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

Abstract

Disclosed herein are genetically engineered Kluyver omyces marxianus cells capable of producing D-allulose with reduced talitol by-product formation. The engineered cell may comprise a deletion or disruption of a native talitol dehydrogenase gene; an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme 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:249-256, 258, and 259; and an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme 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:87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183.

Description

PT-2209-WO-PCTGENETICALLY MODIFIED MICROORGANISM AND FERMENTATION PROCESS FOR THE PRODUCTION OF D-ALLULOSECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 730.531, filed December 11 , 2024, 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-2209- WO-PCT.xml” which is 715,576 bytes in size created on December 5, 2025 and electronically submitted via 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, brow ning 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 com 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.PT-2209-WO-PCTSUMMARY

[0005] The present disclosure provides a genetically engineered Kluyveromyces marxianus cell capable of producing D-allulose, the engineered cell comprising a deletion or disruption of a native talitol dehydrogenase gene; an exogenous polynucleotide sequence encoding an allulose-6- phosphate 3-epimerase enzyme 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:249-256, 258, and 259; and an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme 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:87. 89. 190, 123, 105. 107, 115, 83, 95, 1 13, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183. The native talitol dehydrogenase gene may encode a talitol dehydrogenase enzy me 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:304. The native talitol dehydrogenase gene may encode a talitol dehydrogenase enzyme 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:304. The native talitol dehydrogenase gene encodes a talitol dehydrogenase enzyme at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:304. The native talitol dehydrogenase gene may be 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:305. The native talitol dehydrogenase gene may be 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:305. The native talitol dehydrogenase gene is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:305.

[0006] The allulose-6-phosphate phosphatase may be 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:87, 89, 190, 123, 105, 107, 115, 83, 95, 1 13, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183. The allulose-6-phosphate phosphatase enzyme may be 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:87, 89, 190, 123. 105, 107, and 115. The allulose-6-phosphate phosphatase enzyme may be 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:87, 89, 190, and 105. The allulose-6-phosphate phosphatase enzyme may be 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:87. 89. and 105. The allulose-6-phosphate 3-epimerase may be at least 85%, or at least 90% identical to at least one of SEQ ID NOs: 249-256, 258, and 259. The allulose-6-phosphate epimerase may be at least 85%, or at least 90% identical to at least one ofPT-2209-WO-PCTSEQ ID NOs:249, 250, 251, 254, 255, 256, and 257. The allulose-6-phosphate epimerase may be at least 85%, or at least 90% identical to at least one of SEQ ID NOs:249, 250, 255, or 256. The recombinant cell may include an allulose-6-phosphate 3-epimerase at least 90% identical to at least one of SEQ ID NO:249, 250. 251, 254, 255, 256, and 257 and an allulose-6-phosphate phosphatase at least 90% identical to at least one of SEQ ID NO:87, 89, and 105.

[0007] The recombinant cell may also include a deletion or disruption in a native phosphofructokinase- 1 (PFK1) gene. The native PFK1 gene may encode a PFK1 enzyme 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:244. The native PFK1 gene may encode a PFK1 enzyme 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:244. The native PFK1 gene may encode a PFK1 enzyme at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:244. The native PFK1 gene may be 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:245. The native PFK1 gene may be 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:245. The native PFK1 gene may be at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 245.

[0008] The 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 NOs: 249, 250, 251, 254, 255, 256, and 257; the 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 NOs: 87, 89, 190, 123, 105, 107, and 115; the cell may be capable of producing at least 10 g / L D-allulose, and talitol production is reduced relative to a K. marxianus strain with wild-type of expression of the native talitol dehydrogenase gene.

[0009] The allulose-6-phosphate 3-epimerase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 249, 250, 251, 254, 255, 256, and 257; the allulose-6- phosphate phosphatase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 105; the cell may be capable of producing at least 10 g / L D-allulose; and talitol production is reduced relative to a K. marxianus strain with wild-type of expression of the native talitol dehydrogenase gene.

[0010] One or more of the exogenous polynucleotide sequences in the engineered cell may be operably linked to a heterologous promoter and / or a heterologous terminator. The promoter maybe selected from the group consisting of pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoterPT-2209-WO-PCT(ADHlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFlp); phosphoglucomutase 1 promoter (PGMlp); 3 -phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL 16B; and PDC 1 promoter. The terminator may be selected from the group consisting of GAL10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt): ASNS terminator (ASNSt); EN01 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGK.lt); PGM1 terminator (PGMlt); PYK.1 terminator (PYK.lt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); fructose-bisphosphate aldolase terminator (FBAlt); TEF1; iso- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator.

[0011] 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 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, sucrose, cellulosic biomass, or combinations thereof.

[0012] 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

[0013] 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.

[0014] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0015] FIG. 1 shows a proposed pathway for the production of D-allulose from sucrose, starch, and / or glucose.DETAILED DESCRIPTION

[0016] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosedPT-2209-WO-PCT subject mater will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject mater is not intended to limit the claims to the disclosed subject mater.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] This disclosure relates to various recombinant cells engineered to produce D-allulose with reduced production of talitol. In general, the recombinant cells described herein include a deletion or disruption on a native talitol dehydrogenase gene, 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: 249-256, 258, and 259, or sequences 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: 87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183 or sequences at least 70%, at least 80%, at least 85%, at least 90%, at leastPT-2209-WO-PCT95%, at least 97%, or at least 99% identical thereto. The recombinant cell may additionally include a deletion or disruption in a native PFK1 gene. The disclosure further provides fermentation methods for the production of D-allulose using the genetically engineered cells described herein.

[0021] As used herein, “D-allulose” and “allulose” are used interchangeably and refer to the ketohexose epimer of fructose shown in structure I below. D-allulose is also known in the art as “D-psicose” or “psicose.” D-allulose is found in trace amounts in wheat, figs, raisins, maple sugar, and molasses leading to its designation as a rare sugar.

[0022] In general, recombinant cells described herein are Kluyveromyces marxianus cells. Suitable Kluyveromyces marxianus cells and strains are known and described in the art. An ordinarily skilled artisan can identify strains that would be suitable for use in the generating the recombinant cells described herein. The Kluyveromyces marxianus cell may be a commercially available cell.

[0023] The terms “glucose” and “dextrose” are used interchangeably herein and refer to D- glucose except where expressly indicated otherwise.

[0024] 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.

[0025] 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, a Lactobacillus helveticus lactate dehydrogenase gene is exogenous when introduced into S. cerevisiae.

[0026] 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.

[0027] For the purposes of this application, the Kluyveromyces marxianus cell of Kluyveromyces marxianus CD21 , deposited under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms (BCCM) / Mycotheque de 1'Universite Catholique de Louvain (MUCL), Croix du Sud, box L7.05.06, B-1348 Louvain-la-Neuve. Belgium) on OctoberPT-2209-WO-PCT18, 2024 under accession number 58456, is considered the wild-type Kluyveromyces marxianus cell.

[0028] 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 enzy me, 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

[0029] 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 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.PT-2209-WO-PCT

[0030] As used herein, the phrases “percent 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 uery 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.

[0031] 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.

[0032] 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 aPT-2209-WO-PCT 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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 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 various organisms 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 generatingPT-2209-WO-PCT 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.

[0037] The recombinant cells described herein may include deletions or disruptions in one or more native genes. The phase “deletion or disruption'’ refers to the status of a native gene in the recombinant cell that has either a completely eliminated coding region (deletion) or a modification of the gene, its promoter, or its terminator (such as by a deletion, insertion, or mutation) so that the gene no longer produces an active expression product, produces severely reduced quantities of the expression product (e.g., at least a 75% reduction or at least a 90% reduction) or produces an expression product with severely reduced activity (e.g., at least 75% reduced or at least 90% reduced). The deletion or disruption can be achieved by genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. Deletion or disruption of a native host cell gene can be coupled to the incorporation of one or more polynucleotide sequences (e.g., an exogenous or native polynucleotide sequence) into the host cell at the locus of the host cell gene to be deleted or disrupted. The polynucleotide sequence to be inserted may be designed to replace all or a portion of the host cell gene to be deleted or disrupted. The polynucleotide sequence may encode for a gene product of interest, for example, a polypeptide, an enzyme, and the like. The deletion or disruption can also be accomplished using a deletion construct that does not contain a polynucleotide sequence to be integrated. Other methods for gene disruption or deletion are known and described in the art.

[0038] The recombinant cells described herein may include a deletion or disruption of a native talitol dehydrogenase gene. The native talitol dehydrogenase gene encodes an enzyme that has talitol dehydrogenase activity. As used herein “talitol dehydrogenase activity’” refers to enzymes that catalyze the reversible conversion of D-allulose (or D-allulose-6-phosphate) and NAD(P)H to talitol (or talitol-6-phosphate) and NAD(P)+. The talitol dehydrogenase enzyme catalyzes the production of talitol at the expense of the desired D-allulose product. Without being bound by any particular theory, method, or mode of action, when the activity of talitol dehydrogenase is reduced or eliminated the end titer and yield of D-allulose will be higher because the produced D-allulose cannot be converted to talitol. When the host contains multiple talitol dehydrogenase genes, it is preferred to delete or disrupt at least one of them. When the host contains multiple alleles of a given talitol dehydrogenase gene, it is preferred to delete or disrupt one, both, or all alleles of a given talitol dehydrogenase gene.

[0039] When the recombinant cell is a Kluyveromyces marxianus cell, the recombinant cell may comprise a deletion or disruption of a talitol dehydrogenase gene encoding an amino acidPT-2209-WQ-PCT sequence 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 SEQ ID NO: 304. When the recombinant cell is a Kluyveromyces marxianus cell, the recombinant cell may comprise a deletion or disruption of a talitol dehydrogenase gene with a nucleotide sequence 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 SEQ ID NO:305. When the recombinant cell is a Kluyveromyces marxianus cell, the talitol dehydrogenase gene may be deleted or disrupted by insertion of an exogenous or heterologous nucleic acid sequence at the talitol dehydrogenase gene locus to replace the talitol dehydrogenase gene. For example, in Kluyveromyces marxianus. the talitol dehydrogenase locus is flanked by SEQ ID NQs:302 and 303.

[0040] As used herein, “NAD(P)H” refers to nicotinamide adenine dinucleotide (phosphate) hydrogen and is inclusive of both NADH and NADPH. As is understood in the art, inclusion of the phosphate (or P ' abbreviation) in parentheses indicates that the phosphate may be absent or present and the name and abbreviation are inclusive of both. Similarly, “NAD(H)” or “NADP(H)” refers to both the reduced and oxidized forms of the cofactor.

[0041] The recombinant cells described herein may include a deletion or disruption of a native phosphofructokinase- 1 (PFK1) gene. The native PFK1 gene encodes an enzyme that has phosphofructokinase- 1 activity. As used herein “phosphofmctokinase-1 activity” and "PFK l activity” are used interchangeably and refer to enzymes that catalyze the irreversible conversion of beta-D-fructose 6-phosphate, adenosine triphosphate (ATP) to beta-D-fructose 1,6- bisphosphate, adenosine diphosphate (ADP) and water. PFKl is an enzyme in the gly colytic pathway and is the first irreversible reaction unique to the glycolytic pathway. Without being bound by any particular theory, method, or mode of action, when activity of PFKl is reduced or eliminated more cellular fructose-6-phosphate would be available to produce D-allulose. When the host cell contains multiple PFKl genes, it is preferred to delete or disrupt at least one of them. When the host cell contains, multiple alleles of a given PKF1 gene, it is preferred to delete or disrupt one, two. or all alleles of a given PFKl gene.

[0042] When the recombinant cell is a Kluyveromyces marxianus cell, the recombinant cell may comprise a deletion or disruption of a PFKl gene encoding an amino acid sequence 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 SEQ ID NO:244. When the recombinant cell is a Kluyveromyces marxianus cell, the recombinant cell may comprise a deletion or disruption of a PFKl gene with a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at leastPT-2209-WO-PCT95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:245. When the recombinant cell is a Kluyveromyces marxianus cell, the PFK1 gene may be deleted or disrupted by insertion of an exogenous or native nucleic acid sequence at the PFK1 locus to replace the PFK1 gene. For example, in Kluyveromyces marxianus the PFK1 locus is flanked by SEQ ID NOs:242 and 243.

[0043] The recombinant cells described herein may include one or more genetic modifications in which an exogenous nucleic acid is integrated into the genome of the host cell. One of skill in the art know how to select suitable loci in a yeast genome for integration of the exogenous nucleic acid. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adhl202, ADE2, ADH2556. GAL6, MDH1, SCW1 1, ER1, ER3, pyrF, TRP3, gpdllA, and gpdllB loci. For example, in K marxianus host cell, suitable integration loci may include, but are not limited to, the CYB2 locus (defined as the locus flanked by SEQ ID NO:212 and SEQ ID NO:213), and the PFK1 locus (defined as the locus flanked by SEQ ID NO:242 and 243). The exogenous nucleic acid may also be integrated in an intergenic region or other location in the host cell genome not specifically specified herein. Other suitable integration loci may be determined by one of skill in the art. Furthermore, one of skill in the art would recognize how to use sequences to design primers to verify correct gene integration at the chosen locus.

[0044] The recombinant cell may have one or more copies of a given exogenous nucleic acid sequence integrated in a host chromosome(s) and replicated together with the chromosome(s) into which it has been integrated. For example, the yeast cell may be transformed with nucleic acid construct including a polynucleotide sequence encoding for a polypeptide described herein and the polynucleotide sequence encoding for the polypeptide may be integrated in one or more copies in a host chromosome(s). The recombinant cell may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. The recombinant cell may have one, two, three, four, five, six, seven, eight, nine, ten, or more copies of a polynucleotide sequence encoding a polypeptide described herein integrated into the genome. The multiple copies of said polynucleotide sequence may all be incorporated at a single locus or may be incorporated at multiple loci. The recombinant cell may include one or more copies of a given exogenous nucleic acid sequence including on a plasmid within the recombinant cell.

[0045] The recombinant cells described herein are capable of producing D-allulose, include a deletion or disruption in a native talitol dehydrogenase gene, and include an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme, and may include a deletion or disruption of a native PFK1 gene. The epimerase enzyme may be any suitable enzymePT-2209-WO-PCT with allulose-6-phosphate 3-epimerase activity. The exogenous polynucleotide sequence may be an exogenous allulose-6-phosphate 3-epimerase (epimerase) gene.

[0046] As used herein “allulose-6-phosphate 3-epimerase gene” refers 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 enzy me with increased activity in the presence of cobalt (Co2+), manganese (Mn2+), calcium (Ca2+), magnesium (Mg2+). and / or zinc (Zn2+). The allulose-6-phosphate 3-epimerase enzyme can be from any suitable source organism or may be synthetic. Suitable allulose-6-phosphate 3-epimerase enzymes may include, but are not limited to, enzymes categorized under Enzy me Commission (EC) number 5.1.3. Suitable allulose-6-phosphate 3-epimerase enzymes may be the allulose-6-phosphate 3- epimerase enzymes from Cedecea lapagei, Gilliamella sp., Gilliamella apicola, Klebsiella spallanzanii. Lachnospiraceae bacterium, Oscillospiraceae bacterium. Enterococcus rotai, Tolumonas osonensis Acetcmaerohacterium elongatum, annotated enzymes of unknown origin, and the like. The allulose-6-phosphate 3-epimerase gene may encode an amino acid 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:249-256, 258, and 259.

[0047] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Cedecea lapageigene encoding the amino acid sequence of SEQ ID NO:249. The exogenous polynucleotide may encode an amino acid sequence 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:249.

[0048] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Gilliamella sp. gene encoding the amino acid sequence of SEQ ID NO:250. The exogenous polynucleotide may encode an amino acid sequence 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:250.

[0049] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Gilliamella apicola gene encoding the amino acid sequence of SEQ ID NO:251. The exogenous polynucleotide may encode an amino acid sequence 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:251.PT-2209-WQ-PCT

[0050] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Klebsiella spallanzanii gene encoding the amino acid sequence of SEQ ID NO:252. The exogenous polynucleotide may encode an amino acid sequence 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:252.

[0051] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Lachnospiraceae bacterium gene encoding the amino acid sequence of SEQ ID NO:253. The exogenous polynucleotide may encode an amino acid sequence 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:253.

[0052] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Oscillospiraceae bacterium gene encoding the amino acid sequence of SEQ ID NO: 254. The exogenous polynucleotide may encode an amino acid sequence 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:254.

[0053] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Enterococcus rotai gene encoding the amino acid sequence of SEQ ID NO:255. The exogenous polynucleotide may encode an amino acid sequence 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:255.

[0054] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Tolumonas osonensis gene encoding the amino acid sequence of SEQ ID NO:256. The exogenous polynucleotide may encode an amino acid sequence 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:256.

[0055] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a Acetanaerobacterium elongation gene encoding the amino acid sequence of SEQ ID NO:258. The exogenous polynucleotide may encode an amino acid sequence 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:258.

[0056] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene and may comprise an exogenous polynucleotide sequence that is or may be derived from a gene encoding the amino acid sequence of SEQ ID NO:259. The exogenous polynucleotide mayPT-2209-WO-PCT encode an amino acid sequence 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:259.

[0057] The recombinant cells described herein are capable of producing D-allulose, include a deletion or disruption in a native talitol dehydrogenase gene, include an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme, and may additionally include an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme. The recombinant cell may also include a deletion or disruption of a native PFK1 gene. The allulose-6- phosphate phosphatase enzyme may be any suitable enzyme with allulose-6-phosphate phosphatase activity. The exogenous polynucleotide sequence may be an exogenous allulose-6- phosphate phosphatase gene.

[0058] As used herein “allulose-6-phosphate phosphatase” refers 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 allulose-6-phosphate phosphatase enzy mes active on hexose-6-phosphate substrates are known and described in the art. The allulose-6-phosphate phosphatase enzy me may be derived from any suitable source or may be synthetic. Suitable allulose-6-phosphate phosphatase enzyme may include, but are not limited to, the allulose-6-phosphate phosphatase enzymes from Escherichia coli, Shewanella algae, Tatumella morbirosei. Shewanella loihica, Ferrimonas sediminicola. Shewanella fodinae. Parashewanella spongiae, Wickerhamomyces ciferrii, Trabulsiella odontot rmitis, Shewanella aestuarii, Shewanella pealeana, Shewanella sp., Shewanella denitriflcans, Shewanella woodyi, Ferrimonas lipolytica. Shewanella violacea, Shewanella mangrove. Parashewanella curva, Shewanella frigidimarina, Ferrimonas aestuarii. Shewanella sp. OPT22. The allulose-6- phosphate phosphatase gene may encode an amino acid 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: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 an amino acid 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:87, 89, 190, 123, 105, 107, and 115. The allulose-6-phosphate phosphatase gene may encode an amino acid 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:87, 89, and 190. The allulose-6-phosphate phosphatase gene may encode an amino acid at least 70%, atPT-2209-WO-PCT 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:87 and 89.

[0059] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewcmellci algae gene encoding the amino acid sequence of SEQ ID NO: 87. The exogenous polynucleotide may encode an amino acid sequence 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.

[0060] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may' be derived from a Tatiimella morbirosei gene encoding the amino acid sequence of SEQ ID NO: 89. The exogenous polynucleotide may encode an amino acid sequence 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.

[0061] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and 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: 190. The exogenous polynucleotide may encode an amino acid sequence 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.

[0062] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enz me and may comprise an exogenous polynucleotide that is or may be derived from aShewanella loihica gene encoding the amino acid sequence of SEQ ID NO: 123. The exogenous polynucleotide may encode an amino acid sequence 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.

[0063] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polymucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived from a Ferrimonas sediminicola gene encoding the amino acid sequence of SEQ ID NO: 105. The exogenous polynucleotide may encode an amino acid sequence 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.PT-2209-WO-PCT

[0064] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived from Shewanella fodinae gene encoding the amino acid sequence of SEQ ID NO: 107. The exogenous polynucleotide may encode an amino acid sequence 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.

[0065] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Parashewanella spongiae gene encoding the amino acid sequence of SEQ ID NO: 115. The exogenous polynucleotide may encode an amino acid sequence 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.

[0066] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived from a Wickerhamomyces ciferrii gene encoding the amino acid sequence of SEQ ID NO: 83. The exogenous polynucleotide may encode an amino acid sequence 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.

[0067] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Trabulstella odontotermitis gene encoding the amino acid sequence of SEQ ID NO:95. The exogenous polynucleotide may encode an amino acid sequence 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.

[0068] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella aestuarii gene encoding the amino acid sequence of SEQ ID NO: 1 13. The exogenous polynucleotide may encode an amino acid sequence 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.

[0069] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived fromPT-2209-WO-PCT a Shewanella pealeana gene encoding the amino acid sequence of SEQ ID NO: 117. The exogenous polynucleotide may encode an amino acid sequence 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.

[0070] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella sp. gene encoding the amino acid sequence of SEQ ID NO: 119. The exogenous polynucleotide may encode an amino acid sequence 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.

[0071] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella denitriflcans gene encoding the amino acid sequence of SEQ ID NO: 121. The exogenous polynucleotide may encode an amino acid sequence 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.

[0072] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella wooclyi gene encoding the amino acid sequence of SEQ ID NO: 127. The exogenous polynucleotide may encode an amino acid sequence 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.

[0073] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived from a Ferrimonas lipolytica gene encoding the amino acid sequence of SEQ ID NO: 131. The exogenous polynucleotide may encode an amino acid sequence 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.

[0074] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella violacea gene encoding the amino acid sequence of SEQ ID NO: 137. The exogenous polynucleotide may encode an amino acid sequence 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.PT-2209-WO-PCT

[0075] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella mangrovi gene encoding the amino acid sequence of SEQ ID NO: 145. The exogenous polynucleotide may encode an amino acid sequence 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.

[0076] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Tatumella ptyseos gene encoding the amino acid sequence of SEQ ID NO: 149. The exogenous polynucleotide may encode an amino acid sequence 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.

[0077] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived from a Parashewanella curva gene encoding the amino acid sequence of SEQ ID NO: 169. The exogenous polynucleotide may encode an amino acid sequence 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.

[0078] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Shewanella frigidimarina gene encoding the amino acid sequence of SEQ ID NO: 173. The exogenous polynucleotide may encode an amino acid sequence 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.

[0079] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzyme and may comprise an exogenous polynucleotide that is or may be derived from a Ferrimonas aestuarii gene encoding the amino acid sequence of SEQ ID NO: 179. The exogenous polynucleotide may encode an amino acid sequence 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.

[0080] The recombinant cell includes a deletion or disruption in a native talitol dehydrogenase gene, includes an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3- epimerase enzy me and may comprise an exogenous polynucleotide that is or may be derived fromPT-2209-WO-PCT a Shewanella sp. OPT22 gene encoding the amino acid sequence of SEQ ID NO: 183. The exogenous polynucleotide may encode an amino acid sequence 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.

[0081] 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 promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFp); phosphoglucomutase 1 promoter (PGMlp); 3 -phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B (SEQ ID NO:248); ScPDClp (SEQ ID NO:220); KmTDH3p (SEQ ID NO:222); and / or KmPDCl (SEQ ID NO:306).

[0082] 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, GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt; SEQ ID NO:288); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); fructose-bisphosphate aldolase (FBAlt; SEQ ID NO:289); TEF1; iso- 1 -cytochrome c (CYC 1; SEQ ID NO:218); KmHXK2t (SEQ ID NO:27); KmGPMlt (SEQ ID NO:221); KmPGKlt (SEQ ID NO:223); ScURA3t (SEQ ID NO:217); URA3; ADHL and ScGALlO.

[0083] 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.

[0084] 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 orPT-2209-WO-PCT 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.

[0085] 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.

[0086] 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 com, 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 corn 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.

[0087] 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™PT-2209-WO-PCT(Novozymes, Bagsvaerd, Denmark) and / or the amyloglucosidase from Aspergillus niger sold under the trade name AMG 300L™ by Sigma- Aldrich.

[0088] 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 50 °C, 26 °C to 45 °C, 27 °C to 40 °C, 280to 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, 41, 42, 43, 45, 46. 47. 48. 49. 50°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.

[0089] The pH of a culture medium described herein may be controlled for optimal D-allulose production. The pH of the culture or a fermentation mixture of an engineered cell described herein may be in the range of between 3.0 and 7.5. The pH may be maintained for at least part of the incubation 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. 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.

[0090] The engineered cell (yeast and / or bacteria) may be cultured for approximately 24-96+ 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.

[0091] Reaction parameters can be measured or adjusted during the production of D-allulose. Non-limiting examples of reaction parameters include biological parameters (e.g., grow th 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 anPT-2209-WO-PCT 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 humidity7, molarity7, osmolarity7, 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, turbidity7, 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 ordinary7skill in the art.

[0092] 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.

[0093] The final D-allulose titer may 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, at least 17.5 g / L, or at least 20 g / L.

[0094] In the fermentation, production of talitol will be reduced relative to an equivalent fermentation run with an equivalent K. marxianus cell with wild-type level expression of the native talitol dehydrogenase gene. Talitol production is reduced so the final talitol titer is less than 1 g / L, less than 0.75 g / L, less than 0.5 g / L, less than 0.25 g / L, or less than 0.1 g / L when the fermentation is run for at least 72 hours. Talitol production may be reduced such that the talitol titer is below7the detection limit of the instrument used for analysis. Talitol production may be reduced such that the final fermentation is substantially free of talitol.EXAMPLES

[0095] 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 w ay 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.

[0096] Strain numbering is consistent throughout the Examples. For example, strain 1-5 in Example 1 is the same strain as strain 1-5 in Examples 2 and 3.PT-2209-WO-PCTExample 1 : Genetically Modified Saccharomyces cerevisiae strains

[0097] 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 (S. cerevisiae), the strains described in this, and subsequent examples were built and tested.Strain 1-1

[0098] 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

[0099] Strain 1-2 is an uracil auxotroph derivative of strain 1-1 with an insertion of the Aspergillus nidulans amdS gene at the UR A3 locus.Strain 1-3

[0100] 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: 185; polypeptide SEQ ID NO: 186) 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,’7ACS Synth. Biol., 2018, 7. 6. 1573-1587). with restriction endonuclease Notl. 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.PT-2209-WO-PCTStrain 1-4

[0101] 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

[0102] 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: 186 under the control of the TDH3cp promoter.Strains 1-6 through 1-100

[0103] Polynucleotides encoding the D-allulose-6-phosphate 3-epimerase and D-allulose-6- phosphate phosphatase were cloned into the pSCT036 vector (SEQ ID NO: 187). Sequences encoding the allulose-6-phosphate 3-epimerase enzymes were under the control of the TDH3 promoter (SEQ ID NO: 188) and sequences encoding the allulose-6-phosphate phosphatase enzymes were under the control of the control of the promoter of SEQ ID NO: 189, which includes 8 synthetic transcription factor (sTF) binding sites for the sTF expressed in strain 1-5. The plasmids encoding the allulose-6-phosphate 3-epimerase and allulose-6-phosphate phosphatase enzymes were cloned into parent strain 1-5, as outlined in Table 2.Table 2: Genetically Modified 5. cerevisiae strainsPT-2209-WO-PCTPT-2209-WO-PCTPT-2209-WO-PCTPT-2209-WO-PCTExample 2: Shake Flask Fermentation Assay

[0104] Strains 1-5, 1-7 through 1-31, 1-33 through 1-57, 1-99, and 1-100 were run in duplicate shake flasks to assay D-allulose production.

[0105] Strains were struck on a synthetic complete medium minus uracil (ScD-ura) plates (6.7g / L yeast nitrogen base without amino acids, 1.9 g / L Synthetic Complete amino acid mix, 20PT-2209-WO-PCT g / 1 glucose, and 20g / L agar) and grown until single colonies formed (2-3 days at 30 °C or 3-5 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 4) 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). 1.0 ml samples were withdrawn at 72 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 7.Table 3: ScD-ura with 20g / L dextroseTable 4: DMlu Production Medium - Maltodextrin*GA added just prior to inoculationPT-2209-WO-PCTTable 5: lOOOx trace elementsTable 6: 1OOOX DM1 vitamin solutionTable 7: Shake Flask ResultsPT-2209-WO-PCTnd is not detected, below instrument detection limit*only one shake flask of strain 1-56 was analyzed

[0106] The data shows the expression of the allulose-6-phosphate phosphatases of SEQ ID NOs:83 and 95 produced nearly the same amount of D-allulose as the positive control allulose-6- phosphate phosphatase of SEQ ID NO: 190. However, expression of the allulose-6-phosphate phosphatases of SEQ ID NOs:87 and 89 resulted in increased titer of D-allulose relative to the positive control. Overall, expression of the allulose-6-phosphate phosphatases of SEQ ID NOs: 15, 19, 23, 25, 35, 37, 39, 43, 45, 49, 59, 63, 77, 83. 85. 87. 89. 91. 93. 95, and 101 has higher D- allulose titers than the wild-type strain (1-5) or the negative control strain expressing the allulose- 6-phosphate 3-epimerase of SEQ ID NO: 192 but lacking a allulose-6-phosphate phosphatase.PT-2209-WO-PCTExample 3: Flower Plate Fermentation Assay

[0107] Strains 1-5, 1-6, 1-32, 1-49, 1-50, 1-58 through 1-98, 1-99, and 1-100, were assayed in duplicate to evaluate D-allulose production.

[0108] Strains were struck on a ScD-ura plates and grown until single colonies formed (2-3 days at 30 °C or 3-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a 96 deep well microtiter plate containing 500 microliters of ScD-Ura media (6.7g / L yeast nitrogen base without amino acids, 2.0 g / 1 Synthetic Complete amino acid mix, 20 g / L glucose), and incubated for 24 hours at 30°C and 800 RPM in an orbital shaker. The overnight cultures were transferred to a 48 well flower plate containing 1000 microliters of DMU1 media (lOOg / L maltodextrin and 50ul GA) to an OD600 = 0.2, incubated for 96 hours at 30°C and 800 RPM in an orbital shaker. Samples were taken for analysis by HPLC to determine D-allulose titers. Results are shown in Table 8.Table 8: Shake Flask DataPT-2209-WO-PCT

[0109] Results show that the expression of the allulose-6-phosphate phosphatases of SEQ ID NOs:87 and 145 produced nearly the same amount of D-allulose as the positive control allulose- 6-phosphate phosphatase of SEQ ID NO: 190. However, expression of the allulose-6-phosphate phosphatases of SEQ ID NOs:89, 87, 183, 179, 173, 169, 149, 89, 137, 131, 127, 123, 121, 119, 117, 115, 113, 107, and 105 resulted in increased titer of D-allulose relative to the positive control. Overall, strains expressing the allulose-6-phosphate phosphatases of SEQ ID NOs: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, 1 11, and 109 have higher D-allulose titers than the negative control strain expressing the allulose-6-phosphate 3-epimerase of SEQ ID NO: 192 but lacking a allulose-6-phosphate phosphatase.Example 4: Genetically Modified Kiuweromvces marxianus strains

[0110] 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 (K. marxianus'), the strains described in this, and subsequent examples w ere built and tested.PT-2209-WO-PCTStrain 2-1

[0111] Strain 2-1 is a yeast cell Kluyveromyces marxianus CD21, deposited under Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Uni versite Catholique de Louvain (BCCM MUCL) designation 58456.Stain 2-2

[0112] Strain 2-2 is an uracil auxotroph derivative of strain 2-1 with a deletion of the URA3 locus.Strains 2-3 through 2-21

[0113] Strain 2-2 was transformed according to Table 10 using the indicated transformation fragments (Table 9). Resulting transformants were streaked for single colony isolation on ScD- ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. PCR verified isolates were designated as outline in Table 10.Table 9: Transformation FragmentsPT-2209-WO-PCTPT-2209-WO-PCTTable 10.PT-2209-WO-PCTStrain 2-22

[0114] Strain 2-2 was transformed with SEQ ID NOs: 198 and 199 (Table 9). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the sequence encoding GFP. A PCR verified isolate was designated strain 2-22.Example 5: Shake Flask Fermentation Assay

[0115] Strains 2-3 through 2-22 were assayed to evaluate D-allulose production.

[0116] 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 ScD-Ura production medium (Table 3) 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 72 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 show n in Table 11.Table 11 : 72-hour Shake Flask ResultsPT-2209-WO-PCTnd is not detected, below7instrument detection limit

[0117] Results show that a variety7of promoters and terminators are suitable for expression of epimerase and allulose-6-phosphate phosphatase enzymes in K. marxianus for the production of D-allulose.Example 6: Shake Flask Assay

[0118] Strains 2-3 through 2-22 were assayed to evaluate D-allulose production.

[0119] 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 4) 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 w ithdrawn at 72 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 12.Table 12: 72-hour Shake Flask ResultsPT-2209-WO-PCTnd is not detected, below instrument detection limit

[0120] Results show that a variety of promoters and terminators are suitable for expression of epimerase and allulose-6-phosphate phosphatase enzymes in K. marxianus for the production of D-allulose.Example 7: Genetically Modified marxianus strainsStrain 2-23

[0121] Strain 2-2 was transformed with SEQ ID NO:239. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. SEQ ID NO:239 contained the TDH3 promoter (SEQ ID NO:222), a gene encoding the allulose-6- phosphate epimerase of SEQ ID NO: 192, the Cyc terminator (SEQ ID NO:218), the PGK promoter (SEQ ID NO:223), a gene encoding the allulose-6-phosphate phosphatase of SEQ ID NO: 190, the PDC_Lue2 terminator (SEQ ID NO:225), and a ScURA3 selection marker cassette. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was designated strain 2-23.Strain 2-24

[0122] Strain 2-2 was transformed with SEQ ID NO:203 and SEQ ID NO:237. SEQ ID NO:237 contained a 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), a gene encoding the allulose-6-phosphate phosphatase of SEQ ID NO: 105, the KmPGKl terminator, and a 3' KmCYB2 flanking sequence (SEQ IDNO:213). Resulting transformants were streaked for singlePT-2209-WO-PCT colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was designated strain 2-24.Strain 2-25

[0123] Strain 2-2 was transformed with SEQ ID NO:203 and SEQ ID NO:238. SEQ ID NO:238 contained a 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), a gene encoding the allulose-6-phosphate phosphatase of SEQ ID NO: 107, the KmPGKl terminator, and a 3' KmCYB2 flanking sequence (SEQ IDNO:213). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was designated strain 2-25.Example 8: Flower Plate Assays

[0124] Strains 2-3, 2-14, 2-23, 2-24, and 2-25, were assayed to evaluate D-allulose production.

[0125] Strains were struck on a ScD-ura plates and grown until single colonies formed (2-3 days at 30 °C or 3-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a 250 mL baffled flask containing 40 mL of buffered ScD-Ura media (6.7g / L yeast nitrogen base without amino acids, 1.9 g / 1 Synthetic Complete amino acid mix, 100 g / L glucose, 19.5g / l MES buffer, pH adjusted to 6.0), and incubated for 24 hours at 30°C and 250 RPM in an orbital shaker. 40 pL from the overnight cultures was transferred to a 48 well flower plate containing 1000 microliters of (i) buffered ScD-Ura with glucose, (ii) ScD-Ura with maltodextrin, OR (ii) DMul media (Table 4) and incubated for 92 hours at 30°C and 800 RPM in an orbital shaker. Samples were taken for analysis by HPLC to determine D-allulose titers. Some samples were also tested by high pressure ion chromatography due to a small peak artifact in the HPLC data. Results are shown in Table 13.PT-2209-WO-PCTTable 13.PT-2209-WO-PCTnt = not testedExample 9: Shake Flask Fermentation Assays

[0126] Strains 2-23, 2-3, 2-24, and 2-25 were assayed to evaluate D-allulose production.

[0127] 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 fermentation media (outlined in Table 18, with changes from the base media noted) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.1, and incubated at 30°C and an RPM as designated in Table 17 with 70% humidity. 0.5 ml samples were withdrawn at 236 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 14.PT-2209-WQ-PCT

[0128] Overall, the results indicate that strain 2-24 produced the highest allulose titers across the conditions tested. Strains 2-14, 2-24, and 2-25, which all expressed the allulose-6-phosphate epimerase of SEQ ID NO: 194 and the allulose-6-phosphate phosphatase of SEQ ID NOs:123, 105, and 107, respectively, all produces higher titers of D-allulose than strains 2-3 or 2-23 which both express the allulose-6-phosphate epimerase of SEQ ID NO: 192 and the allulose-6-phosphate phosphatase of SEQ ID NO: 190. The data also show the tested strains produced D-allulose from a variety of feedstocks in different fermentation media. The results demonstrate effects from both the RPM and baffle size that are media dependent. For example, higher RPMs and larger baffles were beneficial to production (i.e., higher D-allulose titers) in the Sc-Ura + glucose media, neutral in Sc-Ura + maltodextrin media, and detrimental in DMul media. However, even though this variability can be seen, all test conditions produced D-allulose, showing that multiple medium, substrates and conditions are suitable for D-allulose fermentation.Table 14.PT-2209-WO-PCT

[0129] Due to a small peak artifact upon HPLC analysis, further analysis by high pressure ion chromatography (HPIC) was performed for the fermentation broths from strain 2-24 (experiments 10.4, 10.9, 10.14, 10.19, 10.24, 10.29, 10.32, 10.34, andl0.36) and results are reported in Table 15.Table 15.PT-2209-WO-PCTExample 10: PFK1 Deletion in marxianus

[0130] PFK1 is an enzyme in the glycolytic pathway and is the first irreversible reaction unique to the glycolytic pathway. Without being bound by any particular theory, method, or mode of action, when activity of PFK1 is reduced or eliminated more cellular fructose-6-phosphate would be available to produce D-allulose. To test this and demonstrate the increased production of D- allulose in a genetically engineered yeast (K marxianus) the strain 2-26 was built and tested.Strain 2-26

[0131] Strain 2-2 was transformed with SEQ ID NO:240 and SEQ ID NO:241. SEQ ID NO:240 contained a 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), a gene encoding the allulose-6-phosphate phosphatase of SEQ ID NO: 105, the KmPGKl terminator, and a 3’ PFK1 flanking sequence (SEQ IDNO:242). SEQ ID NO:241 contained a 5’ PFK1 flanking sequence (SEQ ID NO:243), an ScPDCl promoter (SEQ ID NO:220), a gene encoding the allulose-6- phosphate epimerase of SEQ ID NO: 194, a KmGPMl terminator (SEQ ID NO:221), an ScURA3 promoter (SEQ ID NO:216) and a 5’ portion of the ScURA3 selection marker (SEQ ID NO:215). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was designated strain 2-26.

[0132] Strains 2-23, 2-24, and 2-26 were assayed to evaluate D-allulose production. Strains were struck on a ScD-ura plates and grown until single colonies formed (1-3 days at 30 °C or 2-5 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 50 ml of ScD-ura with 20 g / L glucosePT-2209-WO-PCT media (Table 3) and incubated overnight at 30°C, 250 rpm with 70% humidity. The overnight cultures were diluted into 40 ml fermentation medium (either (i) ScD-ura with a total of 200 g / L glucose or (ii) DMlu in which the maltodextrin is replaced with 200 g / L glucose, as specified in Table 16) in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.1, and incubated at 30°C. 250 rpm with 70% humidity. 0.5 ml samples were withdrawn at 93 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, supernatant collected and stored at -20°C until analysis. Samples were analyzed for D-allulose by HPLC and HPIC. Results are shown in Table 16.

[0133] Strain 2-26. in which the native K. marxianus PFK1 gene was deleted, showed a significant increase in D-allulose production compared to an equivalent strain, strain 2-24, in which the PFK1 gene had not been deleted. In ScD-Ura medium measured by HPLC, D-allulose titer from strain 2-26 was about 15 g / L compared to about 5.5 g / L in strain 2-24, showing an almost 3x increase in titer. In DMlu media measured by HPLC. strain 2-26 produced about 38 g / L D-allulose compared to about 3 g / L from strain 2-24, representing an almost 17x increase in titer. The D-allulose titers from strain 2-26 were also far higher than comparative strain 2-23. The same trends are seen in the HPIC measurements, with D-allulose titer increases in both ScD-Ura (about 3x increase) and DMlu (above a 4x increase) medium. While values are lower in the HPIC measurements, this is due to a peak artifact on the HPLC, and the overall trend of the PFK1 deletion significantly increasing D-allulose titers is still consistent in multiple media conditions assay measurement methods. The D-allulose titers as measured by HPIC from strain 2-26 were also far higher than comparative strain 2-23.Table 16.PT-2209-WO-PCTnd - not detectedExample 11 : PFK1 Deletion in S. cerevisiae

[0134] PFK1 is an enzyme in the glycolytic pathway and is the first irreversible reaction unique to the glycolytic pathway. Without being bound by any particular theory, method, or mode of action, when activity of PFK1 is reduced or eliminated more cellular fructose-6-phosphate would be available to produce D-allulose. To test this and demonstrate the increased production of D- allulose in a genetically engineered yeast (S. cerevisiae) the strains outline below were built and tested.Strain 4-1

[0135] Strain 4-1 is S. cerevisiae strain BY4741 (American Type Culture Collection Deposit No. 4040002), which is a deletion strain derived from S. cerevisiae strain S288C (American Type Culture Collection Deposit No. 204508) in which commonly used genes, including PFK1, were deleted.Strain 4-2

[0136] Upstream (SEQ ID NO:246) and downstream (SEQ ID NO:247) sequences were used to amplify the site of the PFK1 deletion in strain 4-1. This amplification product was then cloned into strain 1-58 to delete the PFK1 gene. This resulted in a strain with a PFK1 knockout and continuing nucleic acid sequences encoding the allulose-6-phosphate epimerase of SEQ ID NO: 192 and the allulose-6-phosphate phosphatase of SEQ ID NO: 105. This strain was designated strain 4-2.PT-2209-WO-PCT

[0137] Strains 1-58, 1-67, and 4-2 were assayed to determine D-allulose production. Strains were struck on a synthetic complete medium minus uracil (ScD-ura) plates (6.7g / L yeast nitrogen base without amino acids, 1.9 g / L Synthetic Complete amino acid mix, 20 g / 1 glucose, and 20g / L agar) and grown until single colonies formed (2-3 days at 30 °C or 3-5 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, with changes in maltodextrin and GA concentrations as noted in Table 17, 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). 1.0 ml samples were withdrawn at 24, 90, 114, 162, 192, and 239 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, supernatant collected and stored at -20°C until analysis. Samples were analyzed for D-allulose by HPLC. Results are shown in Table 17.

[0138] Overall, deletion of the PFK1 gene in Saccharomyces cerevisiae (strain 4-2) didn’t increase D-allulose production relative to the equivalent strain without the deletion (strain 1-58). In fact, under some conditions the D-allulose production in the PFK1 deletion strain was lower than the equivalent strain.Table 17.PT-2209-WO-PCTExample 12: Genetically Modified marxianus strains

[0139] This example describes the engineering of several K. marxianus strains to use the pathway outlined in FIG. 1 to produce D-allulose using various allulose-6-phosphate 3-epimerase enzyme candidate genes.Strain 3-1

[0140] Strain 2-2 was transformed with SEQ ID NO:290 and SEQ ID NO:291. SEQ ID NO:290 contained a 5’ PFK1 flanking sequence (SEQ ID NO:243), the ScURA3 promoter (SEQ ID NO:216), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:291 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:317), and a 3’ PFK.1 flanking sequence (SEQ ID NO:242). Resulting transformants were streaked for single colony isolation on ScD-ura plates and a single colony was selected. The selected colony was evaluated by colony PCR for knockout of the PKF1 gene and designated strain 3-1.PT-2209-WO-PCTStrain 3-2

[0141] Strain 3-1 was transformed with the Cre recombinase plasmid of SEQ ID NO:292. Removal of the ScURA3 selection marker gene was confirmed by lack of grow th on ScD-ura plates. A verified isolate was designated strain 4-2.Strains 3-3 through

[0142] Strain 2-2 and strain 3-2 were transformed according to Table 18 using the indicated transformation fragments. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. PCR verified isolates w ere designated as outlined in Table 18.

[0143] SEQ ID NOs:262-274 contained a 5’ CYB2 flanking sequence (SEQ ID NO:212), the KmTDH3 promoter (SEQ ID NO:222), a polynucleotide encoding the D-allulose-6-phosphate epimerase as outlined in Table 18, the PGK terminator (SEQ ID NO:223), the ScURA3 promoter (SEQ ID NO:216), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215).

[0144] SEQ ID NO:275 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), the ScPDCl promoter (SEQ ID NO:220), a polynucleotide sequence encoding the amino acid of SEQ ID NO: 105, the ENO1 terminator (SEQ ID NO:288), the KmTDH3 promoter (SEQ ID NO:222), a nucleic acid sequence encoding the amino acid of SEQ ID NO: 105, the FBA1 terminator (SEQ ID NO:289), and a 3' CYB2 flanking sequence (SEQ ID NO:213).

[0145] SEQ ID NOs:276-286 contained a 5’ CYB2 flanking sequence (SEQ ID NO:212), the ENO1 terminator (SEQ ID NO:288), a polynucleotide encoding the D-allulose-6-phosphate epimerase as outlined in Table 10, the ScPDCl promoter (SEQ ID NO:220), a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215), and the ScURA3 promoter (SEQ ID NO:216).

[0146] SEQ ID NO:287 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), the ScPDCl promoter (SEQ ID NO:220), a nucleic acid sequence encoding the amino acid of SEQ ID NO: 105, the ENO1 terminator (SEQ ID NO:288), and a 3’ CYB2 flanking sequence (SEQ ID NO:213).PT-2209-WO-PCTTable 18.PT-2209-WO-PCTPT-2209-WO-PCTStrain 3-38

[0147] Strain 2-2 was transformed with SEQ ID NO:293 and SEQ ID NO:294. SEQ ID NO:293 contained a 5' CYB2 flanking sequence (SEQ ID NO:212), the ScPDCl promoter (SEQ ID NO:220), a nucleic acid sequence encoding the D-allulose-6-phosphate epimerase of SEQ ID NO: 194, the ENO1 terminator (SEQ ID NO:288), the ScURA3 promoter (SEQ ID NO:216), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:294 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:215), the ScURA3 terminator (SEQ ID NO:217), the KmTDH3 promoter (SEQ ID NO:222), a nucleic acid sequence encoding the allulose-6-phosphate phosphatase of SEQ ID NO: 105, the KmFBAl terminator (SEQ ID NO:289), and a 3’ CYB2 flanking sequence (SEQ ID NO:213). Transformants were plated on ScD-URA plates, and a single colony was selected. The selected colony was evaluated by colony PCR and the PCR verified isolate was designated strain 3-38.Strain 3-39

[0148] Strain 2-2 was transformed with SEQ ID NO:295 and SEQ ID NO:296. SEQ ID NO:295 contained a 5’ CYB2 flanking sequence (SEQ ID NO:212), the KmENOl terminator (SEQ ID NO:288), a nucleic acid sequence encoding the D-allulose-6-phosphate epimerase of SEQ ID NO: 194, the ScPDCl promoter (SEQ ID NO:220), the ScURA3 promoter (SEQ ID NO:216), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:296 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), the ScPDCl promoter (SEQ ID NO:220), a nucleic acidPT-2209-WO-PCT sequence encoding the D-allulose-6-phosphate phosphatase of SEQ ID NO: 105, the ENO1 terminator (SEQ ID NO:288), and a 3’ CYB2 flanking sequence (SEQ ID NO:213). Transformants were plated on ScD-URA plates, and a single colony was selected. The selected colony was evaluated by colony PCR and the PCR verified isolate was designated strain 3-39.Strain 3-40

[0149] Strain 2-2 was transformed with SEQ ID NO:297 and SEQ ID NO:298. SEQ ID NO:297 contained a 5’ PFK1 flanking sequence (SEQ ID NO:243), the KmENOl terminator (SEQ ID NO:288), a nucleic acid sequence encoding the D-allulose-6-phosphate epimerase of SEQ ID NO: 194, the ScPDCl promoter (SEQ ID NO:220), the ScURA3 promoter (SEQ ID NO:216), a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:298 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:215), the ScURA3 terminator (SEQ ID NO:217), the ScPDCl promoter (SEQ ID NO:220), a nucleic acid sequence encoding the allulose-6-phosphate phosphatase of SEQ ID NO: 105, the KmENOl terminator (SEQ ID NO:288), and a 3’ PFK1 flanking sequence (SEQ ID NO:242). Transformants were plated on ScD-URA plates, and a single colony was selected. The selected colony was evaluated by colony PCR and the PCR verified isolate was designated strain 3-40.Example 13: Flow Injection Analysis

[0150] Strains 3-3 through 3-15, 3-38, and 3-39 were evaluated by Flow Injection Analysis (FIA) and high performance ion chromatography (HPIC) for production of D-allulose, talitol, C7 sugar, and C7 polyol.

[0151] Strains were struck on a ScD-ura plates and grown until single colonies formed (2-3 days at 30 °C or 3-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a well of a 48 well flower plate containing 500 pL of ScD-Ura media (Table 3). Flower plates were incubated overnight at 900 rpm, 30 °C, and 70-80% relative humidity. Following overnight incubation, 1 mL of DMU1 medium (Table 4, maltodextrin replaced with 200 g / L glucose and no glucoamylase was added) was inoculated to an OD600 of 0.2 and incubated for 96 hours at 900 rpm, 30 °C, and 70-80% relative humidity. After 96 hours, 500 pL was removed and the supernatant for FIA and HPIC. FIA results are shown in Table 19 and HPIC results are shown in Table 20.

[0152] Results show that strains expressing several of the allulose-6-phosphate epimerase enzyme candidates (for example, SEQ ID NOs:249-256, 258, and 259) produced D-allulose at aPT-2209-WO-PCT titer near that produced by the two positive control strains, 3-38 and 3-39. Many of these strains produced significantly less talitol (e.g., strains 3-4, 3-5, 3-6, 3-7, 3-9, and 3-12), C7 polyols (e.g., strains 3-3, 3-4, 3-5, 3-6, 3-9, and 3-10), and / or C7 sugars (e.g., strains3-3, 3-4, 3-5, 3-6, 3-9, and 3-10) than the control strains, demonstrating important by-product reduction / elimination while maintaining D-allulose production.Table 19.Table 20.PT-2209-WO-PCTExample 14: Flower Plate Assay

[0153] Strains 3-16 through 3-37, 3-39, and 3-40, were assayed to evaluate D-allulose, talitol, C7 polyol, and C7 sugar production.

[0154] Strains were struck on a ScD-ura plates and grown until single colonies formed (2-3 days at 30 °C or 3-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a well of a 48 well flower plate containing 500 pL of ScD-Ura media (Table 3). Flower plates were incubated overnight at 900 rpm, 30 °C, and 80% relative humidity7. Following overnight incubation, 1 rnL of DMU1 medium (Table 4, maltodextrin replaced with 200 g / L glucose and no glucoamylase was added) was inoculated to an OD600 of 0.2 and incubated at 900 rpm, 30 °C, and 80% relative humidity. After 69 hours, 500 pL was removed from strains 3-16 through 3-26 and 3-39 for HPIC analysis and results are shown in Table 21. After 120 hours, 500 pL was removed from strains 3-27 through 3-37 and 3-40 for HPIC analysis and results are shown in Table 22.

[0155] Results show that strains expressing the D-allulose-6-phosphate epimerase enzymes of SEQ ID NOs:249, 250, 251, 254, 255, 256, and 257 produced significantly more D-allulose than the positive control strains. Likewise, many of the strains tested produced less C7 polyols and less C7 sugars. Strains that also included a knockout of the PFK1 gene showed further improvements in D-allulose production.PT-2209-WO-PCTTable 21.nd - not detectedTable 22.PT-2209-WO-PCTnd - not detectedExample 15: Shake Flask Fermentation Assay

[0156] Strains 2-24 and 2-26 were grown in shake flask to determine viable feedstocks for the production of D-allulose. Strains were assayed for D-allulose, talitol, and allitol production using fructose, sucrose, glycerol, and dextrose feedstocks.

[0157] Strains were struck on a ScD-ura plates and grown until single colonies formed (1-3 days at 30 °C or 2-5 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 50 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 fermentation medium (DMlu media, Table 4, in which the maltodextrin was replaced with either (i) 200 g / L glucose, (ii) 200 g / L fructose, (iii) 200 g / L sucrose, or (iv) 50 g / L glycerol, as specified in Tables 23 and 24) in 250 ml baffled Erlenmey er flasks to achieve an initial cell density of OD600 = 0.1, and incubated at 30°C, 250 rpm with 70% humidity. 0.5 ml samples were withdrawn at 25, 31, 48, 73, and 144 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, supernatant collected and stored at - 20°C until analysis. Samples were analyzed for D-allulose by HPLC and results are shown in Table 23. Samples from the 144 hour time point using the fructose, sucrose, or dextrose carbon sources were also analyzed by HPIC for D-allulose and talitol and results are shown in Table 24.Table 23.PT-2209-WO-PCTPT-2209-WO-PCTnd - not detectedTable 24.Example 16: Kluyveromyces marxianus strain engineering

[0158] Talitol byproduct production in D-allulose producing strains of Kluyveromyces marxianus reduces the final D-allulose titer by pulling carbon away from D-allulose production and / or by conversion of the produced D-allulose to talitol by a talitol dehydrogenase enzyme.PT-2209-WO-PCTDeletions of candidate polyol reductase and dehydrogenase enzymes were genetically engineered into K. marxianus strains to demonstrate the impact on D-allulose and talitol production.Strain 4- 1

[0159] Strain 2-2 was transformed with SEQ ID NO:299 and SEQ ID NO:300. SEQ ID NO:299 contained a 5’ CYB2 flanking sequence (SEQ ID NO:212), the ENO1 terminator (SEQ ID NO:288), a nucleic acid sequence encoding the D-allulose-6-phosphate epimerase of SEQ ID NO: 194, the ScPDCl promoter (SEQ ID NO:220), the KmFBAl terminator (SEQ ID NO:289), a nucleic acid sequence encoding the D-allulose-6-phosphate epimerase of SEQ ID NO: 194, the KmTDH3 promoter (SEQ ID NO:222), the KmHXK2 terminator (SEQ ID NO:227), a nucleic acid sequence encoding the D-allulose-6-phosphate epimerase of SEQ ID NO: 194, the KmPDCl promoter (SEQ ID NO:301), the ScURA3 promoter (SEQ ID NO:216), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:300 contained a 3‘ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), the ScPDCl promoter (SEQ ID NO:220), a nucleic acid sequence encoding the D-allulose-6- phosphate phosphatase of SEQ ID NO: 105, the ENO1 terminator (SEQ ID NO:288), the KmTDH3 promoter (SEQ ID NO:222), a nucleic acid sequence encoding the D-allulose-6- phosphate phosphatase of SEQ ID NO: 105, the KmFBAl terminator (SEQ ID NO:289), the KmPDCl promoter (SEQ ID NO:301), a nucleic acid sequence encoding the D-allulose-6- phosphate phosphatase of SEQ ID NO: 105, the KmHXK2 terminator (SEQ ID NO:227), and a 3’ CYB2 flanking sequence (SEQ ID NO:213). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was designated strain 4-1.Strain 4-2

[0160] Strain 4-1 was transformed with the Cre recombinase plasmid of SEQ ID NO:292. Removal of the ScURA3 selection marker gene was confirmed by lack of growth on ScD-ura plates. A verified isolate was designated strain 4-2.Strain 4-3a-c

[0161] Strain 4-3a-c was engineered to knockout the gene encoding the proposed oxidoreductase / dehydrogenase enzyme of SEQ ID NO:308. Strain 4-2 was transformed with SEQPT-2209-WO-PCTID NO:306 and SEQ ID NOA07. SEQ ID NOA06 contained a 5’ flanking sequence (SEQ ID NO:309) of the proposed oxidoreductase / dehydrogenase gene, the ScURA3 promoter (SEQ ID NO:216), a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:307 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), and a 3’ flanking sequence (SEQ ID NO:310) of the proposed oxidoreductase / dehydrogenase gene. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for deletion of the candidate gene. PCR verified isolates were designated strains 4-3a, 4-3b, and 4-3c.Strain 4-4a-c

[0162] Strains 4-4a-c were engineered to knockout the gene encoding the proposed oxidoreductase / dehydrogenase enzyme of SEQ ID NO:311. Strain 4-2 was transformed with SEQ ID NOAM and SEQ ID NO I5. SEQ ID NOAM contained a 5’ flanking sequence (SEQ ID NOA 12) of the gene encoding the proposed oxidoreductase / dehydrogenase enzyme of SEQ ID NOAH, the ScURA3 promoter (SEQ ID NOAM), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NOAM). SEQ ID NOAM contained a 3‘ portion of the ScURA3 selection marker gene (SEQ ID NOAM), the ScURA3 terminator (SEQ ID NOA17), and 3‘ flanking sequence (SEQ ID NOA 13) of the gene encoding the proposed oxidoreductase / dehydrogenase enzyme of SEQ ID NOAH. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for deletion of the candidate gene. PCR verified isolates were designated strains 4-4a, 4-4b, and 4-4c.Strain 4-5a-c

[0163] Strains 4-5a-c were engineered to knockout the gene encoding the talitol dehydrogenase enzyme of SEQ ID NOA04. Strain 4-2 was transformed with SEQ ID NOA21 and SEQ ID NOA22. SEQ ID NOA21 contained a 5’ flanking sequence (SEQ ID NOA03) of the talitol dehydrogenase gene, the ScURA3 promoter (SEQ ID NOAM), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NOA 15). SEQ ID NOA22 contained a 3‘ portion of the ScURA3 selection marker gene (SEQ ID NOAM), the ScURA3 terminator (SEQ ID NOA17), and a 3' flanking sequence (SEQ ID NOA02) of the talitol dehydrogenase gene. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected.PT-2209-WO-PCTSelected colonies were evaluated by colony PCR for deletion of the taltiol dehydrogenase gene. PCR verified isolates were designated strains 4-5a, 4-5b, and 4-5c.Strain 4-6a-c

[0164] Strain 4-6a-c were engineered to knockout the gene encoding the proposed oxidoreductase / dehydrogenase enzyme of SEQ ID NO:31 . Strain 4-2 was transformed with SEQ ID NO:319 and SEQ ID NO:320. SEQ ID NO:319 contained a 5’ flanking sequence (SEQ ID NO:317) of the gene encoding the proposed oxidoreductase / dehydrogenase enzyme of SEQ ID NO:316, the ScURA3 promoter (SEQ ID NO:216), and a 5' portion of the ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:320 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), and a 3’ flanking sequence (SEQ ID NO:318) of the gene encoding the proposed oxidoreductase / dehydrogenase enzymes of SEQ ID NO:316. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for deletion of the candidate gene. PCR verified isolates were designated strains 4-6a, 4-6b, and 4-6c.Strain 4-7

[0165] Strain 2-26 was transformed with the Cre recombinase plasmid of SEQ ID NO:292. Removal of the ScURA3 selection marker gene was confirmed by lack of growth on ScD-ura plates. A verified isolate was designated strain 4-7.Strain 4-8

[0166] Strain 4-7 was transformed wdth SEQ ID NO:295 and SEQ ID NO:296. Transformants were plated on ScD-URA plates, and a single colony was selected. The selected colony was evaluated by colony PCR and the PCR verified isolate was designated strain 4-8.Strain 4-9

[0167] Strain 4-8 was transformed wdth the Cre recombinase plasmid of SEQ ID NO:292. Removal of the ScURA3 selection marker gene was confirmed by lack of growth on ScD-ura plates. A verified isolate was designated strain 4-9.PT-2209-WO-PCTStrain 4-10a-c

[0168] Strain 4-7 was transformed with SEQ ID NO:323 and SEQ ID NO:342. SEQ ID NO:323 contained a 5' CYB2 flanking sequence (SEQ ID NO:212), the ENO1 terminator (SEQ ID NO:288), a poly nucleotide sequence encoding the talitol dehydrogenase enzyme of SEQ ID NO:304, the ScPDCl promoter (SEQ ID NO:220), the ScURA3 promoter (SEQ ID NO:216), and a 5’ portion ofthe ScURA3 selection marker gene (SEQ ID NO:215). SEQ ID NO:324 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:214), the ScURA3 terminator (SEQ ID NO:217), and a 3’ CYB2 flanking sequence (SEQ ID NO:213). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for deletion of the candidate gene. PCR verified isolates were designated strains 4- 10a, 4- 10b, and 4- 10c.Strain 4-lla-f

[0169] Strain 4-9 was transformed with SEQ ID NO: 321 and SEQ ID NO: 322. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for deletion of the candidate gene. PCR verified isolates were designated strains 4-l la, 4-1 lb, 4-llc, 4-1 Id, 4-l le, and 4-1 If.Example 17: Flower Plate Fermentation Assay

[0170] Strains 4-1, 4-3a-c, 4-4a-c, 4-5a-c, and 4-6a-c were assayed to evaluate production of D-allulose and talitol. Strains were struck on ScD-ura plates and grown until single colonies formed (1-3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a 48-well flower plate containing 1000 microliters of ScD-Ura media (6.7g / L yeast nitrogen base without amino acids, 2.0 g / 1 Synthetic Complete amino acid mix, 20 g / L glucose), and incubated for 24 hours at 30°C and 900 RPM in an orbital shaker. The overnight cultures were transferred to a 48 well flower plate containing 1000 microliters of DMU1 media (Table 4, maltodextrin replaced with 200 g / L dextrose and no glucoamylase added) to an OD600 = 0.2, incubated for 48 hours at 30°C, 80% relative humidity, and 900 RPM in an orbital shaker. Samples were taken for analysis by HPIC to determine D-allulose and talitol titers. Results are shown in Table 25.

[0171] Strains 4-5a-c, in which the native K. marxianus talitol dehydrogenase gene encoding the talitol dehydrogenase enzyme of SEQ ID NO:304 was deleted, showed a significant decrease in talitol production and increased D-allulose production relative to control strain 4-1. TalitolPT-2209-WO-PCT production was reduced to the point that any production was at or below the level of detection of the HPIC instrument. For strain 4-5 a, a small concentration w as detected for talitol, but rounding resulting in a reported value of 0.00.Table 25.nd - not detectedExample 18: Flower Plate Fermentation Assay

[0172] Strains 2-26, 4-8, 4-10a-c, and 4-l la-f w ere assayed to evaluate production of talitol and D-allulose. Strains were struck on ScD-ura plates and grown until single colonies formed (1- 3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a 48-well flower plate containing 1000 microliters of ScD-Ura media (6.7g / L yeast nitrogen base without amino acids, 2.0 g / 1 Synthetic Complete amino acid mix, 20 g / L glucose), and incubated for 24 hours at 30°C and 900 RPM in an orbital shaker. The overnight cultures were transferred to a 48 well flower plate containing 1000 microliters of DMU1 mediaPT-2209-WO-PCT containing (Table 4) (i) 200 g / L dextrose; (ii) 250 g / L dextrose, or (iii) 300 g / L dextrose (indicated in Tables 27 and 26 below) to replace the maltodextrin, with no added glucoamylase, to an OD600 = 0.2, incubated at 30°C, 80% relative humidity, and 900 RPM in an orbital shaker. Samples were taken at 120 hours for analysis by HPIC to determine D-allulose and talitol titers (Table 26). Samples were taken at 196 hours for analysis by HPLC to determine combined D-allulose and talitol titers (Table 27). By HPLC analysis, D-allulose and talitol elute in the same peak, so the concentration value given is for the total of D-allulose and talitol.

[0173] Strains 4-l la-f, in which the native K. marxianus talitol dehydrogenase gene was deleted, produced significantly less talitol production and increased D-allulose production than control strains 2-26 and 4-8. This result was consistent across all fermentation medium tested. Strains 4-10a-c, in which the native ?, marxianus talitol dehydrogenase gene was overexpressed by integration of an additional copy of the gene at the CYB2 locus, shows increased talitol and decreased D-allulose production relative to control strains 2-26 and 4-8.Table 26.PT-2209-WO-PCTTable 27.PT-2209-WO-PCT

Claims

PT-2209-WO-PCTCLAIMSWhat is claimed is:

1. A genetically engineered Kluyveromyces marxianus cell capable of producing D-allulose, the engineered cell comprising a deletion or disruption of a native talitol dehydrogenase gene; an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme 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:249-256, 258. and 259; and an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme 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:87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117. 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183; wherein talitol production is reduced relative to af. marxianus strain with wild-type level expression of the native talitol dehydrogenase gene.

2. The engineered cell of claim 1, wherein the native talitol dehydrogenase gene encodes a talitol dehydrogenase enzyme is 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:304; the native talitol dehydrogenase gene encodes a talitol dehydrogenase enzy me 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: 304; and / or the native talitol dehydrogenase gene encodes a talitol dehydrogenase enzyme at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 304.

3. The engineered cell of any preceding claim, wherein the native talitol dehydrogenase gene is 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:305; wherein the native talitol dehydrogenase gene is 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:305; and / or wherein the native talitol dehydrogenase gene is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 305.PT-2209-WO-PCT4. The engineered cell of any preceding claim, wherein the allulose-6-phosphate phosphatase is 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:87, 89, 190, 123, 105. 107, 115, 83, 95, 113, 117, 119, 121, 127. 131, 137, 145, 169, 173. 179, and 183; wherein the allulose-6-phosphate phosphatase enzyme is 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:87, 89, 190, 123, 105, 107, and 115; wherein the allulose-6-phosphate phosphatase enzyme is 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:87, 89, and 190; or wherein the allulose-6-phosphate phosphatase enzyme is 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:87 and 89.

5. The engineered cell of any preceding claim, wherein the allulose-6-phosphate 3-epimerase is at least 85%, or at least 90% identical to at least one of SEQ ID NOs: 249-256. 258, and 259; the allulose-6-phosphate epimerase is at least 85%, or at least 90% identical to at least one of SEQ ID NOs:249, 250, 251, 254, 255, 256, and 257; and / or the allulose-6-phosphate epimerase is at least 85%, or at least 90% identical to at least one of SEQ ID NOs:

249. 250, 255, or 256.

6. The engineered cell of any preceding claim, wherein the allulose-6-phosphate 3-epimerase is at least 90% identical to at least one of SEQ ID NO:249, 250, 251, 254, 255. 256, and 257; and / or the allulose-6-phosphate phosphatase is at least 90% identical to at least one of SEQ ID NO: 87 and 89.

7. The engineered cell of any preceding claim, wherein the engineered cell comprises a deletion or disruption of a native phosphofructokinase- 1 (PFK1) gene.

8. The engineered cell of claim 7,PT-2209-WO-PCT wherein the native PFK1 gene encodes a PFK1 enzyme 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:244; wherein the native PFK1 gene encodes a PFK1 enzy me 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:244; wherein the native PFK1 gene encodes a PFK1 enzyme at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:244; wherein the native PFK1 gene is 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:245; wherein the native PFK1 gene is 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:245; and / or wherein the native PFK1 gene is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:245.

9. The engineered cell of any preceding claim, 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 NOs: 249, 250, 251, 254, 255, 256, and 257; 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 NOs: 87, 89, 190, 123. 105, 107, and 115; wherein the cell is capable of producing at least 10 g / L D-allulose, and wherein talitol production is reduced relative to a K. mcirxictnus strain with wild-type of expression of the native talitol dehydrogenase gene.

10. The engineered cell of any preceding claim, 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: 249, 250, 251, 254, 255, 256, and 257; wherein the allulose-6-phosphate phosphatase is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 105; wherein the cell is capable of producing at least 10 g / L D-allulose; and wherein talitol production is reduced relative to a K. marxianus strain with wild-type of expression of the native talitol dehydrogenase gene.

11. The engineered cell of any preceding claim, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous promoter and / or a heterologous terminator.

12. The engineered cell of claim 11,PT-2209-WO-PCT 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 (ADHlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFlp); phosphoglucomutase 1 promoter (PGMl p); 3-phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B; and PDC1 promoter; and / or wherein the terminator is selected from the group consisting of GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); fructose-bisphosphate aldolase terminator (FBAlt); TEF1; iso- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator.

13. A method for producing D-allulose, the method comprising: contacting a substrate with the engineered cell of any preceding claim, wherein the engineered cell produces 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, and wherein the final talitol titer is less than 1 g / L, less than 0.75 g / L, less than 0.5 g / L, less than 0.25 g / L, or less than 0. 1 g / L after 72 hours.

14. The method of claim 13, wherein the substrate comprises starch, glucose, sucrose, cellulosic biomass, or combinations thereof.

15. Use of the engineered cell of any one of claims 1-12 to produce D-allulose.

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