Compositions and methods for reducing trehalose during xylitol production

Abstract

This document discloses compositions and methods for reducing trehalose during xylitol production via fermentation. The method involves contacting a substrate containing dextrose in a fermentation broth with engineered cells capable of producing xylitol, wherein the engineered cells ferment the substrate to produce xylitol. During fermentation, trehalase is present in the fermentation broth, such that the trehalose concentration at the end of fermentation is lower than that of an equivalent fermentation run without trehalase.

Description

[0001] RELEVANT APPLIANCES cross reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 594,063, filed October 30, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference via the patent center submitted to the sequence table

[0004] The contents of the sequence list XML file named "PT-1892-WO-PCT.xml", which is 397,770 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 their entirety. 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 chemically catalytic hydrogenation of xylose derived from the hydrolysis of xylan extracted from biomass, are both economically and environmentally costly. These methods require high temperatures and pressures, large quantities of water, and mineable metal catalysts. In contrast, fermentation processes are commercially available on a large scale to produce other organic molecules, such as ethanol, citric acid, and lactic acid, and offer a cost-effective and sustainable alternative to traditional xylitol processing methods.

[0006] In the development of microbial-based fermentation strategies for xylitol production, the production of metabolic pathway intermediates and alternative fermentation products is an important consideration. For example, microbial-based fermentation of xylitol can also lead to the production of degree-of-polymerization 2 (DP2) sugars, such as trehalose. However, trehalose production may deduct carbon from xylitol production, and it is possible to reuse the two glucose molecules that make up trehalose to improve xylitol titers and / or yields. Therefore, this paper presents a genetically modified yeast and fermentation method for xylitol production, wherein trehalose is also reduced or eliminated from the fermentation broth during fermentation. Summary of the Invention

[0007] This disclosure provides a method for producing xylitol, comprising contacting a substrate containing dextran in a fermentation broth with engineered cells capable of producing xylitol, wherein the engineered cells ferment the substrate to produce xylitol, and wherein trehalase is present in the fermentation broth during fermentation, such that the concentration of trehalose at the end of fermentation is lower than that of an equivalent fermentation run without trehalase. The trehalose titer is reduced relative to the equivalent method lacking trehalase, for example, by at least 25%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or 100% relative to the equivalent method lacking trehalase. The trehalose titer may be zero (0) or may be below the detection limit. Trehalase may be exogenously added to the fermentation broth. The engineered cells may contain an exogenous polynucleotide sequence encoding a secretion signal operatively linked to trehalase, and the trehalase may be secreted from the engineered cells. The polypeptide sequence of the secretion signal may be at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 29-32, and the polypeptide sequence of the trehalase may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 25-28, preferably SEQ ID NO: 25 or 26. The polynucleotide sequence encodes a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 33, 35, 36, 38, 39, 40, and 43, preferably SEQ ID NO: 35, 36, 38, and 40, most preferably SEQ ID NO: 35, 36, and 40. The fermentation temperature may be 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, or between these temperatures. Volumetric oxygen uptake (OUR) can be between 5 mmol O2 / (L) h)-80mmol O2 / (L Between h), and between 10 mmol O2 / (L h)-75mmol O2 / (L Between h), and between 15 mmol O2 / (L h)-70mmol O2 / (L Between h), and between 20 mmol O2 / (L h)-60mmol O2 / (L h), 30mmol O2 / (L h)-50mmol O2 / (L Between h) or between 40 mmol O2 / (L h)-50mmol O2 / (L Between h). Xylitol 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 -1 The rate of xylitol production is as follows. When fermentation is carried out at 35°C for 96 hours, the xylitol yield 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.

[0008] This article also provides a genetically engineered yeast cell capable of producing xylitol, the engineered yeast cell comprising an exogenous polynucleotide sequence encoding a secretion signal operatively linked to trehalase, and the trehalase being secreted from the engineered cell. The polypeptide sequence of the secretion signal may be at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 29-32, and the polypeptide sequence of the trehalase may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 25-28, preferably SEQ ID NO: 25 or 26. The polynucleotide sequence encodes a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs 33, 35, 36, 38, and 40, preferably SEQ ID NOs 35, 36, and 40. The yeast cells may be osmotically resistant yeast cells. The yeast cells may be cells of the Ustilaginomycotina subphylum. The yeast cells may be selected from the group consisting of the following species: giant filamentous yeasts (… Trichosporonoides megachiliensis Trisporium globosum ( Trichosporonoides oedocephalis ), blackening filamentous yeast ( Trichosporonoides blackening ), Tsukuba yeast ( Pseudozyma tsukubaensis ), mutant triangular yeast ( Trigonopsis variable ), genus *Platymonas* ( Moniliella ), Ustilago idaeus ( Ustilaginomycetes ), genus *Mycosaccharomyces* ( Trichosporon ), Yarrowia lipolytica ( Yarrowia lipolytica ), Penicillium ( Penicillium ), genus *Cytomyces* ( Torula ), Pichia pastoris ( Peach ), Candida genus ( White ), Candida magnoliae ( White magnolias ) and the genus *Briestros* ( Aureobasidium Yeast cells may be *Saccharomyces cerevisiae* ( ). Moniliella pollen )cell.

[0009] The yeast cell may be a *Saccharomyces cerevisiae* 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: 74 and 76. The cell may have the deletion of at least one allele of the gene encoding erythrose reductase. The cell may have the deletion of both alleles of the gene encoding erythrose reductase.

[0010] The cell may additionally contain an exogenous polynucleotide sequence encoding a xylitol phosphate dehydrogenase (XPDH) enzyme, the XPDH enzyme containing 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, 108-114, and 116. The XPDH enzyme may have at least 85% identical sequence to at least one of SEQ ID NO: 5, 110, 111, or 114. The XPDH enzyme may have at least 90% identical sequence to at least one of SEQ ID NO: 5, 108-114, and 116, or at least one of SEQ ID NO: 5, 110, 111, or 114.

[0011] The cell may additionally contain an exogenous polynucleotide sequence encoding a xylitol kinase (XKS) enzyme, wherein the 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: 103 and 104. The cell may additionally contain an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme, wherein the 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: 117-119.

[0012] 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 *Amygdaloides* yeast cell. The yeast cell may be an *Amygdaloides* yeast cell, and the native RPE enzyme 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: 14 and 107. 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.

[0013] The yeast may additionally contain 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: 24, 101, 105, or 106. The genetic modification includes 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 with 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 NO: 24 and 88-104. Yeast cells may contain a foreign polynucleotide sequence encoding an enzyme with 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 NO: 24, 89-93, 97, and 99. Yeast cells may contain a foreign polynucleotide sequence encoding an enzyme with 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 NO: 24, 90, 91, 93, and 99. X5PP activity in genetically engineered yeast cells can be higher than that in equivalent cells lacking genetic modification or exogenous polynucleotide sequences.

[0014] 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: 120), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 121), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 122), phosphoglucose mutase 1 promoter (PGM1p; SEQ ID NO: 123), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 22), enolase promoter (ENO1p; SEQ ID NO: 124), asparagine synthase promoter (ASNSp; SEQ ID NO: 125), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 126), and RPL16B (SEQ ID NO: 127). 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.

[0015] 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 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, or between these temperatures. The volumetric oxygen uptake (OUR) may be between 5 mmol O2 / (L) h)-80mmol O2 / (L Between h), and between 10 mmol O2 / (L h)-75mmolO2 / (L Between h), and between 15 mmol O2 / (L h)-70mmol O2 / (L h), 20mmol O2 / (L h)-60mmol O2 / (L Between h), and between 30 mmol O2 / (L h)-50mmol O2 / (L Between h) or between 40 mmol O2 / (L h)-50mmol O2 / (L Between h). Xylitol 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 -1 The rate of xylitol production is as follows. When fermentation is carried out at 35°C for 96 hours, the xylitol yield 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.

[0016] This disclosure also provides the use of the engineered cells described herein for the production of xylitol in a trehalose-reduced fermentation process. Attached Figure Description

[0017] This patent or application contains at least one color-drawn drawing. A published copy of this patent or application with a color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0018] The accompanying figures illustrate various aspects of the discussion herein in a manner that is not restrictive, but rather illustrative.

[0019] Figure 1 The natural pentose phosphate pathway (dashed lines and arrows) and natural glycolysis pathway (solid lines and arrows) in *Saccharomyces cerevisiae* are shown.

[0020] Figure 2 The results show the xylitol and trehalose titers (g / L) of the deep-well plate as described in Example 2.

[0021] Figure 3 The results show the titers (g / L) of erythritol and xylitol as described in Example 3.

[0022] Figure 4 The results show the trehalose titer (g / L) as described in Example 3.

[0023] Figure 5 The xylitol yields (%) at 72 and 96 hours are shown as described in Example 3. The black horizontal line shows the xylitol yield of the control strain 8-1 used for comparison. Detailed Implementation

[0024] Reference will now be made in detail to certain aspects of the subject matter disclosed herein, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0025] In this document, unless the context clearly requires otherwise, the terms "an," "a," or "the / described" are used to include one or more. Unless otherwise indicated, the term "or" is used to mean a non-exclusive "or." All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety as if individually cited. In the event of any inconsistency between the usage of this document and those documents so incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of irreconcilable inconsistencies, the usage in this document shall prevail.

[0026] 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".

[0027] 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)%.

[0028] This disclosure relates to compositions and methods for reducing trehalose in fermentation broth during xylitol production. This disclosure includes various recombinant cells engineered to produce and secrete trehalase. Generally, the recombinant cells described herein are capable of producing xylitol and contain exogenous polynucleotide sequences encoding trehalase. Additionally, the recombinant yeast may be characterized by: overexpression of ribulose 5-phosphate epimerase (RPE); containing an exogenous polynucleotide sequence encoding xylitol phosphate dehydrogenase (XPDH); containing an exogenous polynucleotide sequence encoding xylulose kinase (XKS); containing an exogenous polynucleotide sequence encoding xylitol dehydrogenase (XDH); and / or overexpression of xylitol-5-phosphate phosphatase (X5PP). This disclosure also includes compositions and methods for directly adding trehalase to the fermentation broth. This disclosure also provides a fermentation method for producing xylitol from dextran using genetically engineered cells described herein.

[0029] Generally, the recombinant cells described herein are yeast cells. As used herein, “yeast” refers to a eukaryotic unicellular microorganism classified as a member of the fungi kingdom. Yeasts are unicellular organisms that evolved from multicellular ancestors, and some species retain multicellular characteristics, such as the formation of connected budding cell chains called pseudohyphae or pseudohyphae. Yeast cells may also be referred to in the art as yeast-like cells, and as used herein, “yeast cell” encompasses both yeast and yeast-like cells. Suitable yeast and yeast-like host cells for modification may include, but are not limited to, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces yeast ), a certain type of yeast of the genus ( Komagataella sp. Kluyveromyces ( ) Kluyveromyces (For example, Kluyveromyces lactis) Kluyveromyces lactis ), Max Kluyveromycin ( Kluveromyces Marxian Yersinia lipolyticis, Isaac's orientalis ( Issatchenkia orientalis ), Helmet-shaped Pichia pastoris ( Pichia galeiformis Pichia pastoris species YB-4149 (NRRL name), Pichia pastoris ( Peach shepherd ), Candida genus (e.g., Candida magnoliae, Candida ethanolica) Candida ethanolica ), Pichia pastoris ()), desert Pichia pastoris () Pichia deserticola ), Pichia pastoris ( ) Pichia membranafadens ), fermented Pichia pastoris ( Fermenting peach Aspergillus ( ) Aspergillus Trichoderma ( ) Trichoderma ), thermophilic hygrophytes ( Myceliphthora thermophila ), *Branchiolus* (e.g., *Branchiolus* yeast), *Fafia* ( Pfaffia ), Yamada yeast ( Yamadazyma ), Hansenula genus ( Little Hansen ), Kudria zweipichia yeast ( Peach kudriavzevvi ), genus *Phyllospora* ( Trichosporonoides (e.g., *Gastromycium macrocarpa*, *Trichoderma spp.*, *Gastromycium melanogaster*), *Pseudomonas tsukuba*, *Trichoderma mutagenes*, *Penicillium*, and *Cytomyces*. 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 required for transforming and engineering suitable yeast cells.

[0030] Suitable yeast cells can belong to the Basidiomycetes phylum ( Basidiomycota Cells of the Ustilago phylum. Suitable yeasts of the Ustilago phylum include, but are not limited to, *Ustilago* genus. I burn. (For example, Bermuda grass smut) U. cynodontis ), corn smut ( U. maydis ), Coccidial Ustilago maydis ( U. sphaerogena ), Kodal smut ( U. cordal ), sugarcane smut ( U. scitaminea ), Coix seed smut ( U. coicis ), Digitaria smut ( U. syntherismae ), water chestnut smut fungus ( U. esculenta ), Millet Ustilago maydis ( U. neglected ), barnyard grass smut fungus ( U. crus-galli ), oat smut ( Oat scorch )); Ustilago maydis genus ( Sporisory (For example, *Ustilago maydis*) Sporisorium exserted )); genus *Plasmodium* (e.g., *Plasmodium spp.*, *Plasmodium vulgare*). M. tomentose ), Esitosporum tobira ( Moniliella acetoabutans ), Fonseca zoea ( M. fossae ), Moistened Circulatory Saccharomyces ( M. wet Giant Flocculation Yeast () M. megachiliensis ), Snakehead fasciculata ( M. ocedocephalus ), Sterculia nigra ( M. nigrescens )); and the genus *Pseudomonas* ( Pseudozyma(e.g., *Pseudomonas tsukuba*); and *Pseudomonas* species (e.g., *Pseudomonas giantis*, *Pseudomonas spherulites*, *Pseudomonas melanogaster*). Yeasts of the subphylum Ustilagoeae are known and described in the art as potential production organisms for valuable chemicals such as itaconic acid esters, malates, succinates, 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 the genusUstilago, 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).

[0031] 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 pentose phosphate pathway converts phosphate 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 pentoses (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 naturally present in yeast cells or introduced into yeast cells through genetic engineering.

[0032] Yeast cells can be osmotically resistant. As used herein, “osmotically resistant” 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 osmotically resistant yeasts are known and described in the art, including many yeast species used in industrial fermentation processes. Similarly, methods for determining yeast osmotic resistance 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).

[0033] Recombinant yeast cells can be recombinant genus cells, such as *Bacillus moniliforme* cells. Figure 1 The predicted natural pentose phosphate pathway and glycolysis pathway in *Fragaria filamentous fungi* are shown. *Fragaria* has previously been used in the fermentation production of erythritol, and methods for genetic modification and fermentation of *Fragaria* are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb Cell Fact, 16:168).

[0034] Various plasmids and methods for transforming *Branchiolus* are also described in the following examples. For example, *Branchiolus* can be transformed using a dichotomous polynucleotide sequence, wherein, after recombination, the target exogenous polynucleotide is integrated into a specific locus, and a selection marker is expressed intracellularly. Suitable selection markers are known and used in the art. Selection markers may include, but are not limited to, amdS (e.g., broken into 3' portions SEQ ID NO: 68 and 5' portions SEQ ID NO: 69), G418 resistance genes (e.g., broken into 3' portions SEQ ID NO: 9 and 5' portions SEQ ID NO: 8), zeocin resistance genes (e.g., broken into 3' portions SEQ ID NO: 17 and 5' portions SEQ ID NO: 16), norsinosin N-acetyltransferase (NAT) (e.g., broken into 3' portions SEQ ID NO: 70 and 5' portions SEQ ID NO: 71), and invertase genes (SUC2) (e.g., the 3' portion of SEQ ID NO: 72 and the 5' portion of SEQ ID NO: 73).

[0035] The recombinant cells described herein contain one or more polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve glucose fermentation into xylitol via the recombinant cells.

[0036] Unless otherwise expressly stated, the terms “glucose” and “dextrose” are used interchangeably in this document and refer to D-glucose.

[0037] As used herein, "exogenous" refers to genetic material or its expression product originating outside the host organism. For example, exogenous genetic material or its expression product can be a modified form of the host organism's natural genetic material, it can originate from another organism, it can be a modified form of a component derived from another organism, or it can be a component of synthetic origin. For example, when introduced into Saccharomyces cerevisiae, the Kluyveromyces lactis invertase gene is exogenous.

[0038] As used herein, “natural” means the genetic material or its expression products found in the genome of wild-type cells of a host cell, excluding inter-individual mutations that do not affect function or expression. For the purposes of this application, the description of *Saccharomyces cerevisiae* cells in US 6,440,712, which is incorporated herein by reference in its entirety, is provided. Moniliella tomentosa var pollenTCV364” is considered to be a wild-type *Catholique* cell 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; deposited by Eridania Béghin Say, Vilvoorde R&D Centre, located at Havenstraat 84, B-1800 Vilvoorde) in accordance with the Budapest Treaty.

[0039] 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 required to give the macromolecule described its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (independently or as part of a biosynthetic pathway) should be understood to include the cofactors, coenzymes, and metals required for the enzyme to function properly. As understood in the art, an overview of amino acids and their three-letter and one-letter symbols is provided in Table 1. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0040] Table 1: amino acids, three letters, and single letter symbols

[0041] Variants or sequences having substantially the same identity or homology as the polypeptides described herein can be used to practice the disclosed recombinant cells, compositions, and methods. Such sequences may be referred to as variants or modified sequences. That is, the polypeptide sequence may be modified but still retain the ability to exhibit the desired activity. Typically, variants or modified sequences may include sequences having more than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity 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 using computational methods. Alignment can be performed using BLAST (National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using the standard protein BLAST with the following default parameters: Maximum target sequence: 100; Short sequence query: Parameters automatically adjusted for short input sequences; Expected threshold: 10; Word length: 6; Maximum number of matches within the query range: 0; Matrix: BLOSUM62; Void penalty: (Presence: 11, Extension: 1); Composition adjustment: Conditional compositional score matrix adjustment; Filtering: Not selected; Masking: Not selected. The standard nucleotide BLAST algorithm with the following default parameters can be used to determine the percentage of nucleic acid sequence identity between nucleic acid sequences: Maximum target sequence: 100; Short sequence query: Automatically adjusts parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within the query range: 0; Match / mismatch score: 1, -2; Gap penalty: Linear; Filtering: Low complexity region; Masking: Mask only the lookup table. Sequences that have an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently have XX% sequence identity with the reference sequence.

[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 over which a measurable percentage of identity can be measured.

[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 the variant or mutant form. As used herein, “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence different from that of a reference protein or polypeptide molecule. Variants or mutants may have one or more amino acid residues inserted, deleted, or substituted relative to the reference molecule.

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

[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., containing both 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 the expression of polypeptides or proteins in heterologous expression systems. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and artificial manipulation can also be used to generate degenerate coding sequences.

[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., at least 75% or at least 90%), or produces a severely reduced expression product (e.g., at least 75% or at least 90%). Deletions or disruptions can be achieved through genetic engineering methods, directed evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. Deletions or disruptions of natural host cell genes may 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 may be designed to replace all or part of the host cell gene to be deleted or disrupted. The polynucleotide sequence may encode a target gene product, such as a polypeptide, enzyme, etc. Deletions or disruptions may 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 impairs the recombinant cells' ability to produce erythritol and, depending on the deletion or disruption, may 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 gene encodes an enzyme with erythrose reductase activity. As used herein, “erythrose reductase activity” and “ER activity” are used interchangeably and refer to an enzyme that catalyzes the reversible conversion of erythrose or erythrose-4-phosphate to erythritol or erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor. In the art, an enzyme that catalyzes the reversible conversion of erythrose-4-phosphate to erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor may also be described as an erythrose or erythritol phosphate dehydrogenase. When the host cell contains multiple ER genes, it is preferable to delete or disrupt at least one of them. When the host cell contains multiple alleles of a given ER gene, it is preferable to delete or disrupt one or two alleles of the given ER gene.

[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 (or the abbreviation “P”) in parentheses indicates that phosphate may be absent or present, and the name and abbreviation include both. Similarly, “NAD(H)” or “NADP(H)” refers to both the reduced and oxidized forms of the cofactor.

[0053] When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 74. When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 75. See, for example, U.S. Provisional Application No. 63 / 499,990, filed May 4, 2023, and U.S. Provisional Application No. 63 / 499,989, filed May 4, 2023.

[0054] When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 76. When the recombinant cells are *Saccharomyces cerevisiae* cells, the recombinant cells may contain a deletion or disruption of the ER gene, which has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 77. See, for example, U.S. Provisional Application No. 63 / 499,990, filed May 4, 2023, and U.S. Provisional Application No. 63 / 499,989, filed May 4, 2023.

[0055] The recombinant cells described herein may contain one or more genetic modifications in which exogenous nucleic acids are integrated into the host cell genome. Those skilled in the art know how to select suitable loci in the yeast genome for the integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in *Saccharomyces cerevisiae* host cells, suitable interacting loci may include, but are not limited to: ER1 Locus (defined as a locus flanking SEQ ID NO: 3 and SEQ ID NO: 10), ER3 Locus (defined as a locus flanking SEQ ID NO: 13 and SEQ ID NO: 19), PDC1 Locus (defined as the locus flanking SEQ ID NO: 78 and SEQ ID NO: 79), pyrf Locus (defined as a locus flanking SEQ ID NO: 80 and SEQ ID NO: 81), TRP3 Locus (defined as the locus flanking SEQ ID NO: 82 and SEQ ID NO: 83), gpdIIA Locus (defined as the locus flanking SEQ ID NO: 84 and SEQ ID NO: 85) and gpdIIBLoci (defined as loci flanking SEQ ID NO: 86 and SEQ ID NO: 87). 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 integration loci. Furthermore, those skilled in the art will recognize how sequences can be used to design primers to verify that correct gene integration occurs at the selected loci.

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

[0057] The recombinant cells described herein are capable of producing xylitol and may contain an exogenous polynucleotide sequence encoding an enzyme with trehalase activity. This enzyme may be any suitable enzyme with trehalase activity. As used herein, “trehalase” and “trehalase enzyme” are used interchangeably and refer to an enzyme with trehalase activity. Herein, “trehalase activity” refers to the ability to catalyze the hydrolysis of one molecule of trehalose into two molecules of glucose. In the art, trehalase may also be referred to as α,α-trehalose-1-C-glucose hydrolase. Suitable enzymes with trehalase activity may include, but are not limited to, those classified as EC 3.2.1.28. The polynucleotide encoding the trehalase may be derived from any suitable source. For example, the polynucleotide encoding the trehalase may be derived from *Bacillus thuringiensis*, *Bacillus oryzae*, or *Bacillus oryzae*. Magnaporthe grisea ), Candida glabrata ( Candida glabrataTrehalase 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 the amino acid sequence of at least one of SEQ ID NO: 25 and 26. Trehalase may be linked to a secretion signal having an amino acid sequence that is 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: 29, 30, 31, and 32. Trehalase may be linked to a secretion signal such that the recombinant cell contains an exogenous polynucleotide sequence encoding 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 NO: 33, 35, 36, 38, 39, 40, and 43. Trehalase can be linked to a secretion signal, causing recombinant cells to contain a foreign polynucleotide sequence encoding 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: 35, 36, 38, and 40.

[0058] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence, which is or may be derived from *Blastophyte oryzae*, encoding the amino acid sequence of SEQ ID NO: 25. 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: 25. The foreign polynucleotide sequence may encode amino acids containing a sequence that is at least 90% identical to that of SEQ ID NO: 25.

[0059] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence, which is or may be derived from *Candida glabrata*, encoding the amino acid sequence of SEQ ID NO: 26. 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: 26. The foreign polynucleotide sequence may encode amino acids containing at least 90% of the sequence identical to SEQ ID NO: 26.

[0060] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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 that of SEQ ID NO: 33. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 33.

[0061] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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: 35. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 35.

[0062] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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 that of SEQ ID NO: 36. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 36.

[0063] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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: 38. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 38.

[0064] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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 that of SEQ ID NO: 39. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 39.

[0065] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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: 40. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 40.

[0066] Recombinant cells capable of producing xylitol may contain a foreign polynucleotide sequence encoding 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: 43. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 90% identical to SEQ ID NO: 43.

[0067] The recombinant cells described herein may additionally include one or more enzymes in the xylitol biosynthesis pathway. For example, 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.

[0068] The final step in the xylitol pathway, from xylitol 5-phosphate to xylitol, requires a phosphatase. Saccharomyces cerevisiae PYP1 ( Diverse alcohol phosphorus acid enzyme 1 The 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 class of sorbitol-6-phosphatases (Enzyme Committee (EC) 3.1.3.50). Since xylitol 5-phosphate is a molecule similar to a known substrate 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* (… E. coli HxpA ( Hexose alcohol phosphorus acid enzyme AIt is a HAD-like enzyme belonging to EC3.1.3.50, and it has a substrate spectrum similar to PYP1 (Kuznetsova et al., "Genome wide analysis of substrate specificities of the Escherichia coli (HALODI-DEhalogenase-like phosphate family, 2006, J. Biol. Chem., 281, 36149-36161). Therefore, this paper also demonstrates that one or more HAD-like hydrolases or orthologs of HAD-like hydrolases possess xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells described herein.

[0069] The recombinant cells described herein are capable of producing xylitol, comprising an exogenous polynucleotide sequence encoding trehalase, and characterized by overexpression of a native enzyme with xylitol-5-phosphate phosphatase (X5PP) activity, and / or comprising an exogenous polynucleotide sequence encoding a native or exogenous enzyme with xylitol-5-phosphate phosphatase (X5PP) activity. Generally, recombinant cells including those overexpressing or expressing an exogenous X5PP enzyme produce more xylitol compared to equivalent cells lacking an exogenous X5PP enzyme or lacking X5PP enzyme overexpression. 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 Mg. 2+ Mn 2+ or Co 2+ Suitable enzymes possessing 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, the polynucleotide encoding the X5PP enzyme may be derived from *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Darcylachansa*, etc. Lachancea dasiensis ), cockroach tetrasporum ( Tetrapisispora blattae Pasteur yeast ( Saccharomyces pastorianus ), Kazakhstan yeast ( Kazachstania Africana ), *Trichoderma* ( Podospora comata ), white ground mold ( Geotrichum candidum Hagler's Osamuth Yeast ( Ogattaea haglerorum Fabry Barry yeast ( Debaryomyces fabryi), American and Australian type of Sclerotinia sclerotiorum ( Monilinia fructicola ), pale yellow yeast ( Nadsonia fulvescens Elongated variant DSM 6958, Escherichia coli, Wickham Severinia ( Wickerhamomyces ciferrii ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens X5PP 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 the amino acid sequence of at least one of SEQ ID NO: 24, 88, 89, 90, 91, 92, 93, 97, and 99. X5PP 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 the amino acid sequence of at least one of SEQ ID NO: 24, 89, 90, 91, 92, 93, 97, and 99. X5PP enzymes may 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 at least one of SEQ ID NOs: 24, 90, 91, 93, and 99. See, for example, U.S. Provisional Application No. 63 / 499,992, filed May 4, 2023, and incorporated herein by reference in its entirety.

[0070] The recombinant cell is capable of producing xylitol, contains an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* gene, encoding the amino acid sequence of SEQ ID NO: 74. The exogenous 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: 74.

[0071] The recombinant cells are capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae gene, encoding the amino acid sequence of SEQ ID NO: 75. The exogenous 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: 75.

[0072] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may be derived from the *Darcylachansa* 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 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.

[0073] The recombinant cell is capable of producing xylitol, contains an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from the *Tetrasporium cristatum* gene, encoding the amino acid sequence of SEQ ID NO: 77. The exogenous 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.

[0074] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may 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.

[0075] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may be derived from a yeast gene in Kazakhstan, Africa, 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.

[0076] The recombinant cells are capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from a gene of *Trichoderma* encoding the amino acid sequence of SEQ ID NO: 80. The exogenous 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.

[0077] The recombinant cells are capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from the *Geotrichum candida* gene, encoding the amino acid sequence of SEQ ID NO: 81. The exogenous 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.

[0078] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may be derived from the Hagler's Ogata yeast 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.

[0079] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may be derived from the *Fabried barley* 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.

[0080] The recombinant cells are capable of producing xylitol, containing a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may be derived from the *Sclerotinia 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.

[0081] The recombinant cells are capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from the *Narcissus nauplius* elongation variant DSM 6958 gene, encoding the amino acid sequence of SEQ ID NO: 85. The exogenous 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.

[0082] The recombinant cells are capable of producing xylitol, containing a foreign polynucleotide sequence encoding trehalase, 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.

[0083] The recombinant cell is capable of producing xylitol, comprising a foreign polynucleotide sequence encoding trehalase, 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.

[0084] The recombinant cell is capable of producing xylitol, contains an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* gene, encoding the amino acid sequence of SEQ ID NO: 88. The exogenous 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.

[0085] The recombinant cell is capable of producing xylitol, contains a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence that is or may be derived from a 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.

[0086] The recombinant cell is capable of producing xylitol, containing a foreign polynucleotide sequence encoding trehalase, 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.

[0087] The recombinant cell is capable of producing xylitol, containing a foreign polynucleotide sequence encoding trehalase, 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.

[0088] Enzymes with X5PP activity may be native to the host cell. For example, when the host organism is *Saccharomyces cerevisiae*, the X5PP enzyme may be an enzyme having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% with at least one of SEQ ID NO: 24, 101, 105, or 106. Recombinant cells may contain exogenous polynucleotides encoding X5PP enzymes having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% with at least one of SEQ ID NO: 24, 101, 105, or 106. The recombinant cell may contain genetic modifications that increase the expression of the X5PP enzyme, which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to at least one of SEQ ID NO: 24, 101, 105, or 106. Genetic modifications may include, but are not limited to: inserting an extra copy of the nucleic acid encoding the native X5PP enzyme into the cell (e.g., integrating an extra 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 native X5PP enzyme-encoding gene in the host cell genome, and / or modifying an existing promoter upstream of the coding region of the native X5PP enzyme-encoding gene in the host cell genome. Those skilled in the art will recognize that the expression of the native 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.

[0089] 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.

[0090] The recombinant cells described herein that produce xylitol and contain a foreign polynucleotide sequence encoding trehalase are further characterized by overexpression of ribulose 5-phosphate epimerase (RPE). The recombinant cells described herein that produce xylitol and contain a foreign polynucleotide sequence encoding trehalase may also contain a foreign polynucleotide sequence encoding X5PP and / or overexpress natural X5PP, and / or may contain a foreign polynucleotide encoding a natural or foreign RPE, or may have genetic modifications leading to overexpression of natural RPE, as described herein. Generally, recombinant cells including RPE overexpression produce more xylitol compared to equivalent cells lacking RPE or lacking RPE overexpression.

[0091] The recombinant cells described herein are capable of producing xylitol, contain an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have genetic modifications that lead to overexpression of the native RPE enzyme. The RPE enzyme may 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 may be native to the host cell, or the RPE enzyme may be an exogenous RPE enzyme. For example, when the host organism is *Saccharomyces cerevisiae*, the RPE enzyme may 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: 14 and 107. 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: 14 and 107. The recombinant cell may contain genetic modifications that increase RPE enzyme expression, wherein the enzyme is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical with at least one of SEQ ID NO: 14 and 107. Genetic modifications may include, but are not limited to: inserting an additional copy of the nucleic acid encoding the native RPE into the cell; inserting a constitutive promoter upstream of the coding region of the native RPE gene in the host cell genome; and / or modifying an existing promoter upstream of the coding region of the native RPE gene in the host cell genome. Those skilled in the art will recognize that the expression of the native RPE 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 No. PCT / US2023 / 066631 filed on May 5, 2023.

[0092] The recombinant cells described herein are capable of producing xylitol and contain a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide sequence encoding xylitol phosphate dehydrogenase (XPDH) enzyme. The foreign polynucleotide sequence may be the foreign xylitol phosphate dehydrogenase (XPDH) gene. The recombinant cells described herein capable of producing xylitol have a foreign polynucleotide sequence encoding trehalase and may contain a foreign polynucleotide sequence encoding X5PP enzyme and / or overexpress natural X5PP enzyme, may contain a foreign polynucleotide sequence encoding 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 natural RPE enzyme, as described herein.

[0093] 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 originate from any suitable source. For example, the XPDH gene can originate from Clostridium difficile (Clostridium difficile). Clostridium difficile Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), salt-resistant Bacillus ( Bacillus halodurans ), Bacillus ligninophilus ( Alkalihalobacillus ligniniphilus ), soil salt seafood Bacillus ( Jeotgalibacillus soli ), heat-resistant spores of Heindrix bacillus ( Heyndrickxia sporothermodurans Clostridium difficile ( ) Clostridium fungisolvens ) and Cucumber Neosporus ( Neobacillus cucumis The XPDH gene may encode an amino acid 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: 5, 108, 109, 110, 111, 112, 113, 114, 115, and 116. The XPDH gene 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%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NO: 5, 110, 111, or 114. See, for example, PCT application No. PCT / US2023 / 066629, filed May 5, 2023, the entire contents of which are incorporated herein by reference.

[0094] The recombinant cell is capable of producing xylitol, comprising an exogenous polynucleotide sequence encoding trehalase, 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: 108. 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: 108.

[0095] The recombinant cell is capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, 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: 109. 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: 109.

[0096] The recombinant cell is capable of producing xylitol, comprising an exogenous polynucleotide sequence encoding trehalase, 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.

[0097] The recombinant cell is capable of producing xylitol, comprising an exogenous polynucleotide sequence encoding trehalase, 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: 110. 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: 110.

[0098] The recombinant cell is capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide that is or may be derived from the gene of *Bacillus ligninophilus* encoding the amino acid sequence of SEQ ID NO: 111. 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: 111.

[0099] The recombinant cell is capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoded by or derived from the gene of *Bacillus spp.*, encoding the amino acid sequence of SEQ ID NO: 112. 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: 112.

[0100] The recombinant cell is capable of producing xylitol, comprising an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoded by or derived from the gene of *Hydroxella thermophila* encoding the amino acid sequence of SEQ ID NO: 113. 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: 113.

[0101] The recombinant cell is capable of producing xylitol, comprising a foreign polynucleotide sequence encoding trehalase, and may contain a foreign polynucleotide that is or may be derived from the Clostridium difficile gene encoding the amino acid sequence of SEQ ID NO: 114. 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: 114.

[0102] The recombinant cell is capable of producing xylitol, containing an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide that is or may be derived from a *Bacillus cucumberis* gene encoding the amino acid sequence SEQ ID NO: 116. 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: 116.

[0103] The recombinant cells described herein are capable of producing xylitol and contain an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide sequence encoding xylulose kinase (XKS) enzyme. The recombinant cells described herein capable of producing xylitol and containing an exogenous polynucleotide sequence encoding trehalase may additionally contain an exogenous polynucleotide sequence encoding X5PP enzyme and / or overexpress natural X5PP enzyme, an exogenous polynucleotide sequence encoding XKS enzyme, and / or contain an exogenous polynucleotide encoding natural or exogenous RPE enzyme, or may have genetic modifications leading to overexpression of natural RPE enzyme, as described herein. The exogenous polynucleotide sequence may be an exogenous xylulose phosphatase (XKS) gene.

[0104] 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 SEQ ID NO: 104 and 103. Further description of recombinant cells capable of producing xylitol and containing 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.

[0105] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoded by or derived from the *Saccharomyces cerevisiae* DOG1 sugar phosphatase 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.

[0106] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoded by the *Saccharomyces cerevisiae* DOG2 sugar phosphatase gene, which is or may be derived from the amino acid sequence encoding SEQ ID NO: 103. 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.

[0107] The recombinant cells described herein are capable of producing xylitol and possess exogenous polynucleotide sequences encoding trehalase and xylitol dehydrogenase (XDH) enzyme. The recombinant cells described herein capable of producing xylitol and possessing exogenous polynucleotide sequences encoding trehalase 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.

[0108] 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 originate from any suitable source. For example, the XDH gene can originate from Pichia pastoris (…). Pichia stipitis ), Rhodobulbaceae bacteria ( Rhodobacteraceae bacterium ) and silver leaf whitefly ( Bemisia argentofolii The XDH 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 SEQ ID NO: 117, 118, or 119. Further 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.

[0109] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoding the amino acid sequence of SEQ ID NO: 117, which is or may be derived from the Pichia pastoris XDH gene with cofactor switching. 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: 117.

[0110] The recombinant cell is capable of producing xylitol, possesses an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoding the amino acid sequence of SEQ ID NO: 118, which is or may be derived from the SDR family oxidoreductase gene of Rhodobulbusaceae bacteria. 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: 118.

[0111] The recombinant cells are capable of producing xylitol, possess an exogenous polynucleotide sequence encoding trehalase, and may contain an exogenous polynucleotide encoding the amino acid sequence of SEQ ID NO: 119, which is or may be derived from the ketose reductase (sorbitol dehydrogenase) gene of the silver leaf whitefly (Aphidida albicans). 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: 119.

[0112] The exogenous polynucleotides in recombinant cells described herein can be controlled by promoters. 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 decarboxylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 6); 6-phosphoglucose dehydrogenase promoter (6PGDp; SEQ ID NO: 120); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 121); translation elongation factor 1 promoter (TEFp; SEQ ID NO: 122); phosphoglucose mutase 1 promoter (PGM1p; SEQ ID NO: 123); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 22); enolase promoter (ENO1p; SEQ ID NO: 124); asparagine synthase promoter (ASNSp; SEQ ID NO: 124). 125); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 126); RPL16B (SEQ ID NO: 127), glycerol-3-phosphate dehydrogenase 2a promoter (GPDIIap; SEQ ID NO: 138); glycerol dehydrogenase NADP(H) 1 promoter (GDN1p; SEQ ID NO: 139); erythritol-P dehydrogenase 1 promoter (EPDH1; SEQ ID NO: 140), translation elongation factor 1A-like gene 7 promoter (TEF7p; SEQ ID NO: 141); heat shock protein 90 promoter (HSP90p; SEQ ID NO: 142); translation elongation factor 1A-like gene 4 promoter (TEF4p; SEQ ID NO: 143); translation elongation factor 2A-like gene 6 promoter (TEF6p; SEQ ID NO: 144); transaldolase 1 promoter (TAL1p; SEQ ID NO: 145); 145); Citrate synthase 1 promoter (CIT1p; SEQ ID NO: 146); Endoplasmic reticulum chaperone protein BiP (KAR2p; SEQ ID NO: 147); Heat shock protein 88 promoter (HSP88p; SEQ ID NO: 148); Heat shock protein 70 promoter (HPS70p; SEQ ID NO: 149); Tubulin α-1 chain promoter (TUB1p; SEQ ID NO: 150); Glyceraldehyde-3-phosphate dehydrogenase 2 promoter (TDH2p; SEQ ID NO: 151);The promoters of translation elongation factor 1A-like gene 5 (TEF5p; SEQ ID NO: 152); translation elongation factor 3 promoter (TEF3p; SEQ ID NO: 153); thioredoxin peroxidase promoter (TPXp; SEQ ID NO: 154); alginate lyase promoter (ALPp; SEQ ID NO: 155); thiazole biosynthesis 4 promoter (THI4p; SEQ ID NO: 156); phosphogluconosome / phosphogmannose mutase promoter (PMMp; SEQ ID NO: 157); fructose-bisphosphate aldolase 1 promoter (FBA1p; SEQ ID NO: 158); alcohol dehydrogenase 1 promoter (ADH1p; SEQ ID NO: 159); and extracellular endoglucanase 1 promoter (GLX1p; SEQ ID NO: 160).

[0113] The exogenous nucleic acids in the recombinant cells described herein can be controlled by terminators. For example, the exogenous nucleic acids can be operatively linked to heterologous or artificial terminators. Suitable terminators are known and described in the art. Terminators may include, but are not limited to: GAL10 terminator, PDC terminator, transaldolase terminator (TAL), 6PGD terminator (6PGDt; SEQ ID NO: 70); ASNS terminator (ASNSt; SEQ ID NO: 128); ENO1 terminator (ENO1t; SEQ ID NO: 129); hexokinase 1 terminator (HXK1t; SEQ ID NO: 130); PGK1 terminator (PGK1t; SEQ ID NO: 131); PGM1 terminator (PGM1t; SEQ ID NO: 132); PYK1 terminator (PYK1t; SEQ ID NO: 15); RPLA terminator (RPLAt; SEQ ID NO: 133); transaldolase 1 terminator (TAL1t; SEQ ID NO: 134); TDH3 terminator (TDH3t; SEQ ID NO: 135); translation elongation factor 2 terminator (TEF2t; SEQ ID NO: 135). 18); Triose phosphate isomerase 1 terminator (TPI1t; SEQ ID NO: 136); MpTEF1 (SEQ ID NO: 137); TEF7 terminator (TEF7t; SEQ ID NO: 161); HSP90 terminator (HSP90t; SEQ ID NO: 162); TEF4 terminator (TEF4t; SEQ ID NO: 163); TEF6 terminator (TEF6t; SEQ ID NO: 164); CIT1 terminator (CIT11t; SEQ ID NO: 165); KAR2 terminator (KAR2t; SEQ ID NO: 166); HPS88 terminator (HSP88t; SEQ ID NO: 167); HSP70 terminator (HSP70t; SEQ ID NO: 168); TUB1 terminator (TUB1t; SEQ ID NO: 169); TKL1 terminator (TAL1t; SEQ ID NO: 169); NO:170); TDH2 terminator (TDH2t; SEQ ID NO: 171); TEF5 terminator (TEF5t; SEQ ID NO: 172); TGL2 terminator (TGL2t; SEQ ID NO: 173); TEF3 terminator (TEF3t; SEQ ID NO: 174); TPX terminator (TPXt; SEQ ID NO: 175); ALP terminator (ALPt; SEQ ID NO: 176); plasma membrane ATPase terminator (PMA1t;SEQ ID NO: 177); THI4 terminator (THI4t; SEQ ID NO: 178); glucose-6-phosphate isomerase 1 terminator (PGI1t; SEQ ID NO: 179); PMM terminator (PMMt; SEQ ID NO: 180); FBA1 terminator (FBA1t; SEQ ID NO: 181); ADH1 terminator (ADH1t; SEQ ID NO: 182); sodium / potassium transport ATPase α-chain terminator (ATP1t; SEQ ID NO: 183); and GLX1 terminator (GLX1t; SEQ ID NO: 184).

[0114] 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).

[0115] 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 a sense strand or an antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods and may comprise polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Therefore, constructs may include novel modifications to endogenous genes introduced through, for example, genome editing technologies. Constructs may also include recombinant polynucleotides produced using, for example, recombinant DNA methodologies. Constructs may be vectors containing promoters operatively linked to a polynucleotide encoding a polypeptide as described herein. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide linked to it. Vectors may be plasmids, which are circular double-stranded DNA loops into which additional DNA fragments may be integrated.

[0116] This disclosure also provides a method for reducing trehalose in fermentation for xylitol production. Generally, the method involves contacting a substrate with engineered cells capable of producing xylitol. During fermentation, a trehalase is added to the fermentation broth to reduce and / or eliminate trehalose byproducts in the fermentation broth. Unbound by any particular theory or mode of action, the trehalase catalyzing the breakdown of trehalose into two glucose molecules will increase the glucose concentration available for xylitol production in the engineered cells, and the fermentation process also improves the xylitol titer and / or yield relative to an equivalent process without trehalase.

[0117] As fermentation progresses, exogenous trehalase (e.g., not generated in situ during the fermentation reaction) can be added to the fermentation broth. Trehalase can be added after the engineered cells have passed their growth phase. Trehalase can be added once, twice, three times, or more during fermentation. Trehalase can be added to the fermentation broth continuously.

[0118] Trehalase can be secreted from engineered cells. For example, a fermentation method may include the step of using a genetically engineered yeast fermentation substrate as described herein to produce xylitol, whereby trehalase expressed by the engineered yeast is secreted into the fermentation broth, and the concentration of trehalose is reduced.

[0119] As will be understood by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of these 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.

[0120] Fermentation methods can be carried out 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.

[0121] The fermentation process can be operated under a variety of conditions. The fermentation temperature (i.e., the temperature of the fermentation broth during processing) can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained 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 modified as appropriate.

[0122] The fermentation process can operate within a certain oxygen uptake (OUR) range. The volumetric OUR of the fermentation process can be up to 5 mmol O2 / (L). h)-80mmol O2 / (L h), 10mmol O2 / (L h)-75mmol O2 / (L h), 15mmol O2 / (L h)-70mmol O2 / (L h), 20mmol O2 / (L h)-60mmol O2 / (L h), 30mmol O2 / (L h)-50mmol O2 / (L h) or 40mmol O2 / (L h)-50mmol O2 / (L Within the range of h). In some implementations, the ratio of OUR can be 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 9mmol O2 / (g cell dry weight) h), 0.5 mmol O2 / (g cell dry weight) h) to 8mmol O2 / (g cell dry weight) h), 1.0 mmol O2 / (g cell dry weight) h) to 7mmol O2 / (g cell dry weight) h), 1.5 mmol O2 / (g cell dry weight) h) to 6mmol O2 / (g cell dry weight) h), 2mmol O2 / (g cell dry weight) h) to 5mmol O2 / (g cell dry weight) h) or 2.5 mmol O2 / (g cell dry weight) h) to 4mmol O2 / (g cell dry weight) The range of h) is not limited to the volume of the fermentation process. However, the volume of OUR or more than OUR is not limited to any particular rate or range described herein.

[0123] 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 inoculum density or inoculum rate of the fermentation process can vary. In some embodiments, the inoculum 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.

[0124] 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.

[0125] The fermentation process can be operated in a batch feed manner with dextrose. 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.

[0126] Fermentation processes can be associated with a variety of characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be influenced by yeast selection and / or genetic modification 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 selection of fermentation process conditions.

[0127] 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 -1 The 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.

[0128] The xylitol yield of this process can be at least 25%, at least 30%, at least 35%, at least 37%, at least 38%, or at least 40%. The xylitol yield of this process can be higher than that of an equivalent process operating in the absence of trehalase. For example, the xylitol yield of this process can be higher than that of an equivalent method using equivalent engineered cells lacking the polynucleotide sequence encoding trehalase.

[0129] Compared to an equivalent fermentation process without trehalase, this process can reduce trehalose titers by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The trehalase titer can be below the instrument detection limit. The trehalase titer can be 0 g / L.

[0130] Example

[0131] The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be construed as encompassing any and all variations that become apparent from the teachings provided herein.

[0132] Throughout the embodiments, strain numbers and sequence identification numbers were used consistently. For example, strains 1-8 in Example 2 were the same as strains 1-8 in Example 3, and so on.

[0133] Example 1: Trehalase Sequence

[0134] During fermentation, trehalose can accumulate in the fermentation broth. A recommended method for removing trehalose from the fermentation broth during xylitol production is to use trehalase in the fermentation broth. In these examples, the trehalases outlined in Table 2 will be used.

[0135] Table 2.

[0136] Trehalase may contain or be engineered to contain a secretion signal sequence, enabling the trehalase to be secreted from the cell and into the fermentation broth. Trehalase may have its own natural secretion signal, or it may be engineered to contain an exogenous signal sequence. Table 3 lists the various secretion signals used in these examples.

[0137] Table 3.

[0138] Example 2: Genetically modified *Saccharomyces cerevisiae* strain

[0139] strain 1-1

[0140] Strain 1-1 is the host strain of *Saccharomyces cerevisiae* described in US 6,440,712, which is incorporated herein by reference in its entirety. Moniliella tomentosa var pollinisTCV364” was deposited on March 28, 1997, under the number MUCL40385, at BCCM / MUCL (Belgian Coordinating Center for Microbial Collections / KU Leuven Fungal Collection; deposited by Eridania Béghin Say, Vilvoorde R&D Centre, at Havenstraat 84, B-1800 Vilvoorde) in accordance with the Treaty of Budapest.

[0141] Strain 1-2

[0142] Strains 1-1 were transformed with SEQ ID NO: 2 and SEQ ID NO: 1. Protoplast preparation of the parent strain was first performed by adding a mixture containing 0.6 M MgSO4, 7.5 g / L cytokinase, and 12.5 g / L Trichoderma harzianum to the mycelial precipitate of the parent strain. Trichoderma harzianum The enzyme mixture of lysins was then used. The protoplast precipitate was then washed with 0.6 M MgSO4 and resuspended in STC medium (0.6 M sucrose, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). The protoplast mixture was then precipitated by adding approximately 200 μL of the protoplast mixture (10... 8 To prepare a dichotomous transformation system, 100 µg of single-stranded salmon sperm DNA and 1.5 µg to 5 µg each of 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, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, resuscitation medium (0.4 M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L sugar, pH 4.5) was added to the mixture, and the mixture was incubated at 27°C and 100 rpm for 16–24 hours. After incubation, the protoplasts were precipitated by centrifugation and resuspended in 1 mL of PBS. The resuspended protoplasts were plated on PDA + 250 mg / L genistein (G418) selection plates and incubated at 35°C for at least 2 days until transformants grew. The resulting transformants were streaked onto PDA+genimycin (G418) plates to isolate single colonies, which were then selected. The selected colonies were evaluated by colony PCR to confirm the integration of two copies of the *Lactobacillus rhamnosus* XPDH sequence. The PCR-validated isolates were named strains 1-2.

[0143] SEQ ID NO: 1 contains (i) 5' flanking DNA for targeting chromosome integration into the ER1 locus (SEQ ID NO: 3); (ii) a polynucleotide sequence SEQ ID NO: 4 encoding an XPDH homologue of *Lactobacillus rhamnosus* from SEQ ID NO: 5, controlled by the PYK1 promoter of SEQ ID NO: 6 and the PGD terminator of SEQ ID NO: 7; and (iii) a G418-selectively labeled 5' portion (SEQ ID NO: 8). SEQ ID NO: 2 contains (i) a G418-selectively labeled 3' portion (SEQ ID NO: 9); (ii) a polynucleotide sequence SEQ ID NO: 4 encoding an XPDH homologue of *Lactobacillus rhamnosus* from SEQ ID NO: 5, controlled by the PYK1 promoter of SEQ ID NO: 6 and the PGD terminator of SEQ ID NO: 7; and (iii) 3' flanking DNA for targeting chromosome integration into the ER1 locus (SEQ ID NO: 10).

[0144] Strains 1-3

[0145] Using the procedure outlined above, strains 1-2 were transformed with SEQ ID NO: 11 and SEQ ID NO: 12. The transformation fragment of SEQ ID NO: 11 contained, sequentially, a 5' ER3 flanking sequence (SEQ ID NO: 13), an MpPYK1 promoter (SEQ ID NO: 6), a gene encoding the *Saccharomyces cerevisiae* RPE2 polypeptide (SEQ ID NO: 14), an MpPYK terminator (SEQ ID NO: 15), and the 5' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 16). The transformation fragment of SEQ ID NO: 12 contained, sequentially, the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 17), an MpTEF2 terminator (SEQ ID NO: 18), and a 3' ER3 flanking sequence (SEQ ID NO: 19). The resulting transformants were streaked on PDA+bleomycin plates to isolate single colonies, and single colonies were selected. Selected colonies were evaluated by colony PCR to confirm the integration of the *Saccharomyces cerevisiae* RPE2 sequence. The PCR-validated isolates were named strains 1-3.

[0146] Strains 1-4

[0147] UV mutagenesis was used (using a Hoefer UV crosslinker, energy 360 uJ / cm). 3The selection of strains 1-3 was conducted to generate *Saccharomyces cerevisiae* strains exhibiting reduced foaming during shake-flask fermentation. Based on a visual assessment of the degree of foaming during shake-flask fermentation compared to the foaming degree of parental strains 1-3, strains with a low-foaming phenotype were selected. The resulting low-foaming strains were named 1-4, 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: 14) integrated at the ER3 locus.

[0148] Strains 1-5

[0149] Using the transformation method outlined above, strains 1-4 were transformed with the Cre recombinase plasmid of SEQ ID NO: 20. The resulting transformants were evaluated by colony PCR to confirm the removal of the G418 and bleomycin resistance selection markers. The PCR-validated isolates were named strains 1-5.

[0150] Strains 1-6

[0151] Strains 1-5 were non-selectively grown on YPD plates to allow for the loss of the plasmid in SEQ ID NO: 20. Biomass was streaked to isolate single colonies, and plasmid loss was confirmed by PCR evaluation. The PCR-validated isolates were named strains 1-6.

[0152] Strains 1-7

[0153] Using the transformation method described above, strains 1-6 were transformed with SEQ ID NO: 21 and SEQ ID NO: 11. SEQ ID NO: 21 contains, sequentially, the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 17), the MpPGK1 promoter (SEQ ID NO: 22), the gene encoding the *Saccharomyces cerevisiae* X5PP polypeptide (SEQ ID NO: 23), the Mp6PGD terminator (SEQ ID NO: 7), and the 3' ER3 flanking sequence (SEQ ID NO: 19). The resulting transformants were streaked on PDA+bleomycin plates to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the specified sequence. The PCR-validated isolates were named strains 1-7.

[0154] Strains 1-8

[0155] To remove the bleomycin resistance selection marker, strains 1-7 were transformed with the Cre recombinase plasmid of SEQ ID NO: 20 using the transformation method outlined above. The resulting transformants were evaluated by colony PCR to confirm the removal of the bleomycin resistance selection marker. The PCR-validated isolates were named strains 1-8.

[0156] strains 1-9 to 1-26

[0157] Using the transformation method outlined above, strains 1-8 were transformed with SEQ ID NO: 67 and SEQ ID NO: 49-66 according to Table 4. SEQ ID NO: 67 contains, sequentially, a 5' gpdIIB flanking sequence (SEQ ID NO: 86) and the 5' portion of the G418 resistance gene expression cassette (SEQ ID NO: 8). Each of SEQ ID NO: 49-66 contains, sequentially, the 3' portion of the G418 resistance gene expression cassette (SEQ ID NO: 9), the MpTDH3 promoter (SEQ ID NO: 121), the nucleotide sequence encoding one of SEQ ID NO: 27, 28, or 33-48, the Mp6PGD terminator (SEQ ID NO: 7), and the 3' gpdIIB flanking sequence (SEQ ID NO: 87). The resulting transformants were streaked on PDA + 250 mg / L genimycin (G418) plates to isolate single colonies, and single colonies were selected. Selected colonies were evaluated by colony PCR to confirm the integration of the specified sequence. PCR-validated isolates were named as summarized in Table 4.

[0158] For example, strains 1-8 were transformed using the transformation method outlined above with SEQ ID NO: 67 and SEQ ID NO: 49. SEQ ID NO: 49 contains, sequentially, the 3' portion of the G418 resistance gene expression cassette (SEQ ID NO: 9), the MpTDH3 promoter (SEQ ID NO: 121), the nucleotide sequence encoding SEQ ID NO: 33, the Mp6PGD terminator (SEQ ID NO: 7), and the 3' gpdIIB flanking sequence (SEQ ID NO: 87). The resulting transformants were streaked on PDA + 250 mg / L genistein (G418) plates to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR to confirm the integration of the polypeptide sequence encoding SEQ ID NO: 33. The PCR-validated isolates were named strains 1-9a, 1-9b, 1-9c, and 1-9d.

[0159] Table 4.

[0160] Example 3: Deep Hole Plate Measurement

[0161] The strain from Example 2 was added to 48-well plates containing 1 mL of enriched medium (170 g / L glucose, 10 g / L yeast extract) and incubated at 900 RPM and 30 °C for 44 h. 20 μL of the resulting culture was transferred to 48-well plates containing production medium (Table 5), in triplicate (plates A, B, and C). Control strains 1–8 were also added to each plate. Plate A was removed at 48 h, plate B at 72 h, and plate C at 90 h. Xylitol, DP2 sugar, and trehalose in plate C (90 h) were analyzed by high-performance liquid chromatography equipped with a differential refractive index detector. The results are reported in Table 6 and Figure 2 middle.

[0162] Table 5: Production Culture Media

[0163] Table 6.

[0164] The results showed that several strains had reduced DP2 sugar and trehalose levels compared to the control. For example, strains 1-11, 1-12, 1-14, 1-15, 1-16, 1-21, and 1-23 showed reduced levels of both DP2 sugar and trehalose at 90 hours compared to control strain 1-8. These results indicate that the secretion of trehalase into the fermentation broth (e.g., through the use of secretion signals) reduces the amount of trehalose in the fermentation broth.

[0165] Example 4: Shake-flask fermentation assay

[0166] Strains 1-9c, 1-11a-d, 1-12b-d, 1-14b, 1-14d, 1-15b, 1-15c, 1-16a, 1-16c, 1-16d, 1-17a, 1-17c, 1-18a, 1-18c, 1-20d, 1-21b, 1-22a, 1-23c, 1-24d, 1-25s, 1-25d, 1-26c, and 1-8 were run in shake flasks to assess glucose consumption and the production of erythritol, xylitol, glycerol, trehalose, and ethanol.

[0167] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. After incubation, cells were scraped from the YPD plates and transferred to 250 mL baffleless shake flasks containing 40 mL of rich medium (170 g / L glucose, 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) in a 1 cm path length cuvette. The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0168] 0.4 mL of seed culture was inoculated into a 250 mL unbaffled shaker flask containing 20 mL of production medium (Table 5) 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, xylitol, erythritol, glycerol, trehalose, and ethanol in the samples were analyzed by high-performance liquid chromatography (HPLC) equipped with a differential refractive index detector. The results are shown in Table 7, and the xylitol yield is reported in Table 8. Results are also shown in... Figure 3 and Figure 4 Trehalose titers were measured only at the 72-hour and 96-hour time points. Xylitol yield was 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 was 100 g / L, and 20 g / L of xylitol was produced at 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%.

[0169] The results showed that, compared with the control strain 8-1, several tested trehalases could reduce trehalase titers and / or increase xylitol yields. For example, at 96 hours, strains 1-9c, 1-11a, 1-11b, 1-11c, 1-11d, 1-12b, 1-12c, 1-12d, 1-14b, 1-14d, 1-15b, 1-15c, 1-16a, 1-16c, 1-16d, 1-17a, 1-17c, 1-18c, 1-21b, 1-22a, 1-23c, 1-24d, and 1-26c all showed trehalase titers lower than the control strain 1-8 (2.45 g / L). Furthermore, strains 1-11b, 1-16c, and 1-16d showed trehalase titers reduced to below the HPLC detection limit. After 96 hours of fermentation, strains 1-9c, 1-11a, 1-11d, 1-12b, 1-12c, 1-12d, 1-14d, 1-15b, 1-15c, 1-16a, 1-16c, 1-16d, 1-17a, 1-17c, 1-20d, 1-21b, and 1-22a produced higher xylitol yields than the control strain 8-1. Strains 1-9c, 1-11a, 1-15b, 1-17c, and 1-20d showed yield increases exceeding 0.5%, while strains 1-11d, 1-12d, 1-15c, 1-16c, and 1-22a showed yield increases exceeding 1.0%.

[0170] Among the strains tested, some sister strain variability was observed. Although the strains were confirmed by PCR to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, may have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused differences between individual sister strains and other strains. The results here indicate that similar transformation events exist among these sister strains. However, the results still suggest that the expression of the encoded trehalase reduces the trehalose concentration in the fermentation broth and increases the xylitol yield.

[0171]

[0172] Table 8: Xylitol yield at 96 hours

Claims

1. A method for producing xylitol, the method comprising: The fermentation broth containing a substrate containing dextran is contacted with engineered cells capable of producing xylitol, wherein the engineered cells ferment the substrate to produce xylitol. During fermentation, the fermentation broth contains trehalase, which results in a lower concentration of trehalose at the end of fermentation compared to an equivalent fermentation run without trehalase.

2. The method according to claim 1, wherein the trehalase is exogenously added to the fermentation broth.

3. The method of claim 1, wherein the engineered cell comprises an exogenous polynucleotide sequence encoding a secretion signal operatively linked to a trehalase, and the trehalase is secreted from the engineered cell.

4. A genetically engineered cell comprising an exogenous polynucleotide sequence encoding a secretion signal operatively linked to a trehalase, the trehalase being secreted from the engineered cell.

5. The method or cell according to claim 3 or 4, wherein the polypeptide sequence of the secretion signal is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 29-32, and the polypeptide sequence of the trehalase is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NO: 25-28, preferably SEQ ID NO: 25 or 26.

6. The method or cell according to any one of claims 3-5, wherein the polynucleotide sequence encodes a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of 33, 35, 36, 38, 39, 40, and 43, preferably SEQ ID NO: 35, 36, 38, and 40, most preferably SEQ ID NO: 35, 36, and 40.

7. The method according to any one of claims 1-3 or 5-6, wherein the trehalose titer is reduced relative to an equivalent method lacking the trehalase, for example, the trehalose titer is reduced by at least 25%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or 100% relative to an equivalent method lacking the trehalase.

8. The method according to any one of claims 1-3 or 5-7, wherein the method does not produce any trehalose, or the trehalose titer is below a detectable level.

9. The method according to any one of claims 1-3 or 5-8, 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).

10. The method according to any one of claims 1-3 or 5-9, 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 The rate at which it is generated.

11. The method according to any one of claims 1-3 or 5-10, 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.

12. The method according to any one of claims 1-3 or 5-11, wherein the concentration of dextrose is at least 100 g / L.

13. The method or cell according to any of the preceding claims, wherein the engineered cell comprises an exogenous polynucleotide sequence encoding a xylitol phosphate dehydrogenase (XPDH) enzyme, the XPDH enzyme comprising 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, 108-114, and 116, preferably at least one of SEQ ID NO: 5, 110, 111, and 114.

14. The method or cell according to any of the preceding claims, wherein the engineered cell comprises an exogenous polynucleotide sequence encoding a xylulosinase (XKS) enzyme, the XKS enzyme comprising 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: 103 and 104.

15. The method or cell of claim 14, wherein the engineered cell further comprises an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme, the XDH enzyme comprising 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: 117-119.

16. The method or cell according to any of the preceding claims, wherein the cell is a yeast cell, such as an osmotically resistant yeast cell.

17. The method or cell according to claim 16, wherein the yeast cell is a cell of the subphylum Ustilago, for example, a cell selected from the group consisting of: *Gastromycota macrocarpa*, *Tritylosporium spp.*, *Gastromycota melanogaster*, *Pseudomonas tsukuba*, *Trichoderma mutabilis*, *Gastromycota*, *Ustilago*, *Gastromycota*, *Gastromycota*, *Yarrowia lipolytica*, *Saccharomyces cerevisiae*, *Penicillium*, *Cytomyces*, *Pichia pastoris*, *Candida*, *Candida magnoliata*, and *Candida brevis*.

18. The method or cell according to any of the preceding claims, wherein the cell is a *Saccharomyces cerevisiae* cell.

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

20. The method or cell of claim 19, wherein the cell is a *Saccharomyces cerevisiae* cell, and the genetic modification results in the overexpression of a natural X5PP enzyme having 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: 24, 101, 105, or 106, for example, the genetic modification comprising 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.

21. The method or cell according to any of the preceding claims, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having X5PP activity, and is 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: 24 and 88-104, preferably SEQ ID NO: 24, 89-93, 97 and 99, most preferably SEQ ID NO: 24, 90, 91, 93 and 99.

22. The method or cell according to any of the preceding claims, wherein the engineered cell is a *Saccharomyces cerevisiae* cell, and the cell contains 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 natural gene encoding erythrose reductase as at least one of SEQ ID NO: 74 and 76.

23. The method of claim 22, wherein the rate, titer, and / or yield of erythritol production are reduced relative to equivalent fermentation using equivalent yeast cells in which the natural gene encoding the erythritol reductase is not deleted or destroyed.

24. The method according to claim 22 or 23, wherein when the fermentation is carried out at 35°C for 96 hours, the erythritol titer is less than 20 g / L, 18 g / L, 15 g / L, 12 g / L, 10 g / L, 5 g / L, 1 g / L, or 0.5 g / L; wherein the erythritol production rate is less than 0.5 g / L. -1 h -1 0.4g L -1 h -1 0.3g L -1 h -1 0.25g L -1 h -1 0.2g L -1 h -1 0.15g L -1 h -1 Or 0.1gL -1 h -1 ; and / or the erythritol yield is less than 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1%.

25. Use of engineered cells according to any one of claims 4-22 for the production of xylitol in a trehalose-reduced fermentation process.

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