WO2024182327A1

WO2024182327A1 - Genetically modified microorganism and fermentation process for the production of d-allulose

Info

Publication number: WO2024182327A1
Application number: PCT/US2024/017373
Authority: WO
Inventors: Hans H. Liao
Original assignee: Cargill Inc
Current assignee: Cargill Inc
Priority date: 2023-02-28
Filing date: 2024-02-27
Publication date: 2024-09-06
Anticipated expiration: 2025-08-28
Also published as: KR20250154456A; CN120858175A

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 allulose-6-phosphate 3-epimerase (epimerase) enzyme 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 NOs:2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 50.

Description

GENETICALLY MODIFIED MICROORGANISM AND FERMENTATION PROCESS

FOR THE PRODUCTION OF D-ALLULOSE

CROSS-REFERENCE TO RELATED APPLICATIONS

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

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA PATENT CENTER [0002] The content of the Sequence Listing XML file of the sequence listing named “PT-1533- WO-PCT.xml” which is 91,968 bytes in size created on February 26, 2024 and electronically submitted vis Patent Center herewith the application is incorporated by reference in its entirety.

BACKGROUND

[0003] D-allulose, also known in the art as D-psicose, is a low-calorie sweetener used as a food additive and sugar substitute. Used commercially in beverages, yogurt, ice cream, baked goods, and other typically high calorie items, D-allulose has 70% of the sweetness of sucrose but has a caloric value of about 0.2 to 0.4 kcal/g compared to the 4 kcal/g of sucrose. D-allulose is minimally metabolized and is excreted largely unchanged giving it a very low glycemic index. D-allulose is naturally found in low amounts in figs, raisins, and maple syrup leading to its designation as a “rare sugar.” D-allulose is also associated with certain functional benefits such as mouthfeel, browning capability, and freezing point allowing for its use as a sugar substitute in many food and beverage applications.

[0004] Current methods for the production of D-allulose include epimerization of fructose from corn starch and beet sugar. However, this method is expensive due to the low reaction yield and need to separate the D-allulose from residual fructose in the reaction mixture. In contrast, fermentation processes have been used commercially at large scale to produce other organic molecules, such as ethanol, citric acid, lactic acid, and the like, and may offer a cost effective and sustainable alternative to current D-allulose processing methods. Accordingly, provided herein are genetically modified microorganisms and fermentation methods for the production of D- allulose. SUMMARY

[0005] The present disclosure provides genetically engineered yeast cells and/or genetically engineered bacterial cells capable of producing D-allulose. The engineered cells comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3 -epimerase (epimerase) enzyme 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 NOs:2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 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 NOs: 2, 4, 22, 34, 48, and 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 NOs: 2, 48, and 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 engineered cell(s) capable of producing D-allulose may additionally comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphatase (phosphatase) enzyme 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 NOs: 54 and 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 engineered cell(s) described here in may include an encoded epimerase 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 NOs:2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 50 and an encoded phosphatase 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, wherein the yeast is capable of producing at least 0.5 g/L D-allulose.

[0008] The engineered cell(s) described herein may include an encoded epimerase 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 NOs:2, 4, 22, 34, 48, and 50 and an encoded phosphatase 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, wherein the yeast is capable of producing at least 1.0 g/L D-allulose.

[0009] The engineered cell(s) described herein may include an encoded epimerase 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 NOs:2, 48, and 50 and an encoded phosphatase 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, wherein the yeast is capable of producing at least 3.0 g/L D-allulose.

[0010] In the 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-l-cytophrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEF1 terminator, or a GAL 10 terminator.

[0011] The engineered cell(s) describe 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., P achy solen spp., Debaryomyces spp., and Yarrowia spp., Saccharomyces cerevisiae. Issatchenkia orienlalis. Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida elhanolica. Pichia deserlicola. Kluyveromyces marxianus. Kluyveromyces laclis. Pichia membranifadens. Yarrowia lipolylica. or Pichia fermentans. The engineered cell(s) described herein may be a bacterial cell selected from the group consisting of Escherichia coli. Corynebacterium glutamicum, and Bacillus spp.. The engineered cell(s) may be a Saccharomyces cerevisiae cell.

[0012] The disclosure also provides a method for producing D-allulose, the method comprising contacting a substrate with an engineered cell(s) as described herein, wherein the engineered cell produces at least 0.5 g/L, at least 1.0 g/L, or at least 3.0 g/L after 72 hours. The substrate may inlcude starch, glucose, cellulosic biomass, or combinations thereof.

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

BRIEF DESCRIPTION OF THE FIGURES

[0014] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and the payment of the necessary fee. [0015] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0016] FIG. 1 shows a proposed pathway for the production of D-allulose from sucrose, starch, and/or glucose.

[0017] FIG. 2 shows a graph of the production of D-allulose at 24, 48, and 72 hours for the stains outlined in Example 5.

[0018] FIG. 3 shows a graph of the average D-allulose yield at 24, 48, and 72 hours for the strains outlined in Example 6.

DETAILED DESCRIPTION

[0019] 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 exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0020] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0021] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range 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 just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. [0022] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis is also referred to as wt% or % (wt) below.

[0023] This disclosure relates to various recombinant cells engineered to product D-allulose. In general, the recombinant cells described herein include a heterologous polynucleotide encoding an allulose-6-phosphate 3-epimerase, for example the allulose-6-phosphate 3-epimerase enzyme of at least one of SEQ ID NOs: 2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 50 or sequences 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 cell may additionally include a heterologous nucleic acid encoding an allulose-6-phosphate phosphatase, for example the allulose-6-phosphate phosphatase enzyme of at least one of SEQ ID NOs: 54 and 56 or sequences 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 disclosure further provides fermentation methods for the production of D-allulose using the genetically engineered cells described herein.

[0024] 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 found in trace amounts in wheat, figs, raisins, maple sugar, and molasses leading to its designation as a rare sugar.

(i)

[0025] In general, recombinant cells described herein are yeast or bacterial cells. Suitable yeast and bacterial cells are known and described in the art. An ordinarily skilled artisan can identify yeast and bacterial strains that would be suitable for use in the generating the recombinant cells described herein.

[0026] 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 genera Bacillus. An ordinarily skilled artisan would understand the requirements for selection of a suitable bacterial cell, and recombinant bacterial cells of the present disclosure are not limited to those expressly recited herein.

[0027] In general, recombinant cells described herein are yeast cells. Non-limiting examples of yeast cells include yeast cells obtained from, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains. Suitable yeast cells may include, but are not limited to, Saccharomyces cerevisiae, Issatchenkia orienlalis. Pichia galeiformis, Pichia sp. YB-4149 (NR.R.L designation), Candida elhanolica. Pichia deserlicola. Kluyveromyces marxianus. Kluyveromyces laclis, Pichia membranifadens. Yarrowia lipolylica, or Pichia fermentans. The yeast cell may be, for example, a commercially available yeast such as Kluyveromyces marxianus, Kluyveromyces laclis, or Yarrowia lipolytica. An ordinarily skilled artisan would understand the requirements for selection of a suitable yeast cell, and recombinant yeast cells of the present disclosure are not limited to those expressly recited herein.

[0028] The recombinant cells described herein include one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed enable the production of D-allulose by the recombinant cells.

[0029] As used herein, “exogenous” refers to genetic material or an expression product thereof that originates from outside of the host organism. For example, the exogenous genetic material or expression product thereof can be a modified form of genetic material native to the host organism, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component. For example, Lactobacillus helveticus lactate dehydrogenase gene is exogenous when introduced into S. cerevisiae.

[0030] As used herein, “native” refers to genetic material or an expression product thereof that is found, apart from individual-to-individual mutations which do not affect function or expression, within the genome of wild-type cells of the host cell.

[0031] 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 give the recited macromolecule its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function. A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein. Table 1 : Amino Acid three and one letter symbols

[0032] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include or greater than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild-type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.

[0033] 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 of amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An 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: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0034] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, 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. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0035] The polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild-type” is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant,” “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 may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.

[0036] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area 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 bulk of the side chain.

[0037] As used herein, 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 the antisense strand. The DNA polynucleotides may be a cDNA or a genomic DNA sequence.

[0038] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

[0039] Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, the polynucleotides (i.e., polynucleotides encoding a non-heme iron-binding protein polypeptide) may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in coral are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.

[0040] The recombinant cells described herein are capable of producing D-allulose and include an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3 -epimerase (or simply “epimerase”) enzyme. The epimerase enzyme may be any suitable enzyme with allulose-6- phosphate 3-epimerase activity. The exogenous polynucleotide sequence may be an exogenous allulose-6-phosphate 3-epimerase (epimerase) gene.

[0041] An “allulose-6-phosphate 3-epimerase gene” and “epimerase gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with 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 by epimerization of the C3 position of fructose-6-phosphate. The enzyme with allulose- 6-phosphate 3-epimerase activity may be an enzyme with increased activity in the presence of cobalt (Co²⁺) The epimerase enzyme can be from any suitable source organism or may be synthetic. Suitable epimerase enzymes may include, but are not limited to, enzymes categorized under Enzyme Commission (EC) number 5.1.3. Suitable epimerase enzymes may be the epimerase enzymes from Mesorhizobium sp. WSM3873, Alkalibaculum sporogenes OX=2655001, Rahnella woolbedingensis, Bifidobacterium breve, Streptomyces sp., Escherichia coli, Streptomyces angustmyceticus, Streptomyces sp. NR.R.L S-1813, Streptomyces sp. NR.R.L F- 5727, Pseudoleptotrichia goodfello ii, Blautia glucerasea, and the like. The epimerase gene may encode an amino acid 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 NOs:2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 50. The epimerase gene may encode an amino acid 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 NOs:2, 4, 22, 34, 48, and 50. The epimerase gene may encode an amino acid 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 NOs:2, 48, and 50. The epimerase gene may encode an amino acid 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 NOs:48 and 50.

[0042] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from an Escherichia coli gene encoding the amino acid sequence of SEQ ID NO:2. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:2.

[0043] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptomyces angustmyceticus gene encoding the amino acid sequence of SEQ ID NO:4. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NON.

[0044] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from Mesorhizobium sp. WSM3873 gene encoding the amino acid sequence of SEQ ID NO: 8. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:8.

[0045] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Alkalibaculum sporogenes OX=2655001 gene encoding the amino acid sequence of SEQ ID NO:22. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:22.

[0046] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rahnella woolbedingensis gene encoding the amino acid sequence of SEQ ID NO:24. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:24.

[0047] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Bifidobacterium breve gene encoding the amino acid sequence of SEQ ID NO:26. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:26.

[0048] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptomyces sp. gene encoding the amino acid sequence of SEQ ID NO:34. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:34. [0049] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptomyces sp. NRRL S-1813 gene encoding the amino acid sequence of SEQ ID NO:36. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:36.

[0050] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptomyces sp. NRRL F-5727 gene encoding the amino acid sequence of SEQ ID NO:40. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:40.

[0051] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Pseudoleptotrichia goodfellowii gene encoding the amino acid sequence of SEQ ID NO:48. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:48.

[0052] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Blautia glucerasea gene encoding the amino acid sequence of SEQ ID NO:50. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:50.

[0053] The recombinant cells described herein are capable of producing D-allulose, 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 with allulose-6- phosphate phosphatase activity. The exogenous polynucleotide sequence may be an exogenous phosphatase gene.

[0054] An “allulose-6-phosphatase” and “phosphatase” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with 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 to D-allulose and a phosphate ion. Suitable phosphatase enzymes active on hexose-6-phosphate substrates are known and described in the art. The phosphatase enzyme may be derived from any suitable source or may be synthetic. Suitable phosphatase enzyme may include, but are not limited to, the phosphatase enzymes from Escherichia coli and Saccharomyces cerevisiae. The phosphatase gene may encode an amino acid 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 NOs:54 and 56.

[0055] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Escherichia coli gene encoding the amino acid sequence of SEQ ID NO: 54. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:54.

[0056] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO:56. The exogenous polynucleotide may encode an amino acid 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 to SEQ ID NO:56.

[0057] The exogenous nucleic acids in the recombinant cells described herein may be under the control of a promoter. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial promoter. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarboxylase (PDC1), glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (TDH3 herein; annotated in EC 1.2.1.12), translational elongation factor 1 (TEF1), URA3, S-adenosyl methionine transferase 2 (SAM2), alcohol dehydrogenase 1 (ADH1), 3 -phosphoglycerate kinase (PGK1), and synthetic promoters.

[0058] The exogenous nucleic acids 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 known and described in the art. Terminators may include, but are not limited to, i so- 1 -cytochrome c (CYC1), URA3, PDC, ADH1, TEF1, and ScGALlO.

[0059] 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 said polynucleotide is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0060] The polynucleotides described herein may be provided as part of a construct. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. The construct may be a vector including a promoter operably linked to the polynucleotide encoding the allulose-6-phosphate 3 -epimerase. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. The vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.

[0061] The disclosure also provides fermentation methods for the production of D-allulose using the recombinant cells described herein. The fermentation methods include the step of fermenting a substrate using the genetically engineered yeast cells or the genetically engineered bacterial cells described herein to product D-allulose. The fermentation method can include additional steps, as would be understood by a person 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 the ethanol from the fermentation broth.

[0062] The fermentation substrate can comprise a starch. Starch can be obtained from a natural source, such as a plant source. Starch can also be obtained from a feedstock with high starch or sugar content, including, but not limited to corn, sweet sorghum, fruits, sweet potato, rice, barley, sugar cane, sugarbeets, wheat, cassava, potato, tapioca, arrowroot, peas, or sago. The fermentation substrate may be from lignocellulosic biomass such as wood, straw, grasses, or algal biomass, such as microalgae and macroalgae. The fermentation substrate may include cellulosic or lignocellulosic biomass. The fermentation substrate may be from grasses, trees, or agricultural and forestry residues, such as com cobs and stalks, rice straw, sawdust, and wood chips. The fermentation substrate can also comprise a sugar, such as glucose (dextrose) or sucrose, and/or a polysaccharide, such as maltodextrin. The fermentation substrate may be physically (e.g., heat, pressure, and the like) or chemically (e.g., acid, hydrolysis, enzyme treatment, such as glucoamylase, and the like) pretreated prior to or during the fermentation process.

[0063] Media for fermentation of the engineered cells described herein can be supplemented with various components. For example, media for fermentation of the engineered cells described herein can be supplemented with a glucoamylase, e.g., the glucoamylase Spirizyme™ (Novozymes, Bagsvaerd, Denmark) and/or the amyloglucosidase from Aspergillus niger sold under the trade name AMG 300L™ by Sigma- Aldrich. [0064] The fermentation process can be run under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, may be ambient temperature. Alternatively, or additionally, the fermentation temperature may 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 may be maintained at a temperature of, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or any value in between or range thereof. However, a skilled artisan will recognize that the fermentation temperature is not limited to any specific range recited herein and may be modified as appropriate.

[0065] The pH of a culture medium described herein may be controlled for optimal ethanol production. The pH of the culture or a fermentation mixture of an engineered cell described herein may be in the range of between 5.0 and 7.5. The pH may be maintained for at least part of the incubation 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. The pH may be maintained at a range between 6.0 and 7.0, between 6.2 and 6.7, or between 6.3 to 6.6.

[0066] The engineered cell (yeast and/or bacteria) may be cultured for approximately 24-72 hours. For example, the engineered cell 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 engineered cell described herein may be cultured for approximately 48 to 72 hours. A culture (fermentation) time of about 48 hours or 72 hours can be a representative time for similar commercial-scale fermentation processes. Accordingly, a 48-hour or 72-hour time point can be used to compare the fermentation performance of different genetically engineered cell strains.

[0067] 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 state, etc.), chemical parameters (e.g., pH, redox- potential, concentration of reaction substrate and/or product, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, ethanol concentration, fermentation substrate concentration, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, 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, as well as thermodynamic parameters, such as temperature, light intensity/quality, etc.). Sensors to measure the parameters described herein are well known to one of ordinary skill in the art.

[0068] The fermentation process can be associated with various characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of the cell (yeast and/or bacteria) and/or genetic modification of the cell used in the fermentation process. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be adjusted via a combination of cell selection or modification and the selection of fermentation process conditions.

[0069] 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.

EXAMPLES

[0070] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0071] Strain numbering is consistent throughout the Examples. For example, strain 1-5 in Example 1 is the same strain as strain 1-5 in Example 3.

Example 1 - Genetically Modified Saccharomyces cerevisiae strains

[0072] The proposed pathway to produce D-allulose from sucrose, starch, and/or glucose is shown in FIG. 1. In this pathway, glucose and/or fructose are converted to fructose-6-phosphate. The fructose-6-phosphate is converted to D-allulose-6-phosphate by a hexose-6-phosphate epimerase enzyme. Finally, the 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 a genetically engineered yeast, the strains described in this and subsequent examples were built and tested. Strain 1-1

[0073] Strain 1-1 is yeast strain Saccharomyces cerevisiae CEN.PK 113-7D (MATa HIS3 LEU2 TRP1 MAL2-8 SUC2; Taxonomy ID: NCBI:txid889517).

Stain 1-2

[0074] Strain 1-2 is an uracil auxotroph derivative of strain 1-1 with an insertion of the Aspergillus nidulans amdS gene at the URA3 locus.

Strain 1-3

[0075] Strain 1-2 was transformed using the Li-acetate protocol (Gietz, R. D., et al., “Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method,” Methods Enzymol. 350, 87-96, 2002) with a DNA fragment carrying sequences encoding the constitutively expressed synthetic transcription factor (sTF; nucleotide sequence SEQ ID NO:51; polypeptide SEQ ID NO: 52) and a URA+ marker to create strain 1-3. The DNA fragment was generated by digestion of plasmid B6622 (pKlURA3_TDH3cp-BM3Rl-VP16; Rantasalo A., et al., “Synthetic toolkit for complex genetic circuit engineering in Saccharomyces cerevisiae,” ACS Synth. Biol., 2018, 7, 6, 1573-1587), with restriction endonuclease Noth Transformants were selected for on SDA medium without uracil and verified for the insertion of the sTF by colony PCR resulting in strain 1-3.

Strain 1-4

[0076] To generate the G418-resistant uracil auxotroph strain 1-4, the uracil marker in strain 1-3 was replaced with a marker conferring resistance to the antibiotic G418 bounded by LoxP sites.

Strain 1-5

[0077] To generate strain 1-5, the G418-resistance cassette was removed from strain 1-4 by transformation of strain 1-4 with a plasmid expressing the Cre recombinase. Resulting strain 1-5 is ura3‘ and includes the synthetic transcription factor of SEQ ID NO:52 under the control of the TDH3cp promoter. [0078] Polynucleotides encoding the allulose-6-phosphate 3-epimerase and allulose-6- phosphate phosphatase were cloned into the pSCT036 vector (SEQ ID NO:53). Sequences encoding the epimerase enzymes were under the control of the TDH3 promoter (SEQ ID NO: 58) and sequences encoding the phosphatase enzymes were under the control of the control of the promoter of SEQ ID NO:59, which includes 8 synthetic transcription factor (sTF) binding sites for the sTF expressed in strain 1-5. The plasmids encoding the epimerase and phosphatase enzymes were cloned into parent strain 1-5, as outlined in Table 2.

Table 2: Genetically Modified S. cerevisiae strains

Example 2 - Shake Flask Assay

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

[0080] Strains were struck on a synthetic complete medium minus uracil (ScD-ura) plates and grown until single colonies formed (overnight at 30 °C or 2-3 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 25 ml of ScD-ura with 20 g/L glucose media (Table 3) and incubated overnight at 30°C, 250 rpm with 70% humidity. The overnight cultures were diluted into 40 ml DMlu production medium (Table Y) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of optical density at 600nm (OD600) = 0.2, and incubated at 30°C, 250 rpm with 70% humidity. Optical density is measured at a wavelength of 600 nm with a 1 cm path length using a model Genesys 20 spectrophotometer (Thermo Scientific). 0.5 ml samples were withdrawn at 24, 48, and 74 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, and stored at -20°C until analysis. Samples were analyzed for D-allulose by HPLC.

Table 3: ScD-ura with 20g/L dextrose

Table 4: 72 hour shake flask results

Example 3 - Shake Flask Assay

[0081] Strains 1-5, 1-6 and 1-8 were assayed in duplicate to evaluate D-allulose production.

[0082] Strains were struck on a ScD-ura plates and grown until single colonies formed (overnight at 30 °C or 2-3 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 25 ml of ScD- ura with 20 g/L glucose media (Table 3) and incubated overnight at 30°C, 250 rpm with 70% humidity. The overnight cultures were diluted into 40 ml DMlu production medium (Table 5) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.2, and incubated at 30°C, 250 rpm with 70% humidity. 0.5 ml samples were withdrawn at 24, 48, and 74 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, and stored at -20°C until analysis. Samples were analyzed for D-allulose by HPLC. Results are shown in Table 8.

Table 5: DMlu Production Medium - Maltodextrin

*GA added just prior to inoculation

Table 6: lOOOx trace elements

Table 7: 1000X DM1 vitamin solution

Table 8: Shake flask results

Example 4 - Genetically Modified Saccharomyces cerevisiae strains

[0083] 23 additional allulose-6-phosphate 3 -epimerase candidates were identified based on public database annotation of genes as hexose-6-phospate 3 -epimerases and/or by homology to the S. augustmycetus AgmD gene product. The 23 chosen candidates are outlined in Table 9.

Table 9: Epimerase Sequence Identity

[0084] Polynucleotides, codon optimized for expression in S. cerevisiae and encoding the epimerase candidates outlined in Table E, were cloned into pSCT036 vector described in Example 1. Sequences encoding the epimerase enzyme candidates were under the control of the TDH3 promoter (SEQ ID NO:58). 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 control of the promoter of SEQ ID NO:59. Table 10: Genetically Modified S. cerevisiae strains

Example 5 - Shake Flask Assays

[0085] Strains 1-5, 1-6, 1-9, and 1-10 through 1-32 were grown in shake flasks to assay D- allulose production.

[0086] Strains were struck on a ScD-ura plates and grown until single colonies formed (overnight at 30 °C or 2-3 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 25 ml of ScD- ura with 20 g/L glucose media (Table 3) and incubated overnight at 30°C, 250 rpm with 70% humidity. The overnight cultures were diluted into 40 ml DMlu production medium (Table 5) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.2, and incubated at 30°C, 250 rpm with 70% humidity. 0.5 ml samples were withdrawn at 24, 48, and 74 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, and stored at -20°C until analysis. Samples were analyzed for D-allulose by HPLC. Results are shown in Table 11 and FIG. 2.

Table 11 : Shake Flask Assay Results

Example 6 - Shake Flask Assay

[0087] Strains 1-5, 1-6, were assayed in duplicate to evaluate D-allulose production.

[0088] Strains were struck on a ScD-ura plates and grown until single colonies formed (overnight at 30 °C or 2-3 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 25 ml of ScD- ura with 20 g/L glucose media (Table 3) and incubated overnight at 30°C, 250 rpm with 70% humidity. The overnight cultures were diluted into 40 ml DMlu production medium (Table 12) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.2, and incubated at 30°C, 250 rpm with 70% humidity. 0.5 ml samples were withdrawn at 24, 48, and 74 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, and stored at -20°C until analysis. Results are shown in Table 13 and FIG. 3.

[0089] Without wishing to be bound by any particular theory, embodiment, or mode of action, the use of maltodextrin + glucoamylase as the carbon source (Examples 3 & 5) generates glucose in situ at a rate that reduces the formation of ethanol by the S. cerevisiae strain. This experiment confirms that glucose is also a suitable carbon source for these strains, however the overall yields are lower as it is expected the ethanol would predominate under these conditions.

Table 12: DMlu Production Medium - Glucose

Table 13: Shake Flask Results

Claims

CLAIMS What is claimed is:

1. A genetically engineered yeast cell and/or a genetically engineered bacterial cell capable of producing D-allulose, the engineered cell comprising an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3 -epimerase (epimerase) enzyme 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 NOs:2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 50.

2. The 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 NOs: 2, 4, 22, 34, 48, and 50.

3. The engineered cell of claim 1 or claim 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 NOs: 2, 48, and 50.

4. The engineered cell of any preceding claim, wherein the epimerase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:48; and /or the epimerase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:50.

5. The engineered cell of any preceding claim, wherein the engineered yeast cell additionally comprises an exogenous polynucleotide sequence encoding an allulose-6-phosphatase (phosphatase) enzyme 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 NOs: 54 and 56.

6. The engineered cell of claim 5, wherein the phosphatase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:54; and /or the epimerase is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO:56.

7. The engineered cell of any one of claims 5-6, wherein the epimerase 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 NOs:2, 4, 8, 22, 24, 26, 34, 36, 40, 48, and 50, wherein 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 wherein the yeast is capable of producing at least 0.5 g/L D-allulose.

8. The engineered cell of any one of claims 5-7, wherein the epimerase 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 NOs:2, 4, 22, 34, 48, and 50, wherein 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 wherein the yeast is capable of producing at least 1.0 g/L D-allulose.

9. The engineered cell of any one of claims 5-8, wherein the epimerase 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 NOs:2, 48, and 50, wherein 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 wherein the yeast is capable of producing at least 3.0 g/L D-allulose.

10. The engineered cell of any proceeding claim, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous or artificial promoter.

11. The 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-adenosyl methionine transferase 2 (SAM2) promoter; an alcohol dehydrogenase 1 (ADH1) promoter, and a 3 -phosphoglycerate kinase (PGK1) promoter.

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

13. The engineered cell of claim 12, wherein the terminator is selected from the group consisting of an iso-l-cytophrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEF1 terminator, or a GAL 10 terminator.

14. The engineered cell of any proceeding claim, wherein

(i) the engineered cell is 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., P achy solen spp., Debaryomyces spp., and Yarrow ia spp., Saccharomyces cerevisiae. Issatchenkia orienlalis. Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida elhanolica. Pichia deserlicola. Kluyveromyces marxianus. Kluyveromyces laclis. Pichia membranifadens. Yarrowia lipolylica. o Pichia fermentans and/or

(ii) the engineered cell is a bacterial cell selected from the group consisting of Escherichia coli. Corynebacterium glutamicum, and Bacillus spp..

15. The engineered cell of any proceeding claim, wherein the genetically engineered cell is a Saccharomyces cerevisiae cell.

16. A method for producing D-allulose, the method comprising: contacting a substrate with the engineered cell of any proceeding claim, wherein the engineered cell produces at least 0.5 g/L, at least 1.0 g/L, or at least 3.0 g/L after 72 hours.

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

18. Use of the engineered cell of any one of claims 1-15 to produce D-allulose.