Patent Application
20240336902
The present disclosure generally relates to field of enzyme technology. The disclosure relates particularly, though not exclusively, to phytase variants having an improved IP4 degradation activity. The present phytase variants are useful in various applications where degradation of phytate is desired, such as in feedstuffs.
This section illustrates useful background information without admission of any technique described herein representative of the state of the art.
Phytic acid (phytate, inositol hexakisphosphate, IP6) is found in many plants where it functions as storage of phosphate. Phosphate stored in IP6 molecules can be released as inorganic phosphate. When inorganic acid is released from myo-inositol hexakisphosphate (IP6) it is converted first to myo-inositol pentakisphosphate (IP5) and further via myo-inositol tetrakisphosphate (IP4), myo-inositol trisphosphate (IP3), myo-inositol bisphosphate (IP2) to myo-inositol monophosphate (IP1).
Phytases are a group of phosphatase enzymes that catalyse the hydrolysis of phytic acid. Commercially available phytases belong to the histidine acid phosphatase (HAP) protein family. The phytases belonging to the HAP protein family share conserved N-terminal active site hepta-peptide motif RHGXRXP and the catalytically active HD-dipeptide at the C-terminus. Histidine acid phosphatases are part of a larger superfamily of histidine phosphatases. They share a conserved catalytic core centred on a histidine, which becomes phosphorylated during the reaction. PFAM motif His_Phos_2 (PF00328) represents branch 2 of the histidine phosphatase superfamily, the branch containing mainly acid phosphatases and phytases.
Phytases are used in feeds to improve phosphate availability from feed ingredients of plant origin (e.g., wheat, barley, corn, soybean) by phytate degradation. This is in particular of interest for monogastric animals like poultry, fish and pigs, because intestinal phytate degradation in the upper intestinal tract of these animals is limited. This limitation not only restricts utilisation of phosphorus, but also the availability of other nutrients due to the chelating effect of inositol phosphates (IPs), particularly by the high IPs, that is IP6, IP5 and IP4. Consequently, at least IP6 to IP4 should be dephosphorylated as completely as possible, but the dephosphorylation should preferably continue even to IP1 to further increase availability of phosphate and other nutrients.
Phytate breakdown by phytases is associated with stepwise degradation of IP6 to lower inositol phosphate esters (IP5, IP4, IP3, IP2, and IP1). The use of industry standard levels of phytase have, expectedly, shown to significantly reduce IP6 levels in vitro and in vivo. However, with IP6 degradation an increase of IP4 and IP3 has been detected in ileal digesta which shows that the hydrolytic cleavage of the first phosphate group is not the only limiting step in phytate degradation (e.g., Zeller et al, 2015; Menezes-Blackburn et al, 2015). As even these lower inositol phosphates have antinutritive properties due to binding of different nutrients like minerals (Xu et al, 1992), the target in animal feeding is to degrade IP esters up to the terminal ileum (Bedford and Walk, 2016). Another reason for the aim to get inositol fully released is that increasing the content of free inositol has been shown to improve growth performance in animals by different mechanisms, still under investigation (Lee and Bedford, 2016).
The part in the intestinal tract of animals where optimal degradation of inositol phosphate esters takes place with phytase supplements is the stomach due to its low pH, leading to the best substrate (phytate) solubility. Retention time in the stomach is short and part of the content might flush rapidly to the intestinal tract in which the pH is neutral. Therefore, further development of phytases acting quickly and more effectively on IP6 and, also on lower inositol phosphates like IP4 and IP3 isomers, is of relevance for the animal feeding industry. These type of phytases would further improve intestinal availability of phosphate and inositol.
It is an object of the present disclosure to provide phytase variants that exhibit phytase activity and that have improved properties that allow their use in industrial processes. Yet another object of the present disclosure is to provide phytase variants that can be used in enzyme compositions for phytate degradation.
The appended independent claims define the scope of protection. Any example or technical description of a product and/or method in the description and/or drawings not covered by the claims is presented herein not as an embodiment of the invention, but as background art or as an example which is useful for understanding the claimed invention.
According to a first aspect is provided a variant polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1, wherein the variant has:
Advantageously, the present variant polypeptide (also called herein a phytase variant) has improved IP4 activity compared to the parent phytase having the SEQ ID NO: 1, and/or a phytase having the SEQ ID NO: 2. The position 126 has been identified by the inventors to be important for the increased IP4 activity. Improved IP4 degrading activity of the present phytase variant means that the variant is capable of degrading more IP4 than a phytase with the amino acid sequence of SEQ ID NO: 2. The improved IP degrading activity can be used to an advantage when degradation of phytic acid to lower inositol phosphates is needed, such as in feed or food applications. As shown in the examples provided below, the claimed phytase variant has a IP4 degrading activity that is improved compared to the parent phytase enzyme and/or to a phytase having the SEQ ID NO: 2.
In an embodiment the present variant polypeptide has less than 100% sequence identity with the SEQ ID NO: 1.
In the present variant polypeptide the position 126 is substituted to another amino acid, such as to a histidine, and, optionally, at least one further amino acid is substituted. This results into an increase in the IP4 degrading activity. The at least one further amino acid substitution, which is substitute in addition to the substitution in position 126, can be selected from a position shown in Table 1. More preferably, the at least one further amino acid substitution is selected from Table 1 such that at least one property selected from IP4 degrading activity, IP6 degrading activity, both IP4 and IP6 degrading activities, and IP4/IP6, is improved for a variant having said substitution, even when present in the variant polypeptide together with other substitutions, as compared to the parent phytase enzyme and/or to a phytase having the SEQ ID NO: 2. The IP6 and IP4 degrading activity can be measured and compared as described in the examples.
In an embodiment the present phytase variant has at least IP4 degrading activity.
In an embodiment the present phytase variant has at least IP6 degrading activity.
In another embodiment the variant polypeptide has IP4 degrading activity and IP6 degrading activity.
In another embodiment the ratio of the variant's IP4 degrading activity to its IP6 degrading activity is at least about 1.05, or within the range 1-2, such as within the range 1.05-2, preferably within the range 1.05-1.9, more preferably within the range 1.2-2 or within the range 1.2-1.9 compared to the IP4/IP6 ratio of a phytase having the SEQ ID NO: 2. In another embodiment the IP4/IP6 ratio is about 1.5, or about 1.6, or about 1.7, or about 1.8 compared to the IP4/IP6 ratio of a phytase having the SEQ ID NO: 2. The larger IP4/IP6 indicates capability to degrade IPs into lower IPs, which is useful in feed and food applications.
In an embodiment is provided a functional fragment of the present phytase variant, the functional fragment having phytase activity.
In an embodiment the variant polypeptide has a histidine at the position 126, i.e., the position 126 may be substituted to a histidine.
In an embodiment the variant polypeptide comprises at least one further amino acid substitution at a position selected from 121, 204, 212, 216, and 258. The substitution may be made to the variant polypeptide artificially, such as by genetic engineering.
In an embodiment the variant polypeptide is an E. coli 6-phytase. According to this embodiment the amino acid sequence of the present variant polypeptide is derived from an E. coli 6-phytase, or from its variant. The amino acid in position 126 may naturally be histidine, or it is substituted to histidine from another amino acid present in the E. coli 6-phytase.
In an embodiment the at least one further amino acid substitution results into the presence of at least one of the following amino acids: 121K, 204N/T, 212G, 216T, and 258N/A. The variant polypeptide is thus modified such that it contains at least one amino acid as specified above.
In an embodiment the at least one further amino acid substitution is a substitution selected from P121K, D204N/T, S212G, M216T, and Q258N/A.
In an embodiment the variant polypeptide comprises the amino acids:
In an embodiment the variant polypeptide comprises the amino acid substitutions:
In an embodiment the variant polypeptide comprises the amino acid substitutions P121K, N126H, D204N, W211V, S212G, M216T, and V253Y.
In an embodiment the variant polypeptide comprises the amino acid substitutions P121K, N126H, D204T, W211V, S212G, M216T, and V253Y.
In an embodiment the variant polypeptide comprises the amino acid substitutions P121K, N126H, D204N/T, W211V, S212G, M216T, V253Y, and Q258N/A.
In an embodiment the variant polypeptide polypeptide comprises:
In an embodiment the variant polypeptide comprises the substitutions selected from:
In an embodiment the variant polypeptide has asparagine or threonine at the position 204.
In an embodiment the variant polypeptide further comprises the amino acids 121K, 126H, 216T, and 258N/A; preferably the amino acid substitutions P121K, N126H, M216T, and Q258N/A.
In an embodiment the variant polypeptide further comprises the amino acids 75C/V, 114T, 137E, 141T, 142D, 146R, 157G, 204C, 211W, 253Q, 267R, and 341P; or the amino acids D35Y, G70E, S80P, T161P, N176P, L179F, K180N, S187P, K276M, T277A, E315G, and A380P.
In an embodiment the variant polypeptide has an increased IP4 degradation activity, and/or a ratio of IP4 to IP6 activity of higher than 1.3, compared to the SEQ ID NO: 1 and/or SEQ ID NO: 2. It is thus possible to use substitutions in the presently claimed positions to change the enzyme activity of the variant polypeptide such that it has more activity towards IP4 substrates. Additionally, the present variant polypeptide may have IP6 degrading activity.
The increased relative IP4 degrading activity means higher specific IP4 degrading activity (more IP4 activity per mg phytase protein), a higher concentration of IP4 degrading phytase in spent culture medium after cultivation, a more stable protein having IP4 degrading activity, or any combinations of these. The increased relative IP4 degrading activity of the present variant polypeptide can thus be used to an advantage for example in compositions that are used to degrade inositol phosphates, and in particular where degradation of inositol phosphate to lower inositol phosphates is desired. A corresponding explanation is herein given to the relative IP6 degrading activity.
According to a second aspect is provided a recombinant host cell comprising genetic elements that allow producing at least one variant polypeptide, wherein the host cell is preferably selected from the group consisting of:
According to a third aspect is provided a recombinant host cell comprising genetic elements configured to produce at least one variant polypeptide of the present disclosure, and wherein the host cell is a transgenic plant cell.
According to an alternative third aspect is provided an enzyme composition comprising the present variant polypeptide.
According to a fourth aspect is provided a use of the present variant polypeptide or the present enzyme composition in the manufacturing of feedstuff or foodstuff feed additive, a dietary supplement, or a pharmaceutical.
According to a fifth aspect is provided a method of manufacturing the present variant polypeptide comprising:
According to a sixth aspect is provided an animal feed comprising the present variant polypeptide, or the present enzyme composition, and at least one protein source of plant origin, and
According to a seventh aspect is provided a feed supplement comprising the present variant polypeptide or the present enzyme composition; and
According to an eighth aspect is provided a method of degrading or modifying material containing phytic acid and/or phytate, comprising treating said material with an effective amount of the present variant polypeptide or the present enzyme composition.
Some example embodiments will be described with reference to the accompanying figures, in which:
SEQ ID NO: 1 Amino acid sequence of parent phytase without signal peptide having the following two amino acid substitutions compared to SEQ ID NO: 2: 211V and 253Y
SEQ ID NO: 2 Amino acid sequence of parent phytase without signal peptide
As used herein, the term “phytase” means an enzyme having capability to enzymatically degrade phytic acid to lower inositol phosphates.
Phytases are classified into 3-, 5- or 6-phytases (EC 3.1.3.8, EC 3.1.3.72, and EC 3.1.3.26, respectively) based on the carbon position on the inositol ring at which they preferably initiate phosphate hydrolysis. 6-phytases preferably first remove the phosphate group at the C6 position.
The present invention also relates to a polynucleotide comprising a nucleic acid sequence encoding a phytase variant polypeptide according to the first aspect.
In an embodiment the polypeptide comprising the phytase variant comprises at least one further amino acid sequence selected from a signal sequence, a secretory sequence, a carrier polypeptide, binding domain, a tag, a linker, an enzyme, or any combination thereof.
The terms “phytase variant”, “variant of phytase”, or “variant polypeptide” mean a phytase molecule obtained by site-directed or random mutagenesis, insertion, substitution, deletion, recombination and/or any other protein engineering method, and which leads into a genetically modified phytase that differs in its amino acid sequence from the parent phytase such as a wild type phytase. The terms “wild type phytase”, “wild type enzyme”, “wild type”, or “wt” in accordance with the disclosure, describe a phytase enzyme with an amino acid sequence found in nature or a fragment thereof. The variant encoding gene can be synthesised or the parent gene be modified using genetic methods, e.g., by site-directed mutagenesis, a technique in which one or more than one mutation are introduced at one or more defined sites in a polynucleotide encoding the parent polypeptide. The term variant phytase may also be referred to by using a name given to the variant in the examples and in the tables.
As used herein, the term “mature polypeptide” means any polypeptide wherein at least one signal sequence or signal peptide or signal peptide and a putative pro-peptide, or a carrier peptide or a fusion partner is cleaved off. As used herein, a “peptide” and a “polypeptide” are amino acid sequences including a plurality of consecutive polymerized amino acid residues. For purpose of this disclosure, peptides are molecules including up to 20 amino acid residues, and polypeptides include more than 20 amino acid residues. The peptide or polypeptide may include modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues. As used herein, a “protein” may refer to a peptide or a polypeptide of any size. A protein may be an enzyme, a protein, an antibody, a membrane protein, a peptide hormone, regulator, or any other protein.
As used herein, “sequence identity” means the percentage of exact matches of amino acid residues between two optimally aligned sequences over the number of positions where there are residues present in both sequences. When one sequence has a residue with no corresponding residue in the other sequence, the alignment program allows a gap in the alignment, and that position is not counted in the denominator of the identity calculation.
As used herein, “sequence alignment” of the amino acid sequences means, aligning the sequences using Clustal Omega (1.2.4) multiple sequence alignment program (https://www.ebi.ac.uk/Tools/msa/clustalo/) as described by Sievers et al. 2011, and using the default settings.
Unless otherwise specified, all references to a certain amino acid position refer to an amino acid of the SEQ ID NO: 1 in said position, or to an amino acid present or missing in the corresponding position of an amino acid sequence aligned with SEQ ID NO: 1.
As used herein, the term “disulfide bridge”, or “disulfide bond”, or “SS bridge” refers to a bond formed between the sulfur atoms of cysteine residues in a polypeptide or a protein. Disulfide bridges can be naturally occurring, or non-naturally occurring, and, for example, introduced by way of amino acid substitution(s).
As used herein, the term “corresponding positions” or “corresponding amino acid position” means aligning at least two amino acid sequences according to identified regions of similarity or identity as pairwise alignment or as multiple sequence alignment, thereby pairing up the corresponding amino acids. In the present disclosure corresponding positions typically refers to a position corresponding to the position in SEQ ID NO: 1.
As used herein, “amino acid substitution” means an amino acid residue replacement with an amino acid residue that is different than the original amino acid in that specific replacement position. The term “amino acid substitution” can refer to conservative amino acid substitutions and non-conservative amino acid substitutions, which means the amino acid residue is replaced with an amino acid residue having a similar side chain (conservative), or a different side chain (non-conservative), as the original amino acid residue in that place.
The term “functional fragment” means a fragment or portion of the current variant, which retains about the same enzymatic function or effect.
The term “secretory signal sequence” or “signal sequence” or a “secretory peptide” refers to an amino acid sequence which is a component or a part of a larger polypeptide, and which directs the larger polypeptide through a secretory pathway of a host cell in which it is produced. The secretory signal sequence can be native or it can be obtained from another source. Depending on the host cell, the larger polypeptide may be cleaved from the secretory peptide during transit through the secretory pathway, thereby forming a mature polypeptide lacking the secretory peptide.
The term carrier polypeptide or fusion partner refers to a polypeptide into which the protein of interest (phytase) is translationally fused to improve the yield. The carrier/fusion partner can be either homologous or heterologous to production host in its origin and can be a full-length protein or a fragment of a protein (e.g., a core, a CBM or a CBM and linker region). It is preferably encoded by a gene or a nucleotide sequence with good expression level.
“Phytase activity” as used herein, refers to the phytic acid degrading activity. Examples 3 and 4 provide examples of a method for determining phytase activity. Accordingly, IP4 activity refers to the capability to degrade IP4, and IP6 activity refers to the capability to degrade IP6. The ratio of IP4 degrading activity to IP6 degrading activity is sometimes disclosed herein in as IP4/IP6 compared to the IP4/IP6 ratio of a phytase having the SEQ ID NO: 2. IP4 degrading activity and IP6 degrading activity can be determined as described in the Examples.
In an embodiment the term “enzyme composition” means an enzymatic fermentation product, possibly isolated and purified, typically produced by a pure culture of a microorganism. The enzyme composition usually comprises a number of different enzymatic activities produced by the microorganism. In another embodiment the enzyme composition is a mixture of monocomponent enzymes, preferably enzymes derived from bacterial or fungal species by using conventional recombinant production techniques. The enzyme composition may contain for example stabilators and preservatives which prevent microbial growth and improve storage stability.
As used herein, a “host cell” means any cell type that is susceptible to transformation, transfection, transduction, mating, crossing, CRISPR-Cas, or the like with a nucleic acid construct or expression vector comprising a polynucleotide. The term “host cell” encompasses any progeny that is not identical due to mutations that occur during replication. Non-limiting examples of a host cell are fungal cells, preferably a filamentous fungal cell (e.g., Trichoderma or Trichoderma reesei, Aspergillus or Aspergillus oryzae or A. niger, Thermothelomyces or Thermothelomyces heterothallica, Myceliophthora or Myceliophthora thermophila, or Humicola or Humicola insolens or Fusarium or Fusarium venenatum), bacterial cells, preferably gram-positive Bacilli (e.g., Bacillus subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus), gram-negative bacteria (e.g., Escherichia coli), actinomycetales (e.g., Streptomyces sp., Nonomuraea flexuosa) and yeasts (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica).
In another embodiment the phytase variant is obtained by recombinant production in plant cells, i.e., in a transgenic plant.
The recombinant host cell can be used to produce the phytase variant and to contain a polynucleotide encoding it. The recombinant host cell can be operably linked to one or more control sequences that direct the production of the variant, and that make it possible to initiate the production of the present phytase variant by a stimulus, as is known in the field. The recombinant host cell is useful also in preparation of variants with different properties. For example, a host cell can be selected, which provides post-translational modifications beneficial for stability or activity, or which facilitates post-processing of a variant produced in the host cell.
In an embodiment the host cell is non-pathogenic. This is particularly advantageous for using the host cell or the phytase variant produced in it for animal feed.
In an embodiment the composition containing the phytase variant is food or feed, and it may further comprise plant material which contains phytic acid.
In an embodiment the composition is a food additive or a feed additive further comprising at least one of: at least one trace mineral, at least one amino acid, in particular lysine, water soluble vitamin, fat soluble vitamin, prebiotic, and probiotic.
In an embodiment the composition is a food additive or a feed additive complying with the requirements of Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 Sep. 2003 on additives for use in animal nutrition.
In an embodiment the composition is in a form of a liquid composition or a solid composition such as solution, dispersion, paste, pellet, powder, granule, coated granule, tablet, cake, crystal, crystal slurry, gel, extrude, precipitate, premix, or a combination thereof.
The term “promoter” refers to a portion of a gene containing DNA sequence(s) that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.
The term “propeptide” or “pro-peptide” is a part of a protein that is cleaved during maturation or activation. Once cleaved, a propeptide generally has no independent biological function.
As used herein, the terms “domain” and “region” can be used interchangeably with the term “module”.
The following abbreviations are used for amino acids:
Substitutions are described herein by using of the following nomenclature: amino acid residue in the protein scaffold, i.e., the parent sequence; position; substituted amino acid residue(s). According to this nomenclature the substitution of, for instance, a single residue of alanine to tyrosine residue at position 23 is indicated as Ala23Tyr or A23Y. A substitution of any amino acid in position 23 to tyrosine is indicated as Xaa23Tyr or X23Y or 23Y. A substitution of a tyrosine in position 179 to phenylalanine, tryptophan, or leucine is indicated as Y179F/W/L.
As used herein, the term “comprising” includes the broader meanings of “including”, “containing”, and “comprehending”, as well as the narrower expressions “consisting of” and “consisting only of”.
As used herein, the term “expression” includes any step or all steps involved in the production of a polypeptide in a host cell including, but not limited to, transcription, translation, post-translational modification, and secretion. Expression may be followed by harvesting, i.e., recovering, the host cells or the expressed product.
In an embodiment the phytase variant has phytase activity, and an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids of SEQ ID NO: 1. In an embodiment the variant polypeptide does not have 100% sequence identity with amino acids of SEQ ID NO: 1. In an embodiment, the amino acid numbering of the variant polypeptide corresponds to that of SEQ ID NO: 1. In an alternative embodiment, the amino acid numbering of the variant polypeptide corresponds to that of SEQ ID NO: 1 partially.
In an embodiment the total number of the amino acid substitutions in the variant polypeptide, compared to the SEQ ID NO: 1, is at least 2. The at least one further amino acid may be selected from the positions disclosed herein. In another embodiment the total number of substitutions is at least 5, at least 10, at least 15, at least 20 or at least 25; or 5, 10, 15, 20, or 25. In an embodiment the substitution or the substitutions are made in the non-conservative region of the amino acid sequence. In another embodiment the variant polypeptide comprises the substitutions specified in any claim, and additional substitutions in non-conserved region of the amino acid-sequence. The effect these additional substitutions have on the properties can be analyzed as described in the Examples.
In an embodiment the variant polypeptide, or the functional fragment, has a predicted molecular weight between 40 and 60 kDa, preferably between 43-55 kDa. The predicted molecular weight can be determined by calculating the sum of the molecular weights of the individual amino acids in the variant polypeptide, or in its functional fragment. When the predicted molecular weight of the variant polypeptide is within the above range, the structure of the variant polypeptide may be similar with the parent sequence to which the substitutions are made.
In an embodiment the composition is provided in the form of a liquid composition or a solid composition, such as solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel, or pellet.
In an embodiment the phytic acid is degraded in a plant-based material or partly plant based material which contains phytic acid.
In an embodiment the present phytase variant is used in an animal feed, and the animal is a ruminant or a non-ruminant. In another embodiment the animal is a cattle like beef or cow, a sheep or goat. In another embodiment the non-ruminant include poultry (such as broiler, layer and turkey and duck); pigs (such as piglets, growing pigs and sows); fish (such as salmonids, carp, tilapia and catfish) and crustaceans. In an embodiment the feed is animal feed intended to be fed to animals such as any compound feed or mixture. In another embodiment feed comprises or consists of grains such as maize, wheat, oats, barley, sorghum and rice; protein sources like soybean meal, sunflower meal and canola meal as well as of minerals. The feed, wherein the present variant is used, has improved nutritional value compared to a feed without the variant. The present composition and the present phytase variant degrade phytic acid of the feed and thereby increase its nutritional value for the animals. The animal feed, wherein the present phytase variant or the present composition is used, can be formulated in the form of a wet composition or a dry composition.
Implementation and embodiments are further disclosed in the following numbered clauses:
Clause 1. A variant polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1, wherein the variant has:
Clause 2. The variant polypeptide of Clause 1 having a histidine at the position 126, i.e. it has a 126His substitution.
Clause 3. The variant polypeptide of Clause 1 or 2 comprising at least one further amino acid substitution at a position selected from 121, 204, 212, 216, and 258.
Clause 4. The variant polypeptide of Clause 3, wherein the at least one further amino acid substitution results into the presence of at least one of the following amino acids: 121K, 204N/T, 212G, 216T, and 258N/A.
Clause 5. The variant polypeptide of any one of Clauses 3-4, wherein the at least one further amino acid substitution is a substitution selected from P121K, D204N/T, S212G, M216T, and Q258N/A.
Clause 6. The variant polypeptide of any one of Clauses 1-5, wherein the variant polypeptide is an E. coli 6-phytase.
Clause 7. The variant polypeptide of any one of Clauses 1-6, wherein the variant polypeptide comprises the amino acids:
Clause 8. The variant polypeptide of any one of Clauses 1-7, wherein the variant polypeptide comprises the amino acid substitutions:
Clause 9. The variant polypeptide of any one of Clauses 1-8, wherein the variant polypeptide comprises:
Clause 10. The variant polypeptide of any one of Clauses 1-9, wherein the variant polypeptide comprises:
Clause 11. The variant polypeptide of any one of Clauses 1-10, wherein the variant polypeptide comprises the amino acids 121K, 126H, 216T, and 258N/A; preferably the amino acid substitutions P121K, N126H, M216T, and Q258N/A.
Clause 12. The variant polypeptide of any one of Clauses 1-11 further comprises the amino acids 75C/V, 114T, 137E, 141T, 142D, 146R, 157G, 204C, 211W, 253Q, 267R, and 341P; or D35Y, G70E, S80P, T161P, N176P, L179F, K180N, S187P, K276M, T277A, E315G, and A380P.
Clause 13. The variant polypeptide of any one of Clauses 1-12 having an increased IP4 degradation activity, and/or having a ratio of IP4 to IP6 activity of higher than 1.3, compared to the SEQ ID NO: 2.
Clause 14. A recombinant host cell comprising genetic elements that allow producing at least one variant polypeptide of any one of Clauses 1-13, wherein the host cell is preferably selected from the group consisting of
Clause 15. A recombinant host cell comprising genetic elements configured to produce at least one variant polypeptide of any one of Clauses 1-13, and wherein the host cell is a transgenic plant cell.
Clause 16. An enzyme composition comprising the variant polypeptide of any one of Clauses 1-13.
Clause 17. A use of the variant polypeptide of any one of Clauses 1-13 or the enzyme composition of Clause 16 in the manufacturing of feedstuff or foodstuff, feed additive, a dietary supplement, or a pharmaceutical.
Clause 18. A method of manufacturing the variant polypeptide of any one of Clauses 1-13 comprising:
Clause 19. An animal feed comprising the variant polypeptide of any one of Clauses 1-13 or the enzyme composition of Clause 16, and at least one protein source of plant origin, and
Clause 20 A feed supplement comprising the variant polypeptide of any one of Clauses 1-13 or the enzyme composition of Clause 16; and
Clause 21. A method of degrading or modifying material containing phytic acid or phytate, comprising treating said material with an effective amount of the variant polypeptide of any one of Clauses 1-13 or the enzyme composition of Clause 16.
The present invention is further disclosed by the following non-limiting examples.
Computational Design of Variants with Improved IP4 Activity
Molecular structures of the most probable IP4 intermediates of 6-phytases according to Ariza et al. 2013 (doi: 10.1371/journal.pone.0065062) were constructed, which are IP4 with a phosphate at positions 1, 2, 3 and 4 (IP41,2,3,4), IP4 with a phosphate at positions 1, 2, 5 and 6 (IP41,2,5,6) and IP4 with a phosphate at positions 1, 2, 4 and 6 (IP41,2,4,6). All atoms not part of the protein chain in the structural model of 6-phytase were removed and hydrogens were added according to a pH of 5.0. For each of the prepared IP4 intermediates a solvated and neutralized system was constructed by adding the respective IP4 intermediate near the substrate cavity of the enzyme and filling the simulation box with water and ions. Each thus constructed system was then equilibrated at room temperature and a pressure of 1 bar as described above. Enzyme-substrate interactions were sampled from the equilibrated systems with an accelerated molecular dynamics approach. For each of the thus generated simulation trajectories a distribution of productive and unproductive enzyme-substrate conformations was derived based on first principle knowledge of the phytase reaction mechanism. All positions in the substrate cavity were analyzed by geometrical criteria to derive candidates for amino acid substitutions that potentially reduce unproductive binding conformations and potentially enhance productive binding conformations (substitution candidates). For each of the thus derived substitution candidates, a system was constructed, equilibrated, simulated and analyzed as described above to yield distributions of productive and unproductive binding conformations. Substitution candidates were ranked by their improvement in the ratio of productive conformations over unproductive conformations and the most promising candidates were selected for experimental characterization. The designed phytase variants are described in detail in Table 1.
Standard molecular biology methods were used in the isolation and enzyme treatments of DNA (e.g., isolation of plasmid DNA, digestion of DNA to produce DNA fragments), in E. coli transformations, sequencing etc. The basic laboratory methods used were either as described by the enzyme, reagent or kit manufacturer or as described in the standard molecular biology handbooks, e.g., Sambrook and Russell (2001) or as described in the following examples.
The phytase genes encoding the designed phytase variants (Example 1, Table 1) were ordered as synthetic genes using codons optimised for preparation of transformants and for expression in Trichoderma reesei.
The phytases in the constructions were expressed from T. reesei cbh1 (cel7A) promoter using a carrier polypeptide (CBM and linker) encoding sequence from T. reesei cbh2 (cel6A). A Kex2 protease cleavage site was included between the carrier polypeptide and phytase like described in Paloheimo et al. 2003. The transcription was terminated using cbh2 terminator, followed in the construction by a synthetic amdS marker gene. In addition, the constructions contain cbh1 3′ and 5′ flanking regions for optionally targeting the expression vector into the cbh1 locus (
Circular expression plasmids were transformed in T. reesei protoplasts. The transformants were selected on plates containing acetamide as the sole nitrogen source. The host strain used lacks the four major endogenous T. reesei cellulases: CBHI/Cel7A, CBHII/Cel6A, EGI/Cel7B and EGII/Cel5A. The transformations were performed according to Penttilä et al., 1987, with the modifications described in Karhunen et al., 1993. Alternatively, CRISPR-Cas technology can be used in transformations.
The transformants were sporulated on potato dextrose agar (PDA) prior to cultivation.
The transformants were cultivated on 96-well plates (Havukainen et al., 2020) to analyse the phytase production of the transformants. Phytase activity of the recombinant variant phytases was measured from the culture supernatants as release of inorganic phosphate from sodium phytate as described in Example 3. Production of the recombinant protein was also detected from the culture supernatant by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
All the transformants produced phytase activity. Transformants producing variant phytases with the highest phytase activity and the reference strain producing the parent phytase were purified on selection plates through single conidia prior to sporulating them on PDA. The purified, selected transformants were cultivated in shake flasks and/or bioreactors in complex cellulase-inducing medium to obtain material for further characterisation and application tests.
Phytase activity was analysed from culture supernatants using IP6 and isolated IP4 form as a substrate as described below.
The phytic acid dodecasodium salt (IP6) used in the analysis was purchased from LabChem (C6H6Na12O24P6, EE05501). Method to generate mainly IP4 specific isomer fraction was performed in 4 steps. In a first step Quantum Blue phytase was immobilized on 5 ml HiTrap NHS-activated sepharose column from General Electric (Boston, USA) as described in Greiner & Konietzky, (1996). In a second step, the immobilized phytase degraded IP6 in 0.1 M Na-acetate buffer pH 5.0 stepwise to lower inositol phosphates. The flowrate of 5 ml/min achieved the highest portion of IP4. The next step was used to remove phosphate from the solution and separate IP4 from other undesired inositol phosphates. Therefore, a manually packed anion exchange column with AG1-x4 resin from Bio-Rad (Hercules, USA) was loaded with the produced inositol phosphate mix and IP4 was eluted with 0.5 M HCl. In the last step, HCl was removed with a rotating evaporator and IP4 re-dissolved in water.
The activity of samples from the microtiter plate cultivations (Example 2) was screened using Fluent® automation workstation (Tecan Group Ltd, Mannedorf, Switzerland) as follows. The samples used in the assay are diluted in a 0.2 M citrate buffer (pH 4.0) containing 0.01% Tween 20 (Merck 822184). 0.01% of Tween 20 is added also to the substrate solution. The substrate concentration used in this analysis is 6.35 mM. 200 μl of sample dilution and 200 μl of substrate are mixed and incubated at 37° C. After exactly 15 min incubation 400 μl of 15% (w/v) TCA solution (Trichloroacetic acid, CCl3COOH, Merck 807) is added to the mixture to stop the reaction. 25 μl of reaction mixture is transferred into another well and 225 μl of water is added to make 1:10 dilution. 250 μl of colour reagent consisting of three volumes of 1 M sulphuric acid (H2SO4, Merck 731), one volume of 2.5% (w/v) ammonium molybdate ((NH4)6Mo7O24·4 H2O, Merck 1182) and one volume of 10% (w/v) ascorbic acid (C6H8O6, AnalaR Normapur 20150) is added and the colour reaction is incubated for 20 min at 50° C. After the incubation the absorption is measured at 820 nm. The absorbance of the sample is compared to that of a reference sample which is the SEQ ID NO: 2.
The activity from the shake flask and fermentation cultivations (Example 2) was analysed using a phytase activity assay (PPU). In PPU analysis one activity unit is the quantity of enzyme that liberates 1 μmol of inorganic phosphate per one minute from sodium phytate at pH 4.0 and at 37° C. in a 15 min reaction time. The substrate concentration used in this analysis is 12.7 mM
The samples used in the assay are diluted in a reaction buffer (0.2 M citrate buffer, pH 4.0) and 1 ml of enzyme solution is used in the analysis. 1 ml of substrate is added to the enzyme solution and after incubating the mixture at 37° C. for exactly 15 min, the reaction is stopped by adding 2 ml of 15% (w/v) TCA solution (Trichloroacetic acid, Merck 807). The reaction mixture is cooled to room temperature and after this 1:10 dilution is done by mixing 0.2 ml of the mixture and 1.8 ml of water in a test tube. 2.0 ml of freshly made colour reagent is added to the tube and mixed. The colour reagent consists of three volumes of 1 M sulphuric acid (H2SO4, Merck 731), one volume of 2.5% (w/v) ammonium molybdate ((NH4)6Mo7O24·4 H2O, Merck 1182) and one volume of 10% (w/v) ascorbic acid (C6H8O6, AnalaR Normapur 20150). The tubes are incubated at 50° C. for 20 min and cooled to room temperature. After this the absorption is measured at 820 nm against the enzyme blank. For the enzyme blank the substrate is added after the TCA and the 15 min incubation is passed. The amount of liberated phosphate is determined via a standard curve of the color reaction with a phosphate solution of known concentration.
The activity for the samples used in gastrointestinal tract test (GIT) (Example 4) was analysed by an internal validated phytase method (FTU assay). In FTU assay inorganic phosphate released from sodium phytate substrate by the hydrolytic enzymatic action of phytase is detected. Colour formation, which is measured spectrophotometrically, is the result of molybdate and vanadate ions complexing with inorganic phosphate. One phytase unit (FTU) is the quantity of enzyme that liberates 1 μmol of inorganic phosphate per minute from sodium phytate at 37° C., pH 5.50, using 60 min incubation time.
In the assay, 2.0 ml of 1% sodium phytate substrate (LabChem EE05501, in 250 mM sodium acetate buffer, pH 5.5 and including 1 mM CaCl2): 2H2O and 0.01% Tween 20) is pipetted to plastic centrifuge tubes. The substrate tubes are pre-incubated for 5-10 minutes at 37° C. after which 1.0 ml of diluted enzyme sample is added. After exactly 60 min incubation 2.0 ml of colour stop solution is added and tube contents are mixed by vortexing. Enzyme blanks are prepared like the sample but the colour stop solution is added to the substrate tubes prior to addition of the diluted enzyme sample. For colour reaction the tubes are incubated for 20 min at room temperature after which they are centrifuged at 4000 rpm for 10 minutes. The sample absorbance is measured against an enzyme blank at 415 nm. For the activity units, a potassium phosphate standard curve (pH 5.50) is prepared (dried KH2PO4, Merck 1.04873.1 is used for the standard; drying at 105° C. for 2 hours before weighting).
The stop solution is prepared as follows (preparation just prior to use): for 100 ml of colour stop solution, 25 ml of stock ammonium heptamolybdate (20 g of (NH4)6Mo7O24·4H2O, Merck 1182 in 180 ml of water, add 2 ml of ammonium hydroxide (NH4OH, Sigma-Aldrich 221228 28-30%), final volume 200 ml) is mixed with 25 ml of stock ammonium vanadate solution (0.47 g of ammonium vanadate (NH4VO3, Riedel de Haen 31153) in 160 ml of water; once the completely dissolved, 4 ml of 22.75% nitric acid solution is added, final volume 200 ml). Then, 16.5 ml of 22.75% nitric acid solution (HNO3, Merck 1.00456) is added after which distilled water is added to make up the volume to 100 ml in volumetric flask.
The comparison of selected novel phytase candidates in their ability to degrade phytate in feed materials was done using an in vitro gastrointestinal simulation test system (GIT) SEQ ID NO:2 was used as a reference.
Phytases to be tested are added with a defined activity level to a feed ingredient mix (60% corn; 40% soybean meal) following a three-step continuous in vitro test simulating the animal digestive conditions. The reactions are run at 40° C. and at corresponding pHs and changes of pH as in the crop, gizzard and small intestine of broilers. Additionally, digestive proteases are added. To succeed in the GIT assay, the phytase needs to have a combination of beneficial biochemical properties. It needs to resist and act at different pHs at the temperature of the digestive tract while being resistant to proteases of the digestive tract. Details of the in vitro test system are described by Sommerfeld et al., 2017.
At the end of the in vitro test inositol phosphates are extracted and phytase removed from the supernatant before analysis of inositol phosphates (IP6-IP3) using high performance liquid chromatography method (HPLC) according to Blaabjerg et al. 2010.
The results obtained from the GIT test are shown in
The foregoing description has provided by way of non-limiting examples of particular implementations and embodiments a full and informative description of the best mode presently contemplated by the inventors for carrying out the invention.
It is however clear to a person skilled in the art that the invention is not restricted to details of the embodiments presented in the foregoing, but that it can be implemented in other embodiments using equivalent means or in different combinations of embodiments without deviating from the characteristics of the invention.
Furthermore, some of the features of the afore-disclosed example embodiments may be used to obtain an advantage or a technical effect without the corresponding use of other features. As such, the foregoing description shall be considered as merely illustrative of the principles of the present invention, and not in limitation thereof. Hence, the scope of the invention is only restricted by the appended claims
| Number | Date | Country | Kind |
|---|---|---|---|
| 21186223.0 | Jul 2021 | EP | regional |
| 21186231.3 | Jul 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2022/050464 | 6/23/2022 | WO |
