Genetically modified yeast and fermentation process for xylitol production

Abstract

This article discloses genetically engineered yeast cells capable of producing xylitol and containing a foreign polynucleotide sequence encoding a ZWF enzyme that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to at least one of SEQ ID NO: 15, 16, and 23. The genetically engineered yeast cells can be further engineered to overexpress native RPE enzymes, foreign XPDH enzymes, foreign XKS enzymes, foreign XDH enzymes, native X5PP enzymes, and / or foreign X5PP enzymes.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 594,062, filed October 30, 2023, which is incorporated herein by reference in its entirety.

[0003] Refer to the sequence list submitted through the Patent Center

[0004] The contents of the sequence list XML file named "PT-1794-WO-PCT.xml", which is 377,000 bytes in size and was created on October 28, 2024 and submitted electronically with this application through the Patent Centre, are incorporated herein by reference in full. Background Technology

[0005] Xylitol is a low-calorie sweetener used as a food additive and sugar substitute. Commonly used in pharmaceuticals, dietary supplements, confectionery, and toothpaste compositions, xylitol is also associated with anti-caries properties when used in chewing gum. Traditional methods of xylitol production, including the chemocatalytic hydrogenation of xylose from the hydrolysis of xylan extracted from biomass, are expensive both financially and environmentally. These methods require high temperatures and pressures, large quantities of water, and metal catalysts that must be mined. In contrast, fermentation processes are already commercially available on a large scale to produce other organic molecules such as ethanol, citric acid, and lactic acid, and can provide a cost-effective and sustainable alternative to traditional xylitol production methods.

[0006] Therefore, this article provides a genetically modified yeast and fermentation method for the production of xylitol. Summary of the Invention

[0007] This disclosure provides a genetically engineered yeast cell capable of producing xylitol, the engineered yeast cell containing an exogenous polynucleotide sequence encoding a glucose-6-phosphate dehydrogenase (ZWF) enzyme that is 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 at least one of SEQ ID NO: 15, 16, and 23. The ZWD enzyme may be at least 85% identical to SEQ ID NO: 15 or 16. The yeast cell may be an osmotically resistant yeast cell. The yeast cell may be a cell of the Ustilaginomycotina subphylum. Yeast cells can be selected from the group consisting of the following: Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. When engineered cells are used in a fermentation process in the presence of dextran, the yield of polyol production increases relative to the yield of polyols in an equivalent fermentation process using equivalent cells that do not express the ZWF enzyme, wherein the ZWF enzyme is 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 at least one of SEQ ID NO: 15, 16, and 23.

[0008] The yeast cell may be a *Moniliella pollinis* cell, and additionally contains a deletion or disruption of a gene encoding erythrose reductase that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to at least one of SEQ ID NO: 60 and 62. The cell may have the deletion of at least one allele encoding erythrose reductase. The cell may have the deletion of two alleles encoding erythrose reductase. The gene encoding erythrose reductase may be at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to at least one of SEQ ID NO: 61 and 63.

[0009] The cell may additionally contain an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme, which contains 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 sequence to at least one of SEQ ID NO: 5, 95-101, and 103. The XPDH enzyme may have at least 85% identical sequence to at least one of SEQ ID NO: 5, 87, 98, or 101. The XPDH enzyme has at least 90% identical sequence to at least one of SEQ ID NO: 12-15, 28-31, and 33, or at least one of SEQ ID NO: 5, 97, 98, or 101.

[0010] The cell may additionally contain an exogenous polynucleotide sequence encoding a xylulokine (XKS) enzyme, wherein the xylulokine (XKS) enzyme comprises 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 at least one of SEQ ID NO: 90 and 91. The cell may additionally contain an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme, wherein the xylitol dehydrogenase (XDH) enzyme comprises 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 at least one of SEQ ID NO: 104-106.

[0011] The cell may additionally contain genetic modifications that result in the overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity. The yeast cell may be a *Bacillus* genus yeast cell. The yeast cell may be a *Bacillus* genus yeast cell, and the native RPE enzyme contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical sequence to at least one of SEQ ID NO: 46 and 94. Genetic modifications resulting in the overexpression of the native RPE enzyme include the addition of a foreign polynucleotide encoding the native RPE enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the RPE enzyme.

[0012] The yeast may additionally include genetic modifications that result in the overexpression of a native enzyme having xylitol-5-phosphate phosphatase (X5PP) activity; and / or a foreign polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity. The cell may be a *Saccharomyces cerevisiae* cell, and the genetic modification results in the overexpression of a native X5PP enzyme having a sequence that is 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 at least one of SEQ ID NO: 74, 88, 92, and 93. The genetic modification may include the addition of a foreign polynucleotide sequence encoding a native X5PP enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the native X5PP enzyme. Yeast cells may contain a foreign polynucleotide sequence encoding an enzyme having X5PP activity, and a sequence that is 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 at least one of SEQ ID NOs: 74-91. Yeast cells may contain a foreign polynucleotide sequence encoding an enzyme having X5PP activity, and a sequence that is 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 at least one of SEQ ID NOs: 74, 76, 77, 78, 79, 80, 84, and 86. Yeast cells contain an exogenous polynucleotide sequence encoding an enzyme with X5PP activity, and a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 74, 77, 78, 80, and 86. X5PP activity in genetically engineered yeast cells may be higher than that in equivalent cells lacking genetic modification or the exogenous polynucleotide sequence.

[0013] One or more of the above-mentioned exogenous polynucleotide sequences can be operatively linked to a heterologous promoter or an artificial promoter. Heterologous or artificial promoters may be selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 6), 6-phosphoglucuronide dehydrogenase promoter (6PGDp; SEQ ID NO: 107), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 108), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 109), phosphoglucose mutase 1 promoter (PGM1p; SEQ ID NO: 110), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 111), enolase promoter (ENO1p; SEQ ID NO: 112), asparagine synthase promoter (ASNSp; SEQ ID NO: 113), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 114), and RPL16B (SEQ ID NO: 115). The promoter may be a constitutive promoter. One or more exogenous polynucleotide sequences are integrated into the genome of yeast cells at loci selected from ER1, ER3, PDC1, pyrF, TRP3, gpdIIA, and gpdIIB.

[0014] This disclosure also provides a method for producing xylitol using engineered cells described herein, the method comprising contacting a substrate containing dextran with the engineered cells described herein, wherein the engineered cells ferment the substrate to produce xylitol. The fermentation temperature may be from 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, or between therewith. The volumetric oxygen uptake (OUR) may be between 5 mmol O2 / (L • h) and 80 mmol O2 / (L • h), between 10 mmol O2 / (L • h) and 75 mmol O2 / (L • h), between 15 mmol O2 / (L • h) and 70 mmol O2 / (L • h), between 20 mmol O2 / (L • h) and 60 mmol O2 / (L • h), between 30 mmol O2 / (L • h) and 50 mmol O2 / (L • h), or between 40 mmol O2 / (L • h) and 50 mmol O2 / (L • h). Xylitol can be produced at rates of at least 0.2 g L⁻¹ h⁻¹, 0.3 g L⁻¹ h⁻¹, 0.5 g L⁻¹ h⁻¹, 0.75 g L⁻¹ h⁻¹, or at least 1.0 g L⁻¹ h⁻¹. When fermentation is run at 35°C for 96 hours, xylitol yields can be at least 20 g / L, 30 g / L, 50 g / L, 75 g / L, or 100 g / L. The concentration of dextrose can be at least 100 g / L. The xylitol yield from fermentation is higher than that of the same fermentation process run using equivalent yeast lacking the exogenous polynucleotide sequence encoding the ZWF enzyme.

[0015] This disclosure also provides for the use of the engineered cells described herein in the production of xylitol. Attached Figure Description

[0016] This patent or application contains at least one color-drawn drawing. A copy of this patent or patent application publication with a color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0017] The accompanying figures illustrate, in a manner that is not restrictive, various aspects described herein.

[0018] Figure 1 The natural pentose phosphate pathway (dashed lines and arrows) and natural glycolysis pathway (solid lines and arrows) in *Saccharomyces cerevisiae* are shown.

[0019] Figure 2 The erythritol, ribitol, and xylitol titers of the reaction outlined in Example 3 are shown.

[0020] Figure 3 The erythritol, ribitol, and xylitol titers of the reaction outlined in Example 4 are shown.

[0021] Figure 4 The erythritol, ribitol, and xylitol titers of the reaction outlined in Example 6 are shown.

[0022] Figure 5 The erythritol, ribitol, and xylitol titers of the reaction outlined in Example 7 are shown. Detailed Implementation

[0023] Reference will now be made specifically to certain aspects of the subject matter disclosed herein, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0024] In this document, unless the context clearly requires otherwise, the terms “a,” “an,” or “the / described” are used to include one or more. Unless otherwise indicated, the term “or” is used to mean a non-exclusive “or.” All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety as if individually cited. In the event of any inconsistency between the usage in this document and those documents so incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of irreconcilable inconsistencies, the usage in this document shall prevail.

[0025] Values ​​expressed in range format should be interpreted flexibly to include not only the values ​​explicitly listed as limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise stated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise stated, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0026] Unless otherwise specified, ppm (parts per million), percentages, and ratios are based on weight. Percentages based on weight are also referred to below as weight% or (weight)%.

[0027] This disclosure relates to various recombinant cells engineered for the production of xylitol. Generally, the recombinant cells described herein are capable of producing xylitol and contain a foreign polynucleotide sequence encoding a ZWF enzyme. The recombinant yeast may further be characterized by overexpression of a ribulose-5-phosphate epimerase (RPE); containing a foreign polynucleotide sequence encoding a xylitol phosphate dehydrogenase (XPDH); containing a foreign polynucleotide sequence encoding a xylitol kinase (XKS); containing a foreign polynucleotide sequence encoding a xylitol dehydrogenase (XDH); and / or overexpression of a xylitol-5-phosphate phosphatase (X5PP). This disclosure also provides a fermentation method for producing xylitol from dextran using the genetically engineered cells described herein.

[0028] Generally speaking, the recombinant cells described herein are yeast cells. As used herein, “yeast” refers to a eukaryotic unicellular microorganism classified as a member of the kingdom Fungi. Yeasts are unicellular organisms that evolved from multicellular ancestors, and some species retain multicellular characteristics, such as forming connected budding cell chains called pseudohyphae or false hyphae. Yeast cells are also referred to in the art as yeast-like cells, and as used herein, “yeast cell” encompasses both yeast and yeast-like cells. Suitable yeasts and yeast-like host cells used for modification may include, but are not limited to: *Saccharomyces cerevisiae*, *Komagataella* sp., *Kluyveromyces* (e.g., *Kluyveromyces lactis*, *Kluyveromyces marxianus*), oil-producing yeasts, *Issatchenkia orientalis*, *Pichia galeiformis*, *Pichia* YB-4149 (NRRL name), *Pichia pastoris*, *Candida* (e.g., *Candida magnolia*, *Candida ethanolica*), *Pichia deserticola*, *Pichiamembranifadens*, and *Pichia fermentans*. Fermentans, Aspergillus, Trichoderma, Myceliphthora thermophila, Cladosporium (e.g., Cladosporium spp.), Pfaffia, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., Trichosporonoides macrocarpa, Trichosporonoides spp., Trichosporonoides melanogaster), Tsukuba spp., Trichosporonoides var. mutabilis, Penicillium, and Cyclosporium. Those skilled in the art will understand that the requirements for selecting suitable yeast cells and the recombinant yeast cells of this disclosure are not limited to those explicitly listed herein. Methods for genetic engineering yeast cells are known and described in the art, and those skilled in the art will understand the methods necessary for transforming and engineering suitable yeast cells.

[0029] Suitable yeast cells can be Basidiomycota or Ustilago smut. Suitable yeasts of the Ustilago subphylum include, but are not limited to, *Ustilago* (e.g., *U. cynodontis*, *U. maydis*, *U. sphaerogena*, *U. cordal*, *U. scitaminea*, *U. coicis*, *U. syntherismae*, *U. esculenta*, *U. neglecta*, *U. crus-galli*, *Ustilago avenae*), *Sporisorium* (e.g., *Sporisorium exsertum*), and *M. tomentosa* (e.g., *M. acetoabutans*, *M. fonsecae*, *M.*). Madida, *M. megachiliensis*, *M. ocedocephalis*, *M. nigrescens*, and *Pseudozyma* (e.g., *Pseudozyma tsukuba*) and *Pseudozyma* species (e.g., *Pseudozyma megachiliensis*, *Pseudozyma ocedocephalis*, *Pseudozyma nigrescens*). Yeasts of the subphylum Ustilagoeum are known and described in the art as potential production organisms for valuable chemicals such as itaconic acid esters, malate esters, succinate esters, mannitol, and erythritol, as well as other valuable biotechnological applications.See, e.g., Geiser et al. 97(8):3253-65), Guevarra et al. ("Accumulation of itaconic, 2-hydroxyparaconic, itatartaric, and malic acids by strains of thegenus Ustilago, Agric.Biol.Chem., 1990, 54(9), 2353-2358) and Moon et al. ("Biotechnological production of erythritol and its applications,” ApplMicrobiol Biotechnol, 2010, 86:1017-1025).

[0030] Suitable yeast cells will possess an active pentose phosphate pathway for the production of ribulose-5-phosphate. As used herein, the "active pentose phosphate pathway" refers to the expression of one or more functional enzymes that together produce glucose-6-phosphate, NADP... + Or NAD+ (NAD(P)) + The enzyme converts 5-phosphate (NP) and water into NADPH or NADH (NAD(P)H), CO2, and ribulose-5-phosphate. Continuing in the non-oxidative phase, this pathway can also produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway can produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, and combinations thereof. The active pentose phosphate pathway can be natural in yeast cells or can be introduced into yeast cells through genetic engineering.

[0031] Yeast cells can be osmolyzed yeast cells. As used herein, “osmolyzed” means yeast capable of growing and reproducing under conditions of high molar osmolar concentrations, such as at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v), or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), or at least 15% (w / v) sodium chloride. Species and strains of osmolyzed yeast are known and described in the art, including many species of yeast used in industrial fermentation processes. Similarly, methods for determining yeast osmolyzability are known and described in the art. See, for example, Tiwari, S. et al. (“Nectar yeast community of tropical flowering plants and assessment of their osmotolerance and xylitol-producing potential,” Current Microbiology, 2022, 79:28).

[0032] Recombinant yeast cells can be recombinant moniliform cells, such as moniliform yeast cells. Figure 1 The predicted natural pentose phosphate and glycolysis pathways in *Fragaria filamentous fungi* are shown. *Fragaria filamentous fungi* has been previously used in the fermentation production of erythritol, and methods for genetic modification and fermentation of *Fragaria filamentous fungi* are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb CellFact, 16:168).

[0033] Various plasmids and methods for transforming *Strombophytum comosum* are also described in the following examples. For example, *Strombophytum comosum* can be transformed using a dimeric polynucleotide sequence, wherein, after recombination, the exogenous polynucleotide of interest is integrated at a specific locus and a selection marker is expressed intracellularly. Suitable selection markers are known and used in the art. Optional markers may include, but are not limited to, amdS (e.g., broken into 3' parts SEQ ID NO: 54 and 5' parts SEQ ID NO: 55), G418 resistance gene (e.g., broken into 3' parts SEQ ID NO: 9 and 5' parts SEQ ID NO: 8), zeocin resistance gene (e.g., broken into 3' parts SEQ ID NO: 47 and 5' parts SEQ ID NO: 48), nourseothricin N-acetyltransferase (NAT) (e.g., broken into 3' parts SEQ ID NO: 57 and 5' parts SEQ ID NO: 56), and invertase gene (SUC2) (e.g., the 3' part of SEQ ID NO: 58 and the 5' part of SEQ ID NO: 59).

[0034] The recombinant cells described herein contain one or more polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve the recombinant cells' ability to ferment glucose into xylitol.

[0035] The terms “glucose” and “dextrose” are used interchangeably in this document and refer to D-glucose unless otherwise explicitly stated.

[0036] As used herein, “exogenous” refers to genetic material or its expression product originating outside the host organism. For example, exogenous genetic material or its expression product can be a modified form of the host organism’s natural genetic material, it can 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, when introduced into Saccharomyces cerevisiae, the Kluyveromyces lactis invertase gene is exogenous.

[0037] As used herein, “natural” means genetic material or its expression products found in the genome of wild-type cells of a host cell, except for inter-individual mutations that do not affect function or expression. For the purposes of this application, the Moniliella tomentosa var pollinis TCV364 cells described in US 6,440,712 are considered wild-type Moniliella tomentosa cells, which are incorporated herein by reference in their entirety and were deposited on March 28, 1997, under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800Vilvoorde).

[0038] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the total primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the macromolecule its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (independently or as part of a biosynthetic pathway) should be understood to include cofactors, coenzymes, and metals necessary for the enzyme to function properly. An overview of amino acids and their three-letter and one-letter symbols as understood in the art is provided in Table 1. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0039] Table 1: Three-letter and one-letter symbols for amino acids

[0040]

[0041] Variants or sequences having substantially the same identity or homology with the polypeptides described herein can be used to implement the disclosed recombinant cells, compositions, and methods. Such sequences may be referred to as variants or modified sequences. That is, the polypeptide sequence can be modified but still retains the ability to exhibit the desired activity. Typically, variants or modified sequences may include sequence identity greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% with wild-type, naturally occurring polypeptide sequences or with variant polypeptides as described herein.

[0042] As used herein, the phrases “sequence identity %”, “identity %”, and “identity percentage” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a normalized algorithm. Methods for amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and sequence identity generation involve both global and local alignments performed using computational methods. Alignment can be performed using BLAST (National Center for Biotechnology Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using a standard protein BLAST with the following default parameters: Maximum target sequence: 100; Short query: Parameters automatically adjusted for short input sequences; Expected threshold: 10; Word length: 6; Maximum number of matches within the query range: 0; Matrix: BLOSUM62; Empty penalty: (Existence: 11, Expansion: 1); Component adjustment: Conditional component score matrix adjustment; Filter: Not selected; Mask: Not selected. The standard nucleotide BLAST algorithm with the following default parameters can be used to determine the percentage of nucleic acid sequence identity between nucleic acid sequences: Maximum target sequence: 100; Short query: Parameters are automatically adjusted for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within the query range: 0; Match / mismatch score: 1, -2; Gaps penalty: Linear; Filter: Low complexity region; Mask: Mask only for lookup tables. Sequences that have an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently have XX% sequence identity with the reference sequence.

[0043] Peptide or polynucleotide sequence identity can be measured over the entire length of a defined peptide sequence (e.g., as defined by a specific SEQ ID number) or over a shorter length, such as over a fragment taken from a larger defined peptide sequence (e.g., a fragment of at least 15, 20, 30, 40, 50, 70, or 150 consecutive residues). Such lengths are merely exemplary and it should be understood that any fragment length supported by the sequences shown in this document, tables, figures, or sequence listings can be used to describe the length of measurable percentage identity.

[0044] The polypeptides disclosed herein may include “variant” polypeptides, “mutants”, and “derivatives thereof.” As used herein, the term “wild-type” is a term understood by those skilled in the art and refers to the typical form of the polypeptide in nature, as distinct from variant or mutant forms. As used herein, “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence different from that of a reference protein or polypeptide molecule. Variants or mutants may have insertions, deletions, or substitutions of one or more amino acid residues relative to the reference molecule.

[0045] The amino acid sequences of peptide variants, mutants, derivatives, or fragments considered herein may include conserved amino acid substitutions relative to a reference amino acid sequence. For example, variant, mutant, derivative, or fragment peptides may include conserved amino acid substitutions relative to a reference molecule. A “conserved amino acid substitution” is one in which an amino acid is substituted to a different amino acid that is predicted to have the least interference with the properties of the reference peptide. In other words, a conserved amino acid substitution essentially preserves the structure and function of the reference peptide. Conserved amino acid substitutions typically maintain (a) the structure of the peptide backbone in the substituted region, such as a β-sheet or α-helical conformation, (b) the charge and / or hydrophobicity of the molecule at the substitution site, and / or (c) the volume of the side chains.

[0046] As used herein, the terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence” are used interchangeably with “nucleic acid” and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may indicate a sense strand or antisense strand. DNA polynucleotides may be cDNA (e.g., encoding DNA) or genomic DNA sequences (e.g., including introns and exons).

[0047] A polynucleotide is said to encode a polypeptide if, in its natural state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce a polypeptide or a fragment thereof. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0048] Those skilled in the art understand the degeneracy of the genetic code and that multiple polynucleotides can encode the same polypeptide. In some aspects, polynucleotides (e.g., polynucleotides encoding erythrose reductase polypeptides) can be codon-optimized for expression in specific cells, including but not limited to plant cells, bacterial cells, fungal cells, or animal cells. While polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptide described herein may be used. Therefore, sequences not naturally occurring may be used. These sequences may be desired, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and the human hand can also be used to generate degenerate coding sequences.

[0049] The recombinant cells described herein may contain deletions or disruptions in one or more natural genes. The phrase "deletion or disruption" refers to the state of a natural gene in the recombinant cell, having a completely eliminated coding region (deletion) or a modification (such as by deletion, insertion, or mutation) of the gene, its promoter, or its terminator, such that the gene no longer produces an active expression product, produces a severely reduced amount of expression product (e.g., reduced by at least 75% or at least 90%), or produces a severely reduced expression product (e.g., reduced by at least 75% or at least 90%). Deletions or disruptions can be achieved through genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. Deletions or disruptions of natural host cell genes can be coupled with the incorporation of one or more polynucleotide sequences (e.g., exogenous or natural polynucleotide sequences) into the host cell at the locus of the host cell gene to be deleted or disrupted. The polynucleotide sequence to be inserted can be designed to replace all or part of the host cell gene to be deleted or disrupted. The polynucleotide sequence can encode a gene product of interest, such as a polypeptide, enzyme, etc. Deletions or disruptions can also be accomplished using deletion constructs that do not contain the polynucleotide sequence to be integrated. Other methods for gene disruption or deletion are known and described in the art.

[0050] The recombinant cells described herein have deletions or disruptions in one or more natural genes encoding enzymes involved in erythritol fermentation or consumption. Deletion or disruption of one or more of these biosynthetic pathway enzymes reduces the recombinant cells' ability to produce erythritol and, depending on the deletion or disruption, can increase the carbon flux entering the fermentation pathway for xylitol production.

[0051] The recombinant cells described herein may include the deletion or disruption of the native erythrose reductase (ER) gene. The native ER encodes an enzyme with erythrose reductase activity. As used herein, “erythrose reductase activity” and “ER activity” are used interchangeably and refer to an enzyme that catalyzes the reversible conversion of erythrose or erythrose-4-phosphate to erythritol or erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor. In the art, an enzyme that catalyzes the reversible conversion of erythrose-4-phosphate to erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor may also be described as an erythrose or erythritol phosphate dehydrogenase. When the host cell contains multiple ER genes, it is preferred that at least one of them is deleted or disrupted. When the host cell contains multiple alleles of a given ER gene, it is preferred that one or both alleles of the given ER gene are deleted or disrupted.

[0052] As used herein, “NAD(P)H” refers to nicotinamide adenine dinucleotide (phosphate) hydrogen and includes both NADH and NADPH. As understood in the art, the inclusion of phosphate ester (or the abbreviation “P”) in parentheses indicates that the phosphate ester may or may not be present, and the name and abbreviation include both. Similarly, “NAD(H)” or “NADP(H)” refers to both the reduced and oxidized forms of the cofactor.

[0053] When the recombinant cell is a *Bacillus cereus* cell, the recombinant cell may contain a deletion or disruption of the ER gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 60. When the recombinant cell is a *Bacillus cereus* cell, the recombinant cell may contain a deletion or disruption of the ER gene, which has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 61. See, for example, U.S. Provisional Application No. 63 / 499,990 and U.S. Provisional Application No. 63 / 499,989, filed May 4, 2023.

[0054] When the recombinant cell is a *Bacillus cereus* cell, the recombinant cell may contain a deletion or disruption of the ER gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 62. When the recombinant cell is a *Bacillus cereus* cell, the recombinant cell may contain a deletion or disruption of the ER gene, which has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 63. See, for example, U.S. Provisional Application No. 63 / 499,990 and U.S. Provisional Application No. 63 / 499,989, filed May 4, 2023.

[0055] The recombinant cells described herein may contain one or more genetic modifications, wherein exogenous nucleic acids are integrated into the genome of the host cell. Those skilled in the art know how to select suitable loci in the yeast genome for the integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in *Saccharomyces cerevisiae* host cells, suitable interacting loci may include, but are not limited to, the ER1 locus (defined as the locus flanked by SEQ ID NO: 3 and SEQ ID NO: 10), the ER3 locus (defined as the locus flanked by SEQ ID NO: 49 and SEQ ID NO: 50), the PDC1 locus (defined as the locus flanked by SEQ ID NO: 64 and SEQ ID NO: 65), the pyrF locus (defined as the locus flanked by SEQ ID NO: 66 and SEQ ID NO: 67), the TRP3 locus (defined as the locus flanked by SEQ ID NO: 68 and SEQ ID NO: 69), the gpdIIA locus (defined as the locus flanked by SEQ ID NO: 70 and SEQ ID NO: 71), and the gpdIIB locus (defined as the locus flanked by SEQ ID NO: 72 and SEQ ID NO: 73). Exogenous nucleic acids may also be integrated into intergenic regions or other locations in the host cell genome not specifically specified herein. Those skilled in the art can identify other suitable integrating loci. Furthermore, those skilled in the art will recognize how sequences can be used to design primers to verify correct gene integration at the selected locus.

[0056] Recombinant cells may have one or more copies of a given exogenous nucleic acid sequence, which is integrated into the host chromosome and replicates along with the chromosome into which it is integrated. For example, yeast cells can be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide described herein, and the polynucleotide sequence encoding the polypeptide may be integrated into the host chromosome in one or more copies. Recombinant cells may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten or more copies of the polynucleotide sequence encoding a polypeptide described herein integrated into the genome. The multiple copies of the polynucleotide sequence may be incorporated entirely into a single locus or may be incorporated into multiple loci.

[0057] The recombinant cells described herein are capable of producing xylitol and include an exogenous polynucleotide sequence encoding an exogenous enzyme with glucose-6-phosphate dehydrogenase (ZWF) activity. The enzyme can be any suitable enzyme with ZWF activity. As used herein, “ZWF” and “ZWF enzyme” are used interchangeably and refer to an enzyme with ZWF activity. In this document, “ZWF activity” and “glucose-6-phosphate dehydrogenase activity” are used interchangeably and refer to the reversible catalysis of glucose-6-phosphate and NAD(P)… + The ability to convert to phosphogluconide and NAD(P)H. Suitable enzymes with ZWF activity may include, but are not limited to, those classified as EC1.1.1.388, EC 1.1.1.49, and EC 1.1.1.363. The polynucleotide encoding the ZWF enzyme may be derived from any suitable source. For example, the polynucleotide encoding the ZWF enzyme may be derived from *Pseudomonas putida*, *Pseudomonas putida*, *Pantoea sp.* Ap-967, etc. The ZWF enzyme may be a polypeptide having an amino acid sequence that 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 at least one of SEQ ID NO: 15, 16, and 23. ZWF enzymes can be polypeptides having an amino acid sequence that 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 the amino acid sequence of at least one of SEQ ID NO: 15 and 16. ZWF enzymes can be polypeptides having an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NO: 15 and 16.

[0058] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23, derived from the *Saccharomyces cerevisiae* ZWF enzyme sequence. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 23. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 23.

[0059] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence that is, or may be derived from, a *Pseudomonas putida* gene encoding the amino acid sequence of SEQ ID NO: 15. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 15. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 15.

[0060] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence that is, or can be derived from, the pan-Bacteria genus Ap-967 encoding the amino acid sequence of SEQ ID NO: 16. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 16. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 16.

[0061] The recombinant cells described herein may additionally contain one or more enzymes in the xylitol biosynthesis pathway. For example, the recombinant cells may include one or more of the following: (i) genetic modifications leading to overexpression of the native X5PP enzyme; (ii) a foreign polynucleotide sequence encoding a foreign X5PP enzyme; (iii) genetic modifications leading to overexpression of the native RPE enzyme; (iv) a foreign polynucleotide sequence encoding a foreign XPDH enzyme; (v) a foreign polynucleotide sequence encoding a foreign XKS enzyme; and (iv) a foreign polynucleotide sequence encoding a foreign XDH enzyme.

[0062] The final step in the xylitol pathway, from xylitol 5-phosphate to xylitol, requires a phosphatase. Saccharomyces cerevisiae PYP1 (polyol phosphatase) is an example. 1The gene encodes a sugar alcohol phosphatase that hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (D)-glycerol-3-phosphate (Xu et al., “Discovery and functional characterization of a yeast sugaralcohol phosphatase,” ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova et al., “Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae,” J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the sorbitol-6-phosphatase class (Enzyme Commission (EC) 3.1.3.50). Since xylitol 5-phosphate is a molecule similar to known substrates of PYP1, this paper demonstrates that one or more PYP-like enzymes or PYP orthologs possess xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells described herein. *Escherichia coli* HxpA (hexitol phosphatase A) is a HAD-like enzyme belonging to EC3.1.3.50, with a substrate profile similar to PYP1 (Kuznetsova et al., “Genome wide analysis of substrate specificities of the Escherichia coli haloaciddehalogenase-like phosphate family,” 2006, J. Biol. Chem., 281, 36149-36161). Therefore, this paper also demonstrates that one or more HAD-like hydrolases or HAD-like hydrolases or their orthologs possess xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells described herein.

[0063] The recombinant cells described herein are capable of producing xylitol, contain an exogenous polynucleotide sequence encoding a ZWF enzyme, and are characterized by overexpression of a natural enzyme with xylitol-5-phosphate phosphatase (X5PP) activity and / or contain an exogenous polynucleotide sequence encoding a natural or exogenous enzyme with xylitol-5-phosphate phosphatase (X5PP) activity. Generally, recombinant cells including overexpression of X5PP enzymes or expressing exogenous X5PP enzymes produce more xylitol compared to equivalent cells lacking exogenous X5PP enzymes or lacking overexpression of X5PP enzymes. The enzyme can be any suitable enzyme with X5PP activity. As used herein, “X5PP enzyme” and “X5PP” are interchangeable and refer to an enzyme with X5PP activity. As used herein, “xylitol-5-phosphate phosphatase activity” and “X5PP activity” are used interchangeably and refer to the ability to catalyze the conversion of xylitol-5-phosphate to xylitol and phosphate. Suitable X5PP enzymes may include divalent metal cations, such as Mg2+. 2+ Mn 2+ or Co 2+Suitable enzymes with X5PP activity may include, but are not limited to, those classified as EC 3.1.3.50, such as sugar alcohol phosphatases and HAD-like hydrolases. The polynucleotide encoding the X5PP enzyme may be derived from any suitable source. For example, polynucleotides encoding the X5PP enzyme can be derived from *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Lachancea dasiensis*, *Tetrapisispora blattae*, *Saccharomyces pastorianus*, *Kazachstania Africana*, *Podospora comata*, *Geotrichum candidum*, *Ogattaea haglerorum*, *Debaryomyces fabryi*, *Monilinia fructicola*, *Nadsonia fulvescens* longleaf variant DSM 6958, *Escherichia coli*, *Wickerhamomyces ciferrii*, and *Bacillus amyloliquefaciens*. X5PP enzyme can be a polypeptide having an amino acid sequence that 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 the amino acid sequence of at least one of SEQ ID NOs: 74, 75, 76, 77, 78, 79, 80, 84, and 86. X5PP enzymes can be polypeptides having an amino acid sequence that 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 the amino acid sequence of at least one of SEQ ID NOs: 74, 77, 78, 80, and 86. See, for example, U.S. Provisional Application No. 63 / 499,992, filed May 4, 2023, and incorporated herein by reference in its entirety.

[0064] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or can be derived from the *Saccharomyces cerevisiae* gene encoding the amino acid sequence of SEQ ID NO: 74. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 74.

[0065] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or can be derived from a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO: 75. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 75.

[0066] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Saccharomyces d'Arachnium* gene encoding the amino acid sequence of SEQ ID NO: 76. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 76.

[0067] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Tetradisporum praecox* gene encoding the amino acid sequence of SEQ ID NO: 77. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 77.

[0068] The recombinant cells are capable of producing xylitol, containing a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or can be derived from a Pasteurella gene encoding the amino acid sequence of SEQ ID NO: 78. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 78.

[0069] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from an African Kazakhstani yeast gene encoding the amino acid sequence of SEQ ID NO: 79. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 79.

[0070] The recombinant cells are capable of producing xylitol, containing a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Trichoderma longicornis* gene encoding the amino acid sequence of SEQ ID NO: 80. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 80.

[0071] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Geotrichum candida* gene encoding the amino acid sequence of SEQ ID NO: 81. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 81.

[0072] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Hagler's Ogata* gene encoding the amino acid sequence of SEQ ID NO: 82. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 82.

[0073] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Fabried Barry yeast* gene encoding the amino acid sequence of SEQ ID NO: 83. The foreign polynucleotide sequence may encode an amino acid sequence that is 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.

[0074] The recombinant cells are capable of producing xylitol, containing a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Streptococcus sclerotiorum* gene encoding the amino acid sequence of SEQ ID NO: 84. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 84.

[0075] The recombinant cells are capable of producing xylitol, containing a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or can be derived from the gene encoding the amino acid sequence of SEQ ID NO: 85, specifically the *Rhizopus longleaf* variety DSM 6958. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 85.

[0076] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from an *E. coli* gene encoding the amino acid sequence of SEQ ID NO: 86. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 86.

[0077] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding the ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Wickham Severus* gene encoding the amino acid sequence of SEQ ID NO: 87. The foreign polynucleotide sequence may encode an amino acid sequence that is 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.

[0078] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* gene encoding the amino acid sequence of SEQ ID NO: 88. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 88.

[0079] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the Bacillus amyloliquefaciens gene encoding the amino acid sequence of SEQ ID NO: 89. The foreign polynucleotide sequence may encode an amino acid sequence that is 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.

[0080] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* DOG2 gene encoding the amino acid sequence of SEQ ID NO: 90. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 90.

[0081] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* DOG1 gene encoding the amino acid sequence of SEQ ID NO: 91. The foreign polynucleotide sequence may encode an amino acid sequence that is 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: 91.

[0082] Enzymes with X5PP activity can be native to the host cell. For example, when the host organism is *Saccharomyces cerevisiae*, the X5PP enzyme can be an enzyme having a sequence identity of at least 50%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% with at least one of SEQ ID NOs: 74, 88, 92, and 93. Recombinant cells may contain exogenous polynucleotides encoding an X5PP enzyme having a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 97%, or 99% with at least one of SEQ ID NOs: 92, 93, 200, or 221. The recombinant cells may contain genetic modifications that increase the expression of an X5PP enzyme, wherein the X5PP enzyme is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 92, 93, 74, or 88. Genetic modifications may include, but are not limited to, inserting an additional copy of the nucleotide encoding the natural X5PP enzyme into the cell (e.g., integrating an additional copy of the X5PP-encoding polynucleotide into a non-natural locus in the cell), inserting a constitutive promoter upstream of the coding region of the natural X5PP enzyme-encoding gene in the host cell's genome, and / or modifying an existing promoter upstream of the coding region of the natural X5PP enzyme-encoding gene in the host cell's genome. Those skilled in the art will recognize that the expression of the natural X5PP enzyme-encoding gene can be increased by a variety of methods known in the art, and will be able to appropriately select and apply these methods.

[0083] As used herein, “overexpression” means that the expression level of a polypeptide is higher than that of the same polypeptide in equivalent cells in the absence of genetic modification or exogenous polynucleotides encoding the polypeptide.

[0084] The recombinant cells described herein, capable of producing xylitol and possessing an exogenous polynucleotide sequence encoding a ZWF enzyme, are further characterized by overexpression of a ribulose-5-phosphate epimerase (RPE enzyme). These recombinant cells may also contain an exogenous polynucleotide sequence encoding an X5PP enzyme and / or overexpress a native X5PP enzyme, and / or may contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have genetic modifications leading to overexpression of a native RPE enzyme, as described herein. Generally, recombinant cells including RPE enzyme overexpression produce more xylitol compared to equivalent cells lacking RPE enzyme or lacking RPE enzyme overexpression.

[0085] The recombinant cells described herein are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have genetic modifications leading to the overexpression of a native RPE enzyme. The RPE enzyme can be any suitable enzyme with ribulose-5-phosphate epimerase activity. As used herein, “ribulose-5-phosphate epimerase activity” and “RPE activity” are used interchangeably and refer to the ability to catalyze the conversion of ribulose-5-phosphate to xylitose-5-phosphate. The enzyme with RPE activity can be native to the host cell, or the RPE enzyme can be an exogenous RPE enzyme. For example, when the host organism is *Saccharomyces cerevisiae*, the RPE enzyme can be an enzyme having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the sequence identical to at least one of SEQ ID NO: 46 and 94. The recombinant cell may contain an exogenous polynucleotide encoding an RPE enzyme having at least 70%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with at least one of SEQ ID NO: 46 or 94. The recombinant cell may contain genetic modifications that increase the expression of the RPE enzyme, wherein the RPE enzyme is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to at least one of SEQ ID NO: 46 and 94. Genetic modifications may include, but are not limited to, inserting an additional copy of the nucleotide encoding the natural RPE enzyme into the cell, inserting a constitutive promoter upstream of the coding region of the natural RPE enzyme gene in the host cell's genome, and / or modifying an existing promoter upstream of the coding region of the natural RPE enzyme gene in the host cell's genome. Those skilled in the art will recognize that the expression of the natural RPE enzyme gene can be increased by a variety of methods known in the art, and will be able to appropriately select and apply these methods. See, for example, PCT application number PCT / US2023 / 066631 filed on May 5, 2023.

[0086] The recombinant cells described herein are capable of producing xylitol, possess a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide sequence encoding a xylitol-phosphoric acid dehydrogenase (XPDH) enzyme. The foreign polynucleotide sequence may be a foreign xylitol-phosphoric acid dehydrogenase (XPDH) gene. The recombinant cells described herein capable of producing xylitol possess a foreign polynucleotide sequence encoding a ZWF enzyme and may contain a foreign polynucleotide sequence encoding an X5PP enzyme and / or overexpress a natural X5PP enzyme, may contain a foreign polynucleotide sequence encoding an XPDH enzyme, and / or may contain a foreign polynucleotide encoding a natural or foreign RPE enzyme, or may have genetic modifications leading to overexpression of a natural RPE enzyme, as described herein.

[0087] The terms "xylitol-phosphate dehydrogenase gene" and "XPDH gene" are used interchangeably in this document and refer to any gene or polynucleotide encoding a polypeptide with xylitol-phosphate dehydrogenase activity. As used herein, "xylitol-phosphate dehydrogenase activity" refers to the catalytic conversion of xylulose-5-phosphate and NADPH or NADH to xylitol-5-phosphate and NADP. + or NAD + The XPDH gene can be derived from any suitable source. For example, the XPDH gene can be derived from *Clostridium difficile*, *Lactobacillus rhamnosus*, *Bacillushalodurans*, *Alkalihalobacillus ligniniphilus*, *Jeotgalibacillus soli*, *Heyndrickxiasporothermodurans*, *Clostridium fungisolvens*, and *Neobacillus cucumis*. The XPDH gene can encode amino acids having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with at least one of SEQ ID NO: 5 and 95-103. The XPDH gene may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with at least one of SEQ ID NO: 5, 97, 98, and 101. See, for example, PCT application No. PCT / US2023 / 066629, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0088] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from a Clostridium difficile gene encoding the amino acid sequence of SEQ ID NO: 95. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 95.

[0089] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from a Clostridium difficile gene encoding the amino acid sequence of SEQ ID NO: 96. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 96.

[0090] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the Lactobacillus rhamnosus gene encoding the amino acid sequence of SEQ ID NO: 5. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5.

[0091] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from a Bacillus halophilus gene encoding the amino acid sequence of SEQ ID NO: 97. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 97.

[0092] The recombinant cell is capable of producing xylitol, possesses an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from a gene encoding the amino acid sequence of Bacillus ligninophilus encoding SEQ ID NO: 98. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 98.

[0093] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the gene encoding the amino acid sequence of SEQ ID NO: 99 from *Bacillus sphaeroides*. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 99.

[0094] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the Heindrie thermospore gene encoding the amino acids of SEQ ID NO: 100. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 100.

[0095] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from a Clostridium difficile gene encoding the amino acid sequence of SEQ ID NO: 101. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 101.

[0096] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from a gene encoding the amino acid sequence of SEQ ID NO: 103 from *Bacillus cucumberis*. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 103.

[0097] The recombinant cells described herein are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide sequence encoding a xylulose kinase (XKS) enzyme. The recombinant cells described herein capable of producing xylitol and possessing an exogenous polynucleotide sequence encoding a ZWF enzyme may additionally contain an exogenous polynucleotide sequence encoding an X5PP enzyme and / or overexpress a native X5PP enzyme, an exogenous polynucleotide sequence encoding an XKS enzyme, and / or contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have genetic modifications leading to overexpression of a native RPE enzyme, as described herein. The exogenous polynucleotide sequence may be an exogenous xylulose phosphatase (XKS) gene.

[0098] The terms “xylulose kinase gene” and “XKS gene” are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylulose kinase activity. As used herein, “xylulose kinase activity” refers to the ability to catalyze the conversion of xylulose-5-phosphate and ADP to xylulose and ATP. The XKS gene may be derived from any suitable source. For example, the XKS gene may be derived from Saccharomyces cerevisiae. The XKS gene may encode amino acids having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with at least one of the amino acid sequences in SEQ ID NO: 90 and 91. Additional description of recombinant cells capable of producing xylitol and comprising a polypeptide having xylulose kinase activity is provided in PCT application No. PCT / US2023 / 066627, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0099] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the *Saccharomyces cerevisiae* DOG1 sugar phosphatase gene encoding the amino acid sequence of SEQ ID NO: 91. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 91.

[0100] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the *Saccharomyces cerevisiae* DOG2 sugar phosphatase gene encoding the amino acid sequence of SEQ ID NO: 90. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 90.

[0101] The recombinant cells described herein are capable of producing xylitol and possess exogenous polynucleotide sequences encoding both the ZWF enzyme and the xylitol dehydrogenase (XDH) enzyme. The recombinant cells described herein capable of producing xylitol and possessing exogenous polynucleotide sequences encoding the ZWF enzyme may additionally contain exogenous polynucleotide sequences encoding xylitol dehydrogenase (XDH), XKS, X5PP, and / or overexpress natural X5PP and XKS enzymes, and / or contain exogenous polynucleotide sequences encoding natural or exogenous RPE enzymes, or may have genetic modifications leading to overexpression of natural RPE enzymes, as described herein. The exogenous polynucleotide sequence may be an exogenous XDH gene.

[0102] The terms "xylitol dehydrogenase gene" and "XDH gene" are used interchangeably in this document and refer to any gene or polynucleotide encoding a polypeptide with xylitol dehydrogenase activity. As used herein, "xylitol dehydrogenase activity" refers to the catalytic conversion of xylulose and NADH or NADPH into xylitol and NAD. + or NADP + The XDH gene can be derived from any suitable source. For example, the XDH gene can be derived from Pichia stipitis, Rhodobacteraceae bacterium, and Bemisia argentofolii. The XDH gene can encode amino acids having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with at least one of SEQ ID NO: 104, 105, and 106. Additional description of recombinant cells capable of producing xylitol and comprising polypeptides having xylitol kinase activity and polypeptides having xylitol dehydrogenase activity is provided in PCT application No. PCT / US2023 / 066627, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0103] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the cofactor-transforming Pichia pastoris XDH gene encoding the amino acid sequence of SEQ ID NO: 104. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 104.

[0104] The recombinant cell is capable of producing xylitol, possesses a foreign polynucleotide sequence encoding a ZWF enzyme, and may contain a foreign polynucleotide that is or may be derived from a gene encoding an SDR family oxidoreductase of Rhodobulbaceae bacteria encoding the amino acid sequence of SEQ ID NO: 105. The foreign polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 105.

[0105] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding a ZWF enzyme, and may contain an exogenous polynucleotide that is or may be derived from the *Bemisia argentofolii* ketose reductase (sorbitol dehydrogenase) gene encoding the amino acid sequence of SEQ ID NO: 106. The exogenous polynucleotide may encode an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 106.

[0106] The exogenous polynucleotides in the recombinant cells described herein can be controlled by a promoter. For example, exogenous nucleic acids can be operatively linked to heterologous or artificial promoters. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarbonylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 6); 6-phosphogluconic acid dehydrogenase promoter (6PGDp; SEQ ID NO: 107); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 108); translation elongation factor 1 promoter (TEFp; SEQ ID NO: 109); phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO: 110); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 111); enolase promoter (ENO1p; SEQ ID NO: 112); asparagine synthase promoter (ASNSp; SEQ ID NO: 112). 113); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 114); RPL16B (SEQ ID NO: 115); glycerol-3-phosphate dehydrogenase 2a promoter (GPDIIap; SEQ ID NO: 134); glycerol dehydrogenase NADP(H) 1 promoter (GDN1p; SEQ ID NO: 135); erythritol-P dehydrogenase 1 promoter (EPDH1; SEQ ID NO: 136); translation elongation factor 1A-like gene 7 promoter (TEF7p; SEQ ID NO: 137); heat shock protein 90 promoter (HSP90p; SEQ ID NO: 138); translation elongation factor 1A-like gene 4 promoter (TEF4p; SEQ ID NO: 139); translation elongation factor 2A-like gene 6 promoter (TEF6p; SEQ ID NO: 140); transaldolase 1 promoter (TAL1p; SEQ ID NO: 139); 141); Citrate synthase 1 promoter (CIT1p; SEQ ID NO: 142); Endoplasmic reticulum chaperone BiP (KAR2p; SEQ ID NO: 143); Heat shock protein 88 promoter (HSP88p; SEQ ID NO: 144); Heat shock protein 70 promoter (HPS70p; SEQ ID NO: 145); Tubulin α-1 chain promoter (TUB1p; SEQ ID NO: 146); Glyceraldehyde-3-phosphate dehydrogenase 2 promoter (TDH2p; SEQ ID NO: 147);The promoters of the following genes were selected: Translational elongation factor 1A-like gene 5 (TEF5p; SEQ ID NO: 148); Translational elongation factor 3 promoter (TEF3p; SEQ ID NO: 149); Thioredoxin peroxidase promoter (TPXp; SEQ ID NO: 150); Alginate lyase promoter (ALPp; SEQ ID NO: 151); Thiazole biosynthesis 4 promoter (THI4p; SEQ ID NO: 152); Phosphoglucose mutase / phosphomannose mutase promoter (PMMp; SEQ ID NO: 153); Fructose-bisphosphate aldolase 1 promoter (FBA1p; SEQ ID NO: 154); Alcohol dehydrogenase 1 promoter (ADH1p; SEQ ID NO: 155); and Extracellular endoglucanase 1 promoter (GLX1p; SEQ ID NO: 156).

[0107] The exogenous nucleic acids in the recombinant cells described herein can be controlled by terminators. For example, the exogenous nucleic acids can be operatively linked to heterologous or artificial terminators. Suitable terminators are known and described in the art. Terminators may include, but are not limited to, the GAL10 terminator, the PDC terminator, the transaldolase terminator (TAL), the 6PGD terminator (6PGDt; SEQ ID NO: 7), the ASNS terminator (ASNSt; SEQ ID NO: 116), the ENO1 terminator (ENO1t; SEQ ID NO: 117), the hexokinase 1 terminator (HXK1t; SEQ ID NO: 118), the PGK1 terminator (PGK1t; SEQ ID NO: 119), the PGM1 terminator (PGM1t; SEQ ID NO: 120), the PYK1 terminator (PYK1t; SEQ ID NO: 51), the RPLA terminator (RPLAt; SEQ ID NO: 121), the transaldolase 1 terminator (TAL1t; SEQ ID NO: 122), the TDH3 terminator (TDH3t; SEQ ID NO: 123), and the translation elongation factor 2 terminator (TEF2t; SEQ ID NO: 51). 53); Triose phosphate isomerase 1 terminator (TPI1t; SEQ ID NO: 124); MpTEF1 (SEQ ID NO: 125); TEF7 terminator (TEF7t; SEQ ID NO: 157); HSP90 terminator (HSP90t; SEQ ID NO: 158); TEF4 terminator (TEF4t; SEQ ID NO: 159); TEF6 terminator (TEF6t; SEQ ID NO: 160); CIT1 terminator (CIT11t; SEQ ID NO: 161), KAR2 terminator (KAR2t; SEQ ID NO: 162); HPS88 terminator (HSP88t; SEQ ID NO: 163); HSP70 terminator (HSP70t; SEQ ID NO: 164); TUB1 terminator (TUB1t; SEQ ID NO: 165); TKL1 terminator (TAL1t; SEQ ID NO: 165). 166); TDH2 terminator (TDH2t; SEQ ID NO: 167); TEF5 terminator (TEF5t; SEQ ID NO: 168); TGL2 terminator (TGL2t; SEQ ID NO: 169); TEF3 terminator (TEF3t; SEQ ID NO: 170); TPX terminator (TPXt; SEQ ID NO: 171); ALP terminator (ALPt; SEQ ID NO: 172); plasma membrane ATPase terminator (PMA1t;SEQ ID NO: 173); THI4 terminator (THI4t; SEQ ID NO: 174); glucose-6-phosphate isomerase 1 terminator (PGI1t; SEQ ID NO: 175); PMM terminator (PMMt; SEQ ID NO: 176); FBA1 terminator (FBA1t; SEQ ID NO: 177); ADH1 terminator (ADH1t; SEQ ID NO: 178); sodium / potassium transport ATPase α-chain terminator (ATP1t; SEQ ID NO: 179); and GLX1 terminator (GLX1t; SEQ ID NO: 180).

[0108] If the position of a promoter or terminator relative to the polynucleotide in the genome or expression cassette allows the promoter or terminator (as the case may be) to perform its transcriptional control function, then the promoter or terminator is “operably linked” to the given polynucleotide (e.g., a gene).

[0109] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent sense or antisense strands. Recombinant polynucleotides are polynucleotides formed by laboratory methods and may comprise polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Therefore, a construct may include novel modifications to an endogenous gene introduced, for example, by genome editing techniques. A construct may also include recombinant polynucleotides produced using, for example, recombinant DNA methods. A construct may be a vector containing a promoter operatively linked to a polynucleotide encoding the polypeptide described herein. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide linked thereto. A vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA fragments may be integrated.

[0110] This disclosure also provides a fermentation method for producing xylitol using the recombinant cells described herein. The fermentation method includes the step of using a genetically engineered cell fermentation substrate described herein to produce xylitol. As will be understood by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and separating xylitol from the fermentation broth. The fermentation process may be a fully aerobic or partially aerobic process.

[0111] Fermentation methods can be operated using suitable fermentation substrates. Substrates for fermentation methods may include glucose, sucrose, galactose, mannose, molasses, xylose, fructose, starch hydrolysates, lignocellulose hydrolysates, or combinations thereof. Those skilled in the art will recognize which fermentation substrates are suitable for a given fermentation organism and system.

[0112] Fermentation processes can be operated under a variety of conditions. The fermentation temperature (i.e., the temperature of the fermentation broth during processing) can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably about 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to any particular range or temperature described herein and can be appropriately modified.

[0113] The fermentation process can operate within a certain oxygen uptake (OUR) range. The OUR of the fermentation process can be in the range of 5 mmol O2 / (L • h) to 80 mmol O2 / (L • h), 10 mmol O2 / (L • h) to 75 mmol O2 / (L • h), 15 mmol O2 / (L • h) to 70 mmol O2 / (L • h), 20 mmol O2 / (L • h) to 60 mmol O2 / (L • h), 30 mmol O2 / (L • h) to 50 mmol O2 / (L • h), or 40 mmol O2 / (L • h) to 50 mmol O2 / (L • h). In some implementations, the ratio of OUR can range from 0.05 mmol O2 / (g cell dry weight • h) to 10 mmol O2 / (g cell dry weight • h), 0.1 mmol O2 / (g cell dry weight • h) to 9 mmol O2 / (g cell dry weight • h), 0.5 mmol O2 / (g cell dry weight • h) to 8 mmol O2 / (g cell dry weight • h), 1.0 mmol O2 / (g cell dry weight • h) to 7 mmol O2 / (g cell dry weight • h), 1.5 mmol O2 / (g cell dry weight • h) to 6 mmol O2 / (g cell dry weight • h), 2 mmol O2 / (g cell dry weight • h) to 5 mmol O2 / (g cell dry weight • h), or 2.5 mmol O2 / (g cell dry weight • h) to 4 mmol O2 / (g cell dry weight • h). However, the volume of the fermentation process or the ratio of OUR is not limited to any specific rate or range described herein.

[0114] The fermentation process can be operated at various cell concentrations. In some embodiments, the cell dry weight at the end of fermentation can be from 5 g / L to 40 g / L, 8 g / L to 30 g / L, or 10 g / L to 20 g / L. Furthermore, the pitch density or pitting rate of the fermentation process can vary. In some embodiments, the pitch density can be from 0.05 g / L to 11 g / L, 0.1 g / L to 10 g / L, or 0.25 g / L to 8 g / L.

[0115] When operating in batch fermentation mode, the initial dextrose concentration can be at least 100 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L, or at least 400 g / L. When operating in batch fermentation mode, the initial dextrose concentration can be between 100 g / L and 500 g / L, between 150 g / L and 450 g / L, between 400 g / L and 200 g / L, or between 250 g / L and 350 g / L. When operating in fed-batch fermentation, the dextrose concentration can be at least 100 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, or at least 450 g / L.

[0116] The fermentation process can be operated as a dextran feed batch. Furthermore, the fermentation process can be a batch process, a continuous process, or a semi-continuous process, as understood by those skilled in the art.

[0117] Fermentation processes can be associated with a variety of characteristics, including, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be influenced by yeast selection and / or genetic modifications of the yeast used in the fermentation process. These characteristics can also be influenced by adjusting fermentation process conditions. These characteristics can be modulated through a combination of yeast selection or modification and the selection of fermentation process conditions.

[0118] The xylitol production rate of this process can be at least 0.2 g / L. -1 h -1 0.3g L -1 h -1 0.5g L -1 h -1 0.75g L -1 h -1 Or at least 1.0 g L -1 h -1The final xylitol titer of this process can be at least 5 g / L, 10 g / L, 20 g / L, 30 g / L, 50 g / L, 75 g / L, or 100 g / L. The rate, titer, and / or yield of xylitol production can be higher than that of fermentation processes run with equivalent yeast lacking exogenous polynucleotides encoding ZWF enzymes.

[0119] The xylitol yield of this process can be at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 65%, or at least 70%. The xylitol yield of this process can be higher than that of a process run with equivalent yeast cells lacking an exogenous polynucleotide sequence encoding the ZWF enzyme.

[0120] The total polyol yield of this process can be at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 65%, or at least 70%. The total polyol yield of this process can be higher than that of a process run with equivalent yeast cells lacking an exogenous polynucleotide sequence encoding the ZWF enzyme. Without being bound by any particular hypothesis, theory, or mode of action, it is believed that higher ZWF enzyme activity (e.g., through an increase in the ZWF gene copy number) increases the carbon flux into the pentose phosphate pathway and will lead to an increase in polyol yield in this process.

[0121] Example

[0122] The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

[0123] Throughout the embodiments, strain numbers and sequence identification numbers were used consistently. For example, strains 1-2 in Example 2 were the same as strains 1-2 in Example 3, etc.

[0124] Example 1: Diversity of glucose-6-phosphate dehydrogenase

[0125] 330 candidate sequences of glucose-6-phosphatase (ZWF) were obtained from Uniprot based on EC 1.1.1.388 and analyzed using the Geneious protein alignment tool. These sequences showed relatively high sequence identity, with 221 sequences being more than 75% identical to each other. Based on their low sequence identity with each other, a total of 6 sequences were selected for evaluation.

[0126] In addition, the enzyme sequence (UniprotQ43727) of the ZWF1 enzyme from Arabidopsis thaliana was used as the basis for further sequence-based enzyme identification, which used sequence homology against the National Center for Biotechnology Information (NCBI) database. Alignment with the top 100 sequences from this search demonstrated at least 80% sequence identity across the entire collection. Based on randomly altered distances from the original sequence (% homology), five candidate ZWF enzyme sequences from plants were selected for further testing.

[0127] Four candidate ZWF-encoding genes from *Saccharomyces cerevisiae* were also identified and selected for testing: RCSR20949, RCSR22401, RCSR24965, and RCSR02959. Three of these four genes, RCSR20949, RCSR22401, and RCSR24965, were mutated to contain aspartic acid and isoleucine (DI) instead of alanine and arginine (AR) at the proposed cofactor binding site, potentially converting the enzyme to the preferred NADH.

[0128] The 18 total ZWF enzyme candidate sequences were then cloned into *Saccharomyces cerevisiae* as described below to characterize in vivo enzyme activity.

[0129] Example 2: Genetically modified *Saccharomyces cerevisiae* strain

[0130] strain 1-1

[0131] Strain 1-1 is the host strain of *Moniliellatomentosa* var. pollinis TCV364 described in US 6,440,712, which is incorporated herein by reference in its entirety and was deposited on March 28, 1997, under the accession number MUCL40385 at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde).

[0132] Strain 1-2

[0133] The parent strain was first protoplastized by adding an enzyme mixture containing 0.6 M MgSO4, 7.5 g / L lyase, and 12.5 g / L Trichoderma harzianum lyase to the mycelial pellet of the parent strain. The strain 1-1 was then transformed with SEQ ID NO: 2 and SEQ ID NO: 1. The protoplast pellet was then washed with 0.6 M MgSO4 and resuspended in STC medium (0.6 M sucrose, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). Approximately 200 μL of the protoplast mixture (10...) was then added to the mycelial pellet to induce protoplast formation. 8 To prepare dimeric transformations, 100 µg of single-stranded salmon sperm DNA and 1.5 µg to 5 µg each of the 5' and 3' DNA transformation fragments (total 3 µg–10 µg) were added to the salmon sperm DNA, transformation DNA, and protoplast mixture (cells / mL). Then, 1 mL of STC medium containing 50% PEG was added to the mixture of salmon sperm DNA, transformation DNA, and protoplasts, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, the recovered culture medium (0.4 M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L sugar, pH 4.5) was added to the mixture, and the mixture was incubated at 27 °C and 100 rpm for 16–24 hours. After incubation, the protoplasts were precipitated by centrifugation and resuspended in 1 mL of PBS. The resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until transformants grew. The obtained transformants were streaked onto PDA + Genimycin (G418) plates for single-colony isolation, and single colonies were selected. Integration of two copies of the *Lactobacillus rhamnosus* XPDH sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 1-2.

[0134] SEQ ID NO: 1 contains (i) 5' flanking DNA for targeting chromosomal integration into the ER1 locus (SEQ ID NO: 3); (ii) a polynucleotide sequence SEQ ID NO: 4 encoding the XPDH homology of *Lactobacillus rhamnosus* (SEQ ID NO: 5) under the control of the PYK1 promoter of SEQ ID NO: 6 and the PGD terminator of SEQ ID NO: 7; and (iii) the 5' portion of a G418 selectable marker (SEQ ID NO: 8). SEQ ID NO: 2 contains the 3' portion of a G418 selectable marker (SEQ ID NO: 9); (ii) a polynucleotide sequence SEQ ID NO: 4 encoding the XPDH homology of *Lactobacillus rhamnosus* (SEQ ID NO: 5) under the control of the PYK1 promoter of SEQ ID NO: 6 and the PGD terminator of SEQ ID NO: 7; and (iii) a 3' flanking DNA for targeting chromosomal integration into the ER1 locus (SEQ ID NO: 10).

[0135] strains 1-3 to 1-19

[0136] Table 2 below lists various *Saccharomyces cerevisiae* strains, including information on the parental strains, the sequences of the transformed parental strains, and the characterization of the expression cassettes contained in the transformed sequences. Although this table lists the origin of the encoded ZWF polypeptide sequences, the polynucleotide sequences encoding these polypeptide sequences underwent codon optimization prior to cloning and therefore do not match the gene sequences from the source organisms. The gene sequences encoding the polypeptides shown are part of the transformed fragment sequences.

[0137] For example, using the transformation method described for strains 1-2, strains 1-2 were transformed with SEQ ID NO: 28 and SEQ ID NO: 45 to produce strains 1-3a-e. SEQ ID NO: 28 contains (i) the 3' portion of a giomycin resistance marker (SEQ ID NO: 47); (ii) a polynucleotide encoding a candidate ZWF enzyme of SEQ ID NO: 11 under the control of the PYK1 promoter (SEQ ID NO: 6) and the 6PGD terminator (SEQ ID NO: 7); and (ii) a 3' flanking DNA for targeting chromosomal integration into the ER3 locus (SEQ ID NO: 50). SEQ ID NO: 45 contains (i) 5' flanking DNA for targeting chromosomal integration into the ER3 locus (SEQ ID NO: 49); (ii) a polynucleotide encoding the RPE enzyme SEQ ID NO: 46 under the control of the PYK1 promoter (SEQ ID NO: 6) and PYK1 terminator (SEQ ID NO: 51); and (iii) the 5' portion of a giomycin resistance marker (SEQ ID NO: 48). Transformed protoplasts were selected on a PDA + giomycin selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA + giomycin plate for single-colony isolation and single-colony selection. Integration of the ZWF and RPE coding sequences of the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 1-3a, 1-3b, 1-3c, 1-3d, and 1-3e.

[0138] The transformed fragments of SEQ ID NOs:29-44 comprise the same components as SEQ ID NO:28, except that the sequence encoding the polypeptide of SEQ ID NO:11 is replaced by polynucleotide substitutions of the polypeptides encoding SEQ ID NO:12-27, respectively. In some cases, more than one PCR-validated isolate (e.g., “sister” isolates) is indicated by the letter following the strain number. For example, as described in the previous paragraph, strain 1-3 has five sister isolates: strains 1-3a, 1-3b, 1-3c, 1-3d, and 1-3e.

[0139] Table 2

[0140]

[0141] Strains 1-20

[0142] Using the transformation methods outlined above for strains 1-2, strains 1-2 were transformed with SEQ ID NO: 45 and SEQ ID NO: 52. SEQ ID NO: 52 contains (i) the 3' portion of the genomiconine resistance gene expression cassette (SEQ ID NO: 47), the MpTEF2 terminator (SEQ ID NO: 53), and the 3' ER3 flanking sequence (SEQ ID NO: 50). Transformed protoplasts were selected on a PDA + genomiconine selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA + genomiconine plate for single-colony isolation, and single colonies were selected. Integration of the RPE coding sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 1-20a, 1-20b, 1-20c, 1-20d, and 1-20e.

[0143] Example 3: Shake-flask fermentation assay

[0144] Strains 1-2, 1-3a-e, 1-4a-e, 1-5a-e, 1-6a-e, 1-7a-e, 1-8a-e, 1-9a-e, 1-10a-e, 1-11a, 1-11c, 1-20a, and 1-20b were run in shake flasks to evaluate glucose consumption and the production of ribitol, erythritol, glycerol, and ethanol.

[0145] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0146] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 3) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. See Table 4 and... Figure 2The results are shown in Table 5, and the yields of xylitol and polyols are reported. Xylitol yield is calculated by dividing the xylitol concentration (w / v) by the total glucose consumed (w / v) at a given time point. For example, if the starting glucose is 100 g / L, and 20 g / L of xylitol is produced after 48 hours with 20 g / L of glucose remaining, the yield at the 48-hour time point would be the xylitol concentration at 48 hours (20 g / L) divided by the total glucose consumed at 48 hours (100 g / L - 20 g / L), resulting in a yield of 25%. Polyols are calculated using the same method, but using the total concentration of xylitol, ribitol, and erythritol produced at a given time point instead of the xylitol concentration.

[0147] The results showed that several strains produced higher polyol titers than the control strains. For example, strains 1-3a, 1-3e, 1-6d, 1-7a, 1-7b, 1-7c, 1-7d, 1-8c, 1-8d, 1-8e, 1-9a, 1-9c, and 1-10b had higher polyol titers, and strains 1-7c, 1-7d, 1-8c, 1-8d, 1-8e, and 1-9a had higher xylitol titers. Additionally, strains 1-7a and 1-8b had higher xylitol yields, while strains 1-3a, 1-3e, 1-7b, 1-7c, 1-7d, 1-8c, 1-8d, and 1-9a had higher total polyol yields.

[0148] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the specified strains, further analysis showed that in most strains, the RPE and ZWF coding sequences were integrated into the strain but not into the planned ER3 locus. This may explain the presence of some sister strain variability in the results. Nevertheless, the results still demonstrate that expression of the encoded ZWF contributes to increased xylitol yield.

[0149] Table 3: Production Culture Media

[0150]

[0151]

[0152]

[0153] Table 5: Yield (%) at 96 hours

[0154]

[0155] Example 4: Shake-flask fermentation assay

[0156] Strains 1-2, 1-20c, 1-20d, 1-11c, 1-11d, 1-11e, 1-12a-e, 1-13a-e, 1-14-ae, 1-15a-e, 1-16a-d, 1-17a-e, 1-18a-e, and 1-19e-a were run in shake flasks to evaluate glucose consumption and the production of ribitol, erythritol, glycerol, and ethanol.

[0157] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0158] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 3) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. See Table 6 and... Figure 3 The results are shown in Table 7 and the yields of xylitol and polyols are reported. Yields were calculated as outlined in Example 3.

[0159] The results showed that several strains produced higher polyol titers than the control strains. For example, strains 1-11e, 1-12c, 1-12d, 1-13a, 1-13b, 1-13c, 1-13d, 1-13e, 1-14b, 1-14c, 1-14d, 1-14e, 1-15a, 1-15b, 1-15d, 1-15e, 1-16a, 1-16b, 1-16d, 1-18b, 1-18c, and 1-19c had higher polyol titers. Strains 1-13a, 1-13c, 1-14a, 1-14e, 1-16c, 1-17c, 1-19b, 1-19c, 1-19d, and 1-19e had higher xylitol titers than control strains 1-20c and 1-20d. In addition, strains 1-19b and 1-19c had higher xylitol yields, while strains 1-11c, 1-11e, 1-12b, 1-13b, 1-13c, 1-14b, 1-14c, 1-15a, 1-15b, 1-15d, 1-15e, 1-16a, 1-16b, 1-16d, 1-18c, and 1-19c had higher total polyol yields.

[0160] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the specified strains, further analysis showed that in most strains, the RPE and ZWF coding sequences were integrated into the strain but not into the planned ER3 locus. This may explain the presence of some sister strain variability in the results. Nevertheless, the results still demonstrate that expression of the encoded ZWF contributes to increased xylitol yield.

[0161]

[0162]

[0163] Table 7: Yields of xylitol and polyols at 96 hours (%)

[0164]

[0165] Example 5: Genetically modified *Saccharomyces cerevisiae* strain

[0166] strain 2-1

[0167] Using the protocol outlined above, strains 1-2 were transformed with SEQ ID NO: 45 and SEQ ID NO: 52. The transformation fragment of SEQ ID NO: 45 sequentially contained a 5' ER3 flanking sequence (SEQ ID NO: 49), an MpPYK1 promoter (SEQ ID NO: 6), a gene encoding the *Saccharomyces cerevisiae* RPE2 polypeptide (SEQ ID NO: 46), an MpPYK terminator (SEQ ID NO: 51), and the 5' portion of the genomiconine resistance gene expression cassette (SEQ ID NO: 48). The transformation fragment of SEQ ID NO: 52 sequentially contained the 3' portion of the genomiconine resistance gene expression cassette (SEQ ID NO: 47), an MpTEF2 terminator (SEQ ID NO: 53), and a 3' ER3 flanking sequence (SEQ ID NO: 50). The resulting transformants were streaked onto PDA + genomiconine plates for single-colony isolation, and single colonies were selected. The integration of the *Saccharomyces cerevisiae* RPE2 sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolate was named strain 2-1.

[0168] strain 2-2

[0169] UV mutagenesis was used (energy of 360 uJ / cm²). 3 Hoefer UV crosslinking agent and strains 1-3 were selected to produce *Saccharomyces cerevisiae* with reduced foaming during shake-flask fermentation. Based on visual evaluation of foaming during shake-flask fermentation compared to foaming on parental strains 1-3, strains with a low-foaming phenotype were selected. The resulting low-foaming strain was named 2-2, containing two copies of the exogenous polynucleotide sequence encoding XPDH (SEQ ID NO: 5) integrated at the ER1 locus and one copy of the polynucleotide sequence encoding RPE (SEQ ID NO: 46) integrated at the ER3 locus.

[0170] Strain 2-3

[0171] Strains 2-2 were transformed with the Cre recombinase plasmid of SEQ ID NO: 126 using the transformation method described above. The removal of G418 and genomiconine resistance selection markers in the transformed organisms was evaluated by colony PCR. The PCR-validated isolate was named strain 2-3.

[0172] Strain 2-4

[0173] Strain 2-3 was non-selectively grown on YPD plates to allow for the loss of the plasmid in SEQ ID NO: 126. The biomass was subjected to single-colony knockout and evaluated by PCR to confirm the plasmid loss. The PCR-validated isolate was named strain 2-4.

[0174] Strains 2-5

[0175] Using the transformation method outlined above, strains 2-4 were transformed with SEQ ID NO: 127 and SEQ ID NO: 45. SEQ ID NO: 127 contains, in sequence, the 3' portion of the giomycin resistance gene expression cassette (SEQ ID NO: 47), the MpPGK1 promoter (SEQ ID NO: 111), the gene encoding the *Saccharomyces cerevisiae* X5PP polypeptide (SEQ ID NO: 128) encoded by SEQ ID NO: 74, the Mp6PGD terminator (SEQ ID NO: 7), and the 3' ER3 flanking sequence (SEQ ID NO: 50). The resulting transformants were streaked onto PDA + giomycin plates for single-colony isolation, and single colonies were selected. Integration of the indicated sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strain 2-5.

[0176] strains 2-6 to 2-9

[0177] Using the transformation method outlined above, strains 2-5 were transformed with one of SEQ ID NO: 129 and SEQ ID NO: 130-133 according to Table 8. SEQ ID NO: 129 contains, in sequence, the 3' portion of the G418 selectable marker (SEQ ID NO: 9), the MpTEF1 terminator (SEQ ID NO: 125), and the 3' gpdIIB flanking sequence (SEQ ID NO: 73). Each of SEQ ID NO: 130-133 contains, in sequence, the 5' gpdIIB flanking sequence (SEQ ID NO: 72), the MpPGK1 promoter (SEQ ID NO: 111), the nucleotide sequence encoding one of SEQ ID NO: 15, 16, 23, or 24, the MpTDH3 terminator (SEQ ID NO: 123), and the 5' portion of the G418 selectable marker (SEQ ID NO: 8). The resulting transformants were streaked onto PDA plates containing 250 mg / L genimycin (G418) for single-colony isolation, and single colonies were selected. Integration of the indicated sequence into the selected colonies was evaluated by colony PCR. PCR-validated isolates were named as outlined in Table 8.

[0178] For example, using the transformation methods outlined above, strain 2-5 was transformed with SEQ ID NO: 129 and SEQ ID NO: 130. The resulting transformants were streaked onto PDA plates containing 250 mg / L genimycin (G418) for single-colony isolation, and single colonies were selected. Integration of the polypeptide sequence encoding SEQ ID NO: 15 into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 2-6a, 2-6b, 2-6c, 2-6d, and 2-6e.

[0179] Table 8.

[0180]

[0181] Example 6: Shake-flask fermentation assay

[0182] Strains 2-5, 2-6a-e, 2-7e-a, 2-8a, 2-8b, and 2-8e were run in shake flasks to evaluate glucose consumption and the production of ribitol, erythritol, glycerol, and ethanol.

[0183] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0184] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 3) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. See Table 9 and... Figure 4 The results are shown in Table 10 and the yields of xylitol and polyols are reported. Yields were calculated as outlined in Example 3.

[0185] The results showed that expression of the ZWF enzyme from *Pantotheca* spp. (SEQ ID NO: 16) led to increased xylitol yield (strains 2-7a-e). Increased polyol yields were also observed in strains 2-7a, 2-7d, and 2-7e. Expression of the ZWF enzyme from *Pseudomonas putida* spp. (SEQ ID NO: 15) led to increased xylitol and polyol yields (strains 2-6a, 2-6d, and 2-6e).

[0186] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the specified strains, further analysis indicated that in most strains, the ZWF coding sequence was integrated into the strain but not into the planned gpdIIB locus. Strains 2-6a, 2-6d, 2-7e, 2-8e, and 2-9d included coding sequences correctly integrated into the gpdIIB locus. Further analysis of the integration locus for the coding sequence in strain 2-7a was inconclusive. In strains 2-6b, 2-6c, 2-6e, 2-7b, 2-7c, 2-7d, 2-8a, and 2-8b, the coding sequence for the ZWF enzyme was not integrated into the gpdIIB locus. This may explain the presence of some sister strain variability in the results. Nevertheless, the results still demonstrate that expression of the encoded Pantotheca ZWF contributes to increased yields of xylitol and polyols.

[0187] Table 9

[0188]

[0189] Table 10 Yield (%) at 96 hours

[0190]

[0191] Example 7: Shake-flask fermentation determination

[0192] Strains 2-5 and 2-9a-e were run in shake flasks to assess glucose consumption and the production of ribitol, erythritol, glycerol, and ethanol.

[0193] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0194] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 3) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. See Table 11 and... Figure 5 The results are shown in Table 12 and the yields of xylitol and polyols are reported. Yields were calculated as outlined in Example 3.

[0195] The results showed that overexpression of the ZWF enzyme RSCR02929 in the context of 2x Lactobacillus rhamnosus XPDH, 2x RPE2, and SEQ ID NO: 74 in *Saccharomyces cerevisiae* X5PP did not further increase the titer or yield of xylitol, nor did it increase the titer or yield of total polyols during fermentation.

[0196] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the specified strains, further analysis indicated that in most strains, the ZWF coding sequence was integrated into the strain but not into the planned gpdIIB locus. Strain 2-9d included the coding sequence correctly integrated into the gpdIIB locus. In strains 2-9a, 2-9b, 2-9c, and 2-9e, the ZWF enzyme coding sequence was not integrated into the gpdIIB locus. This may explain the presence of some sister strain variability in the results.

[0197] Table 11

[0198]

[0199] Table 12: Yield (%) at 96 hours

[0200]

Claims

1. A genetically engineered yeast cell capable of producing xylitol, said engineered yeast cell comprising: The exogenous polynucleotide sequence encoding glucose-6-phosphate dehydrogenase (ZWF) enzyme is 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 at least one of SEQ ID NO: 15, 16, and 23, preferably at least 85% identical to SEQ ID NO: 15 or 16.

2. The yeast cell according to claim 1, wherein the yeast cell is a permeable yeast cell.

3. The yeast cell according to claim 1 or claim 2, wherein the yeast cell is a cell of the Ustilago phylum.

4. The yeast cell according to any of the preceding claims, wherein the yeast cell is selected from the group consisting of: giant filamentous yeast, Tritylosporium spp., Melanocytosporium spp., Tsukuba spp., Tritylosporium variegatum, Tritylosporium variegatum, Ustilago maydis, filamentous yeast, Yersinia lipolytica, Saccharomyces cerevisiae, Penicillium, Cyclosporium, Pichia pastoris, Candida albicans, Candida magnoliata, and Candida brevis.

5. The yeast cell according to any of the preceding claims, wherein the yeast cell comprises a deletion or disruption of the natural gene encoding 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% of the same erythrose reductase as at least one of SEQ ID NO: 60 and 62.

6. The yeast cell according to any of the preceding claims, wherein the yeast cell is a *Saccharomyces cerevisiae* cell, and the gene encoding the erythrose reductase is 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 at least one of SEQ ID NO: 61 and 63.

7. A yeast cell according to any of the preceding claims, wherein the cell further comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme, the xylitol-phosphate dehydrogenase (XPDH) enzyme comprising 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 at least one of SEQ ID NO: 5, 95-101, and 103, preferably at least 85% identical to at least one of SEQ ID NO: 5, 97, 98, or 101, or most preferably at least 90% identical to at least one of SEQ ID NO: 5, 97, 98, or 101.

8. A yeast cell according to any of the preceding claims, wherein the cell further comprises an exogenous polynucleotide sequence encoding a xylulokine (XKS) enzyme, the xylulokine (XKS) enzyme comprising 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 at least one of SEQ ID NO: 90 and 91.

9. The yeast cell of claim 8, wherein the cell further comprises an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme, the xylitol dehydrogenase (XDH) enzyme comprising 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 at least one of SEQ ID NO: 104-106.

10. The yeast cell according to any of the preceding claims, wherein the cell further comprises a genetic modification that results in the overexpression of a natural enzyme having ribulose-5-phosphate epimerase (RPE) activity.

11. The yeast cell of claim 10, wherein the cell is a *Saccharomyces cerevisiae* cell, and the natural RPE enzyme comprises 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 at least one of SEQ ID NO: 46 and 94.

12. The yeast cell of claim 10 or 11, wherein the genetic modification leading to overexpression of the natural RPE enzyme comprises adding an exogenous polynucleotide encoding the natural RPE enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the RPE enzyme.

13. The yeast cell according to any of the preceding claims, wherein the yeast further comprises a genetic modification that results in the overexpression of a native enzyme having xylitol-5-phosphate phosphatase (X5PP) activity; and / or an exogenous polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity.

14. The yeast cell of claim 13, wherein the cell is a *Saccharomyces cerevisiae* cell, and the genetic modification results in the overexpression of a natural X5PP enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 74, 88, 92, and 93.

15. The yeast cell of claim 13 or 14, wherein the genetic modification comprises adding an exogenous polynucleotide sequence encoding the natural X5PP enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the natural X5PP enzyme.

16. The yeast cell according to any one of claims 13 to 15, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having X5PP activity, and at least one of SEQ ID NO: 74-91, preferably at least one of SEQ ID NO: 74, 76, 77, 78, 79, 80, 84 and 86, most preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of at least one of SEQ ID NO: 74, 77, 78, 80 and 86.

17. The yeast cell according to any of the preceding claims, wherein one or more of the exogenous polynucleotide sequences are operatively linked to a heterologous promoter or an artificial promoter.

18. The yeast cell according to claim 17, wherein the heterologous promoter or artificial promoter is selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 6), 6-phosphoglucuronide dehydrogenase promoter (6PGDp; SEQ ID NO: 107), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 108), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 109), phosphoglucose mutase 1 promoter (PGM1p; SEQ ID NO: 110), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 111), enolase promoter (ENO1p; SEQ ID NO: 112), asparagine synthase promoter (ASNSp; SEQ ID NO: 113), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 114), and RPL16B (SEQ ID NO: 115). 115).

19. The yeast cell according to any of the preceding claims, wherein one or more of the exogenous polynucleotide sequences are integrated into the genome of the yeast cell at a locus selected from the ER1, ER3, PDC1, pyrF, TRP3, gpdIIA, and gpdIIB loci.

20. A method for producing xylitol, the method comprising: A substrate containing dextrose is contacted with engineered yeast cells according to any of the preceding claims, wherein the engineered cells ferment the substrate to produce xylitol.

21. The method of claim 20, wherein the fermentation temperature is 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, or between therewith, and the volumetric oxygen uptake (OUR) is between 5 mmol O2 / (L • h) and 80 mmol O2 / (L • h), between 10 mmol O2 / (L • h) and 75 mmol O2 / (L • h), between 15 mmol O2 / (L • h) and 70 mmol O2 / (L • h), between 20 mmol O2 / (L • h) and 60 mmol O2 / (L • h), between 30 mmol O2 / (L • h) and 50 mmol O2 / (L • h), or between 40 mmol O2 / (L • h) and 50 mmol O2 / (L • h).

22. The method according to claim 20 or 21, wherein the xylitol is at least 0.2 g / L. -1 h -1 0.3g L -1 h -1 0.5g L -1 h -1 0.75g L -1 h -1 Or at least 1.0 g L -1 h -1 Production rate.

23. The method according to any one of claims 20 to 22, wherein when the fermentation is run at 35°C for 96 hours, the xylitol titer is at least 20 g / L, 30 g / L, 50 g / L, 75 g / L, or 100 g / L.

24. The method according to any one of claims 20 to 23, wherein the concentration of dextrose is at least 100 g / L.

25. The method according to any one of claims 20 to 24, wherein the xylitol yield of the fermentation is higher than that of the same fermentation process run using an equivalent yeast lacking the exogenous polynucleotide sequence encoding the ZWF enzyme.

26. Use of the engineered yeast according to any one of claims 1 to 19 for the production of xylitol.

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