Genetically modified yeast and fermentation processes for the production of arabitol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing arabitol and characterized by a genetic modification resulting in overexpression of a native enzyme with arabitol-5-phosphate phosphatase (A5PP) activity and / or an exogenous polynucleotide sequence encoding an enzyme with arabitol-5-phosphate phosphatase (A5PP) activity. The genetically engineered yeast cell may additionally be engineered to overexpress a native RPE enzyme, engineered to express an exogenous APDH enzyme, and / or engineered to express an exogenous ARD2DH enzyme.

Description

PT-1755-WO-PCT GENETICALLY MODIFIED YEAST AND FERMENTATION PROCESSES FOR THE PRODUCTION OF ARABITOL CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 499,991, filed May 4, 2023, 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-1755- WO-PCT.xml” which is 803,466 bytes in size created on April 29, 2024 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. The intermediates and products have their own uses and markets that make their fermentation commercially relevant. Accordingly, provided herein are genetically modified yeast and fermentation methods for the production of arabitol based on closely related pathways for the production of xylitol.PT-1755-WO-PCT 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 arabitol-5-phosphate phosphatase (A5PP) activity; and / or an exogenous polynucleotide sequence encoding an enzyme with arabitol-5-phosphate phosphatase (A5PP) activity. The yeast cell may be an osmotolerant yeast cell. The yeast cell may be a cell of the subphylum Ustilaginomycotina or Saccharomycotina. The yeast cell may be selected form the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Saccharomyces cerevisiae, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. The yeast cell may be a yeast cell of the genus Moniliella.

[0006] The yeast cell may be a Moniliella pollinis cell and the genetic modification results in overexpression of a native A5PP 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:198, 199, 200, or 221.

[0007] The genetic modification may comprise replacement of the native A5PP gene promoter with a heterologous or artificial promoter. The heterologous or artificial promoter may be selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130), glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132), translational elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQ ID NO:131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p ; SEQ ID NO:136), asparagine synthetase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139).

[0008] The genetic modification may comprise addition of an exogenous polynucleotide sequence encoding the native A5PP enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the native A5PP enzyme. The yeast cell may comprise an exogenous polynucleotide sequence encoding an enzyme with A5PP 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:200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188. The yeastPT-1755-WO-PCT call may comprise an exogenous polynucleotide sequence encoding an enzyme with A5PP 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:200, 202, 203, 204, 205, 206, 210, and 213. The yeast cell may comprise an exogenous polynucleotide sequence encoding an enzyme with A5PP 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:200, 203, 204, 206, and 213.

[0009] In general, A5PP activity in the genetically engineered yeast cell is higher than A5PP activity in an equivalent cell lacking the genetic modification or exogenous polynucleotide sequence. When the engineered cell is used in a fermentation process in the presence of dextrose, titer and / or yield of arabitol is increased relative to titer and / or yield of arabitol in an equivalent fermentation process using an equivalent cell lacking the genetic modification or exogenous polynucleotide sequence.

[0010] The engineered yeast cell may additionally comprise a genetic modification resulting in overexpression of a native enzyme with ribulose-5-phosphate epimerase (RPE) activity. The cell may be a Moniliella pollinis cell and the native RPE enzyme may comprise 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:179 and 180. The genetic modification resulting in overexpression of a native RPE enzyme may comprise replacement of the native RPE gene promoter with a heterologous or artificial promoter. The heterologous or artificial promoter may be selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132), translational elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQ ID NO:131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134), 3- phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p ; SEQ ID NO:136), asparagine synthetase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139). The genetic modification resulting in overexpression of a native RPE enzyme may comprise 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.PT-1755-WO-PCT

[0011] The yeast cell may additionally comprise an exogenous polynucleotide sequence encoding an arabitol-phosphate dehydrogenase (APDH) 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:11.

[0012] The cell may additionally comprise an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) 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:193, 194, 195, 196, or 197. The cell may additionally comprise an exogenous polynucleotide sequence encoding an arabitol 2- dehydrogenase (ARD2DH) 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:194, 195, 196, or 197.

[0013] Any of the exogenous polynucleotide sequences 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 (PYK1p; SEQ ID NO:86), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130), glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132), translational elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQ ID NO:131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p ; SEQ ID NO:136), asparagine synthetase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139). Any of the exogenous polynucleotide sequences may be integrated into the genome of the yeast cell at a loci selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdIIA locus, and the gpdIIB locus.

[0014] The disclosure also provides a method for producing arabitol 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 arabitol. 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 0.5 to 40, 1 to 35, 2 to 30, 3 to 25, 4 to 20, or 5 to 15 mmol O2 / (L • h). The arabitol 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. Arabitol 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. Rate and / or yield of arabitol production may be increased relative to an equivalent fermentation run with an equivalentPT-1755-WO-PCT yeast cell lacking the genetic modification to overexpress the A5PP enzyme and lacking an exogenous polynucleotide sequence encoding an exogenous A5PP enzyme. The concentration of dextrose may be at least 100 g / L.

[0015] The disclosure also provides a use of the engineered yeast described herein to produce arabitol. BRIEF DESCRIPTION OF THE FIGURES

[0016] 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.

[0017] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0018] FIG. 1 shows the native pentose phosphate pathway (dotted lines and arrows) and the native glycolysis pathways (solid lines and arrows) in Moniliella pollinis.

[0019] FIG. 2 shows diversity in the galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) sequence space.

[0020] FIG.3 shows the structural characteristics of the NAD or NADP binding pocket located +23 amino acids from the characteristic GXGXXG motif (SEQ ID NO:133) of XPDH enzymes.

[0021] FIG.4 shows diversity in the ribulose-5-phosphate reductase sequence space.

[0022] FIG.5 shows in vitro activity of TarJ’ and XPDH enzymes as outlined in Example 3.

[0023] FIG.6 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 96 hours of shake flask fermentations of strains 1-1, 1-13a-f, and 1-15a-f as outlined in Example 5. Data labels report the concentration (g / L) of xylitol.

[0024] FIG.7 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 96 hours of shake flask fermentations of strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, and 1-39a-f as outlined in Example 5. Data labels report the concentration (g / L) of xylitol.

[0025] FIG.8 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 96 hours of shake flask fermentations of strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e as outlined in Example 6. Data labels report the concentration (g / L) of xylitol.

[0026] FIG.9 shows erythritol, ribitol, arabitol, and xylitol metabolite concentrations (g / L) at 96 hours of shake flask fermentations of strains 1-13c, 1-12a-e, 1-14a-e, and 1-16a-e as outlined in Example 8. Data labels report the concentration (g / L) of xylitol (strains 1-13c, 1-14a-e, and 1- 16a-e) or arabitol (strains 12a-e).PT-1755-WO-PCT

[0027] FIG.10 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 96 hours of shake flask fermentations of strains -13c, 1-36a-e, and 1-40a-e as outlined in Example 9. Data labels report the concentration (g / L) of xylitol.

[0028] FIG.11 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 96 hours of shake flask fermentations of strains 1-30a-e, 1-31a-e, 1-32a-e, and 1-13c as outlined in Example 10. Data labels report the concentration (g / L) of xylitol.

[0029] FIG. 12 shows a comparison of xylitol and ribitol concentrations (g / L) produced in strains with (3-7a-f and 3-8a-f) and without (1-13c and 1-15a) RPE2 overexpression.

[0030] FIG. 13 shows respective yields of xylitol, ribitol, glycerol, and erythritol for strains with (3-8a-f) and without (1-15a) RPE2 overexpression.

[0031] FIG.14 shows xylitol and ribitol titers (g / L) for the indicated strains.

[0032] FIG. 15 shows respective yields of xylitol, ribitol, glycerol, and erythritol for the indicated strains with various configurations of RPE overexpression.

[0033] FIG.16 shows xylitol concentrations (g / L) at 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15.

[0034] FIG.17 shows xylitol concentrations (g / L) at 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15.

[0035] FIG. 18 shows xylitol rate (g / (L•h)) between 48 and 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15.

[0036] FIG. 19 shows xylitol rate (g / (L•h)) between 48 and 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15.

[0037] FIG.20 shows xylitol yield (%) between 48 and 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15.

[0038] FIG.21 shows xylitol yield (%) between 48 and 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15.

[0039] FIG.22 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 18. Data labels report the concentration (g / L) of xylitol.

[0040] FIG.23 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 18. Data labels report the concentration (g / L) of xylitol.PT-1755-WO-PCT

[0041] FIG.24 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 22. Data labels report the concentration (g / L) of xylitol.

[0042] FIG.25 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 22. Data labels report the concentration (g / L) of xylitol.

[0043] FIG.26 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 23. Data labels report the concentration (g / L) of xylitol.

[0044] FIG.27 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 23. Data labels report the concentration (g / L) of xylitol.

[0045] FIG.28 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 24. Data labels report the concentration (g / L) of xylitol.

[0046] FIG.29 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 24. Data labels report the concentration (g / L) of xylitol.

[0047] FIG.30 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 25. Data labels report the concentration (g / L) of xylitol.

[0048] FIG.31 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 27. Data labels report the concentration (g / L) of xylitol.

[0049] FIG.32 shows erythritol, ribitol, and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 27. Data labels report the concentration (g / L) of xylitol.

[0050] FIG. 33 shows erythritol and xylitol metabolite concentrations (g / L) at 72 hours of shake flask fermentations of strains as outlined in Example 28. Data labels report the concentration (g / L) of xylitol.

[0051] FIG. 34 shows erythritol, glycerol, and arabitol metabolite concentration (g / L) at 72 hours of shake flask fermentation of strains as outlined in Example 30. Data labels report the concentration (g / L) of arabitol.PT-1755-WO-PCT

[0052] FIG. 35 shows xylitol and arabitol metabolite concentrations (g / L) of fermentations described in Example 32.

[0053] FIG. 36 shows xylitol and arabitol metabolite concentrations (g / L) as measured by HPIC of fermentations described in Example 32. DETAILED DESCRIPTION

[0054] 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 understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] This disclosure relates to various recombinant cells engineered to produce arabitol. In general, the recombinant cells described herein are capable of producing arabitol and arePT-1755-WO-PCT characterized by overexpression of a phosphatase enzyme. The recombinant yeast may additionally be characterized by overexpression of an X5PP enzyme or by inclusion of an exogenous polynucleotide sequence encoding an X5PP enzyme. The recombinant yeast may additionally be characterized by overexpression of a ribulose 5-phosphatase epimerase (RPE) enzyme and by incorporation of an exogenous polynucleotide sequence encoding an arabitol phosphate dehydrogenase (APDH) and / or an ARD2DH enzyme. The disclosure further provides fermentation methods for the production of arabitol from dextrose using the genetically engineered cells described herein.

[0059] 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, Kluveromyces marxianus), Yarrowia lipolytica, Issatchenkia orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnoliae, Candida ethanolica), Pichia deserticola, Pichia membranifadens, Pichia fermentans, 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.

[0060] 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 PseudozymaPT-1755-WO-PCT (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).

[0061] 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.

[0062] The yeast cell may be an osmotolerant yeast cell. As used herein, “osmotolerant” 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).

[0063] 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 fermentationPT-1755-WO-PCT 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).

[0064] 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 not limited to, amdS (for example broken into a 3’ portion, SEQ ID NO:167, and a 5’ portion, SEQ ID NO:174), G418 resistance gene (for example broken into a 3’ portion, SEQ ID NO:172, and a 5’ portion, SEQ ID NO:175), zeocin resistance gene (for example broken into a 3’ portion, SEQ ID NO:168, and a 5’ portion, SEQ ID NO:169), nourseothricin N-acetyl transferase (NAT) (for example broken into a 3’ portion, SEQ ID NO:171, and a 5’ portion, SEQ ID NO:170), and invertase gene (SUC2) (for example a 3’ portion of SEQ ID NO:173 and a 5’ portion of SEQ ID NO:176).

[0065] 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 arabitol by the recombinant cells.

[0066] The terms “glucose” and “dextrose” are used interchangeably herein and refer to D- glucose except where expressly indicated otherwise.

[0067] 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.

[0068] 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 / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, HavenstraatPT-1755-WO-PCT 84, B-1800 Vilvoorde) on March 28, 1997 under number MUCL40385, is considered the wild- type Moniliella pollinis cell.

[0069] 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 as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein. Table 1: Amino Acid three and one letter symbols Amino Acid Three letter symbol One letter symbol Al i Al APT-1755-WO-PCT Amino Acid Three letter symbol One letter symbol Tyrosine Tyr Y

[0070] Variaith 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.

[0071] 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.PT-1755-WO-PCT

[0072] 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.

[0073] The polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild-type” is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant,” “mutant,” or “derivative” refers to 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.

[0074] 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.

[0075] 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).PT-1755-WO-PCT

[0076] 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.

[0077] 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 A5PP 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 of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for 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.

[0078] 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. The native gene to be deleted or disrupted may be replaced with an exogenous nucleic acid of interest for the expression of an exogenous gene product (e.g., polypeptide, enzyme, and the like).

[0079] 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, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in a M. pollinis host cells, suitable interaction loci may include, but are not limited to, the ER1 locus (defined asPT-1755-WO-PCT the locus flanked by SEQ ID NO:85 and SEQ ID NO:162), the ER3 locus (defined as the locus flanked by SEQ ID NO:155 and SEQ ID NO:165), the PDC1 locus (defined as the locus flanked by SEQ ID NO:152 and SEQ ID NO:164), the pyrF locus (defined as the locus flanked by SEQ ID NO:153 and SEQ ID NO:163), the TRP3 locus (defined as the locus flanked by SEQ ID NO:156 and SEQ ID NO:159), the gpdIIA locus (defined as the locus flanked by SEQ ID NO:157 and SEQ ID NO:161); and the gpdIIB locus (defined as the locus flanked by SEQ ID NO:158 and SEQ ID NO:166). 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.

[0080] The recombinant cell may have one or more copies of a given exogenous nucleic acid sequence integrated in a host chromosome(s) and replicated together with the chromosome(s) into which it has been integrated. For example, the yeast cell may be transformed with nucleic acid construct including a polynucleotide sequence encoding for a polypeptide described herein and the polynucleotide sequence encoding for the polypeptide may be integrated in one or more copies in a host chromosome(s). The recombinant cell may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. The recombinant cell may have one, two, three, four, five, six, seven, eight, nine, ten, or more copies of a polynucleotide sequence encoding a polypeptide described herein integrated into the genome. The multiple copies of said polynucleotide sequence may all be incorporated at a single locus or may be incorporated at multiple loci.

[0081] The final step in the arabitol pathway, from arabitol 5-phosphate to arabitol, 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 of haloacid 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 arabitol 5-phosphate and xylitol 5-phosphate are similar molecules 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 / or arabitol-5-PT-1755-WO-PCT phosphate phosphatase activity and can be used to increase xylitol or arabitol 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 / or arabitol-5-phosphate phosphatase activity and can be used to increase xylitol or arabitol production in the recombinant cells describe here.

[0082] The recombinant cells described herein are capable of producing arabitol and are characterized by overexpression of a native enzyme with arabitol-5-phosphate phosphatase (A5PP) activity and / or include an exogenous polynucleotide sequence encoding a native or exogenous enzyme with arabitol-5-phosphate phosphatase (A5PP) activity. In general, the recombinant cell(s) including overexpression of an A5PP enzyme or expressing an exogenous A5PP enzyme produce more arabitol than an equivalent cell lacking the exogenous A5PP enzyme or lacking overexpression of the A5PP enzyme. The enzyme may be any suitable enzyme with A5PP activity. As used herein, “A5PP enzyme” and “A5PP” are interchangeable and refer to an enzyme with A5PP activity. Herein, “arabitol-5-phosphate phosphatase activity” and “A5PP activity” are used interchangeably and refer to the ability to catalyze the conversion of arabiotl-5- phosphate to arabitol and phosphate. Suitable A5PP enzymes may include a divalent metal cation, for example, Mg2+, Mn2+, or Co2+. Suitable enzymes with A5PP 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 A5PP enzymes may be derived from any suitable source. For example, a polynucleotide encoding an A5PP enzyme may be derived from Moniliella pollinis, Saccharomyces cerevisiae, Lachancea dasiensis, Tetrapisispora blattae, Saccharomyces pastorianus, Kazachstania Africana, Podospora comata, Geotrichum candidum, Ogattaea haglerorum, Debaryomyces fabryi, Monilinia fructicola, Nadsonia fulvescens var. elongata DSM 6958, Escherichia coli, Wickerhamomyces ciferrii, Bacillus amyloliquefaciens, and the like. The A5PP 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:200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188. The A5PP 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 thePT-1755-WO-PCT amino acid sequence of at least one of SEQ ID NOs:200, 202, 203, 204, 205, 206, 210, and 213. The A5PP 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:200, 203, 204, 206, and 213.

[0083] The A5PP enzymes described herein may also have activity on xylitol-5-phosphase and may be described as xylitol-5-phosphate phosphatase enzymes with xylitol-5-phosphate phosphatase 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. Accordingly, polypeptides described herein may be both A5PP and X5PP enzymes as the given polypeptide has both A5PP and X5PP activity. Herein, description of an enzyme as one with X5PP activity or as an X5PP enzymes does not mean that the enzyme is not also an A5PP enzyme.

[0084] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO:200. 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:200.

[0085] The recombinant cell 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:201. 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:201.

[0086] The recombinant cell 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:202. 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:202.

[0087] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Tetrapisispora blattae gene encoding the amino acid sequence of SEQ ID NO:203. 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:203.PT-1755-WO-PCT

[0088] The recombinant cell 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:204. 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:204.

[0089] The recombinant cell 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:205. 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:205.

[0090] The recombinant cell 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:206. 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:206.

[0091] The recombinant cell 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:207. 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:207.

[0092] The recombinant cell 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:208. 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:208.

[0093] The recombinant cell 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:209. 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:209.

[0094] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Monilinia fructicola gene encoding the amino acid sequence of SEQ ID NO:210. The exogenous polynucleotide sequence may encode an amino acid sequence at leastPT-1755-WO-PCT 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:210.

[0095] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Nadsonia fulvescens var. elongata DSM 6958 gene encoding the amino acid sequence of SEQ ID NO:211. 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:211.

[0096] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Escherichia coli gene encoding the amino acid sequence of SEQ ID NO:213. 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:213.

[0097] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Wickerhamomyces ciferrii gene encoding the amino acid sequence of SEQ ID NO:214. 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:214.

[0098] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO:221. 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:221.

[0099] The recombinant cell 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:222. 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:222.

[0100] The recombinant cell 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:189. 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:189.PT-1755-WO-PCT

[0101] The recombinant cell 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:188. 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:188.

[0102] The enzyme with A5PP activity may be native to the host cell. For example, when the host organism is M. pollinis, the A5PP 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:198, 199, 200, or 221. The recombinant cell may comprise an exogenous polynucleotide encoding an A5PP 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: 198, 199, 200, or 221. The recombinant cell may include a genetic modification that increases expression of an A5PP 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: 198, 199, 200, or 221. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native A5PP enzyme into the cell (e.g., integration of additional copies of the A5PP encoding polynucleotide into it non-native locus in the cell), insertion of a constitutive promoter upstream of the coding region of the native A5PP 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 A5PP enzyme encoding gene in the genome of the host cell. One of skill in the art will recognize that expression of a native A5PP 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.

[0103] 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.

[0104] The recombinant cells described herein capable of producing arabitol and including an exogenous polynucleotide sequence encoding an A5PP enzyme and / or overexpression of a native A5PP enzyme may also be characterized by overexpression of a ribulose 5-phosphate epimerase (RPE enzyme). In general, the recombinant cell(s) including overexpression of the RPE enzyme produce more arabitol than an equivalent cell lacking the RPE enzyme or lacking overexpression of the RPE enzyme.PT-1755-WO-PCT

[0105] The recombinant cells described herein are capable of producing arabitol, include an exogenous polynucleotide sequence encoding an A5PP enzyme and / or overexpress a native A5PP 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 M. pollinis, 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:179 and 180. 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:179 and 180. 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:179 and 180. 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.

[0106] The recombinant cells described herein are capable of producing arabitol, include an exogenous polynucleotide encoding an A5PP enzyme and / or overexpress a native A5PP enzyme, and may include an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) enzyme. The recombinant cells described herein are capable of producing arabitol, include an exogenous polynucleotide encoding an A5PP enzyme and / or overexpress a native A5PP enzyme, and may include an exogenous polynucleotide sequence encoding an arabitol 2- dehydrogenase (ARD2DH) enzyme and may include an exogenous polynucleotide encoding a native or exogenous RPE enzyme or may have a genetic modification resulting in overexpressionPT-1755-WO-PCT of a native RPE enzyme, as described herein. The exogenous polynucleotide sequence may be an exogenous ARD2DH gene.

[0107] An “arabitol 2-dehydrogenase gene” and an “ARD2DH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with arabitol 2- dehydrogenase activity. As used herein “arabitol 2-dehydrogenase activity” refers to the ability to catalyze the conversation of D-ribulose and NADH or NADPH to D-arabitol and NAD+or NADP+. Enzymes with arabitol 2-dehydrogenase may be characterized under Enzyme Classification 1.1.1.250. The ARD2DH gene may be derived from any suitable source. For example, the ARD2DH gene may be derived from Beauveria bassiana, Pichia stipitis, Candida albicans, Kwoniella heveanensis, Candida maltosa. The ARD2DH gene may encode a polypeptide 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 at least one of SEQ ID NOs:193, 194, 195, 196, or 197. The ARD2DH gene may encode a polypeptide 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 at least one of SEQ ID NOs:194, 195, 196, or 197. Additional description of recombinant cells capable of producing arabitol and including a polypeptide with arabitol 2-dehydrogenase activity is provided in US Provisional Application No. 63 / 364,359, filed May 9, 2022, which is incorporated herein by reference in its entirety.

[0108] The recombinant cell may include a genetic modification resulting in overexpression of an RPE enzyme and an exogenous polynucleotide that is, or may be derived from, a Beauveria bassiana ARD2DH gene encoding the amino acid of SEQ ID NO:193. 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:193.

[0109] The recombinant cell may include a genetic modification resulting in overexpression of an RPE enzyme and an exogenous polynucleotide that is, or may be derived from, a Pichia stipitis ARD2DH gene encoding the amino acid of SEQ ID NO:194. 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:194.

[0110] The recombinant cell may include a genetic modification resulting in overexpression of an RPE enzyme and an exogenous polynucleotide that is, or may be derived from, a CandidaPT-1755-WO-PCT albicans ARD2DH gene encoding the amino acid of SEQ ID NO:195. 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:195.

[0111] The recombinant cell may include a genetic modification resulting in overexpression of an RPE enzyme and an exogenous polynucleotide that is, or may be derived from, a Kwoniella heveanensis ARD2DH gene encoding the amino acid of SEQ ID NO:196. 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:196.

[0112] The recombinant cell may include a genetic modification resulting in overexpression of an RPE enzyme and an exogenous polynucleotide that is, or may be derived from, a Candida maltosa ARD2DH gene encoding the amino acid of SEQ ID NO:197. 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:197.

[0113] The recombinant cells described herein are capable of producing arabitol, include an exogenous polynucleotide sequence encoding an A5PP enzyme and / or overexpress a native A5PP enzyme, and may include an exogenous polynucleotide sequence encoding an arabitol phophtase dehydrogenase (APDH) enzyme. The exogenous polynucleotide sequence may be an exogenous arabitol-phosphate dehydrogenase (APDH) gene. A recombinant cell described herein capable of producing arabitol, including an exogenous polynucleotide sequence encoding an A5PP enzyme and / or overexpress a native A5PP enzyme, and including an exogenous polynucleotide sequence encoding an APDH enzyme may also 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, as described herein.

[0114] An “arabitol-phosphate dehydrogenase gene” and an “APDH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with arabitol-phosphate dehydrogenase activity. As used herein “arabitol-phosphate dehydrogenase activity” refers to the ability to catalyze (i) the conversion of xylulose 5-phosphate and NADPH or NADH to arabitol-1-phosphate and NADP+or NAD+and / or (ii) the conversion of ribulose-5- phosphate and NADPH or NADH to arabitol-5-phosphate and NADP+or NAD+. The APDH genePT-1755-WO-PCT may be derived from any suitable source. For example, the ARDH gene may be derived from Lactobacillus salivarius cp400.

[0115] The recombinant cell may include a genetic modification resulting in overexpression of an RPE enzyme and an exogenous polynucleotide that is, or may be derived from, a Lactobacillus salivarius cp400 gene encoding the amino acid of SEQ ID NO:11. 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:11.

[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 (PYK1p; SEQ ID NO:86); 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132); translational elongation factor 1 promoter (TEFp; SEQ ID NO:133); modified TEFp (SEQ ID NO:131); phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135); enolase promoter (ENO1p ; SEQ ID NO:136); asparagine synthetase promoter (ASNSp; SEQ ID NO:137); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138); and RPL16B (SEQ ID NO:139).

[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, GAL10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt; SEQ ID NO:140); ASNS terminator (ASNSt; SEQ ID NO:141); ENO1 terminator (ENO1t; SEQ ID NO:142); hexokinase 1 terminator (HXK1t; SEQ ID NO:143); PGK1 terminator (PGK1t; SEQ ID NO:144); PGM1 terminator (PGM1t; SEQ ID NO:145); PYK1 terminator (PYK1t; SEQ ID NO:146); RPLA terminator (RPLAt: SEQ ID NO:147); transaldolase 1 terminator (TAL1t; SEQ ID NO:148); TDH3 terminator (TDH3t; SEQ ID NO:149); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:150); triosephosphate isomerase 1 terminator (TPI1t; SEQ ID NO:151); and MpTEF1 (SEQ ID NO:289).PT-1755-WO-PCT

[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 arabitol 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 arabitol. The fermentation method can include additional steps, as would be understood by a person skilled in the art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and isolating the arabitol from the fermentation broth. The fermentation process may be a fully aerobic or a partially aerobic process.

[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 inPT-1755-WO-PCT 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 0.5 to 40, 1 to 35, 2 to 30, 3 to 25, 4 to 20, or 5 to 15 mmol O2 / (L • h). In some embodiments, the specific OUR can be in the range of 0.05 to 10, 0.1 to 8, 0.15 to 5, 0.2 to 1, or 0.3 to 0.75 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. The initial dextrose concentration may be between 100 to 400, 150 to 350, or 250 to 325 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 arabitol production rate of the process may be at least at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L-1h-1. The arabitol mass yield of the process may be at least 25, at least 30, at least 35, at least 40, at least 50, at least 55 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, or at least 85 percent. The final arabitol titer of the process may be at least 5, 10, 20, 30, 50, 75, or 100 g / L.

[0128] 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.PT-1755-WO-PCT EXAMPLES

[0129] 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.

[0130] Throughout the Examples, strain numbering and sequence identification numbers are used consistently. For example, strain 1-1 in Example 4 is the same as strain 1-1 in Example 5, etc. Example 1: Xylitol-Phosphate Dehydrogenase Diversity

[0131] Roughly three thousand galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol- phosphate dehydrogenase (XPDH) enzyme sequences were obtained from Uniprot and analyzed. FIG.2 illustrates the natural sequence diversity for this set of sequences. This set is diverse, with ~25% of the enzymes having no homologue more than 75% identical. As these enzymes tend to prefer NAD to NADP as a cofactor, the cofactor binding preferences of the homologs were assessed in a manner similar to that described by Duax et al., (“Rational proteomics I. Fingerprinting identification and cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme family,” Proteins, 2003, 53(4):931-943). Cofactor binding pockets were identified by proximity to the Rossman fold (+23 to +30 amino acids from the GXGXXG motif (SEQ ID NO:129)) and scored on the basis of total charge in an 8-residue window. The top 8 candidates that were predicted to use NADP were selected for further characterization, along with 4 candidates predicted to use NAD, and 3 controls.

[0132] Upon further review of the structural characteristics of the predicted binding pocket for factors that may influence cofactor preference, an important aspartate residue was identified. See FIG. 3. The polypeptide of SEQ ID NO:34 and substitutions thereof were used to construct a structural homology model to predict cofactor binding pocket confirmations FIG.3 shows the C- terminal end of the penultimate β-strand on the outside of the Rossman Fold domain. Without wishing to be bound by any particular theory, it is predicted that enzymes in which the first residue in this region (residue 198 relative to SEQ ID NO:34) is an aspartate and the second residue (residue 199 relative to SEQ ID NO:34) is a large hydrophobic amino acid (for example, isoleucine) will prefer an NAD cofactor due to the hydrogen bonding of the aspartate to the hydroxyl groups of the NAD ribose. However, enzymes in which that first residue (residue 198PT-1755-WO-PCT relative to SEQ ID NO:34) is an alanine, glycine, or serine and the second residue (residue 199 relative to SEQ ID NO:34) is lysine or arginine will prefer an NADP cofactor as the positive charge on the lysine or arginine residue will interact with the negative charge of the phosphate of the NADP and the smaller residue in the first position allows space in the binding pocket for said phosphate. Based on this analysis, 12 additional enzymes were selected for their predicted preference for NADP. Finally, 6 additional enzymes with sequence similarity to active XPDH enzymes were selected for testing. Example 2: TarJ’ Diversity

[0133] Roughly eight hundred ribulose 5-phosphate reductase sequences were obtained from Uniprot and analyzed. FIG. 3 illustrates the natural sequence diversity for this set of sequences. Overall, the diversity in this set is low, as only 10% of the enzymes have no sequence similarity more than 75% identical. As these enzymes tend to prefer NADP to NAD as a cofactor, no scoring was performed, and the sequences were simply aligned in Geneious (ClustalW, default settings). Eight enzymes were selected for further analysis based on sequence similarity. Example 3: In vitro Enzyme Assays

[0134] Polynucleotides encoding suspected XPDH homologs (Table 2) or TarJ’ homologs (Table 3) were cloned into a vector containing a T7 promoter and terminator for cell-free protein expression (New England Biolabs, PURExpress® In Vitro Protein Synthesis). Cell-free synthesized proteins were analyzed for activity on four substrates (ribulose 5-phosphate, xylulose 5-phosphate, ribulose, and xylulose) with either NADP or NAD cofactors. Seven enzymes (XPDH of SEQ ID NOs:12 and 34, TarJ’ of SEQ ID NOs:36, 37, 38, 40, and 42), were able to catalyze the reduction of either ribulose 5-phosphate or xylulose 5-phosphate (FIG.5) but not the reduction of xylulose or ribulose (data not shown). Table 2: XPDH Homologs Source Organism Gene Name / Accession Numbers Polypeptide O:PT-1755-WO-PCT Acidobacteria bacterium DMG39_01315 4 Spirochaetaceae bacterium DCP56_03760 5Table 3: TarJ’ Homologs Source Organism Gene Name / Accession Numbers Polypeptide O:PT-1755-WO-PCT Pradoshia sp. D12 F7984_17520 37 Lactobacillus plantarum EGD-AQ4 N692_02285 38

[0135] 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 / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) on March 28, 1997 under number MUCL40385. Table 4 below lists various Moniliella pollinis strains, including information on the parent strain, the sequence with which the parent strain was transformed, and characterizations of the expression cassette(s) contained on the transformed sequence. Each “XPDH / TarJ’ Homolog Expression Cassette” contained, in order, a 5’ ER1 flanking sequence (SEQ ID NO:85), a MpPYK1 promoter (SEQ ID NO:86), a gene encoding the indicated XPDH or TarJ’ homolog (one of SEQ ID NOs:87-128), a Mp6PGD terminator (SEQ ID NO:140), and a 5’ portion of a G418 resistance gene expression cassette (SEQ ID NO:175). Each “Selectable Marker Cassette” contained, in order, a 3’ portion of a G418 resistance gene expression cassette (SEQ ID NO:172), an MpTEF2 terminator (SEQ ID NO:150), and a 3’ ER1 flanking sequence (SEQ ID NO:160). Upon bipartite transformation with both the XPDH / TarJ’ Homolog Expression Cassette and the Selectable Marker Cassette, the two cassettes recombine for integration of both the nucleotide sequence encoding the XPDH or TarJ’ homolog and the G418 resistance marker at the ER1 locus.

[0136] The indicated Moniliella pollinis parent strain was transformed with the indicated sequence(s) 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 CaCl2, 10 mM Tris-HCl, pH 7.5). BipartitePT-1755-WO-PCT transformations were prepared by adding 100 µg single stranded salmon sperm DNA and 1.5 to 5 µg each of the 5’ and 3’ DNA transformation fragments (3-10 µg total; see Table 4 for list of fragments) to approximately 200 μL 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 ºC, 100 rpm, for 16 to 24 hours. Following the incubation, protoplasts were pelleted by centrifugation and resuspended in 1 mL PBS.

[0137] The resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30-35 ºC for at least 2-4 days until transformants grew. Resulting transformants were evaluated by colony PCR for integration of the indicated sequence. 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 1-2 has 5 sister isolates, strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.

[0138] For example, Strain 1-1 was transformed with SEQ ID NO:43 and SEQ ID NO:44. SEQ ID NO:43 contains (i) 3’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:162), and (ii) a 3’ portion of the G418 resistance gene selectable marker (SEQ ID NO:172). SEQ ID NO:44 contains (i) an expression cassette for the XPDH homolog from M. sediminis, SEQ ID NO:87 encoding the amino acid sequence of SEQ ID NO:1, under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; (ii) 5’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:85); and (iii) a 5’ portion of the G418 resistance gene selectable marker (SEQ ID NO:175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30-35 ºC 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 the indicated sequence. PCR verified isolates were designated strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.PT-1755-WO-PCT Table 4. Selectable Marker XPDH / TarJ’ Homolog Expression Cassette CassettePT-1755-WO-PCT 28 (Alkalihalobacillus ligniniphilus XPDHExample 5: Shake Flask Fermentation Assay

[0139] Strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, 1-39a-f, 1-42a-f, 1-13a-f, and 1-15a-f (outlined in Table 4 above), were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.PT-1755-WO-PCT

[0140] 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.

[0141] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 6 and FIGS.6 and 7. Table 5: Production Medium Component Concentration (units)PT-1755-WO-PCT Choline-Cl 100 (mg / L) Antifoam CF-32 1.00 (g / L)

[0142] While PCRnucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-35a, 1-37a-d, 1- 38a-c, 1-39d-f, 1-42a-b, 1-42d, 1-13a-b, 1-13d-e, 1-15b-c, and 1-15e-f include the transformed polynucleotide sequence, but it is not integrated at the ER1 locus. Table 6: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Gl Ribit l X lit l Er thrit l Gl r l Eth n lPT-1755-WO-PCT 1-39f 0.8569 91.5653 11.1004 24.3341 6.1712 37.6166 1-42a 53.2303 1.3617 0.1586 62.1601 16.1305 44.7835 Example 6: Shake Flask Fermentation Assay

[0143] Strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e (outlined in Table 4 above), were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0144] 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.PT-1755-WO-PCT

[0145] A 250 ml non-baffled flask containing production medium (Table 5) 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 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 7 and FIG.8.

[0146] As seen in FIG. 8, while sister strains 1-34c and 1-34d produced 15.8 and 18.6 g / L xylitol, respectively, strains 1-34a, 1-34b, and 1-34e did not produce significantly more xylitol than wild-type (strain 1-1, FIG. 6). While strains 1-34a, 1-34b, and 1-34e were initially PCR verified, it was later determined that the integrated polynucleotide, which should encode the N. cucumis XPDH homolog, contained a frameshift mutation and no functional XPDH was expressed. Therefore, while the results appear varied, they are in fact consistent given that strains 1-34a, 1-34b, and 1-34e did not contain a polynucleotide that encoded a functional XPDH. Table 7: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) StrainPT-1755-WO-PCT 1-34e 92.4856 0.434 0.22 47.7303 9.2374 32.5853

[0147] Strains 1-13c, 1-17a-e, 1-18a-e, 19a-e, 1-21a-e, 1-22a-e, 1-23a-e, 1-24a-e, 1-25a-e, and 1-27a-d (outlined in Table 4 above), were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0148] 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.

[0149] A 250 ml non-baffled flask containing production medium (Table 5) 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 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 8. Table 8: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) StrainPT-1755-WO-PCT 1-18c 88.21 0.6902 0.0625 59.74 14.7915 31.5115 1-18d 76.4267 0.8183 0.0692 57.1505 16.2282 34.0999PT-1755-WO-PCT 1-25e 78.0893 0.6945 0.0792 61.3866 15.5101 31.7923 1-27a 54.0049 0.8002 0.0653 73.3789 15.1766 36.3078

[0150] Strains 1-13c, 1-3a-e, 1-10a-e, 1-11a-e, 1-12a-e, 1-14a-e, 1-16a-e, 1-28a-e, and 1-2a-e (outlined in Table 4 above), were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0151] 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.

[0152] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 9 and FIG.9.

[0153] While PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strain 1-16b-e includes the transformed polynucleotide sequence, but it is not at the ER1 locus. Further analysis was inconclusive on the integration location in strains 1-2c and 1-2d.PT-1755-WO-PCT Table 9: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Glucose Ribitol Xylitol Erythritol Glycerol Ethanol Arabitol 5 6 8 9 1PT-1755-WO-PCT 1-16d 45.5429 5.6345 22.6578 36.7881 11.2977 41.0565 1-16e 41.996 2.3924 19.2866 43.8223 17.3556 40.5198 Exap y

[0154] Strains 1-13c, 1-8a-d, 1-26a-e, 1-36a-e, 1-41a-e, 1-40a-e, and 1-20a-e (outlined in Table 4 above), were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0155] 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.

[0156] A 250 ml non-baffled flask containing production medium (Table 5) 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 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 10 and FIG.10.

[0157] While PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence wasPT-1755-WO-PCT not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-8c, 1-8d and 1- 41c include the transformed polynucleotide sequence, but it is not integrated at the ER1 locus. Further analysis was inconclusive on integration locus in strains 1-36a, 1-41b, 1-41e, and 1-20a- e. Table 10: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Glucose Ribitol Xylitol Erythritol Glycerol EthanolPT-1755-WO-PCT 1-40e 38.1811 32.5265 0.2004 31.8564 10.647 51.5381 1-20a 43.2545 0.7591 0.0973 60.0985 13.8425 44.7316

[0158] Strains 1-13c, 1-30a-e, 1-31a-e, 1-32a-e, 1-4a-e, 1-5a-e, 1-6a-e, 1-7a-e, and 1-9a-e (outlined in Table 4 above), were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0159] 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.

[0160] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 11 and FIG.11.

[0161] While PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-30c and 1-30d include the transformed polynucleotide sequence, but it is not integrated at the ER1 locus. Further analysis was inconclusive on the integration locus in strain 1-6c.PT-1755-WO-PCT Table 11: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Glucose Ribitol Xylitol Erythritol Glycerol EthanolPT-1755-WO-PCT 1-6c 62.2098 0.4975 0.1159 50.7104 13.912 46.5117 1-6d 88.3493 0.392 0.0963 46.7061 15.7906 39.0682 Ea pe : wo opy o e on e a po n s a s

[0162] Strain 1-1 was transformed as described in Example 4 with SEQ ID NO:55 and SEQ ID NO:177. SEQ ID NO:177 contains (i) a 3’ portion of the G418 selectable marker (SEQ ID NO:172); ii) an expression cassette for the XPDH homolog from Clostridium difficile, SEQ ID NO:98 encoding the amino acid sequence of SEQ ID NO:12, under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (iii) a 3’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:162). SEQ ID NO:55 contains i) a 5’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:85), ii) an expression cassette for the XPDH homolog from Clostridium difficile, SEQ ID NO:98 encoding the amino acid sequence of SEQ ID NO:12, under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (iii) a 5’ portion of the G418 selectable marker (SEQ ID NO:175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 ºC 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 C. difficile XPDH sequence. PCR verified isolates were designated strains 2-1a, 2-1b, 2-1c, 2-1d, and 2-1e.

[0163] Strain 1-1 was transformed as described in Example 4 with SEQ ID NO:57 and SEQ ID NO:178. SEQ ID NO:178 contains (i) a 3’ portion of the G418 selectable marker (SEQ IDPT-1755-WO-PCT NO:172); ii) an expression cassette for the XPDH homolog from Lactobacillus rhamnosus, SEQ ID NO:100 encoding the amino acid sequence of SEQ ID NO:14, under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (iii) a 3’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:162). SEQ ID NO:57 contains (i) 5’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:85); (ii) an expression cassette for the XPDH homolog from Lactobacillus rhamnosus, SEQ ID NO:100 encoding the amino acid sequence of SEQ ID NO:14, under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (iii) a 5’ portion of the G418 selectable marker (SEQ ID NO:175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 ºC 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. PCR verified isolates were designated strains 2-2a, 2-2b, 2-2c, 2-2d, and 2-2e.

[0164] Strains 1-13c, 2-1a-e, and 2-2a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0165] 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.

[0166] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 12.

[0167] While PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence wasPT-1755-WO-PCT not correctly targeted to the ER1 locus. Further analysis indicated that strain 2-2e includes the transformed polynucleotide sequence, but it is not targeted to the ER1 locus. Table 12: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Glucose Ribitol Xylitol Erythritol Glycerol Ethanol Example 12: Genetically Modified Moniliella pollinis Strains

[0168] Strain 1-1 was transformed with NO:186 and SEQ ID NO:187 as described in Example 4. SEQ ID NO:186 contained (i) 5’DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:85), (ii) an MpPYK1 promoter (SEQ ID NO:86), (iii) a gene encoding the Staphylococcus aureus xylitol dehydrogenase of SEQ ID NO:34, (iv) an Mp6PGD terminator (SEQ ID NO:140), and (v) a 5’ portion of a G418 resistance gene expression cassette (SEQ ID NO:175). SEQ ID NO:187 contained (i) a 3’ portion of a G418 resistance gene expression cassette (SEQ ID NO: 172), (ii) an MpTEF2 terminator (SEQ ID NO:150), (iii) an MpPGK1 promoter (SEQ ID NO:135), (iv) a gene encoding the Saccharomyces cerevisiae DOG1 sugar phosphatase of SEQ ID NO:188, (v) an MpENO1 terminator (SEQ ID NO:142), and (vi) 3’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:162).Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 ºC for at least 2 days until transformants grew. Resulting transformants were streaked for single colony isolation on PDA + geneticin (G418) plates and single colonies werePT-1755-WO-PCT selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate were designated strain 3-1.

[0169] Table 13 below lists various Moniliella pollinis strains, including information on the parent strain, the sequence with which the parent strain was transformed, and characterizations of the expression cassette(s) contained on the transformed sequence. The transformation fragment of SEQ ID NO:181 contained, in order, a 5’ ER3 flanking sequence (SEQ ID NO:155), a MpPYK1 promoter (SEQ ID NO:86), a gene encoding the M. pollinis RPE2 polypeptide of SEQ ID NO:180, a MpPYK terminator (SEQ ID NO:146), and a 5’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:169). The transformation fragment of SEQ ID NO:182 contained, in order, a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:168), an MpTEF2 terminator (SEQ ID NO:150), and a 3’ ER3 flanking sequence (SEQ ID NO:165). The transformation fragment of SEQ ID NO:183 contained, in order, a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:168), a Mp6PGD promoter (SEQ ID NO:130), a gene encoding the M. pollinis RPE1 polypeptide of SEQ ID NO:179, an Mp6PGD terminator (SEQ ID NO:140), and a 3’ ER3 flanking sequence (SEQ ID NO:165). The transformation fragment of SEQ ID NO:184 contained, in order, a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:168), a Mp6PGD promoter (SEQ ID NO:130), a gene encoding the M. pollinis RPE2 polypeptide od SEQ ID NO:180, an Mp6PDG terminator (SEQ ID NO:140), and a 3’ ER3 flanking sequence (SEQ ID NO:165). The transformation fragment of SEQ ID NO:185 contained, in order, a 5’ ER3 flanking sequence (SEQ ID NO:155), a MpTEF1 promoter (SEQ ID NO:133), and a 5’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:169).

[0170] The indicated Moniliella pollinis parent strain was transformed with the indicated sequence(s) as described in Example 4 using either zeocin or G418 selection corresponding to the indicated selection marker. Resulting transformants were evaluated by colony PCR for integration of the indicated sequence. 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 3-2 has 4 sister isolates, strains 3-2a, 3-2b, 3-2c, and 3-2d (collectively 3-2a-d).PT-1755-WO-PCT Table 13. First Bipartite Cassette Second Bipartite Cassette Encoded EncodedExample 13: Shake Flask Fermentation Assay

[0171] Strains 1-1, 3-1, 3-2a-c, 3-3a-c, 3-4a-c, 3-5a-c, and 3-6a-c were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, and ethanol production.

[0172] 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 Genesys20PT-1755-WO-PCT spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0173] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 14 and FIGS.14 and 15. Table 14: 96 Hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Gl Ribit l X lit l Er thrit l Gl r l Eth n lPT-1755-WO-PCT Example 14: Shake Flask Fermentation Assay

[0174] Strains 1-1, 1-13c, 1-15a, 3-7a-f, and 3-8a-f, were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, erythritol, and ethanol production.

[0175] 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.

[0176] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 15 and FIGS.12 and 13. Table 15: 96 Hour Shake Flask Results (g / L) Strain Glucose Ribitol Xylitol Erythritol Glycerol EthanolPT-1755-WO-PCT 1-13c 60.56 3.20 35.39 25.66 16.35 38.60 1-13c 59.43 3.27 35.70 26.11 16.39 37.46 Exam

[0177] Strains 1-13c, 1-29c, 1-34d, 1-30b, 1-31d, 1-32d, 1-14d, 1-16a, 2-1c, 2-2b, 3-8b, 3-9a- d, 3-10a-e, 3-11a-e, 3-12a-e, 3-13a-e, 3-14a-e, 3-15a-e, 3-16a-e, and 3-17a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, glycerol, erythritol, and ethanol production.

[0178] 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.

[0179] A 250 ml non-baffled flask containing production medium (Table 5) 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Tables 16-18 and FIGS.16-21.

[0180] Yield was calculated as the ratio of product (i.e., xylitol) to consumed substrate (i.e., the difference between one time point and a second time point) expressed as a percentage.PT-1755-WO-PCT Table 16: Shake Flask Yield (48-96 hours) Yield (%) Yield (%) ll l l43 61 60 19 80 02 12 91 25 13 35 47 25 08 61 67 29 62 95 79 14 42 10 58 54 73 50PT-1755-WO-PCT Yield (%) Yield (%) lonaSthtE1- 5 15.6 1- 5 15.4 3- 4 14.4 3- 3 13.3 3- 8 14.1 3- 3 15.4 3-16e 5.746 5.071 0.366 13.030 3-17e 23.978 6.848 2.443 16.01lor lecStylGt1- 78 0.4 1- 91 0.4 3 76 0.4 3 72 0.4 1- 04 0.51-29c 3.370 0.620 0.427 0.093 0.409 3-11b 3.119 0.782 0.192 0.077 0.434 3-9a 3.093 0.821 0.245 0.107 0.429 3-11c 3.020 0.778 0.158 0.081 0.419 3-9b 2.781 0.746 0.109 0.150 0.416 3-11d 3.024 0.749 0.154 0.077 0.457 3-9c 3.094 0.759 0.184 0.082 0.495 3-11e 2.811 0.711 0.129 0.107 0.411 3-9d 3.135 0.747 0.182 0.089 0.508 2-2b 3.332 0.630 0.382 0.073 0.457 1-34d 2.960 0.504 0.396 0.068 0.410 2-2b 3.431 0.631 0.397 0.077 0.426 1-34d 2.992 0.511 0.424 0.072 0.398 3-12a 2.596 0.675 0.145 0.050 0.312 3-10a 2.722 0.625 0.226 0.070 0.378 3-12b 2.888 0.779 0.159 0.105 0.443 3-10b 2.792 0.646 0.224 0.067 0.396 3-12c 2.905 0.823 0.186 0.084 0.417 3-10c 2.667 0.605 0.132 0.067 0.408 3-12d 3.012 0.851 0.169 0.083 0.420PT-1755-WO-PCT Rate (g / (L•h)) Rate (g / (L•h)) elsol loltcoitr orouilht ec naStrainlGy yXr yEl hGtE3-12e 2.9720.7810.156 0.101 0.45 1-30b 3.0810.4970.316 0.064 0.40 1-30b 2.9390.4860.300 0.064 0.45 3-13a 2.7480.5250.180 0.078 0.42 3-13b 2.9580.6830.139 0.078 0.48 3-13c 2.9400.5820.148 0.083 0.46 3-13d 2.5600.3900.269 0.073 0.348- . . . . . 3-13e 2.9830.5960.187 0.066 0.487 3-16a 3.8480.2170.189 0.013 0.554 1-13c 3.4940.6550.397 0.070 0.466 1-13c 3.4000.6460.383 0.064 0.44 1-31d 3.1370.4290.492 0.072 0.42 1-31d 3.0410.4240.455 0.068 0.44 3-14a 2.7640.4990.266 0.077 0.38 3-14b 3.2750.5920.275 0.083 0.45 3-14c 3.0040.6580.240 0.090 0.42 3-14d 2.8600.5440.234 0.084 0.37 3-14e 2.9190.5370.237 0.086 0.40 1-32d 3.3970.6670.320 0.066 0.501-32d 3.6700.7210.463 0.085 0.402 3-17e 3.1430.7540.215 0.077 0.50300.79. 04. 44. 86. 90. 69. 12. 95. 34. 543. 040. 137. 105. 47.stluseRksalFekahS:81elbaTan 9643729316134 1 6033009 477584A h4t.5.2.2.2.9. 38. 43.9. 080.3. 6.6.0o 8 8 4 4 9 8 6 4 4.40 06 6.7r1 1Bn2 17 43 9 794 1 3 2 6 7o0 02i7 129736109717 037504448609539392tat5.8.n8 8 .0.1011.7 0.7 5..8091.8 6.88.15.56.8 0.8.8emre1872 6 1458 7 668 83 2 692 45F25. 00510222177611212121 3935306321 420.0.0.0.0.0.0.0.0.0.0.0.0 2.0.0105 2 10612940272514932624138 953857.7 8939915 5 7.1860580779 3357..6. . . . . .0604151204 401.461.6.4.6.8402 2 2 2 202122 122 0202020228 8 8 8 8 8 8 8 8 8 8 8 84 4 4 4 4 4 4 4 4 4 4 4 48484c3c131b8bc c8929 a9b9c9d9 d4d4a0b0c0-1-1-3-3-21-1-3-3-3-3 3-3 11-1-1 13-3-3PT-1755-WO-PCT71492261957526458956878 6 2 6 5 1 9 5 2 838 7 7 0 4 2 1 0 9 97.138026.011060.612397.917369.06038.431279502.815128.8312.97.5 1.38.8 4.4 4.5 9.4 9.4 7. 54.00. 5 2 9 8 28.7. 9 7 2101.4.4.4.4.40101.6.5.660 5954 1104082 6 9 451 58158 5 1 1 6 7 63 9 734057 4 5 4 7 6 2 8 6 2 4 9 3 8 9 01.7.9.2.1. 8.2.3.2.0 74. 84. 4.12. 0.6.28. 72106861507 6 8 9 9010101 1 8 801 91131 8.6.6.6.9.7238 7 6 9 9 8 8 5 5 5 4 9 4 121993567154683910413868801998539 7 68394806 728883.10.4 4 10.0.0.0 1.00.0 0.10.0 5.2 4.22.0 5.20.0 2.40.0 8.0 8.10.0 14.10.0417036896 607275915 3 3 1 1 5 7 1 4 6 5 80. 9. 8. 09.8. 5. 9. 73.90.74 0 8 2 4 8 9 9 5 12 9 1 66. 0 6 5 5 6 5. 04. 30. 29. 80. 87. 9048351933217 8 704 7 7 9 9 1 3 4 8 7 5.0. .1. .8222020222 1212120291021212120212062910220202848484848484848484848484848484848484848484d0e a c e a c e10-1c1c11b1111d11111b2b22b12 2d12 2b b a10 0 3b c13 313-3-2-2-3-3-3-3-3-2-2-13-3-13-3-3 33-1-1-13-3-3PT-1755-WO-PCT6895656859 431119647 956 585 42 8 8 2 5 910 7 5 1 7 23.937696.314947.226565.611281.26271.20127917.421364.119967.141.6 0.8 0. 98.2. 8 4 8 5 8 3 5.1 6 0 2 5 5 4 5131.7.7.6.7.7.7 8.4.4.4.4.4.5.5.533331646788953721742 9606 5 3 8 1 5 4 3 64 9 6 1 7 6 5 6 1 0 4 783850 2 4 6 1 4 85.4.3.5.9.0.8.7.7.0.9.1.9.1. 33. 33. 44. 16. 37. 37975 7 8 8 4 5 5 5 8 7 6 8 7 9 8 9 9 9 3.3.28 1 8 5 5 4 23614 6 5 3 6 2 3 578807479791 8 1 6 9.275957 8 815918 65153850036213199740733240.0.0.0 4.0 4.0.0.0 1.0.0 1.0 5.0 6.10.0 2.10.0 0.10.0 .4.14.1512157535164787358628240188229637 7 6 3 5 90. 5 4 2 8 9 7 8 0 4 0 4 6 8 734839 6 5 48 2 4 9 0 3 3 4 5 4 0 3 4 7 5 0 090231 46. . . . . . . . . . . . . . . . . .5.5.801010 1 8 3 5 9 3 4 3 0 4 3 4 6 1 1 1 21 2 2120291121291020212123222222222020202848484848484848484848484848484848484848484d3e c c d a c e a c e a13-131311d3134b1414d1414d d1232 5b15 5d15 5d d14 4 613-3-1-1-1-1-3-3-3-3-3-31-1-13-3-13-3-1 13-1-1-3PT-1755-WO-PCT387 6 1 6 9 4 699 8 9 5 9 3 6 947 7 1 3 9 8 6 4 4 2 4 45.547948.73023.415196.99306.84675.27275607.32633.74174.6 3.5 7.5 8.5 2.8 4.8 4.5 3.6 0.5 8.5 8..8442 .4 6. .8. .5. 57.5.20101829281.93131621 9 77 3 9 1 6 8 4 9 8801. 884. 6420. 259 90. 168. 361. 0561 34929704614420576436939320.7. 01. 48. 65. 0.2.6.6.9.8.9.5.3.5 5.3 2 3 3 5 6 6 7 8 7 77373848463637464 4446856 2. 92 685085396715939953713 532223 98058 00 47 0 3 9 7 8 5 7 3 622 3 6 0 4 5827.15 91.5 6. 7.161 0 7.20.0 1.10.0 1.10.70.82.2 0.1 0.01.14.54.40.0 6.0 6.0447883283598 1 5 6 5 1 1 3 9 5 8 6 7 9 7 12. 59351446587 0 3 3 8722233 8 5250635806420721584740.92.17.98.93.22.25.130.37 0.328.20 43.3 0.9.8.6.6.1.0.4.4.6366 7 2 2 6 7 6 61 2 1 1 2 2 2 2 2 2 27 7 4 4 6 7 6 6848484848484848484848469696969696969696969b6c e a a a c e c16d-16 6161617b17 7d17 713c131b8bc c89292a9b9c9d93-13-3-3-1-1-13-3-13-3-3-1-1-3-3-1-1-3-3-3-3PT-1755-WO-PCT455 3 7 5 9 7 8 3134 5 52888 2 142.63964.11549.92948.33404.3959.450216202.045039.019920.79.5.2037.3.1713.7.1327.12. 5222. 69.54.12.9.1210 8. .18292 .1 8.9.1113.12 71.1116 7 5 4 5 6 2 9 1 2 5 1 3 3 3.30256516359048045304258792771894493125 38598936.50.1.3.6.4.3.1.2.9 4.5.2.3.4 7.7.8.6.5.4.2.383937363638383 3847464 4838324640545647 7 1 9 2 74 86951102193139389175563 72750 40 356663 44 464120889.2 2.03.1 7.70.0 6.51.40.42.2 4.0 72.30.30. 004. 70.3 4. 3. 5303141 1.2. 4136.31.2628 2 1 2 6 2 2 6 7 3 1 1 2 8 3 5 3 7105. 0830 2 6 3 8 4 8 3 5 3 5 7 1 1 7 9 0 5434 2.1. 7 1 6 3 3 2 9 4 8 6 2 8 0 9 8 9 0 363.4 7.6 2.9.7.2.7.3.6.4.6.7.9.7.3.4.5.1.6 6 7 7 707982636974627274793638857972637696969696969696969696969696969696969696969d4d a c e a c e a c e34-30b1010d10101c1c11b1111d11 11b2b22b12 2d12 211-1-3-3-3-3-3-2-2-3-3-13-3-3-2-2-13-3-13-3-3PT-1755-WO-PCT987 3 6 3 2 824449387183051288655126475129.33277.2145.627.23410.4298.840211086.14402.219140..5.3.3.6.3.0. .5.9.5.6.3.7. .7.7.6.7.5625251213142517262634302028181912203215.12172 419 652846388744510712120534 897583158231485335331.0 3.09.2 9.1 9.4 2.14.86.59.9 5.45. 058. 691. 824. 101. 0703.2 2. 605. 64. 86. 90.3 2 3 4 3 2 3 3 3 2 2 2 3 4 3 3 424845484626 481688 1 8 4 5 8 682 7 6 9 4 4 5 14 6063704247962 7 3 131 9 66.3 5.33. 1078.50. 7073.32. 141. 633. 137.61. 914. 1048.40. 7037.300.0 6. .2323. 4001.41.012 1 5 7 5 1 6 3 2 7 8 1 6 2 42 8. 78744642925233970 1.42748781421414787281241519428782 8 42133050079825 .5.59. . .606763676 .2. . . . . . . . . . . .6.474052508855566360543261656696969696969696969696969696969696969696969b0b a c e c c a c e a c30-33b1313d13131313d d113134b14 4d14 4d d12 2 5b15 511-1-3-3-3-3-3-1-1-1-1-13-3-13-3-3 33-1-1-13-3-3PT-1755-WO-PCT62651685399892678 4 8 732 204 6 0 8 2 0 06.048825.114202.21100.753232.05445.7302133460. 2 4 9 1 6 77 27 7 64 5.1 2.46 8.46 1.9.7.3. .4 4 4 4 46.2 1.9.9.4 32 7.1.37 9.48 8 6 74 42 53637322353865811955384 91196141 8 4 7 10752.3.2389980157638 7 708323005 4682550111.7.6.6.8.5.8 2.7 .0.10.12.12.11.11.111751521502023981 9 3 2 9 1 1 4 24 7 6 55 1 5 9 7 6 9 2 41.42.15 6.3. 26. 7141414 0. 54. 01515 4. 71. 21415 6. 111 4. 532 3. 730 2. 79. 27. 12.2915161514356 2728057 932 26027508 191 3830.49. 11. 47. 02. 213.0. 273.5. 53. 44. 208. 88. 370. 68. 37.4059181313121213143430444744434283 7280679 08179175 5 5388385884243378 463 4 767 6 91.7 .00.016 .0 6.1.0.6.9. 2 0 2.514244368617962607.4.4 1.0.0.0.050608881483 3 8 9 3 1 9 6 8 19 2 7 191 926279336238266678 95.9 2.9.5.8.835.9 5 8 8 5 7 36905594913271.98.12.25.14.52.52.71.82.79.79769696969696969696969696969696969d5e15d4d4a6b6c6d e6a6a6a7b c7d e7-13-1 1 13-1-1-1 163 -3-1 13-3-13-11-171 171 11-3-3-3-3-3PT-1755-WO-PCT Example 16: Phosphatase Diversity

[0181] 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 of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae,” J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the enzyme class of sorbitol-6-phosphatases (EC 3.1.3.50). As xylitol 5-phosphate is a similar molecule to the known substrates of PYP1, an ortholog of PYP1 in Moniliella is likely responsible for the final catalytic step in the pathway for xylitol production. A query of the Moniliella pollinis genome of strain 1-1 found two genes with high homology to PYP1; RCSR00371 and RCSR15215. These two genes are 40% and 38% identical to ScPYP1, respectively, and 89% identical to each other.

[0182] 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). HxpA and PYP1 share low sequence similarity, and therefore the M. pollinis genome was searched for HxpA orthologs as alternative phosphatase candidates. RCSR21016 was identified as 38% identical to HxpA and independently identified as a sugar phosphatase with sequence similarity to S. cerevisiae DOG1 and DOG2. As shown in Examples 12-15, S. cerevisiae DOG1 and DOG2 expression increases xylitol production in M. pollinis.

[0183] Based on enzyme classification and / or sequence identity to the M. pollinis RCSR00371, RCSR15215, and RCSR21016 phosphatases and the S. cerevisiae PYP1 phosphatase, 26 additional phosphatase candidates were chosen for further analysis, as outlined in Table 19. Table 19: Phosphatase Source Organism Gene Name / Accession Numbers PolypeptidePT-1755-WO-PCT Moniliella pollinis RCSR21016 200 Saccharomyces cerevisiae PYP1 201Example 17: Genetically Modified Moniliella pollinis Strains

[0184] The indicated Moniliella strain was transformed with the bipartitetransformation fragments as indicated using the transformation method outlined in Example 4. Resulting transformants were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was then designated as the indicated strain number in Table 20. In some instances, more than one PCR verified isolate, e.g., “sister” isolates, arePT-1755-WO-PCT indicated by letters following the strain number. For example, strain 4-1 has 5 sister isolates, strains 4-1a, 4-1b, 4-1c, 4-1d, and 4-1e.

[0185] For example, Strain 2-2b was transformed with SEQ ID NO:223 and SEQ ID NO:181. SEQ ID NO:181 is described in Example 12 and contains, in order, a 5’ ER3 flanking sequence (SEQ ID NO:155), a MpPYK1 promoter (SEQ ID NO:86), a gene encoding the M. pollinis RPE2 polypeptide of SEQ ID NO:180, a MpPYK terminator (SEQ ID NO:146), and a 5’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:169). SEQ ID NO:223 contains a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:168), a MpPGK1 promoter (SEQ ID NO:135), a gene (SEQ ID NO:224) encoding the S. cerevisiae PYP1 polypeptide of SEQ ID NO:201, a Mp6PGD terminator (SEQ ID NO:140), and a 3’ ER3 flanking sequence (SEQ ID NO:165). Transformants were selected on PDA + zeocin selection plates and incubated at 35 ºC 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 4-1a, 4-1b, 4-1c, 4-1d, and 4-1e.

[0186] Transformation cassettes of the phosphatase homologs outlined in Table 20 have the same components as SEQ ID NO:223 above but included the indicated nucleotide sequence encoding the indicated polypeptide sequence. Table 20. Phosphatase Homolog Expression Cassette Second Bipartite Cassette e O:PT-1755-WO-PCT 5-1a-e 1-1 223 224 201 185 na 5-2a-e 1-1 225 226 198 185 naPT-1755-WO-PCT 5-33 5-34a-cExample 18: Shake Flask Fermentation Assay

[0187] Strains 2-2b, 1-14d, 3-12c, 3-12d, 4-1a-e, 4-2a-e, 4-3a-e, 4-4a-e, 4-5a-e, 4-6a-b, 4-7a- e, and 4-8a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0188] 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.

[0189] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol,PT-1755-WO-PCT glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results are reported in Table 21 and FIGS.22 and 23. Results from the 96-hour time point are not shown as some of the reactions consumed the entire glycerol feedstock leading to inaccurate estimates of reaction rate. Results demonstrate that overexpression of the X5PP enzymes of SEQ ID NOs:198, 199, and 200 increases xylitol titers relative to the parent strain 2- 2b and to the control strain expressing RPE and XPDH but lacking an X5PP (strains 3-12c and 3- 12d). Results also show that expression of the exogenous X5PP enzyme of SEQ ID NO:201 also increased xylitol titer relative to strains 2-2b, 3-12c, and 3-12d.PT-1755-WO-PCTPT-1755-WO-PCT93.5267.6143.3308.8442.520.362785.3140.5105.2121. 27 2. 015 6. 228 7. 013 1. 527 8. 23. 24. 25.29181313152 1. 4. 28. 35. 08. 12. 46. 68. 20. 78.69336323131311112121 0. 458.92 0 3 8 0 5 5 7 8 8 8 3 4 0 1818029043482.2.3.5.5.4.2.2.9.2 1 1 1 1 4 . . . . .1.1 0 1 0 0 0 0 0 0.3.0.0.0.0.032622232512363. 04 6. 947. 760. 331. 358. 154. 613. 025. 544. 690. 286. 545. 269. 081. 69. 49. 10. 47. 77. 33.9 9 9 8 8 8 9 9 78 4 6 0 9 1 41 1 1 1 1 1 1 1 1716151815161413151417141848484848484848484848484848484848484848484a2b2c2d2e2a3b3c3d3e3a4b4c4d4e4a b c d e a- - - - - - - - - -5 5 5 5 5 64 4 4 4 4 4 4 4 4 4-4-4-4-4-4-4-4-4-4-4-4PT-1755-WO-PCT22.5393.3147.1206.6283.8662 1 1 6 9428. 1.7 0.7 6.4 2.5 6.9 98. 2. 4.4 12. 44.3 2 3 1 2 3 5012133552727272727272727272727175 5 4 0 8 7 6 8 9.4 3.0 4.9 9.4 0.4 0.9 0.2 9.2 01. 4.2 1.2 2 1 2 2 1 2 2 5 25144. 3285.54194996042 301 517.18.13.16. . .1715141 8..541 9.821. 942.10 561 42.. 591 5.52101 1. 3. 57.4190101 6. 5.8301 0.70 5 3 9 1 4438 2 59.7 8 5 0 7 .4.429.8.8.6.5.5.6.54141.4210138. 87. 67. 18. 26. 73. 8573 5360772129182629.16.1781..37.10161. 921 6 45.6 3.40.1. 19. 85. 61. 64. 40. 55.4 6 764943706850 0 41 1 1 1 1 1 1 16261028484848484848484848484b6a b c d e a b c d e-7-7-7-7-7-8 8 8 8 84 4 4 4 4 4-4-4-4-4-4PT-1755-WO-PCT

[0190] The top xylitol producing strain(s) for each phosphatase enzymes were sequenced. All strains contained two copies of the L. rhamnosus XPDH as expected; however, there was variability in the gene copy number for the phosphatase and RPE2, as outlined in Table 22. Table 22: Gene copy number Strain Phosphatase SEQ ID NO: Phosphatase gene copy number RPE2 gene copy number 4-4c 200 5 about 7p y

[0191] Strains 1-1, 5-1a-e, 5-2a-e, 5-3a-e, and 5-4a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, 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 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the final 96-hour time point are reported in Tables 23 and 24. Results from the 24-, 48-, and 72-hour time points are not shown. Results demonstrate that the phosphatases of SEQ ID NOs:198-201 do not have the same effect on erythritol production that they do on xylitol production.PT-1755-WO-PCT Table 23: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) Strain Glucose Ribitol Xylitol Erythritol Glycerol EthanolTable 24: Erythritol Yield (%) Average ErythritolPT-1755-WO-PCT 5-1a 32.2 5-1b 32.4Example 20: Shake Flask Fermentation Assay

[0194] Strains 2-2b, 4-1a, 4-1c, 4-4b, 4-4c, 5-1a-e, 5-2a-e, 5-3a-e, and 5-4a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0195] 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 Genesys20PT-1755-WO-PCT spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15-20 in about 32-50 hours.

[0196] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the final 96-hour time point are reported in Tables 25 and 26. Results from the 24-, 48-, and 72-hour time points are not shown. Table 25: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) St i Gl Ribit l X lit l E th it l Gl l Eth lPT-1755-WO-PCT Table 26: 96-hour Shake Flask Yield Average Xylitol Strain Description Xylitol )

[0197] This example demonstrates shake flask results comparing two X5PP enzymes (SEQ ID NOs:200 & 201) with and without expression of the RPE of SEQ ID NO:180. The average xylitol yields in strains containing an overexpressed X5PP but no overexpressed RPE had yields of 14% and 18.3% with phosphatases of SEQ ID Nos:200 and 201, respectively, in comparison to 11.7%PT-1755-WO-PCT yield in the control strain (see Table 26). These yields are significantly lower than the yields of 33.7% and 37.7% seen in strains containing both the X5PP and RPE expression. Example 21: Shake Flask Fermentation Assay

[0198] Strains 1-13c, 1-15a, 1-29c, 1-32d, 1-16a, 3-7a, 3-8b, 3-9c, 3-15d, 3-17d, 5-7a-e, 5-8a- e, 5-9a-e, 5-10a-e, and 5-11a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0199] 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.

[0200] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the final 96-hour time point are reported in Tables 27 and 28. Results from the 24-, 48-, and 72-hour time points are not shown. Table 27: 96-hour Shake Flask Results Fermentation Broth Analyte (g / L) lPT-1755-WO-PCT 1-32d 67.72 2.53 29.81 36.53 21.10 16.27 1-32d 68.42 2.51 29.26 36.25 20.95 16.63PT-1755-WO-PCT 5-11d 0.99 0.46 87.05 27.17 14.00 23.53 5-11e 0.13 1.21 83.35 27.06 10.80 27.4896-hour XPDH SEQ RPE SEQ ID Phosphatase Average Strain Xylitol ldPT-1755-WO-PCT 5-9a 26.5 5-9b 28.4

[0201] This shake flask fermentation assay looks at combinations of the phosphatase of SEQ ID NO:200 with overexpression of RPE2 (SEQ ID NO:180) and 5 different XPDH enzymes. Table 28 shows the xylitol yields for the strains tested. Significant yield increases were evident for all of the XPDH candidates tested in conjunction with overexpression of RPE2 and the X5PP enzyme of SEQ ID NO:200. Strains with the XPDHs of SEQ ID NOs:14 and 15 had the highest 96-hour xylitol yields with 27% and 29.5% respectively. Strains tested in this Example all have 1 copy of the indicated XPDH, while strain 2-2b and its progeny have 2 copies of the sequence encoding the XPDH of SEQ ID NO:14, potentially explaining why strains 4-4b and 4-4c have an average yield of 37.7% (see Table 26). Example 22: Shake Flask Fermentation Assay

[0202] Strains 2-2b, 3-12d, 4-4c, 5-12a-e, 5-13a-d, 5-14a-e, 5-15a-e, 5-16a-e, 5-17a-e, 5-18a- e, 5-19a-d, and 5-20a-d were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0203] 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 48-72PT-1755-WO-PCT 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.

[0204] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the 48- and 72-hour time point are reported in Table 29. Results from the 24- and 96-hour time points are not shown. Titers at the 24-hour time point are low, and after 96 hours, many of the cultures included low or no concentrations of glucose. There was an error in the running of the shake flask with strain 5-15d so no results are reported for this strain. Results for the 72-hour time point are also shown in FIGS.24 and 25.)L / g(etylanAhtorBnoitatnemreF)L / g(etylan 4 1 9 9 2 6A29. 34 86 06 63 7 7 0 8 3 9 2. . . .6 7.4 5.8 5.7 2.0 5.2 1.2 98. 2.3 0.9.6.hto 8 1 2 1 1 1 2 2 1 2 2 4 2423271rBn3o853908304129141390 4 4 4 2 2 4it. . . . . . . . .2.2.1.2.1.1.7.a2 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1tne6 9 3 8 1mr 5.1 8.7 2.6 1.2 93.6 10.3 44.8 50.1 63.3 84.1 99.11.72.83.73.0.eF819102919103 0 3 6 8 227181029181328191918188 8 8 8 8 8 84 4 4 4 4 4 4 48484848484848484bd2 ca2b2c2d2e2a3b3c3d a b c d2-1-4-1-1 1 1 1 1 1 131414141412 3 4 5-5-5-5-5-5-5-5-5-5-5-5-5PT-1755-WO-PCT89. 543.242534.6 9.17159. 59 9.27354. 113.63314. 13 4.984. 558.4652727273.3 3.15144. 65 7.16163. 641.131552518203352 07954215071 20019177 5 8 4.1.1.0.0.8.16..1.6. . . 9 . . .0.6.6.487.2 1 2 3 1 2 6 1 2825242.0026202626181.91208. 7 00 1.2 2. 301. 100. 813. 601. 228. 522. 801. 603. 701. 107. 808. 101. 002. 302. 107. 105. 904. 122.021. 45 60.0 09.4 46 7 3 6 5 6 9 7 5 2 4 6 4 4 6 7 8.4 6.7 6.4 8.0 9.5 5.1 0.2 1.8 0.2 5.0 1.2 8.8.0.0.8.4.9.9 9 8 8 8 0 1 8 9 8 89 8 6 3 6 3 41 1 1 1 1 2 2 1 1 1 191818191919191819181848484848484848484848484848484848484848484e4a5b5c5e5a6b6c6d6e6a7b7c7d e a b c d e a1 1 1 1 1 1 1 1 17 7 8 8 8 8 8 9- - - - - -1 1 1 1 1 1 1 1 1 1 1 15 5 5 5 5 5-5-5-5-5-5-5-5-5-5-5-5-5-5-5-5PT-1755-WO-PCT27. 303.262450.8 4.18184. 82 1.28269. 00. 79. 20. 8890853 9 7. . .5 5 60266566666. 71 1. 031. 820. 254. 622. 227.107. 77. 00. 77. 10. 05782 2 3 0. .6 4 4 974745527272727272727019 8 1 0 4 7.5 4.5.5.7.9.2.121414131312120. 58 0. 553. 557. 48. 85. 98.1 1 122414131447 8 4 2 6 6.9 7.9 9.9 9.9 5.9 6.9 9.7020006 631140.8.7.18..1.5.1 1 2 6 2020235. 80 7. 105. 601. 116. 605. 504.084. 58 41.0 13.3 79.3 56.6 99.5 8.8 0 9 9 921 2 1 1 1910284848484848484b9a9d a b c d19 0 0 0 0-1 1 2 2 2 25-5-5-5-5-5-5PT-1755-WO-PCT

[0205] As shown in FIGS.24 and 25, and the results shown in Table 29, strains expressing the X5PP enzymes of SEQ ID NOs:201-209 demonstrated increased titers of xylitol compared to the control strain 2-2b as well as a strain expressing RPE (3-12d). Strains expressing the X5PP enzymes of SEQ ID NOs:201-207 also had at least one sister isolate with xylitol titers equivalent to or above the xylitol titer of strain 4-4c, which expresses the X5PP of SEQ ID NO:200. There is also sister to sister variability in several of the strains assayed. As demonstrated in several examples above, while a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, 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, but the results do demonstrate the effectiveness of the indicated X5PP enzymes.

[0206] For example, while PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further whole genome sequencing analysis indicated that for strains 5-12a-e, 5-15a-c, and 5-15d, the sequence was integrated in more than one copy and / or was not correctly integrated at the ER3 locus. Whole genome sequencing results for these strains are reported below in Table 30. Results show the sister to sister variability is likely due to differences in gene copy number (of the XPDH, RPE2, and / or the phosphatase genes) and integration loci, but even with copy number and integration variability the effectiveness of the indicated X5PP enzymes is consistent. Table 30. Phosphatase geneDIDIies E2PT-1755-WO-PCT The construct was designed to insert the sequence encoding SEQ ID NO:202 at of 1 ut ed he t rst a al e se ePT-1755-WO-PCT Example 23: Shake Flask Fermentation Assay

[0207] Strains 2-2b, 4-4b, 4-4c, 5-21a-c, 5-22, 5-23a-c, 5-24a-b, 5-25a-e, 5-26a-d, 5-27, 5-28a- d, 5-29, 5-30a-b, 5-31a-b, 5-32a-c, and 5-33 were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0208] 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.

[0209] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the 48- and 72-hour time point are reported in Table 31. Results from the 24- and 96-hour time points are not shown. Titers at the 24-hour time point are low, and after 96 hours, many of the cultures included low or no concentrations of glucose. Results from the 72- hour time point are also shown in FIGS.26 and 27.0 2 2 4 1 8 6 2 8 4 6 2 3 3) / (etlatritatere) / (etylan 117 9A. 8h1 3. 019. 296.9 6.4 07.6 6. 8740.4. 115.3. 404 9.8.6.t1 1 3 3 3 3 363920292725242orB0n 7277 6 0 0 6 9 6 8 2 8 3 7o . .1.1.1.1.1.3.6.1.5.1 2.2.it 3 3 0 0 0 0 0 0 1 0 0.0 0 0atne4mr 3. 6 7 0 0857166 830 221 16173 6.0 5.1 9. . . .4. .9. .6. . .e 6 6 44407373594531883079 9F1 1 1 1 1 1 1 1 1 1 1 151518484848484848484848484848484bb b b c ca1b1c1a b c a2-2-4-4 4 4 2 2 222323232422 2 4-4-4-4-5-5-5-5-5-5-5-5PT-1755-WO-PCT90.6237.3227.5169.7447.030.672773.6104.0227.7198317 7 4971666355 7 5 5 3 1 5 8.6. . 510131.5 7.. . . .8.0.5.7.4.7.5.7.67614273020432113232528192 4.99203232 4 0 4 6 9 7 2 5 1 4 5 0 2 3.0.2.0 3.1 3.0 4.0 6.1 1.0 9.1 0.0 2.0 1.0 1.0 2.0 1.0 2.0 2.0 3.187. 088 1 3 74.9 8.32.4 9.60881.7.7 2. 4240.6. 29. 90. 01. 81. 29. 14. 68. 69.7161811281714152153159192101263166151199144191848484848484848484848484848484848484b4a5b5c5d5e5a6b6c6d6 7a8b8c8d8a b2 2 2 2 2 2 2 2 2 2 29 0 0- - - - - - -2 2 2 2 2 3 35 5 5 5 5 5 5-5-5-5-5-5-5-5-5-5-5-5PT-1755-WO-PCT15.4261.8173.5317. 82 4. 7453422745.76.31.24.2436 9 46 528.6 1.0 7..3.401101 0.124. 7 8 218 .4 1.2 1.627.3. 76 21186560127272727272732629778998.1.4.4.7.0.2 2 1 1916155. 99 5. 1688896115.18.10.28.10225. 446 97.3121 28..7.874161 1.720. 21 3. 403.5 496. 5. 41.2 3 1 801419292 02 7 1 4. .0 3.0 4.3 6.3 4.061. 64 3. 915. 322. 167. 75.4 4 85 11 1 1615181848484848484a1b1a b c32 2 2 3-3 3 3 3 35-5-5-5-5-5PT-1755-WO-PCT

[0210] As shown in FIGS.26 and 27, and the results shown in Table 31, strains expressing the X5PP enzymes of SEQ ID NOs:210, 211, 213, 214, and 189 demonstrated increased titers of xylitol compared to the control strain 2-2b. Strains expressing the X5PP enzymes of SEQ ID NOs:210 and 213 also had at least one sister isolate with xylitol titers equivalent to or above the xylitol titer of strains 4-4b and 4-4c, which expresses the X5PP of SEQ ID NO:200. There is also sister to sister variability in several of the strains assayed. As demonstrated in several examples above, while a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, 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, but the results do demonstrate the effectiveness of the indicated X5PP enzymes. Example 24: Shake Flask Fermentation Assay

[0211] Strains 2-2b, 4-4b, 4-4c, 5-34a-c, 5-35a-c, 5-36a-b, 5-37a-c, 5-38, 5-39a-b, 5-40a-c, 5- 41a-b, 5-42a-d, 5-43a-f, 5-44a-b, 5-45a-d, 5-46, and 5-47a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0212] 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.

[0213] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the 48- and 72-hour time point are reported in Table 32. Results from the 24- and 96-hour time points are not shown. Titers at the 24-hour time point are low, and after 96 hours, many of the cultures included low or no concentrations of glucose. Results from the 72- hour time point are also shown in FIGS.28 and 29.) L / g( etylanA htorBnoitatns etlumrseeFRksalFekahS:23elbaT)L / g(etylan 42256 4 9 1 9 8 3 2 6A. .9.5.7.261.2.0.987 4 0.0.7 2.ht2o174939321.9014131.50.37.811171.831r Bn68it. 62. 31. 61. 82. 39. 34. 379650052947525307oa4 0 0 0 0 0 3.0.0.0.1.1.5.5.3.6tne25519172208 6 5 8 8 8 8 4 8 2 6mr. . . . .1.4.5.3.9.5.4.2.2.9.7.e94334626293037589938399 3 1 6 1F1 1 1 1 1 2 1 1 1 2 1915161714184848484848484848484848484848484bb c ca4b4c4a5b5c5a b a b c2 4 4 4 3 3 36 6 7 7 7 8-2-4-4-4-3 3 3 3 3 3 3 3 35-5-5-5-5-5-5-5-5-5-5-5PT-1755-WO-PCT03. 11 3.22235. 35 7.21228. 221.22349. 98 2.15203. 603.750.2 92 7.1982727701.3 4.17168. 01 1.20284. 20 2.161116 7 0979496213962 627 8 618 0 588265 95..27.8..0.7.7.8.3.04..74 4 . 3 1 . .1.3.7 1 6 8 1 1 1 1 1 1 8 2.5.621.9.64233331370. 4 3 5 81 9.2 6.1 5.2 1. 355. 504. 804. 204. 801. 425. 636. 602. 255. 435. 464. 413. 241. 003. 202. 202.04302 12802680860462 8 7 3 0 5 6 1 9 7 2 3 7 7 9. . . . . .1 7.8 0.5 5.2 5.1 3.6 7.6 4.0 1.8 9.2 6.4 5.6.6.8.8.0 6 1 7 5 8 7 7 9 8 7 6587 4 3 92 1 2 1 1 1 1 1 1 1 1 181614181 151515131848484848484848484848484848484848484848484a9b9a0b0c0a1b1a2b2c2d2a3b3c d e f a b a b3 3 4 4 4 4 4 4 43 3 3 3 4 4 5 5- - - - - -4 4 4 4 4 4 4 4 4 4 4 45 5 5 5 5 5-5-5-5-5-5-5-5-5-5-5-5-5-5-5-5PT-1755-WO-PCT69. 12 9.22287. 37 4.27298. 997.36360. 32 0.. . . . . .291532353233323647.3 2.4 2 4 4 7 11171.30.70.60.40.91.111 5 5 4 2 85. 3 2.3.5.0.2.3.6 9.34141411 5 67 8 1 1 12101012727272727272727620.7 7. 0635.4 9. 642. 145. 339. 942.1 1 1 1 1 1 15165. 03 6. 537. 323. 60. 83. 67. 76.2 2 2523222021265. 97 9.6 46. 6585096591.1 1 8.8.9.8.7012011 13 2 5 2 7. 1. 7.7 2.5 5.8 1.6 2.5 0.1 9 1 1 1 1 16152. 55 1. 143. 901. 504. 603. 507. 608.0430.4 13.0 87.4 69.7 48.3 80.1 97.7.6 7 7 7 71 41 1 1 1 18181718484848484848484c5d5 6a b c d e4 47 7 7 7 7-4 4 4 4 4 45-5-5-5-5-5-5-5PT-1755-WO-PCT

[0214] As shown in FIGS.28 and 29, and the results shown in Table 32, strains expressing the X5PP enzymes of SEQ ID NOs:221, 222, and 188 demonstrated increased titers of xylitol compared to the control strain 2-2b. None of the strains tested had xylitol titers equivalent to or above the xylitol titer of strains 4-4b and 4-4c, which expresses the X5PP of SEQ ID NO:200. There is also sister to sister variability in several of the strains assayed. As demonstrated in several examples above, while a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, 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, but the results do demonstrate the effectiveness of the indicated X5PP enzymes. Example 25: Shake Flask Fermentation Assay

[0215] Strains 4-4b, 4-4c, 5-12d, 5-12e, 5-14b, 5-14c, 5-15c, 5-16d, 5-16e, 5-17e, 5-21a, 5- 21b, 5-26b, and 5-26d were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0216] 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.

[0217] A 250 ml non-baffled flask containing 20mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the 48- and 72-hour time point are reported in Table 33. Results from the 24- and 96-hour time points are not shown. Titers at the 24-hour time point are low, and after 96 hours, many of the cultures included low or no concentrations of glucose. Results for the 72-hour time point are also shown in FIG.30.)L / g(etylanAhtorBnoitatnemreF)L / g(etylan 3 3 8 7 1 5A5.5 1.5 2.8 1.9 6.1 4. 619. 178. 664. 944. 989. 604. 17. 06. 93. 39.hto 3 3 2 2 3 3 2 2 3 2 30313921343rBn7o14111704141313 6 5 7 0 8 4 5 8it. . . . . . .0.1.3.1.1.1.4.1.0.a0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0tne243938805784670389642849671 1 1mr.8.6.9.8.5.3.6. . . . . .0.3.3.eF616193 1 2 1 6 7 7 4 519161718191718171817171717188 8 8 8 84 4 4 4 4 484848484848484848484bb c cd2e2b4c4c5d6e6e a b b d4-4-4-4-1 1 1 1 1 1 171121262624 4 4 4-5-5-5-5-5-5-5-5-5-5-5-5PT-1755-WO-PCT

[0218] The results of this example are consistent with the results of previous examples, demonstrating that strains expressing the X5PP enzymes of SEQ ID NOs:202, 203, 204, 205, 206, 210, and 213 produce xylitol titers that are roughly equivalent to or greater than the xylitol titers produced by control strains expressing the X5PP of SEQ ID NO:200. As demonstrated in several examples above, while a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, 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, but the results do demonstrate the effectiveness of the indicated X5PP enzymes. Example 26: Genetically Modified Moniliella pollinis Strains

[0219] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 3-12d was used to generate a 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 on the parent 3- 12d strain. The resulting low-foaming strain, containing two copies of an exogenous polynucleotide sequence encoding the XPDH of SEQ ID NO:14 integrated at the ER1 locus and one copy of a polynucleotide sequence encoding the RPE of SEQ ID NO:180 integrated at the ER3 locus, was designated 6-1.

[0220] Strain 6-1 was transformed with the Cre recombinase plasmid of SEQ ID NO:288 using the transformation method outlined in Example 4. The resulting transformants were evaluated by colony PCR for removal of the zeocin resistance selection marker. A PCR verified isolate was designated strain 6-2.

[0221] Strain 6-2 was grown non-selectively on YPD plates to allow for the loss of the plasmid of SEQ ID NO:288. Biomass was struck for single colonies and evaluated by PCR to confirm loss of the plasmid. A PCR verified isolate was designated strain 6-3.

[0222] The indicated Moniliella pollinis parent strain, 6-3, was transformed with the bipartite transformation fragments as indicated in Table 34 using the transformation method outlined in Example 4. Resulting transformants were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was then designated as the indicated strain number in Table 34. In some instances, more than one PCR verified isolate, e.g., “sister” isolates, arePT-1755-WO-PCT indicated by letters following the strain number. For example, strain 6-4 has 5 sister isolates, strains 6-4a, 6-4b, 6-4c, 6-4d, and 6-4e.

[0223] For example, Strain 6-3 was transformed with SEQ ID NO:280 and SEQ ID NO:279. SEQ ID NO:279 contains, in order, a 3’ portion of a G418 resistance gene expression cassette (SEQ ID NO:172), a MpTEF1 terminator (SEQ ID NO:289), and a 3’ gpdIIB flanking sequence (SEQ ID NO:166). SEQ ID NO:280 contains a 5’ gpdIIB flanking sequence (SEQ ID NO:158), a PGK1 promoter (SEQ ID NO:135), a gene (SEQ ID NO:230) encoding the Moniliella pollinis phosphatase polypeptide of SEQ ID NO:210, a TDH3 terminator (SEQ ID NO:149), and a 5’ portion of a G418 resistance gene expression cassette (SEQ ID NO:175). Transformants were selected on PDA + G418 selection plates and incubated at 35 ºC 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 6-4a, 6- 4b, 6-4c, 6-4d, and 6-4e.

[0224] The transformation cassettes of SEQ ID NOs:281-287 for the phosphatase homologs outlined in Table 34 have the same components as SEQ ID NO:280 but included the indicated nucleotide sequence encoding the indicated polypeptide sequence. SEQ ID NO:181 was described in Example 12 and contained, in order, a 5’ ER3 flanking sequence (SEQ ID NO:155), a MpPYK1 promoter (SEQ ID NO:86), a gene encoding the M. pollinis RPE2 polypeptide of SEQ ID NO:180, a MpPYK terminator (SEQ ID NO:146), and a 5’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:169). Table 34. Phosphatase Homolog Expression Cassette Second Bipartite CassettePT-1755-WO-PCT 6-9a-e 6-3 285 238 204 279 6-10a-e 6-3 286 232 202 279ted polynucleotides integrated and what the final copy number of the given genes are. The parent strain 6-3 included 1 copy of a polynucleotide encoding the RPE of SEQ ID NO:180 integrated at an ER3 loci on a single allele. In strain 6-12b, the second copy of the RPE encoding sequence integrated at the ER3 locus on the second allele resulting in a complete ER3 knockout. Strain 6- 12b also was confirmed to have two copies of SEQ ID NO:230 encoding the X5PP enzyme of SEQ ID NO:200. However, in strains 6-12a and 6-12c, the transformation resulted in replacement of previously integrated sequence with new sequences encoding both the RPE of SEQ ID NO:180 and the X5PP of SEQ ID NO:200. Example 27: Shake Flask Fermentation Assay

[0226] Strains 6-3, 6-4a-e, 6-5a-e, 6-6a-e, 6-7a-e, 6-8a-e, 6-9a-e, 6-10a-e, and 6-11a-e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0227] 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.

[0228] A 250 ml non-baffled flask containing 20 mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector.PT-1755-WO-PCT Fermentation results for the 48- and 72-hour time points are reported in Table 35. Results from the 24- and 96-hour time points are not shown. Titers at the 24-hour time point are low, and after 96 hours, many of the cultures included low or no concentrations of glucose. Results of the 72- hour time point are also shown in FIGS.31 and 32.

[0229] There was an error running the shake flask fermentation for strain 6-7b. While the results show little glucose consumption and low metabolite production, it’s likely this was due to an error in the fermentation set up and not a defect of the strain.) L / g( etylanAhtorBnoitatns etlumrseeFRksalFekahS:53elbaT )L / g(etylan 0A2. 97. 75. 75. 77. 66. 87. 12. 09. 29192111138362ht6 6 2 5 3. . . . . . .6 1 3 3 6 3 7 7 1 6 5o1 1 1 1 1 2 3 2 2 2 2 1 1 2 2 1r Bn7 6 8 4 1o4.0 4.0 3.0 4.0 42.0 24.0 21.23.24.23.24.26.45.24.22.4.ita0 0 0 0 0 0 0 0 0 0tne3mr 2. 559. 978. 389. 588. 24. 08. 37. 17. 87. 51. 95. 23. 87. 91. 84.e6 6 1 5 2 5 2 0 3 9 5 1F7171817181514161615161817161518184848484848484848484848484848484ab c d e a b c3- 3-4-4 4 4 4 5 5 5d5e5a6b6c6d66 6 6-6-6-6-6-6-6-6-6-6-6-6-6-6PT-1755-WO-PCT61. 72 5.22230. 74 0.35243. 867.11107. 17 7.35484. 833.1513. 1.95801272798. 91 3.12189. 746.29155. 57 7.561. 22.429. 58. 47. 84. 22. 092112951432016684706165800 39.. . . . . . . . . . . . .1 1 06272424382626292229232524281020202022754502024023020022022022 0 6 2 5 7 8 1 3 4 4 7.0.0. . . . . . .0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 3.0 3.0 3.0 3.0 3.013. 39 2. 56. 69. 09. 98. 82. 44. 9945945189624983627874790498 2 5 0 2 6 3.8.3.3.5.1.6.6.9.2.9.7.7.910203515161415151615161516151517161616161848484848484848484848484848484848484848484e a b c d e a b c d e a b c da b c d e6 7 7 7 7 7 8 8 8 8 8e 0 0 0 0 0- - - - - - -9 9 9 9 9 1 1 1 1 16 6 6 6 6 6 6-6-6-6-6-6-6-6-6-6-6-6-6-6-6PT-1755-WO-PCT21. 29 6.28240. 86 8.25245. 91. 24. 72. 1322223.1712269. 19 7. 571. 1 225 5.4.9 9 9 85964. 64. 67. 26440 0 0.0.046 98. 14 2. 4.4.687182 5.8272727272786. 952421849. . . .18102818132. 827. 07. 64. 39.27242036224. 19 0. 698. 5567.7 7.835. 568780281.39.20.38.282817 6 5 8.0 1.0 2.0 2.0 1.091. 6 5 9 00 0.1.8.2.561581551561518484848484a1b d e f11-11111116-6-6-6-6PT-1755-WO-PCT

[0230] The results demonstrate that the same increase in xylitol titer and yield can be achieved when the X5PP encoding sequence is integrated at the gpdIIB locus as was seen in previous examples where the sequence was integrated at the ER3 locus. As demonstrated in several examples above, while a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, 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, but the results do demonstrate the effectiveness of the indicated X5PP enzymes. Example 28: Shake Flask Fermentation Assay

[0231] Strains 6-3, 6-12a, 6-12b, 6-12c, 6-4d, 6-4e, 6-7d, 6-8a, 6-8e, 6-9b, 6-9d, 6-11a, 6-11b, 6-11c, 6-11d, 6-11e were run in shake flasks to assess glucose consumption as well as ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0232] 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.

[0233] A 250 ml non-baffled flask containing 20 mL production medium (Table 5) was inoculated with 0.4 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, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for the 48- and 72-hour time points are reported in Table 36. Results from the 24- and 96-hour time points are not shown. Titers at the 24-hour time point are low, and after 96 hours, many of the cultures included low or no concentrations of glucose. Results for the 72- hour time point are also shown in FIG.33.PT-1755-WO-PCT

[0234] There was an error running the shake flask fermentation for strain 6-7b. While the results show little glucose consumption and low metabolite production, it’s likely this was due to an error in the fermentation set up and not a defect of the strain.)L / g(etylanAhtorBnoitatnemreF)L / g(etylan 81854151462748461280082198698A.1.2.5.6.8.8.5.6. . . . . .8.hto 1 1 2 2 2 2 2 252521232133272rBn0o303417 6 6 2 4 2 4 6 1 5 4 4it. . .1.0.0.1.1.1.1.1.1 1.1.1a0 0 0 0 0 0 0 0 0 0 0.0 0 0.0tne96748227277490761 8 5 5 9 0 7mr.7.5. . . . . .3.9.9.6.8.1.1.e 9 99555469675459595763 8 3 2F1 1 1 1 1 1 1 1 1 1 161416151848484848484848484848484848484a32a2b2b2c2c2 de c-3-1-1-1 1 1 1 4 4 7d7a8e8b96 6 6 6-6-6-6-6-6-6-6-6-6-6-6PT-1755-WO-PCT881.6 4.252394.9 8.32322. 112.262829304976 9.1.2.1.8.2.7 9 978861995. 93. 525573140 0.0.0.0.090. 50 4. 804. 309. 065. 881.3 1 1 2 5012727272727272294 20794 5 9. . .5.2.0.1 17171718140. 50221537006. . . . .323232313237491.. 71.6 91080101 5.6 6..701403 0 8 0 5.9 7.9.4.8.9.1727292526212906161022.0.0.0.0.0 1.03956274 5 3.0.6.2.4.6.7 54 9 9 51 151515151848484848484da b c d e91-1111111116-6-6-6-6-6PT-1755-WO-PCT Example 29: Genetically Modified Moniliella pollinis Strains

[0235] The indicated Moniliella pollinis parent strain, 1-12a, was transformed with the bipartite transformation fragments as indicated in Table 37 using the transformation method outlined in Example 4. The transformation fragments of SEQ ID NOs:181, 182, 185, and 229 are described in previous examples. Resulting transformants were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was then designated as the indicated strain number in Table 37. In some instances, more than one PCR verified isolate, e.g., “sister” isolates, are indicated by letters following the strain number. For example, strain 7-1 has 5 sister isolates, strains 7-1a, 7-1b, 7-1c, 7-1d, and 7-1e. Table 37. First Bipartite Cassette Second Bipartite Cassette E d d E d d QExample 30: Shake Flask Fermentation Assay

[0236] Strains 1-12a, 7-1a-e, 7-2a-e, and 7-3a-e were run in shake flasks to assess glucose consumption as well as arabitol, ribitol, erythritol, glycerol, and ethanol production.

[0237] 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.

[0238] A 250 ml non-baffled flask containing 20 mL production medium (Table 5) was inoculated with 0.4 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,PT-1755-WO-PCT 48, 72, and 96 hours of incubation. Samples were analyzed for glucose, ribitol, arabitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with refractive index detector. Fermentation results for all time points are reported in Table 38. Data on arabitol yield is provided in Table 39. Data for the 72-hour time point is also shown in FIG.34.

[0239] There was an error running the shake flask fermentation for strain 7-1b. While the results show little glucose consumption and low metabolite production, it’s likely this was due to an error in the fermentation set up and not a defect of the strain.

[0240] Results demonstrate the overexpression of a native RPE enzyme and / or overexpression of a native A5PP enzyme increase both arabitol titer and arabitol yield relative to the parent strain. As demonstrated in several examples above, while a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, 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, but the results do demonstrate the effectiveness of the indicated RPE and A5PP enzymes.) L / g(etylanAhtorBnoitatnestmlruesFeRksalFekahS:83elbaT )L / g(etylanA86. 28. 73. 66. 905173520779632992252663hto3 3 2 0.2.2.2.2.2.2.3.1.3.1.1.2r Bn70it. 70. 30. 00. 20. 20. 20. 11. 50. 50702080203020oa0 0 0 0 0 0 0 0 0.0.0.0.0.0.0.0tne2mr 6. 79. 34. 168 7 4 4 7 0 4 5 5 0 6 1.8.7.8.6.4.0.6.5.9.2.7.5.e08082879282818782 3 2 5 9 1 8 9F2 2 2 2 2 2 2 2828282827292828244 4242424242424242 2 2424242424242a2a2 a b c d e a b c d e1 1 1 1 1 1 1 2 2a b d c-1-1-7-7-7-2 2 2 3 3 3 37-7-7-7-7-7-7-7-7-7-7PT-1755-WO-PCT053.7 6.22444. 55 1.22280. 95 8.151697.1 4.18700. 40 6.043.4951 0.08469090. 2.173464. 4.0231392.08282824493838383149223631460 3 9 7 2 5 9 0 1 2 7 7 0 0 793..1.0.44919122 78..2.3.9 6.3.6.4.4.9.0.1.6.2423252 8.9618151416171517141828. 42 7 2 4 1 9 1 9 6 8 3 8 9 5 2.1.9.554.0.8.0.2.2.0.3.0.5.3.1.4.1374753.16262727234546545228164430220. 80 3. 903. 001. 000. 000. 000. 000. 609. 533. 015. 200. 821. 020. 000. 304. 000. 000.062.7 47. 99. 8024.262959570861 7 9 27164 8 182412. 9 . . . . . .13492050505729171 3.3 5.6 1.. .55815 4.4 2.4422727272727272727272727272727272727ea a32-121a1b1c1d1e1a2b2c2d2e2a3b3d3c3e37-1-1-7-7-7-7-7-7-7-7-7-7-7-7-7-7-7PT-1755-WO-PCT Table 39: Arabitol Yield Strain Arabitol Yield % 1-12a 14.26 Example 31: Genetically Modified Moniliella pollinis strains Strain 8-1

[0241] Strain 6-12b was transformed with SEQ ID NO:290 using the transformation protocol outlined in Example 4 and positive transformants were selected using nourseothricin selection plates. SEQ ID NO:290 contains a deletion construct to remove one copy of the RSCR18717 gene. A PCR verified isolate, in which one copy of the RCSR18717 was knocked out, was designated strain 8-1. Strain 8-2

[0242] Strain 8-1 was transformed with SEQ ID NO:291 using the transformation protocol outlined in Example 4. SEQ ID NO:291 contains a construct to loop out the zeocin and nourseothricin resistance selection markers. A PCR verified isolate, in which the zeocin and nourseothricin resistance selection markers were removed, was designated strain 8-1.PT-1755-WO-PCT Strain 8-3

[0243] Strain 8-2 was transformed with SEQ ID NO:290 using the transformation protocol outlined in Example 4 and positive transformants were selected using nourseothricin selection plates. SEQ ID NO:299 contains deletion construct to remove one copy of the RSCR18717 gene. A PCR verified isolate, in which both copies of the RCSR18717 gene were knocked out, was designated strain 8-3. Strain 8-4

[0244] Strain 8-3 was transformed with SEQ ID NO:292 and SEQ ID NO:279 using the transformation protocol outlined in Example 4 and positive transformants were selected on geneticin (G418) selection plates. A PCR verified isolate, containing two copies of the gene encoding the X5PP of SEQ ID NO:200, was designated strain 8-4. Strain 8-5

[0245] Strain 8-4 was transformed with SEQ ID NO:293 using the transformation protocol outlined in Example 4. SEQ ID NO:293 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 8- 5. Strain 8-6

[0246] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 8-5 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 9-3 strain. The resulting strain with improved xylitol rate, which contained 2 copies of the gene encoding the XPDH of SEQ ID NO:14, 2 additional copies of the gene encoding the RPE of SEQ ID NO:180 (for 3 copies total), 3 additional copies of the gene encoding the X5PP of SEQ ID NO:200 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, was designated strain 8-6.PT-1755-WO-PCT Strain 8-7

[0247] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 8-6 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 9-4 strain. The resulting strain with improved xylitol rate, which contained 2 copies of the gene encoding the XPDH of SEQ ID NO:14, 2 additional copies of the gene encoding the RPE of SEQ ID NO:180 (for 3 copies total), 3 additional copies of the gene encoding the X5PP of SEQ ID NO:200 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, was designated strain 8-7. Strains 8-10 and 8-11

[0248] To test arabitol production in a xylitol producing strain with reduced erythritol production and increased rate, the polynucleotide encoding the XPDH of SEQ ID NO:14 at the ER1 was replaced with a polynucleotide sequence encoding the APDH of SEQ ID NO:11 as outlined in the strains below.

[0249] The parent strains were transformed with the transformation fragments as indicated in Table 40 using the transformation method outlined in Example 4. The remaining transformation fragments are outline below: a. SEQ ID NO:294 contained a 5’ ER1 flanking sequence (SEQ ID NO:85), an MpPYK1 promoter (SEQ ID NO:86), a gene encoding the APDH of SEQ ID NO:11, a Mp6PGD terminator (SEQ ID NO:140), and a 5’ portion of a nourseothricin resistance gene expression cassette (SEQ ID NO:170). b. SEQ ID NO:295 contained a 3’ portion of a nourseothricin resistance gene expression cassette (SEQ ID NO:171), an MpPYK1 promoter (SEQ ID NO:86), a gene encoding the APDH of SEQ ID NO:11, a Mp6PGD terminator (SEQ ID NO:140), and a 3’ ER1 flanking sequence (SEQ ID NO:162). c. SEQ ID NO:296 contained a 5’ ER1 flanking sequence (SEQ ID NO:85); an MpPYK1 promoter (SEQ ID NO:86); a nucleic acid sequence encoding the APDH of SEQ ID NO:11; an Mp6PGD terminator (SEQ ID NO:140); and a 5’ portion of a G418 resistance gene expression cassette (SEQ ID NO:175). d. SEQ ID NO:298 contained a 3’ portion of a G418 resistance gene expression cassette (SEQ ID NO:172) and a 3’ ER1 flanking sequence (SEQ ID NO:162).PT-1755-WO-PCT

[0250] Resulting transformants were evaluated by colony PCR for integration of the indicated sequence. A PCR verified isolate was then designated as the indicated strain number in Table 37. In some instances, more than one PCR verified isolate, e.g., “sister” isolates, are indicated by letters following the strain number. For example, strain 8-9 has 3 sister isolates, strains 8-9a, 8- 9b, and 8-9c. Table 40. First Bipartite Cassette Second Bipartite Cassette Encoded Encodedp y

[0251] Strains 8-9a-j, 8-10a-j, 1-12a, 7-3a, and 8-7 were run in shake flasks to assess glucose consumption as well as arabitol, xylitol, erythritol, glycerol, and ethanol production.

[0252] 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.

[0253] A 250 ml non-baffled flask containing 20 mL production medium (Table 5) was inoculated with 0.4 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, arabitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography (HPLC) with refractive index detector and results for the 96-hour time point are reported in Table 41 and FIG.35. Additionally, for strains 7-3a, 8-9h, 8-10b, 8-10c, and 8-10d, the samples collected at 96 hours were analyzedPT-1755-WO-PCT by high performance ion chromatography (HPIC) to resolve slight peak overlaps seen in the HPLC results and results are reported in Table 42 and FIG.36. Table 41. Strain Analyte Concentration (g / L) Arabitol Glucose Xylitol Erythritol Glycerol Ethanol ArabitolYieldPT-1755-WO-PCT Table 42. Strain Xylitol (g / L) Arabitol (g / L) 7-3a 1.08 84.72:14 at the ER1 locus was incomplete in most of the assayed strains. In general, the transformation using two copies of the sequence encoding the APDH of SEQ ID NO:11 (strains 8-10a-j) were able to produce arabitol, indicating at least partial replacement of the XPDH encoding sequence. Strains 8-9h and 8-10a-j all showed arabitol production, indicating expression of the APDH at a level that competes with the XPDH to produce less xylitol than parent strain 8-7. As demonstrated in the HPIC results strain 8-10c didn’t produce any xylitol and produced significantly more arabitol than control strain 7-3a.

Claims

PT-1755-WO-PCT CLAIMS What is claimed is:

1. A genetically engineered yeast cell capable of producing arabitol, the engineered yeast cell comprising: a genetic modification resulting in overexpression of a native enzyme with arabitol-5- phosphate phosphatase (A5PP) activity; and / or an exogenous polynucleotide sequence encoding an enzyme with arabitol-5-phosphate phosphatase (A5PP) activity.

2. The yeast cell of claim 1, wherein the yeast cell is an osmotolerant yeast cell.

3. The yeast cell of claim 1 or claim 2, wherein the yeast cell is a cell of the subphylum Ustilaginomycotina or Saccharomycotina.

4. 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 tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Saccharomyces cerevisiae, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium 5. The yeast cell of any preceding claim, wherein the cell is a Moniliella pollinis cell and the genetic modification results in overexpression of a native A5PP 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:198, 199, 200, or 221.

6. The yeast cell of any preceding claim, wherein the genetic modification comprise replacement of the native A5PP gene promoter with a heterologous or artificial promoter.

7. The yeast cell of claim 6, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130), glyceraldehyde-3-PT-1755-WO-PCT phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132), translational elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQ ID NO:131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p ; SEQ ID NO:136), asparagine synthetase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139).

8. The yeast cell of any preceding claim, wherein the genetic modification comprises addition of an exogenous polynucleotide sequence encoding the native A5PP enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding the native A5PP enzyme.

9. The yeast cell of any preceding claim, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with A5PP 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:200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188.

10. The yeast cell of any preceding claim, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with A5PP 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:200, 202, 203, 204, 205, 206, 210, and 213.

11. The yeast cell of any preceding claim, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme with A5PP 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:200, 203, 204, 206, and 213.

12. The yeast cell of any preceding claim, wherein A5PP activity in the genetically engineered yeast cell is higher than A5PP activity in an equivalent cell lacking the genetic modification or exogenous polynucleotide sequence.PT-1755-WO-PCT 13. 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 arabitol is increased relative to titer and / or yield of arabitol in an equivalent fermentation process using an equivalent cell lacking the genetic modification or exogenous polynucleotide sequence.

14. 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.

15. The yeast cell of claim 14, wherein the cell is 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:179 and 180.

16. The yeast cell of claim 14 or claim 15, wherein the genetic modification resulting in overexpression of a native RPE enzyme comprises replacement of the native RPE gene promoter with a heterologous or artificial promoter.

17. The yeast cell of any one of claims 14-16, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130), glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132), translational elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQ ID NO:131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p ; SEQ ID NO:136), asparagine synthetase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139).

18. The yeast cell of any one of claims 14-17, 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.PT-1755-WO-PCT 19. The yeast cell of any preceding claim, wherein the cell additionally comprises an exogenous polynucleotide sequence encoding an arabitol-phosphate dehydrogenase (APDH) 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:

11.

20. The yeast cell of any preceding claim, wherein the cell additionally comprise an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) 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:193, 194, 195, 196, or 197.

21. The yeast cell of any preceding claim, wherein the cell additionally comprise an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) 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:194, 195, 196, or 197.

22. 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.

23. The yeast cell of claim 22, wherein the promoter is a constitutive promoter.

24. The yeast cell of claim 22 or 23, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6- phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:130), glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:132), translational elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQ ID NO:131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p ; SEQ ID NO:136), asparagine synthetase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139).PT-1755-WO-PCT 25. 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 gpdIIA locus, and the gpdIIB locus.

26. A method for producing arabitol 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 arabitol.

27. The method of claim 26, 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 0.5 to 40, 1 to 35, 2 to 30, 3 to 25, 4 to 20, or 5 to 15 mmol O2 / (L • h).

28. The method of claim 26 or 27, wherein the arabitol 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.

29. The method of any one of claims 26-28, wherein the arabitol titer is at least at least 20, 30, 50, 75, or 100 g / L when the fermentation is run at 35 °C for 96 hours.

30. The method of any one of claims 26-29, wherein rate, titer, and / or yield of arabitol production is increased relative to an equivalent fermentation run with an equivalent yeast cell lacking the genetic modification to overexpress the A5PP enzyme and lacking an exogenous polynucleotide sequence encoding an exogenous A5PP enzyme.

31. The method of any one of claims 26-30, wherein the concentration of dextrose is at least 100 g / L.

32. Use of the engineered yeast of any one of claims 1-25 to produce arabitol.

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