Nutritional compositions comprising recombinant polypeptides having a nutritionally complete amino acid profile - Patent Application 20070122999

Abstract

The present invention provides nutritional compositions for general and specialized nutrition, comprising recombinant polypeptides having a nutritionally complete amino acid profile suitable for daily human intake. The present invention further provides recombinant polypeptides, as well as variants and truncations thereof, suitable for providing a nutritionally complete amino acid profile when provided in a nutritional composition. The present invention also provides polynucleotides encoding the recombinant polypeptides, nucleic acid constructs, vectors and host cells comprising the polynucleotides, and methods for producing the recombinant polypeptides.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable format, which is incorporated herein by reference.

[0002] The present invention relates to nutritional compositions comprising a recombinant polypeptide of the invention, the recombinant polypeptide of the invention, polynucleotides encoding the recombinant polypeptide, nucleic acid constructs, vectors and host cells comprising the polynucleotides, and methods for producing said recombinant polypeptides. [Background technology]

[0003] The inclusion of high-quality dietary protein sources is a general requirement for all human diets. The body cannot synthesize certain amino acids necessary for health and growth. These "essential" amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Dietary proteins that provide all essential amino acids are referred to as high-quality proteins or complete proteins. Foods with higher protein quality are considered more beneficial in mammalian diets than proteins that do not. Complete proteins promote the maintenance of muscle mass, a healthy body mass index, and blood sugar balance. Additionally, by increasing the amount of complete protein in the diet, total protein intake can be reduced compared to diets primarily consisting of lower-quality proteins.

[0004] In general nutrition, there is a general demand for foods that are high in protein and have an amino acid composition that meets the daily requirements of essential amino acids. In addition, there is a demand for foods that meet the protein needs of people with special nutritional needs. Special nutrition includes the nutritional needs of sick or elderly patients, or patients 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.

[0005] Protein-enriched dietary products, whether for general nutrition or special nutrition, are typically enriched with proteins that are relatively easy to obtain and inexpensive to refine.Such proteins include whey protein, casein, and albumin.Although these proteins are good sources of many essential amino acids, they cannot be adjusted to provide the desired amount of a specific amino acid.In addition, patients with metabolic disorders often need to exclude at least one essential amino acid from their diet and highly enrich other amino acids.To address at least part of this problem, dietary products often contain free amino acids added in amounts that provide a complete nutritional amino acid profile.Unfortunately, free amino acids have a very bitter taste that cannot be completely masked by the addition of other ingredients such as sugars and / or flavorings.Therefore, these dietary products are not desirable for people who need to consume them. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for protein sources that can be tailored to provide a desired nutritional profile and that can also be provided in a cost-effective manner. [Means for solving the problem]

[0007] The present invention provides recombinant polypeptides that can be modified to be suitable for nutritional compositions as a complete protein source for general or specialized nutrition. For general nutritional compositions, recombinant polypeptides can be modified to provide variants with a nutritionally complete amino acid profile as determined by an amino acid score of 0.94 or greater. The present invention also provides polypeptide variants that can be tailored for specialized nutrition by enriching and / or reducing specific amino acids depending on the amino acid needs of the targeted group of amino acids in the nutritional composition. Because the recombinant polypeptides of the present invention have a complete nutritional profile, there is no need to add free amino acids to the composition. Therefore, the compositions do not have the bitter taste associated with many dietary products available today. The recombinant polypeptides of the present invention are highly expressed and relatively easy to purify, making them extremely cost-effective to produce. Additionally, the recombinant polypeptides of the present invention can tolerate a wide pH range and high temperature treatments, making them compatible with a variety of foods and food manufacturing processes.

[0008] definition Following the detailed description, the following definitions apply: Please note that the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0009] Unless otherwise defined or clearly indicated by 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.

[0010] This disclosure refers to amino acids. The full names of amino acids are used interchangeably with the standard three-letter and one-letter abbreviations, respectively. For the avoidance of doubt, they 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), and valine (Val, V).

[0011] Amino acid composition: The amino acid composition of a polypeptide is calculated as milligrams of amino acids per gram of polypeptide. The amino acid composition can be calculated for a specific amino acid of interest, such as the amount of leucine in a given polypeptide, or for a group of amino acids, such as the amount of branched-chain amino acids in a given polypeptide. Calculations 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 an amino acid residue of interest is determined by multiplying the number of said residues in the polypeptide by the molecular weight of that residue in the polypeptide (which is different from the molecular weight of the free amino acid).

[0012] cDNA: The term "cDNA" refers to 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 emerging as a mature, spliced ​​mRNA.

[0013] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0014] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., derived from the same gene) or heterologous (i.e., derived from different genes) to the polynucleotide encoding the variant, and can be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include a promoter and transcription and translation stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the variant.

[0015] Expression: The term "expression" includes all steps involved in the production of a variant (for example, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion).

[0016] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a variant, the coding sequence operably linked to a suitable control sequence capable of effecting the 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, an enhancer, and sequences that control the termination of transcription and translation.

[0017] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxy terminus of a polypeptide.

[0018] Fragment: The term "fragment" refers to a variant in which one or more amino acids are missing from the amino and / or carboxy terminus of a polypeptide.

[0019] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which a polypeptide is fused at the N-terminus and / or C-terminus of a variant of the present invention. Fusion polypeptides are 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 to each other. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding polypeptides such that the coding sequences 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 a fusion polypeptide is created post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). Fusion polypeptides can further include a cleavage site between the two polypeptides. Upon secretion of the fusion protein, this site is cleaved to release the two polypeptides. Examples of cleavage sites include Martin et al., 2003, J.Ind.MicrobiolBiotechnol.3:568-576;Svetina et al.,2000, J.Biotechnol.76:245-251;Rasmussen-Wilson et al. al., 1997, Appl.Environ.Microbiol.63:3488-3493; Ward et al., 1995, 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. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0020] Heterologous: The term "heterologous" with respect to a host cell means that the polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that the regulatory sequences of the polypeptide or nucleic acid, e.g., a promoter, are not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequences are derived from a gene other than the gene encoding the mature polypeptide.

[0021] 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 containing a polynucleotide encoding a variant has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0022] Improved trait: The term "improved trait" refers to a characteristic associated with a variant that is improved compared to the parent.

[0023] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction," as known in the art.

[0024] Isolated: The term "isolated" refers to a variant, nucleic acid, cell, or other specified substance or component that is separated from at least one other substance or component, including, but not limited to, other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other substance is in a form that does not occur in nature. Isolated polypeptides include, but are not limited to, culture broth containing the secreted variant expressed in a host cell.

[0025] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0026] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide.

[0027] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0028] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0029] Nucleic Acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules that can encode variants. Nucleic acids can be single- 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 particular amino acid, and the compositions and methods of the present invention encompass multiple nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0030] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner not normally found in nature, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0031] Operably linked: The term "operably linked" means that the particular components are in a relationship (including, but not limited to, a juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0032] Parent or Parent Polypeptide: The term "parent" or "parent polypeptide" refers to a polypeptide to which modifications are made to generate a polypeptide variant of the present invention.

[0033] Purified: The term "purified" refers to a nucleic acid, variant, or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, and usually 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., percent by weight or molar concentration). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule after application of the purification or enrichment technique. The term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other particular substance or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0034] In one aspect, the term "purified," as used herein, refers to a variant or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a variant that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which it is obtained. In one aspect, the variant has been separated from a portion of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0035] Thus, a variant can be purified so that only trace amounts of other proteins (particularly other polypeptides) are present. The term "purified," as used herein, can refer to the removal of other components, particularly other proteins, most particularly other enzymes, present in the cell from which the polypeptide originates. A variant can be "substantially pure," i.e., free from other components from the organism from which it is produced, e.g., the host organism of a recombinantly produced variant. In one embodiment, the polypeptide is at least 40% pure by weight of total polypeptide material present in the preparation. In one embodiment, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains up to 10%, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and even most preferably up to 0.5% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0036] Thus, 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, by weight of total polypeptide material present in the preparation. The variants of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the variant by well-known recombinant methods or by classical purification methods.

[0037] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form sequence configurations that differ from those found in nature. The term recombinant refers to a cell, nucleic acid, variant, polypeptide, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions that are different from those found in nature. Recombinant polypeptides are the products of these genes expressed in recombinant cells. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0038] Recover: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified substances, for example, recovery of a polypeptide from a whole fermentation broth or from a cell-free fermentation broth, by collecting polypeptide crystals, by filtration, for example, depth filtration (using filter aids or packed filter materials, chamber filter fabric filtration, rotary drum filtration, drum filtration, rotary vacuum drum filter, candle filter, horizontal leaf filter, or similar, with seed or pad filtration in a frame or modular configuration), or membrane filtration (using sheet filtration, modular filtration, candle filtration, microfiltration, ultrafiltration, either in cross-flow, dynamic cross-flow, or dead-end operation), or by centrifugation (using a decanter centrifuge, disc centrifuge, hydrocyclone, or similar), or by precipitating the polypeptide and using particle size classification separation using an appropriate solid-liquid separation method. Recovery encompasses isolation and / or purification of the polypeptide.

[0039] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0040] For the purposes of the present invention, sequence identity between two amino acid sequences is preferably determined as a "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified on the command line. The "longest identity" output displayed by Needle is calculated as follows: (Identical residues × 100) / (length of alignment − total number of gaps in the alignment)

[0041] For purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as a "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified on the command line. The "longest identity" output displayed by Needle is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment − total number of gaps in alignment)

[0042] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0043] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more nucleotides are missing from the 5' and / or 3' end of the mature polypeptide coding sequence.

[0044] Truncate: The term "truncate" refers to a polypeptide that is smaller than a full-length polypeptide. Truncates may be truncated at the C-terminus, the N-terminus, or both. Truncates may also, or alternatively, include internal truncations, where an internal portion of the full-length sequence may be removed.

[0045] Mutant: The term "mutant" refers to a polypeptide that contains a substitution, insertion (including extension) and / or deletion (including, for example, a truncation) at one or more positions compared to its parent. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid, a deletion refers to the removal of an amino acid occupying a position, and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0046] Wild-type: The term "wild-type" with respect 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 something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modifications of a wild-type sequence).

[0047] Sufficient amount: The term "sufficient amount" refers to an amount of a polypeptide or amino acid sufficient to elicit a desired effect. A sufficient amount of a polypeptide or amino acid can be provided directly, i.e., by administering the polypeptide or amino acid to a subject, or can be provided as part of a composition comprising the polypeptide or amino acid.

[0048] 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 each age group ("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 pattern for protein quality assessment was determined by calculating the ratio of essential amino acids to protein requirements, expressed as mg of amino acids per gram of protein. The AAS was established using the scoring pattern determined for children's protein requirements (6 months to 3 years; see Table 5 in the FAO report cited as a reference). To calculate the AAS for a given polypeptide, the ratio of the amount of that essential amino acid present in the polypeptide compared to the amount of that essential amino acid recommended in the FAO report is determined for each essential amino acid. The AAS for that polypeptide is the lowest ratio determined for any of the essential amino acids. Polypeptides with an AAS greater than 0.94 satisfy the essential amino acid requirements for all essential amino acids and are generally considered to be complete protein sources.

[0049] Branched Chain Amino Acids (BCAAs): The term "branched chain amino acids" refers to amino acids selected from leucine, isoleucine, and valine.

[0050] Essential amino acid: The term "essential amino acid" refers to an amino acid selected from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

[0051] Large neutral amino acids (LNAA): The term "large neutral amino acids" refers to amino acids selected from phenylalanine, arginine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, tyrosine, and valine. These amino acids utilize the same transport systems in the brain and intestinal mucosa.

[0052] Nutritional composition: The term "nutritional composition" refers to a composition comprising a recombinant polypeptide of the present invention containing a desired amount of amino acids. The nutritional composition may also contain any number of optional additional ingredients, including conventional food additives (synthetic or natural), such as one or more acidulants, additional thickeners, buffers or pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavors, minerals, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizing agents, minerals, and / or vitamins. Optional ingredients can be added in any suitable amount. The nutritional composition may be a complete or incomplete source of nutrition. The nutritional composition may provide the daily dietary requirements for protein or essential amino acids.

[0053] 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 it is administered. A patient can receive 100 percent of their nutritional needs from such a complete nutritional composition.

[0054] Effective amount: The term "effective amount" refers to an amount that prevents a deficiency, treats an individual's disease or medical condition, or more generally reduces the symptoms, manages the progression of a disease, or provides an individual with a nutritional, physiological, or medical benefit. An effective amount can also be an amount that satisfies the daily dietary requirements of protein or essential amino acids for a generally healthy person based on recommendations published by the FAO (FAO Food Nutr Paper, 92:1-66, 2013).

[0055] Patient or Individual: The terms "patient" or "individual" are often 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 can benefit from the nutritional compositions of the present invention, or any animal, mammal, or human without a medical condition that can benefit from the nutritional compositions of the present invention for general well-being.

[0056] Rules for naming variants In describing the variants of the present invention, the nomenclature described below will be adopted for ease of reference: Recognized IUPAC one-letter or three-letter amino acid abbreviations are used.

[0057] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine with alanine at position 226 is designated "Thr226Ala" or "T226A." Multiple mutations are separated by adding a symbol ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent a substitution of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0058] Deletions: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 is designated "Gly195*" or "G195*". Multiple deletions are separated by adding a symbol ("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*".

[0059] Insertions: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of lysine after glycine at position 195 is designated as "Gly195GlyLys" or "G195GK." Multiple amino acid insertions are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, an insertion of lysine and alanine after glycine at position 195 is designated as "Gly195GlyLysAla" or "G195GKA."

[0060] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. In the above example, the sequence would therefore be:

[0061] [Table 1]

[0062] Multiple modifications: Variants containing multiple modifications are separated by adding a symbol ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively.

[0063] Variant modifications: Where a variety of modifications can be introduced at a position, the variants are separated by commas, for example, "Arg170Tyr,Glu" denotes the substitution of arginine with tyrosine or glutamic acid at position 170. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" denotes the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly" and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION OF THE INVENTION

[0064] A recombinant polypeptide has been identified that is expressed at very high levels when recombinantly expressed in Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis and has also been found to be relatively easily purified to high levels using methods well known in the art. Surprisingly, this polypeptide also has an amino acid composition that provides a desirable AAS value and contains neither toxic domains nor amino acid motifs known to be involved in protein allergens, making it suitable for nutritional compositions. While the polypeptide has mannanase activity (as described in WO 2021 / 152123, incorporated herein by reference), inactive mutants can be generated by introducing substitutions at positions E426 and / or E334, using the amino acid sequence of SEQ ID NO:49 for numbering purposes. Examples of inactive mutants are SEQ ID NOs:50 and 51. The inventors found that the inactive mutants were still highly expressible and easy to purify.

[0065] Additionally, the mannanase polypeptide variants of the polypeptides of the present invention are resistant to heat treatment and a wide range of pH, making them suitable as components of nutritional compositions that often have a neutral or acidic pH, and suitable for withstanding production and processing of nutritional compositions that may require heat during production and may also require some form of heat treatment for sterilization.

[0066] The present invention further relates to nutritional compositions containing mannanase polypeptide variants suitable as a complete or nearly complete protein source for human daily consumption. The nutritional compositions of the present invention may contain mannanase polypeptide variants suitable for individuals with increased medical needs and / or receiving long-term care, including the elderly, pregnant women, cancer patients, and individuals with long-term conditions such as diabetes. The nutritional compositions of the present invention may contain mannanase polypeptide variants suitable as a complete protein source for patients with metabolic disorders. The polypeptides of the present invention are recombinant and include SEQ ID NOS: 3-101 and any variants thereof. The variants have 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 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 the polypeptides of any one of SEQ ID NOS: 3-101.

[0067] Polypeptides of the invention may comprise substitutions, insertions or deletions at one or more positions in the polypeptide of any one of SEQ ID NOs: 3 to 101. Polypeptides of the invention may further comprise an extension of one or more amino acids at the N-terminal and / or C-terminal end.

[0068] In some embodiments, a polypeptide of the present invention may be a fusion polypeptide or a cleavable fusion polypeptide. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide to a polynucleotide encoding a polypeptide of the present invention. Fusion polypeptides may comprise a fragment of a polypeptide of the present invention or a full-length polypeptide of the present invention. In some embodiments, a fusion polypeptide may 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.

[0069] Techniques for generating fusion polypeptides are known in the art and involve ligating coding sequences encoding polypeptides such 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 generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0070] The fusion polypeptide may further comprise a cleavage site between the two polypeptides, which is cleaved to release the two polypeptides upon secretion of the fusion protein. Examples of cleavage sites include Martin et al.,2003, J.Ind.Microbiol.Biotechnol.3:568-576;Svetina et al.,2000, J. Biotechnol.76:245-251;Rasmussen-Wilson et al. al., 1997, Appl.Environ.Microbiol.63:3488-3493; Ward et al., 1995, 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. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0071] Alternatively, the polypeptides of the present invention may be truncated variants comprising one or more amino acid truncations at the N- and / or C-terminus relative to SEQ ID NO: 51, or internal truncations in which the N- or C-terminal amino acids are preserved and some internal amino acids are removed. Polypeptides of the present invention include truncated variants of any one of SEQ ID NOs: 3 to 101. Polypeptides of the present invention may be truncated variants 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 polypeptides of the invention are truncated variants 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 truncated version of any one of SEQ ID NOs: 3-101.

[0072] 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 is 1 to 350 amino acids, 1 to 340 amino acids, 1 to 330 amino acids, 1 to 320 amino acids, 1 to 310 amino acids, 1 to 320 amino acids, 1 to 330 amino acids, 1 to 340 amino acids, 1 to 35 ... 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, 1-255 amino acids, 1-250 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, 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 and a C-terminal truncation of 1 to 95 amino acids, 1 to 90 amino acids, 1 to 85 amino acids, 1 to 80 amino acids, 1 to 75 amino acids, 1 to 70 amino acids, 1 to 65 amino acids, 1 to 60 amino acids, 1 to 55 amino acids, 1 to 50 amino acids, 1 to 45 amino acids, 1 to 40 amino acids, 1 to 35 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, or 1 to 5 amino acids.

[0073] 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 is 1 to 350 amino acids, 1 to 340 amino acids, 1 to 330 amino acids, 1 to 320 amino acids, 1 to 310 amino acids, 1 to 320 amino acids, 1 to 330 amino acids, 1 to 340 amino acids, 1 to 35 ... 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, 1-255 amino acids, 1-250 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, 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 and an N-terminal truncation of 1 to 95 amino acids, 1 to 90 amino acids, 1 to 85 amino acids, 1 to 80 amino acids, 1 to 75 amino acids, 1 to 70 amino acids, 1 to 65 amino acids, 1 to 60 amino acids, 1 to 55 amino acids, 1 to 50 amino acids, 1 to 45 amino acids, 1 to 40 amino acids, 1 to 35 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, or 1 to 5 amino acids.

[0074] 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 is 1 to 350 amino acids, 1 to 340 amino acids, 1 to 330 amino acids, 1 to 320 amino acids, 1 to 310 amino acids, or a combination thereof, relative to the amino acid sequence of SEQ ID NO: 51. 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, 1-255 amino acids, 1-250 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 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, 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 to 95 amino acids, 1 to 90 amino acids, 1 to 85 amino acids, 1 to 80 amino acids, 1 to 75 amino acids, 1 to 70 amino acids, 1 to 65 amino acids, 1 to 60 amino acids, 1 to 55 amino acids, 1 to 50 amino acids, 1 to 45 amino acids, 1 to 40 amino acids, 1 to 35 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, or 1 to 5 internal truncations.

[0075] 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 truncates can be N-terminal truncations in which amino acids 1-265 are removed relative to SEQ ID NO:3, such as SEQ ID NO:10. In some embodiments, the truncates can be N-terminal truncations in which amino acids 1-158 are removed relative to SEQ ID NO:3, such as SEQ ID NOs:4-8, 11-43, and 45-48. In some embodiments, the truncates can be N-terminal truncations in which amino acids 1-157 are removed relative to SEQ ID NO:3, such as SEQ ID NO:9. In some embodiments, the truncates can have the first 11 amino acids from the N-terminus, followed by internal truncations at positions 12-160 according to the numbering positions of the amino acid sequence of SEQ ID NO:3, such as internal truncation variants SEQ ID NOs:52 and 53.

[0076] 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 NOs: 3-101 or truncations thereof, and further comprise a substitution at a position corresponding to E334 and / or E426 of SEQ ID NO: 49. The substitution at position 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 substitution at position 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.

[0077] Amino acid composition The polypeptides of the present invention have an amino acid score (AAS) of 0.94 or greater (≥ 0.94). The AAS is based on the scoring pattern for essential amino acid requirements by age group published by the Food and Agriculture Organization of the United Nations (FAO) ("Dietary protein quality evaluation in human nutrition: Report of an FAO Expert Consultation," FAO Food Nutr Paper, 92:1-66, 2013), particularly for the protein requirements of children (6 months to 3 years old). To calculate the AAS of a given polypeptide, the ratio of the amount of that essential amino acid present in the polypeptide compared to the amount of that essential amino acid recommended in the FAO report is determined for each essential amino acid. The AAS of that polypeptide is the lowest ratio determined for any of the essential amino acids. Polypeptides with an AAS of 0.94 or greater satisfy the essential amino acid requirements for all essential amino acids and are generally considered complete protein sources. In some embodiments, the polypeptides of the invention have an AAS of 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or more than 1.

[0078] Polypeptides suitable for patients with metabolic disorders are considered complete protein sources after their amino acid composition is adjusted to suit the patient's needs. For example, phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are inherited metabolic disorders in which phenylalanine (Phe) cannot be properly processed. Therefore, phenylalanine in the diet should be avoided as much as possible. The recombinant polypeptides of the present invention suitable as complete protein sources for PKU or HPA patients do not contain phenylalanine residues and have an AAS of ≥ 0.94 for all amino acids other than phenylalanine.

[0079] In addition to being deficient in certain amino acids, recombinant polypeptides that serve as the main protein source for patients with metabolic disorders may need or preferably have high levels of certain other amino acids.For example, PKU patients are often recommended to consume proteins that are particularly high in large neutral amino acids such as threonine, tryptophan, tyrosine and leucine.Therefore, polypeptides suitable for PKU patients may have higher amounts of these amino acids than the amount that is usually considered sufficient.In some embodiments, the polypeptides of the invention contain at least 50 mg of threonine per gram of polypeptide (e.g., 50 mg of threonine per gram of polypeptide, at least 60 mg of threonine per gram of polypeptide, at least 70 mg of threonine per gram of polypeptide, at least 80 mg of threonine per gram of polypeptide, at least 90 mg of threonine per gram of polypeptide, or at least 100 mg of threonine per gram of polypeptide); at least 9 mg of tryptophan per gram of polypeptide (e.g., 9 mg of tryptophan per gram of polypeptide; at least 15 mg of tryptophan per gram of polypeptide, at least 20 mg of tryptophan per gram of polypeptide, at least 25 mg of tryptophan per gram of polypeptide, at least 30 mg of tryptophan per gram of polypeptide, at least 35 mg of tryptophan per gram of polypeptide, at least 40 mg of tryptophan per gram of polypeptide, at least 45 mg of tryptophan per gram of polypeptide, at least 50 ... at least 40 mg tryptophan per gram polypeptide, at least 45 mg tryptophan per gram polypeptide, or at least 50 mg tryptophan per gram polypeptide; at least 55 mg tyrosine per gram polypeptide (e.g., 55 mg tyrosine per gram polypeptide, at least 65 mg tyrosine per gram polypeptide, at least 75 mg tyrosine per gram polypeptide, at least 85 mg tyrosine per gram polypeptide, at least 95 mg tyrosine per gram polypeptide); and at least 66 mg leucine per gram polypeptide (e.g., 66 mg leucine per gram polypeptide, at least 70 mg leucine per gram polypeptide, at least 80 mg leucine per gram polypeptide, at least 90 mg leucine per gram polypeptide, at least 100 mg leucine per gram polypeptide, or at least 110 mg leucine per gram polypeptide).

[0080] In further embodiments, polypeptides of the present invention suitable as a complete protein source for PKU patients contain at least 60 mg of threonine per gram of polypeptide; at least 15 mg of tryptophan per gram of polypeptide; at least 65 mg of tyrosine per gram of polypeptide; and at least 76 mg of leucine per gram of polypeptide. In some embodiments, polypeptides of the present invention suitable as a complete protein source for PKU patients contain at least 70 mg of threonine per gram of polypeptide; at least 20 mg of tryptophan per gram of polypeptide; at least 75 mg of tyrosine per gram of polypeptide; and at least 86 mg of leucine per gram of polypeptide. In some embodiments, polypeptides of the present invention suitable as a complete protein source for PKU patients contain at least 80 mg of threonine per gram of polypeptide; at least 25 mg of tryptophan per gram of polypeptide; at least 85 mg of tyrosine per gram of polypeptide; and at least 96 mg of leucine per gram of polypeptide.

[0081] Those skilled in the art will understand that similar strategies to those described above can be used to create nutritional compositions for patients suffering from metabolic disorders other than PKU. Such diseases 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 may contain a polypeptide with low or no tyrosine and otherwise an AAS of ≥ 0.94.

[0082] In some embodiments, the polypeptides of the present invention have an AAS of ≥ 0.94, with no additional constraints on the amount of any amino acid. Such recombinant polypeptides may generally be useful in nutritional compositions. The nutritional compositions may be used as a protein source for individuals without metabolic disorders that limit protein intake. Such nutritional compositions may be useful for patients with reduced appetite or reduced ability to eat.

[0083] In some embodiments, the polypeptides of the present invention contain specific amino acids in amounts greater than those required to achieve an AAS of 0.94. For example, the amino acid leucine is an important factor in stimulating muscle protein synthesis, and more generally, branched-chain amino acids (BCAAs; including valine, leucine, and isoleucine) are rich in muscle protein, stimulate muscle growth in the body, and provide energy during exercise. Nutritional compositions high in leucine and / or BCAAs may be needed and / or desired by groups interested in increasing and / or preserving muscle mass, such as professional athletes (which may be referred to as "sports nutrition") and elderly or hospitalized individuals with poor appetites who tend to lose muscle mass as part of aging (which may be referred to as "medical nutrition"). Leucine may also be involved in managing blood glucose levels and help regulate appetite. Therefore, nutritional compositions high in leucine may be needed and / or desired by groups interested in better maintaining blood glucose levels and / or body weight, such as diabetics, pre-diabetic individuals, and individuals struggling with weight management.

[0084] In some embodiments, the polypeptides of the invention have an AAS > 0.94 and, additionally, at least 105 mg leucine per gram polypeptide, e.g., 105 mg leucine per gram polypeptide, at least 110 mg leucine per gram polypeptide, at least 115 mg leucine per gram polypeptide, at least 120 mg leucine per gram polypeptide, at least 125 mg leucine per gram polypeptide, at least 130 mg leucine per gram polypeptide, at least 135 mg leucine per gram polypeptide, at least 140 mg leucine per gram polypeptide, at least 145 mg leucine per gram polypeptide, at least 150 mg leucine per gram polypeptide, at least 155 mg leucine per gram polypeptide. In some embodiments, the polypeptide comprises at least 160 mg leucine per gram polypeptide, at least 170 mg leucine per gram polypeptide, at least 180 mg leucine per gram polypeptide, at least 190 mg leucine per gram polypeptide, at least 200 mg leucine per gram polypeptide, at least 210 mg leucine per gram polypeptide, at least 220 mg leucine per gram polypeptide, at least 230 mg leucine per gram polypeptide, at least 240 mg leucine per gram polypeptide, at least 250 mg leucine per gram polypeptide, at least 275 mg leucine per gram polypeptide, at least 300 mg leucine per gram polypeptide, or at least 350 mg leucine per gram polypeptide.

[0085] In some embodiments, the polypeptides of the invention have an AAS > 0.94 and comprise at least 210 mg of branched chain amino acids per gram of polypeptide, e.g., 210 mg of branched chain amino acids per gram of polypeptide, at least 215 mg of branched chain amino acids per gram of polypeptide, at least 220 mg of branched chain amino acids per gram of polypeptide, at least 225 mg of branched chain amino acids per gram of polypeptide, at least 230 mg of branched chain amino acids per gram of polypeptide, at least 235 mg of branched chain amino acids per gram of polypeptide, at least 240 mg of branched chain amino acids per gram of polypeptide, at least 245 mg of branched chain amino acids per gram of polypeptide, at least 250 mg of branched chain amino acids per gram of polypeptide, at least 255 mg of branched chain amino acids per gram of polypeptide, at least 260 mg of branched chain amino acids per gram of polypeptide, at least 265 mg of branched chain amino acids per gram of polypeptide, at least 270 mg of branched chain amino acids per gram of polypeptide, at least 280 mg of branched chain amino acids per gram of polypeptide, at least 285 mg of branched chain amino acids per gram of polypeptide, at least 290 mg of branched chain amino acids per gram of polypeptide, at least 300 mg of branched chain amino acids per gram of polypeptide, at least 310 mg of branched chain amino acids per gram of polypeptide, at least 320 mg of branched chain amino acids per gram of polypeptide, at least 330 mg of branched chain amino acids per gram of polypeptide, at least 340 mg of branched chain amino acids per gram of polypeptide, at least 345 mg of branched chain amino acids per gram of polypeptide, at least 350 mg of branched chain amino acids per gram of polypeptide, at least 355 mg of branched chain amino acids per gram of polypeptide, at least 360 mg of branched chain at least 275 mg branched chain amino acids, at least 280 mg branched chain amino acids per gram polypeptide, at least 285 mg branched chain amino acids per gram polypeptide, at least 290 mg branched chain amino acids per gram polypeptide, at least 300 mg branched chain amino acids per gram polypeptide, at least 310 mg branched chain amino acids per gram polypeptide, at least 320 mg branched chain amino acids per gram polypeptide, at least 330 mg branched chain amino acids per gram polypeptide, at least 340 mg branched chain amino acids per gram polypeptide, at least 350 mg branched chain amino acids per gram polypeptide, at least 360 mg branched chain amino acids per gram polypeptide, at least 370 mg branched chain amino acids per gram polypeptide, at least 380 mg branched chain amino acids per gram polypeptide, at least 390 mg branched chain amino acids per gram polypeptide, at least 400 mg branched chain amino acids per gram polypeptide, at least 425 mg branched chain amino acids per gram polypeptide,The polypeptide may contain at least 450 mg of branched chain amino acids per gram, at least 475 mg of branched chain amino acids per gram, or at least 500 mg of branched chain amino acids per gram of polypeptide. Branched chain amino acids include leucine, isoleucine, and valine.

[0086] In a further embodiment, the polypeptides of the invention have an AAS > 0.94, contain at least 105 mg leucine per gram of polypeptide, and further contain at least 210 mg branched chain amino acids per gram of polypeptide.

[0087] The polypeptides of the present invention may have an amino acid composition optimized to provide an ideal amino acid composition for a nutritional composition suitable for the needs of any group of proteins and amino acids. For example, greater amounts of protein and fortification with specific amino acids may also be needed during pregnancy. 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 is recognized that amino acid needs may change throughout pregnancy. The polypeptides of the present invention may have an amino acid composition optimized to provide an ideal amino acid composition for a nutritional composition suitable for each stage of pregnancy.

[0088] Mutants Polypeptides of the invention include variants of any one of SEQ ID NOS: 3-101, where variants include amino acid sequences 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 truncations thereof, including polypeptides having sequence identity from 3 to 101 or truncations 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, etc.

[0089] A variant has a different amino acid composition compared to its parent polypeptide, which may be optimized to provide a complete protein source for daily intake for any one of several different amino acid requirements, as described above. In some embodiments, the variant has an AAS of ≥ 0.94 but a different amino acid composition compared to any one of SEQ ID NOS: 3-101. Such variants may be useful in nutritional compositions.

[0090] In some embodiments, variants may have improved properties compared to the parent polypeptide, for example, improved stability under storage conditions or improved heat tolerance.

[0091] In some embodiments, the variant has improved ease of purification compared to the parent polypeptide. In some embodiments, the improved ease of purification may result in a higher yield when following the same purification steps as the parent polypeptide. In some embodiments, the improved ease of purification may require fewer steps. In some embodiments, the improved ease of purification may result in a less resource-intensive purification process, which may mean that less energy is used, less time is used, or lower cost reagents or materials are used.

[0092] In one embodiment, 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.

[0093] In one aspect, the number of modifications in a variant of the invention is 1 to 20, for example 1 to 10 or 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications.

[0094] In another aspect, the variant comprises a substitution, insertion or deletion at one or more positions in 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 in 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 in 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 in the amino acid sequence of any one of SEQ ID NOs: 3 to 101.

[0095] The amino acid changes can be minor, 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 to 30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that alter net charge or serve another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding domain, to facilitate purification.

[0096] Examples of conservative substitutions are those within the group of 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, in H. Neurath and R.L. Hill, 1979, In: The Proteins, Academic Press, New York. Common substitutions include 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.

[0097] Instead, the amino acid changes are of such a nature that they alter the physicochemical properties of the polypeptide, e.g., they may improve the thermal stability of the polypeptide, alter its pH optimum, improve purification yield, etc.

[0098] Polypeptides of the present invention are desirable for their amino acid composition and their ease of purification. Amino acids in a polypeptide that are essential for stability (which may affect ease of purification) or for ease of purification itself can be identified using 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 essential amino acid residues. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Amino acid residues essential for stability can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., 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. Identification of amino acids essential for stability or ease of purification can also be inferred from alignment with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or within polypeptide or protein families, typically with polypeptides / proteins derived from a common ancestor with 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 essential for stability or ease of purification. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold," Nature 596:583-589.

[0099] The mutant 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.

[0100] The polypeptide of the present invention may be a fusion polypeptide comprising a variant of the present invention.

[0101] nucleic acid construct The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the 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.

[0102] Polynucleotides can be manipulated in a variety of ways to result in expression of variants. It may be desirable or necessary to manipulate the polynucleotide relative to the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0103] promoter The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a variant of the invention. The promoter contains transcriptional control sequences that mediate expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell, including mutated promoters, truncated promoters, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0104] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as the promoters described by Mukherjee et al., 2013, "Trichoderma: Biology and Applications" and Schmoll and Dattenbock, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0105] Terminator The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. This terminator is operably linked to the 3' end of the polynucleotide encoding the variant. Any terminator functional in the host cell may be used in the present invention.

[0106] Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as those obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described by Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenbock, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0107] mRNA stabilizing factor A regulatory sequence can also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0108] Examples of suitable mRNA stabilization 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).

[0109] Examples of mRNA stabilization regions for 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.

[0110] Leader sequence The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0111] Preferred leaders for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0112] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of a polynucleotide that, upon transcription, is recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0113] 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.

[0114] signal peptide The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant, directing the variant into the secretory pathway of the cell. The 5' end of the coding sequence of the polynucleotide may naturally contain a signal peptide coding sequence that is naturally linked in translation reading frame with the segment of the coding sequence encoding the variant. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of the host cell may be used.

[0115] Effective signal peptide coding sequences for filamentous fungal host cells include those obtained from the genes encoding 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.

[0116] Propeptide The regulatory sequence may also be a propeptide coding sequence encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or sometimes a zymogen). Propolypeptides are generally inactive and can be converted to active variants by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences can be obtained, for example, from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, or Saccharomyces cerevisiae alpha-factor.

[0117] When both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the variant, and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence.

[0118] Regulatory sequences It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that turn gene expression on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are sequences that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified by heavy metals.

[0119] transcription factors A regulatory sequence can also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which often binds to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a protein of interest directly, i.e., by activating the transcription of a gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of an additional transcription factor that regulates the transcription of the gene encoding the protein of interest, for example, by binding to the promoter of the additional transcription factor. Transcription factors suitable 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.

[0120] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. Various nucleotides and control sequences can be linked to one another to generate a recombinant expression vector that contains one or more convenient restriction sites, allowing for the insertion or substitution of a polynucleotide encoding the variant at such sites. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a vector suitable for expression. In creating an expression vector, the coding sequence is placed within the vector so that it is operably linked to suitable control sequences for expression.

[0121] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a 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 may be a linear or closed circular plasmid.

[0122] The vector may be an autonomously replicating vector, i.e., a vector whose replication exists as an extrachromosomal entity independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means for ensuring autonomous replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids or transposons that together contain the total DNA to be introduced into the genome of the host cell, may be used.

[0123] Vectors preferably contain one or more selectable markers which permit easy selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0124] The vector preferably contains at least one element that allows for integration of the vector into the genome of the host cell or autonomous replication of the vector within the cell independent of the genome.

[0125] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide 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).

[0126] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously within the host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0127] Two or more copies of a polynucleotide of the present invention can be inserted into a host cell to increase production of the polypeptide. For example, two, three, four, five or more copies are inserted into the host cell. Increasing the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, where cells containing an amplified copy of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selection agent.

[0128] host cell 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 the variants of the present invention.

[0129] Once the construct or vector containing the polynucleotide is introduced into a host cell, the construct or vector may be maintained as a chromosomal integrant or a self-replicating extrachromosomal vector, as described above. The choice of host cell will largely depend on the gene encoding the variant and its source. The recombinant host cell may contain a single copy of the polynucleotide of the invention or may contain at least two copies, e.g., three, four, five or more copies.

[0130] The host cell can be any cell useful for the recombinant production of the variants of the invention, for example, a prokaryotic or fungal cell.

[0131] The host cell can be any microbial cell useful for the recombinant production of the polypeptides of the invention, for example, a prokaryotic or fungal cell.

[0132] The host cell may be a fungal cell. "Fungi," as used herein, includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0133] Fungal cells can be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistics, and shock wave-mediated transformation, which are reviewed in Li et al., 2017, Microbial Cell Factories 16:168, and procedures described 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 can be used, and the DNA can be introduced as a linear or circular polynucleotide.

[0134] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial cell wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular body, and carbon catabolism can be fermentative.

[0135] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nelumbo nucifera, and others. The cell may be a cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium. In a more preferred embodiment, the filamentous fungal host cell is a cell of Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum. For example, the filamentous fungal host cell is a cell of Aspergillus awamori, Aspergillus foetidus, or Aspergillus niger.foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis ribulosa rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium cloakwellenscrookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolorThe cell may be a cell of Trichoderma versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0136] The host cell can be a prokaryotic cell. The prokaryotic host cell can be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0137] Bacterial host cells include, but are 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 spp. The cell may be any Bacillus cell, including Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0138] A bacterial host cell can also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0139] A bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0140] Introduction of DNA into Bacillus cells can be carried out by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), competent cell transformation (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 can be 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 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 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). DNA can be introduced into Streptococcus cells by natural transformability (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.

[0141] In some embodiments, the host cells are isolated. In some embodiments, the host cells are purified.

[0142] Generation method The present invention also relates to a method for producing a polypeptide of the present invention, comprising (a) culturing a recombinant host cell of the present invention under conditions suitable for production of the polypeptide, and (b) recovering the polypeptide.

[0143] The host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells can be cultured by shake flask cultures or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors under conditions that allow for expression and / or isolation of the polypeptide in a suitable medium. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, the polypeptide can be recovered from cell lysates.

[0144] Polypeptides may be detected using methods known in the art that are specific for the polypeptides, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or enzyme assays to determine the relative or actual activity of the polypeptide.

[0145] The polypeptide may be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, 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.

[0146] 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).

[0147] In an alternative embodiment, 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.

[0148] Nutritional Composition Nutritional compositions of the present invention comprise the polypeptides disclosed herein. To be suitable for nutritional compositions, the polypeptides of the present invention must be soluble and remain intact at a variety of pHs and temperatures.

[0149] Nutritional compositions typically have a pH between 3 and 8. Polypeptides of the present invention should remain soluble at the pH of the nutritional composition in which they are contained. In some embodiments, 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, 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, polypeptides of the present invention remain soluble at a given pH before, during, and after heat treatment.

[0150] The nutritional composition may be heat-treated for pasteurization or sterilization. The 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, as it eliminates microorganisms in the beverage that may spoil the beverage by causing spoilage or reducing the quality of the beverage.

[0151] In some embodiments, the heat treatment is at 85-95°C for 10 seconds to 30 minutes. In some embodiments, the heat treatment is high temperature, short time (HTST) pasteurization. For this, the composition is heated to a temperature between 71.5-74°C for 15-30 seconds or to a temperature between 74-76°C for 15-20 seconds. After heat treatment, the nutritional composition may then be rapidly cooled to 4-5.5°C. In some embodiments, the acidic probiotic beverage is heat treated by ultra-high temperature (UHT) treatment. The UHT treatment may be direct or indirect. In some embodiments, the UHT treatment is at 135-154°C for 1-10 seconds. In some embodiments, the nutritional composition is heat treated by ultra pasteurization. For this, the composition is heated to a temperature between 70-75°C for 20-30 minutes.

[0152] In some embodiments, the polypeptides of the invention have qualities, such as a high melting temperature, that indicate they will remain intact through heat treatment.

[0153] 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 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 dairy fat. In some embodiments, the fat source is derived from a vegetable source, such as fractionated vegetable oil. In some embodiments, the fat source provides about 20%-70% of the energy in the nutritional composition. In further embodiments, the fat source provides about 25%-60% of the energy in the nutritional composition.

[0154] In some embodiments, the nutritional composition includes 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%-70% of the energy in the nutritional composition. In further embodiments, the carbohydrate source provides about 30%-60% of the energy in the nutritional composition.

[0155] In some embodiments, the nutritional composition includes dietary vitamins and / or minerals. Vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B12 (various cobalamins; commonly 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.

[0156] In some embodiments, the nutritional composition includes additional components that may be beneficial to gut health and / or overall health, such as omega-3 fatty acids, such as α-linolenic acid, stearidonic acid, docosahexaenoic acid, and eicosapentaenoic acid; phytonutrients, such as carotenoids, plant sterols, quercetin, curcumin, and limonin; compounds, such as α-ketoglutaric acid and L-carnitine; or antioxidants, such as astaxanthin, coenzyme Q10, flavonoids, glutathione, hesperidin, lactowberry, lignans, lutein, lycopene, polyphenols, selenium, or zeaxanthin, or any combination thereof.

[0157] In some embodiments, the nutritional composition comprises a prebiotic, including acacia gum, alpha glucan, arabinogalactan, beta glucan, dextran, fructooligosaccharides, fucosyllactose, galactooligosaccharides, galactomannan, gentiooligosaccharides, glucooligosaccharides, guar gum, inulin, isomaltooligosaccharides, lactoneotetraose, lactosucrose, lactulose, levan, maltodextrin, milk oligosaccharides, partially hydrolyzed guar gum, pectin oligosaccharides, resistant starch, retrograded starch, sialooligosaccharides, sialyllactose, soy oligosaccharides, sugar alcohols, xylooligosaccharides, hydrolysates thereof, or any combination thereof.

[0158] In some embodiments, the nutritional composition comprises a probiotic, such as a bacterium of the genus Aerococcus, Aspergillus, Bacteroides, Bacillus, Bifidobacterium, Brevibacillus, Candida, Clostridium, Debaromyces, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, or the like. us, Mucor, Oenococcus, Paenibacillus, Pediococcus, Penicillium, Peptostrepococcus, Pichia, Propionibacterium, Pseudocatenulatum, Rhizopus, Saccharomyces, Staphylococcus, Streptococcus, Torulopsis, Weissella, non-replicating microorganisms, or any combination thereof.

[0159] In some embodiments, the nutritional compositions 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 pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavors, minerals, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizing agents, minerals, and / or vitamins. The optional ingredients may be added in any suitable amount.

[0160] The nutritional compositions of the present invention can be prepared by mixing the recombinant polypeptide in powder form, optionally with a fat or carbohydrate source or other additional components. The nutritional compositions 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 compositions of the present invention include tablets, dispersible powders, granules, capsules, liquids, suspensions, and syrups.

[0161] Inert diluents and carriers for tablets include, for example, calcium carbonate, sodium carbonate, lactose, and talc. Tablets may also contain granulating and disintegrating agents, 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 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, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, such as ethyl-p-hydroxybenzoate.

[0162] The nutritional composition of the present invention can be added to foods normally consumed by patients. 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 receiving long-term care, including, for example, the elderly, pregnant women, cancer patients, and individuals with long-term illnesses such as diabetes. In some embodiments, the nutritional composition can be suitable as a complete protein source for patients with metabolic disorders. In further embodiments, the nutritional composition can be suitable as a complete protein source for patients with PKU and / or HPA.

[0163] Preferred Embodiments The present invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to be illustrative of some aspects of the invention. Any equivalent embodiments 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 apparent 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 case of conflict, the present disclosure, including definitions, will control.

[0164] The present invention is further defined by the following numbered embodiments: 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 truncations thereof. 2. The nutritional composition of claim 1, wherein the recombinant polypeptide comprises amino acids 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 truncations thereof. 3. A nutritional composition according to claim 1 or 2, wherein the recombinant polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 3 to 101. 4. The nutritional composition of any one of claims 1 to 3, 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 to 11, 45 to 48, 50 or 51 or truncations thereof. 5. A nutritional composition according to any one of claims 1 to 4, 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 an internal truncation. 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, and wherein the truncation has an N-terminal truncation of at least one amino acid and up to 350 amino acids compared to the amino acid sequence of SEQ ID NO:50. 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, and wherein the truncation has a C-terminal truncation of at least one amino acid and up to 350 amino acids compared to the amino acid sequence of SEQ ID NO:50. 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, and wherein the truncation has an internal truncation of at least 1 amino acid and up to 350 amino acids compared to the amino acid sequence of SEQ ID NO:50. 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. 10. A nutritional composition according to any one of claims 1 to 9, wherein the recombinant polypeptide has an amino acid score (AAS) of 0.94 or greater. 11. The recombinant polypeptide is a) at least 50 mg of threonine per gram of polypeptide; b) at least 9 mg of tryptophan per gram of polypeptide; c) at least 55 mg of tyrosine per gram of polypeptide; and d) at least 66 mg of leucine per gram of polypeptide The nutritional composition according to any one of claims 1 to 10, comprising: 12. A nutritional composition according to any one of claims 1 to 11, wherein the composition does not contain added free amino acids. 13. A nutritional composition according to any one of claims 1 to 12, further comprising vitamins and minerals. 14. A nutritional composition according to any one of claims 1 to 13, further comprising a carbohydrate source and / or a fat source. 15. A nutritional composition according to any one of claims 1 to 14, wherein the composition is a tablet, dispersible powder, granules, capsule, liquid, suspension or syrup. 16. A nutritional composition according to any one of claims 1 to 15, wherein the recombinant polypeptide comprises a nutritionally complete amino acid profile sufficient for a patient with a metabolic disorder. 17. The nutritional composition of any one of claims 1 to 16, wherein the recombinant polypeptide comprises a nutritionally complete amino acid profile sufficient for patients with phenylketonuria, hyperphenylalaninemia, tyrosinemia, maple syrup urine disease, methylmalonic acidemia, homocystinuria, glutaric aciduria, isovaleric acidemia and / or hyperlysinemia. 18. The nutritional composition of claim 16 or 17, wherein the recombinant polypeptide does not contain amino acids that are harmful to patients with metabolic disorders. 19. A nutritional composition according to any one of claims 16 to 18, wherein the recombinant polypeptide has an AAS of 0.94 or greater, excluding amino acids that are harmful to patients with metabolic disorders. 20. A nutritional composition according to any one of claims 16 to 19, wherein the recombinant polypeptide does not contain any phenylalanine residues. 21. The nutritional composition of claim 20, wherein the recombinant polypeptide has an AAS, excluding phenylalanine, of 0.94 or greater. 22. The nutritional composition of any one of claims 16 to 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 to 48. 23. The nutritional composition of any one of claims 1-15, wherein the recombinant polypeptide has an AAS of 0.94 or greater, and further wherein the recombinant polypeptide contains at least 105 mg of leucine per gram of polypeptide. 24. The nutritional composition of any one of claims 1-15 or 23, wherein the recombinant polypeptide has an AAS of 0.94 or greater, and further wherein the recombinant polypeptide comprises at least 210 mg of branched chain amino acids per gram of polypeptide. 25. The nutritional composition of any one of claims 1 to 15, 23 or 24, wherein the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 78 to 101. 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. 27. The recombinant polypeptide of claim 26, comprising amino acids 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 truncated version thereof, and further comprising a substitution at a position corresponding to positions E334 and / or E426 of SEQ ID NO:49. 28. The recombinant polypeptide of claim 26 or claim 27, wherein the substitution at the position corresponding to E334 of SEQ ID NO: 49 is A, I, G, L, M, Q, or V. 29. The recombinant polypeptide of claim 26 or claim 27, wherein the substitution at the position corresponding to E426 of SEQ ID NO: 49 is A, I, G, L, M, Q, or V. 30. The recombinant polypeptide of any one of claims 26 to 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 NO: 3 to 11, 45 to 48, 50, or 51, or a truncated version thereof. 31. The recombinant polypeptide of any one of claims 26 to 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 an internal truncation. 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, and wherein the truncation has an N-terminal truncation of at least one amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO:50. 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, and wherein the truncation has a C-terminal truncation of at least one amino acid, up to a maximum of 350 amino acids, relative to the amino acid sequence of SEQ ID NO:50. 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, and wherein the truncation has an internal truncation of at least one amino acid and up to 350 amino acids relative to the amino acid sequence of SEQ ID NO:50. 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%, or at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4 to 48, 52, 53, 58, 61, 62, 63, 66, 67, and 70 to 75. 36. The recombinant polypeptide of any one of claims 26 to 35, wherein the recombinant polypeptide has an amino acid score (AAS) of 0.94 or greater. 37. The recombinant polypeptide is a) at least 50 mg of threonine per gram of polypeptide; b) at least 9 mg of tryptophan per gram of polypeptide; c) at least 55 mg of tyrosine per gram of polypeptide; and d) A recombinant polypeptide according to any one of claims 26 to 36, comprising at least 66 mg of leucine per gram of polypeptide. 38. The recombinant polypeptide of any one of claims 26-34 or 37, wherein the recombinant polypeptide does not contain any phenylalanine residues. 39. The recombinant polypeptide of claim 38, wherein the recombinant polypeptide has an AAS of 0.94 or greater, excluding phenylalanine. 40. The recombinant polypeptide of claim 38 or 39, wherein the recombinant polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 3 to 11 and 45 to 48. 41. The recombinant polypeptide of any one of claims 26-36, wherein the recombinant polypeptide has an AAS of 0.94 or greater, and further wherein the recombinant polypeptide contains at least 105 mg of leucine per gram of polypeptide. 42. The recombinant polypeptide of any one of claims 26-36 or 41, wherein the recombinant polypeptide has an AAS of 0.94 or greater, and further wherein the recombinant polypeptide comprises at least 210 mg of branched chain amino acids per gram of polypeptide. 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 NO: 78-101. 44. Use of a recombinant polypeptide according to any one of claims 26 to 43 in a nutritional composition. 45. Use of a recombinant polypeptide according to any one of claims 26 to 43 in a nutritional composition for patients with metabolic disorders. 46. ​​An isolated polynucleotide encoding the polypeptide of any one of claims 26 to 43. 47. A nucleic acid construct or expression vector comprising the polynucleotide of claim 46. 48. A recombinant host cell transformed with the polynucleotide of claim 46. 49. A method for producing a polypeptide, comprising culturing a recombinant host cell of claim 48 under conditions suitable for expression of the polypeptide, and recovering the polypeptide.

[0165] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0166] Example 1: Production of recombinant polypeptides for nutritional compositions for general nutrition We identified a recombinant polypeptide that was found to be expressed at very high levels when recombinantly expressed in A. niger, A. oryzae, and Bacillus licheniformis. Unexpectedly, this polypeptide has an amino acid composition similar to that of desirable AASs and contains neither toxic domains nor amino acid motifs known to be involved in protein allergens, making it suitable for use in nutritional compositions. While the polypeptide has mannanase activity (as described in WO 2021 / 152123, incorporated herein by reference), inactive mutants can be generated by introducing substitutions at positions 334 and / or 426 (relative to SEQ ID NO: 49).

[0167] A polypeptide with a desirable amino acid composition, no known toxic or allergenic domains, and high expression with no detectable nonspecific carryover from purification is unpredictable and highly desirable for a recombinant polypeptide for nutritional compositions. The high AAS of this polypeptide also makes it suitable as a nutritious protein in categories other than general nutrition, such as sports drinks, or as medical nutrition for elderly and hospitalized patients with poor appetites. Therefore, it was determined whether the inactive mutant polypeptide still possessed the same useful qualities of ease of purification and sample purity.

[0168] 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 either SEQ ID NO:49 or 50 were introduced into strains of the filamentous fungus A. 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. Expression and purification of the recombinant polypeptides were analyzed using SDS-PAGE and MS proteomics after trypsin digestion. Both recombinant polypeptides of SEQ ID NO:49 and SEQ ID NO:50 were found to be expressed at high levels.

[0169] Yield and purity data from two different fermentations that produced the polypeptide of SEQ ID NO:49 and a single fermentation that produced the polypeptide of SEQ ID NO:50 are provided in Table 1. SDS-PAGE analysis confirmed the identification of the protein of interest, and densitometric scanning of the SDS-PAGE gel allowed for relative quantification of the purified protein. The results show robust yields and excellent purity.

[0170] [Table 2]

[0171] In addition to the purity estimated by SDS-PAGE, the expression of the protein variants relative to the host strain protein was estimated using LC-MS / MS. Tryptic digests were prepared by the filter-aided sample preparation (FASP) method. Briefly, after tryptic digestion, extracted peptides were analyzed using a nanoLC-MS / MS system: Evosep One (Evosep, Denmark) / timsTOF Pro (Bruker Daltonik, Massachusetts, USA). For protein identification, Genedata Expressionist software was used with a 1% false discovery rate cutoff, and data were searched against available internal and public databases using the Mascot search engine (Matrix Science, London, UK). Relative protein concentrations were calculated by label-free quantification of peptide volumes in Genedata Expressionist. The results are shown in Table 2.

[0172] [Table 3]

[0173] The LC-MS / MS data in Table 2 confirm the conclusions from the SDS-PAGE analysis about the high ratio of target protein to host strain protein, indicating that the purification process was successful in removing fermentation-derived cell debris and other non-proteinaceous components that can affect food product functionality and the flavor profile of the final product.

[0174] Example 2: Production of recombinant polypeptides for nutritional compositions for metabolic disorders As shown in Example 1, inactive mutants of mannanase can be produced with high expression and purity levels. Therefore, additional mutations were introduced to test whether other mutants of the polypeptide still possess ease of purification and high yield and purity.

[0175] The polypeptides of SEQ ID NOs: 49 and 50 have a relatively low phenylalanine composition. Mutant polypeptides were generated in which phenylalanine was replaced with other amino acids. Substitutions were also introduced to balance the amino acid composition so that, except for the lack of phenylalanine, the polypeptide had an AAS of ≥ 0.94. An example of the nucleic acid sequence of an inactive Phe-free recombinant polypeptide having an AAS of ≥ 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 from which the signal peptide has been removed is SEQ ID NO: 3.

[0176] A nucleic acid construct containing a nucleotide sequence encoding SEQ ID NO:3 was introduced into a strain of the filamentous fungus A. oryzae. The recombinant strain was fermented using standard protocols (3-4 days, 30°C) and then purified by chromatography using standard ion exchange techniques. Expression and purification of the recombinant polypeptide were analyzed using SDS-PAGE. The recombinant polypeptide of SEQ ID NO:3 was found to be expressed at high levels.

[0177] Data from the fermentation and purification of the recombinant polypeptide of SEQ ID NO: 3 using four different media are shown in Table 3. SDS-PAGE analysis confirmed the identification of the protein of interest, and densitometric scanning of the SDS-PAGE gels allowed for relative quantification of the purified protein. The results show robust yields and excellent purity in the various media.

[0178] [Table 4]

[0179] Phenylanine levels in purified samples were estimated by total amino acid analysis. While the polypeptide of SEQ ID NO:3 did not contain phenylalanine, phenylalanine may be present in purified samples as a result of carryover of background phenylalanine from the expression host, such as in cellular debris. The amount of residual phenylalanine present in the purified protein was determined using standard amino acid analysis methods. Amino acids were derivatized with AccQ-Tag Ultra Reagent (Waters Corp., Milford, MA) and separated using reversed-phase UPLC (UPLC®, Waters Corp., Milford, MA). Derivatives were quantified based on UV absorbance. The detection limit for phenylalanine using this method is 2 pmol. No phenylalanine was detected, indicating that little or no phenylalanine was present in the purified samples.

[0180] Example 3: In vitro digestibility The digestibility of the purified recombinant polypeptides described in Examples 1 and 2 was determined using the method published by Minekus et al. ("A standardized static in vitro digestion method suitable for food—an international consensus," Food Funct, 5:1113-1124, 2014). The processed samples were then analyzed by SDS-PAGE. The results are shown in Table 4.

[0181] [Table 5]

[0182] While none of the variants were degraded in the simulated oral phase, all variants showed extensive degradation in 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 in the simulated intestinal phase. In conclusion, the in vitro digestibility assays demonstrate the complete digestibility of these molecules and therefore the high bioavailability of the individual amino acids.

[0183] Example 4: Thermal Stability and Solubility The successful use of polypeptides for food applications requires more than just high nutritional value: functionality such as heat stability and solubility is important to enable quality finished nutritional compositions, including processed foods such as powders, drinks, and bars.

[0184] The thermal stability of purified polypeptides containing the amino acid sequence of SEQ ID NO: 49 or 50 was examined by nano-differential scanning fluorimetry (nano-DSF) using a NanoTemper Technologies (Munich, Germany) instrument. Nano-DSF is a biophysical characterization technique used to determine the conformational stability of biological samples. It uses the intrinsic fluorescence of proteins to monitor how they respond to stress inputs such as temperature and chaotropes. This information is used to determine the conformational stability of the protein and to rank candidate or buffer formulations based on their effect on this stability.

[0185] The thermal stability of a protein is determined by the onset of unfolding (T on ) and the melting temperature at which 50% of the protein is unfolded (T m The results are shown in Table 5.

[0186] [Table 6]

[0187] The high melting temperatures of the purified polypeptides indicate that these polypeptides can remain intact through heat treatments such as high temperature, short time (HTST) pasteurization or similar heat treatments for pasteurization or sterilization typically used in the food industry.

[0188] Solubility is another important factor in determining the suitability of a polypeptide for use in the production of nutritional compositions. The pH dependence of solubility was determined for the polypeptides of SEQ ID NOs: 49 and 50, with and without brief heat treatment. The solubility of solutions containing the polypeptides of SEQ ID NOs: 49 or 50 (10 mg / mL) at various pHs from 3 to 8 was determined before and after heat treatment at 95°C for 3 minutes. The percent solubility was determined by measuring the absorbance at 280 nm before and after centrifugation (to remove precipitate) and calculating the ratio. The percent solubility was determined before and after heat treatment. The results are shown in Table 6.

[0189] [Table 7]

[0190] Before heat treatment, the samples showed near 100% protein solubility, except at pH 5, which is close to the pI of the polypeptides. In water at neutral pH, the polypeptides encoding the amino acid sequences of SEQ ID NOs: 49 and 50 showed high solubility (greater than 10% w / w). After heat treatment, solubility decreased for both polypeptides. However, the inactive mutant polypeptide containing SEQ ID NO: 50 showed a more favorable solubility profile after heat treatment, indicating that the inactive mutants are good candidates for use in nutritional compositions.

[0191] The thermal stability of several mutants is shown in Table 7. The thermal stability was determined by nanoDSF as described above. The mutants have unfolding temperatures (Tm) that vary by more than 20°C, indicating that a wide range of recombinant polypeptide variants can be generated for specific applications with different requirements for thermal stability.

[0192] [Table 8]

[0193] Example 5: Production of additional recombinant polypeptides for nutritional compositions Additional protein variants of SEQ ID NO:3 and SEQ ID NO:50 were generated. The variants contain N-terminal truncations, with the length of the truncations ranging from 15 to 158 amino acids. These constructs encode polypeptides containing the amino acids of SEQ ID NO:4-53. Expression of these variants is evaluated in A. oryzae and B. licheniformis. A recombinant polypeptide containing SEQ ID NO:52 was highly expressed in B. licheniformis, demonstrating the broad versatility of this protein scaffold for use in the food industry.

[0194] Example 6: Production of recombinant polypeptides for specialized nutritional compositions It was desired to produce polypeptides containing 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 specialized nutrition such as medical nutrition and / or sports nutrition.

[0195] Substitutions were introduced into the parent, SEQ ID NO: 51, inactive mutant to generate polypeptides with leucine contents of 115-137 mg / g polypeptide and branched chain amino acid contents of 221-250 mg / g polypeptide and overall amino acid scores of > 1. The data are shown in Table 8.

[0196] [Table 9]

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 NO: 3-101 or truncations thereof.

2. 2. The nutritional composition of claim 1, 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 an internal truncation.

3. 3. The nutritional composition of claim 1 or 2, wherein the recombinant polypeptide has an amino acid score (AAS) of 0.94 or greater.

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 truncated version thereof, and further comprising a substitution at a position corresponding to position E334 and / or E426 of SEQ ID NO:

49.

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

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

7. 7. The recombinant polypeptide of any one of claims 4 to 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 NO: 3 to 11, 45 to 48, 50 or 51 or truncations thereof.

8. 8. The recombinant polypeptide of any one of claims 4 to 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 an internal truncation.

9. The recombinant polypeptide of any one of claims 4 to 8, wherein the recombinant polypeptide has an amino acid score (AAS) of 0.94 or greater.

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

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

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

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

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

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

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