Genetically modified microorganisms and fermentation processes for the production of d-psicose

Abstract

This article discloses genetically engineered yeast cells and genetically engineered bacterial cells capable of producing D-allulose. These genetically engineered cells contain a foreign polynucleotide sequence encoding 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% of allulose-6-phosphate 3-epimerase, which is identical to at least one of SEQ ID NO: 192, 194, and 196; and a foreign polynucleotide sequence encoding 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% of allulose-6-phosphate phosphatase, which is identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183.

Description

Cross-reference to related applications

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 558,139, filed February 27, 2024; U.S. Provisional Application No. 63 / 568,011, filed March 21, 2024; and U.S. Provisional Application No. 63 / 724,932, filed November 26, 2024, the entire contents of each of which are incorporated herein by reference. Citations of sequence listings submitted through the Patent Center

[0002] The contents of the sequence list XML file named “PT-1883-WO-PCT.xml” (506,703 bytes in size, created on February 24, 2025 and electronically submitted with this application via the Patent Centre) are incorporated herein by reference in their entirety. Background Technology

[0003] D-allulose (also known in the art as D-psicose) is a low-calorie sweetener used as a food additive and sugar substitute. Commercially used in beverages, yogurts, ice cream, baked goods, and other typically high-calorie items, D-allulose has 70% the sweetness of sucrose, but its calorific value is approximately 0.2 kcal / g to 0.4 kcal / g compared to 4 kcal / g sucrose. D-allulose is metabolized very little and is essentially unchanged upon excretion, resulting in a very low glycemic index. D-allulose is naturally found in small 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 taste, browning ability, and freezing point, allowing it to be used as a sugar substitute in many food and beverage applications.

[0004] Current methods for producing D-allulose include the metaisomerization of fructose from corn starch and beet sugar. However, these methods are expensive due to low reaction yields and the need to separate D-allulose from residual fructose in the reaction mixture. In contrast, fermentation processes are already commercially available on a large scale for producing other organic molecules such as ethanol, citric acid, and lactic acid, and can provide a cost-effective and sustainable alternative to current D-allulose production methods. Therefore, this paper presents genetically modified microorganisms and fermentation methods for the production of D-allulose. Summary of the Invention

[0005] This disclosure provides genetically engineered yeast cells and / or genetically engineered bacterial cells capable of producing D-allulose, wherein the engineered yeast cells comprise 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 allulose-6-phosphate phosphatase to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183; and an exogenous polynucleotide sequence encoding 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% of the allulose-6-phosphate phosphatase. The exogenous polynucleotide sequence of allulose-6-phosphate 3-epimerase 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: 192, 194, and 196. Allulose-6-phosphate phosphatase 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: 87, 89, 190, 123, 105, 107, and 115. Allulose-6-phosphate phosphatase 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:87, 89, and 105. Allulose-6-phosphate phosphatase 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:87 and 105. Allulose-6-phosphate 3-epimerase may be at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO:192, 194, and 196.Allulose-6-phosphate phosphatase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, and 115. Allulose-6-phosphate 3-epimerase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO: 192, 194, and 196. Allulose-6-phosphate phosphatase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO: 87 and 105.

[0006] The engineered cells described in this article can be yeast cells 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., *Yarrowia* spp., *Saccharomyces cerevisiae*, and *Issatchenkia*. The engineered cells described herein can be bacterial cells selected from the group consisting of: *Pichiagaleiformis*, *Pichiagaleiformis*, *Pichiagaleiformis* species YB-4149 (NRRL designation), *Candida ethanolica*, *Pichia deserticola*, *Kluyveromyces marxianus*, *Kluyveromyces lactis*, *Pichia membranifadens*, *Yarrowia lipolytica*, or *Pichia fermentans*.

[0007] Allulose-6-phosphate 3-epimerase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 192, 194, and 196, allulose-6-phosphate phosphatase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183, and the cell may be capable of producing at least 5 g / L of allulose.

[0008] Allulose-6-phosphate 3-epimerase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 192, 194, and 196, allulose-6-phosphate phosphatase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, and 115, and the cell may be capable of producing at least 10 g / L D-allulose.

[0009] The engineered cell can be a *Saccharomyces cerevisiae* cell, the allulose-6-phosphate 3-epimerase can be at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:196, the allulose-6-phosphate phosphatase can be at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO:87 and 89, and the cell can produce at least 15 g / L D-allulose.

[0010] In the cells described herein, one or more exogenous polynucleotide sequences are operatively linked to a heterologous promoter and / or a heterologous terminator. The promoter can be selected from the group consisting of the following: pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoter (ADH1p), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p); 6-phosphogluconic acid dehydrogenase promoter (6PGDp); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p); translation elongation factor 1 promoter (TEFp); phosphoglucose mutase 1 promoter (PGM1p); 3-phosphoglycerate kinase promoter (PGK1p); enolase promoter (ENO1p); asparagine synthase promoter (ASNSp); 50S ribosomal protein L1 promoter (RPLAp); RPL16B; and PDC1 promoter. The terminator can be selected from the group consisting of the following: GAL10 terminator, PDC terminator, transaldolase terminator (TAL), 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENO1t); hexokinase 1 terminator (HXK1t); PGK1 terminator (PGK1t); PGM1 terminator (PGM1t); PYK1 terminator (PYK1t); RPLA terminator (RPLAt); transaldolase 1 terminator (TAL1t); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triose phosphate isomerase 1 terminator (TPI1t); TEF1; iso-1-cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGAL10 terminator.

[0011] This disclosure also provides a method for producing D-allulose, the method comprising contacting a substrate with engineered cells as described herein, wherein the engineered cells produce at least 1.0 g / L, at least 5.0 g / L, at least 10.0 g / L, or at least 15 g / L after 72 hours. The substrate may include starch, glucose, cellulose biomass, or combinations thereof.

[0012] This disclosure also provides the use of engineered cells, as described herein, for the production of D-allulose. Attached Figure Description

[0013] This patent or application contains at least one color-drawn drawing. A copy of this patent or patent application publication with a color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0014] The accompanying figures illustrate, in a manner that is not restrictive, various aspects described herein.

[0015] Figure 1 A recommended pathway for producing D-allulose from sucrose, starch, and / or glucose is shown.

[0016] Figure 2 A graph showing the allulose titer at 96 hours as outlined in Example 8 is displayed. Detailed Implementation

[0017] Reference will now be made in detail to certain aspects of the subject matter disclosed herein, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0018] In this document, unless the context clearly requires otherwise, the terms "an," "a," or "the / described" are used to include one or more. Unless otherwise indicated, the term "or" is used to mean a non-exclusive "or." All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety as if individually cited. In the event of any inconsistency between the usage of this document and those documents so incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of irreconcilable inconsistencies, the usage in this document shall prevail.

[0019] Values ​​expressed in range format should be interpreted flexibly to include not only the values ​​explicitly listed as limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. 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 specified range. Unless otherwise stated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise stated, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0020] Unless otherwise specified, ppm (parts per million), percentages, and ratios are based on weight. Percentages based on weight are also referred to below as weight% or (weight)%.

[0021] This disclosure relates to various recombinant cells engineered to produce D-allulose. Generally, the recombinant cells described herein comprise allulose-6-phosphate 3-epimerases encoding at least one of SEQ ID NO: 192, 194, and 196 or allulose-6-phosphate 3-epimerases having a 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 sequence thereon. The recombinant cells may additionally include a heterologous nucleic acid encoding allulose-6-phosphate phosphatase, such as at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183, or an allulose-6-phosphate phosphatase having a 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 that sequence. This disclosure also provides a fermentation method for producing D-allulose using the genetically engineered cells described herein.

[0022] As used herein, “D-allulose” and “allulose” are used interchangeably and refer to the hexulose epimer of fructose as shown in Structure I below. D-allulose is also referred to in the art as “D-pseudofructose” or “pseudofructose”. D-allulose is present in trace amounts in wheat, figs, raisins, maple syrup, and molasses, leading to its designation as a rare sugar.

[0023] (I)

[0024] Generally, the recombinant cells described herein are yeast cells or bacterial cells. Suitable yeast cells or bacterial cells are known and described in the art. Those skilled in the art can identify yeast strains and bacterial strains suitable for producing the recombinant cells described herein.

[0025] The recombinant cells described herein can be bacterial cells. Non-limiting examples of suitable bacterial cells include *Escherichia coli*, *Corynebacterium glutamicum*, and *Bacillus* spp. Those skilled in the art will understand that the requirements for selecting suitable bacterial cells and the recombinant bacterial cells of this disclosure are not limited to those expressly listed herein.

[0026] Generally, the recombinant cells described herein are yeast cells. Non-limiting examples of yeast cells include those obtained from, for example, *Saccharomyces*, *Fissula*, *Pichia pastoris*, *Pichia phagnum*, *Kluyveromyces*, *Candida*, *Basilella*, *Saccharomyces*, *Candida ...

[0027] Recombinant yeast cells can be recombinant filamentous fungi cells, such as *Fragaria filamentous* yeast cells. *Fragaria* has previously been used in the fermentation production of erythritol, and methods for genetically modifying and fermenting *Fragaria* are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb Cell Fact, 16:168).

[0028] Various plasmids and methods for transforming *Branchiolus* are also described in the following examples. For example, *Branchiolus* can be transformed using a two-component polynucleotide sequence, wherein, after recombination, the target exogenous polynucleotide is integrated into a specific locus, and a selection marker is expressed intracellularly. Suitable selection markers are known and used in the art. Optional markers may include, but are not limited to, amdS (e.g., broken into 3' parts SEQ ID NO: 237 and 5' parts SEQ ID NO: 238), G418 resistance gene (e.g., broken into 3' parts SEQ ID NO: 239 and 5' parts SEQ ID NO: 240), bleomycin (zeocin) resistance gene (e.g., broken into 3' parts SEQ ID NO: 241 and 5' parts SEQ ID NO: 242), norsinosin N-acetyltransferase (NAT) (e.g., broken into 3' parts SEQ ID NO: 243 and 5' parts SEQ ID NO: 244), and invertase gene (SUC2) (e.g., the 3' part of SEQ ID NO: 245 and the 5' part of SEQ ID NO: 246).

[0029] Unless otherwise expressly stated, the terms “glucose” and “dextrose” are used interchangeably in this document and refer to D-glucose.

[0030] The recombinant cells described herein contain one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, enable the recombinant cells to produce D-allulose.

[0031] As used herein, "exogenous" refers to genetic material or its expression product originating outside the host organism. For example, exogenous genetic material or its expression product can be a modified form of the host organism's natural genetic material, it can originate from another organism, it can be a modified form of a component derived from another organism, or it can be a component of synthetic origin. For example, when introduced into *Saccharomyces cerevisiae*, the lactate dehydrogenase gene from *Lactobacillus helveticus* is exogenous.

[0032] As used in this article, “natural” refers to genetic material or its expression products found in the genome of wild-type cells of a host cell, except for inter-individual mutations that do not affect function or expression.

[0033] For the purposes of this application, the *Kluyveromyces maculae* CD21 cells deposited on October 18, 2024, under accession number 58456 at BCCM / MUCL (Belgian Coordinated Collections of Microorganisms / Mycothèque de l'Université Catholique de Louvain; address: Croix du Sud, box L7.05.06, B-1348 Louvain-la-Neuve, Belgium) are considered wild-type *Kluyveromyces maculae* cells.

[0034] For the purposes of this application, the Moniliella tomentosa var pollinis TCV364 cells described in US 6,440,712, which is incorporated herein by reference in its entirety, are considered wild-type Moniliella tomentosa cells and were deposited on March 28, 1997, under the number MUCL40385, at BCCM / MUCL (Belgian Coordinated Collections of Microorganisms / Mycothèque de l'Université Catholique de Louvain; deposited by Erdania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800Vilvoorde).

[0035] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the total primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the macromolecule described its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (independently or as part of a biosynthetic pathway) should be understood to include the cofactors, coenzymes, and metals necessary for the enzyme to function properly. As understood in the art, an overview of 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.

[0036] Table 1: Three-letter and single-letter symbols for amino acids

[0037]

[0038] Variants or sequences having substantially the same identity or homology as the polypeptides described herein can be used to carry out the disclosed compositions and methods. Such sequences may be referred to as variants or modified sequences. That is, the polypeptide sequence may be modified but still retain the ability to exhibit the desired activity. Typically, variants or modified sequences may include sequence identity of about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% with wild-type, naturally occurring polypeptide sequences or with variant polypeptides as described herein.

[0039] As used herein, the phrases “sequence identity %”, “identity %”, and “identity percentage” 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 normalized algorithm. Methods for amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and sequence identity generation involve both global and local alignments using computational methods. Alignment can be performed using BLAST (National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using the standard protein BLAST with the following default parameters: Maximum target sequence: 100; Short sequence query: Parameters automatically adjusted for short input sequences; Expected threshold: 10; Word length: 6; Maximum number of matches within the query range: 0; Matrix: BLOSUM62; Void penalty: (Presence: 11, Extension: 1); Composition adjustment: Conditional compositional score matrix adjustment; Filtering: Not selected; Masking: Not selected. The standard nucleotide BLAST algorithm with the following default parameters can be used to determine the percentage of nucleic acid sequence identity between nucleic acid sequences: Maximum target sequence: 100; Short sequence query: Automatically adjusts parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within the query range: 0; Match / mismatch score: 1, -2; Gap penalty: Linear; Filtering: Low complexity region; Masking: Mask only the lookup table. Sequences that have an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently have XX% sequence identity with the reference sequence.

[0040] Peptide or polynucleotide sequence identity can be measured over the entire length of a defined peptide sequence (e.g., as defined by a specific SEQ ID number) or over a shorter length, such as over a fragment taken from a larger defined peptide sequence (e.g., a fragment of at least 15, 20, 30, 40, 50, 70, or 150 consecutive residues). Such lengths are merely exemplary and it should be understood that any fragment length supported by the sequences shown in this document, tables, figures, or sequence listings can be used to describe the length over which a measurable percentage of identity can be measured.

[0041] The polypeptides disclosed herein may include “variant” polypeptides, “mutants” and “derivatives thereof.” As used herein, the term “wild-type” is a term understood by those skilled in the art and refers to the typical form of the polypeptide in nature, as distinct from the variant or mutant form. As used herein, “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence different from that of a reference protein or polypeptide molecule. Variants or mutants may have one or more amino acid residues inserted, deleted, or substituted relative to the reference molecule.

[0042] The amino acid sequences of peptide variants, mutants, derivatives, or fragments covered herein may include conserved amino acid substitutions relative to a reference amino acid sequence. For example, variant, mutant, derivative, or fragment peptides may include conserved amino acid substitutions relative to a reference molecule. A “conserved amino acid substitution” is one in which an amino acid is substituted for a different amino acid, wherein the substitution is predicted to have minimal interference with the properties of the reference peptide. In other words, a conserved amino acid substitution substantially preserves the structure and function of the reference peptide. Conserved amino acid substitutions typically maintain (a) the structure of the peptide backbone in the substitution region, such as a β-sheet or α-helical conformation, (b) the charge and / or hydrophobicity of the molecule at the substitution site, and / or (c) the bulk portion of the side chains.

[0043] As used herein, the terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence” are used interchangeably with “nucleic acid” and refer to a nucleotide sequence 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 indicate a sense strand or antisense strand. DNA polynucleotides can be cDNA or genomic DNA sequences.

[0044] A polynucleotide is said to encode a polypeptide if, in its natural state or when manipulated by methods known to those skilled 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 considered to encode the sequence.

[0045] Those skilled in the art understand the degeneracy of the genetic code and that multiple polynucleotides can encode the same polypeptide. In some respects, polynucleotides (i.e., polynucleotides encoding non-heme iron-binding protein polypeptides) can be codon-optimized for expression in specific cells, including but not limited to plant cells, bacterial cells, fungal cells, or animal cells. Although polypeptides encoded by polynucleotide sequences found in corals are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptide described herein can be used. Therefore, sequences that are not naturally occurring can be used. These sequences may be desired, for example, to enhance the expression of polypeptides or proteins in heterologous expression systems. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and artificial manipulation can also be used to generate degenerate coding sequences.

[0046] The recombinant cells described in this article have deletions or disruptions in one or more natural genes encoding enzymes involved in erythritol fermentation or consumption. Deletion or disruption of one or more enzymes in these biosynthetic pathways reduces the recombinant cells' ability to produce erythritol.

[0047] The recombinant cells described herein may include the deletion or disruption of the native erythrose reductase (ER) gene. The native ER gene encodes an enzyme with erythrose reductase activity. As used herein, “erythrose reductase activity” and “ER activity” are used interchangeably and refer to an enzyme that catalyzes the reversible conversion of erythrose or erythrose-4-phosphate to erythritol or erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor. In the art, an enzyme that catalyzes the reversible conversion of erythrose-4-phosphate to erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor may also be described as an erythrose or erythritol phosphate dehydrogenase. When the host cell contains multiple ER genes, it is preferable to delete or disrupt at least one of them. When the host cell contains multiple alleles of a given ER gene, it is preferable to delete or disrupt one or two alleles of the given ER gene.

[0048] As used herein, “NAD(P)H” refers to nicotinamide adenine dinucleotide (phosphate) hydrogen and includes both NADH and NADPH. As understood in the art, the inclusion of phosphate (or the abbreviation “P”) in parentheses indicates that phosphate may be absent or present, and the name and abbreviation include both. Similarly, “NAD(H)” or “NADP(H)” refers to both the reduced and oxidized forms of the cofactor.

[0049] When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO:286. When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO:287. See, for example, U.S. Provisional Application No. 63 / 499,990, filed May 4, 2023, and U.S. Provisional Application No. 63 / 499,989, filed May 4, 2023.

[0050] When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO:288. When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO:289. See, for example, U.S. Provisional Application No. 63 / 499,990, filed May 4, 2023, and U.S. Provisional Application No. 63 / 499,989, filed May 4, 2023.

[0051] The recombinant cells described herein may contain one or more genetic modifications in which exogenous nucleic acids are integrated into the host cell genome. Those skilled in the art know how to select suitable loci in the yeast genome for the integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in *Saccharomyces cerevisiae* host cells, suitable integration loci may include, but are not limited to: the ER1 locus (defined as a locus flanked by SEQ ID NO:247 and SEQ ID NO:248), the ER3 locus (defined as a locus flanked by SEQ ID NO:249 and SEQ ID NO:250), the PDC1 locus (defined as a locus flanked by SEQ ID NO:251 and SEQ ID NO:252), the pyrF locus (defined as a locus flanked by SEQ ID NO:253 and SEQ ID NO:254), the TRP3 locus (defined as a locus flanked by SEQ ID NO:255 and SEQ ID NO:256), the gpdIIA locus (defined as a locus flanked by SEQ ID NO:257 and SEQ ID NO:258); the gpdIIB locus (defined as a locus flanked by SEQ ID NO:259 and SEQ ID NO:260); and the RSCR26640 locus (defined as a locus flanked by SEQ ID NO:261 and SEQ ID NO:260). (Section 262). In Kluyveromyces martensii host cells, suitable integration loci may include, but are not limited to, the CYB2 locus (defined as the locus flanking SEQ ID NO:212 and SEQ ID NO:213). Exogenous nucleic acids may also be integrated into intergenic regions or other locations in the host cell genome not specifically specified herein. Other suitable integration loci can be identified by those skilled in the art. Furthermore, those skilled in the art will recognize how sequences can be used to design primers to verify that correct gene integration occurs at the selected locus.

[0052] Recombinant cells may have one or more copies of a given exogenous nucleic acid sequence, which are integrated into the host chromosome and replicate together with the integrated chromosome. For example, yeast cells may be transformed with a nucleic acid construct containing a polynucleotide sequence encoding a polypeptide described herein, and one or more copies of the polynucleotide sequence encoding the polypeptide may be integrated into the host chromosome. Recombinant cells may contain multiple (two or more) copies of a given polynucleotide sequence encoding a polypeptide described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten or more copies of the polynucleotide sequence encoding the polypeptide described herein, integrated into the genome. The multiple copies of the polynucleotide sequence may be incorporated entirely into a single locus or may be incorporated into multiple loci.

[0053] The recombinant cells described herein are capable of producing D-allulose and contain an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase. The epimerase can be any suitable enzyme with allulose-6-phosphate 3-epimerase activity. The exogenous polynucleotide sequence can be an exogenous allulose-6-phosphate 3-epimerase (epimerase) gene.

[0054] As used herein, "allulose-6-phosphate 3-epimerase gene" refers 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 via epimerization at the C3 position of fructose-6-phosphate. Enzymes with allulose-6-phosphate 3-epimerase activity can be cobalt (Co)-containing enzymes. 2+ ), manganese (Mn) 2+ ), calcium (Ca 2+ ), magnesium (Mg) 2+ ) and / or zinc (Zn 2+The activity of an enzyme increases in the presence of allulose-6-phosphate 3-epimerase. Allulose-6-phosphate 3-epimerase can be derived from any suitable biological source or can be synthetic. Suitable allulose-6-phosphate 3-epimerases may include, but are not limited to, enzymes classified under Enzyme Committee (EC) number 5.1.3. Suitable allulose-6-phosphate 3-epimerases may be allulose-6-phosphate 3-epimerases derived from *Escherichia coli*, *Pseudoleptotrichia goodfellowii*, *Blautia glucerasea*, etc. The allulose-6-phosphate 3-epimerase gene may encode 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% of the amino acids identical to at least one of SEQ ID NO: 192, 194, and 196.

[0055] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from an E. coli gene encoding the amino acid sequence of SEQ ID NO:192. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:192.

[0056] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Pseudococcus guillier* gene encoding the amino acid sequence of SEQ ID NO:194. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:194.

[0057] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Glucosidobacterium bratianae* gene encoding the amino acid sequence of SEQ ID NO:196. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:196.

[0058] The recombinant cell described herein is capable of producing D-allulose and contains a foreign polynucleotide sequence encoding allulose-6-phosphate 3-epimerase, and may additionally contain a foreign polynucleotide sequence encoding allulose-6-phosphate phosphatase. Allulose-6-phosphate phosphatase can be any suitable enzyme with allulose-6-phosphate phosphatase activity. The foreign polynucleotide sequence can be a foreign allulose-6-phosphate phosphatase gene.

[0059] As used herein, “alulose-6-phosphate phosphatase” refers to any gene or polynucleotide encoding a polypeptide having allulose-6-phosphate phosphatase activity. As used herein, “alulose-6-phosphate phosphatase activity” refers to the ability to catalyze the hydrolysis of D-alulose-6-phosphate to D-alulose and phosphate ions. Suitable allulose-6-phosphate phosphatases active against hexose-6-phosphate substrates are known and described in the art. Allulose-6-phosphate phosphatases may be derived from any suitable source or may be synthetic. Suitable allulose-6-phosphate phosphatases may include, but are not limited to, those from the following: *Escherichia coli*, *Shewanella algae*, *Tatumella morbirosei*, *Shewanella loihica*, *Ferrimonas sediminicola*, *Shewanella fodinae*, *Parashewanella spongiae*, *Wickerhamomyces ciferrii*, *Trabulsiella odontotermitis*, *Shewanella aestuarii*, *Shewanella pealeana*, *Shewanella sp.*, *Shewanella denitrificans*, and *Shewanella wulliganensis*. *Ferrimonas lipolytica*, *Shewanella violacea*, *Shewanella mangrove*, *Parashewanella curva*, *Shewanella frigidimarina*, *Ferrimonas aestuarii*, and *Shewanella* species OPT22. The allulose-6-phosphate phosphatase gene may encode 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% of the amino acids that are identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183.The allulose-6-phosphate phosphatase gene may encode 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% of the amino acids identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, and 115. The allulose-6-phosphate phosphatase gene may encode 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% of the amino acids identical to at least one of SEQ ID NO: 87, 89, and 190. The allulose-6-phosphate phosphatase gene may encode 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% of the amino acids identical to at least one of SEQ ID NO: 87 and 89.

[0060] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella algae genes encoding the amino acid sequence of SEQ ID NO:87. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:87.

[0061] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Mobirosseta* gene encoding the amino acid sequence of SEQ ID NO:89. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:89.

[0062] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from E. coli genes encoding the amino acid sequence of SEQ ID NO:190. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:190.

[0063] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella photovoltaicis genes encoding the amino acid sequence of SEQ ID NO:123. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:123.

[0064] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Isodon ferruginosa* gene encoding the amino acid sequence of SEQ ID NO:105. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:105.

[0065] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella vulgaris genes encoding the amino acid sequence of SEQ ID NO:107. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:107.

[0066] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from a *Pseudomonas sponge* gene encoding the amino acid sequence of SEQ ID NO:115. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:115.

[0067] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the Wickham Severus gene encoding the amino acid sequence of SEQ ID NO:83. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:83.

[0068] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from a *Terrabus* gene encoding the amino acid sequence of SEQ ID NO:95. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:95.

[0069] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella estuarineis genes encoding the amino acid sequence of SEQ ID NO:113. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:113.

[0070] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Shewanella perensis* gene encoding the amino acid sequence of SEQ ID NO:117. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:117.

[0071] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella species genes encoding the amino acid sequence of SEQ ID NO:119. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:119.

[0072] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from a denitrifying Shewanella gene encoding the amino acid sequence of SEQ ID NO:121. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:121.

[0073] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella velutipes genes encoding the amino acid sequence of SEQ ID NO:127. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:127.

[0074] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Ferrodiola cricolyticus* gene encoding the amino acid sequence of SEQ ID NO:131. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:131.

[0075] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from a *Shewanella violaceum* gene encoding the amino acid sequence of SEQ ID NO:137. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:137.

[0076] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the Shewanella mangrove gene encoding the amino acid sequence of SEQ ID NO:145. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:145.

[0077] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Temminckia sputum* gene encoding the amino acid sequence of SEQ ID NO:149. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:149.

[0078] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Pseudomonas lanceolata* gene encoding the amino acid sequence of SEQ ID NO:169. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:169.

[0079] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from Shewanella glabrata genes encoding the amino acid sequence of SEQ ID NO:173. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:173.

[0080] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the *Ferructus estuarineis* gene encoding the amino acid sequence of SEQ ID NO:179. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:179.

[0081] The recombinant cells may contain exogenous polynucleotides, which are or may be derived from the Shewanella OPT22 gene encoding the amino acid sequence of SEQ ID NO:183. The exogenous polynucleotides 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%, or at least 99% identical to SEQ ID NO:183.

[0082] The exogenous nucleic acids in the recombinant cells described herein can be controlled by promoters. For example, the exogenous nucleic acids can be operatively linked to heterologous promoters or artificial promoters. Suitable promoters are known and described in the art. Promoters may include, but are not limited to: pyruvate decarboxylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:263); 6-phosphogluconic acid dehydrogenase promoter (6PGDp; SEQ ID NO:264); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:265); translation elongation factor 1 promoter (TEFp; SEQ ID NO:266); phosphoglucose mutase 1 promoter (PGM1p; SEQ ID NO:267); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:268); enolase promoter (ENO1p; SEQ ID NO:269); asparagine synthase promoter (ASNSp; SEQ ID NO:269). SEQ ID NO: 270); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 271); RPL16B (SEQ ID NO: 272); ScPDC1p (SEQ ID NO: 220); KmTDH3p (SEQ ID NO: 222).

[0083] The exogenous nucleic acids in the recombinant cells described herein can be controlled by terminators. For example, the exogenous nucleic acids can be operatively linked to heterologous or artificial terminators. Suitable terminators are known and described in the art. Terminators may include, but are not limited to: GAL10 terminator, PDC terminator, transaldolase terminator (TAL), 6PGD terminator (6PGDt; SEQ ID NO:273); ASNS terminator (ASNSt; SEQ ID NO:274); ENO1 terminator (ENO1t; SEQ ID NO:275); hexokinase 1 terminator (HXK1t; SEQ ID NO:276); PGK1 terminator (PGK1t; SEQ ID NO:277); PGM1 terminator (PGM1t; SEQ ID NO:278); PYK1 terminator (PYK1t; SEQ ID NO:279); RPLA terminator (RPLAt; SEQ ID NO:280); transaldolase 1 terminator (TAL1t; SEQ ID NO:281); TDH3 terminator (TDH3t; SEQ ID NO:282); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:282). NO:283); triose phosphate isomerase 1 terminator (TPI1t; SEQ ID NO:284); MpTEF1 (SEQ ID NO:285); iso-1-cytochrome c (CYC1; SEQ ID NO:218); KmHXK2t (SEQ ID NO:27); KmGPM1t (SEQ ID NO:221); KmPGK1t (SEQ ID NO:223); ScURA3t (SEQ ID NO:217); URA3; ADH1; and ScGAL10.

[0084] If the position of a promoter or terminator relative to the polynucleotide in the genome or expression cassette allows the promoter or terminator (as the case may be) to perform its transcriptional control function, then the promoter or terminator is “operably linked” to the given polynucleotide (e.g., a gene).

[0085] 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 a sense strand or an antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods and may comprise polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Therefore, constructs may include novel modifications to endogenous genes introduced, for example, through genome editing technologies. Constructs may also include recombinant polynucleotides produced using, for example, recombinant DNA methodologies. Constructs may be vectors comprising a promoter operatively linked to a polynucleotide encoding an allulose-6-phosphate-3-epimerase. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide linked to it. Vectors may be plasmids, which are circular double-stranded DNA loops into which additional DNA fragments may be integrated.

[0086] This disclosure also provides a fermentation method for producing D-allulose using the recombinant cells described herein. The fermentation method includes the step of fermenting a substrate using genetically engineered yeast cells or genetically engineered bacterial cells described herein to produce D-allulose. As will be understood by those skilled in the art, the fermentation method may include additional steps. 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 separating ethanol from the fermentation broth.

[0087] Fermentation substrates may include starch. Starch can be obtained from natural sources, such as plants. Starch can also be obtained from feedstocks with high starch or sugar content, including but not limited to corn, sweet sorghum, fruit, sweet potato, rice, barley, sugarcane, sugar beet, wheat, cassava, potato, tapioca, calathea, peas, or sago. Fermentation substrates may be derived from lignocellulosic biomass (such as wood, straw, grass) or algal biomass (such as microalgae and macroalgae). Fermentation substrates may include cellulose or lignocellulosic biomass. Fermentation substrates may be derived from grass, trees, or agricultural and forestry residues, such as corn cobs and corn stalks, straw, sawdust, and wood chips. Fermentation substrates may also include sugars, such as glucose (dextrose) or sucrose, and / or polysaccharides, such as maltodextrin. Fermentation substrates may be pretreated physically (e.g., by heat, pressure, etc.) or chemically (e.g., by acid, hydrolysis, enzymatic treatment, such as glucosylamylase, etc.) before or during the fermentation process.

[0088] The culture medium used for fermenting the engineered cells described herein can be supplemented with various components. For example, the culture medium used for fermenting the engineered cells described herein can be supplemented with glucosylamylase, such as Spirizyme. ™(Novozymes, Bagsvaerd, Denmark) and / or from Aspergillus niger, produced by Sigma-Aldrich under the trade name AMG 300L ™ Amylase for sale.

[0089] The fermentation process can be operated under a variety of conditions. The fermentation temperature (i.e., the temperature of the fermentation broth during processing) can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained within the range of 25°C to 50°C, 26°C to 45°C, 27°C to 40°C, 28°C to 35°C, or 29°C to 32°C. The fermentation temperature can be maintained at, for example, any value or range of 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or between these values. However, those skilled in the art will recognize that the fermentation temperature is not limited to any specific range described herein and can be modified as appropriate.

[0090] The pH of the culture medium described herein can be controlled for optimal D-allulose production. The pH of the engineered cell culture or fermentation mixture described herein can be in the range of 3.0 to 7.5. The pH can be maintained at 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.8, 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 for at least a portion of the incubation time. The pH can be maintained in the range between 6.0 and 7.0, 6.2 and 6.7, or 6.3 and 6.6.

[0091] Engineered cells (yeast and / or bacteria) can be cultured for approximately 24 hours to 96+ hours. For example, engineered cells can be cultured for approximately 12 hours, 18 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 78 hours, 80 hours, 90 hours, 96 hours, or more than 96 hours. The engineered cells described in this article can be cultured for approximately 48 to 72 hours. A culture (fermentation) time of approximately 48 or 72 hours can be representative of a similar commercial-scale fermentation process. Therefore, the 48-hour or 72-hour time point can be used to compare the fermentation performance of different genetically engineered cell strains.

[0092] Reaction parameters can be measured or adjusted during D-allulose production. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state), chemical parameters (e.g., pH, redox potential, concentration of reaction substrates and / or products, concentration of dissolved gases such as oxygen and CO2, 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, molar concentration, molar osmotic pressure, and other chemicals such as buffers, adjuvants, or reaction byproducts), and 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 / mass, etc.). Sensors for measuring the parameters described herein are well known to those skilled in the art.

[0093] Fermentation processes can be associated with a variety of characteristics, such as, 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) and / or genetic modifications of the cells used in the fermentation process. These characteristics can also be influenced by regulating fermentation process conditions. These characteristics can be modulated through a combination of cell selection or modification and the selection of fermentation process conditions.

[0094] The final D-allulose titer can be at least 0.5 g / L, at least 1.0 g / L, at least 2.0 g / L, at least 5 g / L, at least 7.5 g / L, at least 10 g / L, at least 15 g / L, or at least 17.5 g / L.

[0095] Example

[0096] The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be construed as encompassing any and all variations that become apparent from the teachings provided herein.

[0097] The strain numbering is consistent throughout the examples. For example, strains 1-5 in Example 1 are the same strains as strains 1-5 in Examples 2 and 3.

[0098] Example 1 - Genetically Modified Saccharomyces cerevisiae strain

[0099] Figure 1 The proposed pathway for the production of D-allulose from sucrose, starch, and / or glucose is shown. 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. 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 (Saccharomyces cerevisiae), the strains described in this and subsequent examples were constructed and tested.

[0100] strain 1-1

[0101] Strain 1-1 is the yeast strain Saccharomyces cerevisiae CEN.PK 113-7D (MATa HIS3 LEU2 TRP1 MAL2-8SUC2; Classification ID: NCBI:txid889517).

[0102] Strain 1-2

[0103] Strain 1-2 is a uracil auxotrophic strain derivative of strain 1-1, with the Aspergillus nidulans amdS gene inserted at the URA3 locus.

[0104] Strains 1-3

[0105] Strains 1-2 were transformed using the Li-acetate protocol (Gietz, RD 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 the sequence encoding a constitutively expressed synthetic transcription factor (sTF; nucleotide sequence SEQ ID NO: 185; polypeptide SEQ ID NO: 186) and a URA+ marker to produce strains 1-3. The DNA fragment was generated by digesting plasmid B6622 (pKlURA3_TDH3cp-BM3R1-VP16; Rantasalo A. et al., “Synthetic toolkit for complex genetic circuit engineering in Saccharomyces cerevisiae,” ACS Synth. Biol., 2018, 7, 6, 1573-1587) using the restriction endonuclease Notl. Transformants were selected on SDA medium without uracil, and the insertion of sTF was verified by colony PCR to generate strains 1-3.

[0106] Strains 1-4

[0107] To generate G418-resistant uracil auxotrophic strains 1-4, the uracil markers in strains 1-3 were replaced with markers that confer resistance to the antibiotic G418, which binds to the LoxP site.

[0108] Strains 1-5

[0109] To generate strains 1-5, strains 1-4 were transformed with a plasmid expressing Cre recombinase, thus removing the G418 resistance cassette from strains 1-4. The resulting strains 1-5 are ura3. - And includes the synthetic transcription factor SEQ ID NO:186 controlled by the TDH3cp promoter.

[0110] strains 1-6 to 1-100

[0111] The polynucleotides encoding D-allulose-6-phosphate 3-epimerase and D-allulose-6-phosphate phosphatase were cloned into the pSCT036 vector (SEQ ID NO: 187). The sequence encoding allulose-6-phosphate 3-epimerase is controlled by the TDH3 promoter (SEQ ID NO: 188), and the sequence encoding allulose-6-phosphate phosphatase is controlled by the promoter of SEQ ID NO: 189, which includes eight synthetic transcription factor (sTF) binding sites for sTF expression in strains 1-5. The plasmids encoding allulose-6-phosphate 3-epimerase and allulose-6-phosphate phosphatase were cloned into parental strains 1-5, as outlined in Table 2.

[0112] Table 2: Genetically modified Saccharomyces cerevisiae strains

[0113]

[0114]

[0115]

[0116] Example 3 - Shake Flask Fermentation Determination

[0117] Strains 1-5, 1-7 to 1-31, 1-33 to 1-57, 1-99 and 1-100 were run in duplicate in shake flasks to determine D-allulose production.

[0118] The strain was bumped onto synthetic complete medium minus uracil (ScD-ura) plates (6.7 g / L yeast nitrogen without amino acids, 1.9 g / L synthetic complete amino acid mixture, 20 g / L glucose, and 20 g / L agar) and allowed to grow until single colonies formed (2 to 3 days at 30°C or 3 to 5 days at room temperature (approximately 25°C)). Cells from the ScD-ura plates were scraped into sterile seed containers (250 ml Erlenmeyer flasks with baffles) containing 25 ml of ScD-ura and 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted in 40 ml of DM1u production medium (Table 4) in a 250 ml baffled Erlenmeyer flask to achieve an initial cell density of 0.2 at 600 nm (OD600) and incubated at 30 °C, 250 rpm, and 70% humidity. Optical density was measured at 600 nm and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). At 72 hours, 1.0 ml of the sample was transferred to a microcentrifuge tube, centrifuged at 14,000 x g for 2 min, and stored at -20 °C until analysis. D-allulose in the sample was analyzed by HPLC. The results are shown in Table 7.

[0119] Table 3: ScD-ura with 20 g / L dextrose

[0120]

[0121] Table 4: DM1u Production Medium - Maltodextrin

[0122]

[0123] GA added before vaccination

[0124] Table 5: Trace Elements at 1000x

[0125]

[0126] Table 6: 1000X DM1 Vitamin Solution

[0127]

[0128]

[0129] Data showed that expression of allulose-6-phosphate phosphatases in SEQ ID NO: 83 and 95 produced almost the same amount of D-allulose as the positive control allulose-6-phosphate phosphatases in SEQ ID NO: 190. However, expression of allulose-6-phosphate phosphatases in SEQ ID NO: 87 and 89 resulted in an increase in D-allulose titers relative to the positive control. Overall, expression of allulose-6-phosphate phosphatases in SEQ ID NO: 15, 19, 23, 25, 35, 37, 39, 43, 45, 49, 59, 63, 77, 83, 85, 87, 89, 91, 93, 95, and 101 had higher D-allulose titers than wild-type strains (1-5) or negative control strains expressing allulose-6-phosphate 3-epimerase in SEQ ID NO: 192 but lacking allulose-6-phosphate phosphatases.

[0130] Example 3 - Fermentation determination of flower-shaped plates

[0131] D-allulose production was evaluated by testing strains 1-5, 1-6, 1-32, 1-49, 1-50, 1-58 to 1-98, 1-99 and 1-100 in duplicate.

[0132] The strain was bumped onto ScD-ura plates and grown until single colonies formed (2 to 3 days at 30°C or 3 to 5 days at room temperature (approximately 25°C)). A small piece of biomass was used to inoculate a deep 96-well microtiter plate containing 500 μL of ScD-Ura medium (6.7 g / L amino acid-free yeast nitrogen, 2.0 g / L synthetic complete amino acid mixture, 20 g / L glucose) and incubated for 24 hours at 30°C and 800 RPM in a fixed-track shaker. The overnight culture was transferred to a 48-well flower-shaped plate containing 1000 μL of DMU1 medium (100 g / L maltodextrin and 50 μL GA) to OD600 = 0.2 and incubated for 96 hours at 30°C and 800 RPM in a fixed-track shaker. Samples were taken for HPLC analysis to determine the D-allulose titer. The results are shown in Table 8.

[0133]

[0134] The results showed that expression of allulose-6-phosphate phosphatases in SEQ ID NO:87 and 145 produced almost the same amount of D-allulose as the positive control allulose-6-phosphate phosphatases in SEQ ID NO:190. However, expression of allulose-6-phosphate phosphatases in SEQ ID NO:89, 87, 183, 179, 173, 169, 149, 89, 137, 131, 127, 123, 121, 119, 117, 115, 113, 107, and 105 caused an increase in the D-allulose titer relative to the positive control. Overall, strains expressing allulose-6-phosphate phosphatases of SEQ ID NO:89, 87, 183, 179, 173, 169, 149, 89, 137, 131, 127, 123, 121, 119, 117, 115, 113, 107, 105, 87, 145, 53, 175, 171, 167, 165, 163, 157, 153, 147, 141, 111, and 109 had higher D-allulose titers than negative control strains expressing allulose-6-phosphate 3-epimerase of SEQ ID NO:192 but lacking allulose-6-phosphate phosphatases.

[0135] Example 4 - Genetically Modified Kluyveromyces Strain

[0136] Figure 1 The proposed pathway for the production of D-allulose from sucrose, starch, and / or glucose is shown. 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. 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 (Kluyveromyces martensii), the strains described in this and subsequent examples were constructed and tested.

[0137] strain 2-1

[0138] Strain 2-1 is a yeast cell, *Kluyveromyces maculae* CD21, deposited at the Belgian Coordinated Collection of Microorganisms / Kouven Catholic University Fungal Collection (BCCMMUCL) 58456.

[0139] strain 2-2

[0140] Strain 2-2 is a uracil auxotrophic derivative of strain 2-1, with the URA3 locus missing.

[0141] strains 2-3 to 2-21

[0142] According to Table 10, strain 2-2 was transformed using the specified transformation fragments (Table 9). The resulting transformants were streaked on ScD-ura plates to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the specified sequence. The PCR-validated isolates were named as outlined in Table 10.

[0143] Table 9: Transformed Fragments

[0144]

[0145]

[0146] Table 10 :

[0147]

[0148] strain 2-22

[0149] Strain 2-2 was transformed using SEQ ID NO: 198 and 199 (Table 9). The resulting transformants were streaked on ScD-ura plates to isolate single colonies, and these colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the GFP-encoding sequence. The PCR-validated isolates were named strain 2-22.

[0150] Example 5 - Shake Flask Fermentation Determination

[0151] Strains 2-3 to 2-22 were tested to evaluate D-allulose production.

[0152] The strain was bumped 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 and 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted in 40 ml of ScD-Ura production medium (Table 3) 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. At 72 hours, 0.5 ml of the sample was transferred to a microcentrifuge tube, centrifuged at 14,000 x g for 2 minutes, and stored at -20°C until analysis. D-allulose in the sample was analyzed by HPLC. The results are shown in Table 11.

[0153] Table 11: Results of 72-hour shaking flask operation

[0154]

[0155] nd not detected, below the instrument's detection limit

[0156] The results showed that multiple promoters and terminators were suitable for the expression of epimerase and allulose-6-phosphate phosphatase in *Kluyveromyces martensii* to produce D-allulose.

[0157] Example 6: Shaking Flask Test

[0158] Strains 2-3 to 2-22 were tested to evaluate D-allulose production.

[0159] The strain was bumped 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 and 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted in 40 ml of DM1u production medium (Table 4) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.2 and incubated at 30°C, 250 rpm, and 70% humidity. At 72 hours, 0.5 ml of the sample was transferred to a microcentrifuge tube, centrifuged at 14,000 x g for 2 minutes, and stored at -20°C until analysis. D-allulose in the sample was analyzed by HPLC. The results are shown in Table 12.

[0160] Table 12: Results of 72-hour shaking flask operation

[0161]

[0162] nd not detected, below the instrument's detection limit

[0163] The results showed that multiple promoters and terminators were suitable for the expression of epimerase and allulose-6-phosphate phosphatase in *Kluyveromyces martensii* to produce D-allulose.

[0164] Example 7 – Genetically Modified *Saccharomyces cerevisiae* Strain

[0165] strain 3-1

[0166] Strain 3-1 is the host strain of *Moniliella tomentosa* var. *polinis* TCV364 described in US 6,440,712, which is incorporated herein by reference in its entirety, and was deposited on March 28, 1997, under the accession number MUCL40385, at BCCM / MUCL (Belgian Coordinating Center for Microbial Collections / Fungal Collections of the Catholic University of Leuven; deposited by Erdania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde).

[0167] strains 3-2 to 3-14

[0168] According to Table 14, for strain 3-1, protoplast preparation of the parent strain was first performed by adding an enzyme mixture containing 0.6 M MgSO4, 7.5 g / L lysis enzyme, and 12.5 g / L Trichoderma harzianum lysin to the mycelial precipitate of the parent strain. The protoplast precipitate was then washed with 0.6 M MgSO4 and resuspended in STC medium (0.6 M sucrose, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). The protoplast mixture was then precipitated by adding approximately 200 μL of the protoplast mixture (10... 8 A two-component transformation system was prepared by adding 100 µg of single-stranded salmon sperm DNA and 1.5 µg to 5 µg of 5' and 3' DNA transformation fragments (total 3 µg–10 µg) to the mixture of salmon sperm DNA, transforming DNA, and protoplasts (cells / mL). The transformation fragments are described in Table 13. Then, 1 mL of STC medium containing 50% PEG was added to the mixture of salmon sperm DNA, transforming DNA, and protoplasts, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, resuscitation medium (0.4 M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L sugar, pH 4.5) was added to the mixture, and the mixture was incubated at 27 °C and 100 rpm for 16–24 hours. After incubation, the protoplasts were precipitated by centrifugation and resuspended in 1 mL of PBS. The resuspended protoplasts were plated on PDA + 250 mg / L genimycin (G418) selection plates and incubated at 35 °C for at least 2 days until transformants grew. The resulting transformants were streaked onto PDA+genimycin (G418) plates to isolate single colonies, which were then selected. The selected colonies were evaluated by colony PCR to confirm the integration of the relevant genes.

[0169] For example, strain 3-1 was transformed with SEQ ID NO:235 and SEQ ID NO:228 as outlined above. The resulting transformants were streaked on PDA+genycin (G418) plates to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the relevant genes. The PCR-validated isolates were named strains 3-2a and 3-2b.

[0170] Table 13

[0171]

[0172] Table 14

[0173]

[0174] Example 8: Shaking Flask Test

[0175] Strains 3-1 to 3-14 were run in shake flasks to assess glucose consumption and the production of erythritol, glycerol, ethanol, and allulose.

[0176] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. After incubation, cells were scraped from the YPD plates and transferred to 250 mL baffleless shake flasks containing 40 mL of rich medium (170 g / L glucose, 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) in a 1 cm path length cuvette. The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0177] 0.4 mL of seed culture was inoculated into a 250 mL unbaffled shaker flask containing 20 mL of production medium (Table 15) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation for 24, 48, 72, and 96 hours. Glucose, erythritol, glycerol, ethanol, and allulose in the samples were analyzed by high-performance liquid chromatography equipped with a differential refractive index detector. The results are shown in Table 16. The results are also... Figure 2 As shown in the image.

[0178] The results showed that strains expressing only allulose-6-phosphate 3-epimerase or only allulose-6-phosphate phosphatase (strains 3-2, 3-3, 3-4, 3-5, and 3-6) did not produce allulose titers significantly higher than the negative control wild-type strain (3-1). However, a sister strain, 3-6b, did show a 96-hour allulose titer of 3.04 g / L. As summarized in Table 14, strains expressing both allulose-6-phosphate 3-epimerase and allulose-6-phosphate phosphatase produced higher allulose titers, with some strains producing allulose above 10 g / L at the 96-hour time point. For example, strains 3-7b, 3-8c, 3-10e, 3-11a, 3-11b, 3-11c, 3-11d, 3-12c, 3-13b, 3-13c, and 3-14d all produced allulose greater than 10 g / L after 96 hours. Furthermore, strains 3-8b, 3-10b, 3-10c, 3-12a, 3-12b, 3-13a, 3-13c, 3-14a, and 3-14c produced allulose greater than 5 g / L after 96 hours.

[0179] Among the strains tested, some sister strain variability was observed. Although the strains were validated by PCR to contain the expected polynucleotide sequence, the sequence may not have been integrated at the correct locus, may have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sister strains to differ from other strains. The results here indicate that similar transformation events exist among these sister strains. However, the results still demonstrate that the expression of the encoded allulose-6-phosphate 3-epimerase and allulose-6-phosphate phosphatase increases allulose titers.

[0180] Table 15: Production Culture Media

[0181]

[0182] Table 16

[0183]

[0184]

[0185] Example 9: Genetically modified Kluyveromyces macrocarpa strain

[0186] strain 2-23

[0187] Strain 2-2 was transformed with SEQ ID NO:292. The resulting transformants were streaked onto ScD-ura plates to isolate single colonies, which were then selected. SEQ ID NO:292 contains the TDH3 promoter (SEQ ID NO:222), the gene encoding allulose-6-phosphate epimerase (SEQ ID NO:192), the Cyc terminator (SEQ ID NO:218), the PGK promoter (SEQ ID NO:223), the gene encoding allulose-6-phosphate phosphatase (SEQ ID NO:190), the PDC_Lue2 terminator (SEQ ID NO:225), and the ScURA3 selection marker cassette. Selected colonies were evaluated by colony PCR to confirm the integration of the specified sequences. The PCR-validated isolates were named strain 2-23.

[0188] Strain 2-24

[0189] Strain 2-2 was transformed using SEQ ID NO: 203 and SEQ ID NO: 290. SEQ ID NO: 290 contains the 3' portion of the ScURA3 selection marker (SEQ ID NO: 214), the ScURA3 terminator (SEQ ID NO: 217), the KmTDH3 promoter (SEQ ID NO: 222), the gene encoding allulose-6-phosphate phosphatase (SEQ ID NO: 105), the KmPGK1 terminator, and the 3' KmCYB2 flanking sequence (SEQ ID NO: 213). The resulting transformants were streaked on ScD-ura plates to isolate single colonies, and these colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the specified sequences. The PCR-validated isolates were named strain 2-24.

[0190] Strain 2-25

[0191] Strain 2-2 was transformed using SEQ ID NO: 203 and SEQ ID NO: 291. SEQ ID NO: 291 contains the 3' portion of the ScURA3 selection marker (SEQ ID NO: 214), the ScURA3 terminator (SEQ ID NO: 217), the KmTDH3 promoter (SEQ ID NO: 222), the gene encoding allulose-6-phosphate phosphatase (SEQ ID NO: 107), the KmPGK1 terminator, and the 3' KmCYB2 flanking sequence (SEQ ID NO: 213). The resulting transformants were streaked on ScD-ura plates to isolate single colonies, and these colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the specified sequences. The PCR-validated isolates were named strain 2-25.

[0192] Example 10: Measurement of Flower-Shaped Plates

[0193] The D-allulose production of strains 2-3, 2-14, 2-23, 2-24, and 2-25 was evaluated.

[0194] The strain was bumped onto ScD-ura plates and allowed to grow until single colonies formed (2 to 3 days at 30°C or 3 to 5 days at room temperature (approximately 25°C)). A small piece of biomass was used to inoculate a 250 mL baffled flask containing 40 mL of buffered ScD-Ura medium (6.7 g / L amino acid-free yeast nitrogen, 1.9 g / L synthetic complete amino acid mixture, 100 g / L glucose, 19.5 g / L MES buffer, pH adjusted to 6.0) and incubated for 24 hours at 30°C and 250 RPM in a shaker. 40 μL of the overnight culture was transferred to 48-well flower-shaped plates containing 1000 μL of (i) buffered ScD-Ura with glucose, (ii) ScD-Ura with maltodextrin, or (ii) DMu1 medium (Table 4) and incubated for 92 hours at 30°C and 800 RPM in a shaker. Samples were taken for HPLC analysis to determine the D-allulose titer. Due to the presence of small peak artifacts in the HPLC data, some samples were also tested by high-pressure ion chromatography. The results are shown in Table 17.

[0195] Table 17 .

[0196]

[0197] nt = Not detected

[0198] Example 11: Shake Flask Fermentation Determination

[0199] The D-allulose production of strains 2-23, 2-3, 2-24, and 2-25 was evaluated.

[0200] The strain was bumped 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 and 20 g / L glucose medium (Table 3) and incubated overnight at 30°C, 250 rpm, and 70% humidity. The overnight culture was diluted in 40 ml of fermentation medium (summarized in Table 18) in a 250 ml baffled Erlenmeyer flask to achieve an initial cell density of OD600 = 0.1 and incubated at 30°C and RPM as specified in Table 17, and 70% humidity. At 236 hours, 0.5 ml of the sample was transferred to a microcentrifuge tube, centrifuged at 14,000 x g for 2 minutes, and stored at -20°C until analysis. The D-allulose in the samples was analyzed by HPLC. The results are shown in Table 18.

[0201] Overall, the results indicate that strain 2-24 produced the highest allulose titer under the tested conditions. Compared to strains 2-3 or 2-23, which expressed both allulose-6-phosphate epimerase (SEQ ID NO:192) and allulose-6-phosphate phosphatase (SEQ ID NO:190), strains 2-14, 2-24, and 2-25, all expressing allulose-6-phosphate epimerase (SEQ ID NO:194) and allulose-6-phosphate phosphatases (SEQ ID NO:123, 105, and 107), all produced higher D-allulose titers. The data also showed that the tested strains produced D-allulose from a variety of feedstocks in different fermentation media. The results demonstrated that both RPM and baffle size effects were medium-dependent. For example, higher RPM and larger baffles are beneficial for production in Sc-Ura + glucose medium (i.e., higher D-allulose titers), neutral in Sc-Ura + maltodextrin medium, and detrimental in DMu1 medium. However, even with this variability, all tested conditions produced D-allulose, demonstrating that a variety of media, substrates, and conditions are suitable for D-allulose fermentation.

[0202] Table 18

[0203]

[0204] Due to the presence of small peak artifacts during HPLC analysis, the fermentation broth from strain 2-24 was further analyzed by high-pressure ion chromatography (HPIC) (Experiments 10.4, 10.9, 10.14, 10.19, 10.24, 10.29, 10.32, 10.34 and 10.36), and the results are reported in Table 18.

[0205] Table 19 .

[0206] PCT / RO / 134 form

Claims

1. A genetically engineered yeast cell and / or genetically engineered bacterial cell capable of producing D-allulose, said engineered cell comprising... The exogenous polynucleotide sequence of allulose-6-phosphate 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 at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183; and The exogenous polynucleotide sequence encoding 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: 192, 194, and 196.

2. The engineered cell according to claim 1, wherein the allulose-6-phosphate 3-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: 192, 194, and 196.

3. The engineered cell according to claim 1 or claim 2, wherein the allulose-6-phosphate 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 at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183; The allulose-6-phosphate phosphatase described therein 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: 87, 89, 190, 123, 105, 107, and 115; The allulose-6-phosphate phosphatase described therein 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: 87, 89, and 105; or The allulose-6-phosphate phosphatase described therein 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:87 and 105.

4. The engineered cell according to any of the preceding claims, wherein... The allulose-6-phosphate 3-epimerase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO: 192, 194, and 196; and / or The allulose-6-phosphate phosphatase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO:87, 89, 190, 123, 105, 107, and 115.

5. The engineered cell according to any of the preceding claims, The allulose-6-phosphate 3-epimerase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO: 192, 194, and 196; and / or The allulose-6-phosphate phosphatase is at least 80%, at least 85%, or at least 90% identical to at least one of SEQ ID NO:87 and 105.

6. The engineered cell according to any of the preceding claims, wherein (i) The engineered cells are yeast cells selected from the group consisting of: *Saccharomyces*, *Schizosaccharomyces*, *Pichia pastoris*, *Phaeodactyces*, *Kluyveromyces*, *Candida*, *Basilella*, *Saccharomyces*, *Bacillus*, *Tetranychus*, *Debarella*, *Bacillus*, *Bacillus*, *Yersinia*, *Saccharomyces cerevisiae*, *Issaccharomyces orientalis*, *Pichia galbana*, *Pichia pastoris* species YB-4149 (NRRL designation), *Candida albicans*, *Pichia desertica*, *Kluyveromyces martensii*, *Kluyveromyces lactis*, *Pichia membranaceus*, *Yersinia lipophila*, *Bacillus* or *Pichia fermentata*; and / or (ii) The engineered cells are bacterial cells selected from the group consisting of: Escherichia coli, Corynebacterium glutamicum and Bacillus species.

7. The engineered cell according to any of the preceding claims, wherein the genetically engineered cell is a Saccharomyces cerevisiae cell, a Kluyveromyces maculvidifolia cell, or a Sterculia foetida cell.

8. The engineered cell according to any of the preceding claims, wherein the allulose-6-phosphate 3-epimerase is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 192, 194, and 196; wherein the allulose-6-phosphate phosphatase is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183; and wherein the cell is capable of producing at least 5 g / L D-allulose.

9. The engineered cell according to any of the preceding claims, wherein the allulose-6-phosphate 3-epimerase is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 192, 194, and 196; wherein the allulose-6-phosphate phosphatase is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO: 87, 89, 190, 123, 105, 107, and 115; and wherein the cell is capable of producing at least 10 g / L D-allulose.

10. The engineered cell according to any of the preceding claims, wherein the engineered cell is a *Saccharomyces cerevisiae* cell; wherein the allulose-6-phosphate 3-epimerase is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:196; wherein the allulose-6-phosphate phosphatase is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NO:87 and 89; and wherein the cell is capable of producing at least 15 g / L D-allulose.

11. The engineered cell according to any of the preceding claims, wherein one or more of the exogenous polynucleotide sequences are operatively linked to a heterologous promoter and / or a heterologous terminator.

12. The engineered cell of claim 10, wherein the promoter is selected from the group consisting of: pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoter (ADH1p), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p); 6-phosphogluconic acid dehydrogenase promoter (6PGDp); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p); translation elongation factor 1 promoter (TEFp); phosphoglucose mutase 1 promoter (PGM1p); 3-phosphoglycerate kinase promoter (PGK1p); enolase promoter (ENO1p); asparagine synthase promoter (ASNSp); 50S ribosomal protein L1 promoter (RPLAp); RPL16B; and PDC1 promoter.

13. The engineered cell of claim 11, wherein the terminator is selected from the group consisting of: GAL10 terminator, PDC terminator, transaldolase terminator (TAL), 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENO1t); hexokinase 1 terminator (HXK1t); PGK1 terminator (PGK1t); PGM1 terminator (PGM1t); PYK1 terminator (PYK1t); RPLA terminator (RPLAt); transaldolase 1 terminator (TAL1t); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triose phosphate isomerase 1 terminator (TPI1t); TEF1; iso-1-cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGAL10 terminator.

14. A method for producing D-allulose, the method comprising: The substrate is contacted with engineered cells according to any of the preceding claims, wherein the engineered cells produce at least 1.0 g / L, at least 5.0 g / L, at least 10.0 g / L, or at least 15 g / L after 72 hours.

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

16. Use of engineered cells according to any one of claims 1 to 13 for the production of D-allulose.

Images