Nutritional compositions comprising recombinant polypeptides having nutritionally intact amino acid profile

Abstract

The present invention provides a nutritional composition comprising a recombinant polypeptide having a nutritionally intact amino acid profile suitable for daily consumption by humans for general and special nutrition. The invention further provides a recombinant polypeptide, and variants and truncations thereof, which, when provided in nutritional compositions, are suitable for providing a nutritionally intact amino acid profile. The invention also provides polynucleotides encoding the recombinant polypeptides, nucleic acid constructs, vectors, and host cells comprising these polynucleotides, and methods of producing the recombinant polypeptides.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a nutritional composition comprising the recombinant polypeptide of the present invention, the recombinant polypeptide of the present invention, polynucleotides encoding the recombinant polypeptide, nucleic acid constructs, vectors and host cells comprising these polynucleotides, and a method for producing the recombinant polypeptide. Background Art

[0004] It is a universal requirement for all human diets to include a source of high-quality dietary protein. The body cannot synthesize certain amino acids that are essential for health and growth. These "essential" amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Dietary proteins that provide all the essential amino acids are referred to as high-quality proteins or complete proteins. Compared to foods that do not have a higher protein content, foods with a higher protein content are considered more beneficial in a mammalian diet. Complete proteins promote the maintenance of muscle mass, a healthy body mass index, and blood sugar balance. Additionally, the total amount of protein consumed can be reduced by increasing the amount of complete protein in the diet compared to a diet that primarily consumes low-quality protein.

[0005] There is a general need for foods that are high in protein and have an amino acid profile that meets the daily requirement for essential amino acids for general nutrition. Additionally, there is a need for foods that meet the protein needs of those with special nutritional requirements. Specialty nutrition includes the nutritional needs of sick or elderly patients, as well as those with specific conditions such as pregnancy, short-term medical needs such as hospitalization, or long-term medical needs such as chronic conditions such as diabetes or metabolic disorders.

[0006] Protein-enriched dietary products (for general nutrition or special nutrition) are typically enriched with proteins that are relatively easy to obtain and that purification cost is low. Such protein includes whey protein, casein and albumin. Although these proteins are good sources of many essential amino acids, they can not be customized to provide the specific amino acids of desired amount. Additionally, patients suffering from metabolic disorders require to exclude at least one essential amino acid from their diet usually, and require other amino acids of high enrichment. In order to solve at least part of this problem, dietary products generally comprise free amino acids, which are added to provide a complete nutritional amino acid profile. Unfortunately, free amino acids have an extremely bitter taste, which is not fully covered by adding other ingredients (such as sugar and / or spices). Therefore, these dietary products are undesirable for those crowds that must consume them.

[0007] There is a need for a protein source that can be tailored to provide a desired nutritional profile and that can also be provided in a cost-effective manner. Summary of the Invention

[0008] The present invention provides a recombinant polypeptide that can be modified to be suitable for use in nutritional compositions as a complete protein source for general or special nutrition. For general nutritional compositions, the recombinant polypeptide can be modified to provide one or more variants with a nutritionally complete amino acid profile, as determined by an amino acid score greater than or equal to 0.94. The present invention also provides polypeptide variants that can be customized for special nutrition by enriching and / or consuming certain amino acids, depending on the amino acid requirements of the group targeted by the nutritional composition. Because the recombinant polypeptide of the present invention comprises a complete nutritional profile, there is no need to add free amino acids to the composition. Therefore, the composition does not have the bitter taste associated with many dietary products currently available. The recombinant polypeptide of the present invention is highly expressed and relatively easy to purify, making their manufacture extremely cost-effective. Additionally, the recombinant polypeptide of the present invention can tolerate a wide range of pH ranges and high temperature treatments, making them compatible in a variety of foods and in food manufacturing processes.

[0009] definition

[0010] In light of this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0012] The disclosure refers to amino acids. The full names of these amino acids can be used interchangeably with their respective standard three-letter and single-letter abbreviations. For the avoidance of doubt, those are alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V).

[0013] Amino Acid Composition: The amino acid composition of a polypeptide is calculated as milligrams of amino acids per gram of polypeptide. Amino acid composition can be calculated for a specific amino acid of interest (e.g., the amount of leucine in a given polypeptide) or for a group of amino acids (e.g., the amount of branched-chain amino acids in a given polypeptide). The calculation can be based on the known sequence of the polypeptide, for example, by dividing the total molecular weight of the amino acid residues of interest by the total molecular weight of the polypeptide. The total molecular weight of the amino acid residues of interest is determined by multiplying the number of such residues in the polypeptide by the molecular weight of the residues in the context of the polypeptide (other than the molecular weight of the free amino acids).

[0014] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA, which is processed through a series of steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0015] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0016] Control sequence: the term "control sequence" means a nucleic acid sequence that is directed to regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) for the polynucleotide encoding the variant, and is native or heterologous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, prepropeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, the control sequence includes a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction sites that promote the connection of the control sequence to the coding region of the polynucleotide encoding the variant, the control sequence can be provided with a joint.

[0017] Expression: The term "expression" includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0018] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant operably linked to appropriate control sequences capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers, and sequences that control termination of transcription and translation.

[0019] Extension: The term "extension" means the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide.

[0020] Fragment: The term "fragment" means a variant of a polypeptide having one or more amino acids deleted from the amino and / or carboxyl terminus.

[0021] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide is fused to the N-terminus and / or C-terminus of a variant of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which fusion polypeptides are produced after translation (Cooper et al., 1993, EMBO J. [Journal of the European Molecular Biology Association] 12:2575-2583; Dawson et al., 1994, Science [Science] 266:776-779). The fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1998, J. Biotechnol. 76:245-251; and 95, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0022] Heterologous: With respect to a host cell, the term "heterologous" means that the polypeptide or nucleic acid is not naturally present in the host cell. With respect to a polypeptide or nucleic acid, the term "heterologous" means that the control sequences (e.g., promoter) of the polypeptide or nucleic acid are not naturally associated with the polypeptide or nucleic acid, i.e., the control sequences are from a gene other than the gene encoding the mature polypeptide.

[0023] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct (including a polynucleotide encoding a variant) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by a cell.

[0024] Improved property: The term "improved property" means a characteristic associated with a variant that is improved relative to a parent.

[0025] Introduced: In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection," "transformation," or "transduction," as known in the art.

[0026] Isolated: The term "isolated" means a variant, nucleic acid, cell, or other designated material or component that is separated from at least one other material or component, including but not limited to other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form not found in nature. An isolated polypeptide includes, but is not limited to, a culture medium containing a secreted variant expressed in a host cell.

[0027] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form following N-terminal processing and / or C-terminal processing (eg, removal of a signal peptide).

[0028] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide.

[0029] Mutant: The term "mutant" means a polynucleotide encoding a variant.

[0030] Native: The term "native" means a nucleic acid or polypeptide that occurs naturally in a host cell.

[0031] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the present compositions and methods encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5' to 3' orientation.

[0032] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner not originally found in nature to contain a segment of nucleic acid or is synthetic and comprises one or more control sequences operably linked to the nucleic acid sequence.

[0033] Operably linked: The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0034] Parent or parent polypeptide: The term "parent" or "parent polypeptide" means a polypeptide to which an alteration is made to produce a polypeptide variant of the present invention.

[0035] Purified: The term "purified" means a nucleic acid, variant, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified variants or nucleic acids can form discrete bands in electrophoretic gels, chromatography eluates, and / or culture medium subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., by weight or by mole). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, variant, cell, nucleic acid, amino acid or other designated material or component in a composition at a relative or absolute concentration greater than that of the starting composition.

[0036] In one aspect, the term "purified" as used herein refers to a variant or cell that is substantially free of components (especially insoluble components) from the producing organism. In other aspects, the term "purified" refers to a variant that is substantially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some soluble components of the organism and culture medium from which it is recovered. The variant can be purified (i.e., isolated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0037] Accordingly, variants can be purified so that only small amounts of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" can refer to the removal of other components present in the cell of the polypeptide source, particularly other proteins and most particularly other enzymes. Variants can be "substantially pure," i.e., free of other components from the organism from which they were produced (e.g., a host organism for recombinantly producing the variant). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation containing at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1% and even most preferably at most 0.5% of the polypeptide and other polypeptide materials associated with its natural or recombinant origin.

[0038] Thus, it is preferred that a substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure, based on the weight of the total polypeptide material present in the preparation. The variants of the present invention are preferably in a substantially pure form (i.e., the preparation is substantially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the variant via well-known recombinant methods or via classical purification methods.

[0039] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation (e.g., cleavage and rejoining) of a nucleic acid sequence to form a sequence population that is different from that found in nature. The term recombinant refers to a cell, nucleic acid, variant, polypeptide, or vector that has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found within the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. Recombinant polypeptides are the products of these genes expressed in the recombinant cell. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0040] Recovery: The term "recover" or "recovery" means removing a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, e.g., recovering the polypeptide from a whole fermentation broth or from a cell-free fermentation broth by harvesting the polypeptide crystals, by filtration (e.g., depth filtration (by using filter aids or packed filter media, cloth filtration in box filters, drum filtration, drum filtration, rotary vacuum drum filters, candle filters, horizontal leaf filters or the like, using sheet or pad filtration in frame or modular devices) or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, ultrafiltration in crossflow, dynamic crossflow or dead-end operation)) or by centrifugation (using a horizontal centrifuge, a disk stack centrifuge, a hyrdo cyclone or the like) or by precipitating the polypeptide and using related solid-liquid separation methods to harvest the polypeptide from a broth culture by using size fractionation. Recovery encompasses isolation and / or purification of the polypeptide.

[0041] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0042] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of " longest identity " using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453), as implemented in the Needle program of EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 6.6.0 version or later). The parameters used are gap open penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order to make the Needle program report the longest identity, it is necessary to specify the non-simplified (-nobrief) option in the command line. The output of " longest identity " of the Needle mark is calculated as follows:

[0043] (Identical residues x 100) / (Alignment length - Total number of gaps in the alignment)

[0044] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of " longest identity " using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same), which is implemented in the Needle program of EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (preferred 6.6.0 version or later). The parameters used are gap open penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. In order to make the Needle program report the longest identity, non-simplified options must be specified in the command line. The output of " longest identity " marked by Needle is calculated as follows:

[0045] (number of identical deoxyribonucleotides x 100) / (length of alignment – ​​total number of gaps in the alignment)

[0046] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0047] Subsequence: The term "subsequence" means a polynucleotide having one or more nucleotides deleted from the 5' end and / or 3' end of the mature polypeptide coding sequence.

[0048] Truncation: The term "truncation" means a polypeptide that is smaller than the full-length polypeptide. Truncation can be truncated at the C-terminus, the N-terminus, or both. Truncation can also or alternatively comprise internal truncations, wherein one or more internal portions of the full-length sequence are removed.

[0049] Variant: The term "variant" means a polypeptide comprising a substitution, insertion (including extension) and / or deletion (including truncation) at one or more positions compared to its parent. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a certain position.

[0050] Wild-type: The term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of a wild-type sequence).

[0051] Sufficient amount: The term "sufficient amount" is an amount of a polypeptide or amino acid that is sufficient to cause a desired effect. The sufficient amount of the polypeptide or amino acid can be provided directly, i.e., by administering the polypeptide or amino acid to the subject, or it can be provided as part of a composition comprising the polypeptide or amino acid.

[0052] Amino acid score (AAS): The term "amino acid score" (AAS) is based on the essential amino acid requirements published by the Food and Agriculture Organization of the United Nations (FAO) for different age groups ("Dietary protein quality evaluation in human nutrition: Report of an FAO Expert Consultation", FAO Food Nutr Paper, 92: 1-66, 2013). In this report, the scoring model for protein quality evaluation is determined by calculating the ratio of essential amino acids to protein requirements, expressed in mg amino acids / g protein. AAS is defined using the scoring model determined for the protein requirements of children (6 months to 3 years; see Table 5 of the reference FAO report). To calculate the AAS of a given polypeptide, for each essential amino acid, the ratio of the amount of the essential amino acid present in the polypeptide to the amount of that essential amino acid recommended in the FAO report is determined. The AAS of the polypeptide is the lowest ratio determined for any essential amino acid. Polypeptides with an AAS greater than 0.94 meet the essential amino acid requirements for all essential amino acids and are generally considered to be complete protein sources.

[0053] Branched chain amino acids (BCAA): The term "branched chain amino acids" are amino acids selected from the group consisting of leucine, isoleucine and valine.

[0054] Essential amino acids: The term "essential amino acids" are amino acids selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

[0055] Large neutral amino acids (LNAAs): The term "large neutral amino acids" is an amino acid selected from the group consisting of phenylalanine, arginine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, tyrosine, and valine. These amino acids share the same transport system in the brain and intestinal mucosa.

[0056] Nutritional composition: The term "nutritional composition" is a composition comprising a recombinant polypeptide of the present invention containing a desired amount of amino acids. The nutritional composition may also include any number of optional additional ingredients, including conventional food additives (synthetic or natural), such as one or more acidulants, additional thickeners, buffers or reagents for pH adjustment, chelating agents, coloring agents, emulsifiers, excipients, spices, minerals, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, minerals, and / or vitamins. The optional ingredients may be added in any suitable amount. The nutritional composition may be a source of complete nutrition or may be a source of incomplete nutrition. The nutritional composition may provide the daily dietary requirements for protein or essential amino acids.

[0057] Complete Nutrition: The term "complete nutrition" includes nutritional products and compositions that contain sufficient types and levels of macronutrients (protein, fat, and carbohydrates) and micronutrients to be the sole source of nutrition for the animal to which they are administered. A patient can obtain 100% of their nutritional requirements from such complete nutritional compositions.

[0058] Effective amount: The term "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally alleviates symptoms, manages disease progression, or provides a nutritional, physiological, or medical benefit to an individual. An effective amount can also be an amount that meets the daily dietary requirements of an average healthy person for protein or for essential amino acids based on recommendations published by the FAO (FAO Food Nutr Paper, 92: 1-66, 2013).

[0059] Patient or individual: The terms "patient" or "individual" are generally used herein to refer to a human. However, in some embodiments, the terms "individual" and "patient" refer to any animal, mammal, or human with a medical condition that may benefit from the nutritional compositions of the present invention, or any animal, mammal, or human without a medical condition who may benefit from the nutritional compositions of the present invention for general health.

[0060] Variant naming conventions

[0061] In describing the variants of the present invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0062] Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 by alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent that glycine (G) and serine (S) at positions 205 and 411 are replaced by arginine (R) and phenylalanine (F), respectively.

[0063] Deletion: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is represented as "Gly195*" or "G195*." Multiple deletions are separated by a plus sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*."

[0064] Insertions: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK." Multiple amino acid insertions are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA."

[0065] In such cases, the inserted amino acid residues are numbered by adding lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue. In the above example, the sequence would therefore be:

[0066] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0067] Multiple alterations: Variants containing multiple alterations are separated by plus signs ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine at position 170 and glycine at position 195 with tyrosine and glutamic acid, respectively.

[0068] Different changes: Where different changes can be introduced at one position, the different changes are separated by commas, e.g., "Arg170Tyr,Glu" represents a substitution of arginine at position 170 by tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants:

[0069] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”. DETAILED DESCRIPTION

[0070] A recombinant polypeptide was identified that was found to be expressed at very high levels when recombinantly expressed in Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis, and can also be purified to high levels relatively easily using methods well known in the art. Surprisingly, the polypeptide is also suitable for nutritional compositions because it has an amino acid composition that provides a value close to the desired AAS and does not contain toxic domains or amino acid motifs known to play a role in protein allergens. Although the polypeptide has mannanase activity (described in WO 2021 / 152123, incorporated herein by reference), inactive variants can be produced by introducing substitutions at positions E426 and / or E334 (numbered using the amino acid sequence of SEQ ID NO: 49). Examples of inactive variants are SEQ ID NOs: 50 and 51. The inventors have found that inactive variants still have high expression and ease of purification.

[0071] Additionally, variants of the mannanase polypeptides of the present invention are resistant to heat treatment and a wide pH range. This indicates that they are suitable as components of nutritional compositions, which typically have a neutral or acidic pH, and are also suitable for withstanding the production and processing of nutritional compositions, which may require heat during production and may also require some form of heat treatment for sterilization.

[0072] The present invention further relates to a nutritional composition comprising variants of mannanase polypeptides suitable as complete or near-complete protein sources for daily human consumption. The nutritional composition of the present invention may comprise variants of mannanase polypeptides suitable for individuals with increased medical needs and / or long-term care, including the elderly, pregnant women, cancer patients, and individuals with long-term diseases such as diabetes. The nutritional composition of the present invention may comprise variants of mannanase polypeptides suitable as a complete protein source for patients with metabolic disorders. The polypeptides of the present invention are recombinant and comprise SEQ ID NOs: 3 to 101 and any variants thereof. The variants have at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or at least 100% sequence identity with the polypeptide of any one of SEQ ID NOs: 3 to 101.

[0073] The polypeptide of the present invention may comprise a substitution, insertion or deletion at one or more positions of the polypeptide of any one of SEQ ID NOs: 3 to 101. The polypeptide of the present invention may further comprise an extension of one or more amino acids at the N-terminus and / or C-terminus.

[0074] In certain embodiments, the polypeptide of the present invention can be a fusion polypeptide or a cleavable fusion polypeptide. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide encoding a polypeptide of the present invention. Fusion polypeptides can comprise a fragment of a polypeptide of the present invention or a full-length polypeptide of the present invention. In certain embodiments, the fusion polypeptide can comprise 490 to 450, 450 to 400, 400 to 350, 350 to 300, 300 to 250, 250 to 200, 200 to 150, 150 to 100, or 100 to 50 amino acids of any one of SEQ ID NOs: 3 to 101.

[0075] Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is produced after translation (Cooper et al., 1993, EMBO J. [Journal of the European Molecular Biology Association] 12:2575-2583; Dawson et al., 1994, Science [Science] 266:776-779).

[0076] The fusion polypeptide may further comprise a cleavage site between the two polypeptides. When the fusion protein is secreted, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1997 ...3:568-576; Svetina et al., 2000, J. Biotechnol. 3:568-576; Svetina et al. 95, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0077] Alternatively, the polypeptide of the present invention may be a truncation variant comprising one or more amino acids at the N-terminus and / or C-terminus relative to SEQ ID NO: 51, or an internal truncation wherein the amino acids at the N-terminus or C-terminus are retained and a number of internal amino acids are removed. The polypeptides of the present invention include truncation variants of any one of SEQ ID NOs: 3 to 101. The polypeptides of the present invention may be truncations comprising at least 100, at least 150, at least 200, at least 250, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 420, at least 430, at least 440, at least 450, at least 460, at least 470 or at least 480 consecutive amino acid residues of any one of SEQ ID NOs: 3 to 101. In some embodiments, the polypeptide of the invention is a truncation variant comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or at least 100% sequence identity to a truncation of any one of SEQ ID NOs: 3 to 101.

[0078] In some embodiments, a polypeptide of the invention comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 3, wherein the truncation has 1-350 amino acids, 1-340 amino acids, 1-330 amino acids, 1-320 amino acids, 1-310 amino acids, 1-300 amino acids, 1-290 amino acids, 1-285 amino acids, 1-280 amino acids, 1-275 amino acids, 1-270 amino acids, 1-265 amino acids, 1-260 amino acids, 1-275 ... 250 amino acids, 1-245 amino acids, 1-240 amino acids, 1-235 amino acids, 1-230 amino acids, 1-225 amino acids, 1-220 amino acids, 1-215 amino acids, 1-210 amino acids, 1-205 amino acids, 1-200 amino acids, 1-195 amino acids, 1-190 amino acids, 1-185 amino acids, 1-180 amino acids, 1-175 amino acids amino acids, 1-170 amino acids, 1-165 amino acids, 1-160 amino acids, 1-155 amino acids, 1-150 amino acids, 1-145 amino acids, 1-140 amino acids, 1-135 amino acids, 1-130 amino acids, 1-125 amino acids, 1-120 amino acids, 1-115 amino acids, 1-110 amino acids, 1-95 amino acids, 1-90 amino acids, 1-85 amino acids, 1-80 amino acids, 1-75 amino acids, 1-70 amino acids, 1-65 amino acids, 1-60 amino acids, 1-55 amino acids, 1-50 amino acids, 1-45 amino acids, 1-40 amino acids, 1-35 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids, 1-15 amino acids, 1-10 amino acids, or a C-terminal truncation of 1-5 amino acids.

[0079] In some embodiments, a polypeptide of the invention comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 3, wherein the truncation has 1-350 amino acids, 1-340 amino acids, 1-330 amino acids, 1-320 amino acids, 1-310 amino acids, 1-300 amino acids, 1-290 amino acids, 1-285 amino acids, 1-280 amino acids, 1-275 amino acids, 1-270 amino acids, 1-265 amino acids, 1-260 amino acids, 1-275 ... 250 amino acids, 1-245 amino acids, 1-240 amino acids, 1-235 amino acids, 1-230 amino acids, 1-225 amino acids, 1-220 amino acids, 1-215 amino acids, 1-210 amino acids, 1-205 amino acids, 1-200 amino acids, 1-195 amino acids, 1-190 amino acids, 1-185 amino acids, 1-180 amino acids, 1-175 amino acids amino acids, 1-170 amino acids, 1-165 amino acids, 1-160 amino acids, 1-155 amino acids, 1-150 amino acids, 1-145 amino acids, 1-140 amino acids, 1-135 amino acids, 1-130 amino acids, 1-125 amino acids, 1-120 amino acids, 1-115 amino acids, 1-110 amino acids, 1-95 amino acids, 1-90 amino acids, 1-85 amino acids, 1-80 amino acids, 1-75 amino acids, 1-70 amino acids, 1-65 amino acids, 1-60 amino acids, 1-55 amino acids, 1-50 amino acids, 1-45 amino acids, 1-40 amino acids, 1-35 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids, 1-15 amino acids, 1-10 amino acids, or an N-terminal truncation of 1-5 amino acids.

[0080] In some embodiments, a polypeptide of the invention comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 3, wherein the truncation has 1-350 amino acids, 1-340 amino acids, 1-330 amino acids, 1-320 amino acids, 1-310 amino acids, 1-300 amino acids, 1-290 amino acids, 1-285 amino acids, 1-280 amino acids, 1-275 amino acids, 1-260 amino acids, 1-265 amino acids, 1-270 amino acids, 1-27 ... -250 amino acids, 1-245 amino acids, 1-240 amino acids, 1-235 amino acids, 1-230 amino acids, 1-225 amino acids, 1-220 amino acids, 1-215 amino acids, 1-210 amino acids, 1-205 amino acids, 1-200 amino acids, 1-195 amino acids, 1-190 amino acids, 1-185 amino acids, 1-180 amino acids, 1-175 or an internal truncation of 1-5 amino acids, 1-170 amino acids, 1-165 amino acids, 1-160 amino acids, 1-155 amino acids, 1-150 amino acids, 1-145 amino acids, 1-140 amino acids, 1-135 amino acids, 1-130 amino acids, 1-125 amino acids, 1-120 amino acids, 1-115 amino acids, 1-110 amino acids, 1-95 amino acids, 1-90 amino acids, 1-85 amino acids, 1-80 amino acids, 1-75 amino acids, 1-70 amino acids, 1-65 amino acids, 1-60 amino acids, 1-55 amino acids, 1-50 amino acids, 1-45 amino acids, 1-40 amino acids, 1-35 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids, 1-15 amino acids, 1-10 amino acids, or 1-5 amino acids.

[0081] SEQ ID NOs: 4-48, 52, 53, 58, 61, 62, 63, 66, 67, and 70-75 are truncated variants of SEQ ID NO: 3. In some embodiments, the truncations can be N-terminal truncations wherein amino acids 1-265 are removed relative to SEQ ID NO: 3, such as, for example, SEQ ID NO: 10. In some embodiments, the truncations can be N-terminal truncations wherein amino acids 1-158 are removed relative to SEQ ID NO: 3, such as, for example, SEQ ID NOs: 4-8, 11-43, and 45-48. In some embodiments, the truncations can be N-terminal truncations wherein amino acids 1-157 are removed relative to SEQ ID NO: 3, such as, for example, SEQ ID NO: 9. In some embodiments, the truncations can have the first 11 amino acids at the N-terminus, followed by internal truncations at positions 12-160, wherein positions are numbered according to the amino acid sequence of SEQ ID NO: 3, such as, for example, the internal truncation variants SEQ ID NOs: 52 and 53.

[0082] The polypeptides of the present invention are recombinant polypeptides. The polypeptides of the present invention comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3-101 or a truncation thereof, and further comprise substitutions at positions corresponding to positions E334 and / or E426 of SEQ ID NO: 49. The substitutions at positions E334 and / or E426 can be A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, the substitutions at positions E334 and / or E426 can be A, I, G, L, M, Q, or V. In some embodiments, only position E334 has a substitution. In other embodiments, only position E426 has a substitution. In some embodiments, both positions E334 and E426 are substituted.

[0083] Amino acid composition

[0084] The polypeptides of the present invention have an amino acid score (AAS) greater than or equal to 0.94 (≥0.94). The AAS is based on the essential amino acid requirements published by the Food and Agriculture Organization of the United Nations for different age groups ("Dietary protein quality evaluation in human nutrition: Report of an FAO Expert Consultation", FAO Food Nutr Paper, 92: 1-66, 2013), in particular based on a scoring model for protein requirements for children (6 months to 3 years old). To calculate the AAS of a given polypeptide, for each essential amino acid, the ratio of the amount of the essential amino acid present in the polypeptide to the amount of that essential amino acid recommended in the FAO report is determined. The AAS of the polypeptide is the lowest ratio determined for any essential amino acid. Polypeptides with an AAS greater than or equal to 0.94 meet the essential amino acid requirements for all essential amino acids and are generally considered to be a complete protein source. In some embodiments, the AAS of the polypeptides of the invention is greater than or equal to 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.

[0085] After adjusting the amino acid composition to suit the patient's needs, polypeptides suitable for use in patients with metabolic disorders are considered complete protein sources. For example, phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are inherited metabolic disorders in which phenylalanine (Phe) is not properly processed. Therefore, dietary phenylalanine needs to be avoided as much as possible. Recombinant polypeptides of the present invention that are suitable as complete protein sources for patients with PKU or HPA do not have phenylalanine residues and, excluding phenylalanine, have an AAS of ≥ 0.94.

[0086] In addition to lacking certain amino acids, recombinant polypeptides used as the primary protein source for patients with metabolic disorders may need to be or preferably be enriched in certain other amino acids. For example, it is generally recommended that PKU patients consume proteins that are particularly rich in large neutral amino acids (such as threonine, tryptophan, tyrosine and leucine). Therefore, polypeptides suitable for use in PKU patients can have an amount greater than what would otherwise be considered sufficient for these amino acids. In some embodiments, the polypeptides of the present invention comprise at least 50 mg threonine / gram polypeptide (e.g., 50 mg threonine / gram polypeptide, at least 60 mg threonine / gram polypeptide, at least 70 mg threonine / gram polypeptide, at least 80 mg threonine / gram polypeptide, at least 90 mg threonine / gram polypeptide, or at least 100 mg threonine / gram polypeptide); at least 9 mg tryptophan / gram polypeptide (e.g., 9 mg tryptophan / gram polypeptide, at least 15 mg tryptophan / gram polypeptide, at least 20 mg tryptophan / gram polypeptide, at least 25 mg tryptophan / gram polypeptide, at least 30 mg tryptophan / gram polypeptide, at least 35 mg tryptophan / gram polypeptide, at least 40 mg tryptophan / gram polypeptide); / gram polypeptide, at least 45 mg tryptophan / gram polypeptide, or at least 50 mg tryptophan / gram polypeptide); at least 55 mg tyrosine / gram polypeptide (e.g., 55 mg tyrosine / gram polypeptide, at least 65 mg tyrosine / gram polypeptide, at least 75 mg tyrosine / gram polypeptide, at least 85 mg tyrosine / gram polypeptide, at least 95 mg tyrosine / gram polypeptide); and at least 66 mg leucine / gram polypeptide (e.g., 66 mg leucine / gram polypeptide, at least 70 mg leucine / gram polypeptide, at least 80 mg leucine / gram polypeptide, at least 90 mg leucine / gram polypeptide, at least 100 mg leucine / gram polypeptide, or at least 110 mg leucine / gram polypeptide).

[0087] In further embodiments, a polypeptide of the invention suitable as a complete protein source for PKU patients comprises at least 60 mg threonine / gram polypeptide; at least 15 mg tryptophan / gram polypeptide; at least 65 mg tyrosine / gram polypeptide; and at least 76 mg leucine / gram polypeptide. In some embodiments, a polypeptide of the invention suitable as a complete protein source for PKU patients comprises at least 70 mg threonine / gram polypeptide; at least 20 mg tryptophan / gram polypeptide; at least 75 mg tyrosine / gram polypeptide; and at least 86 mg leucine / gram polypeptide. In some embodiments, a polypeptide of the invention suitable as a complete protein source for PKU patients comprises at least 80 mg threonine / gram polypeptide; at least 25 mg tryptophan / gram polypeptide; at least 85 mg tyrosine / gram polypeptide; and at least 96 mg leucine / gram polypeptide.

[0088] Those skilled in the art will appreciate that strategies similar to those described above can be used to create nutritional compositions intended for use in patients with metabolic disorders other than PKU. Such disorders include tyrosinemia, maple syrup urine disease, methylmalonic acidemia, homocystinuria, glutaric aciduria, isovaleric acidemia, and hyperlysinemia. For example, a nutritional composition suitable as a complete protein source for patients with tyrosinemia can include a polypeptide having low to no tyrosine and additionally an AAS of ≥ 0.94.

[0089] In some embodiments, the polypeptides of the present invention have an AAS of ≥ 0.94, without any further restrictions on the amount of any amino acid. Such recombinant polypeptides can generally be used in nutritional compositions. Such nutritional compositions can be used as a protein source for people with metabolic disorders who do not have restricted protein intake. Such nutritional compositions can be used for patients with reduced appetite or reduced ability to eat.

[0090] In some embodiments, the polypeptide of the present invention comprises certain amino acids in an amount higher than the amount required to achieve AAS≥0.94. For example, the amino acid leucine is an important factor in stimulating muscle protein synthesis, and more generally, branched-chain amino acids (BCAA; including valine, leucine and isoleucine) are abundant in muscle protein, stimulate muscle growth in the body, and provide energy during exercise. For groups interested in increasing and / or maintaining muscle mass, such as professional athletes (which can be referred to as "sports nutrition") and elderly people who tend to lose muscle mass during the aging process, or hospitalized patients with limited appetite (which can be referred to as "medical nutrition"), nutritional compositions rich in leucine and / or BCAA may be needed and / or desired. Leucine can also play a role in managing blood sugar levels and helping to control appetite. Therefore, groups interested in better maintaining their blood sugar levels and / or weight (such as diabetics, prediabetic individuals, and individuals struggling with weight control) may need and / or desire nutritional compositions rich in leucine.

[0091] In some embodiments, the polypeptides of the invention have an AAS of ≥ 0.94 and additionally comprise at least 105 mg leucine / gram polypeptide, e.g., 105 mg leucine / gram polypeptide, at least 110 mg leucine / gram polypeptide, at least 115 mg leucine / gram polypeptide, at least 120 mg leucine / gram polypeptide, at least 125 mg leucine / gram polypeptide, at least 130 mg leucine / gram polypeptide, at least 135 mg leucine / gram polypeptide, at least 140 mg leucine / gram polypeptide, at least 145 mg leucine / gram polypeptide, at least 150 mg leucine / gram polypeptide, at least 155 mg leucine / gram polypeptide, leucine / gram polypeptide, at least 160 mg leucine / gram polypeptide, at least 170 mg leucine / gram polypeptide, at least 180 mg leucine / gram polypeptide, at least 190 mg leucine / gram polypeptide, at least 200 mg leucine / gram polypeptide, at least 210 mg leucine / gram polypeptide, at least 220 mg leucine / gram polypeptide, at least 230 mg leucine / gram polypeptide, at least 240 mg leucine / gram polypeptide, at least 250 mg leucine / gram polypeptide, at least 275 mg leucine / gram polypeptide, at least 300 mg leucine / gram polypeptide, or at least 350 mg leucine / gram polypeptide.

[0092] In some embodiments, the polypeptides of the invention have an AAS of ≥ 0.94 and comprise at least 210 mg branched-chain amino acids / gram polypeptide, for example, 210 mg branched-chain amino acids / gram polypeptide, at least 215 mg branched-chain amino acids / gram polypeptide, at least 220 mg branched-chain amino acids / gram polypeptide, at least 225 mg branched-chain amino acids / gram polypeptide, at least 230 mg branched-chain amino acids / gram polypeptide, at least 235 mg branched-chain amino acids / gram polypeptide, at least 240 mg branched-chain amino acids / gram polypeptide, at least 245 mg branched-chain amino acids / gram polypeptide, at least 250 mg branched-chain amino acids / gram polypeptide, at least 255 mg branched-chain amino acids / gram polypeptide, at least 260 mg branched-chain amino acids / gram polypeptide, at least 265 mg branched-chain amino acids / gram polypeptide, at least 270 mg branched-chain amino acids / gram polypeptide, at least 275 mg branched-chain amino acids / gram polypeptide, at least 280 mg branched-chain amino acids / gram polypeptide.

[0014] In some embodiments, the amount of branched-chain amino acids present in the polypeptide may be at least 285 mg branched-chain amino acids per gram of polypeptide, at least 290 mg branched-chain amino acids per gram of polypeptide, at least 300 mg branched-chain amino acids per gram of polypeptide, at least 310 mg branched-chain amino acids per gram of polypeptide, at least 320 mg branched-chain amino acids per gram of polypeptide, at least 330 mg branched-chain amino acids per gram of polypeptide, at least 340 mg branched-chain amino acids per gram of polypeptide, at least 350 mg branched-chain amino acids per gram of polypeptide, at least 360 mg branched-chain amino acids per gram of polypeptide, at least 370 mg branched-chain amino acids per gram of polypeptide, at least 380 mg branched-chain amino acids per gram of polypeptide, at least 390 mg branched-chain amino acids per gram of polypeptide, at least 400 mg branched-chain amino acids per gram of polypeptide, at least 425 mg branched-chain amino acids per gram of polypeptide, at least 450 mg branched-chain amino acids per gram of polypeptide, at least 475 mg branched-chain amino acids per gram of polypeptide, or at least 500 mg branched-chain amino acids per gram of polypeptide. Branched-chain amino acids include leucine, isoleucine, and valine.

[0093] In further embodiments, the polypeptides of the invention have an AAS > 0.94 and comprise at least 105 mg leucine / gram of polypeptide and further comprise at least 210 mg branched chain amino acids / gram of polypeptide.

[0094] The polypeptides of the present invention may have an optimized amino acid composition to provide an ideal amino acid composition for a nutritional composition suitable for any group of protein and amino acid requirements. For example, during pregnancy, a higher amount of protein and enrichment of certain amino acids may also be required. In some embodiments, the nutritional composition of the present invention comprises a recombinant polypeptide of the present invention having an amino acid composition optimized for pregnant women. It should be recognized that amino acid requirements may change throughout pregnancy. The polypeptides of the present invention may have an optimized amino acid composition to provide an ideal amino acid composition for a nutritional composition suitable for each stage of pregnancy.

[0095] Variants

[0096] The polypeptides of the present invention include variants of any one of SEQ ID NOs: 3 to 101, wherein the variant comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3 to 101 or a truncation thereof. Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0097] Variants have different amino acid compositions compared to their parent polypeptides, which can be optimized as complete protein sources for daily consumption for any of a number of different amino acid requirements, as described above. In some embodiments, the variant has an AAS of ≥ 0.94, but has a different amino acid composition compared to any of SEQ ID NOs: 3 to 101. Such variants can be used in nutritional compositions.

[0098] In some embodiments, a variant may have improved properties compared to the parent polypeptide, such as improved stability under storage conditions or improved thermal stability.

[0099] In certain embodiments, variants have improved purification ease compared to parent polypeptides. In certain embodiments, when following the same purification steps as parent polypeptides, improved purification ease can be higher productive rates. In certain embodiments, improved purification ease may require fewer steps. In certain embodiments, improved purification ease can be that purification process resource intensity is lower, which means that it can use less energy, can spend less time or can use reagents or materials with lower costs.

[0100] In embodiments, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 3 to 101.

[0101] In one aspect, the number of alterations in a variant of the invention is 1-20, such as 1-10 or 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

[0102] In another aspect, the variant comprises a substitution, insertion or deletion at one or more positions of the amino acid sequence of any one of SEQ ID NOs: 3 to 101. In another aspect, the variant comprises a substitution, insertion or deletion at two or more positions of the amino acid sequence of any one of SEQ ID NOs: 3 to 101. In another aspect, the variant comprises a substitution, insertion or deletion at three or more positions of the amino acid sequence of any one of SEQ ID NOs: 3 to 101. In another aspect, the variant comprises a substitution, insertion or deletion at four or more positions of the amino acid sequence of any one of SEQ ID NOs: 3 to 101.

[0103] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0104] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0105] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are altered. For example, amino acid changes can improve the thermal stability of the polypeptide, change the optimal pH, improve purification yield, etc.

[0106] The polypeptides of the present invention are desirable in terms of their amino acid composition and their ease of purification. Amino acids that are critical for stability (which may affect ease of purification) or for ease of purification themselves can be identified in polypeptides according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for stability or ease of purification to identify critical amino acid residues. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Amino acid residues that are critical for stability can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutating putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of amino acids critical for stability or ease of purification can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a family of polypeptides or proteins that are derived from a common ancestor (typically having similar three-dimensional structure, function, and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify amino acids critical for stability or ease of purification. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold,” Nature 596:583-589.

[0107] The variant may consist of 500 to 450, 450 to 400, 400 to 350, 350 to 300, 300 to 250, 250 to 200, 200 to 150, 150 to 100, or 100 to 50 amino acids of any one of SEQ ID NOs: 3 to 101.

[0108] The polypeptide of the present invention may be a fusion polypeptide comprising a variant of the present invention.

[0109] Nucleic acid constructs

[0110] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0111] Polynucleotides can be manipulated in a variety of ways to provide expression of variants. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0112] promoter

[0113] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell to express the polynucleotide encoding the variant of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter can be any polynucleotide that shows transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0114] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications,” and in Schmoll et al., 2014. 2016. “Gene Expression Systems in Fungi: Advancements and Applications.” Fungal Biology.

[0115] terminator

[0116] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3'-end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

[0117] Preferred terminators for filamentous fungal host cells are obtainable from Aspergillus or Trichoderma species, such as the genes for Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase 1, and Trichoderma reesei endoglucanase 1, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications,” and Schmoll et al., 2014. 2016. “Gene Expression Systems in Fungi: Advancements and Applications.” Fungal Biology.

[0118] mRNA stabilizer

[0119] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0120] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0121] Examples of mRNA stabilizer regions of fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0122] leader sequence

[0123] The control sequence can also be a leader sequence, i.e., a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell can be used.

[0124] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0125] polyadenylation sequence

[0126] The control sequence may also be a polyadenylation sequence, i.e., a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0127] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0128] signal peptide

[0129] The control sequence can also be a signal peptide coding region, a signal peptide that is connected to the N-terminal of the coding region encoding the variant, and instructs the variant to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide can inherently contain a signal peptide coding sequence, and this signal peptide coding sequence is naturally linked together in the translation reading frame with the section of the coding sequence of the variant of the coding. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence, so as to strengthen the secretion of the variant. However, any signal peptide coding sequence that the variant of instructing expression can be used to enter the secretory pathway of the host cell.

[0130] An effective signal peptide coding sequence for a filamentous fungal host cell is the signal peptide coding sequence obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0131] propeptide

[0132] The control sequence can also be a propeptide coding sequence encoding the propeptide at the N-terminal end of the variant. The resulting polypeptide is referred to as a proenzyme (proenzyme) or propolypeptide (or in some cases as a zymogen). Propolypeptide is typically inactive and can be converted into an active variant by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained, for example, from the gene of: bacillus subtilis alkaline protease (aprE), bacillus subtilis neutral protease (nprT), thermophilic myceliophthora laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease or saccharomyces cerevisiae α-factor.

[0133] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0134] Regulatory sequence

[0135] It is also desirable to add regulatory sequences that regulate the expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are regulatory sequences that cause gene expression to be turned on or off in response to a chemical or physical stimulus (including the presence of a regulatory compound). In filamentous fungi, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter and Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals.

[0136] transcription factors

[0137] Control sequence can also be a transcription factor, i.e. a polynucleotide encoding a polynucleotide specific DNA binding polypeptide, which controls the transcription rate of genetic information from DNA to mRNA by being combined with a specific polynucleotide sequence. Transcription factors can play a role alone and / or together with one or more other polypeptides or transcription factors in the complex by promoting or blocking the recruitment of RNA polymerase and play a role. Transcription factors are characterized in that they comprise at least one DNA binding domain, which is generally attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can directly (i.e., activate the transcription of the gene encoding the target protein by combining with its promoter) or indirectly (i.e., as by combining with the promoter of another transcription factor (which regulates the transcription of the gene encoding the target protein) to activate the transcription of another transcription factor) regulate the expression of the target protein. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0138] expression vector

[0139] The present invention still further relates to the recombinant expression vector that comprises the polynucleotide of coding variant of the present invention, promoter and transcription and translation termination signal.Various Nucleotide and control sequence can be linked together to produce recombinant expression vector, and this recombinant expression vector can comprise one or more suitable restriction sites to allow to insert or replace the polynucleotide of coding variant at such site.Alternately, can express this polynucleotide by polynucleotide or the nucleic acid construct that comprises this polynucleotide being inserted in the suitable vector for expression.When producing expression vector, coding sequence is so positioned in carrier, makes coding sequence be operably connected with the suitable control sequence for expression.

[0140] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0141] The carrier can be an autonomous replicating vector, i.e. a carrier existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can contain any means for ensuring self-replication. Alternatively, the carrier can be a carrier that is integrated into the genome and replicates with the chromosome into which it has been integrated when it is introduced into the host cell. Moreover, a single carrier or plasmid or two or more carriers or plasmids can be used that contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0142] The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0143] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0144] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0145] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0146] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of the polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into the host cell. The increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene and thus an additional copy of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0147] host cells

[0148] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.

[0149] The construct or vector comprising the polynucleotide is introduced into the host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described earlier. The choice of host cell will depend to a large extent on the gene encoding the variant and its source. The recombinant host cell may comprise a single copy or at least two copies, such as three, four, five or more copies, of the polynucleotide of the present invention.

[0150] The host cell may be any cell useful for the recombinant production of a variant of the invention, such as a prokaryotic or fungal cell.

[0151] The host cell can be any microbial cell, such as a prokaryotic cell or a fungal cell, that can be used to recombinantly produce the polypeptides of the present invention.

[0152] The host cell may be a fungal cell. As used herein, "fungi" includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitospore fungi (as defined in Hawksworth et al., Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0153] Fungal cells can be transformed by processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (reviewed in Li et al., 2017, Microbial Cell Factories 16:168), as well as in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474; Christensen et al., 1988, Bio / Technology 6:1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27:53-75. However, any method known in the art for introducing DNA into fungal host cells may be used, and the DNA may be introduced as a linearized or circular polynucleotide.

[0154] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subphylum Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is carried out by hyphal extension, and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeast (such as Saccharomyces cerevisiae) is carried out by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0155] The filamentous fungal host cell can be Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallima In some embodiments, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium. In other preferred embodiments, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium. In other preferred embodiments, the filamentous fungal host cell is a cell of Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum.

[0156] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetida, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneurina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium stoloniferum, Chrysosporium truncat ... merdarium), Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium nigra sambucinum), Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0157] The host cell can be a prokaryotic cell. The prokaryotic host cell can be any gram-positive or gram-negative bacteria. Gram-positive bacteria include but are not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter (Campylobacter), Escherichia coli, Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), Helicobacter (Helicobacter), Ilyobacter (Ilyobacter), Neisseria (Neisseria), Pseudomonas (Pseudomonas), Salmonella (Salmonella) and Ureaplasma (Ureaplasma).

[0158] The bacterial host cell can be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0159] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equi, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0160] The bacterial host cell can also be any Streptomyces cell, including but not limited to Streptomyces chromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0161] Introduction of DNA into Bacillus cells can be achieved by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), transformation of competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). Introduction of DNA into E. coli cells can be achieved by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be achieved by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells can be achieved by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).Introduction of DNA into Streptococcus cells can be achieved by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0162] In some embodiments, the host cell is isolated. In some embodiments, the host cell is purified.

[0163] Generation method

[0164] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0165] Host cell is cultivated in the nutrient medium that is suitable for using methods known in the art to produce polypeptide.For example, can pass through shake flask culture, or in suitable substratum and under the condition of allowing polypeptide expression and / or separation, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) to cultivate cell in laboratory or industrial fermentor tank.Suitable substratum can be obtained from commercial supplier or can be prepared according to disclosed composition (for example, in the catalogue of American Type Culture Collection).If polypeptide is secreted in nutrient medium, then can directly reclaim this polypeptide from this substratum.If polypeptide is not secreted, then can be reclaimed from cell lysate.

[0166] The polypeptide can be detected using methods known in the art that are specific for the polypeptide, including but not limited to the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or enzymatic assays to determine the relative or actual amount of the polypeptide.

[0167] The polypeptide can be recovered from the culture medium using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0168] Polypeptides can be purified by various procedures known in the art to obtain substantially pure polypeptides and / or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0169] In alternative aspects, the polypeptide is not recovered, but rather a host cell of the invention expressing the variant is used as a source of the variant, for example.

[0170] Nutritional composition

[0171] The nutritional compositions of the present invention comprise the polypeptides disclosed herein.To be suitable for use in nutritional compositions, the polypeptides of the present invention need to be soluble and remain intact over a range of pH and temperature.

[0172] Nutritional compositions are typically comprised at a pH between 3 and 8. The polypeptides of the present invention need to remain soluble at the pH of the nutritional composition in which they are comprised. In some embodiments, the polypeptides of the present invention remain soluble at a pH of at least about pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, or pH 8.0. In some embodiments, the polypeptides of the present invention remain soluble at a pH of about pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, or pH 8.0. In some embodiments, the polypeptides of the present invention remain soluble at a given pH before, during, and after heat treatment.

[0173] The nutritional composition may be heat treated for pasteurization or sterilization. Heat treatment is intended to make the nutritional composition safe for consumption by killing harmful microorganisms, thereby reducing the risk of food poisoning. Heat treatment also increases the shelf life or shelf stability of the nutritional composition because it eliminates microorganisms in the beverage that could degrade the beverage by causing it to spoil or otherwise reducing its quality.

[0174] In some embodiments, the heat treatment is 85°C-95°C for 10 seconds to 30 minutes. In some embodiments, the heat treatment is high temperature short time (HTST) pasteurization. To this end, the composition is heated to a temperature between 71.5°C-74°C for 15-30 seconds, or to a temperature between 74°C-76°C for 15-20 seconds. After heat treatment, the nutritional composition can then be quickly cooled to 4°C-5.5°C. In some embodiments, the acidic probiotic beverage is heat treated by ultra-high temperature (UHT) treatment. UHT treatment can be direct or indirect. In some embodiments, UHT treatment is between 135°C-154°C for 1-10 seconds. In some embodiments, the nutritional composition is heat treated by ultra-pasteurization. To this end, the composition is heated to a temperature between 70°C-75°C for 20-30 minutes.

[0175] In some embodiments, the polypeptides of the invention have qualities that indicate they will remain intact throughout heat treatment, such as, for example, a high melting temperature.

[0176] The nutritional composition may include additional ingredients. In some embodiments, the nutritional composition of the present invention includes a fat source. The fat source can be any suitable fat or fat mixture. In some embodiments, the fat source is a vegetable fat. The vegetable fat can be soybean oil, palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canola oil, lecithin, or any suitable vegetable fat. In some embodiments, the fat source is an animal source, such as milk fat. In some embodiments, the fat source is derived from a plant source, such as fractionated vegetable oil. In some embodiments, the fat source provides approximately 20% to 70% of the energy in the nutritional composition. In other embodiments, the fat source provides approximately 25% to 60% of the energy in the nutritional composition.

[0177] In some embodiments, the nutritional composition comprises a carbohydrate source. The carbohydrate source can be any suitable carbohydrate, including sucrose, lactose, glucose, fructose, corn syrup, corn syrup solids, and / or maltodextrin. In some embodiments, the carbohydrate source provides about 20% to 70% of the energy in the nutritional composition. In other embodiments, the carbohydrate source provides about 30% to 60% of the energy in the nutritional composition.

[0178] In some embodiments, the nutritional composition comprises vitamins and / or minerals required in the diet. Vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid) and vitamin B12 (various cobalamin; usually cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 and K2 (i.e., MK-4, MK-7), folic acid, biotin, choline or any combination thereof. Minerals include boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or any combination thereof.

[0179] In some embodiments, the nutritional composition comprises additional components that may benefit intestinal health and / or overall health. These additional components include omega-3 fatty acids, such as α-linolenic acid, stearidonic acid, docosahexaenoic acid, and eicosapentaenoic acid; phytonutrients, such as carotenoids, plant sterols, quercetin, curcumin, limonin, compounds such as α-ketoglutarate and L-carnitine; or antioxidants, such as astaxanthin, coenzyme Q10, flavonoids, glutathione, hesperidin, emulsified goji berries, lignans, lutein, lycopene, polyphenols, selenium, or zeaxanthin, or any combination thereof.

[0180] In some embodiments, the nutritional composition comprises prebiotics. Prebiotics include gum arabic, alpha glucan, arabinogalactan, beta glucan, dextran, fructooligosaccharides, fucosyllactose, galacto-oligosaccharides, galactomannan, gentio-oligosaccharides, glucose oligosaccharides, guar gum, inulin, isomaltooligosaccharides, lactose neotetraose, lactofructooligosaccharides, lactulose, fructans, maltodextrin, milk oligosaccharides, partially hydrolyzed guar gum, pectin oligosaccharides, resistant starch, retrograded starch, sialo-oligosaccharides, sialyllactose, soy oligosaccharides, sugar alcohols, xylo-oligosaccharides, hydrolyzates thereof, or any combination thereof.

[0181] In some embodiments, the nutritional composition comprises a probiotic. Probiotics include microorganisms such as Aeromonas, Aspergillus, Bacteroides, Bacillus, Bifidobacterium, Brevibacillus, Candida, Clostridium, Debaromyces, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Apis, Micrococcus, Mucor, Oenococcus, Paenibacillus, Pediococcus, Penicillium, Peptostreptococcus, Pichia, Propionibacterium, Pseudocatenulatum, Rhizopus, Saccharomyces, Staphylococcus, Streptococcus, Torulopsis, Weissella, non-replicating microorganisms, or any combination thereof.

[0182] In some embodiments, the nutritional composition may further include any number of optional additional ingredients, including conventional food additives (synthetic or natural), such as one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavors, minerals, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, minerals, and / or vitamins. The optional ingredients may be added in any suitable amount.

[0183] The nutritional composition of the present invention can be prepared by mixing the recombinant polypeptide in powder form, optionally with a fat source or a carbohydrate source or other additional components. The nutritional composition can also be prepared by adding the ingredients together in liquid form and then spray-drying them into a powder. Suitable dosage forms for the nutritional composition of the present invention include tablets, dispersible powders, granules, capsules, liquids, suspensions and syrups.

[0184] Inert diluents and carriers used in tablets include, for example, calcium carbonate, sodium carbonate, lactose, and talc. Tablets may also contain granulating agents and disintegrants, such as starch and alginic acid; binders, such as starch, gelatin, and gum arabic; and lubricants, such as magnesium stearate and stearic acid. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption. Inert diluents and carriers that can be used in capsules include, for example, calcium carbonate, calcium phosphate, and kaolin. Suspensions, liquids, and syrups may contain conventional excipients, such as methylcellulose, gum tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, such as ethyl paraben.

[0185] The nutritional composition of the present invention can be added to the food that patients usually eat. In some embodiments, the nutritional composition can be suitable as a complete protein source. In some embodiments, the nutritional composition can be suitable for individuals with increased medical needs and / or long-term care, including, for example, the elderly, pregnant women, cancer patients, and individuals with long-term diseases such as diabetes. In some embodiments, the nutritional composition can be suitable as a complete protein source for patients with metabolic disorders. In other embodiments, the nutritional composition can be suitable as a complete protein source for patients with PKU and / or HPA.

[0186] Preferred embodiment

[0187] The invention described and claimed herein is not limited in scope to the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become clear to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail.

[0188] The invention is further defined by the following numbered examples.

[0189] 1. A nutritional composition comprising a recombinant polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3-101 or a truncation thereof.

[0190] 2. The nutritional composition of claim 1 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3-101 or a truncation thereof.

[0191] 3. The nutritional composition of claim 1 or claim 2, wherein the recombinant polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 3-101.

[0192] 4. The nutritional composition of any of the preceding claims, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 3-11, 45-48, 50, or 51, or a truncation thereof.

[0193] 5. The nutritional composition of any of the preceding claims, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an N-terminal truncation, a C-terminal truncation, and / or internal truncations.

[0194] 6. The nutritional composition of claim 5, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an N-terminal truncation of at least 1 amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO: 50.

[0195] 7. The nutritional composition of claim 5, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the truncation has a C-terminal truncation of at least 1 amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO: 50.

[0196] 8. The nutritional composition of claim 5, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an internal truncation of at least 1 amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO: 50.

[0197] 9. The nutritional composition of claim 5, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4-48, 52, 53, 58, 61, 62, 63, 66, 67, and 70-75.

[0198] 10. The nutritional composition of any one of the preceding claims, wherein the recombinant polypeptide has an amino acid score (AAS) greater than or equal to 0.94.

[0199] 11. The nutritional composition of any one of the preceding claims, wherein the recombinant polypeptide comprises:

[0200] a) at least 50 mg threonine per gram of polypeptide;

[0201] b) at least 9 mg tryptophan per gram of polypeptide;

[0202] c) at least 55 mg tyrosine per gram of polypeptide; and

[0203] d) at least 66 mg of leucine per gram of polypeptide.

[0204] 12. The nutritional composition according to any one of the preceding claims, wherein the composition does not comprise added free amino acids.

[0205] 13. The nutritional composition of any one of the preceding claims, further comprising vitamins and minerals.

[0206] 14. The nutritional composition according to any one of the preceding claims, further comprising a carbohydrate source and / or a fat source.

[0207] 15. The nutritional composition of any one of the preceding claims, wherein the composition is a tablet, a dispersible powder, a granule, a capsule, a liquid, a suspension or a syrup.

[0208] 16. The nutritional composition of any of the preceding claims, wherein the recombinant polypeptide comprises a nutritionally complete amino acid profile sufficient for use in patients suffering from metabolic disorders.

[0209] 17. The nutritional composition of any of the preceding claims, wherein the recombinant polypeptide comprises a nutritionally complete amino acid profile sufficient for use in patients with phenylketonuria, hyperphenylalaninemia, tyrosinemia, maple syrup urine disease, methylmalonic acidemia, homocystinuria, glutaric aciduria, isovaleric acidemia, and / or hyperlysinemia.

[0210] 18. The nutritional composition of claim 16 or 17, wherein the recombinant polypeptide does not contain amino acids that are harmful to the patient with the metabolic disorder.

[0211] 19. The nutritional composition of any one of claims 16-18, wherein the AAS of the recombinant polypeptide, excluding amino acids harmful to the patient with metabolic disorders, is equal to or greater than 0.94.

[0212] 20. The nutritional composition of any one of claims 16-19, wherein the recombinant polypeptide does not comprise phenylalanine residues.

[0213] 21. The nutritional composition of claim 20, wherein the AAS of the recombinant polypeptide, excluding phenylalanine, is equal to or greater than 0.94.

[0214] 22. The nutritional composition of any one of claims 16-21, wherein the recombinant polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3-48.

[0215] 23. The nutritional composition of any one of claims 1-15, wherein the AAS of the recombinant polypeptide is equal to or greater than 0.94, and further wherein the recombinant polypeptide comprises at least 105 mg leucine / gram of polypeptide.

[0216] 24. The nutritional composition of any one of claims 1-15 or 23, wherein the AAS of the recombinant polypeptide is equal to or greater than 0.94, and further wherein the recombinant polypeptide comprises at least 210 mg branched chain amino acids per gram of polypeptide.

[0217] 25. The nutritional composition of any one of claims 1-15, 23 or 24, wherein the recombinant polypeptide comprises the amino acid sequence of SEQ ID NOs: 78-101.

[0218] 26. A recombinant polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3-101 or a truncation thereof, and further comprising a substitution at a position corresponding to position E334 and / or E426 of SEQ ID NO: 49.

[0219] 27. The recombinant polypeptide of claim 26, comprising an amino acid sequence having at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3-101, or a truncation thereof, and further comprising a substitution at a position corresponding to position E334 and / or E426 of SEQ ID NO: 49.

[0220] 28. The recombinant polypeptide of claim 26 or claim 27, wherein the substitution at the position corresponding to position E334 of SEQ ID NO: 49 is to A, I, G, L, M, Q or V.

[0221] 29. The recombinant polypeptide of claim 26 or claim 27, wherein the substitution at the position corresponding to position E426 of SEQ ID NO: 49 is to A, I, G, L, M, Q or V.

[0222] 30. The recombinant polypeptide of any one of claims 26-29, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or at least 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3-11, 45-48, 50, 51, or a truncation thereof.

[0223] 31. The recombinant polypeptide of any one of claims 26-30, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an N-terminal truncation, a C-terminal truncation, and / or internal truncations.

[0224] 32. The recombinant polypeptide of claim 31 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an N-terminal truncation of at least 1 amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO: 50.

[0225] 33. The recombinant polypeptide of claim 31 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the truncation has a C-terminal truncation of at least 1 amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO: 50.

[0226] 34. The recombinant polypeptide of claim 31 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an internal truncation of at least 1 amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO: 50.

[0227] 35. The recombinant polypeptide of claim 31 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4-48, 52, 53, 58, 61, 62, 63, 66, 67, and 70-75.

[0228] 36. The recombinant polypeptide of any one of claims 26-35, wherein the recombinant polypeptide has an amino acid score (AAS) greater than or equal to 0.94.

[0229] 37. The recombinant polypeptide of any one of claims 26-36, wherein the recombinant polypeptide comprises:

[0230] a) at least 50 mg threonine per gram of polypeptide;

[0231] b) at least 9 mg tryptophan per gram of polypeptide;

[0232] c) at least 55 mg tyrosine per gram of polypeptide; and

[0233] d) at least 66 mg of leucine per gram of polypeptide.

[0234] 38. The recombinant polypeptide of any one of claims 26-34 or 37, wherein the recombinant polypeptide does not comprise a phenylalanine residue.

[0235] 39. The recombinant polypeptide of claim 38, wherein excluding phenylalanine, the AAS of the recombinant polypeptide is equal to or greater than 0.94.

[0236] 40. The recombinant polypeptide of claim 38 or claim 39, wherein the recombinant polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 3-11 and 45-48.

[0237] 41. The recombinant polypeptide of any one of claims 26-36, wherein the AAS of the recombinant polypeptide is equal to or greater than 0.94, and further wherein the recombinant polypeptide comprises at least 105 mg leucine / gram of polypeptide.

[0238] 42. The recombinant polypeptide of any one of claims 26-36 or 41, wherein the AAS of the recombinant polypeptide is equal to or greater than 0.94, and further wherein the recombinant polypeptide comprises at least 210 mg branched-chain amino acids / gram of polypeptide.

[0239] 43. The recombinant polypeptide of any one of claims 26-36, 41 or 42, wherein the recombinant polypeptide comprises the amino acid sequence of SEQ ID NOs: 78-101.

[0240] 44. Use of the recombinant polypeptide according to any one of claims 26 to 43 in a nutritional composition.

[0241] 45. Use of the recombinant polypeptide according to any one of claims 26 to 43 in a nutritional composition for a patient suffering from a metabolic disorder.

[0242] 46. ​​An isolated polynucleotide encoding the polypeptide of any one of claims 26-43.

[0243] 47. A nucleic acid construct or expression vector comprising the polynucleotide of claim 46.

[0244] 48. A recombinant host cell transformed with the polynucleotide of claim 46.

[0245] 49. A method of producing a polypeptide, the method comprising culturing the recombinant host cell of claim 48 under conditions suitable for expression of the polypeptide and recovering the polypeptide.

[0246] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0247] Examples

[0248] Example 1: Production of recombinant polypeptides for nutritional compositions for general nutrition

[0249] A recombinant polypeptide was identified that was found to be expressed at very high levels when recombinantly expressed in Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis. Surprisingly, the polypeptide is also suitable for nutritional compositions because it has an amino acid composition close to the desired AAS and does not contain toxic domains or amino acid motifs known to play a role in protein allergens. Although the polypeptide has mannanase activity (described in WO 2021 / 152123, incorporated herein by reference), inactive variants can be generated by introducing substitutions at positions 334 and / or 426 (relative to the position of SEQ ID NO: 49).

[0250] The combination of a polypeptide with a desired amino acid composition and no known toxic or allergenic domains, and also highly expressed without detectable nonspecific residues from purification, is unpredictable and highly desirable for recombinant polypeptides used in nutritional compositions. The high AAS of this polypeptide also makes it suitable as a nutritional protein in categories beyond general nutrition, such as sports drinks or as medical nutrition for the elderly and hospitalized patients with limited appetite. Therefore, it was determined whether the inactive variant polypeptide still possessed the same useful qualities with respect to ease of purification and sample purity.

[0251] The polypeptide encoded by SEQ ID NO:49 is an active mannanase. The polypeptide encoded by SEQ ID NO:50 is an inactivated mannanase. Nucleic acid constructs encoding SEQ ID NO:49 or 50 were introduced into a strain of the filamentous fungus Aspergillus niger. These recombinant strains were fermented using standard protocols (3-4 days, 30°C), and the recombinant polypeptides were purified by chromatography using standard ion exchange techniques. After trypsin digestion, expression and purification of the recombinant polypeptides were analyzed using SDS-PAGE and MS proteomics. It was found that the recombinant polypeptides of both SEQ ID NO:49 and 50 were expressed at high levels.

[0252] Table 1 provides yield and purity data from two different fermentations producing the polypeptide of SEQ ID NO: 49 and a single fermentation producing the polypeptide of SEQ ID NO: 50. SDS-PAGE analysis unambiguously identified the target protein, and density scanning of the SDS-PAGE gel enabled relative quantification of the purified protein. The results demonstrate robust yields and excellent purity.

[0253] Table 1: Yield and purity of recombinant peptides

[0254] Fermentation Fermentation supernatant Purified samples SEQ ID NO: 49, Fermentation 1 87％-93％ 100％ SEQ ID NO: 49, Fermentation 2 95％ 100％ SEQ ID NO:50 94％ 100％

[0255] In addition to the purity estimated by SDS-PAGE, LC-MS / MS was used to estimate the expression of protein variants relative to the host strain protein. Tryptic digests were prepared by filter-assisted sample preparation (FASP) method. In brief, after trypsin digestion, extracted peptides were analyzed on a nanoLC-MS / MS system: Evosep One (Evosep, Denmark) / timsTOF Pro (Bruker Daltonik, Massachusetts, USA). For protein identification, the Mascot search engine (Matrix Science, London, UK) was used to search data for available internal and public databases using Genedata Expressionist software with a 1% false discovery rate cutoff. Relative protein concentration was calculated by label-free quantitative analysis according to peptide volume in Genedata Expressionist. The results are shown in Table 2.

[0256] Table 2: LC-MS / MS data of recombinant peptide yield and purity

[0257] Fermentation Fermentation supernatant Purified samples SEQ ID NO: 49, Fermentation 1 94％ 97％ SEQ ID NO: 49, Fermentation 2 93％ 98％ SEQ ID NO:50 87％ 98％

[0258] The LC-MS / MS data in Table 2 confirm the conclusion from SDS-PAGE analysis regarding the high ratio of target protein to host strain protein. This indicates that the purification process successfully removed cell debris and other non-protein components from the fermentation that could affect the functionality of the food product and the flavor profile of the final product.

[0259] Example 2: Production of recombinant polypeptides for nutritional compositions for metabolic disorders

[0260] Inactive variants of mannanase can be produced with high expression levels and high purity levels as shown in Example 1. Therefore, additional mutations were introduced to test whether other variants of the polypeptide would still be easy to purify with high yield and high purity.

[0261] The polypeptides of SEQ ID NOs: 49 and 50 have a relatively low phenylalanine composition. Variant polypeptides were generated in which phenylalanine was substituted with other amino acids. Substitutions were also introduced to balance the amino acid composition such that, in addition to the absence of phenylalanine, the polypeptide had an AAS ≥ 0.94. An example of the nucleic acid sequence of an inactive, Phe-free recombinant polypeptide with an AAS ≥ 0.94 is SEQ ID NO: 1. The corresponding amino acid sequence of the recombinant polypeptide is SEQ ID NO: 2, and the corresponding amino acid sequence of the mature polypeptide without the signal peptide is SEQ ID NO: 3.

[0262] A nucleic acid construct comprising the nucleotide sequence encoding SEQ ID NO:3 was introduced into a filamentous fungus strain of Aspergillus oryzae. The recombinant strain was fermented using a standard protocol (3-4 days at 30°C) and then purified by chromatography using standard ion exchange techniques. SDS-PAGE was used to analyze the expression and purification of the recombinant polypeptide. The recombinant polypeptide of SEQ ID NO:3 was found to be expressed at high levels.

[0263] Table 3 shows data from the fermentation and purification of the recombinant polypeptide of SEQ ID NO: 3 using four different culture media. SDS-PAGE analysis clearly identified the target protein, and density scanning of the SDS-PAGE gel enabled relative quantification of the purified protein. The results demonstrate that robust yields and excellent purity can be achieved across a range of culture media.

[0264] Table 3: Yield and purity using different fermentation media

[0265] Fermentation supernatant Purified samples Medium 1 More than 95% 100％ Medium 2 More than 95% 100％ Medium 3 More than 95% 100％ Medium 4 More than 95% 100％

[0266] The level of phenylalanine in the purified samples was estimated by total amino acid analysis. Although the polypeptide of SEQ ID NO: 3 does not contain phenylalanine, phenylalanine may be present in the purified samples due to background phenylalanine residues carried by the expression host (e.g., cell debris). The amount of residual phenylalanine present in the purified protein was determined using standard amino acid analysis methods, wherein amino acids were derivatized using AccQ-Tag Ultra reagent (Waters Corp., Milford, MA) and reversed-phase UPLC ( Amino acids were separated using a 5-mercaptoethanol (Waters Corporation, Milford, MA). Derivatives were quantified based on UV absorbance. The detection limit for phenylalanine using this method was 2 pmol. No phenylalanine was detected, indicating that little or no phenylalanine was present in the purified sample.

[0267] Example 3: In vitro digestibility

[0268] The digestibility of the purified recombinant polypeptides described in Examples 1 and 2 was determined using the method published by Minekus et al. ("A standardised static in vitro digestion method suitable for food – an international consensus," FoodFunct, 5:1113-1124, 2014). The treated samples were then analyzed by SDS-PAGE. The results are shown in Table 4.

[0269] Table 4: In vitro digestibility

[0270] peptides oral cavity Stomach intestinal SEQ ID NO:3 - +++ +++ SEQ ID NO:49 - +++ +++ SEQ ID NO:50 - +++ +++

[0271] None of the variants were degraded during the simulated oral phase, while all variants showed extensive degradation during the simulated gastric phase, with only a few peptide fragments detectable by SDS-PAGE. The few fragments detectable after the simulated gastric phase were completely degraded during the simulated intestinal phase. Overall, the in vitro digestibility assays indicate complete digestibility of these molecules, and therefore high bioavailability of the individual amino acids.

[0272] Example 4: Thermal stability and solubility

[0273] The use of polypeptides in food applications requires more than just high nutritional value to be successful. Functionalities like heat stability and solubility are important for achieving good quality in finished nutritional compositions such as processed foods like powders, beverages and bars.

[0274] The thermal stability of purified polypeptides comprising the amino acid sequence of SEQ ID NO: 49 or 50 was studied by nanodifferential scanning fluorimetry (nanoDSF) using equipment from NanoTemper Technologies, Munich, Germany. nanoDSF is a biophysical characterization technique used to assess the conformational stability of biological samples. It uses the intrinsic fluorescence of proteins to monitor their response to stress inputs such as temperature or chaotropes. This information is used to determine the conformational stability of proteins and rank candidates based on their impact on stability, either by the candidate or the buffer formulation.

[0275] The thermal stability of a protein is determined by its unfolding onset temperature (T on ) and the melting temperature (T m The results are shown in Table 5.

[0276] Table 5: Thermal stability

[0277] peptides <![CDATA[T on (℃)]]> <![CDATA[T m (℃)]]> SEQ ID NO:49 73.6 78.7 SEQ ID NO:50 66.2 78.7

[0278] The high melting temperatures of the purified polypeptides indicate that these polypeptides can remain intact throughout heat treatments, such as high temperature short time (HTST) pasteurization or similar heat treatments typically used in the food industry for pasteurization or sterilization.

[0279] Solubility is another key factor in determining the suitability of a polypeptide in producing a nutritional composition. For the polypeptides of SEQ ID NO: 49 and 50, the pH dependence of solubility was determined respectively in the case of and without short-term heat treatment. Before and after heat treatment at 95°C for 3 min, the solubility of a solution containing the polypeptide of SEQ ID NO: 49 or 50 (10 mg / mL) was determined in the range of pH 3 to 8. The solubility percentage was determined by measuring the absorbance at 280 nm before and after centrifugation (to remove precipitation) and calculating the ratio. The solubility percentage was determined before and after heat treatment. The results are shown in Table 6.

[0280] Table 6: Solubility in the pH range after heat treatment

[0281]

[0282]

[0283] Prior to heat treatment, samples showed near 100% protein solubility, except at pH 5, which is close to the pI of the polypeptide. In water at neutral pH, polypeptides encoding the amino acid sequences of SEQ ID NOs: 49 and 50 showed high solubility (greater than 10% w / w). After heat treatment, the solubility of both polypeptides decreased. However, the inactive variant polypeptide comprising SEQ ID NO: 50 exhibited a more favorable solubility profile after heat treatment, indicating that the inactive variant is a good candidate for use in nutritional compositions.

[0284] The thermal stability of many variants is shown in Table 7. The thermal stability was determined by nanoDSF as described above. These variants have an unfolding temperature (Tm) range greater than 20°C, thus demonstrating that variants of the recombinant polypeptide can be generated over a wide range for specific applications with various thermal stability requirements.

[0285] Table 7: Thermal stability of polypeptide variants

[0286]

[0287]

[0288] Example 5: Production of additional recombinant polypeptides for use in nutritional compositions

[0289] Additional protein variants of SEQ ID NO:3 and SEQ ID NO:50 were generated. The variants included N-terminal truncations, with these truncations ranging in length from 15 to 158 amino acids. These constructs encoded polypeptides comprising the amino acid sequences of SEQ ID NOs:4-53. Expression of these variants was evaluated in Aspergillus oryzae and Bacillus licheniformis. The recombinant polypeptide comprising SEQ ID NO:52 was highly expressed in Bacillus licheniformis, demonstrating the broad versatility of this protein backbone for use in the food industry.

[0290] Example 6: Production of recombinant polypeptides for special nutritional compositions

[0291] It is desirable to produce polypeptides comprising at least 105 mg leucine per gram of polypeptide and at least 210 mg branched chain amino acids per gram of polypeptide for use in nutritional compositions for general nutrition and / or specialty nutrition, such as medical nutrition and / or sports nutrition.

[0292] Substitutions were introduced into the parent SEQ ID NO: 51 (which is an inactive variant) to generate polypeptides having a leucine content of 115-137 mg / g polypeptide and a branched chain amino acid content of 221-250 mg / g polypeptide, and a total amino acid score > 1. These variants are shown in Table 8.

[0293] Table 8: Peptide variants for general nutrition

[0294]

[0295]

Claims

1. A nutritional composition comprising a recombinant polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3-101 or a truncation thereof.

2. The nutritional composition of any one of the preceding claims, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an N-terminal truncation, a C-terminal truncation, and / or internal truncations.

3. The nutritional composition of claim 1 or claim 2, wherein the recombinant polypeptide has an amino acid score (AAS) greater than or equal to 0.

94.

4. A recombinant polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3-101 or a truncation thereof, and further comprising a substitution at a position corresponding to position E334 and / or E426 of SEQ ID NO:

49.

5. The recombinant polypeptide of claim 4, wherein the substitution at the position corresponding to position E334 of SEQ ID NO: 49 is substituted with A, I, G, L, M, Q or V.

6. The recombinant polypeptide of claim 4, wherein the substitution at the position corresponding to position E426 of SEQ ID NO: 49 is substituted with A, I, G, L, M, Q or V.

7. The recombinant polypeptide of any one of claims 4-6, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or at least 100% sequence identity to the amino acid sequence of SEQ ID NOs: 3-11, 45-48, 50, 51, or a truncation thereof.

8. The recombinant polypeptide of any one of claims 4-7, wherein the recombinant polypeptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a truncation of the amino acid sequence of SEQ ID NO: 50, wherein the truncation has an N-terminal truncation, a C-terminal truncation, and / or internal truncations.

9. The recombinant polypeptide of any one of claims 4-8, wherein the recombinant polypeptide has an amino acid score (AAS) greater than or equal to 0.

94.

10. Use of the recombinant polypeptide according to any one of claims 4 to 9 in a nutritional composition.

11. Use of the recombinant polypeptide according to any one of claims 4 to 9 in a nutritional composition for patients suffering from metabolic disorders.

12. An isolated polynucleotide encoding the polypeptide of any one of claims 4-9.

13. A nucleic acid construct or expression vector comprising the polynucleotide according to claim 12.

14. A recombinant host cell transformed with the polynucleotide according to claim 12.

15. A method of producing a polypeptide, the method comprising culturing the recombinant host cell of claim 14 under conditions suitable for expression of the polypeptide and recovering the polypeptide.

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