Genetically engineered microorganisms and fermentation processes for the production of D-allulose

Abstract

Disclosed herein are genetically engineered yeast cells and genetically engineered bacterial cells capable of producing D-allulose. The genetically engineered cells comprise an exogenous polynucleotide sequence encoding an allose-6-phosphate 3-epimerase (epimerase) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:50.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 487,275, filed February 28, 2023, and U.S. Provisional Patent Application No. 63 / 487,636, filed March 1, 2023, each of which is incorporated by reference herein in its entirety.

[0002] (Reference to sequence listing submitted via Patent Center) The contents of the Sequence Listing XmL file entitled "PT-1533-WO-PCT.xmL", created on February 26, 2024, and submitted electronically with this application through the Patent Center, having a size of 91,968 bytes, are incorporated herein by reference in their entirety. [Background technology]

[0003] D-Allulose, also known as D-psicose, is a low-calorie sweetener used as a food additive and sugar substitute. When used commercially in beverages, yogurt, ice cream, baked goods, and other typical high-calorie items, D-allulose has 70% of the sweetness of sucrose but a caloric value of approximately 0.2–0.4 kcal / g compared to sucrose's 4 kcal / g. D-Allulose is minimally metabolized and excreted largely unchanged, resulting in a very low glycemic index. D-Allulose is classified as a "rare sugar" because it is naturally found in small amounts in figs, raisins, and maple syrup. D-Allulose is also associated with certain functional benefits, such as mouthfeel, browning ability, and freezing point, enabling its use as a sugar substitute in many food and beverage applications.

[0004] Current methods for producing D-allulose involve epimerizing fructose from corn starch and sugar beet. However, this method is expensive due to the low reaction yield and the need to separate D-allulose from the residual fructose in the reaction mixture. In contrast, fermentation processes are commercially used on a large scale to produce other organic molecules (e.g., ethanol, citric acid, lactic acid, etc.), and may provide a cost-effective and sustainable alternative to traditional D-allulose processing methods. Therefore, the present specification provides genetically modified microorganisms and fermentation methods for producing D-allulose. Summary of the Invention

[0005] The present invention provides genetically engineered yeast cells and / or genetically engineered bacterial cells capable of producing D-allulose. The genetically engineered cells comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase (epimerase) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:50. The encoded epimerase enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:50. The encoded epimerase enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO: 2, SEQ ID NO: 48, and SEQ ID NO: 50. The encoded epimerase may be at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 48. The encoded epimerase may be at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 50.

[0006] The genetically engineered cell(s) capable of producing D-allulose may additionally comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphatase (phosphatase) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO: 54 and SEQ ID NO: 56. The encoded phosphatase may be at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 54. The encoded phosphatase may be at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 56.

[0007] The genetically engineered cell(s) described herein may include an encoded epimerase that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:50, and an encoded phosphatase that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:54, and the yeast is capable of producing at least 0.5 g / L of D-allulose.

[0008] The genetically engineered cell(s) described herein may include an encoded epimerase that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:50, and an encoded phosphatase that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:54, and the yeast is capable of producing at least 1.0 g / L of D-allulose.

[0009] The genetically engineered cell(s) described herein may include an encoded epimerase that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:48, and SEQ ID NO:50, and an encoded phosphatase that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:54, and the yeast is capable of producing at least 3.0 g / L of D-allulose.

[0010] In the genetically engineered cell(s) described herein, one or more of the exogenous polynucleotide sequences may be operably linked to a heterologous or artificial promoter and / or a heterologous or artificial terminator. The promoter may be selected from the group consisting of a pyruvate decarboxylase (PDC) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (TDH3) promoter, a translation elongation factor 1 (TEF1) promoter, a URA3 promoter, an S-adenosyl methionine transferase 2 (SAM2) promoter, an alcohol dehydrogenase 1 (ADH1) promoter, and a 3-phosphoglycerate kinase (PGK1) promoter. The terminator may be selected from the group consisting of an iso-1-cytophrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEF1 terminator, or a GAL10 terminator.

[0011] The genetically engineered cell(s) described herein may be a yeast cell selected from the group consisting of Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., and Yarrowia spp., Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia galeiformis, Pichia spp. YB-4149 (NRRL designation), Candida ethanolica, Pichia deserticola, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia membranifadens, Yarrowia lipolytica, or Pichia fermentans. The genetically engineered cell(s) described herein may be bacterial cells selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, and Bacillus species. The genetically engineered cell(s) may be Saccharomyces cerevisiae cells.

[0012] The present disclosure also provides a method for producing D-allulose, the method comprising contacting a substrate with a genetically engineered cell(s) as described herein, wherein the genetically engineered cells produce at least 0.5 g / L, at least 1.0 g / L, or at least 3.0 g / L after 72 hours. The substrate can include starch, glucose, cellulosic biomass, or a combination thereof.

[0013] The present disclosure further provides the use of a genetically engineered cell(s) as described herein for the production of D-allulose. [Brief explanation of the drawings]

[0014] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. [Figure 1] 1 shows a proposed pathway for producing D-allulose from sucrose, starch, and / or glucose. [Figure 2] 1 shows a graph of D-allulose production at 24, 48, and 72 hours for the strains outlined in Example 5. [Figure 3] 1 shows a graph of average D-allulose yields at 24, 48, and 72 hours for the strains outlined in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0016] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0017] In this document, the words "a," "an," or "the" are used to include one or more unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents referenced in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of inconsistent usage between this document and a document so incorporated by reference, the usage in the incorporated reference should be construed as supplementary to the usage in this document. In the case of irreconcilable conflicts, the usage in this document shall control.

[0018] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the stated range. The statement "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the statement "about X, Y, or about Z" has the same meaning as "about X, Y, or about Z" unless otherwise indicated.

[0019] Unless expressly stated, ppm (parts per million), percentages and ratios are by weight. Percentages by weight are also referred to below as % by weight or % (by weight).

[0020] The present disclosure relates to various recombinant cells genetically engineered to produce D-allulose. Generally, the recombinant cells described herein contain a heterologous polynucleotide encoding an allulose-6-phosphate 3-epimerase enzyme, such as at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:50, or a sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The recombinant cells can additionally comprise a heterologous nucleic acid encoding an allulose-6-phosphate phosphatase enzyme, such as at least one of SEQ ID NO: 54 and SEQ ID NO: 56, or an allulose-6-phosphate phosphatase enzyme with a sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The present disclosure further provides fermentation methods for producing D-allulose using the genetically engineered cells described herein.

[0021] As used herein, "D-allulose" and "allulose" are used interchangeably and refer to the ketohexose epimer of fructose, shown in Structure I below. D-allulose is also known in the art as "D-psicose" or "psicose." D-allulose is designated as a rare sugar because it is found in trace amounts in wheat, figs, raisins, maple sugar, and molasses.

[0022] [ka]

[0023] Generally, the recombinant cells described herein are yeast or bacterial cells. Suitable yeast and bacterial cells are well known and described in the art. Those skilled in the art will be able to identify suitable yeast and bacterial strains for use in producing the recombinant cells described herein.

[0024] The recombinant cells described herein may be bacterial cells. Non-limiting examples of suitable bacterial cells include Escherichia coli, Corynebacterium glutamicum, and bacteria of the genus Bacillus. Those skilled in the art will understand the requirements for selecting a suitable bacterial cell, and the recombinant bacterial cells of the present disclosure are not limited to those explicitly listed herein.

[0025] Generally, the recombinant cells described herein are yeast cells. Non-limiting examples of yeast cells include, for example, yeast cells obtained from Saccharomyces species, Schizosaccharomyces species, Pichia species, Paffia species, Kluyveromyces species, Candida species, Talaromyces species, Brettanomyces species, Pachysolen species, Debaryomyces species, Yarrowia species, and industrial polyploid yeast strains. Suitable yeast cells may include, but are not limited to, Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia galeiformis, Pichia species YB-4149 (NRRL designation), Candida ethanolica, Pichia desertinicola, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia membranifadens, Yarrowia lipolytica, or Pichia fermentans. The yeast cells may be, for example, commercially available yeast such as Kluyveromyces marxianus, Kluyveromyces lactis, or Yarrowia lipolytica. One of skill in the art will understand the considerations for selecting a suitable yeast cell, and the recombinant yeast cells of the present disclosure are not limited to those explicitly listed herein.

[0026] The recombinant cells described herein contain one or more exogenous polynucleotide sequences that encode one or more exogenous polypeptides that, when expressed, enable the recombinant cell to produce D-allulose.

[0027] As used herein, "exogenous" refers to genetic material or its expression products that originate outside the host organism. For example, exogenous genetic material or its expression products can be a modified form of genetic material native to the host organism, can be derived from another organism, can be a modified form of a component derived from another organism, or can be a synthetically derived component. For example, the Lactobacillus helveticus lactate dehydrogenase gene becomes exogenous when introduced into S. cerevisiae.

[0028] As used herein, "naturally occurring" refers to genetic material or its expression products that is found in the genome of a wild-type cell of a host cell apart from inter-individual variations that do not affect function or expression.

[0029] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to confer its function and properties on the recited macromolecule. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. Recitation of any particular enzyme is understood to include cofactors, coenzymes, and metals necessary for the enzyme to function properly, either independently or as part of a biosynthetic pathway. A summary of art-recognized amino acids and their three-letter and one-letter symbols is provided in Table 1. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0030] [Table 1]

[0031] Variants or sequences having substantial identity or homology to the polypeptides described herein can be utilized in practicing the disclosed compositions and methods. Such sequences can be referred to as variant or modified sequences. That is, a polypeptide sequence can be modified while still retaining the ability to exhibit a desired activity. Generally, a variant or modified sequence can comprise or exceed about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a wild-type, naturally occurring polypeptide sequence, or a variant polypeptide described herein.

[0032] As used herein, the phrases "% sequence identity," "% identity," and "percent identity" are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods for amino acid and nucleic acid sequence alignment are well known. Sequence alignment and sequence identity generation include global and local alignments, which typically use computational approaches. Alignment can be performed using BLAST (National Center for Biological Information, NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word size: 6; Maximum match in query range: 0; Matrix: BLOSUM62; Gap cost: (Presence: 11, Extension: 1); Composition adjustment: Conditional composition score matrix adjustment; Filter: None; Mask: None. The percent nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word size: 28; Maximum match in query range: 0; Match / Mismatch score: 1, -2; Gap cost: Linear; Filter: Low complexity region; Mask: Mask for lookup table only. A sequence with an identity score of XX% (e.g., 80%) to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical to the reference sequence, or equivalently, to have XX% sequence identity.

[0033] Polypeptide or polynucleotide sequence identity may be measured over the length of the entire defined polypeptide sequence, for example, as defined by a particular SEQ ID NO:, or over a shorter length, for example, over the length of a fragment derived from the larger defined polypeptide sequence, for example, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It is understood that such lengths are merely exemplary, and that any fragment length supported by the sequences shown in the specification, tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured.

[0034] Polypeptides disclosed herein can include "variant" polypeptides, "mutants," and "derivatives thereof." As used herein, the term "wild-type" is a term of art understood by those skilled in the art and refers to the typical form of a polypeptide as it occurs in nature, as distinguished from variant or mutant forms. As used herein, a "mutant," "mutant," or "derivative" refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant can have one or more insertions, deletions, or substitutions of amino acid residues compared to the reference molecule.

[0035] The amino acid sequence of a polypeptide variant, mutant, derivative, or fragment contemplated herein may contain conservative amino acid substitutions compared to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may contain conservative amino acid substitutions compared to a reference molecule. A "conservative amino acid substitution" is a substitution in which an amino acid is substituted with a different amino acid, such that the substitution is predicted to least interfere with the properties of the reference polypeptide. In other words, a conservative amino acid substitution substantially preserves the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of the substitution, for example, as a beta-sheet or alpha-helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulkiness of the side chain.

[0036] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence," and "nucleic acid" are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand. A DNA polynucleotide may be a cDNA or genomic DNA sequence.

[0037] A polynucleotide is said to encode a polypeptide if, in its natural state, or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce a polypeptide or a fragment thereof. The antisense strand of such a polynucleotide is also said to encode a sequence.

[0038] Those skilled in the art understand the degeneracy of the genetic code and that various polynucleotides can encode the same polypeptide. In some aspects, a polynucleotide (i.e., a polynucleotide encoding a non-heme iron-binding protein polypeptide) may be codon-optimized for expression in a particular cell, including, but not limited to, a plant cell, a bacterial cell, a fungal cell, or an animal cell. While polypeptides encoded by polynucleotide sequences found in corals are disclosed herein, any polynucleotide sequence encoding a desired form of the polypeptide described herein may be used. Thus, non-naturally occurring sequences can be used. These may be desirable, for example, to enhance expression of the polypeptide or protein in a heterologous expression system. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and the human hand can also be used to generate degenerate coding sequences.

[0039] The recombinant cells described herein are capable of producing D-allulose and comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase (or simply "epimerase") enzyme. The epimerase enzyme may be any suitable enzyme having allulose-6-phosphate 3-epimerase activity. The exogenous polynucleotide sequence may be an exogenous allulose-6-phosphate 3-epimerase (epimerase) gene.

[0040] The terms "allulose-6-phosphate 3-epimerase gene" and "epimerase gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having allulose-6-phosphate epimerase activity. As used herein, "allulose-6-phosphate 3-epimerase activity" refers to the ability to catalyze the conversion of fructose-6-phosphate to allulose-6-phosphate through an epimerization reaction at the C3 position of fructose-6-phosphate. An enzyme having allulose-6-phosphate 3-epimerase activity can be produced by the addition of cobalt (Co 2+The epimerase enzyme may be an enzyme whose activity is increased in the presence of a soluble ... The encoded epimerase enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:50. The encoded epimerase enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:50. The encoded epimerase gene may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:48, and SEQ ID NO:50.The encoded epimerase gene may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:48 and SEQ ID NO:50.

[0041] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from an Escherichia coli gene that encodes the amino acid sequence of SEQ ID NO: 2. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0042] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Streptomyces angustmyceticus gene that encodes the amino acid sequence of SEQ ID NO: 4. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0043] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Mesorhizobium sp. WSM3873 gene that encodes the amino acid sequence of SEQ ID NO: 8. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0044] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from an Alkalibaculum sporogenes OX=2655001 gene that encodes the amino acid sequence of SEQ ID NO: 22. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22.

[0045] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Rahnella woolbedingensis gene that encodes the amino acid sequence of SEQ ID NO: 24. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24.

[0046] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Bifidobacterium breve gene that encodes the amino acid sequence of SEQ ID NO: 26. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 26.

[0047] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Streptomyces sp. gene that encodes the amino acid sequence of SEQ ID NO: 34. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0048] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Streptomyces sp. NRRL S-1813 gene that encodes the amino acid sequence of SEQ ID NO: 36. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36.

[0049] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Streptomyces sp. NRRL F-5727 gene that encodes the amino acid sequence of SEQ ID NO: 40. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 40.

[0050] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Pseudoleptotrichia goodfellowii gene that encodes the amino acid sequence of SEQ ID NO: 48. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 48.

[0051] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Blautia glucerase gene that encodes the amino acid sequence of SEQ ID NO: 50. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 50.

[0052] The recombinant cells described herein are capable of producing D-allulose and include an exogenous polynucleotide sequence encoding an epimerase enzyme, and may additionally include an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase ("phosphatase") enzyme. The phosphatase enzyme may be any suitable enzyme having allulose-6-phosphate phosphatase activity. The exogenous polynucleotide sequence may be an exogenous phosphatase gene.

[0053] "Allulose-6-phosphate phosphatase" and "phosphatase" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having allulose-6-phosphate phosphatase activity. As used herein, "allulose-6-phosphate phosphatase activity" refers to the ability to catalyze the hydrolysis of D-allulose-6-phosphate into D-allulose and phosphate ions. Suitable phosphatase enzymes active on hexose-6-phosphate substrates are well known and described in the art. The phosphatase enzyme may be derived from any suitable source or may be synthesized. Suitable phosphatase enzymes may include, but are not limited to, phosphatase enzymes from Escherichia coli and Saccharomyces cerevisiae. The phosphatase gene may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:54 and SEQ ID NO:56.

[0054] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from an Escherichia coli gene that encodes the amino acid sequence of SEQ ID NO: 54. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 54.

[0055] The recombinant cells described herein can include an exogenous polynucleotide that can be or be derived from a Saccharomyces cerevisiae gene that encodes the amino acid sequence of SEQ ID NO: 56. The exogenous polynucleotide can encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 56.

[0056] The exogenous nucleic acid in the recombinant cells described herein can be under the control of a promoter. For example, the exogenous nucleic acid can be operably linked to a heterologous or artificial promoter. Suitable promoters are well known and described in the art. Promoters can include, but are not limited to, pyruvate decarboxylase (PDC1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (herein TDH3, annotated in EC 1.2.1.12), translation elongation factor 1 (TEF1), URA3, S-adenosylmethionine transferase 2 (SAM2), alcohol dehydrogenase 1 (ADH1), 3-phosphoglycerate kinase (PGK1), and synthetic promoters.

[0057] The exogenous nucleic acid in the recombinant cells described herein may be under the control of a terminator. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial terminator. Suitable terminators are well known and described in the art. Terminators may include, but are not limited to, iso-1-cytochrome c (CYC1), URA3, PDC, ADH1, TEF1, and ScGAL10.

[0058] A promoter or terminator is "operably linked" to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to that polynucleotide is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0059] The polynucleotides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide (including, but not limited to, DNA and RNA), which may be single-stranded or double-stranded and may represent the sense or antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that contain polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Thus, a construct may contain new modifications to an endogenous gene introduced, for example, by genome editing techniques. A construct may also include recombinant polynucleotides produced, for example, using recombinant DNA methodologies. A construct may be a vector containing a promoter operably linked to a polynucleotide encoding allulose-6-phosphate 3-epimerase. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide to which it is linked. A vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be incorporated.

[0060] The present invention also provides a fermentation method for producing D-allulose using the recombinant cells described herein. The fermentation method includes fermenting a substrate using the genetically engineered yeast cells or genetically engineered bacterial cells described herein to produce D-allulose. The fermentation method can include additional steps as understood by those skilled in the art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and isolating ethanol from the fermentation broth.

[0061] The fermentation substrate may include starch. The starch may be obtained from natural sources, such as plant sources. The starch may also be obtained from raw materials with a high starch or sugar content, including, but not limited to, corn, sorghum, fruit, sweet potato, rice, barley, sugarcane, sugar beet, wheat, cassava, potato, tapioca, arrowroot, pea, or sago. The fermentation substrate may be derived from lignocellulosic biomass, such as wood, straw, or grass, or algal biomass, such as microalgae and macroalgae. The fermentation substrate may include cellulosic biomass or lignocellulosic biomass. The fermentation substrate may be from grass, wood, or agricultural and forestry residues, such as corn cobs and stalks, rice straw, sawdust, and wood chips. The fermentation substrate may also include sugars, such as glucose (dextrose) or sucrose, and / or polysaccharides, such as maltodextrin. The fermentation substrate may be pretreated physically (e.g., heat, pressure, etc.) or chemically (e.g., with acid, hydrolysis, enzymatic treatment such as glucoamylase, etc.) before or during the fermentation process.

[0062] The medium for fermentation of the genetically engineered cells described herein can be supplemented with various components. For example, the medium for fermentation of the genetically engineered cells described herein can be supplemented with glucoamylase, such as glucoamylase Spirizyme™ (Novozymes, Bagsvaerd, Denmark) and / or amyloglucosidase from Aspergillus niger sold under the trade name AMG 300L™ by Sigma-Aldrich.

[0063] The fermentation process can be carried out under a variety of conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, can be ambient temperature. Alternatively, or in addition, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25°C to 40°C, 26°C to 38°C, 28°C to 35°C, or 29°C to 32°C. The fermentation temperature can be maintained at, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or any value therebetween or within the range. However, those skilled in the art will recognize that the fermentation temperature is not limited to the specific ranges described herein and can be varied as needed.

[0064] The pH of the culture media described herein may be controlled for optimal ethanol production. The pH of the culture or fermentation mixture of the genetically engineered cells described herein may be in the range of 5.0 to 7.5. The pH may be maintained at 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and / or 7.5 during at least a portion of the incubation. The pH may be maintained in the range of 6.0 to 7.0, 6.2 to 6.7, or 6.3 to 6.6.

[0065] The genetically engineered cells (yeast and / or bacteria) may be cultured for approximately 24 to 72 hours. For example, the genetically engineered cells may be cultured for approximately 12, 18, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 80, 90, 96 hours, or more than 96 hours. The genetically engineered cells described herein may be cultured for approximately 48 to 72 hours. A culture (fermentation) time of about 48 or 72 hours can be representative of a similar commercial-scale fermentation process, and therefore the 48 or 72 hour time point can be used to compare the fermentation performance of different engineered cell lines.

[0066] Reaction parameters can be measured or adjusted during the production of D-allulose. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell condition, etc.), chemical parameters (e.g., pH, redox potential, reaction substrate and / or product concentration, dissolved gas concentration, such as oxygen concentration and CO2 concentration, nutrient concentration, metabolite concentration, ethanol concentration, fermentation substrate concentration, oligopeptide concentration, amino acid concentration, vitamin concentration, hormone concentration, additive concentration, serum concentration, ionic strength, ion concentration, relative humidity, molarity, osmolarity, other chemicals such as buffers, adjuvants, or reaction by-products), physical / mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, and thermodynamic parameters such as temperature, light intensity / quality, etc.). Sensors for measuring the parameters described herein are well known to those skilled in the art.

[0067] Fermentation processes can be associated with various characteristics, including, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be influenced by the selection of cells (yeast and / or bacteria) used in the fermentation process and / or genetic modification of the cells. These characteristics can be influenced by adjusting fermentation process conditions. These characteristics can be modulated through a combination of cell selection or cell modification and selection of fermentation process conditions.

[0068] The final D-allulose titer may be at least 0.25 g / L, at least 0.5 g / L, at least 0.75 g / L, at least 1.0 g / L, at least 1.5 g / L, at least 2.0 g / L, at least 2.5 g / L, at least 3.0 g / L, at least 4 g / L, or at least 5 g / L. [Example]

[0069] The present invention will be described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0070] Strain numbering is consistent throughout the examples, e.g., strains 1-5 in Example 1 are the same strains as strains 1-5 in Example 3.

[0071] Example 1 - Genetically modified Saccharomyces cerevisiae strains A proposed pathway for producing D-allulose from sucrose, starch, and / or glucose is shown in Figure 1. In this pathway, glucose and / or fructose are converted to fructose-6-phosphate. Fructose-6-phosphate is converted to D-allulose-6-phosphate by a hexose-6-phosphate epimerase enzyme. Finally, D-allulose-6-phosphate is converted to D-allulose by a hexose-6-phosphate phosphatase. To test this pathway and demonstrate the production of D-allulose in genetically engineered yeast, the strains described in this and subsequent examples were constructed and tested.

[0072] Stock 1-1 Strain 1-1 is a yeast strain Saccharomyces cerevisiae CEN.PK 113-7D (MATaHIS 3 LEU2 TRP1 MAL2-8 SUC2; Classification ID: NCBI: txid889517).

[0073] Dyeing 1-2 Strain 1-2 is a uracil auxotrophic derivative of strain 1-1 with the Aspergillus nidulans amdS gene inserted at the URA3 locus.

[0074] Stocks 1-3 Strain 1-2 was transformed with a DNA fragment carrying a sequence encoding a constitutively expressed synthetic transcription factor (sTF; nucleotide sequence (SEQ ID NO: 51; polypeptide sequence (SEQ ID NO: 52)) and a URA+ marker using a Li-acetate protocol (Gietz, R.D., et al., "Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method," Methods Enzymol. 350, pp. 87-96, 2002) to generate strain 1-3. The DNA fragment was transfected into plasmid B6622 (pKlURA3_TDH3cp-BM3R1-VP16; Rantasalo A., et al., "Synthetic toolkit for complex genetic circuit engineering in Saccharomyces cerevisiae," ACS Biochem. 2002) using the restriction endonuclease NotI. Synth. Biol., 2018, 7, 6, 1573-1587). Transformants were selected on SDA medium lacking uracil and confirmed for insertion of sTF by colony PCR, yielding strains 1-3.

[0075] Stocks 1-4 To generate the G418-resistant uracil auxotroph 1-4, the uracil marker in strain 1-3 was replaced with a marker conferring resistance to the antibiotic G418 bounded by a LoxP site.

[0076] Stocks 1-5 To generate strain 1-5, the G418 resistance cassette was removed from strain 1-4 by transforming it with a plasmid expressing Cre recombinase. The resulting strain, 1-5, is ura3 - and comprises the synthetic transcription factor of SEQ ID NO: 52 under the control of the TDH3cp promoter.

[0077] Polynucleotides encoding allulose-6-phosphate 3-epimerase and allulose-6-phosphate phosphatase were cloned into the pSCT036 vector (SEQ ID NO: 53). The sequence encoding the epimerase enzyme was under the control of the TDH3 promoter (SEQ ID NO: 58), and the sequence encoding the phosphatase enzyme was under the control of the promoter of SEQ ID NO: 59, which contains eight synthetic transcription factor (sTF) binding sites for the sTFs expressed in strains 1-5. Plasmids encoding the epimerase and phosphatase enzymes were cloned into parent strains 1-5 as outlined in Table 2.

[0078] [Table 2]

[0079] Example 2 - Shake Flask Assay Strains 1-6, 1-7, 1-8, and 1-9 were run in duplicate shake flasks to assay for D-allulose production.

[0080] Strains were plated onto synthetic complete medium without uracil (ScD-ura) plates and grown until single colonies formed (overnight at 30°C or 2–3 days at room temperature (approximately 25°C)). Cells from the ScD-ura plates were scraped into sterile seed containers (250 mL baffled Erlenmeyer flasks) containing 25 mL of ScD-ura with 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted into 40 mL of DM1u production medium (Table Y) in a 250 mL baffled Erlenmeyer flask to achieve an initial cell density of optical density at 600 nm (OD600) = 0.2 and incubated at 30°C, 250 rpm, and 70% humidity. Optical density was measured at 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm pathlength. 0.5 mL samples were collected in microcentrifuge tubes at 24, 48, and 74 hours, centrifuged at 14,000 x g for 2 minutes, and stored at -20°C until analysis. Samples were analyzed for D-allulose by HPLC.

[0081] [Table 3]

[0082] [Table 4]

[0083] Example 3 - Shake Flask Assay To assess D-allulose production, strains 1-5, 1-6, and 1-8 were assayed in duplicate.

[0084] Strains were plated onto ScD-ura plates and grown until single colonies formed (overnight at 30°C or 2-3 days at room temperature (approximately 25°C)). Cells from the ScD-ura plates were scraped into sterile seed containers (250 mL baffled Erlenmeyer flasks) containing 25 mL of ScD-ura with 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted into 40 mL of DM1u production medium (Table 5) in a 250 mL baffled Erlenmeyer flask to achieve an initial cell density of OD600 = 0.2 and incubated at 30°C, 250 rpm, and 70% humidity. 0.5 mL samples were collected into microcentrifuge tubes at 24, 48, and 74 hours, centrifuged at 14,000 × g for 2 minutes, and stored at -20°C until analysis. The samples were analyzed for D-allulose by HPLC and the results are shown in Table 8.

[0085] [Table 5]

[0086] [Table 6]

[0087] [Table 7]

[0088] [Table 8]

[0089] Example 4 - Genetically modified Saccharomyces cerevisiae strains Twenty-three additional allulose-6-phosphate 3-epimerase candidates were identified based on public database annotation of the genes as hexose-6-phosphate 3-epimerases and / or by homology to the S. augustmycetus AgmD gene product. The 23 selected candidates are summarized in Table 9.

[0090] [Table 9]

[0091] Polynucleotides obtained from S. cerevisiae and plasmids encoding the candidate epimerases outlined in Table E were cloned into the pSCT036 vector described in Example 1. The sequences encoding the candidate epimerase enzymes were under the control of the TDH3 promoter (SEQ ID NO:58). The strains outlined in Table 10 were also transformed with the vector described in Example 1 containing the sequence encoding the phosphatase enzyme of SEQ ID NO:54 under the control of the promoter of SEQ ID NO:59.

[0092] [Table 10]

[0093] Example 5 - Shake Flask Assay To assay for D-allulose production, strains 1-5, 1-6, 1-9, and 1-10 through 1-32 were grown in shake flasks.

[0094] Strains were plated onto ScD-ura plates and grown until single colonies formed (overnight at 30°C or 2-3 days at room temperature (approximately 25°C)). Cells from the ScD-ura plates were scraped into sterile seed containers (250 mL baffled Erlenmeyer flasks) containing 25 mL of ScD-ura with 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted into 40 mL of DM1u production medium (Table 5) in a 250 mL baffled Erlenmeyer flask to achieve an initial cell density of OD600 = 0.2 and incubated at 30°C, 250 rpm, and 70% humidity. 0.5 mL samples were collected into microcentrifuge tubes at 24, 48, and 74 hours, centrifuged at 14,000 × g for 2 minutes, and stored at -20°C until analysis. The samples were analyzed for D-allulose by HPLC, and the results are shown in Table 11 and Figure 2.

[0095] [Table 11]

[0096] Example 6 - Shake Flask Assay To assess D-allulose production, strains 1-5 and 1-6 were assayed in duplicate.

[0097] Strains were plated onto ScD-ura plates and grown until single colonies formed (overnight at 30°C or 2-3 days at room temperature (approximately 25°C)). Cells from the ScD-ura plates were scraped into sterile seed containers (250 mL baffled Erlenmeyer flasks) containing 25 mL of ScD-ura with 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted into 40 mL of DM1u production medium (Table 12) in a 250 mL baffled Erlenmeyer flask to achieve an initial cell density of OD600 = 0.2 and incubated at 30°C, 250 rpm, and 70% humidity. 0.5 mL samples were collected into microcentrifuge tubes at 24, 48, and 74 hours, centrifuged at 14,000 × g for 2 minutes, and stored at -20°C until analysis. The results are shown in Table 13 and Figure 3.

[0098] Without wishing to be bound by any particular theory, embodiment, or mode of action, it is believed that the use of maltodextrin plus glucoamylase as a carbon source (Examples 3 and 5) produces glucose in situ at a rate that reduces ethanol production by S. cerevisiae strains. This experiment confirms that glucose is also a suitable carbon source for these strains, although ethanol is expected to predominate under these conditions, resulting in lower overall yields.

[0099] [Table 12]

[0100] [Table 13]

Claims

1. A genetically engineered yeast cell and / or a genetically engineered bacterial cell capable of producing D-allulose, the genetically engineered cell comprising:

1. A genetically engineered cell comprising an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase (epimerase) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:

50.

2. 2. The genetically engineered cell of claim 1, wherein the epimerase enzyme is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:

50.

3. 3. The genetically engineered cell of claim 1 or 2, wherein the epimerase enzyme is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:48, and SEQ ID NO:

50.

4. the epimerase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 48; and / or 4. The genetically engineered cell of any one of claims 1 to 3, wherein the epimerase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:

50.

5. 5. The genetically engineered cell of any one of claims 1 to 4, wherein the genetically engineered yeast cell additionally comprises an exogenous polynucleotide sequence encoding an allulose-6-phosphatase (phosphatase) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:54 and SEQ ID NO:

56.

6. the phosphatase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 54; and / or 6. The genetically engineered cell of claim 5, wherein the epimerase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:

56.

7. 7. The genetically engineered cell of claim 5, wherein the epimerase is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:50, and the phosphatase is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:54, and the yeast is capable of producing at least 0.5 g / L of D-allulose.

8. 8. The genetically engineered cell of any one of claims 5-7, wherein the epimerase is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:50; the phosphatase is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:54; and the yeast is capable of producing at least 1.0 g / L of D-allulose.

9. 9. The genetically engineered cell of any one of claims 5-8, wherein the epimerase is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2, SEQ ID NO:48, and SEQ ID NO:50; the phosphatase is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:54; and the yeast is capable of producing at least 3.0 g / L of D-allulose.

10. 10. The genetically engineered cell of any one of claims 1 to 9, wherein one or more of the exogenous polynucleotide sequences are operably linked to a heterologous or artificial promoter.

11. 11. The genetically engineered cell of claim 10, wherein the promoter is selected from the group consisting of a pyruvate decarboxylase (PDC) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (TDH3) promoter, a translation elongation factor 1 (TEF1) promoter, a URA3 promoter, an S-adenosylmethionine transferase 2 (SAM2) promoter, an alcohol dehydrogenase 1 (ADH1) promoter, and a 3-phosphoglycerate kinase (PGK1) promoter.

12. 12. The genetically engineered cell of any one of claims 1 to 11, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous or artificial terminator.

13. 13. The genetically engineered cell of claim 12, wherein the terminator is selected from the group consisting of an iso-1-cytochrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEF1 terminator, or a GAL10 terminator.

14. (i) the genetically engineered cell is selected from the group consisting of Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., and Yarrowia spp., Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia galeiformis, Pichia spp. YB-4149 (NRRL designation), Candida ethanolica, Pichia deserticola, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia membranifadensis, Yarrowia lipolytica, or Pichia fermentans, and / or (ii) The genetically engineered cell of any one of claims 1 to 13, wherein the genetically engineered cell is a bacterial cell selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, and a Bacillus species.

15. The genetically engineered cell of any one of claims 1 to 14, wherein the genetically engineered cell is a Saccharomyces cerevisiae cell.

16. 1. A method for producing D-allulose, said method comprising:

16. A method comprising contacting a substrate with the genetically engineered cells of any one of claims 1-15, wherein the genetically engineered cells produce at least 0.5 g / L, at least 1.0 g / L, or at least 3.0 g / L after 72 hours.

17. 17. The method of claim 16, wherein the substrate comprises starch, glucose, cellulosic biomass, or a combination thereof.

18. 16. Use of the genetically engineered cell of any one of claims 1 to 15 for producing D-allulose.

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