Synthetic phytase variants

Abstract

The present invention relates to a synthetic phytase which comprises (a) the amino acid sequence as set forth in SEQ ID NO. 1, (b) a variant of (a) having at least 70% sequence identity to SEQ ID NO. 1, or (c) a fragment, fraction or shuffled variant of (a) or (b) maintaining phytase activity, with the proviso that the phytase comprises one or more amino acid substitutions at one or more positions relative to SEQ ID NO. 1 selected from the group consisting of V13, T30, N37, K45, L46, T81, N83, A89, A113, Q121, Q122, A123, H128, K131, K139, A142, H143, Q162, S164, A166, S170, Q182, N184, G186, K187, L188, A194, M195, A200, I201, N202, D204, N206, K207, A209, S218, T219, L225, H228, K234, N239, E243, S248, Q256, F257, M260, S261, N270, P288, P292, A311, A314, L319, S320, W321, K344, K347, M355, L359, Q365, P367, E372, T388, Q404, and N405.

Description

[0001] Synthetic phytase variants

[0002] Reference to sequence listing submitted as a compliant xml 1.0 format file (.xml)

[0003] Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an XML 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference. For the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

[0004] Field of the invention

[0005] The present invention relates to the field of phytases.

[0006] Background

[0007] Phytases (myo-inositol hexakisphosphate phosphohydrolase) are phosphatase enzymes that catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate) - an indigestible, organic form of phosphorus that is found in grains and oil seeds - and releases a usable form of inorganic phosphorus. Phytases have been found to occur in animals, plants, fungi and bacteria. Phytases used in feed applications were initially of fungal origin but today are dominated by bacterial enzymes. Phytases can be grouped based on their pH activity optima, as acid or alkaline phytases, their catalytic mechanism and enzyme fold, as histidine acid, cysteine, or purple acid phosphatases, or B-propeller phytases, or by the stereoselectivity of phytate degradation as 3- or 6- phytase. The EC database accounts for this stereoselectivity by defining two classes, 3.1.3.8 and 3.1.3.26, the two classes that are used commercially. Phytases belonging to the group 3.1.3.72 (PTP-like phytases) and 3.1.3.2 (purple acid phosphatase) are rare und not exploited commercially.

[0008] Phytic acid and its metabolites have several important roles in seeds and grains, most notably, phytic acid functions as a phosphorus store, as an energy store, as a source of cations and as a source of myo-inositol (a cell wall precursor). Phytic acid is the principal storage form of phosphorus in plant seeds and the major source of phosphorus in the grain-based diets used in intensive livestock operations. The organic phosphate found in phytic acid is largely unavailable to the animals that consume it, but the inorganic phosphate that phytases release can be easily absorbed. Ruminant animals can use phytic acid as a source of phosphorus because the bacteria that inhabit their gut are well-characterized producers of many types of phytases.

[0009] However, monogastric animals cannot use phytic acid as a major source of phosphorus, due to a lack of sufficient amount of phytase produced by the gastro intestinal microbiota, and it is excreted in the feces.

[0010] In ruminants (cattle, sheep), phytase is produced by bacteria found in the gut making it possible for them to use the phytic acid found in grains as a source of phosphorus. Non-ruminants (monogastric animals) like humans, dogs, birds, etc. do not produce phytase. Research in the field of animal nutrition has put forth the idea of supplementing feed with phytase so as to make available to the animal phytate-bound nutrients like calcium, phosphorus, other minerals, carbohydrates, and proteins.

[0011] Phytase is used as an animal feed supplement - often in poultry and swine - to enhance the nutritive value of plant material by liberation of inorganic phosphate from phytic acid (myoinositol hexakisphosphate). Phytase can be purified from transgenic microbes and has been produced recently in transgenic canola, alfalfa and rice plants. Phytase can also be produced on a large scale through cellulosic biomass fermentation using genetically modified (GM) yeast. In many applications, improved stability of the enzyme is a significant advantage. Improved thermostability helps to increase the processability of the respective phytase, because the latter oftentimes undergoes thermal treatment during the manufacturing process.

[0012] This applies, inter alia, for the use of phytases in animal feed. During feed processing, the feed is often subjected to heat, e.g., by application of steam, to reduce or eliminate pathogens, increase storage life of the feed and optimized utilization of the ingredients leading to improved feed conversion. The conditioning time can vary from a few seconds up to several minutes depending on the type and formulation of the feed. The temperature during conditioning typically ranges from 70°C to 100°C. After conditioning, the feed is sometimes extruded through a pelleting die, which for a short time raises the temperature of the feed incrementally due to heat dissipation caused by friction.

[0013] Yet in other applications, phytase enzymes are exposed to heat as well. Because phytases are proteins, they are susceptible to denaturation by heat and pressure. Denaturing essentially alters the structure of the enzyme, resulting in decreased activity levels and decreased efficacy of the enzyme.

[0014] There are different ways to improve phytase stability or protect phytases from thermal impact. In animal feed applications, one option is post-pellet liquid application, which is relatively complex and expensive because it requires the purchase and installation of specialized equipment, space in which to store the liquid enzyme and careful calculation of the amount of enzyme to apply.

[0015] Another option is the application of a protective coating before pelleting of the synthetic phytase with other ingredients (e.g., in feed). This approach, which is for example described in Partridge (2007), may reduce the efficacy of the enzyme because the coating may not fully dissolve, e.g., in the digestive tract of the animal. It is furthermore difficult to achieve a coating design that can withstand the high heat and moisture content of the pelleting process, but subsequently dissolve in the lower temperature and higher moisture conditions, e.g., in the animal’s gut.

[0016] Another option is to use intrinsically thermostable phytases. These phytases can theoretically be derived from thermophilic and hyperthermophilic organisms and have unique structure and function properties of high thermostability. However, these phytases may suffer from other limitations, like suboptimal activity, specificity, bioavailability, pH-range or processability.

[0017] In WO2022106483 Al the inventors of the present invention have shown that wildtype phytases of Obesumbacterium proteus and Hafnia alvei can be used to create synthetic phytase variants having increased stability compared to the respective wildtype enzymes. The content of WO2022106483 Al is incorporated herein by reference in its entirety. However, the resulting synthetic phytases may still suffer from insufficient thermostability and other limitations, like suboptimal activity, stability, specificity, bioavailability, pH-range or processability.

[0018] It is hence one object of the present invention to provide synthetic phytase variants which do not suffer from the above discussed limitations.

[0019] Summary of the invention

[0020] These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.

[0021] Brief Description of the Figures

[0022] Figure 1 : Structure of Phytate

[0023] Figure 2: Scheme depicting the reaction catalysed by phytase

[0024] Figure 3: Properties of the synthetic phytase backbone (OHO-phytase) used in the present invention vs the respective wildtype phytases. See examples 1 - 4 for the respective experimental description.

[0025] Embodiments of the invention

[0026] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and / or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word “comprising” does not exclude other elements or steps.

[0027] It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

[0028] It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

[0029] According one aspect of the invention, a synthetic phytase is provided which comprises

[0030] (a) the amino acid sequence as set forth in SEQ ID NO. 1,

[0031] (b) a variant of (a) having at least 70% sequence identity to SEQ ID NO. 1, or

[0032] (c) a fragment, fraction or shuffled variant of (a) or (b) maintaining phytase activity, with the proviso that the phytase comprises one or more amino acid substitutions at one or more positions relative to SEQ ID NO. 1 selected from the group consisting of

[0033] V13, T30, N37, K45, L46, T81, N83, A89, A113, Q121, Q122, A123, H128, K131, K139, A142, H143, Q162, S164, A166, S170, Q182, N184, G186, K187, L188, A194, M195, A200, 1201, N202, D204, N206, K207, A209, S218, T219, L225, H228, K234, N239, E243, S248, Q256, F257, M260, S261, N270, P288, P292, A311, A314, L319, S320, W321, K344, K347, M355, L359, Q365, P367, E372, T388, Q404, and N405.

[0034] In WO2022106483 Al the inventors of the present invention have shown that wildtype phytases of Obesumbacterium proteus and Hafnia alvei can be used to create synthetic phytase variants having increased stability compared to the respective wildtype enzymes. The content of WO2022106483 Al is incorporated herein by reference in its entirety.

[0035] One of the synthetic phytase variants, called OHO-phytase, is used as backbone in the present invention and corresponds to SEQ ID NO 1 of the present invention. Some properties of the synthetic phytase backbone (OHO-phytase) and the respective wildtype phytases are shown in figure 3.

[0036] Note that, while the numbering set forth above refers to SEQ ID NO 1, the claimed synthetic phytase can be a fragment, fraction or shuffled variant thereof maintaining phytase activity. In such case, the resulting amino acid sequence is shorter than that of SEQ ID NO 1, while the numbering of the mutant residues still refers to the full length SEQ ID NO 1.

[0037] The term “shuffled variant” relates to a combination of such fragment or fraction with one or more fragments from other homologous enzymes, as long as such combination maintains phytase activity.

[0038] The term “homologous enzyme” describes enzymes belonging to the same structural fold as the phytase and at least 40% sequence identity.

[0039] As used herein, the term “maintaining its phytase activity” means, for example, that the respective variant has a phytase activity of >50 % compared to that of the phytase of SEQ ID NO 1.

[0040] In some embodiments, the synthetic phytase variant has > 71, > 72, > 73, > 74, > 75, > 76, > 77, > 78, > 79, > 80, > 81, > 82, > 83, > 84, > 85, > 86, > 87, > 88, > 89, > 90, > 91, > 92, > 93, > 94, > 95, > 96, > 97, > 98, most preferably > 99 % sequence identity to SEQ ID No. 1. “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence (e.g., a polypeptide), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0041] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (z.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0042] One suitable algorithm to determine sequence identities is the BLAST algorithm. Another algorithm to determine sequence identities is the Clustal Omega algorithm.

[0043] The disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. Optionally, the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein. According to one embodiment of the invention a synthetic phytase is provided, which comprises one or more amino acid substitutions selected from the group consisting of V13 A, T30L, T30M, T30C, N37H, N37Y, K45P, L46W, T81H, N83V, A89T, A113P, Q121E, Q121S, Q122W, A123I, H128G, K131T, K131S, K131G, K139P, A142V, A142I, A142L, H143A, Q162S, S164E, S164N, A166V, S170R, Q182K, N184Y, N184F, G186T, K187R, L188D, L188E, A194M, A194E, M195F, A200C, A200V, A200E, 1201W, N202S, D204A, N206R, K207C, K207Q, A209C, S218G, T219E, L225M, H228Y, K234V, K234C, N239K, E243Y, E243F, S248C, Q256Y, Q256H, Q256D, F257I, F257T, M260T, M260E, S261E, S261C, N270E, N270Q, P288W, P292D, A311H, A314G, L319Y, S320D, W321Y, K344S, K347F, M355I, L359M, Q365C, P367T, E372A, E372M, T388L, Q404M, and N405T.

[0044] These individual amino acid substitutions are shown in Tables 1-3. Note that, while the numbering set forth above refers to SEQ ID NO 1, the claimed phytase can be a fragment, fraction or shuffled variant thereof maintaining phytase activity. In such case, the resulting amino acid sequence is shorter, or longer, than that of SEQ ID NO 1, while the numbering of the mutant residues still refers to the full length SEQ ID NO 1.

[0045] Some of these substitutions cause a high AIT50 when introduced individually into the phytase as set forth in SEQ ID NO 1, and are therefore preferred, while others have a high occurrence in the combinatorial and distinct clones of Table 5 and some combinations, which have a combination of individual substitutions with a high overall AIT50.

[0046] Some can interchangeably be used to stabilize the enzyme and some combinations results in other traits that are relevant for the production or performance in feed, like fermentation titers, pH profile, pH and pepsin stability, or stability against and performance under higher ionic strength.

[0047] In some embodiments, the synthetic phytase variant has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 or 83 amino acid substitutions relative to the phytase of SEQ ID NO 1. According to one or more embodiments of the invention, at least one amino acid substitution as discussed above is a conservative amino acid substitution.

[0048] A conservative amino acid substitution44has a smaller effect on enzyme function than a nonconservative substitution. Although there are many ways to classify amino acids, they are often sorted into six main groups on the basis of their structure and the general chemical characteristics of their R groups.

[0049] In one embodiment, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with

[0050] • basic side chains (e.g., lysine, arginine, histidine),

[0051] • acidic side chains (e.g., aspartic acid, glutamic acid),

[0052] • uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),

[0053] • nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),

[0054] • beta-branched side chains (e.g., threonine, valine, isoleucine) and

[0055] • aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0056] Other conserved amino acid substitutions can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide. Thus, a predicted nonessential amino acid residue in a HR domain polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family or homologues across families (e.g. asparagine for aspartic acid, glutamine for glutamic acid). Conservative changes can further include substitution of chemically homologous nonnatural amino acids (i.e. a synthetic non-natural hydrophobic amino acid in place of leucine, a synthetic non-natural aromatic amino acid in place of tryptophan).

[0057] According to one embodiment of the invention a synthetic phytase is provided, which comprises two or more of the recited amino acid substitutions. According to one embodiment of the invention a synthetic phytase is provided, which comprises one or more, preferably two or more, more preferably three or more, more preferably four or more, more preferably five or more, most preferably six or more amino acid substitutions selected from the group consisting of T30L, N37H, K45P, Q121E, H143A, S164E, L188E, A194M, M195F, I201W, N202S, H228Y, K234V, E243Y, Q256Y, M260T, S261E, N270E, P288W, A311H, M355I, L359M, and Q365C.

[0058] Table 5 shows so-called “distinct clones” or “combinatorial clones” which have combinations of the individual mutations set forth above.

[0059] As used herein, the term “combinatorial clone or variant” means a clone or variant screened from a recombination library. Such a recombination library contains a population carrying different amounts and mutations selected from the group of table 1.

[0060] As used herein, the term “distinct clone or variant” means a clone constructed containing a defined set of mutations selected from the group of table 1 in a rational approach.

[0061] According to one embodiment of the invention a synthetic phytase is provided, which comprises one or more amino acid substitutions selected from the group consisting of S164E, M195F, S218G, L225M, Q256D, and S261E.

[0062] According to one embodiment of the invention a synthetic phytase is provided, which comprises a set of substitutions at selected residues set forth in SEQ ID NO. 1, which set is selected from the group consisting of a) VBA, A89T, S164E, M195F, S261E; b) L46W, S164E, M195F, S261E; c) VBA, S164E, M195F, S261E; d) VBA, S164E, S170R, M195F, S261E; e) S164E, Q182K, M195F, S261E; f) A113P, S164E, M195F, S261E; and / or g) VBA, S164E, K187R, M195F, S261E. These sets of simultaneously substituted residues occur in specific distinct or combinatorial clones which are particularly preferred (consensus mutations). See Table 5. For these reasons, these sets of simultaneously substituted residues seem to be particularly synergistic when it comes to improvement of stability.

[0063] According to one embodiment of the invention a synthetic phytase is provided, which demonstrates at least one altered or improved characteristic compared to phytases that lack said one or more amino acid substitutions.

[0064] According to one embodiment of the invention said altered or improved characteristic is increased thermostability (IT50) compared to phytases that lack said one or more amino acid substitutions.

[0065] The term "thermostability" as used herein refers to the ability of a synthetic phytase variant to have equal or greater activity compared to synthetic phytase backbone (OHO-phytase) under standard conditions, and an increased ability to retain activity after exposure to increased temperatures.

[0066] The thermostability of an enzyme is usually determined by measuring the inactivation temperature (IT 50). The "inactivation temperature" is defined as the temperature at which the residual activity of the enzyme after incubation for a certain duration and subsequent cooling to room temperature is 50% of the residual activity of the same enzyme incubated for the same duration under the same conditions at room temperature.

[0067] According to one embodiment a synthetic phytase is provided, which has an IT50 of between

[0068] > 60°C and < 100°C, between > 80°C and < 100°C, between > 90°C and < 100°C, or between

[0069] > 90°C and < 95°C.

[0070] According to one embodiment of the invention said altered or improved characteristic is increased specific activity compared to phytases that lack said one or more amino acid substitutions.

[0071] Phytase activity is commonly expressed in phytase units (FTU). One phytase unit (FTU) is defined as the amount of enzyme required to release 1 micromole of inorganic phosphate from a Sodium phytate substrate in one minute under the reaction conditions as defined in example 1.

[0072] The specific activity of an enzyme generally refers to the purity of an enzyme in a protein mixture, measuring the activity of an enzyme in one milligram of total protein. The term “specific phytase activity”, also called “specific activity”, as used herein was defined as described in example 5.

[0073] According to one embodiment of the invention a synthetic phytase is provided, which has an increased specific activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%.

[0074] According to one embodiment, said altered or improved characteristic is increased stability against protease digestion (also called “protease resistance”) compared to the synthetic phytase backbone (OHO-phytase).

[0075] The stability against protease digestion can be measured by exposure to the protease KI (Kumamolisin AS1, as described in WO2017220776A1). Such stability is quite important because very often phytases are combined with proteases and then used in a specific purpose. Hence, the risk exists that the protease will deactivate, or even digest, the concomitantly added phytase.

[0076] In addition or as an alternative, the stability against protease digestion can be measured by exposure to the protease pepsin. Such stability is quite important because when used in feed applications, the phytase will have to survive and be active in the gastric environment where pepsin is present and active. Hence, the risk exists that the protease will deactivate, or even digest, the concomitantly added phytase.

[0077] As a result of any enzyme optimization process, the optimization of one parameter can affect other parameters. For example, increasing thermostability can at the same time also increase or reduce protease stability or pH stability.

[0078] In the present case of a phytase for animal feed purposes, in one embodiment, thermostability may have priority over protease stability or pH stability. This means that in case that, in a given shuffling product, a substantial increase in IT50 is achieved, slight losses in protease stability or pH stability may be acceptable (e.g., losses of -10 or -20 %).

[0079] According to one embodiment, said increased stability is increased pH stability (also called “pH resistance”) compared to the synthetic phytase backbone (OHO-phytase).

[0080] The term "pH stability " as used herein refers to the ability of an enzyme to have equal or greater activity compared to the wild type under standard conditions, and an increased ability to retain activity after exposure to acidic conditions (e.g., pH 4.0 or lower).

[0081] According to one other aspect of the invention, a nucleic acid molecule is provided which encodes a synthetic phytase according to the above description.

[0082] In one embodiment, such nucleic acid molecule is codon optimized for the expression in eukaryotic hosts, preferably from the group of yeasts and fungi, most preferably for Pichia pastoris, Aspergillus sp. and Trichoderma sp.

[0083] Codon optimization is a useful technology for improving the expression of heterologous proteins. Indeed, there is a large difference between the codon usage of the host cell genome sequence and the native heterologous protein encoding sequence, which will obviously affect the expression of recombinant proteins. Some reports have shown that the production of the target proteins was often increased an average of 1- to 5-fold by optimizing the heterologous protein-encoding sequence based on the codon bias of the host cell.

[0084] Codon optimization is a species-specific process, and the skilled artisan is able to derive, from respective literature reference, suitable codon optimizations schemes for a given expression host. Respective teachings are provided, e.g., in Yu et al (2013), Rosemary et al (2017) and Yang and Liu (2010), the contents of which are incorporated herein by reference for enablement purposes.

[0085] According to one other aspect of the invention, a plasmid or vector system comprising said nucleic acid molecule is provided. According to one other aspect of the invention, a host cell capable of heterologous protein expression is provided, said host cell comprising a nucleic acid molecule or a plasmid or vector system according to the above description.

[0086] According to yet one other aspect of the invention, the heterologous protein expression is achieved by insertion of the said nucleic acid molecule into the genome of the said host cell. The genomic insertion is achieved using homologous recombination based state-of-the-art techniques and genome-editing using programmable nucleases, e.g. CRISPR-Cas, Zn-finger or TAL endonucleases.

[0087] In different embodiments, such host cell is at least one selected from a bacterial cell, fungal cell or a yeast cell.

[0088] Exemplary bacterial cells include E. coll and other Enterob acteriaceae, Escherichia sp., Campylobacter sp., Wolinella sp., Desulfovibrio sp., Vibrio sp., Pseudomonas sp. Bacillus sp., Bacteroides sp., Listeria sp., Staphylococcus sp., Streptococcus sp., Peptostreptococcus sp., Megasphaera sp., Pectinatus sp., Selenomonas sp., Zymophilus sp., Actinomyces sp., Arthrobacter sp., Frankia sp., Micromonospora sp., Nocardia sp., Propionibacterium sp., Streptomyces sp., Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Acetobacterium sp., Agrobacterium sp., Aliivibrio sp., Eubacterium sp., Haloarcula sp., Halobacterium sp., Heliobacterium sp., Heliospirillum sp., Sporomusa sp., Spiroplasma sp., Ureaplasma sp., Erysipelothrix, sp., Corynebacterium sp. Enterococcus sp., Clostridium sp., Mycoplasma sp., Mycobacterium sp., Actinobacteria sp., Salmonella sp., Shigella sp., Moraxella sp., Helicobacter sp, Paracoccidioides sp., Stenotrophomonas sp., Micrococcus sp., Neisseria sp., Bdellovibrio sp., Hemophilus sp., Thermus sp., Klebsiella sp., Proteus sp., Enter obacter sp., Serratia sp., Citrobacter sp., Pseudomonas sp., Proteus sp., Rhodobacter sp., Rhodopseudomonas sp., Rhodospirillum sp., Serratia sp., Yersinia sp., Acinetobacter sp., Actinobacillus sp. Bordetella sp., Brucella sp., Capnocytophaga sp., Cardiobacterium sp., Eikenella sp., Francisella sp., Haemophilus sp., Kingella sp., Pasteurella sp., Flavobacterium sp. Xanthomonas sp., Burkholderia sp., Aeromonas sp., Plesiomonas sp., Legionella sp. and alpha-proteobacteria such as Wolbachia sp., Comamonas sp., Pyrobaculum sp., Sinorhizobium sp., Cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria, Gram-negative cocci, Gram negative bacilli. In a preferred embodiment the bacterial cell is Bacillus subtilis. Exemplary fungi or yeast cells include Pichia sp., Aspergillus sp., Kluyveromyces sp., Saccharomyces sp., Candida sp., Trichoderma sp., Penicillium sp., Neurospora sp., Chrysosporium sp., Cladosporium sp., Phytophthora sp., Scytalidium sp., more preferred Saccharomyces cerevisiae, Pichia pastoris, Aspergillus nidulans, Aspergillus niger, Trichoderma reesei, Kluyveromyces lactis, Kluyveromyces marxianus, and Neurospora crassa .

[0089] In one embodiment the yeast cell is Pichia pastoris. This methanol utilizing yeast is a widely used eukaryotic production host for the expression of recombinant proteins, known also as Komagataella phaffii.

[0090] In further embodiments, such host cell is a eukaryotic host cell selected from the group consisting of plant cells, insect cells or mammalian cells.

[0091] According to one other aspect of the invention, a feed additive, feed ingredient, feed supplement, and / or feedstuff comprising a synthetic phytase according to the above description is provided.

[0092] According to one other aspect of the invention, the use of a synthetic phytase according to the above description for the manufacture of a feedstuff is provided.

[0093] Such feed additive, feed ingredient, feed supplement, and / or feedstuff is preferably meant for monogastric poultry, pig, fish and aquaculture, where it helps to increase protein digestion and absorbance from the feedstuff, plus degrade proteinogenic compounds which are detrimental for animal health or digestion.

[0094] According to one other aspect of the invention, a process of developing a synthetic phytase according to the above description is provided, which process comprises: a) mutagenizing a DNA, cDNA, RNA or mRNA sequence encoding for the amino acid sequence as set forth in SEQ ID NO. 1, to obtain one or more mutated sequences, b) expressing one or more mutated sequences thus obtained, in a suitable expression system, to obtain one or more mutated phytase enzymes, and c) testing the one or more mutated phytase enzymes sequences for stability, preferably thermostability, pH stability or stability against protease digestion. Preferably, in said method, the encoding nucleic acid sequence and / or the amino acid sequence of one or more phytase variants is determined. For this purpose, routine methods from the prior art can be used.

[0095] Experiments and Figures

[0096] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope. All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0097] Example 1: Phytase activity assay

[0098] Assay buffer: 100 mM Na- Acetate pH 5.5

[0099] 10 mM sodium phytate

[0100] 5 mM CaCh

[0101] Stop solution: 20% (w / v) trichloroacetic acid (TCA)

[0102] Color reagent solution: 1 part iron sulfate solution (388 mM FeSO4x 7H2O in 2.5 M H2SO4)

[0103] 4 parts ammonium molybdate solution

[0104] (12 mM (NH4)6MO7O24 X 4H2O in 2.5 M H2SO4)

[0105] Samples were diluted in ddH2O containing 0.01% Triton-X-100 according to their volumetric activity. Diluted samples were than assayed in Nunc 384 well clear flat bottom plate. The reaction was started by adding 1 part assay buffer to one part diluted sample and incubation at 37°C for 30 min in a thermoshaker. The reaction was subsequently stopped by adding an equal volume of stop solution. The color reaction was started by adding an equal volume of color reagent solution and incubation at 25°C in the dark for 20 minutes. The absorbance at 650 nm was measured and the amount of phosphate liberated by the phytase was evaluated against an inorganic phosphate standard curve measured under the same conditions.

[0106] Example 2: IT50

[0107] IT50 defines the temperature where 50% of the activity is inactivated under the conditions described below. Although not equivalent to, it is a measure for the thermal stability in the application, e. g. pelleting conditions.

[0108] Thermal incubation buffer: 100 mM Na- Acetate pH 5.5

[0109] 0.5 mg mL'1digested BSA

[0110] 5 mM CaCl2

[0111] 0.01% Triton-X-100

[0112] The supernatants of submerse cultivation of bacterial or fungal strains producing the phytase or phytase variant of interest were diluted in thermal incubation buffer. Aliquots of 50 pl were incubated for 10 minutes in a PCR machine at an appropriate thermal gradient. A second aliquot was incubated at 25°C. After the thermal incubation the sample is kept at 25°C and assayed for residual activity by the phytase activity assay described under example 1. Results are shown in tables 1 and 5.

[0113] Example 3: pH activity profile

[0114] Dilution buffer: 0.5 mg mL'1digested BSA 0.001% Triton-X-100

[0115] Assay buffer pH 2.0-3.5: 250 mM Glycin-HCl

[0116] 5.6 mM phytate

[0117] 5.6 mM CaCl2

[0118] Assay buffer pH 4.0-5.5: 250 mM sodium acetate

[0119] 5.6 mM phytate

[0120] 5.6 mM CaCl2

[0121] Assay buffer pH 6.0-7.0: 250 mM MES

[0122] 5.6 mM phytate

[0123] 5.6 mM CaCl2 Supernatant of submerse cultivation of bacterial or fungal strains producing the phytase or phytase variant of interest were diluted according to their volumetric activity in dilution buffer. The activity was measured according to the assay described under example 1, with modification of the assay buffers for the required pH. The reaction was started by adding 8 parts of one of the above buffers to provide activity measurements at ambient pH from pH 2.0 to 7.0. The reaction was stopped, and inorganic phosphate was measured as described in example 1. Results are shown in table 5.

[0124] Example 4: pH / Pepsin / KI stability

[0125] The procedure provides information about the resistance against acid proteases, as residual activity. Acid proteases tested are pepsin and the acid protease Kumamolysin AS (KI; see for example European patent publications EP 16206367, EP 16176044, the contents of which are incorporated herein by reference) as a representative for the acid protease of the protease families S53 and Gl. pH 2.5 assay buffer: 100 mM Glycine-HCl pH 2.5

[0126] 5 mg mL'1digested BSA

[0127] 10 mM CaCh pH 2.5 / Pepsin Assay: 100 mM Glycine-HCl pH 2.5

[0128] 0,5 mg mL'1pepsin

[0129] 5 mg mL'1digested BSA

[0130] 10 mM CaCh pH 2.5 Kumamolysin AS Assay: Glycine-HCl pH 2.5

[0131] 12 U mL'1Kumamolysin As

[0132] 5 mg mL'1digested BSA

[0133] 10 mM CaCh

[0134] One part of the supernatants of submerse cultivation of bacterial or fungal strains producing the phytase or phytase variant of interest were diluted with 1 one part of the above buffers, to test for the stability against acid pH, acid pH and pepsin or acid pH and protease KI. Assays were incubated for 30 minutes at 37°C. The residual activity is measured immediately after the incubation as described in example 1. Results are shown in tables 3 and 5.

[0135] Example 5: Specific activity Assay buffer: Phytate Assay pH 2.0

[0136] 1 M Glycine-HCl pH 2.0

[0137] 10 mM Phytate

[0138] 1 mM CaCh

[0139] Phytate Assay pH 2.5

[0140] 1 M Glycine-HCl pH 2.5

[0141] 10 mM Phytate

[0142] 1 mM CaCh

[0143] Phytate Assay pH 3.0

[0144] 1 M Glycine-HCl pH 3.0

[0145] 10 mM Phytate

[0146] 1 mM CaC12

[0147] Phytate Assay pH 3.5

[0148] 1 M Glycine-HCl pH 3.5

[0149] 10 mM Phytate

[0150] 1 mM CaCh

[0151] Phytate Assay pH 4.0

[0152] 300 mM Na-Acetate pH 4.0

[0153] 10 mM Phytate

[0154] 1 mM CaCh

[0155] Phytate Assay pH 5.5

[0156] 100 mM Na-Acetate pH 5.5

[0157] 10 mM Phytate

[0158] 1 mM CaC12

[0159] Stop solution: 20% (w / v) trichloroacetic acid (TCA)

[0160] Color reagent solution: 1 part iron sulfate solution (388 mM FeSCU x 7H2O in 2.5 N H2SO4) 4 parts ammonium molybdate solution

[0161] (12 mM (NH4)6MO7O24 X 4H2O in 2.5 N H2SO4)

[0162] All C-terminally His-tagged phytases were purified to apparent homogeneity using Ni-IMAC and protein content of each phytase preparation was determined based on A280 using a NanoQuant plate (Tecan, Switzerland). All samples were diluted in ddH2O containing 0.01% Triton-X-100 and 0.5 mg / mL digested BSA according to their volumetric activity. Diluted samples were than assayed in Nunc 384 well clear flat bottom plate. The reaction was started by adding 1 part assay buffer to one part diluted sample and incubation at 37°C for 30-60 min in a thermoshaker. The reaction was subsequently stopped by adding an equal volume of stop solution. The color reaction was started by adding an equal volume of color reagent solution and incubation at 25°C in the dark for 20 minutes. The absorbance at 650 nm was measured and the amount of phosphate liberated by the phytase was evaluated against an inorganic phosphate standard curve measured under the same conditions. Results are shown in tables 2 and 5.

[0163] Example 6: Generation of genetic diversity

[0164] Initial genetic diversity was introduced by randomizing each position of the synthetic phytase backbone (OHO-phytase) having the sequence of SEQ ID NO 1. Mutant enzyme single site saturation libraries were introduced in the gene carried on an E.coli / Bacillus shuttle vector using mutagenesis methods as described in Green & Sambrook (eds), Molecular Cloning, 4thedition, CSHL and suitable mutagenic PCR methods as disclosed in Cadwell and Joyce (PCR Methods Appl. 3

[0194] , 136-140. Phytase enzyme variants were characterized after heterologous expression in Bacillus subtilis and phenotypically optimized variants selected by the screening procedure outlined in Example 7.

[0165] In general, methods to mutagenize a protein, like an enzyme, to obtain a library of mutated proteins members of which may have altered characteristics, are well established. Methods to mutagenize a protein encompass site directed mutagenesis and others, as described e.g. in Hsieh & Vaisvila (2013), content of which incorporated herein by reference for enablement purposes.

[0166] Such methods are sometimes called “directed evolution”, namely when the established library is then screened for particular features. Packer & Liu (2015) provide an overview of the respective methodology, content of which incorporated herein by reference for enablement purposes.

[0167] Example 7: Phenotypically screening for enzyme variants with an optimized phenotype

[0168] The generated genetic diversity either in the initial stage in form of single site saturation libraries or in the subsequent stage in the form of recombination libraries or distinct clones was screened for variants with an optimized phenotype, i.e. increased thermal stability or increased specific activity using the method as described in examples 2 and 5 with adaptations required to run them in a fully automated robotic workstation at high throughput. These were mainly adaptation in incubation times, volumes, substrate and the main adaptation was to select optimized variants not by the thermal inactivation profile on a temperature gradient but by the residual activity after incubation at a single temperature, the temperature which was set to discriminate optimized variants from the average of the genetic diversity. Phytase variants were derived which differed in one or more amino acid positions from SEQ ID NO 1, including two positions, three positions, n positions. Appropriate iterative rounds of the procedures described herein were performed to satisfy the demands of the application

[0169] Example 8:

[0170] The following individual mutations which increase the IT50 compared to the used backbone were identified. The IT50 was analyzed as described above and compared to the IT50 of the used synthetic phytase backbone (OHO-phytase) characterizing the variant by the corresponding AIT50. The synthetic phytase backbone (OHO-phytase) has an IT50 of 65,4°C.

[0171] Table 1: Synthetic Phytase single amino acid substitutions relative to SEQ ID NO 1 and their AIT50 compared to the synthetic phytase backbone (OHO-phytase).

[0172] Several distinct clones and combinatorial clones as shown in Table 5 have substitutions in these positions, leading to synergistic effects in thermal stabilization, when two or more residues thereof are mutated simultaneously.

[0173] Example 9: The following individual mutations which increase the specific activity compared to the used synthetic phytase backbone (OHO-phytase) were identified. The specific activity was analyzed as described above and compared to the specific activity of the used synthetic phytase backbone (OHO-phytase) characterizing the variant by the corresponding change in specific activity.

[0174] Table 2: Synthetic Phytase single amino acid substitutions relative to SEQ ID NO 1 and their spec, activity at pH 4 compared to the synthetic phytase backbone (OHO-phytase)

[0175] Several distinct clones and combinatorial clones as shown in Table 5 have substitutions in these positions, leading to synergistic effects in specific activity, when two or more residues thereof are mutated simultaneously.

[0176] Example 10:

[0177] The following individual mutations were tested for their stability against acid pH, acid pH and pepsin or acid pH and protease KI. The results are given in residual activity which was measured immediately after the incubation.

[0178] Table 3: Synthetic Phytase single amino acid substitutions relative to SEQ ID NO 1 and their stability against acid pH, acid pH and pepsin or acid pH and protease KI.

[0179] Example 11 Distinct variants were generated by introducing selected distinct mutations into the phytase sequence of SEQ ID NO 1 via site-directed mutagenesis. Suitable mutagenic PCR methods known in the art and standard cloning techniques as described in Green & Sambrook (eds), Molecular Cloning, 4thedition, CSHL were used. Phytase enzyme variants were characterized after heterologous expression in Bacillus subtilis and phenotypically analysis using the methods described above.

[0180] Combinatorial libraries, combining mutations identified in the examples provided above and outlined in Tables 1-3 were generated by well-known PCR methods as described in Yolov and Shabarova (1990) and standard cloning techniques as described in Green & Sambrook (eds), Molecular Cloning, 4th edition, CSHL were used. Combinatorial libraries were screened for optimized variants as described in example 3.

[0181] Distinct clones and combinatorial clones comprising two or more substitutions from tables 1-3 were identified, the IT50 and / or the specific activity analyzed as described above and compared to the IT50 and / or the specific activity of the used synthetic phytase backbone (OHO-phytase) characterizing the variant by the corresponding change in IT50 and / or the specific activity. Results are shown in the following Tables 4-6.

[0182] Table 4: Distinct clones comprising selected combinations of substitutions from table 1

[0183] Table 5: Distinct clones comprising selected combinations of substitutions from table 1, and their IT50, specific activity at different pH values (pH-profile) and stability (pH 2,5, pepsin, KI). Specific activity values are given relative to the specific activity of the synthetic phytase backbone (OHO-phytase) as shown in table 6. Stability against acid pH, acid pH and pepsin or acid pH and protease KI is given as residual activity as described in example 4.

[0184] Table 6: Specific activity of the synthetic phytase backbone (OHO-phytase) at different pH values (pH-profile)

[0185] References

[0186] The content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

[0187] Cadwell and Joyce, Mutagenic PCR. PCR Methods Appl. 3, 1994, 136-140

[0188] Hsieh & Vaisvila, Protein engineering: single or multiple site-directed mutagenesis. Methods Mol Biol. 2013;978: 173-86

[0189] Jiangke Yang, Liying Liu, Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris, Journal of Molecular Catalysis B: Enzymatic, Volume 63, Issues 3-4, May 2010, Pages 164-169

[0190] Packer & Liu, Methods for the directed evolution of proteins. Nature Reviews Genetics 16, 379-394 (2015)

[0191] Partridge G, New generation phytase with high thermostability. Pig Progress Vol 23 (4) 2007

[0192] Rosemary A. Cripwell et al., Expression and comparison of codon optimised Aspergillus tubingensis amylase variants in Saccharomyces cerevisiae, FEMS Yeast Research, Volume 17, Issue 4, June 2017, fox040

[0193] Yu, P et al., Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris. Sci Rep 3, 3043 (2013).

[0194] Sequence Listing

[0195] The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in the below table shall be deemed to be the correct ones.

[0196] In some cases, signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php7SignalP. The same applies to His tags or C-Myc tags, if existing.

Claims

What is claimed is:

1. A synthetic phytase comprising(a) the amino acid sequence as set forth in SEQ ID NO. 1,(b) a variant of (a) having at least 70% sequence identity to SEQ ID NO. 1, or(c) a fragment, fraction or shuffled variant of (a) or (b) maintaining phytase activity, with the proviso that the phytase comprises one or more amino acid substitutions at one or more positions relative to SEQ ID NO. 1 selected from the group consisting ofV13, T30, N37, K45, L46, T81, N83, A89, A113, Q121, Q122, A123, H128, K131, K139, A142, H143, Q162, S164, A166, S170, Q182, N184, G186, K187, L188, A194, M195, A200, 1201, N202, D204, N206, K207, A209, S218, T219, L225, H228, K234, N239, E243, S248, Q256, F257, M260, S261, N270, P288, P292, A311, A314, L319, S320, W321, K344, K347, M355, L359, Q365, P367, E372, T388, Q404, and N405.

2. The synthetic phytase according to claim 1, which phytase comprises one or more amino acid substitutions selected from the group consisting ofV I 3 A, T30L, T30M, T30C, N37H, N37Y, K45P, L46W, T81H, N83V, A89T, Al 13P, Q121E, Q121S, Q122W, A123I, H128G, K131T, K131 S, K131G, K139P, A142V, A142I, A142L, H143A, Q162S, S164E, S164N, A166V, S170R, Q182K, N184Y, N184F, G186T, K187R, L188D, L188E, A194M, A194E, M195F, A200C, A200V, A200E, I201W, N202S, D204A, N206R, K207C, K207Q, A209C, S218G, T219E, L225M, H228Y, K234V, K234C, N239K, E243Y, E243F, S248C, Q256Y, Q256H, Q256D, F257I, F257T, M260T, M260E, S261E, S261C, N270E, N270Q, P288W, P292D, A311H, A314G, L319Y, S320D, W321Y, K344S, K347F, M355I, L359M, Q365C, P367T, E372A, E372M, T388L, Q404M, and N405T.

3. The synthetic phytase according to any one of the aforementioned claims, which synthetic phytase comprises two or more of the recited amino acid substitutions.

4. The synthetic phytase according to any one of the aforementioned claims, which synthetic phytase comprises one or more, preferably two or more, more preferably three or more, morepreferably four or more, more preferably five or more, most preferably six or more amino acid substitutions selected from the group consisting ofT30L, N37H, K45P, Q121E, H143A, S164E, L188E, A194M, M195F, I201W, N202S, H228Y, K234V, E243Y, Q256Y, M260T, S261E, N270E, P288W, A311H, M355I, L359M, and Q365C.

5. The synthetic phytase according to any one of the aforementioned claims, which synthetic phytase comprises one or more amino acid substitutions selected from the group consisting ofS164E, M195F, S218G, L225M, Q256D, and S261E.

6. The synthetic phytase according to any one of the aforementioned claims, which synthetic phytase comprises a set of substitutions at selected residues set forth in SEQ ID NO. 1, which set is selected from the group consisting of a) VBA, A89T, S164E, M195F, S261E; b) L46W, S164E, M195F, S261E; c) VBA, S164E, M195F, S261E; d) VBA, S164E, S170R, M195F, S261E; e) S164E, Q182K, M195F, S261E; f) A113P, S164E, M195F, S261E; and / or g) VBA, S164E, K187R, M195F, S261E.

7. The synthetic phytase according to any one of the aforementioned claims, which synthetic phytase demonstrates at least one altered or improved characteristic compared to phytases that lack said one or more amino acid substitutions.

8. The synthetic phytase according to claim 7, wherein said altered or improved characteristic is increased thermostability (IT50) compared to phytases that lack said one or more amino acid substitutions.

9. The synthetic phytase according to claim 8, which has an IT50 of between > 60°C and < 100°C, between > 80°C and < 100°C, between > 90°C and < 100°C, or between > 90°C and < 95°C.

10. The synthetic phytase according to claim 7, wherein said altered or improved characteristic is increased specific activity compared to phytases that lack said one or more amino acid substitutions.

11. The synthetic phytase according to claim 10, which has an increased specific activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%.

12. A nucleic acid molecule encoding a synthetic phytase according to any one of the aforementioned claims.

13. A plasmid or vector system comprising the nucleic acid molecule according to claim 12.

14. A host cell capable of heterologous protein expression, said host cell comprising the nucleic acid molecule according to claim 12 or the plasmid or vector system according to claim 13.

15. A feed additive, feed ingredient, feed supplement, and / or feedstuff comprising a synthetic phytase according to any one of claims 1 - 11.

16. Use of a synthetic phytase according to any one of claims 1 - 11 for the manufacture of a feedstuff17. A process of developing a synthetic phytase according to any one of claims 1 - 11, which process comprises: a) mutagenizing a DNA, cDNA, RNA or mRNA sequence encoding for the amino acid sequence as set forth in SEQ ID NO. 1, to obtain one or more mutated sequences, b) expressing one or more mutated sequences thus obtained, in a suitable expression system, to obtain one or more mutated phytase enzymes, andc) testing the one or more mutated phytase enzymes sequences for stability, preferably thermostability, pH stability or stability against protease digestion.

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