WO2025111274A2 - Variants d'épimérase et polynucléotides les codant - Google Patents

Variants d'épimérase et polynucléotides les codant Download PDF

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WO2025111274A2
WO2025111274A2 PCT/US2024/056535 US2024056535W WO2025111274A2 WO 2025111274 A2 WO2025111274 A2 WO 2025111274A2 US 2024056535 W US2024056535 W US 2024056535W WO 2025111274 A2 WO2025111274 A2 WO 2025111274A2
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variant
psicose
polypeptide
epimerase
variants
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WO2025111274A3 (fr
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Steven Joseph Walsh
Annette Helle Johansen
Nikolaj Spodsberg
Julie Bille RANNES
Jr. Bernardo Vidal
Martin GUDMAND
Roland Alexander Pache
Frank Winther Rasmussen
Allan Noergaard
Brian Manning
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Novozymes AS
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Novozymes AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)

Definitions

  • D-psicose is almost non-metabolizable in the human body and, thus, has a calorie value of nearly zero. Consequently, D-psicose is attractive as a dietary sweetener.
  • D-psicose exists naturally in very small amounts in, for example, edible mushrooms, jackfruit, wheat, and Itea plants, and is difficult to chemically synthesize. There is a need in the art for developing a method for efficiently producing D-psicose. Interconversion between D-fructose and D-psicose by enzymatic epimerization catalyzed by psicose 3-epimerase is an attractive way for producing D-psicose.
  • WO 2019/043088 discloses an anaerobic marine mud metagenome D-psicose 3-epimerase.
  • the D-psicose 3-epimerase achieved conversion equilibrium 10-12 hours at each of the temperatures of 50°C, 55°C and 60°C.
  • the present invention provides epimerase variants with improved properties compared to its parent. Particularly increased thermostability.
  • the present invention relates to a D-psicose 3-epimerase variant polypeptide, comprising a substitution at one or more positions corresponding to positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, wherein the variant has D-psicose 3-epimerase activity (EC 5.1.3.30) activity and wherein 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 2, wherein the variant optionally comprises an extension of one or more amino acids at the N-terminal and/or C-terminal ends.
  • the present invention also relates to isolated polynucleotides encoding the D-psicose 3- epimerase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides, nucleic acid constructs, or vectors; and methods of producing the variants.
  • the present invention also relates to a composition comprising the D-psicose 3-epimerase variants of the invention.
  • the present invention also relates to methods of producing D-psicose comprising contacting a fructose containing substrate with a variant D-psicose 3-epimerase polypeptide of the invention under conditions suitable for the polypeptide having D-psicose 3-epimerase activity to convert D- fructose to D-psicose; and optionally (b) recovering the produced D-psicose.
  • the invention relates to a use of the D-psicose 3-epimerase variants of the invention for converting fructose to D-psicose. Definitions In accordance with this detailed description, the following definitions apply.
  • D-psicose means the monosaccharide (3R,4R,5R)-1,3,4,5,6- pentahydroxyhexan-2-one. D-psicose is also known as D-allulose, allulose, psicose, or D-ribo-2- hexulose.
  • D-Psicose 3-epimerase means an enzyme having D- psicose 3-epimerase activity (EC 5.1.3.30) that catalyzes the epimerization of D-psicose to D- fructose.
  • D-psicose 3-epimerase activity can be determined according to the procedures described in Mu et al., 2011, J. Agric. Food Chem.59: 7785-7792, or Kim et al., 2006, J. Mol. Biol.361, 920- 931.
  • D-fructose to D-psicose is a revesible reaction and thus activity may also be determined by measuring the conversion of D-psicose to D-fructose.
  • conversion of fructose to allulose is disclosed as a conversion improvement factor (IF) relative to the parent epimerase of SEQ ID NO: 2.
  • the variant polypeptides of the present invention have at least 20%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 100%, at least 110%, such as at least 120 % epimerase activity compared to the parent epimerase enzyme of SEQ ID NO: 2.
  • the epimerase variants of the invention have a conversion improvement factor (IF) of at least 1.01, 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, such as 2.0, and particularly when epimerase activity is measured at pH 6, at a temperature of 55°C, an incubation time of 16-24 hours (e.g., 16 hours or 24 hours), in presence of manganese chloride, e.g., 0.2 mM manganese chloride.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell.
  • cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • Coding sequence means a polynucleotide, which 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 may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the variant, and 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, the control sequences include a promoter, and transcriptional and translational stop signals.
  • control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.
  • expression includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
  • Expression vector An "expression vector” refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
  • control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
  • Extension means an addition of one or more amino acids to the amino and/or carboxyl terminus of a variant, wherein the “extended” variant has D-psicose 3-epimerase activity.
  • Fragment means a variant having one or more amino acids absent from the amino and/or carboxyl terminus of the variant; wherein the fragment has D-psicose 3- epimerase activity.
  • Fusion polypeptide is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a variant of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together.
  • Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J.12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.
  • heterologous means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell.
  • heterologous means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.
  • Host Strain or Host Cell is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a variant has been introduced.
  • Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides.
  • the term “host cell” includes protoplasts created from cells.
  • Improved property means a characteristic associated with a variant that is improved compared to the parent.
  • Such improved properties include, but are not limited to, increased thermostability, increased conversion rate, reduced or no need for added cofactors such as Mg 2+ , Mn 2+ .
  • the improved property is increased thermostability.
  • the increased thermostability may be determined as denaturing temperature, TmD using the TSA assay as shown in the examples. All variants according to the invention has increased thermostability and particularly the increase in melting temperature is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, such as at least 15 oC, when determined by TSA assay at pH 6, and 1mM MnCl 2 .
  • the variants may preferably have at least equivalent or increased conversion of fructose to allulose compared to the parent enzyme.
  • all variants tested had increased TmD as well as at least an equivalent D-Psicose 3-epimerase activity when determined as allulose conversion improvement factor (IF).
  • the improvement factor was at least 1.01, 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, such as 2.0, and particularly when epimerase activity is measured at pH 6, at a temperature of 55°C, an incubation time of 16-24 hours (e.g., 16 hours or 24 hours), in presence of manganese chloride, e.g., 0.2 mM manganese chloride.
  • 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.
  • Isolated means a variant, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc.
  • An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature.
  • An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted variant expressed in a host cell.
  • Mature polypeptide The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing and/or C-terminal processing (e.g., removal of signal peptide).
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having D-psicose 3-epimerase activity. Mutant: The term “mutant” means a polynucleotide encoding a variant. Native: The term “native” means a nucleic acid or polypeptide naturally occurring in a host cell. Nucleic acid: The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a variant. Nucleic acids may be single stranded or double stranded, and may be chemical modifications.
  • nucleic acid and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation. Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.
  • Operably linked means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
  • a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.
  • Parent or parent D-psicose 3-epimerase The term “parent” or “parent D-psicose 3- epimerase” means a D-psicose 3-epimerase to which an alteration is made to produce the enzyme variants of the present invention.
  • purified means 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 discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
  • a purified nucleic acid or variant is at least about 50% pure, 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 on a molar basis).
  • a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
  • enriched refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
  • purified refers to the variant or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects.
  • purified refers to the variant being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained.
  • the variant is separated from some 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.
  • the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present.
  • purified as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide.
  • the variant may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced variant.
  • the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation.
  • the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein.
  • a "substantially pure polypeptide” may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.
  • the 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, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation.
  • the variant of the present invention is preferably in a substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the variant by well-known recombinant methods or by classical purification methods.
  • Recombinant is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature.
  • the term recombinant refers to a cell, nucleic acid, variant or vector that has been modified from its native state.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
  • the term “recombinant” is synonymous with “genetically modified” and “transgenic”.
  • Recover means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” 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), preferably 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.
  • the -nobrief option In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
  • the output of Needle labeled “longest identity” is calculated as follows: (Identical Residues x 100)/(Length of Alignment – Total Number of Gaps in Alignment)
  • sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably 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 (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the nobrief option In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line.
  • Needle labeled “longest identity” is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment – Total Number of Gaps in Alignment)
  • sequence identity between two polynucleotide sequences can be determined using the same Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably 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 (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • a "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell.
  • the mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.
  • Subsequence means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having D-psicose 3-epimerase activity.
  • Variant means a polypeptide having D-psicose 3-epimerase activity comprising a substitution, an insertion (including extension), and/or a deletion (e.g., truncation), at one or more positions.
  • Wild-type in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence.
  • naturally-occurring refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature.
  • non-naturally occurring refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild- type sequence).
  • Conventions for Designation of Variants For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid positions in another D-Psicose 3-epimerase.
  • the amino acid sequence of another D-Psicose 3-epimerase is aligned with the polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 is determined 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), preferably version 6.6.0 or later.
  • Insertions are separated by addition marks (“+”) or by commas, e.g., “X195* + X411*” or “X195*, X411*”. Insertions.
  • Original amino acid, position, original amino acid, inserted amino acid the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after the amino acid at position 195 is designated “X195XK”.
  • An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after the amino acid at position 195 is indicated as “X195XKA”.
  • the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s).
  • the sequence would thus be: Parent: Variant: 195 195195 195b
  • an insert o o a a o ac es ue suc as lysine after the amino acid at position 195 may be indicated by “195aK”
  • the insertion of two or more additional amino acid residues such as lysine and alanine after the amino acid at position 195 may be indicated by “195aK, 195bA”.
  • the present invention relates to D-psicose 3-epimerase variant polypeptides, comprising a substitution at one or more positions corresponding to positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, wherein 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 2, wherein the variant optionally comprises an extension of one or more amino acids at the N-terminal and/or C-terminal ends or a truncation of one or more amino acids at the N-terminal and/or C-terminal ends, and wherein the variant has D-psicose 3-epimerase activity
  • the variants may further comprise an extension of one or more amino acids at the N-terminal and/or C-terminal ends.
  • the variants may further comprise a truncation of one or more amino acids at the N-terminal and/or C-terminal ends.
  • the variant has a sequence identity of 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%, to the amino acid sequence of the parent D-psicose 3-epimerase polypeptides.
  • 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%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 2.
  • the number of alterations in the variants of the present invention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.
  • a variant comprises a substitution, at one or more positions corresponding to positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286. In another aspect, a variant comprises a substitution at two positions corresponding to any of positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286. In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286.
  • a variant comprises a substitution at four positions corresponding to any of positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286. In another aspect, a variant comprises a substitution at five positions corresponding to any of positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286. In another aspect, a variant comprises a substitution at each position corresponding to positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286. In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 15.
  • the amino acid at a position corresponding to position 15 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile.
  • the variant comprises or consists of the substitution D15I of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 43.
  • the amino acid at a position corresponding to position 43 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably withTrp.
  • the variant comprises or consists of the substitution M43W of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 99.
  • the amino acid at a position corresponding to position 99 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu.
  • the variant comprises or consists of the substitution A99L of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitutionat a position corresponding to position 103.
  • the amino acid at a position corresponding to position 103 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu.
  • the variant comprises or consists of the substitution A103L of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 155.
  • the amino acid at a position corresponding to position 155 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Tyr.
  • the variant comprises or consists of the substitution F155Y of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 213.
  • the amino acid at a position corresponding to position 213 is substituted with Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn.
  • the variant comprises or consists of the substitution C213N of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 246.
  • the amino acid at a position corresponding to position 246 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Tyr.
  • the variant comprises or consists of the substitution F246Y of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 259.
  • the amino acid at a position corresponding to position 259 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Leu or Ile.
  • the variant comprises or consists of the substitution V259L, I of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 278.
  • the amino acid at a position corresponding to position 278 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Trp.
  • the variant comprises or consists of the substitution E278W of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 286.
  • the amino acid at a position corresponding to position 286 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val.
  • the variant comprises or consists of the substitution E286V of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 86.
  • the amino acid at a position corresponding to position 86 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val.
  • the variant comprises or consists of the substitution I86V of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 140.
  • the amino acid at a position corresponding to position 140 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Phe.
  • the variant comprises or consists of the substitution A140F of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of a substitution at a position corresponding to position 240.
  • the amino acid at a position corresponding to position 240 is substituted with Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile.
  • the variant comprises or consists of the substitution C240I of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at two positions corresponding to positions selected from: 15+99 15+43 15+86 15+103 15+140 15+155 15+213 15+240 15+246 15+259 15+278 15+286 99+43 99+86 99+103 99+140 99+155 99+213 99+240 99+246 99+259 99+278 99+286 43+86 43+103 43+140 43+155 43+213 43+240 43+246 43+259 43+278 43+286 86+103 86+140 86+155 86+213 86+240 86+246 86+259 86+278 86+286 103+140 103+155 103+213 103+240 103+246 103+259 103+278 103+286 140+155 140+213 140+240 140+246 86+259 86+278 86+286 103+140 103+155 103+213
  • the D-psicose 3-epimerase variant polypeptide comprises a substitution at one or more positions corresponding to positions 15, 99, 43, 86, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, wherein the substitutions or combination of substitutions are selected from the group consisting of: I86V; A99L; A140F; C240I; V259L; V259I; C213N; A103L; E278W; D15I; M43W; E286V; F246Y; F155Y; F155Y, C213N; D15I, A99L, C213N; A99L, V259L, E286V; D15I, F155Y, C213N; D15I, A99L, F155Y; D15I, A99L, F155Y; D15I, A140F, C213N, E286V; D15I, C213N,
  • the variant comprises or consists of the substitutions D15I+A99L+F155Y of the polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions A99L+A103L+C213N+E278W of the polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions A99L+C213N+E278W+E286V of the polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions D15I+A99L+V259L+E286V of the polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of the substitutions D15I+M43W+C213N+E286V of the polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions D15I+V259L+E278W+E286V of the polypeptide of SEQ ID NO: 2.
  • amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly- histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the groups 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 do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for D-psicose 3-epimerase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol.224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides/proteins descending from a common ancestor, typically having similar three- dimensional structures, functions, and significant sequence similarity.
  • protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and/or active sites of polypeptides. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.
  • the variant has improved thermostability compared to the parent enzyme.
  • the polypeptide may be a fusion polypeptide comprising a variant of the invention.
  • the variant may in one embodiment have an N-terminal and/or C-termial His-tag, preferably C-terminal.
  • the His-tag may comprise 3 to 7 histidines, particularly 5 to 6 Histidines, such as 6xHis.
  • the variant is isolated.
  • the variant is purified.
  • Parent D-psicose 3-epimerases The parent D-Psicose 3-epimerase may be a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 2.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO: 2 of 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%, at least 99%, or 100%, which have D-psicose 3-epimerase activity.
  • the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 2.
  • the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2.
  • the parent D-Psicose 3-epimerase is encoded by the polynucleotide of SEQ ID NO: 1.
  • the parent may be a fusion polypeptide or cleavable fusion polypeptide.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol.3: 568-576; Svetina et al., 2000, J.
  • the parent may be obtained from microorganisms of any genus.
  • the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the parent is secreted extracellularly.
  • the parent is a marine mud metagenome D-psicose 3-epimerase, e.g., the D-psicose 3-epimerase of SEQ ID NO: 2.
  • the present invention also relates to methods for obtaining a variant having D-psicose 3- epimerase activity, comprising: (a) introducing into a parent D-psicose 3-epimerase a substitution at one or more positions corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, wherein the variant has D-psicose 3-epimerase activity; and (b) recovering the variant.
  • the 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.
  • Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent. Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation.
  • Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide.
  • the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci.
  • Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US 2004/0171154; Storici et al., 2001, Nature Biotechnol.19: 773-776; Kren et al., 1998, Nat. Med.4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett.43: 15-16. Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.
  • Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • PCR error-prone PCR
  • phage display e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5,223,409; WO 92/06204
  • region- directed mutagenesis region- directed mutagenesis
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896).
  • Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi- synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques.
  • regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled. Polynucleotides
  • the present invention also relates to polynucleotides encoding a variant of the present invention.
  • the polynucleotide is the DNA sequence of SEQ ID NO: 1 having modifications in specific codons leading to specific amino acid substitutions as disclosed herein.
  • the polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.
  • the polynucleotide is isolated.
  • the polynucleotide is purified.
  • Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • the polynucleotide may be manipulated in a variety of ways to provide for expression of a variant.
  • the control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a variant of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the variant.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier, and Song et al., 2016, PLOS One 11(7): e0158447.
  • promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3’-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
  • aprH Bacillus clausii alkaline protease
  • AmyL Bacillus licheniformis alpha amylase
  • rrnB Escherichia coli ribosomal RNA
  • Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as 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 in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
  • Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
  • mRNA Stabilizers The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • mRNA stabilizer regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol.177: 3465-3471). Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Cell 5(11): 1838-1846. Leader Sequences 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. Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Bläsi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.
  • Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
  • ENO-1 Saccharomyces cerevisiae enolase
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
  • Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase ADH2/GAP
  • control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
  • 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.
  • Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol.15: 5983-5990.
  • the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the Nterminus of a variant and directs the variant into the cell’s secretory pathway.
  • the 5’end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant.
  • the 5’end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence.
  • a foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
  • a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant.
  • any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.
  • Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955
  • Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active variant by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor. Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. 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.
  • regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • filamentous fungi the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha- amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used.
  • regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. Transcription Factors The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence.
  • the transcription factor may 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 comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor.
  • the transcription factor may regulate the expression of a protein of interest either directly, i.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor.
  • Suitable transcription factors for fungal host cells are described in WO 2017/144177.
  • Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52: 7-23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.
  • Expression Vectors 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.
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the 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.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • the vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non-homologous recombination, such as non-homologous end-joining (NHEJ).
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • oil of replication or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo. More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • Host Cells also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier.
  • the choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.
  • the recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.
  • the host cell may be any cell useful in the recombinant production of a variant of the invention, e.g., a prokaryotic cell or a fungal cell.
  • the host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.
  • the prokaryotic host cell may 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.
  • the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
  • the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.
  • Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol.70: 406-438.
  • the bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
  • the bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
  • Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus.
  • the host cell may be a fungal cell.
  • “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic 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).
  • Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by 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.
  • yeast any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.
  • the fungal host cell may be a yeast cell.
  • “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No.9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell ( Komagataella phaffii).
  • the fungal host cell may be a filamentous fungal cell.
  • “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • the filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.
  • the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zona
  • the host cell is isolated. In another aspect, the host cell is purified.
  • Methods of Production also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.
  • the host cell is cultivated in a nutrient medium suitable for production of the variant using methods known in the art.
  • the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the variant to be expressed and/or isolated.
  • Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates. The variant may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the variant. The variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the whole fermentation broth is recovered.
  • a cell-free fermentation broth comprising the polypeptide is recovered.
  • the variant may be purified by a variety of procedures known in the art to obtain substantially pure variants 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).
  • the variant is not recovered.
  • Enzyme Compositions The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in such a polypeptide.
  • compositions may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition.
  • the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of alpha-galactosidase, alpha-glucosidase, alpha-amylase, glucoamylase, glucose isomerase, invertase.
  • compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the compositions may be stabilized in accordance with methods known in the art.
  • Granules The present invention also relates to enzyme granules/particles comprising a variant of the invention.
  • the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.
  • the core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 ⁇ m, particularly 50-1500 ⁇ m, 100-1500 ⁇ m or 250-1200 ⁇ m.
  • the core diameter measured as equivalent spherical diameter, can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under ISO13320 (2020).
  • the core comprises a variant of the present invention.
  • the core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • the core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
  • the core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core may include an inert particle with the variant absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • the core may have a diameter of 20-2000 ⁇ m, particularly 50-1500 ⁇ m, 100-1500 ⁇ m or 250- 1200 ⁇ m.
  • the core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
  • the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • PEG polyethylene glycol
  • MHPC methyl hydroxy-propyl cellulose
  • PVA polyvinyl alcohol
  • the coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%.
  • the coating is preferably at least 0.1 ⁇ m thick, particularly at least 0.5 ⁇ m, at least 1 ⁇ m or at least 5 ⁇ m. In some embodiments, the thickness of the coating is below 100 ⁇ m, such as below 60 ⁇ m, or below 40 ⁇ m.
  • the coating should encapsulate the core unit by forming a substantially continuous layer.
  • a substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas.
  • the layer or coating should, in particular, be homogeneous in thickness.
  • the coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • a salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
  • the salt coating is preferably at least 0.1 ⁇ m thick, e.g., at least 0.5 ⁇ m, at least 1 ⁇ m, at least 2 ⁇ m, at least 4 ⁇ m, at least 5 ⁇ m, or at least 8 ⁇ m.
  • the thickness of the salt coating is below 100 ⁇ m, such as below 60 ⁇ m, or below 40 ⁇ m.
  • the salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 ⁇ m, such as less than 10 ⁇ m or less than 5 ⁇ m.
  • the salt coating may comprise a single salt or a mixture of two or more salts.
  • the salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
  • the salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate.
  • Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum.
  • Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate.
  • alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
  • the salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
  • the salt coating may be as described in WO 00/01793 or WO 2006/034710.
  • the salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595.
  • Specific examples include anhydrous sodium sulfate (Na 2 SO 4 ), anhydrous magnesium sulfate (MgSO 4 ), magnesium sulfate heptahydrate (MgSO 4 . 7H 2 O), zinc sulfate heptahydrate (ZnSO 4 .
  • the salt is applied as a solution of the salt, e.g., using a fluid bed.
  • the coating materials can be waxy coating materials and film-forming coating materials.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol
  • the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • Preparation methods include known feed and granule formulation technologies, e.g., (a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol.71; pages 140-142; Marcel Dekker).
  • the liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme.
  • agglomerate and particles will build up, forming granulates comprising the enzyme.
  • various high-shear mixers can be used as granulators. Granulates consisting of variant, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so-called T-granulate. Reinforced particles, are more robust, and release less enzymatic dust.
  • the cores may be subjected to drying, such as in a fluid bed drier.
  • drying preferably takes place at a product temperature of from 25 to 90°C.
  • the cores comprising the variant contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the enzyme negatively.
  • the cores preferably contain 0.1-10% w/w water.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661,452 and may optionally be coated by methods known in the art.
  • the granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes.
  • Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
  • Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.
  • the present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.
  • the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
  • the one or more additional enzymes are preferably selected from the group consisting of alpha-amylase, glucoamylase, glucose isomerase, invertase or any combination thereof.
  • Liquid Formulations The present invention also relates to liquid compositions comprising a variant of the invention.
  • the composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • an enzyme stabilizer examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid.
  • filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and
  • Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials. In an aspect, the liquid formulation comprises 20-80% w/w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative.
  • the invention relates to liquid formulations comprising: (A) 0.001-25% w/w of a variant of the present invention; (B) 20-80% w/w of polyol; (C) optionally 0.001-2% w/w preservative; and (D) water.
  • the invention relates to liquid formulations comprising: (A) 0.001-25% w/w of a variant of the present invention; (B) 0.001-2% w/w preservative; (C) optionally 20-80% w/w of polyol; and (D) water.
  • the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate.
  • a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA,
  • the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.
  • MPG propylene glycol
  • the liquid formulation comprises 20-80% polyol (i.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol.
  • the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600.
  • MPG propylene glycol
  • the liquid formulation comprises 20-80% polyol (i.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).
  • the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
  • the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1% w/w preservative or 0.1-0.5% w/w preservative.
  • the liquid formulation comprises 0.001-2% w/w preservative (i.e., total amount of preservative), e.g., 0.02-1.5% w/w preservative, 0.05-1% w/w preservative, or 0.1-0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
  • the liquid formulation further comprises one or more additional enzymes, e.g., alpha-amylase, glucoamylase, glucose isomerase, invertase or any combination thereof.
  • the present invention also relates to methods for producing D-psicose, the method comprising: (a) contacting a composition comprising a variant polypeptide having D-psicose 3- epimerase activity of the present invention with D-fructose under conditions suitable for the polypeptide having D-psicose 3-epimerase activity to convert D-fructose to D-psicose; and optionally (b) recovering the produced D-psicose.
  • the D-fructose is previously produced from D-glucose by a glucose isomerase.
  • the D-fructose is simultaneously produced from D-glucose by a glucose isomerase.
  • the glucose isomerase is also known as xylose isomerase (EC. 5.3.1.5).
  • D-fructose can be in the form of a high fructose syrup, and, in particular a high fructose corn syrup. Such high fructose corn syrups are commercially available.
  • Simultaneously produced D-fructose preferably uses D-glucose in the form of a glucose syrup, in particular, a glucose corn syrup.
  • D-fructose is produced by contacting starch with an alpha-amylase and a glucoamylase to release D-glucose, which is then isomerized to D-fructose by a glucose isomerase.
  • the glucose isomerase can be immobilized (see, for example, U.S. Patent Nos. 3,868,304, 3,982,997, 4,208,482, 4,687,742, and 5,177,005).
  • glucose isomerases include, but are not limited to, Streptomyces murinus (e.g., Sweetzyme® IT Extra, Novozymes), and Streptomyces rubiginosus (GensweetTM IGI, DuPont).
  • the reaction between the polypeptide having psicose 3-epimerase activity and D-fructose may be performed using D-fructose at a concentration of 5% to 95% (w/w), at a pH of about 5.0 to about 9.0, preferably about 6 to about 7, more preferably about 6.5, and at a temperature in the range of 45°C to 75°C, 50°C to 70 °C, e.g., such as about 60°C, or about 55°C for a suitable time.
  • a suitable time is about 8 to about 24 hours, preferably about 10 to about 12 hours.
  • the reaction between the polypeptide having psicose 3-epimerase activity and D-fructose is performed in the presence of a divalent metal ion as a cofactor.
  • the divalent metal ion is selected from the group consisting of Co 2+ , Mg 2+ , Mn 2+ , and Ni 2+ .
  • the divalent metal ion is Mg 2+ or Mn 2+ .
  • the divalent metal ion is Mg 2+ .
  • the divalent metal ion is Mn 2+ .
  • the concentration of the metal cation cofactor can be about 0.1 to about 5 mM, for example, 1 mM, for improving the production yield of D-psicose.
  • the processes of the present invention can be conducted in batch or using an immobilized epimerase.
  • the reaction between the D-psicose 3-epimerase and D-fructose to produce D-psicose may be performed in a batch reactor.
  • the reaction between the D-psicose 3-epimerase and D-fructose to produce D-psicose may also be performed by immobilizing the psicose 3-epimerase on a carrier.
  • both glucose isomerase and D-psicose 3-epimerase can be immobilized.
  • the microorganisms producing the enzymes are immobilized.
  • a carrier for immobilization may be any of the carriers known for their use in enzyme immobilization.
  • the enzyme(s) or microorganisms can be immobilized, for example, on any suitable support, such as sodium alginate, Amberlite resin, Sephadex resin, or Duolite resin.
  • the immobilized enzyme(s) or microorganisms can be packed into a suitable column and the glucose or fructose liquid or syrup is continuously introduced into the column. Alternatively, the process may be a batch process.
  • D-psicose 3-epimerases on a support to produce D-psicose are well known to the person skilled in the art (see, for example, WO 2011/040708).
  • the D-psicose thus produced can be used as a dietary or pharmaceutical additive.
  • the D-psicose may be used as a sweetener in e.g., drinks, low-calorie products replacing sucrose (table sugar) in e.g., cakes, drinks etc.
  • the resulting product can be a mixture of D-fructose and D-psicose, and even a mixture of D-glucose, D-fructose and D-psicose.
  • Example 1 Thermostability of epimerase variants measured as melting temperature by Thermal Shift Assay (TSA). Thermal shift assay (TSA) for determination of TmD
  • TSA Thermal shift assay
  • TSA reaction buffers were prepared by diluting Sypro Orange protein stain (#S6650, Invitrogen) 200-fold into pH-multi buffers (100 mM acetic acid, 100 mM MES, 100 mM HEPES, 100 mM Glycine adjusted to pH 5,6, 7 and 8 with HCl or NaOH) with and without 1 mM MnCl2.5 ⁇ l diluted samples were mixed with 10 ⁇ l TSA reaction buffer in a 384-well qPCR plate (# A36931, Applied Biosystems/Thermo Fischer Scientific) and the plate sealed with optical adhesive film (#4311971, Applied Biosystems/ Thermo Fischer Scientific).
  • the melt curve was determined by running a melt curve protocol (temperature ramp 3.2 °C/min, excision/emission filters: x1 (470 +/- 15 nm)/m3586.5 +/- 10 nm) in the QuantStudio 7 Flex Real-time 384-well PCR system.
  • the melting temperature (TmD) was extracted from the melt curves by the Protein Thermal Shift Software (Applied Biosystems, version 1.4). Table 1.
  • the reaction mixture consisted of 25% fructose, 25% glucose, 50 mM acetate buffer (pH 6.0), 0.2 mM manganese chloride, and 0.1 mg epimerase protein per gram of fructose.
  • the plate was centrifuged for 1 min at 1000 g to ensure all components were at the bottom of the well. The plate was then placed into a thermocycler where the reactions were held at 55°C for 24 hours. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C.
  • the quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations. Based on the HPLC data, a ratio of fructose to allulose conversion by the variants and the Marine Mud parent backbone (SEQ ID NO: 2) was calculated and expressed as an Improvement Factor (IF). An IF > 1 corresponds to improved performance at the described conditions. Table 2.
  • IF Improvement factor of variants compared to parent epimerase Mutations IF A103L 1.45 E278W 1.30
  • Example 3 Performance of epimerase variants in conversion of fructose to allulose Epimerase Performance Assay II
  • Epimerase application assays were run in 96-well PCR plates containing a total of 100- 200 ⁇ l of reaction mixture. Before adding the epimerase, the reaction mixture was adjusted to pH 6.0. The reaction mixture consisted of 25% fructose, 25% glucose, 0.2 mM manganese chloride, and 0.1 mg epimerase protein per gram of fructose. Upon adding all the components to the PCR plate, the plate was centrifuged for 1 min at 1000 g to ensure all components were at the bottom of the well.
  • the plate was then placed into a thermocycler where the reactions were held at 55°C for 16 hours. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C.
  • the quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations. Based on the HPLC data, a ratio of fructose to allulose conversion by the variants and the Marine Mud parent backbone (SEQ ID NO: 2) was calculated and expressed as an Improvement Factor (IF).
  • IF Improvement Factor
  • the plate Upon adding all the components to the PCR plate, the plate was centrifuged for 1 min at 1000 g to ensure all components were at the bottom of the well. The plate was then placed into a thermocycler where the reactions were held at 55°C for 24 hours. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C. The quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations.
  • HPLC using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column
  • a ratio of fructose to allulose conversion by the variants and the Marine Mud parent backbone was calculated and expressed as an Improvement Factor (IF).
  • IF Improvement Factor
  • Table 4a Improvement factor (IF) of variants compared to parent epimerase (buffered reaction).
  • I F vs cofactor
  • Table 4b Improvement factor (IF) of variants compared to parent epimerase (un-buffered reaction).
  • the plate Upon adding all the components to the PCR plate, the plate was centrifuged for 1 min at 1000 g to ensure all components were at the bottom of the well. The plate was then placed into a thermocycler where the reactions were held at 55-70°C for 24 hours. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C. The quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations.
  • HPLC using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column
  • a ratio of fructose to allulose conversion by the variants and the Marine Mud parent backbone was calculated and expressed as an Improvement Factor (IF).
  • IF Improvement Factor
  • Table 5 Improvement Factor (IF) of variants compared to parent epimerase.
  • I F D15I, A99L, V259L, E286V 1.02 1.07 1.07 1.07 1.05 1.03 1.02 A99L C213N
  • Example 6 Performance of epimerase variants in conversion of fructose to allulose Epimerase Performance Assay V Epimerase application assays were run in 96-well PCR plates containing a total of 100- 200 uL of reaction mixture.
  • the reaction mixture was adjusted to pH 6.0.
  • the reaction mixture consisted of 25% fructose, 25% glucose, added, 0.2 mM manganese chloride, and 0.06 mg epimerase protein per gram of fructose.
  • the plate was centrifuged for 1 min at 1000 g to ensure all components were at the bottom of the well. The plate was then placed into a thermocycler where the reactions were held at 55°C for 24 hours. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C.
  • the quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations.
  • the improvement factor (IF) (fructose to allulose conversion improvement factor) for each variant was calculated as the ratio of allulose converted by the variant and the Marine Mud A backbone.
  • An Allulose_conv_IF > 1 corresponds to improved performance at the described conditions. Table 6. Improvement factor (IF) of variants compared to parent epimerase.
  • the plate Upon adding all the components to the PCR plate, the plate was centrifuged for 1 min at 1000 g to ensure all components were at the bottom of the well. The plate was then placed into a thermocycler where the reactions were held at 55°C for 6 and 24 hours. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C. The quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations.
  • HPLC using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column
  • Allulose_conv_IF allulose conversion improvement factor
  • the Allulose_conv_IF (allulose conversion improvement factor) for each variant was calculated as the ratio of allulose converted by the variant and the Marine Mud A backbone at the same assay conditions.
  • An Allulose_conv_IF > 1 corresponds to improved performance at the described conditions.
  • Samples with a “His-tag” have been added six additional histidines to the C-terminal of the protein.
  • Table 7a Improvement factor (IF) of variants compared to parent epimerase with and without His- tag after 6 hours. IF - 6 hours Mutations His-tag No His-tag His-tag no His-tag SEQ ID NO: 2 1.00 1.00 1.00 1.00 Table 7b.
  • IF Improvement factor
  • Example 8 Performance of epimerase variants in conversion of fructose to allulose after incubation at temperatures in the range from 55-70°C.
  • Epimerase enzyme was diluted to a concentration of 1 mg/mL in 50 mM acetate buffer (pH 6.0) and 0.2 mM manganese chloride. The diluted epimerase enzymes were then divided, and samples were held on a gradient of 55-70°C in a thermocycler for 6 and 24 hours.
  • epimerase activity assays were run in 96-well PCR plates containing a total of 100- 200 ⁇ l of reaction mixture.
  • the reaction mixture consisted of 10% fructose, 50 mM acetate buffer (pH 6.0), 0.2 mM manganese chloride, and 1 mg epimerase protein per gram of fructose.
  • the plate was then placed into a thermocycler where the reactions were held at 55°C for 5 minutes. At the end of the reaction the thermocycler was set to increase the temperature to 95°C for 10 minutes to inactivate the enzyme, then hold the plate at 4°C.
  • the quenched reactions were then diluted into deionized water and analyzed by HPLC (using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column) for allulose, fructose, and glucose concentrations.
  • HPLC using an isocratic gradient with water as the mobile phase through a Benson Polymetric, BP-800 Ca, 300 x 7.8 mm column
  • a ratio of fructose to allulose conversion by the variants and the Marine Mud parent wild type backbone SEQ ID NO: 2
  • the relative activity of each variant and the Marine Mud parent backbone was determined by comparing the allulose conversion at each temperature to the allulose conversion of the enzyme that was not subjected to the temperature gradient (held at 4°C).
  • a D-psicose 3-epimerase variant polypeptide comprising a substitution at one or more positions corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, wherein the variant has D-psicose 3-epimerase activity (EC 5.1.3.30) activity and wherein 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 2, and wherein the variant has D-psicose 3-epimerase activity (EC 5.1.3.30) activity.
  • any one of paragraphs 1-6 which comprises a substitution of the amino acid residue at position 213 with Asn, preferably C213N.
  • the variants of any one of paragraphs 1-7 which comprises a substitution of the amino acid residue at position 246 with Tyr, preferably F246Y.
  • the variants of any one of paragraphs 1-8 which comprises a substitution of the amino acid residue at position 259 with Leu or Ile, preferably V259L,I.
  • the variants of any one of paragraphs 1-9 which comprises a substitution of the amino acid residue at position 278 with Trp, preferably E278W.
  • the variants of any one of paragraphs 1-10 which comprises a substitution of the amino acid residue at position 286 with Val, preferably E286V.
  • any one of paragraphs 1-14 which comprises a substitution at three positions corresponding to any of positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286. 17.
  • the variants of any one of paragraphs 1-14 which comprises a substitution at four positions corresponding to any of positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286.
  • the variants of any one of paragraphs 1-14 which comprises a substitution at each position corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286. 19.
  • the variant comprises a substitution at two, three, four, or five positions corresponding to positions selected from the group consisting of: 155, 213; 15, 99, 213; 99, 259, 286; 15, 155, 213; 15, 99, 155; 15, 140, 213, 286; 15, 213, 278, 286; 15, 213, 240, 286; 15, 213, 240, 278; 15, 213, 259, 286; 15, 213, 259, 278; 15, 99, 103, 278; 99, 103, 278, 286; 99, 213, 240, 286; 99, 103, 213, 278; 99, 213, 278, 286; 15, 99, 259, 286; 15, 43, 213, 286; 15, 259, 278, 286; 86, 99, 259, 286; 99, 213, 259, 286; 99, 213, 259, 278; 15, 99, 140, 155; 15, 99, 155, 246; and 15, 86, 21
  • variants of any one of paragraphs 1-20 wherein the variant comprises a substitution at at least at two positions corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, and wherein the substitutions or combination of substitutions are selected from the group consisting of: D15I +M43W; D15I +A99L; D15I +I86V; D15I +A103L; D15I +A140F; D15I +F155Y; D15I +C213N; D15I +C240I; D15I +F246Y; D15I +V259L,I; D15I +E278W; D15I +E286V; M43W+A99L; M43W+I86V; M43W+A103L; M43W+A140F; M43W+F155Y; M43W+C213N; M43W+C240I;
  • variants of any one of paragraphs 1-20 wherein the variant comprises a substitution at at least at three positions corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, and wherein the substitutions or combination of substitutions are selected from the group consisting of: D15I +M43W+A99L; D15I +M43W+I86V; D15I +M43W+A103L; D15I +M43W+A140F; D15I +M43W+F155Y; D15I +M43W+C213N; D15I +M43W+C240I; D15I +M43W+F246Y; D15I +M43W+V259L,I; D15I +M43W+E278W; D15I +M43W+E286V; D15I +A99L+I86V; D15I +A99L+A99L+
  • variants of any one of paragraphs 1-20 wherein the variant comprises a substitution at one or more positions corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, and wherein the substitutions or combination of substitutions are selected from the group consisting of: I86V; A99L; A140F; C240I; V259L,I; C213N; A103L; E278W; D15I; M43W; E286V; F246Y; F155Y; F155Y, C213N; D15I, A99L, C213N; A99L, V259L, E286V; D15I, F155Y, C213N; D15I, A99L, F155Y; D15I, A99L, F155Y; D15I, A140F, C213N, E286V; D15I, C213N, E278W,
  • variants of any one of paragraphs 1-28 wherein the variant polypeptides have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%, at least 110%, such as at least 120 % of the D-psicose 3-epimerase activity of the polypeptide of SEQ ID NO: 2. 30.
  • variants of any one of paragraphs 1-29 wherein the variant’s parent D-psicose 3-epimerase 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 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • the variants of any one of paragraphs 1-30 which has an improved property relative to the parent, wherein the improved property is selected from the group consisting of increased thermostability, increased conversion improvement factor (IF), reduced or no need for added cofactors such as Mg 2+ , Mn 2+ . 32.
  • thermostability measure as melting temperature (TmD) thermostability measure as melting temperature (TmD)
  • TmD melting temperature
  • the increase in melting temperature is at least 1 oC, at least 2 oC, at least 3 oC, at least 4 oC, at least 5 oC, at least 6 oC, at least 7 oC, at least 8 oC, at least 9 oC, such as at least 10 oC, at least 11 oC, at least 12 oC, at least 13 oC, at least 14 oC, such as at least 15 oC, when determined by TSA assay at pH 6, and 1mM MnCl 2 . 33.
  • conversion IF is measured at pH 6, at a temperature of 55°C, an incubation time of 16-24 hours (e.g., 16 hours or 24 hours), in presence of manganese chloride, e.g., 0.2 mM manganese chloride.
  • a fusion polypeptide comprising the variants of any one of paragraphs 1-34 and a second polypeptide. 36.
  • variants or fusion polypeptides of any one of paragraphs 1-35 which are isolated. 37. The variants or fusion polypeptides of any one of paragraphs 1-35, which are purified. 38. A granule, which comprises: (a) a core comprising the variants or fusion polypeptides of any one of paragraphs 1-35, and, optionally (b) a coating consisting of one or more layer(s) surrounding the core. 39. A granule, which comprises: (a) a core, and (b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the variant or fusion polypeptide of any one of paragraphs 1-35. 40.
  • a liquid composition comprising the variants or fusion polypeptides of any one of paragraphs 1- 35 and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid.
  • an enzyme stabilizer e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid.
  • a composition comprising the variants or fusion polypeptides of any one of paragraphs 1-35, the granule of paragraphs 38 or 39, or the liquid compositions of any one of paragraphs 40-41.
  • 44. A polynucleotide encoding the variant or fusion polypeptide of any one of paragraphs 1-35 45. The polynucleotide of paragraph 44, which is isolated. 46. The polynucleotide of paragraph 44 or 45, which is purified. 47. A nucleic acid construct or expression vector comprising the polynucleotide of any one of paragraphs 44-46. 48. A recombinant host cell transformed with the polynucleotide of any one of paragraphs 44-46 or the nucleic acid construct or expression vector of paragraph 47.
  • the recombinant host cell of paragraph 48 which comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of any one of paragraphs 44-46. 50.
  • the recombinant host cell of paragraph 48 or 49 which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • the recombinant host cell of paragraph 48 or 49 which is a filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Asper
  • the recombinant host cell of paragraph 48 or 49 which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E.
  • a prokaryotic recombinant host cell e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E.
  • coli Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp.
  • Bacillus alkalophilus Bacillus amyloliquefaciens
  • Bacillus brevis Bacillus circulans, Bac
  • a method for obtaining a D-psicose 3-epimerase variant comprising introducing into a parent D-psicose 3-epimerase a substitution at one or more positions corresponding to positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 and 286 of the polypeptide of SEQ ID NO: 2, wherein the variant has D-psicose 3-epimerase activity; and recovering the variant. 57.
  • a method of producing D-psicose comprising contacting a fructose containing subtrate with a variant D-psicose 3-epimerase polypeptide of any of paragraphs 1-34 under conditions suitable for the polypeptide having D-psicose 3-epimerase activity to convert D-fructose to D-psicose; and optionally (b) recovering the produced D-psicose (allulose).
  • the temperature is in the range from 50-70°C, such as 55, 60, 65°C, and pH is 5-7, such as around pH 6, and optionally in the presence of around 0.2 mM manganese chloride.

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Abstract

La présente invention concerne des polypeptides variants de la D-psicose 3-épimérase. En particulier, les polypeptides variants de la D-psicose 3-épimérase comprennent une substitution en une ou plusieurs positions correspondant aux positions 15, 43, 86, 99, 103, 140, 155, 213, 240, 246, 259, 278 et 286 du polypeptide de SEQ ID NO : 2, et le variant ayant une activité de D-psicose 3-épimérase (EC 5.1.3.30). La présente invention concerne également des polynucléotides codant les variants ; des constructions d'acides nucléiques, des vecteurs et des cellules hôtes comprenant les polynucléotides ; ainsi que des procédés d'utilisation des variants.
PCT/US2024/056535 2023-11-20 2024-11-19 Variants d'épimérase et polynucléotides les codant Pending WO2025111274A2 (fr)

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