Genetically modified yeast and fermentation processes for the production of xylitol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing xylitol and characterized by overexpression of a native glycerol kinase enzyme and overexpression of a native transketolase enzyme. The genetically engineered yeast cell may additionally have a deletion or disruption of a native gene encoding an erythrose reductase and may be engineered to overexpress a native RPE enzyme, express an exogenous XPDH enzyme, express an exogenous XKS enzyme, express an exogenous XDH enzyme, overexpress a native X5PP enzyme, and / or express an exogenous X5PP enzyme.

Description

GENETICALLY MODIFIED YEAST AND FERMENTATION PROCESSES FOR THEPRODUCTION OF XYLITOLCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 640,918, filed May 1, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA PATENT CENTER

[0002] The content of the Sequence Listing XML file of the sequence listing named “PT-2009- WO-PCT.xml” which is 345,628 bytes in size created on April 1, 2025 and electronically submitted via Patent Center herewith the application is incorporated by reference in its entirety.BACKGROUND

[0003] Xylitol is a low-calorie sweetener used as a food additive and sugar substitute. Commonly used in drug, dietary supplement, confectionary, and toothpaste compositions, xylitol has also been associated with anticariogenic properties when used in chewing gums. Traditional methods of xylitol production, including chemically catalyzed hydrogenation of xylose hydrolyzed from biomass extracted xylan, are both monetarily and environmentally costly. These methods require high temperatures and pressures, large amounts of water, and metal catalysts that must be mined. In contrast, fermentation processes have been used commercially at large scale to produce other organic molecules, such as ethanol, citric acid, lactic acid, and the like, and may offer a cost effective and sustainable alternative to traditional xylitol processing methods.

[0004] In the development of microorganism-based fermentation strategies for the production of xylitol, production of metabolic pathway intermediates and alternative fermentation products are important considerations. For example, metabolic pathways active in the production of xylitol may have overlap with the metabolic pathways for the production of arabitol, erythritol, ribitol, and the like. Similarly, alternative fermentation products such as ethanol and glycerol may be produced. However, production of other metabolites and fermentation products pulls carbon away from the production of xylitol. Accordingly, provided herein are genetically modified yeast and fermentation methods for the production of xylitol, wherein the production of glycerol is also reduced or eliminated.SUMMARY

[0005] The present disclosure provides a genetically engineered yeast cell capable of producing xylitol, the engineered yeast cell comprising a genetic modification resulting in overexpression of a native enzyme with glycerol kinase activity, for example a glycerol kinase enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110; and a genetic modification resulting in overexpression of a native enzyme with transketolase activity, for example a transketolase enzyme comprising a sequence 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 SEQ ID NO: 129. The genetic modification resulting in overexpression of a native glycerol kinase enzyme may comprise addition of an exogenous polynucleotide encoding the native glycerol kinase enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the glycerol kinase enzyme. The genetic modification resulting in overexpression of a native transketolase enzyme may comprise an addition of an exogenous polynucleotide encoding the native transketolase enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the transketolase enzyme. The yeast cell may comprise at least one additional copy of a polynucleotide sequence encoding a glycerol kinase enzyme 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 SEQ ID NO:96. The yeast cell may comprise at least one additional copy of a polynucleotide sequence encoding a glycerol kinase enzyme 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 SEQ ID NO: 110. The yeast cell may comprise at least one additional copy of a polynucleotide sequence encoding a transketolase enzyme 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 SEQ ID NO: 129.When the engineered cell is used in a fermentation process in the presence of dextrose, titer and / or yield of glycerol production is decreased relative to titer and / or yield of glycerol in an equivalent fermentation process using an equivalent cell in which there has been no overexpression of a native gene encoding a glycerol kinase at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110 and no over expression of a native gene encoding atransketolase enzyme 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 SEQ ID NO: 129.

[0006] The yeast cell may be an osmotol erant yeast cell. The yeast cell may be a cell of the subphylum Ustilaginomycotina. The yeast cell may be selected form the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis. Trychosporonoides nigrescens. Pseudozyma Isiikubaensis. Trigonopsis variabilis. Moniliella. Ustilaginomycetes, Trichosporon. Yarrowia lipolylica. Penicillium. Torula, Pichia. Candida, Candida magnolias, and Aureobasidium .

[0007] The cell may have a deletion or disruption of a native gene encoding an erythrose reductase enzyme. The cell may have a deletion of at least one allele of the gene encoding the erythrose reductase enzyme. The cell may have a deletion of both alleles of the gene encoding the erythrose reductase enzyme. When the engineered cell is used in a fermentation process in the presence of dextrose, titer and / or yield of erythritol production is decreased relative to titer and / or yield of erythritol in an equivalent fermentation process using an equivalent cell in which there has been no disruption or deletion of the native gene encoding an erythrose reductase gene. The cell may be Moniliella pollinis cell and the gene encoding the erythrose reductase enzyme is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 11 and 15.

[0008] The cell may additionally comprise an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:55-64. The XPDH enzyme may have a sequence at least 85% identical to at least one of SEQ ID NOs:55-62, and 64, or to at least one of SEQ ID NOs:57, 58, 59, or 62. The XPDH enzyme has a sequence at least 90% identical to at least one of SEQ ID NOs: 55-62, and 64, or to at least one of SEQ ID NOs:57, 58, 59, or 62.

[0009] The cell may additionally comprise an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 50 and 49. The cell may additionally comprise an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:65, 66, and 67.

[0010] The cell may additionally comprise a genetic modification resulting in overexpression of a native enzyme with ribulose-5-phosphate epimerase (RPE) activity. The yeast cell may be a yeast cell of the genus Moniliella. The yeast cell may be a Moniliella pollinis cell and the native RPE enzyme comprises a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:53 and 54. The genetic modification resulting in overexpression of a native RPE enzyme comprises addition of an exogenous polynucleotide encoding the native RPE enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the RPE enzyme.

[0011] The yeast may additionally comprise a genetic modification resulting in overexpression of a native enzyme with xylitol-5-phosphate phosphatase (X5PP) activity; and / or an exogenous polynucleotide sequence encoding an enzyme with xylitol-5-phosphate phosphatase (X5PP) activity. The cell may be a Moniliella pollinis cell and the genetic modification results in overexpression of a native X5PP enzyme with a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:51, 52, 33, or 47. The genetic modification comprises addition of an exogenous polynucleotide sequence encoding the native X5PP enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the native X5PP enzyme. The yeast cell may comprise an exogenous polynucleotide sequence encoding an enzyme with X5PP activity and a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:33-50. The yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with X5PP activity and a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:33, 35, 36, 37, 38, 39, 43, and 45. The yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with X5PP activity and a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:33, 36, 37, 39, and 45. X5PP activity in the genetically engineered yeast cell may be higher than X5PP activity in an equivalent cell lacking the genetic modification or exogenous polynucleotide sequence.

[0012] One or more exogenous polynucleotide sequence(s) described above may be operably linked to a heterologous or artificial promoter. The heterologous or artificial promoter may be selected from the group consisting of pyruvate kinase 1 promoter (PYKlp; SEQ ID NO:68), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 69), glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:70), translational elongation factor 1 promoter (TEFp; SEQ ID NO:71), phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72), 3 -phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73), enolase promoter (ENOlp ; SEQ ID NO:74), asparagine synthetase promoter (ASNSp; SEQ ID NO:75), 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76), and RPL16B (SEQ ID NO:77). The promoter may be a constitutive promoter. One or more exogenous polynucleotide sequence(s) may be integrated into the genome of the yeast cell at a locus selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdllA locus, the gpdllB locus, the RCSR26640 locus, and the RCSR18717 locus.

[0013] The disclosure also provides a method for producing xylitol using the engineered cells described herein, the method comprising contacting a substrate comprising dextrose with an engineered cell described herein, wherein fermentation of the substrate by the engineered cell produces xylitol. The fermentation temperature may be at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C. The volumetric oxygen uptake rate (OUR) may be between 5-80, 10- 75, 15-70, 20-60, 30-50, or 40-50 mmol O2 / (L • h). The xylitol may be produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h'1. Xylitol production may be at least at least 20, 30, 50, 75, or 100 g / L when the fermentation is run at 35 °C for 96 hours. The concentration of dextrose may be at least 100 g / L. Titer and / or yield of glycerol production is decreased relative to an equivalent fermentation run with an equivalent yeast cell in which the native gene encoding the glycerol kinase has not been over expressed. The glycerol titer may be less than 10, 8, 6, 5, 4, 3, 2, 1, or 0.5 g / L when the fermentation is run at 35 °C for 96 hours. The glycerol yield may be less than 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%.

[0014] The disclosure also provides use of the engineered cells described herein to produce xylitol.BRIEF DESCRIPTION OF THE FIGURES

[0015] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and the payment of the necessary fee.

[0016] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0017] FIG. 1 shows the native pentose phosphate pathway (dotted lines and arrows) and the native glycolysis pathways (solid lines and arrows) \nMoniliella pollinis.

[0018] FIG. 2 shows glycerol titers (g / L) for the fermentations outlined in Example 2.

[0019] FIG. 3 shows glycerol titers (g / L) for the fermentations outlined in Example 3.

[0020] FIG. 4 shows glycerol titers (g / L) for the fermentations outlined in Example 5.

[0021] FIG. 5 shows glycerol titers (g / L) for the fermentations outlined in Example 6.

[0022] FIG. 6 shows glycerol titers (g / L) for the fermentations outlined in Example 7.

[0023] FIG. 7 shows glycerol titers (g / L) for the fermentations outlined in Example 7.

[0024] FIG. 8 shows glycerol titers (g / L) for the fermentations outlined in Example 8.

[0025] FIG. 9 shows glycerol titers (g / L) for fermentations outlined in Example 10.

[0026] FIG. 10 shows xylitol titers (g / L) for fermentations outlined in Example 10.

[0027] FIG. 11 shows glycerol and xylitol titers (g / L) for fermentations outlined in Example10.

[0028] FIG. 12 shows glycerol and xylitol titers (g / L) for fermentations outlined in Example10.

[0029] FIG. 13 shows DP2 sugar accumulation (g / L) for fermentations outlined in Example10.

[0030] FIG. 14 shows glycerol and xylitol titers (g / L) for fermentations outlined in Example11.

[0031] FIG. 15 shows glycerol and xylitol titers (g / L) for fermentations outlined in Example11.

[0032] FIG. 16 shows glycerol and xylitol titers (g / L) for fermentations outlined in Example11.

[0033] FIG. 17 shows DP2 sugar accumulation (g / L) for fermentations outlined in Example11.DETAILED DESCRIPTION

[0034] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understoodthat the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0035] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0036] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0037] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis is also referred to as wt% or % (wt) below.

[0038] This disclosure relates to various recombinant cells engineered to produce xylitol. In general, the recombinant cells described herein are capable of producing xylitol and are characterized by overexpression of a glycerol kinase enzyme and expression of a transketolase enzyme. The recombinant yeast may additionally be characterized by deletion or disruption of a native gene encoding an erythrose reductase enzyme; overexpression of a ribulose 5-phosphatase epimerase (RPE) enzyme; inclusion of an exogenous polynucleotide sequence encoding a xylitol phosphate dehydrogenase (XPDH) enzyme; and / or overexpression of a xylitol-5-phosphate phosphatase (X5PP) enzyme. The disclosure further provides fermentation methods for the production of xylitol from dextrose, with reduced glycerol production, using the genetically engineered cells described herein.

[0039] In general, recombinant cells described herein are yeast cells. As used herein, “yeast” refers to eukaryotic single celled microorganisms classified as members of the fungus kingdom. Yeast are unicellular organisms which evolved from multicellular ancestors with some species retaining multicellular characteristics such as forming strings of connected budding cells known as pseudo hyphae or false hyphae. 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. Komagataella sp., Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus . Yarrowia lipolytica, Issatchenkia orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NR.R.L designation), Pichia pastoris, Candida (e.g., Candida magnolias, Candida elhanolica , Pichia deserlicola, Pichia memhranifadens, Pichia fermenlans, Aspergillus, Trichoderma, Myceliphthora thermophila, Moniliella (e.g., Moniliella pollinis), Pfaffia, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens), Pseudozyma tsukubaensis, Trigonopsis variabilis, Penicillium, and Torula. An ordinarily skilled artisan would understand the requirements for selection of a suitable yeast cell, and recombinant yeast cells of the present disclosure are not limited to those expressly recited herein. Methods for genetic engineering of yeast cells are known and described in the art and a skilled artisan would understand the methods necessary to transform and engineer a suitable yeast cell.

[0040] A suitable yeast cell may be a cell of the phylum Basidiomycota and the subphylum Ustilaginomycotina. Suitable yeast of the subphylum Ustilaginomycotina include, but are not limited to, Ustilago (e.g., U cynodontis, U maydis, U sphaerogena, U cordal, U scitaminea, U coicis, U syntherismae, U esculenta, U. neglecta, U crus-galli, Ustilago avenae), Sporisorium (e.g., Sporisorium exsertum), Moniliella (e.g., M. pollinis, M. tomentosa, M. acetoabutans, M. fonsecae, M. madida, M. megachiliensis, M. ocedocephalis, M. nigrescens), and Pseudozyma (e.g., Pseudozyma tsukubaensis), and Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens). Yeast of the subphylum Ustilaginomycotina have been known and described in the art as potential production organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol and other valuable biotechnological applications. See, for example, Geiser et al. (Prospecting the biodiversity of the fungal family Ustilaginacceae for the production of value-added chemicals,” Fungal Biol Biotechnol, 2014, 1 :2), Feldbrugge et al., (“The biotechnological use and potential of plant pathogenic smut fungi,” Appl Microbiol Biotechnol, 2013, 97(8):3253-65), Guevarra et al.,(“Accumulation of itaconic, 2-hydroxyparaconic, itatartaric, and malic acids by strains of the genus Ustilago, Agric. Biol. Chem., 1990, 54(9), 2353-2358), and Moon et al., (“Biotechnological production of erythritol and its applications,” Appl Microbiol Biotechnol, 2010, 86: 1017-1025).

[0041] A suitable yeast cell will have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein “active pentose phosphate pathway” refers to expression of one or more functional enzymes which, together, convert glucose-6-phosphate, NADP+or NAD+, and water to NADPH or NADH, CO2, and ribulose-5-phosphate. Continuing in a non-oxidative phase, the pathway may also produce other pentose (i.e., 5-carbon) sugars. For example, the pentose phosphate pathway may produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5- phosphate, fructose 6-phosphate, combinations thereof, and the like, depending on the enzymatic activities present. The active pentose phosphate pathway may be native to the yeast cell or it may be introduced into the yeast cell by genetic engineering.

[0042] The yeast cell may be an osmotol erant yeast cell. As used herein, “osmotol erant” refers to a yeast capable of growth and reproduction under conditions of high osmolarity, 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), at least 15% (w / v) sodium chloride. Species and strains of osmotolerant yeast are known and described in the art, including many species of yeast used in industrial fermentation processes. Likewise, methods for assaying yeast osmotolerance 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).

[0043] The recombinant yeast cell may be a recombinant Moniliella cell, for example, a Moniliella pollinis cell. FIG. 1 shows the predicted native pentose phosphate and glycolysis pathways in Moniliella pollinis. Moniliella has previously been used in the fermentation production of erythritol and methods for genetically modifying and fermenting Moniliella 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).

[0044] Various plasmids and methods for transformation of Moniliella are also described in the Examples below. For example, Moniliella may be transformed using a bipartite polynucleotide sequence in which, following recombination, the exogenous polynucleotide of interest is integrated at the specified locus and the selection marker is expressible within the cell. Suitable selection markers are known and used in the art. The selectable marker may include, but is notlimited to, amdS (for example broken into a 3’ portion, SEQ ID NO: 1, and a 5’ portion, SEQ ID NO:2), G418 resistance gene (for example broken into a 3’ portion, SEQ ID NO:3, and a 5’ portion, SEQ ID NO:4), zeocin resistance gene (for example broken into a 3’ portion, SEQ ID NO:5, and a 5’ portion, SEQ ID NO:6), nourseothricin N-acetyl transferase (NAT) (for example broken into a 3’ portion, SEQ ID NO: 7, and a 5’ portion, SEQ ID NO: 8), and invertase gene (SUC2) (for example a 3’ portion of SEQ ID NO:9 and a 5’ portion of SEQ ID NO: 10).

[0045] The terms “glucose” and “dextrose” are used interchangeably herein and refer to D- glucose except where expressly indicated otherwise.

[0046] The recombinant cells described herein include one or more exogenous polynucleotide sequences encoding one or more polypeptides that, when expressed, improve the fermentation of glucose to xylitol by the recombinant cells.

[0047] As used herein, “exogenous” refers to genetic material or an expression product thereof that originates from outside of the host organism. For example, the exogenous genetic material or expression product thereof can be a modified form of genetic material native to the host organism, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component. For example, a K. lactis invertase gene is exogenous when introduced into S. cerevisiae.

[0048] As used herein, “native” refers to genetic material or an expression product thereof that is found, apart from individual-to-individual mutations which do not affect function or expression, within the genome of wild-type cells of the host cell. For the purposes of this application, the Moniliella pollinis cell “Moniliella tomentosa var pollinis TCV364” described in US 6,440,712, which is incorporated herein by reference in its entirety, and deposited under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) on March 28, 1997 under number MUCL40385, is considered the wildtype Moniliella pollinis cell.

[0049] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequences and structure necessary to give the recited macromolecule its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function. A summary of the amino acids and their three and one letter symbols asunderstood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.Table 1 : Amino Acid three and one letter symbols

[0050] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed recombinant cells, compositions, and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include greater than about 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.

[0051] As used herein, the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0052] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0053] The polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild-type” is a term of the art understood by skilledpersons and means the typical form and expression level of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.

[0054] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain.

[0055] As used herein, terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence,” and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. The DNA polynucleotides may be a cDNA (e.g., coding DNA) or a genomic DNA sequence (e.g., including both introns and exons).

[0056] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

[0057] Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, the polynucleotides (e.g., polynucleotides encoding an erythrose reductase polypeptide) may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in various species are disclosed herein any polynucleotide sequences may be used which encodes a desired form ofthe polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.

[0058] The recombinant cells described herein may include deletions or disruptions in one or more native genes. The phase “deletion or disruption” refers to the status of a native gene in the recombinant cell that has either a completely eliminated coding region (deletion) or a modification of the gene, its promoter, or its terminator (such as by a deletion, insertion, or mutation) so that the gene no longer produces an active expression product, produces severely reduced quantities of the expression product (e.g., at least a 75% reduction or at least a 90% reduction) or produces an expression product with severely reduced activity (e.g., at least 75% reduced or at least 90% reduced). The deletion or disruption can be achieved by genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. Deletion or disruption of a native host cell gene can be coupled to the incorporation of one or more polynucleotide sequences (e.g., an exogenous or native polynucleotide sequence) into the host cell at the locus of the host cell gene to be deleted or disrupted. The polynucleotide sequence to be inserted may be designed to replace all or a portion of the host cell gene to be deleted or disrupted. The polynucleotide sequence may encode for a gene product of interest, for example, a polypeptide, an enzyme, and the like. The deletion or disruption can also be accomplished using a deletion construct that does not contain a polynucleotide sequence to be integrated. Other methods for gene disruption or deletion are known and described in the art.

[0059] The recombinant cells described herein may have a deletion or disruption in one or more native genes encoding an enzyme involved in erythritol fermentation or consumption. Deletion or disruption of one or more of these biosynthetic pathway enzymes decreases the ability of the recombinant cell to produce erythritol and may, depending on the deletion or disruption, increasing carbon flux into the fermentation pathway for the production of xylitol.

[0060] The recombinant cells described herein may include a deletion or disruption of a native erythrose reductase (ER) gene. The native ER gene encodes an enzyme that has erythrose reductase activity. As used herein “erythrose reductase activity” and “ER activity” are used interchangeably and refer to enzymes that catalyze the reversible conversion of erythrose or erythrose-4-phosphate to erythritol or erythritol-4-phosphate using a nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor. In the art, enzymes that catalyze thereversible conversion of erythrose-4-phosphate to erythritol-4-phosphate using a nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor may also be described as erythrose or erythritol phosphate dehydrogenases. When the host cell contains multiple ER genes, it is preferred to delete or disrupt at least one of them. When the host cell contains multiple alleles of a given ER gene, it is preferred to delete or disrupt one allele or both alleles of the given ER gene. Additional description of recombinant cells capable of producing xylitol and including a deletion or disruption of a native erythrose reductase gene is provided in US Provisional Patent Application No. 63 / 499,990, filed May 4, 2023, which is incorporated herein by reference in its entirety.

[0061] As used herein, “NAD(P)H” refers to nicotinamide adenine dinucleotide (phosphate) hydrogen and is inclusive of both NADH and NADPH. As is understood in the art, inclusion of the phosphate (or “P” abbreviation) in parentheses indicates that the phosphate may be absent or present and the name and abbreviation are inclusive of both. Similarly, “NAD(H)” or “NADP(H)” refers to both the reduced and oxidized forms of the cofactor.

[0062] When the recombinant cell is a Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER gene encoding an amino acid sequence 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 SEQ ID NO: 11, also referred to as the RCSR26640 enzyme herein. When the recombinant cell is Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER gene with a nucleotide sequence 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 SEQ ID NO: 12, also referred to as the RCSR26640 gene herein. The recombinant Moniliella pollinis cell may include a heterologous or native polynucleotide sequence incorporated at the RCSR26640 locus defined by the flanking sequences of SEQ ID NO: 13 and SEQ ID NO: 14 such that the native RCSR26640 gene is deleted or disrupted.

[0063] When the recombinant cell is a Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER gene encoding an amino acid sequence 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 SEQ ID NO: 15, also referred to as the RCSR18717 enzyme herein. When the recombinant cell is Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER gene with a nucleotide sequence 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 least98%, at least 99%, or 100% identical to SEQ ID NO: 16, also referred to as the RCSR18717 gene herein. The recombinant Moniliella pollinis cell may include a heterologous or native polynucleotide sequence incorporated at the RCSR18717 locus defined by the flanking sequences of SEQ ID NO: 17 and SEQ ID NO: 18 such that the native RCSR18717 gene is deleted or disrupted.

[0064] The recombinant cells described herein may include one or more genetic modifications in which an exogenous nucleic acid is integrated into the genome of the host cell. One of skill in the art know how to select suitable loci in a yeast genome for integration of the exogenous nucleic acid. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adhl202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdllA, and gpdllB loci. For example, in a pollinis host cells, suitable interaction loci may include, but are not limited to, the ER1 locus (defined as the locus flanked by SEQ ID NO: 19 and SEQ ID NO:20), the ER3 locus (defined as the locus flanked by SEQ ID NO:21 and SEQ ID NO:22), the PDC1 locus (defined as the locus flanked by SEQ ID NO:23 and SEQ ID NO:24), the pyrF locus (defined as the locus flanked by SEQ ID NO:25 and SEQ ID NO:26), the TRP3 locus (defined as the locus flanked by SEQ ID NO:27 and SEQ ID NO:28), the gpdllA locus (defined as the locus flanked by SEQ ID NO:29 and SEQ ID NO:30); the gpdllB locus (defined as the locus flanked by SEQ ID NO:31 and SEQ ID NO:32); the RCSR18717 locus (defined as the locus flanked by SEQ ID NO: 17 and SEQ ID NO: 18); and the RCSR26640 locus (defined as the locus flanked by SEQ ID NO: 13 and SEQ ID NO: 14). The exogenous nucleic acid may also be integrated in an intergenic region or other location in the host cell genome not specifically specified herein. Other suitable integration loci may be determined by one of skill in the art. Furthermore, one of skill in the art would recognize how to use sequences to design primers to verify correct gene integration at the chosen locus.

[0065] The recombinant cell may have one or more copies of a given exogenous nucleic acid sequence integrated in a host chromosome(s) and replicated together with the chromosome(s) into which it has been integrated. For example, the yeast cell may be transformed with nucleic acid construct including a polynucleotide sequence encoding for a polypeptide described herein and the polynucleotide sequence encoding for the polypeptide may be integrated in one or more copies in a host chromosome(s). The recombinant cell may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. The recombinant cell may have one, two, three, four, five, six, seven, eight, nine, ten, or more copies of a polynucleotide sequence encoding a polypeptide described herein integrated into the genome. The multiple copiesof said polynucleotide sequence may all be incorporated at a single locus or may be incorporated at multiple loci.

[0066] Glycerol is a common byproduct of fermentation using yeast and is generally produced when yeast cells face osmotic stress to prevent dehydration of the cell by balancing the intracellular osmolarity with that of the fermentation broth. The production of glycerol is also important for redox balancing during fermentations and ensuring continued metabolism by recycling NADH. However, the synthetic pathway to glycerol diverts carbon away from the pentose phosphate pathway limiting the potential for xylitol production. Accordingly, upregulation or overexpression of the enzymes in the glycerol catabolic pathway can re-consume the produced glycerol and convert it to dihydroxyacetone phosphate and ultimately glyceraldehyde-3 -phosphate which can then enter the pentose phosphate pathway. This glycerol recycle process can begin with the conversion of glycerol to glycerol-3 phosphate using a glycerol kinase enzyme. Without wishing to be bound by any particular theory, method, or mode of action, overexpressing a glycerol kinase enzyme will decrease the residual concentration of glycerol and increase the production of glycerol-3 phosphate that can then be converted to glyceraldehyde-3 - phopshate and recycle carbon back into the pentose phosphate pathway to increase xylitol production. Even in the absence of a xylitol synthetic pathway, over expression of a glycerol kinase enzyme should decrease the accumulation of glycerol.

[0067] The recombinant cells described herein are capable of producing xylitol and include an exogenous polynucleotide encoding a native glycerol kinase and / or may have a genetic modification resulting in overexpression of a native glycerol kinase. In general, cells overexpressing a glycerol kinase enzyme will accumulate less glycerol during fermentation than an equivalent cell lacking the glycerol kinase overexpression (e.g., wild-type expression of the glycerol kinase). The glycerol kinase enzyme may be any suitable enzyme with glycerol kinase activity. As used herein, “glycerol kinase enzyme” refers to an enzyme with glycerol kinase activity. As used herein, “glycerol kinase activity” refers to the ability of the polypeptide to catalyze the irreversible conversion of glycerol and adenosine triphosphate (ATP) to glycerol-3 phosphate and adenosine diphosphate (ADP). The glycerol kinase enzyme may be native to the host cell. For example, when the host cell MoniUella pollinis, the glycerol kinase enzyme may be an enzyme with a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110. The enzyme of SEQ ID NO: 110 is encoded by the M. pollinis gene RCSR17702 and the enzyme of SEQ ID NO:96 is encoded by the M. pollinis geneRCSR09543. The recombinant cell may comprise an exogenous polynucleotide encoding a glycerol kinase enzyme at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110. The recombinant cell may include a genetic modification that increases expression of a glycerol kinase enzyme at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native glycerol kinase enzyme into the cell, insertion of a constitutive promoter upstream of the coding region of the native glycerol kinase gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native glycerol kinase gene in the genome of the host cell. One of skill in the art will recognize that expression of a native glycerol kinase gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate.

[0068] As used herein, “overexpression” refers to an expression level of a polypeptide that is higher than the expression level of the same polypeptide in the absence of a genetic modification or exogenous polynucleotide encoding said polypeptide in an equivalent cell.

[0069] Xylitol is a product of the pentose phosphate pathway, and increasing carbon flux into the pentose phosphate pathway should help in increasing xylitol yield and titer in fermentation. As demonstrated in FIG. 1 carbon can enter the pentose phosphate pathway from the “top” through the reaction of glucose-6-phosphate to gluconate-6-phosphate, or from the “bottom” through the transketolase (TKL) and transaldolase (TAL) enzyme catalyzed reactions of the glycolysis pathway intermediates fructose-6-phosphate and glyceraldehyde-3 -phosphate. Without being bound by any particular theory, method, or mode of action, increasing expression of the transketolase enzyme (labeled TKL in FIG. 1) should increase carbon flux into the pentose phosphate pathway and subsequently increase xylitol rate, titer and / or yield.

[0070] The recombinant cells described herein are capable of producing xylitol and are characterized by overexpression of a native enzyme with transketolase activity and / or include an exogenous polynucleotide sequence encoding a native or exogenous enzyme with transketolase activity. As used herein “transketolase enzyme” and “TKL enzyme” are used interchangeably and refer to an enzyme with transketolase activity. As used here in, “transketolase activity” refers to the ability to reversibly catalyze (i) the conversion of D-xylulose-5-phosphate and D-ribose-5- phosphate to sedoheptulose-7-phosphate and glyceraldehyde-3 -phosphate using a thiaminepyrophosphate (TPP) cofactor; and (ii) the conversion of D-xylulose-5-phosphate and erythrose- 4-phosphate to fructose-6-phosphate and glyceraldehyde-3 -phosphate. Polynucleotides encoding transketolase enzymes may be derived from any suitable source. For example, a polynucleotide encoding a transketolase enzyme may be derived from Moniliella pollinis.

[0071] The recombinant cell has an exogenous polynucleotide sequence that is or may be derived from Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO: 129. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO: 129.

[0072] The enzyme with transketolase activity may be native to the host cell. For example, when the host organism is M. pollinis, the transketolase enzyme may be an enzyme with a sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 129. The recombinant cell may comprise an exogenous polynucleotide encoding a transketolase enzyme with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 129. The recombinant cell may include a genetic modification that increases expression of a transketolase enzyme 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 SEQ ID NO: 129. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native transketolase enzyme into the cell (e.g., integration of additional copies of the transketolase encoding polynucleotide into a non-native locus in the cell), insertion of a constitutive and / or strong promoter upstream of the coding region of the native transketolase enzyme encoding gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native transketolase enzyme encoding gene in the genome of the host cell. One of skill in the art will recognize that expression of a native transketolase enzyme encoding gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate.

[0073] The final step in the xylitol pathway, from xylitol 5-phosphate to xylitol, requires a phosphatase enzyme. The Saccharomyces cerevisiae PYP1 (polyol phosphatase 1) gene encodes a sugar alcohol phosphatase that hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (D)- glycerol-3-phosphase (Xu et al., “Discovery and functional characterization of a yeast sugar alcohol phosphatase,” ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova, et a., “Functional diversity ofhaloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae.'' J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the enzyme class of sorbitol-6-phosphatases (Enzyme Commission (EC) 3.1.3.50). As xylitol 5-phosphate is a similar molecule to the known substrates of PYP1 it is demonstrated herein that one or more PYP-like enzymes or PYP orthologs have xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells described herein. E. coli HxpA (hexitol phosphatase A) is a HAD-like enzyme belonging to EC 3.1.3.50 with a similar substrate profile to PYP1 (Kuznetsova et al., “Genome wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase- like phosphate family,” 2006, J. Biol. Chem., 281, 36149-36161). Accordingly, it is also demonstrated herein that one or more HAD-like hydrolase enzymes or HAD-like hydrolase orthologs have xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells describe here.

[0074] The recombinant cells described herein are capable of producing xylitol, are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may be characterized by overexpression of a native enzyme with xylitol-5-phosphate phosphatase (X5PP) activity and / or include an exogenous polynucleotide sequence encoding a native or exogenous enzyme with xylitol-5-phosphate phosphatase (X5PP) activity. In general, the recombinant cell(s) including overexpression of an X5PP enzyme or expressing an exogenous X5PP enzyme produce more xylitol than an equivalent cell lacking the exogenous X5PP enzyme or lacking overexpression of the X5PP enzyme. The enzyme may be any suitable enzyme with X5PP activity. As used herein, “X5PP enzyme” and “X5PP” are interchangeable and refer to an enzyme with X5PP activity. 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 a divalent metal cation, for example, Mg2+, Mn2+, or Co2+. Suitable enzymes with X5PP activity may include, but are not limited to, those classified under EC 3.1.3.50, for example, sugar alcohol phosphatases and HAD-like hydrolases. Polynucleotides encoding X5PP enzymes may be derived from any suitable source. For example, a polynucleotide encoding an X5PP enzyme may be derived from Moniliella pollinis. Saccharomyces cerevisiae, Lachancea dasiensis, Tetrapisispora blaUae, Saccharomyces pastorianus, Kazachstania Africana, Podospora comala, Geotrichum candidum, Ogattaea haglerorum, Debaryomyces fabryi, Monilinia friiclicola, Nadsonia fulvescens var. elongata DSM 6958, Escherichia coli, Wicker hamomyces ciferrii, Bacillus amyloliquefaciens, and the like. The X5PP enzyme may be apolypeptide with an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NOs:33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. The X5PP enzyme may be a polypeptide with an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NOs:33, 35, 36, 37, 38, 39, 43, and 45. The X5PP enzyme may be a polypeptide with an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NOs:33, 36, 37, 39, and 45. Additional description of recombinant cells capable of producing xylitol and including a polypeptide with xylitol-5-phosphate phosphatase activity is provided in US Provisional Patent Application No. 63 / 499,992, filed May 4, 2023, which is incorporated herein by reference in its entirety.

[0075] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO:33. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:33.

[0076] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO:34. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:34.

[0077] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Lachancea dasiensis gene encoding the amino acid sequence of SEQ ID NO:35. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:35.

[0078] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenouspolynucleotide sequence that is or may be derived from a Tetrapisispora blattae gene encoding the amino acid sequence of SEQ ID NO:36. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:36.

[0079] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces pastorianus gene encoding the amino acid sequence of SEQ ID NO:37. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:37.

[0080] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Kazachstania africana gene encoding the amino acid sequence of SEQ ID NO:38. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:38.

[0081] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Podospora comata gene encoding the amino acid sequence of SEQ ID NO:39. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:39.

[0082] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Geotrichum candidum gene encoding the amino acid sequence of SEQ ID NO:40. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:40.

[0083] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Ogattaea haglerorum gene encoding the amino acid sequence of SEQ ID NO:41. The exogenous polynucleotide sequence may encodean amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:41.

[0084] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Debaryomyces fabryi gene encoding the amino acid sequence of SEQ ID NO:42. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:42.

[0085] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from Monilinia fructicola gene encoding the amino acid sequence of SEQ ID NO:43. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:43.

[0086] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from Nadsonia fulvescens var. elongata DSM 6958 gene encoding the amino acid sequence of SEQ ID NO:44. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:44.

[0087] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from an Escherichia coli gene encoding the amino acid sequence of SEQ ID NO:45. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:45.

[0088] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Wicker hamomyces ciferrii gene encoding the amino acid sequence of SEQ ID NO:46. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:46.

[0089] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO:47. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:47.

[0090] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Bacillus amyloliquefaciens gene encoding the amino acid sequence of SEQ ID NO:48. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:48.

[0091] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae DOG2 gene encoding the amino acid sequence of SEQ ID NO:49. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:49.

[0092] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae DOG1 gene encoding the amino acid sequence of SEQ ID NO:50. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:50.

[0093] The enzyme with X5PP activity may be native to the host cell. For example, when the host organism is M. pollinis, the X5PP enzyme may be an enzyme with a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to at least one of SEQ ID NOs:51, 52, 33, or 47. The recombinant cell may comprise an exogenous polynucleotide encoding an X5PP enzyme with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of SEQ ID NOs: 51, 52, 33, or 47. The recombinant cell may include a genetic modification that increases expression of an X5PP enzyme 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 least98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 51, 52, 33, or 47. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native X5PP enzyme into the cell (e.g., integration of additional copies of the X5PP encoding polynucleotide into it non-native locus in the cell), insertion of a constitutive promoter upstream of the coding region of the native X5PP enzyme encoding gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native X5PP enzyme encoding gene in the genome of the host cell. One of skill in the art will recognize that expression of a native X5PP enzyme encoding gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate. Additional description of recombinant cells capable of producing xylitol and overexpressing a native enzyme with xylitol-5-phosphate phosphatase activity is provided in US Provisional Patent Application No. 63 / 499,992, filed May 4, 2023, which is incorporated herein by reference in its entirety.

[0094] The recombinant cells described herein are capable of producing xylitol, are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may also be characterized by overexpression of a ribulose 5-phosphate epimerase (RPE enzyme). The recombinant cells described herein capable of producing xylitol, characterized by overexpression of a native glycerol kinase enzyme, and overexpression of a native transketolase enzyme may also include a deletion or disruption in a native ER gene; an exogenous polynucleotide sequence encoding an X5PP enzyme; overexpression of a native X5PP enzyme; an exogenous polynucleotide encoding a native or exogenous RPE enzyme; and / or a genetic modification resulting in overexpression of a native RPE enzyme, as described herein. In general, the recombinant cell(s) including overexpression of the RPE enzyme produce more xylitol than an equivalent cell lacking the RPE enzyme or lacking overexpression of the RPE enzyme.

[0095] The recombinant cells described herein are capable of producing xylitol, are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may include an exogenous polynucleotide encoding a native or exogenous RPE enzyme or may have a genetic modification resulting in overexpression of a 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 xylulose-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 A7. polUnis.the RPE enzyme may be an enzyme with a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to at least one of SEQ ID NOs:53 and 54. The recombinant cell may comprise an exogenous polynucleotide encoding an RPE enzyme with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of SEQ ID NOs:53 and 54. The recombinant cell may include a genetic modification that increases expression of an RPE enzyme at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:53 and 54. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native RPE into the cell, insertion of a constitutive promoter upstream of the coding region of the native RPE gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native RPE gene in the genome of the host cell. One of skill in the art will recognize that expression of a native RPE gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate. Additional description of recombinant cells capable of producing xylitol and including an exogenous polynucleotide encoding a native or exogenous RPE enzyme or a genetic modification resulting in overexpression of a native RPE enzyme is provided in PCT Patent Publication No. PCT / US2023 / 066631, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0096] The recombinant cells described herein are capable of producing xylitol, are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may include an exogenous polynucleotide sequence encoding a xylitol- phosphate dehydrogenase (XPDH) enzyme. The exogenous polynucleotide sequence may be an exogenous xylitol-phosphate dehydrogenase (XPDH) gene. A recombinant cell described herein capable of producing xylitol is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may include an exogenous polynucleotide sequence encoding an X5PP enzyme; overexpress a native X5PP enzyme; include an exogenous polynucleotide sequence encoding an XPDH enzyme; include an exogenous polynucleotide encoding a native or exogenous RPE enzyme; and / or have a genetic modification resulting in overexpression of a native RPE enzyme, as described herein.

[0097] A “xylitol-phosphate dehydrogenase gene” and an “XPDH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with xylitol-phosphate dehydrogenase activity. As used herein, “xylitol-phosphate dehydrogenaseactivity” refer to the ability to catalyze the conversion of xylulose-5-phosphate and NADPH or NADH to xylitol 5-phosphate and NADP+or NAD+. The XPDH gene may be derived from any suitable source. For example, the XPDH gene may be derived from Clostridium difficile, Lactobacillus rhamnosus, Bacillus halodurans, Alkalihalobacillus ligniniphilus, Jeotgalibacillus soli, Heyndrickxia sporothermodurans, Clostridium fungisolvens, or Neobacillus cucumis. The XPDH gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:55-64. The XPDH gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:57, 58, 59, or 62. Additional description of recombinant cells capable of producing xylitol and including an exogenous polynucleotide encoding an enzyme with xylitol- phosphate dehydrogenase activity is provided in PCT Patent Publication No. PCT / US2023 / 066629, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0098] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Clostridium difficile gene encoding the amino acid of SEQ ID NO:55. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:55.

[0099] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Clostridium difficile gene encoding the amino acid of SEQ ID NO:56. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:56.

[0100] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Lactobacillus rhamnosus gene encoding the amino acid of SEQ ID NO:57. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:57.

[0101] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Bacillus halodurans gene encoding the amino acid of SEQ ID NO:58. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:58.

[0102] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Alkalihalobacillus ligniniphilus gene encoding the amino acid of SEQ ID NO:59. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:59.

[0103] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Jeotgalibacillus soli gene encoding the amino acid of SEQ ID NO:60. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:60.

[0104] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, Heyndrickxia sporothermodurans gene encoding the amino acid of SEQ ID NO:61. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:61.

[0105] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Clostridium fungisolvens gene encoding the amino acid of SEQ ID NO:62. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:62.

[0106] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Neobacillus cucumis gene encoding the amino acid of SEQ ID NO:64. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:64.

[0107] The recombinant cells described herein are capable of producing xylitol, are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and and may include an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme. A recombinant cell described herein capable of producing xylitol are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may additionally include an exogenous polynucleotide sequence encoding an X5PP enzyme; overexpress a native X5PP enzyme; include an exogenous polynucleotide sequence encoding an XKS enzyme; include an exogenous polynucleotide encoding a native or exogenous RPE enzyme; and / or have a genetic modification resulting in overexpression of a native RPE enzyme, as described herein. The exogenous polynucleotide sequence may be an exogenous xylulose sugar phosphatase (XKS) gene.

[0108] A “xylulokinase gene” and an “XKS gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with xylulokinase activity. As used herein, “xylulokinase activity” refer 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 an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:50 and 49. Additional description of recombinant cells capable of producing xylitol and including a polypeptide with xylulokinase activity is provided in PCT Application Publication No. PCT / US2023 / 066627, with a filing date of May 5, 2023, which is incorporated herein by reference in its entirety.

[0109] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Saccharomyces cerevisiae DOG1 sugar phosphatase gene encoding the amino acid of SEQ ID NO:50. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:50.

[0110] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Saccharomyces cerevisiae DOG2 sugar phosphatase gene encoding the amino acid of SEQ ID NO:49. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:49.

[0111] The recombinant cells described herein are capable of producing xylitol, are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may comprise an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme. A recombinant cell described herein capable of producing xylitol are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and may additionally include an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme; include an exogenous polynucleotide encoding an XKS enzyme; include an exogenous polynucleotide sequence encoding an X5PP enzyme; overexpress a native X5PP enzyme; include an exogenous polynucleotide sequence encoding an XKS enzyme; include an exogenous polynucleotide encoding a native or exogenous RPE enzyme; and / or have a genetic modification resulting in overexpression of a native RPE enzyme, as described herein. The exogenous polynucleotide sequence may be an exogenous XDH gene.

[0112] A “xylitol dehydrogenase gene” and an “XDH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with xylitol dehydrogenase activity. As used herein, “xylitol dehydrogenase activity” refer to the ability to catalyze the conversion of xylulose and NADH or NADPH to xylitol and NAD+or NADP+. The XDH gene may be derived from any suitable source. For example, the XDH gene may be derived from Pichia stipitis, Rhodobacteraceae bacterium, or Bemisia argentofolii . The XDH gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:65, 66, or 67. Additional description of recombinant cells capable of producing xylitol and including a polypeptide with xylulokinase activity and a polypeptide with xylitol dehydrogenase activity is provided in PCT Application Publication No.PCT / US2023 / 066627, with a filing date of May 5, 2023, which is incorporated herein by reference in its entirety.

[0113] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a cofactor switched Pichia stipitis XDH gene encoding the amino acid of SEQ ID NO:65. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0114] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, a Rhodobacteraceae bacterium SDR family oxidoreductase gene encoding the amino acid of SEQ ID NO:66. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:66.

[0115] The recombinant cell is are characterized by overexpression of a native glycerol kinase enzyme, overexpression of a native transketolase enzyme, and include an exogenous polynucleotide that is, or may be derived from, Bemisia argentofolii (Silverleaf Whitefly) ketose reductase (sorbitol dehydrogenase) gene encoding the amino acid of SEQ ID NO:67. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:67.

[0116] The exogenous polynucleotides in the recombinant cells described herein may be under the control of a promoter. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial promoter. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarboxylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp; SEQ ID NO: 68); 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 69); glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:70); translational elongation factor 1 promoter (TEFp; SEQ ID NO:71); phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72); 3 -phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73); enolasepromoter (ENOlp ; SEQ ID NO:74); asparagine synthetase promoter (ASNSp; SEQ ID NO:75); 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76); and RPL16B (SEQ ID NO:77).

[0117] The exogenous nucleic acids in the recombinant cells described herein may be under the control of a terminator. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. Terminators may include, but are not limited to, GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt; SEQ ID NO:78); ASNS terminator (ASNSt; SEQ ID NO:79); ENO1 terminator (ENOlt; SEQ ID NO:80); hexokinase 1 terminator (HXKlt; SEQ ID NO:81); PGK1 terminator (PGKlt; SEQ ID NO:82); PGM1 terminator (PGMlt; SEQ ID NO:83); PYK1 terminator (PYKlt; SEQ ID NO:84); RPLA terminator (RPLAt: SEQ ID NO:85); transaldolase 1 terminator (TALlt; SEQ ID NO:86); TDH3 terminator (TDH3t; SEQ ID NO:87); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:88); triosephosphate isomerase 1 terminator (TPIlt; SEQ ID NO:89); and MpTEFl (SEQ ID NO:90).

[0118] A promoter or terminator is “operably linked” to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to said polynucleotide is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0119] The polypeptides described herein may be provided as part of a construct. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. The construct may be a vector including a promoter operably linked to the polynucleotide encoding a polypeptide as described herein. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. The vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.

[0120] The disclosure also provides fermentation methods for the production of xylitol using the recombinant cells described herein. The fermentation methods include the step of fermenting a substrate using the genetically engineered yeasts described herein to produce xylitol. The fermentation method can include additional steps, as would be understood by a person skilled inthe art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and isolating the xylitol from the fermentation broth. The fermentation process may be a fully aerobic or a partially aerobic process. The fermentation process may include a suitable level of agitation for the desired outcome in relationship to other fermentation process parameters.

[0121] The fermentation method can be run using a suitable fermentation substrate. The substrate of the fermentation method can include glucose, sucrose, galactose, mannose, molasses, xylose, fructose, hydrolysates of starch, lignocellulosic hydrolysates, or a combination thereof. One skilled in the art will recognize what fermentation substrate is suitable for a given fermentation organism and system.

[0122] The fermentation process can be run under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, may be ambient temperature. Alternatively, or additionally, the fermentation temperature may be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C, preferably about 35 °C. However, a skilled artisan will recognize that the fermentation temperature is not limited to any specific range or temperature recited herein and may be modified as appropriate.

[0123] The fermentation process can be run within certain oxygen uptake rate (OUR) ranges. The volumetric OUR of the fermentation process can be in the range of 5-80, 10-75, 15-70, 20- 60, 30-50, or 40-50 mmol O2 / (L • h). In some embodiments, the specific OUR can be in the range of 0.05 to 10, 0.1 to 9, 0.5 to 8, 1.0 to 7, 1.5 to 6, 2 to 5, or 2.5 to 4 mmol O2 / (g cell dry weight • h). However, the volumetric or specific OURs of the fermentation process are not limited to any specific rates or ranges recited herein.

[0124] The fermentation process can be run at various cell concentrations. In some embodiments, the cell dry weight at the end of fermentation can be 5 to 40, 8 to 30, or 10 to 20 g cell dry weight / L. Further, the pitch density or pitching rate of the fermentation process can vary. In some embodiments, the pitch density can be 0.05 to 11, 0.1 to 10, or 0.25 to 8 g cell dry weight / L.

[0125] The initial dextrose concentration of the fermentation may be at least 100, 200, 250, 300, 350, or at least 400 g / L dextrose when run as a batch fermentation. The initial dextrose concentration may be between 100 to 500, 150 to 450, 400 to 200, or 250 to 350 g / L when run as a batch fermentation. When run in a fed batch fermentation process, the dextrose concentration may be at least 100, 200, 250, 300, 350, 400, or at least 450 g / L.

[0126] The fermentation process can be associated with various characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of the yeast and / or genetic modification of the yeast used in the fermentation process. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be adjusted via a combination of yeast selection or modification and the selection of fermentation process conditions.

[0127] The xylitol production rate of the process may be at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h’1. The xylitol mass yield of the process may be at least 25, at least 30, at least 32, at least 34, at least 35, at least 38, or at least 40 percent. The final xylitol titer of the process may be at least 5, 10, 20, 30, 50, 75, 100, 110, or 120 g / L.

[0128] The glycerol mass yield of the process may be less than 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. The final glycerol titer, e.g., after 96 hours at 35 °C, of the process may be less than 10, 8, 6, 5, 4, 3, 2, 1, or 0.5 g / L.

[0129] The fermentation process can be run as a dextrose-fed batch. Further, the fermentation process can be a batch process, continuous process, or semi-continuous process, as would be understood by a person skilled in the art.EXAMPLES

[0130] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0131] Throughout the Examples, strain numbering and sequence identification numbers are used consistently. For example, strain 1-2 in Example 1 is the same as strain 1-2 in Examples 2, 3, etc.Example 1 - Genetically Modified Afo / ie / Za / w / / / w.s Strains

[0132] Strain 1-1 is the Moniliella pollinis host strain “Moniliella tomentosa var pollinis TCV364” described in US 6,440,712, which is incorporated herein by reference in its entirety, and deposited under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain by Eridania Beghin Say,Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) on March 28, 1997 under number MUCL40385.Strain 1-2

[0133] SEQ ID NO:91 contained a 5’ flanking DNA for targeted chromosomal integration into the ER1 loci (SEQ ID NO: 19); an expression cassette for the XPDH homolog from Lactobacillus rhamnosus encoding the amino acid sequence of SEQ ID NO:57, under the control of the PYK1 promoter of SEQ ID NO:68 and the PGD terminator of SEQ ID NO:78; and a 5’ portion of the G418 selectable marker (SEQ ID NO:4). SEQ ID NO:92 contains a 3’ portion of a G418 resistance marker (SEQ ID NO:3), an MpPGKl promoter (SEQ ID NO:73), a polynucleotide encoding the RPE of SEQ ID NO: 54, an MpPYKl terminator (SEQ ID NO: 84), and a 3’ ER1 flanking sequence (SEQ ID NO:20).

[0134] Strain 1-1 was transformed with SEQ ID NO:91 and SEQ ID NO:92 by first protoplasting the parent strain by adding an enzyme mixture containing 0.6M MgSO4, 7.5 g / L driselase, and 12.5 g / L Trichoderma harzianum lysing enzyme to a mycelial pellet of the parent strain. Protoplasts were then pelleted, washed with 0.6M MgSO4, and resuspended in STC medium (0.6M sucrose, 50 mM CaC12, 10 mM Tris-HCl, pH 7.5). Bipartite transformations were prepared by adding 100 pg single stranded salmon sperm DNA and 1.5 to 5 pg each of the 5’ and 3’ DNA transformation fragments (3-10 pg total; see Table 4 for list of fragments) to approximately 200 pL protoplast mixture (108cells / mL). 1 mL 50% PEG in STC medium was then added to the salmon sperm DNA, transformation DNA, and protoplast mixture and the resulting combination was incubated for 15 minutes at room temperature. Following incubation, recovery broth (0.4M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L glucose, pH 4.5) was added to the mixture and incubated at 27 °C, 100 rpm, for 16 to 24 hours. Following the incubation, protoplasts were pelleted by centrifugation and resuspended in 1 mL PBS.

[0135] The resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30-35 °C for at least 2-4 days until transformants grow. Resulting transformants were streaked for single colony isolation on PDA + geneticin (G418) plates and a single colony was selected. The selected colony was evaluated by colony PCR for integration of one copy of the L. rhamnosus XPDH encoding sequence and one additional copy (beyond the existing wild-type copies) of the RPE encoding sequences. A PCR verified isolate was designated strain 1-2.Strain 1-3

[0136] Strain 1-1 was transformed with SEQ ID NO:91 and SEQ ID NO:97 as outlined above. The resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until transformants grew. Resulting transformants were streaked for single colony isolation on PDA + geneticin (G418) plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of two copies of the L. rhamnosus XPDH sequence. A PCR verified isolate was designated strains 1-3.

[0137] SEQ ID NO:97 contained (i) a 3’ portion of the G418 selectable marker (SEQ ID NO:3); ii) an expression cassette encoding the XPDH homolog from Lactobacillus rhamnosus of SEQ ID NO:57, under the control of the PYK1 promoter of SEQ ID NO:68 and the PGD terminator of SEQ ID NO:78; and (iii) a 3’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:20).Strain 1-4

[0138] Strain 1-3 was transformed as outlined above with SEQ ID NO:98 and SEQ ID NO:99. The transformation fragment of SEQ ID NO: 98 contained, in order, a 5’ ER3 flanking sequence (SEQ ID NO:21), a MpPYKl promoter (SEQ ID NO:68), a gene encoding the AT. pollinis RPE2 polypeptide of SEQ ID NO: 54, a MpPYK terminator (SEQ ID NO: 84), and a 5’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:6). The transformation fragment of SEQ ID NO:99 contained, in order, a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:5), and a 3’ ER3 flanking sequence (SEQ ID NO:22). Transformants were selected on PDA + zeocin selection plates and incubated at 35 °C for at least 2 days until transformants grow. Resulting transformants were streaked for single colony isolation on PDA + zeocin plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the gene encoding the M. pollinis RPE2 polypeptide. A PCR verified isolate was designated strain 1-4.Strain 1-5

[0139] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 1-4 was used to generate Moniliella pollinis strain with reduced foaming during shake flask fermentation. Strains with low-foaming phenotypes were selected based on visual evaluation of foaming in a shake flask fermentation compared to foaming of the parent 1-4 strain. The resulting low-foaming strain, containing two copies of an exogenous polynucleotidesequence encoding the XPDH of SEQ ID NO:57 integrated at the ER1 locus and one copy of a polynucleotide sequence encoding the RPE of SEQ ID NO: 54 integrated at the ER3 locus, was designated 1-5.Strain 1-6

[0140] Strain 1-5 was transformed with the Cre recombinase plasmid of SEQ ID NO: 100 using the transformation method outlined above. The resulting transformants were evaluated by colony PCR for removal of the zeocin and geneticin (G418) resistance selection markers. A PCR verified isolate was designated strain 1-6.Strain 1-7

[0141] Strain 1-6 was grown non-selectively on YPD plates to allow for the loss of the plasmid of SEQ ID NO: 100. Biomass was struck for single colonies and evaluated by PCR to confirm loss of the plasmid. A PCR verified isolate was designated strain 1-7.Strain 1-8

[0142] Strain 1-7 was transformed with SEQ ID NO: 101 and SEQ ID NO: 98 as outlined above. SEQ ID NO: 101 contained a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:5), a MpPGKl promoter (SEQ ID NO:73), a gene encoding the X5PP polypeptide of SEQ ID NO:33, a Mp6PGD terminator (SEQ ID NO:78), and a 3’ ER3 flanking sequence (SEQ ID NO:22). Transformants were selected on PDA + zeocin selection plates and incubated at 35 °C for at least 2 days until transformants grow. Resulting transformants were streaked for single colony isolation on PDA + zeocin plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the AT. pollinis X5PP gene at the ER3 locus. A PCR verified isolate was designated strain 1-8.Strain 1-9

[0143] Strain 1-8 was transformed with SEQ ID NO: 102 using the transformation protocol outlined above and positive transformants were selected using nourseothricin selection plates. SEQ ID NO: 102 contains a deletion construct to remove one copy of the RSCR18717 gene (SEQ ID NO: 16) and a nourseothricin resistance gene expression cassette. A PCR verified isolate, in which one copy of the RCSR18717 gene was knocked out, was designated strains 1-9.Strain 1-10

[0144] Strain 1-9 was transformed with SEQ ID NO: 103 using the transformation protocol outlined above. SEQ ID NO: 103 contained a construct to loop out the zeocin and nourseothricin resistance selection markers. A PCR verified isolate, in which zeocin and nourseothricin resistance selection markers were removed, was designated strain 1-10.Strain 1-11

[0145] Strain 1-10 was transformed with SEQ ID NO: 102 using the transformation protocol outlined above and positive transformants were selected using nourseothricin selection plates. A PCR verified isolate, in which both copies of the RCSR18717 gene were knocked out, was designated strains 1-11.Strain 1-12

[0146] Strain 1-11 was transformed with SEQ ID NO: 104 and SEQ ID NO: 105 using the transformation protocol outlined above and positive transformants were selected using geneticin (G418) selection plates. SEQ ID NO: 105 contained a 3’ portion of a G418 resistance gene expression cassette (SEQ ID NO:3), and a 3’ gpdllB flanking sequence (SEQ ID NO:32). SEQ ID NO: 104 contained a 5’ gpdllB flanking sequence (SEQ ID NO:31), an MpPYKl promoter (SEQ ID NO:68), a polynucleotide encoding the M. pollinis X5PP enzyme of SEQ ID NO:33, an MpENOl terminator (SEQ ID NO:80), an MpPGKl promoter (SEQ ID NO:73), a polynucleotide sequence encoding the M. pollinis X5PP enzyme of SEQ ID NO:33, an MpTDH3 terminator (SEQ ID NO:87), and a 5’ portion of a G418 resistance marker (SEQ ID NO:4). A PCR verified isolate, containing two copies of the gene encoding the X5PP of SEQ ID NO:33, was designated strain 1- 12.Strain 1-13

[0147] Strain 1-12 was transformed with SEQ ID NO: 106 using the transformation protocol outlined above. SEQ ID NO: 106 contains a construct to loop out the nourseothricin and geneticin (G418) resistance selection markers. A PCR verified isolate, in which both the nourseothricin and geneticin resistance selection markers were removed, was designated strain 1-13.Strains 1-14 through 1-32

[0148] Table 2 below lists various Moniliella pollinis strains, including information on the parent strain, the sequences with which the parent strain was transformed, and characterizations of the expression cassette(s) contained on the transformed sequences.

[0149] For the sequences outlined in Table 2, the transformation fragments are as follows. a. SEQ ID NO:93 contained a 5’ gpdllA flanking sequence (SEQ ID NO:29); an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO:95; an Mp6PGD terminator (SEQ ID NO:78); and a 5’ portion of a zeocin resistance selection marker (SEQ ID NO:6). b. SEQ ID NO:94 contained a 3’ portion of a zeocin resistance selection marker (SEQ ID NO:5); an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 96; an MpPYKl terminator (SEQ ID NO: 84); and a 3’ gpdllA flanking sequence (SEQ ID NO:30). c. SEQ ID NO: 107 contained a 5’ gpdllA flanking sequence (SEQ ID NO:29); and a 5’ portion of a zeocin resistance selection marker (SEQ ID NO:6). d. SEQ ID NO: 108 contained a 5’ gpdllA flanking sequence (SEQ ID NO:29); an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110; an Mp6PGD terminator (SEQ ID NO:78); and a 5’ portion of a zeocin resistance selection marker (SEQ ID NO:6). e. SEQ ID NO: 109 contained a 3’ portion of a zeocin resistance selection marker (SEQ ID NO: 5); an MpPYKl promoter (SEQ ID NO: 68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110; an MpPYKl terminator (SEQ ID NO:84); and a 3’ gpdllA flanking sequence (SEQ ID NO: 30). f. SEQ ID NO: 111 contained a 5’ gpdllA flanking sequence (SEQ ID NO:29); and a 5’ portion of a G418 resistance marker (SEQ ID NO:4). g. SEQ ID NO: 112 contained a 5’gpdIIa flanking sequence (SEQ ID NO:29), an MpPYKl promoter (SEQ ID NO: 68), a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110, an Mp6PGD terminator (SEQ ID NO:78), and a 5’ portion of a G418 resistance marker (SEQ ID NO:4). h. SEQ ID NO: 113 contained a 3’ portion of a G418 resistance selection marker (SEQ ID NO:3); an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110; an MpPYKl terminator (SEQ ID NO: 84); and a 3’ gpdllA flanking sequence (SEQ ID NO:30).i. SEQ ID NO: 114 contained a 5’gpdIIA flanking sequence (SEQ ID NO:29), an MpPYKl promoter (SEQ ID NO: 68), a nucleic acid sequence encoding the amino acid of SEQ ID NO:96, an Mp6PGD terminator (SEQ ID NO:78), and a 5’ portion of a G418 resistance marker (SEQ ID NO:4). j. SEQ ID NO: 115 contained a 3’ portion of a G418 resistance selection marker (SEQ ID NO:3); an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 96; an MpPYKl terminator (SEQ ID NO: 84); and a 3’ gpdllA flanking sequence (SEQ ID NO:30). k. SEQ ID NO: 116 contained a 5’ RCSR18717 flanking sequence (SEQ ID NO: 17), an MpPYKl promoter (SEQ ID NO:68); a nucleic acid encoding the amino acids of SEQ ID NO: 110, an Mp6PGD terminator (SEQ ID NO:78), and a 5’ portion of a G418 resistance marker gene (SEQ ID NO:4). l. SEQ ID NO: 117 contained a 3’ portion of a G418 resistance fragment (SEQ ID NO:3) and a 3’ RCSR18717 flanking sequence (SEQ ID NO: 18). m. SEQ ID NO: 118 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13); and a 5’ portion of a G418 resistance marker gene (SEQ ID NO:4). n. SEQ ID NO: 119 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13), an MpPYKl promoter (SEQ ID NO: 68), a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110, an Mp6PGD terminator (SEQ ID NO:78), and a 5’ portion of a G418 resistance marker gene (SEQ ID NO:4). o. SEQ ID NO: 120 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13), an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110; an MpTDH3 terminator (SEQ ID NO:87); and a 5’ portion of a G418 resistance marker gene (SEQ ID NO:4). p. SEQ ID NO: 121 contained a 3’ portion of a G418 resistance marker gene (SEQ ID NO:3); an MpPGKl promoter (SEQ ID NO:73); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110, an MpENOl terminator (SEQ ID NO:80); and a 3’ RCSR26640 flanking sequence (SEQ ID NO: 14). q. SEQ ID NO: 122 contained a 3’ portion of a G418 resistance marker gene (SEQ ID NO:3); an MpPYKl promoter (SEQ ID NO: 68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 110; an MpPYKl terminator (SEQ ID NO:84); and a 3’ RCSR26640 flanking sequence (SEQ ID NO: 14)r. SEQ ID NO: 123 contained 3’ portion of a zeocin resistance marker (SEQ ID NO:5); and a 3’ gpdllA flanking sequence (SEQ ID NO: 30). s. SEQ ID NO: 124 contained 3’ portion of a G418 resistance marker (SEQ ID NO:3); and a 3’ gpdllA flanking sequence (SEQ ID NO: 30). t. SEQ ID NO: 125 contained a 3’ portion of a G418 resistance marker gene (SEQ ID NO:3); and a 3’ RCSR26640 flanking sequence (SEQ ID NO: 14). u. SEQ ID NO: 127 contained a 5’ gpdllA flanking sequence (SEQ ID NO:29); an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO:96; an Mp6PGD terminator (SEQ ID NO:78); and a 5’ portion of a zeocin resistance selection marker (SEQ ID NO:6).

[0150] The indicated Moniliella pollinis parent strain was transformed with the indicated sequence(s) in Table 2. Transformants were selected on PDA + selection plates (selection markers for each transformation are outlined in Table 2) and incubated at 30-35 °C for at least 2 days until transformants grew. Resulting transformants were streaked for single colony isolation on PDA + selection plates (selection markers for each transformation are outlined in Table 2) and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequences. A PCR verified isolate was then designated as the indicated strain number. In some instances, more than one PCR verified isolate, e.g., “sister” isolates, are indicated by letters following the strain number. For example, strain 1-15 has 8 sister isolates, strains l-15a, 1- 15b, l-15c, l-15d, l-15e, l-15f, l-15g, and l-15h.

[0151] For example, Strain 1-2 was transformed with SEQ ID NO: 109 and SEQ ID NO: 107. Transformants were selected on PDA + zeocin selection plates and incubated at 30-35 °C for at least 2 days until transformants grew. Resulting transformants were streaked for single colony isolation on PDA + zeocin plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. PCR verified isolates were designated strains l-15a, l-15b, l-15c, l-15d, l-15e, l-15f, l-15g, and l-15h.Table 2.Strain l-33a-b

[0152] Strain 1-13 was transformed with SEQ ID NO: 126 using the transformation protocol outlined above and positive transformants were selected on geneticin (G418) resistance selection plates. PCR verified isolates, in which one copy of the RSCR26640 gene was deleted, were designated strains l-33a-b. SEQ ID NO: 126 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13), the G418 resistance gene expression cassette, and a 3’ RCSR26640 flanking sequence (SEQ ID NO: 14).Example 2 - Shake Flask Fermentation Assay

[0153] Strains 1-14, 1-15, 1-16, 1-17, 1-18, and 1-2 (control) , were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0154] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0155] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 and 96 hours of incubation. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96- hours are reported in Table 4 and FIG. 2.Table 3: Production MediumTable 4: 96-hour Shake Flask Results

[0156] Results show that overexpression of the glycerol kinase RCSR17702 (SEQ ID NO: 110, see strains 1-14 and 1-15) results in significantly decreased glycerol titer and yield compared to control strain 1-2. While strain 1-15e did not show a decrease in glycerol, this strain encountered an error during fermentation causing the results to be incorrect. A number of sister strains also showed an increase in xylitol yield as well (e.g., l-14b, l-14d, l-14e, l-14g, l-15a-d, 1-15f, and 1 - 15h). Likewise, overexpression of the glycerol kinase RCSR09543 (SEQ ID NO:96, see strains 1-16) results in significantly decreased glycerol titer and yield compared to control strain 1-2. Strains l-16a, l-16b, and l-16e also showed an increase in xylitol yield. Overexpression of the glycerol phosphate dehydrogenase RC SRI 6725 (SEQ ID NO: 95), did not result in a decrease in glycerol production nor did any of the strains show an increase in xylitol production. Strains 1- 18, which overexpress both RCSR16725 and RCSR17702, generally showed some decrease in glycerol production with a few strains also showing an increase in xylitol production. Given that the strains with only overexpression of RCSR16725 did not show a decrease in glycerol, the decrease in glycerol production on strains l-18a-d and l-18g-h can be attributed to the overexpression of RCSR17702.Example 3 - Shake Flask Fermentation Assay

[0157] Strains 1-15, 1-34, and 1-2 (control), were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0158] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0159] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the 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. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96-hours are reported in Table 5. Due to errors in the shake flasks for 1 -15e and 1 - 15h (Table 5), these two strains were rerun, and the results are shown in Table 6 and FIG. 3.Table 5: 96-hour Shake Flask ResultsTable 6: 96-hour Shake Flask Results

[0160] These results confirm that overexpression of the glycerol kinase of RCSR17702 (SEQ ID NO: 110) decreases glycerol production, as demonstrated in strains l-15a-h. Several of these strains (1 - 15b-h) also showed an increase in xylitol yield. Strains l-34a-f, which overexpress both RCSR16725 and RCSR09543, all showed some slight decrease in glycerol production. Given that the strains with only overexpression of RCSR16725 did not show a decrease in glycerol (See Example 2 Table 4), the decrease in glycerol production in strains l-34a-f can be attributed to the overexpression of RCSR09543.Example 4 - Shake Flask Fermentation Assay

[0161] Strains l-35a-f and 1-2 (control), were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0162] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0163] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 and 96 hours of incubation. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96- hours are reported in Table 7.Table 7: 96-hour Shake Flask Results

[0164] Strains 1-35c, l-35d, and l-35e showed a decrease in glycerol production relative to the control stain 1-2. Strains 1-35a and l-35c-f showed an increase in xylitol yield. There is some si ster-to- sister strain variability in several of the strains assayed. While a strain is PCR verified to contain the desired polynucleotide sequence (e.g., encoding RCSR09543), said sequence may not be integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters. However, the results still demonstrate that overexpression of RCSR09543 produces more xylitol but produces less glycerol than strains with wild-type expression of the native RCSR09543 gene.Example 5 - Shake Flask Fermentation Assay

[0165] Strains 1-19, 1-20, 1-21, 1-22, 1-23 and 1-8 (control), were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0166] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0167] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the 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. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96- hours are reported in Tables 8, 9, and 10 and FIG. 4.Table 8: 96-Hour Shake Flask ResultsTable 9: 96-Hour Shake Flask ResultsTable 10: 96-Hour Shake Flask Results

[0168] In general, strains overexpressing the glycerol kinase of SEQ ID NO: 110 (RCSR17702), e.g., strains l-19a-d, l-21a-d, l-20a-b, and l-20d-f, showed a reduction in glycerol titer and yield compared to parent strain 1-8. These strains also showed an increase in the titer and yield of xylitol production.

[0169] There is some sister-to-sister strain variability in several of the strains assayed. While a strain is PCR verified to contain the desired polynucleotide sequence (e.g., encoding RCSR17702 or RCSR09543), said sequence may not be integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters. However, the results still demonstrate that overexpression of RCSR17702 produces more xylitol but produces significantly less glycerol than strains with wild-type expression of the native RCSR17702 gene.Example 6 - Shake Flask Fermentation Assay

[0170] Strains 1-24 and 1-13 (control) were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0171] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical densityis measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0172] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 24, 48, 72 and 96 hours of incubation. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96-hours are reported in Table 11 and FIG. 5.Table 11 : 96-hour Shake Flask Results

[0173] Compared to parent strain 1-13, strains l-24a-c, l-24e, l-24f, l-24h, l-24i, l-24k, 1- 24m, and l-24o, which overexpress RCSR17702, showed both an increase in xylitol titer and yield and a decrease in glycerol titer and yield relative to strain 1-13. All of strains l-24a-o showed an increase in xylitol yield.

[0174] There is some sister-to-sister strain variability in several of the strains assayed. While a strain is PCR verified to contain the desired polynucleotide sequence (e.g., encoding RCSR17702), said sequence may not be integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters. However, the results still demonstrate that overexpression of RCSR17702 produces more xylitol but produces significantly less glycerol than strains with wild-type expression of the native RCSR17702 gene.Example 7 - Shake Flask Fermentation Assay

[0175] Strains 1-25, 1-26, 1-27, and 1-28, along with controls 1-13, l-33a, and l-33b, were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0176] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0177] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 24, 48, 72, and 98 hours of incubation. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 98 hours are reported in Tables 12 and 13 and FIGS. 6 and 7.Table 12: 98-hour Shake Flask ResultsTable 13: 98-hour Shake Flask Results

[0178] Compared to the parent strain 1-13, all of strains 1-25, 1-26, 1-27, and 1-28 (including all sister) showed a decrease in glycerol production (titer and yield) when the gene encoding RCSR17702 was incorporated at the RCSR26640 locus. Some of those strains also showed an increase in xylitol yield (strains l-25a, l-25b, l-25d-f, l-26c, l-26e-h, l-26k-r, l-26u, l-27a, 1- 27f, l-27g, l-27m, and l-27t). Compared to control strains l-33a and l-33a, with a deletion of one copy of RCSR26640, all of strains 1-25, 1-26, 1-27, and 1-28 (including all sisters) showed a decrease in glycerol production (titer and yield), however none showed a significant increase in xylitol titer or yield. Therefore, while the overexpression of RCSR17702 decreased the titer and yield of glycerol, it alone may not be enough to significantly increase xylitol titer and yield.Example 8 - Shake Flask Fermentation Assay

[0179] Strains 1-29, 1-30, 1-31, 1-32, and 1-1 (control) were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, and ethanol production.

[0180] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / Lglucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0181] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the 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. Samples were analyzed for glucose, xylitol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96 hours are reported in Tables 14 and 15 and FIG. 8.Table 14: 96-hour Shake Flask ResultsTable 15: 96-hour Shake Flask Results

[0182] Strains overexpressing either RCSR17702 (strains l-29a-h and l-30a-g) or RCSR09543 (strains l-31a-g and l-32a-g) shows a significant decrease in glycerol titer and yield compared to control strain 1-1. This demonstrates that overexpress of either RCSR17702 or RCSR09543 will reduce glycerol production in wild-type background as well as other engineered backgrounds.Example 9 - Moniliella pollinis Strain EngineeringStrain 2-la-b

[0183] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 1-13 was used to generate a Moniliella pollinis strain with improved xylitol production rate in shake flask fermentation. Strains with high xylitol rates were selected based onxylitol production (as measured by HPLC) in a shake flask fermentation compared to xylitol production in the parent 1-13 strain. Two resulting strains with improved xylitol rate, each of which contained 2 copies of the gene encoding the XPDH of SEQ ID NO:57, 2 additional copies of the gene encoding the RPE of SEQ ID NO: 54 (for 3 copies total), 3 additional copies of the gene encoding the X5PP of SEQ ID NO:33 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, were designated strains 2-la and 2-lb.Strain 2-2

[0184] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 2-la was used to generate Moniliella pollinis strain with improved xylitol production rate in shake flask fermentation. Strains with high xylitol rates were selected based on xylitol production (as measured by HPLC) in a shake flask fermentation compared to xylitol production in the parent 2-la strain. The resulting strain with improved xylitol rate, which contained 2 copies of the gene encoding the XPDH of SEQ ID NO: 57, 2 additional copies of the gene encoding the RPE of SEQ ID NO: 54 (for 3 copies total), 3 additional copies of the gene encoding the X5PP of SEQ ID NO:33 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, was designated strain 2-2.Strain 2-3

[0185] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 2-lb was used to generate a Moniliella pollinis strain with improved xylitol production rate in shake flask fermentation. Strains with high xylitol rates were selected based on xylitol production (as measured by HPLC) in a shake flask fermentation compared to xylitol production in the parent 2-lb strain. Two resulting strains with improved xylitol rate, each of which contained 2 copies of the gene encoding the XPDH of SEQ ID NO:57, 2 additional copies of the gene encoding the RPE of SEQ ID NO: 54 (for 3 copies total), 3 additional copies of the gene encoding the X5PP of SEQ ID NO:33 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, were designated strains 2-3a and 2-3b.Strains 2-4 through 2-15

[0186] Strains 2-4 through 2-15 were designed to evaluate the expression of the glycerol kinase of SEQ ID NO: 110 with either a transketolase enzyme (SEQ ID NO: 129) or a trehalase enzyme (SEQ ID NO: 131).

[0187] Table 16 below lists various Moniliella pollinis strains, including information on the parent strain, the sequences with which the parent strain was transformed, and characterizations of the expression cassette(s) contained on the transformed sequences.

[0188] For the sequences outlined in Table 16, the transformation fragments are as follows. a. SEQ ID NO: 128 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13), an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 129; an MpTDH3 terminator (SEQ ID NO:87); and a 5’ portion of a G418 resistance marker gene (SEQ ID NON). b. SEQ ID NO: 130 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13), an MpPYKl promoter (SEQ ID NO:68); a nucleic acid sequence encoding the amino acid of SEQ ID NO: 131; an MpTDH3 terminator (SEQ ID NO:87); and a 5’ portion of a G418 resistance marker gene (SEQ ID NON).

[0189] The indicated Moniliella pollinis parent strain was transformed with the indicated sequence(s) in Table 16. Transformants were selected on PDA + G418 plates and incubated at 30-35 °C for at least 2 days until transformants grew. Resulting transformants were streaked for single colony isolation on PDA + G418 plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequences. A PCR verified isolate was then designated as the indicated strain number. In some instances, more than one PCR verified isolate, e.g., “sister” isolates, are indicated by letters following the strain number. For example, strain 2-4 has 3 sister isolates, strains 2-4a, 2-4b, and 2-4c.

[0190] For example, Strain 2-2 was transformed with SEQ ID NO: 118 and SEQ ID NO: 121. Transformants were selected on PDA + G418 selection plates and incubated at 30-35 °C for at least 2 days until transformants grew. Resulting transformants were streaked for single colony isolation on PDA + G418 plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. PCR verified isolates were designated strains 2-4a, 2-4b, and 2 -4c.Table 16.Example 10 - Shake Flask Fermentation Assays

[0191] Strains 2-4a-b, 2-5a, 2-6a, 2-7, 2-8a-d, 2-9a-b, 2-10a-h, 2-l la-e, 2-12a-g, 2-13a-c, and controls 1-13, 2-2, 2-3a, and 2-3b were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, degree of polymerization 2 (DP2) sugars, and ethanol production.

[0192] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0193] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 24, 48, 72 and 96 hours of incubation. Samples were analyzed for glucose, xylitol, glycerol, DP2 sugars, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96-hours are reported in Table 17 and FIGS. 9-12.Table 17.

[0194] Results show that strains overexpressing the TKL3 enzyme of SEQ ID NO: 129 and the glycerol kinase enzyme of SEQ ID NO: 110 (strains 2-6a, 2- 10a, 2- 10c, 2-10e, 2- 10g, 2-9a, and 2- 9b) accumulated significantly less glycerol than the control strains (1-13, 2-2, and 2-3 a, see FIG. 11). Several of those strains also accumulated significantly less glycerol than strains with only one additional copy of the glycerol kinase of SEQ ID NO:110 (2-4a, 2-4b, and 2-13b) and / or showed an increase in xylitol production relative to the control strains. Similarly, strains overexpressing the trehalase enzyme of SEQ ID NO: 131 and the glycerol kinase enzyme of SEQ ID NO: 110 (strains 2-l la-e, see FIGS. 12 & 13) accumulated significantly less glycerol and less DP2 sugars than control strains 1-13 and 2-3b. Results also show further reduction of glycerol accumulation in strains containing two additional copies of nucleotide sequences encoding the glycerol kinase of SEQ ID NO: 110 compared to strains with only one additional copy (See FIG. 9).

[0195] There is some sister-to-sister strain variability in several of the strains assayed, for example strains 2-10a-h. While a strain is PCR verified to contain the desired polynucleotide sequence (e.g., encoding the glycerol kinase of SEQ ID NO: 110), said sequence may not be integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters. However,the results still demonstrate that overexpression of the combination of SEQ ID NO: 129 and SEQ ID NO: 110 results in significantly reduced glycerol accumulation.Example 11 - Shake Flask Fermentation Assays

[0196] Strains 2-10i-j, 2-1 If, 2-12h, 2-13d-e, and controls 1-13, 2-2, 2-3a, and 2-3b were run in shake flasks to assess glucose consumption as well as xylitol, glycerol, DP2 sugars, and ethanol production.

[0197] Strains were streaked out for biomass growth on YPD plates (bacteriological peptone 20g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) and incubated at 30 °C for 48-72 hours. Cells from the incubated YPD plates were scraped into 40 mL rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL non-baffled flask. Cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15-20 to form the seed culture. Optical density is measured at a wavelength of 600 nm with a 1 cm path length cuvette using a model Genesys20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0198] A 250 ml non-baffled flask containing production medium (Table 3) was inoculated with 0.8 mL of the seed culture to form the production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 24, 48, 72 and 96 hours of incubation. Samples were analyzed for glucose, xylitol, glycerol, DP2 sugars, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results at 96-hours are reported in Table 18 and FIGS. 13-15.Table 18.

[0199] Results show that strains overexpressing the TKL3 enzyme of SEQ ID NO: 129 and the glycerol kinase enzyme of SEQ ID NO: 110 (strains 2-10i, 2-10j, 2-6b, 2-6c, and 2-6d) accumulated significantly less glycerol than the control strains (1-13, 2-2, and 2-3b, see FIG. 15). Several of those strains also showed an increase in xylitol production relative to the control strains. Similarly, strains overexpressing the trehalase enzyme of SEQ ID NO: 131 and the glycerol kinase enzyme of SEQ ID NO: 110 (strains 2-1 If, 2- 14c, 2-14d, and 2-14e, see FIGS. 16 & 17)accumulated significantly less glycerol than control strains 1-13, 2-2, and 2-3b. Results also show further reduction of glycerol accumulation in strains containing two additional copies of nucleotide sequences encoding the glycerol kinase of SEQ ID NO: 110 compared to strains with only one additional copy (See FIG. 14).

[0200] There is some sister-to-sister strain variability in several of the strains assayed, for example strains 2-13e and 2-14c. While a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters. However, the results still demonstrate that overexpression of the combination of SEQ ID NO: 129 and SEQ ID NO: 110 or the combination of SEQ ID NO: 131 and SEQ ID NO: 110 results in significantly reduced glycerol and / or DP2 sugar accumulation.

Claims

CLAIMSWhat is claimed is:

1. A genetically engineered yeast cell capable of producing xylitol, the engineered yeast cell comprising a genetic modification resulting in overexpression of a native enzyme with glycerol kinase activity, for example a glycerol kinase enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110; and a genetic modification resulting in overexpression of a native enzyme with transketolase activity, for example a transketolase enzyme comprising a sequence 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 SEQ ID NO: 129.

2. The yeast cell of claim 1, wherein the genetic modification resulting in overexpression of a native glycerol kinase enzyme comprises addition of an exogenous polynucleotide encoding the native glycerol kinase enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the glycerol kinase enzyme; and / or wherein the genetic modification resulting in overexpression of a native transketolase enzyme comprises addition of an exogenous polynucleotide encoding the native transketolase enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the transketolase enzyme.

3. The yeast cell of any preceding claim, wherein the yeast cell comprises at least one additional copy of a polynucleotide sequence encoding a glycerol kinase enzyme 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 SEQ ID NO:96.

4. The yeast cell of any preceding claim, wherein the yeast cell comprises at least one additional copy of a polynucleotide sequence encoding a glycerol kinase enzyme 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 SEQ ID NO: 110.

5. The yeast cell of any preceding claim, wherein the yeast cell comprises at least one additional copy of a polynucleotide sequence encoding a transketolase enzyme 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 SEQ ID NO: 129.

6. The yeast cell of any preceding claim, wherein the yeast cell is an osmotolerant yeast cell.

7. The yeast cell of any preceding claim, wherein the yeast cell is a cell of the subphylum Ustilaginomycotina.

8. The yeast cell of any preceding claim, wherein the yeast cell is selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis. Trychosporonoides nigrescens. Pseudozyma Isiikubaensis. Trigonopsis variabilis. Moniliella. Ustilaginomycetes, Trichosporon. Yarrowia lipolylica. Saccharomyces cerevisiae. Penicillium. Torula, Pichia. Candida, Candida magnolias, and Aureobasidium.

9. The yeast cell of any preceding claim, wherein the yeast cell comprises a deletion or disruption of a native gene encoding an erythrose reductase enzyme at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 11 and 15.

10. The yeast of claim 9, wherein the cell has a deletion of both alleles of the gene encoding the erythrose reductase enzyme.

11. The yeast cell of any preceding claim, wherein the cell additionally comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:55-64.

12. The yeast cell of claim 11, wherein the XPDH enzyme has a sequence at least 85% identical to at least one of SEQ ID NOs: 55-62 and 64 or to at least one of SEQ ID NOs:57, 58, 59, or 62.

13. The yeast cell of claim 11 or claim 12, wherein the XPDH enzyme has a sequence at least 90% identical to at least one of SEQ ID NOs: 55-62, and 64, or to at least one of SEQ ID NOs:57, 58, 59, or 62.

14. The yeast cell of any one of claims 1-10, wherein the cell additionally comprises an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:50 and 49.

15. The yeast cell of claim 14, wherein the cell additionally comprises an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:65, 66, and 67.

16. The yeast cell of any preceding claim, wherein the cell additionally comprises a genetic modification resulting in overexpression of a native enzyme with ribulose-5-phosphate epimerase (RPE) activity.

17. The yeast cell of claim 16, wherein the cell is Moniliella pollinis cell and the native RPE enzyme comprises a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:53 and 54.

18. The yeast cell of claim 16 or 17, wherein the genetic modification resulting in overexpression of a native RPE enzyme comprises addition of an exogenous polynucleotide encoding the native RPE enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the RPE enzyme.

19. The yeast cell of any preceding claim, wherein the yeast additionally comprises a genetic modification resulting in overexpression of a native enzyme with xylitol-5-phosphate phosphatase(X5PP) activity; and / or an exogenous polynucleotide sequence encoding an enzyme with xylitol- 5 -phosphate phosphatase (X5PP) activity.

20. The yeast cell of claim 19, wherein the cell is a Moniliella pollinis cell and the genetic modification results in overexpression of a native X5PP enzyme with a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:51, 52, 33, or 47.

21. The yeast cell of claim 19 or claim 20, wherein the genetic modification comprises addition of an exogenous polynucleotide sequence encoding the native X5PP enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the native X5PP enzyme.

22. The yeast cell of any one of claims 19-21, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with X5PP activity and a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:33-50.

23. The yeast cell of any one of claims 19-22, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with X5PP activity and a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:33, 35, 36, 37, 38, 39, 43, and 45.

24. The yeast cell of any one of claims 19-23, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with X5PP activity and a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:33, 36, 37, 39, and 45.

25. The yeast cell of any one of claims 19-24, wherein X5PP activity in the genetically engineered yeast cell is higher than X5PP activity in an equivalent cell lacking the genetic modification or exogenous polynucleotide sequence.

26. The yeast cell of any preceding claim, wherein, when the engineered cell is used in a fermentation process in the presence of dextrose, titer and / or yield of glycerol production is decreased relative to titer and / or yield of glycerol in an equivalent fermentation process using an equivalent cell in which there has been no overexpression of a native gene encoding a glycerol kinase at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:96 and 110.

27. The yeast cell of any preceding claim, wherein one or more of the exogenous polynucleotide sequence(s) is operably linked to a heterologous or artificial promoter selected from the group consisting of pyruvate kinase 1 promoter (PYKlp; SEQ ID NO:68), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 69), glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:70), translational elongation factor 1 promoter (TEFp; SEQ ID NO:71), phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72), 3 -phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73), enolase promoter (ENOlp ; SEQ ID NO:74), asparagine synthetase promoter (ASNSp; SEQ ID NO:75), 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76), and RPL16B (SEQ ID NO:77).

28. The yeast cell of any preceding claim, wherein one or more of the exogenous polynucleotide sequence(s) is integrated into the genome of the yeast cell at a locus selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdllA locus, the gpdllB locus, the RCSR26640 locus, and the RCSR18717 locus.

29. A method for producing xylitol comprising contacting a substrate comprising dextrose with the engineered yeast cell of any preceding claim, wherein fermentation of the substrate by the engineered cell produces xylitol.

30. The method of claim 29, wherein the fermentation temperature is at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C and the volumetric oxygen uptake rate (OUR) is between 5-80, 10-75, 15-70, 20-60, 30-50, or 40-50 mmol O2 / (L • h).

31. The method of claim 29 or 30, wherein the xylitol is produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h'1.

32. The method of any one of claims 29-31, wherein the xylitol titer is at least 20, 30, 50, 75, or 100 g / L when the fermentation is run at 35 °C for 96 hours.

33. The method of any one of claims 29-32, wherein titer, and / or yield of glycerol accumulation is decreased relative to an equivalent fermentation run with an equivalent yeast cell in which the native gene encoding the glycerol kinase enzyme has not been over expressed.

34. The method of any one of claims 29-33, wherein the concentration of dextrose is at least 100 g / L.

35. The method of any one of claims 29-33, wherein the glycerol titer is less than 10, 8, 6, 5, 4, 3, 2, 1, or 0.5 g / L when the fermentation is run at 35 °C for 96 hours; and / or wherein the glycerol yield is less than 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%.

36. Use of the engineered yeast of any one of claims 1-28 to produce xylitol.

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