feed components, the mixture steam conditioned in a pellet mill such that at least 60% of the pre-heat treated enzymatic activity is retained, and the feed extruded through a pellet dye. The phytase may thus be used as a supplement in animal feed by itself, in addition with vitamins, minerals, other feed enzymes, agricultural co-products (e.g., wheat middling or corn gluten meal), or in a combination therewith. The phytase of the invention may also be added to mash diets, i.e., diets that have not been through a pelletizer.
Because the currently available commercial phytase enzymes are not thermotolerant, they are often applied after pelleting, generally via spraying an solution of the phytase onto the surface of pelleted feed. Problems found with spraying methods are that only a low percentage of the pellets are contacted with the enzyme, the enzyme is only present on the surface of the coated pellets, and feed mills need to invest in and operate complex spraying machinery. In contrast, the thermotolerant phytase of the invention, which has high specific activity of 3146 U/mg, may be added prior to pelleting, thereby facilitating production of a feed with an improved distribution of the enzyme.
Microbial Recombinant Expression
The genes and gene products of the instant sequences may be produced in heterologous host cells, particularly in the cells of microbial hosts. Expression in recombinant microbial hosts may be useful for the expression of various pathway intermediates; for the modulation of pathways already existing in the host, or for the synthesis of new products heretofore not possible using the host.
Preferred heterologous host cells for expression of the instant genes and nucleic acid fragments are microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. For example, it is contemplated that any of bacteria, yeast, and filamentous fungi will be suitable hosts for expression of the present nucleic acid fragments. Because of transcription, translation and the protein biosynthetic apparatus is the same irrespective of the cellular feedstock, functional genes are expressed irrespective of carbon feedstock used to generate cellular biomass. Large-scale microbial growth and functional gene expression may utilize a wide range of simple or complex carbohydrates, organic acids and alcohols, saturated hydrocarbons such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts. However, the functional genes may be regulated, repressed or depressed by specific growth conditions, which may include the form and amount of nitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrient including small inorganic ions. In addition, the regulation of functional genes may be achieved by the presence or absence of specific regulatory molecules that are added to the culture and are not typically considered nutrient or energy sources.
Examples of host strains include but are not limited to bacterial, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Methylobacterium, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, Myxococcus, and Staphylococcus. In another embodiment, suitable host strains are selected from the group consisting of Aspergillus, Saccharomyces, Pichia, Candida, Hansuela, Bacillus, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Escherichia, Pseudomonas, Methylomonas, Synechocystis, and Klebsiella. In a further embodiment, suitable host strains are selected from the group consisting of Bacillus, Rhodococcus, Escherichia, Pseudomonas, Klebsiella, and Methylomonas. 
Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for expression of the phytases of the present invention. These chimeric genes could then be introduced into appropriate a host via transformation to provide high-level expression of the enzyme
Accordingly it is expected, for example, that introduction of chimeric genes encoding the instant bacterial phytase under the control of an appropriate promoter, will demonstrate increased phytate to phosphate and myo-inositol conversion. It is contemplated that it will be useful to express the instant genes both in natural host cells as well as in a heterologous host. Introduction of the present genes into native hosts will result in altered levels of existing phytase activity.
Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the instant ORF in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IP.sub.L, IP.sub.R, T7, tac, and trc (useful for expression in Escherichia coli) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus. Additionally, the deoxy-xylulose phosphate synthase or methanol dehydrogenase operon promoter (Springer et al., FEMS Microbiol Lett 160:119 124 (1998)), the promoter for polyhydroxyalkanoic acid synthesis (Foellner et al., Appl. Microbiol. Biotechnol. 40:284 291 (1993)), promoters identified from native plasmids in methylotrophs (EP 296484), promoters identified from methanotrophs (PCT/US03/33698), and promoters associated with antibiotic resistance [e.g., kanamycin (Springer et al., supra; Ueda et al., Appl. Environ. Microbiol. 57:924 926 (1991)) or tetracycline (U.S. Pat. No. 4,824,786)] are suitable for expression of the present coding sequences, especially in C1 metabolizers.
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
Methods of manipulating genetic pathways are common and well known in the art. Selected genes in a particularly pathway may be up-regulated or down-regulated by variety of methods. Additionally, competing pathways may be eliminated or sublimated by gene disruption and similar techniques.
Once a key genetic pathway has been identified and sequenced, specific genes may be up-regulated to increase the output of the pathway. For example, additional copies of the targeted genes may be introduced into the host cell on multicopy plasmids such as pBR322. Alternatively the target genes may be modified so as to be under the control of non-native promoters. Where it is desired that a pathway operate at a particular point in a cell cycle or during a fermentation run, regulated or inducible promoters may used to replace the native promoter of the target gene. Similarly, in some cases the native or endogenous promoter may be modified to increase gene expression. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868).
Alternatively, it may be necessary to reduce or eliminate the expression of certain genes in a pathway or in competing pathways that may serve as competing sinks for energy or carbon. Methods of down-regulating genes for this purpose have been explored. Where sequence of the gene to be disrupted is known, one of the most effective methods of gene down regulation is targeted gene disruption where foreign DNA is inserted into a structural gene so as to disrupt transcription. This can be effected by the creation of genetic cassettes comprising the DNA to be inserted (often a genetic marker) flanked by sequence having a high degree of homology to a portion of the gene to be disrupted. Introduction of the cassette into the host cell results in insertion of the foreign DNA into the structural gene via the native DNA replication mechanisms of the cell (Hamilton et al., J. Bacteriol. 171:4617 4622 (1989); Balbas et al., Gene 136:211 213 (1993); Gueldener et al., Nucleic Acids Res. 24:2519 2524 (1996); and Smith et al., Methods Mol. Cell. Biol. 5:270 277(1996)).
Antisense technology is another method of down regulating genes where the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the protein of interest. The person skilled in the art will know that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
Although targeted gene disruption and antisense technology offer effective means of down regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence based. For example, cells may be exposed to a UV radiation and then screened for the desired phenotype. Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect non-replicating DNA such as HNO.sub.2 and NH.sub.2OH, as well as agents that affect replicating DNA such as acridine dyes, notable for causing frameshift mutations. Specific methods for creating mutants using radiation or chemical agents are well documented in the art. (See for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass. (hereinafter “Brock”), or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992) (hereinafter “Deshpande”).
Another non-specific method of gene disruption is the use of transposable elements or transposons. Transposons are genetic elements that insert randomly in DNA but can be latter retrieved on the basis of sequence to determine where the insertion has occurred. Both in vivo and in vitro transposition methods are known. Both methods involve the use of a transposable element in combination with a transposase enzyme. When the transposable element or transposon, is contacted with a nucleic acid fragment in the presence of the transposase, the transposable element will randomly insert into the nucleic acid fragment. The technique is useful for random mutagenesis and for gene isolation, since the disrupted gene may be identified on the basis of the sequence of the transposable element. Kits for in vitro transposition are commercially available (see for example The Primer Island Transposition Kit, available from Perkin Elmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1 element; The Genome Priming System, available from New England Biolabs, Beverly, Mass.; based upon the bacterial transposon Tn7; and the EZ::TN Transposon Insertion Systems, available from Epicentre Technologies, Madison, Wis., based upon the Tn5 bacterial transposable element).
Biocatalytic Conversion of Phytate to Phosphate and Myo-Inositol
An aqueous reaction mixture containing the phytate is prepared by mixing the myo-inositol hexakisphosphate with an aqueous suspension of the appropriate phytase enzyme. The specific activity of the phytase enzyme (U/milligram enzyme, “U/mg”) is determined by measuring the rate of conversion of a 5.0 mmol/L solution of a phytate substrate (sodium phytate) to the desired phosphate and myo-inositol product. Determination of phytase activity is based on the colorimetrical quantification at 700 nm of free phosphorus released by the hydrolysis of phytate using ammonium molybdate as color reagent. An U is the amount of phytase that liberates 1 μmol inorganic orthophosphate per minute under test conditions (pH 5.0; temperature 37° C.; and substrate concentration, sodium phytate at 0.005 mol/L).
The temperature of the hydrolysis reaction is chosen to optimize both the reaction rate and the stability of phytase. The temperature of the reaction may range from just above the freezing point of the reaction mixture (ca. 0) to 65, with a preferred range of reaction temperature of from 5 to 45. An phytase solution may be prepared by suspending the phytase in distilled water, or in an aqueous reaction mixture of a buffer that will maintain the initial pH of the reaction between 5.0 and 10.0, preferably between 6.0 and 8.0, or by suspending the immobilized phytase in a similar mixture, or by preparing a solution of a cell extract, partially purified or purified phytase(s), or a soluble form of the supernatant of cell culture in a similar mixture. After the substrata is added and as the reaction proceeds, the pH of the reaction mixture may change due to the formation of product. The reaction can be run to completely convert the phytate with no pH control, or a suitable acid or base can be added over the course of the reaction to maintain the desired pH.
EXAMPLES
The present invention is further described in the following Examples that indicate preferred embodiments of the invention. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, make adaptive changes and modifications for it to various uses and conditions.
General Methods
Standard method for phytase activity assay was used for thermotolerant phytase screening from different Escherichia coli cell strains. Escherichia coli cell strains were grown in LB media at 37° C. with shaking for 16 hours. Cells were broken by using French press and then suspended in buffers for phytase activity assay under designed conditions.
The other way for thermotolerant phytase screening is to identify the nova phytase genes by genomic DNA sequencing technique using the designed sequencing primers according to the phytase gene sequences in database.
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art. For example, pertinent specification on the operations and conditions can be found in Handbook of Molecular Cloning 3 (Joseph Sambrook & David W. Russell, Cold Spring Harbor Laboratory).
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillip Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C. (1994)) or in Brock.
The following abbreviations in the specification correspond to units of measure, techniques, properties, or compounds as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “mL” means milliliters, “L” means liters, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “rpm” means revolutions per minute, “slpm” means standard liters per minute, “psig” means pounds per square inch, and “wt” means weight. “HPLC” means high performance liquid chromatography, “ca” means approximately, “O.D.” means optical density at a specific wavelength, “dcw” means dry cell weight, and “IU” means International Units.
Example 1
Cloning of Non-K12 Escherichia coli Phytase
Escherichia coli (ATCC 9637, purchased from ATCC) was grown in a LB medium (LB Nutrient, Beijing Luqiao Technology Co. Ltd.) at 37° C. with shaking overnight. Genomic DNA was prepared using a Puregene DNA Isolation Kit according to the manufacturer's specification (Gentra Systems, Minneapolis, Minn.). PCR primers (OLIGO ID NOs.: 1 ̃6, FIG. 3) were designed and synthesized (Invitrogen Biotechnology Co., Ltd) for cloning the genomic sequence of non-K12 Escherichia coli phytase. A PCR reaction was run using InsT/AcloneTM PCR Product Cloning Kit (Fermentas Life Sciences) to obtain the genomic sequence. PCR reaction mixture: 0.1 mM dNTP, 0.05 mM Primer, 10 ng genomic template DNA, 2 units Taq DNA polymerase, 5 mM MgCl2, 10 mM Tris-HCl buffer pH 7.5 to a total liquid volume of 0.1 ml. The above reaction (0.1 ml) was mixed in a reaction tube (0.2 ml) and set in the DNA Thermal Cycler (Perkin-Elmer Thermocycler Type 2400) to be subjected to PCR with the following temperature conditions: 95° C. for 3 min, followed by 35 cycles of 1 min at 94° C., 1 min at 58° C., 1 min at 72° C., then 1 cycle of 7 min at 94° C.
Tow oligomers (synthesized by Invitrogen Biotechnology Co., Ltd) were used to amplify the Non-K12 phytase coding sequence from the obtained genomic DNA of Escherichia coil strain ATCC 9637 by PCR. The 5′ primer encoded the N-terminal of the mature peptide, MQSEPELKL, and included the ribosome binding site and the restriction site NcoI (OLIGO ID NO. 7, FIG. 3). The 3′ primer encoded the C-terminal of the phytase peptide, IPACSL, and included the stop codon TAA and the restriction site SnaBI (OLIGO ID NO. 8, FIG. 3). The amplified product was identified on 1.0% agarose gel electrophoresis as a DNA fragment of 1.4 kb. The fragment was isolated and purified using the DNA gel purification system (Qiagen Biochemical Co.). The purified PCR product was digested by NcoI/SnaBI (Fermentas Life Sciences) and ligated into plasmid pTrcHis2 (Invitrogen Biotechnology Co., Ltd) to obtain the recombinant plasmid pTrcHis2-PhE (see FIG. 5). Sequencing of the obtained plasmid pTrcHis2-PhE showed correct insertion of an insert having the sequence as set forth in SEQ ID NO: 1 excluding the sequence for the signal peptide.
1 microliter of ligation reaction was mixed with 50 microliters of electrocompetent E. coli MG1655 cells (ATCC 700926, purchased from ATCC). The mixture was subjected to a high voltage pulse (Bio-Rad electroporation system). The reaction was then incubated in 0.45 ml a SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37° C. with shaking for 1 hour. The culture was then spread on LB agar plates containing 100 mg/ml ampicillin sulfate for growth overnight. Transformed colonies (pTrcHis2-PhE/MG1655) were then selected and used for phytase gene expression.
Example 2
Expression of Non-K12 Escherichia coli Phytase (W-PhE) in Escherichia coli 
The E. coli transformant obtained in Example 1 was cultured with shaking for 20 hours at 37° C. in a culture medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 50 μg/ml ampicillin). The overnight culture was added, at a ratio of 1/100 volume, into a fresh medium the same as the above, and was incubated under the same condition. When the culture reached OD550 0.5, 1 M IPTG was added to a final concentration of 1 mM. The incubation was continued for another 20 hours to induce the phytase gene expression. The cells were harvested by centrifugation, washed twice with distilled water, and then suspended at 100 mg wet cells/ml in HAC-Na buffer (0.1 M, pH 5.8). The harvested cells were then disrupted using an ultrasonic disintegrator (COSMO BIO CO., LTD). The supernatant was separated from the debris and subjected to SDS-polyacrylamide gel electrophoresis. The expressed protein was confirmed by the electrophoresis, which showed the existence of a protein having a molecular weight of 47 kD as expected. Further, N-terminal sequencing of the protein purified as described in Example 7 below by Edman degradation indicated a correct amino acid sequence as set forth in SEQ ID NO: 3.
Phytase activity of the harvested cells was assayed. The harvested cells (50 mg/mL), sodium phytate (0.3 M) and buffer (0.1 M HAC-Na, pH 5.8), was mixed at ambient temperature. The phytate conversion was detected in the HPLC assay as previously taught (Chen Q C, Li B W (2003) Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications. J Chromatogr A 1018: 41-52).
Example 3
Production of Non-K12 Escherichia coli Phytase (W-PhE) Via Fermentation of Escherichia coli 
The production of non-K12 E. coli phytase in a 14 L Braun Biostat C fermentor (B. Braun Biotech International Gmbh, Melsungen, Germany) used the mineral medium containing glucose, ammonia, and yeast extract (OXOID LTD., BASINFSTOKE HAMPSHIRE, ENGLAND).
E. coli strain pTrcHis2-PhE/MG1655 harboring plasmid pTrcHis2-PhE prepared in Example 1 was grown in a LB medium to prepare the seed culture for inoculation. The 500 mL seed culture was grown in a 2 L flask at 37° C., 300 rpm until OD (λ.=550)>2.0. This may take about 10 hrs.
The vessel medium was prepared in an initial batch volume of 7.5 L, which contained 32 g KH2PO4, 8.0 g MgSO4.7H2O, 8.0 g (NH4)2SO4, 50 g yeast extract, and 10 mL Mazu DF204 antifoam (BASF Corporation, Mount Olive, N.J.) in distilled water. The fermentor filled with the medium was then steam sterilized. Following sterilization, 369 g glucose aqueous solution (60% w/w), 160 mL trace element solution (Table 1), and 100 mg/L ampicillin were added. NH4OH (40% w/v) and 20% w/v H2SO4 were used to adjust pH to 6.8.







TABLE 1







Trace element solution











Concentration 

Concentration 


Trace Element
(g/L)
Trace Element
(g/L)













MnSO4•5H2O
0.001
H3BO4
0.0005


CoCl2•6H2O
0.004
FeSO4•7H2O
0.02


Na2MoO4•2H2O
0.002
CaCl2•2H2O
0.02


ZnCl2
0.002
MgSO4•7H2O
0.3


CuSO4•5H2O
0.001









The seed culture was added into the fermentor filled with the initial batch Vessel medium prepared above at the ratio of 5%(v/v).
The control of agitation, aeration, pH, pressure, dissolved oxygen concentration (DO), and temperature were as described in Table 2 below. The dissolved oxygen concentration was controlled at 25% of air saturation by adjusting the agitation and aeration according to the change in oxygen demand. Glucose feed was started at <5 g/L at time 0, and the feeding rate was well controlled to have the specific cell growth rate controlled at about 10-35% per hour. Glucose feed rate was reduced if glucose accumulated above 2 g/L.







TABLE 2





Several settings of fermentation



















agitation
200 
rpm
pressure
1 atm


aeration
0.4 
L/min
dissolved oxygen 
85-90%












concentration (DO)



pH
6.8
temperature
37° C.









When the culture densities in the fermentor reached at OD. (λ=550) of 20-30, an additional aliquot of ampicillin was added to a final concentration of 100 mg/L. When the culture densities reached OD. (λ.=550) of 30-35, IPTG was added to 1 mM.
About five hours after the IPTG addition, the broth was chilled to 5-10° C. and discharged. The cells were harvested by centrifugation at 25000 g, 10° C., 10 mins. 490 g of wet cells was harvested.
The harvested cells were then disrupted using an ultrasonic disintegrator (COSMO BIO CO., LTD). The supernatant was separated from the debris and subjected to SDS-polyacrylamide gel electrophoresis. The electrophoresis showed the existence of the protein having a molecular weight of 47 kD as expected.
Example 4
Codon Optimization for Yeast Expression Systems
The Non-K12 phytase amino acid sequence (as shown in FIG. 2, SEQ ID NO: 3) was back-translated using the preferred codons in yeast systems (NCBI database). Codons with low usage percentage (<10%) were replaced by the codons used with higher frequency. The designed coding sequence was as shown in FIG. 2 (SEQ ID NO: 2), and was synthesized by Invitrogen Biotechnology Co.
Example 5
Construction of Phytase Gene Expression Strains of Yeasts
Construction of a Phytase Gene Expression Strain of Pichia Pastoris (S-Ph/Pp)
Two restriction sites, EcoRI and NotI, were respectively added at the 5′ and the 3′ ends of the synthetic sequence prepared in Example 4 using the synthetic primers of OLIGO ID NOs: 9 and 10 (FIG. 3). The obtained sequence was digested with the EcoRI and NotI enzymes, and then ligated into pPIC9K (Invitrogen Biotechnology Co., Ltd). The obtained plasmid was transformed into Escherichia coli strain (Escherichia coli strain MG1655, ATCC 700926, purchased from ATCC) for DNA amplification. The transformed E. coli cell colonies were selected by culturing in a LB medium containing 50 mg/L ampicillin overnight. The recombinant plasmid DNA was prepared from the cell culture using Mini spin DNA preparation kit (Qiagen Biochemical Co.). Sequencing showed correct insertion.
The recombinant plasmid prepared above (pPIC9K-PhE, FIG. 5) was digested by BglII and transformed into P. pastoris strain SMD1168 (Invitrogen Biotechnology Co., Ltd) via electroporation. Transformants (S-Ph/Pp) with multiple-copy inserts of phytase gene expression cassette was screened via G418 resistant selection as previously taught (Scorer, C. A., Clare, J. J., McCombie, W. R., Romanos, M. A. and Sreekrishna, K. (1994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology 12, 181-184).
Construction of a Phytase Gene Expression Strain of Pichia Methanolica (S-Ph/Pm)
The strain was constructed substantially in the same way as the above. Two restriction sites, PstI and NotI, were respectively added at the 5′ and the 3′ ends of the synthetic sequence prepared in Example 4 using the synthetic primers of OLIGO ID NO: 11 and 12 (FIG. 3). The obtained sequence was digested with the PstI and NotI enzymes, and then ligated into plasmid pMETalphaA (Novagen, Inc.). The obtained plasmid was amplified in Escherichia coli strain MG1655 and recovered as said above. Sequencing showed correct insertion.
The recombinant plasmid prepared above (pMET-PhE, FIG. 5) was digested by AscI and transformed into Pichia methanolica PMD16 (Novagen, Inc.) via electroporation. Transformants (S-Ph/Pm) were selected as said above.
Construction of a Phytase Gene Expression Strain of Kluyeromyces lactis (S-Ph/Kill)
The strain was constructed substantially in the same way as the above. Two restriction sites, BglII and StuI, were respectively added at the 5′ and the 3′ ends of the synthetic sequence prepared in Example 4 using the synthetic primers of OLIGO ID NO: 13 and 14 (FIG. 3). The obtained sequence was digested with the BglII and StuI enzymes, and then ligated into plasmid pKLAC1 (New England Biolabs, Inc.). The obtained plasmid was amplified in Escherichia coli strain MG1655 and recovered as said above. Sequencing showed correct insertion.
The recombinant plasmid prepared above (pKLAC-PhE, FIG. 5) was digested by SacII and transformed into Kluyeromyces lactis GG799 (New England Biolabs, Inc.) via electroporation. Transformants (S-Ph/Kill) were selected as said above.
Example 6
Fermentation of P. pastoris Strain (S-Ph/Pp)
This example demonstrated a process of high density fermentation of the transformed Pichia pastoris (S-Ph/Pp) of the invention in a 5 liter fermentor (Gaoji biotech Co. Ltd., Shanghai, China). The fermentation process included three major stages.
In stage I (Cell growth stage), 200 mL of a culture of P. pastoris (S-Ph/Pp, prepared in Example 5) in a YPD medium (Glucose 2%, peptone 1%, Yeast Extract 0.5%, grown at 30° C. until OD550 0.3 ̃0.4, about 20 hrs) was inoculated (5 v/v %) into a Basal salts medium (2000 ml) (phosphoric acid 26.7 ml/L, calcium sulfate 0.93 g/L, potassium sulfate 18.2 g/L, Magnesium Sulfate 14.9 g/L, potassium hydrate 4.13 g/L, Glucose 40 g/L), which had been adjusted to pH 5.0 prior to inoculation using 28% NH3.H2O. PTM1 (Cupric sulfate 24 mM, Sodium iodide 0.534 mM, Manganese sulfate 17.8 mM, Sodium molybdate 0.827 mM, boric acid 0.323 mM, Cobalt chloride 2.1 mM, zine chloride 0.147 mM, ferrous sulfate 0.234 mM, biotin 1.64 mM, sulfuric acid 0.188 M) was added to the medium at the volume ratio of 4.37 mL/L medium. The culturing was run with agitation and aeration for about 18-24 hrs, The dissolved oxygen reduced to below 100% due to the growth of cells, and increased to above 80% when the carbon resource was exhausted. At that time, the density of cells reached 90 ̃110 g/L (wet weight).
In stage II (carbon feeding stage), a solution of 25% (w/v)glucose and 12 mL/L PTM1 in distilled water was added at the rate of 28 mL/h over 4 h. The dissolved oxygen was maintained above 20% by adjusting the aeration. At the end of this stage, the density of cells reached 180 ̃220 g/L (wet weight).
Finally, in stage III (induction stage), 12 mL/L PTM1 in methanol was added at the rate of 20 ̃40 ml/h. The concentration of methanol was kept not more than 0.3% (v/v), and the dissolved oxygen was maintained above 20%. Samples of the culture were collected every  ̃10 hrs during the induction stage for enzymatic activity analysis and SDS-PAGE (see FIG. 9).
The cell density reached an OD600 of about 150 at 194th hour, and the phytase activity reached 4,770 U/mL at 209th hour (See FIG. 8).
The culture samples were centrifuged at 25,000 g for 10 min. The obtained supernatants were run on SDS-PAGE and analyzed for protein concentration. The protein concentration was measured by Folin-phenol method as previously described (Lowry O H et al. PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT. J Biol Chem. 1951, 193 (1):265-75). The protein concentration of the final sample at about 198th hour reached 2.7 g/L. The end point and discharge time were determined by monitoring the growth and production rates as commonly known and practiced in the art.
As shown in FIG. 9, the richest band on the SDS-PAGE (12% gel) was identified via phytase activity assay (see example 7) to be the expressed phytase secreted into the culture. The concentration of the expressed phytase increased with the progress of the fermentation. The protein had a molecular weight of around 52 kDa.
Example 7
Purification of the Expressed Phytase
All the following operations were carried out at 4° C. The Non-K12 phytase was expressed in Pichia pastoris (Example 6). The culture was then centrifuged at 25,000 g for 10 min. The supernatant was mixed with 75% saturated ammonium sulfate under agitation for 2 hrs, and the mixture was centrifuged at 25,000 g for 15 mins. The pellet was then resuspended in 20 ml 20 mM acetate buffer, pH 5.0, and dialyzed overnight against the same buffer.
The dialysate was loaded onto a CM-Sephadex C-50 column (Pharmacia) equilibrated with 20 mM acetate buffer, pH 5.0. After the column was washed with the same buffer to remove unbound compounds, the bound phytase was eluted with a linear gradient of 0-500 mM NaCl in the starting buffer (pH 5.0 20 mM HAC-NaAC buffer).
The fractions exhibiting the highest phytase activity (see below) were pooled and dialyzed against deionized water overnight. The dialysate was used in the following studies.
Sequencing of the purified protein indicated a correct amino acid sequence as set forth in SEQ ID NO: 3.
Example 8
Characterization of the Non-K12 Phytase Activity
Determination of Phytase Activity
The protein concentration of phytase was measured as previously described (Lowry O H et al. PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT. J Biol Chem. 1951, 193 (1):265-75).
Determination of the phytase activity is based on the colorimetrical quantification at 700 nm of free phosphorus released by the hydrolysis of phytate using ammonium molybdate as the color reagent. One “U” is the amount of enzyme that liberates 1 μmol inorganic orthophosphate per minute under standard assay conditions (pH 5.0; temperature 37° C.; and substrate concentration, sodium phytate at 0.005 mol/L; also see GB/T18634-2002, and Study On The Determination Condition Of Phytase Activity By Molybdenum Yellow And Molybdenum Blue Method, Zou DaQiong. CHINA FEED, 2005 (03)).
The specific enzyme activity was calculated by the following formula:

Uc=U/C 

    
    
        Uc—phytase specific activity, U/mg;
        U—phytase activity, U/ml;
        C—the protein concentration of phytase, mg/ml
    
    


The specific activity of the phytase from Example 6 purified above was measured to be 3146 U/mg
Optimum pH and Temperature
The phytase activity was measured at different pH under standard assay conditions (see above), using pH 3.0 ̃6.0 in a 0.05 mol/L sodium acetate/acetic acid buffer and pH 6.0 ̃9.0 in a 0.04 mol/L Barbital Sodium-HCl buffer. The phytase activity was measured at different temperatures under standard assay conditions (see above). The measurements were carried out with the purified phytase in Example 7. The results were shown in FIGS. 6 and 7.
The optimum pH of the purified phytase was 3 to 6 (See FIG. 6). The optimum temperature of the purified phytase was around 55° C. (See FIG. 7).
Effects of pH and Temperature on the Expressed Phytase
The Non-K12 phytase purified in Example 7 was diluted in buffers with varying pH values (pH 3.0 ̃6.0, 0.05 mol/L HAC/NaAC buffer and pH 6.0 ̃9.0, 0.04 mol/L Barbital Sodium-HCl buffer) to a final concentration of 1.5 mg/ml, and incubated in 37° C. for 1 hr. The phytase was diluted in a 0.05 mol/L HAC-NaAC buffer at pH 5.0 and incubated at different temperatures for 1 h. The phytase activity was measured under standard assay conditions (see above). The results were shown in FIGS. 12 and 13.
As shown in FIG. 12, the Non-k12 phytase of the invention was stable against low pH of 2-5.5 (as shown in FIG. 12). The thermo-stability of the Non-k12 phytase of the invention was shown in FIG. 13, at least 17% activity was maintained at a temperature up to 80° C. after 1 hr, at least 33% activity at a temperature up to 90° C. after 1 hr, and at least 26% activity at a temperature up to 100° C. after 1 hr (FIG. 13).
Phytase Resistance to Pepsin and Pancreatic Protease
The Non-k12 phytase of the invention purified in Example 7 was incubated with pepsin (300 U/ml, pH 2.5, 37° C., 2 hs) and pancreatic protease (30 U/ml, at pH 7.0, 37° C., 2 hs), respectively. In either treatment, at least 91% activity of the purified Non-K12 phytase was maintained (as showed in FIG. 10). The control (100% activity) was a parallel incubation without the protease treatment.
Size of the Expressed Phytase
The SDS-PAGE (12% gel) indicated that the phytase expressed in P. pastoris (Example 6) showed a molecular weight around 52 kDa. The expressed phytase in P. pastoris was believed to be partially glycosylated, as shown by the blurred band on SDS-PAGE (as shown in FIG. 11).
Effects of Temperature on the Dried Phytase Formulation
The Non-K12 phytase purified in Example 7 was formulated (25% starch, 12.5% dextrin, 5% sodium chloride, 5% potassium sorbate, 1.5% calcium sulfite (w/w)), and spray-dried. The dried phytase formulation was incubated at different temperatures for 1 hr. The phytase activity was measured under standard assay conditions (see above).
The thermo-stability of formulated lyophilized phytase was shown in FIG. 14, 70% activity remained even at a temperature as high as 100° C. after 1 hr.
Example 9
Comparison of Non-K12 Phytase with E. Coli K12 Phytase and the Fungal Phytase from Aspergillus niger 
The fungal phytase from Aspergillus niger (Robert F. M. Van Gorcom et al. Cloning and expression of phytase from aspergillus. U.S. Pat. No. 5,436,156) and E. coli K12 phytase (Jay M. Short et al. Recombinant bacterial phytases and uses thereof. U.S. Pat. No. 6,855,365) were tested on their stability under varying pH values and temperatures as above (Example 8, “Effects of pH and temperature on the expressed phytase”). The obtained results were compared with those obtained with the Non-K12 phytase of the invention prepared in Example 6 and purified as in example 7. The comparisons were shown in FIGS. 15 and 16. As indicated, the Non-K12 phytase of the invention was significantly more thermostable (FIG. 15) and more tolerant to acid environment (FIG. 16).

## Claims

1. An isolated cDNA comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; and
b) the nucleotide sequence completely complementary to a).

2. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, wherein the nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence consisting of nucleotides 67-1296 of SEQ ID NO: 1;
b) the nucleotide sequence consisting of nucleotides 1-1230 of SEQ ID NO: 2; and
c) the nucleotide sequence completely complementary to a) or b).

3. An expression vector, comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 3; and
b) the nucleotide sequence completely complementary to a).

4. The vector according to claim 3, wherein said vector is selected from the group consisting of pTrcHis2-PhE, pPIC9K-PhE, pMET-PhE and pKLAC-PhE.

5. An isolated cell comprising an expression vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 3; and
b) the nucleotide sequence completely complementary to a).

6. The cell according to claim 5, wherein said cell is a yeast cell.

7. The cell according to claim 5, wherein the cell is of the species selected from the group consisting of Escherichia coli, Pichia pastoris, Pichia methanolica and Kluyeromyces lactis.

8. The cell according to claim 5, wherein the cell is derived from a strain selected from the group consisting of E. coli MG1655, P. pastoris GS115, P. methanolica PMAD16 and Kluyeromyces lactis GG799.

9. The cell according to claim 5, wherein the cell is a strain selected from the group consisting of E. coli MG1655 transformed with pTrcHis2-PhE, P. pastoris SMD1168 transformed by pPIC9K-PhE, P. methanolica PMD16 transformed by pMET-PhE and Kluyeromyces lactis GG799 transformed by pKLAC-PhE.

10. An isolated_polypeptide comprising the amino acid sequence of SEQ ID NO: 3, and having phytase activity.

11. A process for production of a Non-K12 Escherichia coli phytase by fermentation, comprising a step of cultivating a cell according to claim 5, that is a strain selected from the group consisting of E. coli MG1655 transformed with pTrcHis2-PhE, P. pastoris SMD1168 transformed by pPIC9K-PhE, P. methanolica PMD16 transformed by pMET-PhE and Kluyeromyces lactis GG799 transformed by pKLAC-PhE, under a condition effective for expression to obtain a polypeptide having phytase activity.