Genetically modified yeast and fermentation process for production of polyols

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing polyols and characterized by overexpression of an enzyme having ribulose 5-phosphate epimerase (RPE) activity. For example, the cell may be a Paureopsis cell, and the natural RPE enzyme comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 179 and 180.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 364,382, filed on May 9, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listing submitted via Patent Center

[0004] The contents of the sequence listing XML file named "PT-1386-WO-PCT.xml", which was created on May 4, 2023 and electronically submitted with this application via the Patent Center, and is 512,801 bytes in size, are incorporated herein by reference in its entirety. Background Art

[0005] Xylitol is a low-calorie sweetener used as a food additive and sugar substitute. Xylitol, which is commonly used in medicines, dietary supplements, candies and toothpaste compositions, is also associated with anti-caries properties when used in chewing gum. The traditional methods of xylitol production (including chemical catalytic hydrogenation of xylose by xylan hydrolysis extracted from biomass) are expensive both monetarily and environmentally. These methods require high temperature and high pressure, a large amount of water and metal catalysts that must be mined. On the contrary, fermentation processes have been used commercially on a large scale to produce other organic molecules, such as ethanol, citric acid, lactic acid, etc., and can provide cost-effective and sustainable substitutes for traditional xylitol production methods.

[0006] In the development of the fermentation strategy based on microorganisms for the production of xylitol, the production of metabolic pathway intermediates and alternative fermentation products is an important consideration. For example, metabolic pathways activated in xylitol production may overlap with metabolic pathways for the production of arabitol, erythritol, ribitol, etc. Intermediates and products have their own uses and markets, which makes their fermentation commercially relevant. Therefore, this paper provides genetically modified yeast and fermentation methods for the production of polyols (including xylitol, arabitol and ribitol). Summary of the invention

[0007] The disclosure provides a genetically engineered yeast cell capable of producing a polyol, the engineered yeast cell comprising a genetic modification resulting in overexpression of a native enzyme with ribulose-5-phosphate epimerase (RPE) activity. The yeast cell can be an osmotic-resistant yeast cell. The yeast cell can be a cell of the subdivision Ustilaginomycotina. The yeast cell may be selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. The yeast cell may be a yeast cell of the genus Moniliella. The yeast cell can be a Moniliella pollinis cell, and the native RPE enzyme comprises a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 179 and 180. The RPE activity in the engineered yeast cell can be higher than the RPE activity in the equivalent cell lacking the genetic modification. When the engineered cell is used in a fermentation process in the presence of dextrose, the yield of polyols can be increased relative to the yield of polyols in the equivalent fermentation process using the equivalent cell lacking the genetic modification.

[0008] The genetic modification may include replacement of the native RPE gene promoter with a heterologous or artificial promoter. The heterologous promoter or artificial promoter can 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), translation 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 may comprise the addition of an exogenous polynucleotide encoding a native RPE enzyme such that the genetically engineered cell comprises at least one additional copy of a sequence encoding a native RPE enzyme.

[0009] The cell can be capable of producing ribitol and comprises an exogenous polynucleotide sequence encoding an enzyme having ribulose-5-phosphate reductase activity, the enzyme comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 13, 34, 35, 36, 37, 38 and 39.

[0010] The cell can be capable of producing xylitol and comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH), the XPDH enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31 and 33.

[0011] The cell may be capable of producing xylitol and comprises an exogenous polynucleotide sequence encoding a sugar phosphatase comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 188 and 189. The cell may further comprise an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 190, 191, and 192.

[0012] The cell can be capable of producing arabitol and comprises (i) an exogenous polynucleotide sequence encoding an arabitol-phosphate dehydrogenase (APDH) 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, and / or (ii) an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 193-197.

[0013] Any of the exogenous polynucleotide sequences may be operably linked to a heterologous promoter or an artificial promoter. The heterologous promoter or artificial promoter can 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), translation elongation factor 1 promoter (TEFp; SEQ ID NO:133), modified TEFp (SEQID 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 (SEQID NO:139). Any of the exogenous polynucleotide sequences may be integrated into the genome of the yeast cell at a locus selected from the group consisting of 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 polyols using engineered cells as described herein, the method including contacting a substrate comprising dextrose with engineered cells as described herein, wherein the fermentation of the engineered cells to the substrate produces polyols. The disclosure also provides a method for producing polyols (e.g., xylitol, arabitol, ribitol), the method including contacting a substrate comprising dextrose with an engineered yeast cell comprising a genetic modification resulting in overexpression of a natural enzyme with ribulose-5-phosphate epimerase (RPE) activity. The cell can be a spore cell, and the natural RPE enzyme comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical sequence to at least one of SEQ ID NO:179 and 180. The fermentation temperature can be 25°C to 45°C, 30°C to 40°C or 32°C to 37°C or between them. The volumetric oxygen uptake rate (OUR) may be between 0.5 mmol O2 / (L·h) and 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) and 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) and 30 mmol O2 / (L·h), 3 mmol O2 / (L·h) and 25 mmol O2 / (L·h), 4 mmol O2 / (L·h) and 20 mmol O2 / (L·h), or 5 mmol O2 / (L·h) and 15 mmol O2 / (L·h). The polyol may be at least 0.2 gL -1 h -1 , 0.3 g L -1 h -1 , 0.5 g L -1 h -1 , 0.75 g L -1 h -1 or at least 1.0 g L -1 h -1 When the fermentation is run at 35°C for 96 hours, the polyol production can be at least 20 g / L, 30 g / L, 50 g / L, 75 g / L or 100 g / L. The rate and / or yield of the polyol can be increased relative to an equivalent fermentation run with an equivalent yeast cell lacking the genetic modification. The concentration of dextrose can be at least 100 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] The drawings illustrate generally by way of example and not by way of limitation various aspects described herein.

[0017] Figure 1 The native pentose phosphate pathway (dashed line and arrows) and the native glycolytic pathway (solid line and arrows) in the yeast Mycorrhizal sp. are shown.

[0018] Figure 2 Diversity in the galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) sequence space is shown.

[0019] Figure 3 Structural features of the NAD or NADP binding pocket located at the +23 amino acids from the characteristic GXGXXG motif of the XPDH enzyme (SEQ ID NO: 133) are shown.

[0020] Figure 4 The diversity in ribulose-5-phosphate reductase sequence space is shown.

[0021] Figure 5 The in vitro activities of TarJ' and XPDH enzymes as outlined in Example 3 are shown.

[0022] Figure 6 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-1, 1-13a-f, and 1-15a-f as outlined in Example 5. Data markers report the concentration of xylitol (g / L).

[0023] Figure 7 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour 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 markers report the concentration of xylitol (g / L).

[0024] Figure 8 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e as outlined in Example 6. Data markers report the concentration of xylitol (g / L).

[0025] Fig. 9 Shown are the concentrations (g / L) of erythritol, ribitol, arabitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-13c, 1-12a-e, 1-14a-e, and 1-16a-e as outlined in Example 8. Data markers report the concentration (g / L) of xylitol (strains 1-13c, 1-14a-e, and 1-16a-e) or arabitol (strains 12a-e).

[0026] Fig.10 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains-13c, 1-36a-e, and 1-40a-e as outlined in Example 9. Data markers report the concentration of xylitol (g / L).

[0027] Fig.11 Shown are the concentrations of erythritol, ribitol, and xylitol metabolites (g / L) from 96 hour shake flask fermentations of strains 1-30a-e, 1-31a-e, 1-32a-e, and 1-13c as outlined in Example 10. Data markers report the concentration of xylitol (g / L).

[0028] Fig.12 Shown is 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.

[0029] Fig.13 The corresponding yields of xylitol, ribitol, glycerol and erythritol for strains with (3-8a-f) and without (1-15a) RPE2 overexpression are shown.

[0030] Fig.14 Xylitol and ribitol titers (g / L) are shown for the indicated strains.

[0031] Fig.15 The corresponding yields of xylitol, ribitol, glycerol and erythritol for the indicated strains with RPE overexpression of different configurations are shown.

[0032] Fig.16 Xylitol concentrations (g / L) over 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15 are shown.

[0033] Fig.17 Xylitol concentrations (g / L) over 96 hours of shake flask fermentations of the indicated strains as outlined in Example 15 are shown.

[0034] Fig.18 Xylitol rates (g / (L·h)) between 48 and 96 hour shake flask fermentations for the indicated strains as outlined in Example 15 are shown.

[0035] Fig.19 Xylitol rates (g / (L·h)) between 48 and 96 hour shake flask fermentations for the indicated strains as outlined in Example 15 are shown.

[0036] Fig. 20Xylitol yields (%) between 48 and 96 hour shake flask fermentations for the indicated strains as outlined in Example 15 are shown.

[0037] Fig.21 Xylitol yields (%) between 48 and 96 hour shake flask fermentations for the indicated strains as outlined in Example 15 are shown.

[0038] Fig. 22 Shown are erythritol, glycerol, and ethanol production over a 96-hour shake flask fermentation as outlined in Example 17. Data markers report erythritol concentration (g / L).

[0039] Fig.23 Shown are erythritol, xylitol, and glycerol production over a 96-hour shake flask fermentation as outlined in Example 18. Data markers report the concentration of xylitol (g / L). DETAILED DESCRIPTION

[0040] Reference will now be made in detail to certain aspects of the presently disclosed subject matter, examples of which are partially illustrated in the accompanying drawings.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0041] 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 non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of an inconsistency between the usages of this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0042] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

[0043] Unless expressly stated, ppm (parts per million), percentages and ratios are based on weight. Percentages based on weight are also referred to below as wt% or (wt)%.

[0044] The present disclosure relates to various recombinant cells engineered to produce polyols, and these polyols include but are not limited to xylitol, arabitol and ribitol. Generally, the recombinant cells described herein can produce polyols (e.g., xylitol, arabitol and / or ribitol), and are characterized by the overexpression of ribulose 5-phosphatase epimerase (RPE). The present disclosure also provides a fermentation method for producing the polyols by dextrose using the genetically engineered cells described herein.

[0045] In general, the recombinant cells described herein are yeast cells. As used herein, "yeast" refers to eukaryotic unicellular microorganisms classified as members of the kingdom Fungi. Yeast is a unicellular organism that evolved from a multicellular ancestor, some of which retain multicellular characteristics, such as forming connected strings of budding cells known as pseudohyphae or false hyphae. Yeast cells may also be referred to as yeast-like cells in the art, and as used herein, "yeast cells" encompass both yeast and yeast-like cells. Suitable yeast and yeast-like host cells for modification can include, but are not limited to, Saccharomyces cerevisiae, Komagataella sp., Kluyveromyces (e.g., Kluyveromyces lactis, Kluveromyces marxianus), oleaginous yeast, Issatchenkia orientalis, Pichia galeiformis, Pichia YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnolii, Candida ethanolica), Pichia deserticola, Pichia membranifadens, Pichia fermentans (e.g., fermentans), Aspergillus, Trichoderma, Myceliphthora thermophila, spores (e.g., spores yeast), Pfaffia, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., Trichosporonoides megafacies, Trichosporonoides globosum, Trichosporonoides nigrofacies), Pseudomonas tsukubaensis, Trichosporon variabilis, Penicillium, and Torula yeast. One of ordinary skill will appreciate that the requirements for selecting suitable yeast cells and recombinant yeast cells of the present disclosure are not limited to those explicitly listed herein. Methods for genetic engineering of yeast cells are known and described in the art, and one of ordinary skill will appreciate the methods necessary to transform and engineer suitable yeast cells.

[0046] Suitable yeast cells can be Basidiomycota and Ustilago. Suitable yeasts of the Ustilago 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 spores), Sporisorium spores. exsertum), spores (e.g., spores yeast, M. tomentosa, M. acetoabutans, M. fonsecae, M. madida, M. megachiliensis, M. ocedocephalis, M. nigrescens), and Pseudozyma (e.g., Pseudozyma tsukubae), and Trichosporon (e.g., Trichosporon megatus, Trichosporon sphericalus, Trichosporon nigrescens). Yeasts of the subdivision Ustilagomyces are known and described in the art as potential production organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol, as well as other valuable biotechnological applications.See, for example, Geiser et al. Biotechnol, 2013, 97(8): 3253-65), Guevarra et al. ("Accumulation of itaconic, 2-hydroxyparaconic, itataric, and malic acids by strains of the genus Ustilago, Agric.Biol.Chem., 1990, 54(9), 2353-2358) and Moon et al. ("Biotechnological productionof erythritol and its applications,” Appl Microbiol Biotechnol, 2010,86:1017-1025).

[0047] Suitable yeast cells will have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein, "active pentose phosphate pathway" refers to the expression of one or more functional enzymes that together convert glucose-6-phosphate, NADP + Or NAD+ and water are converted into NADPH or NADH, CO2 and ribulose-5-phosphate. Continuing in the non-oxidative stage, the pathway can also produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway can produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, combinations thereof, etc. The active pentose phosphate pathway can be natural to yeast cells or can be introduced into yeast cells by genetic engineering.

[0048] Yeast cells can be osmotic yeast cells. As used herein, "osmotic resistance" refers to yeast that can grow and reproduce under conditions of high molar osmotic pressure concentrations, such as at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v) or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), at least 15% (w / v) sodium chloride. The species and strains of osmotic yeasts are known and described in the art, including many types of yeasts used in industrial fermentation processes. Similarly, methods for determining yeast osmotic resistance are known and described in the art. See, for example, Tiwari, S. et al. ("Nectaryeast community of tropical flowering plants and assessment of theirosmotolerance and xylitol-producing potential," Current Microbiology, 2022, 79: 28).

[0049] The recombinant yeast cell can be a recombinant Myristica spp. cell, such as a Myristica spp. yeast cell. Figure 1 The predicted native pentose phosphate and glycolytic pathways in the yeast of the spores are shown. The spores have been previously used in the fermentation production of erythritol, and methods for genetic modification and fermentation of the spores 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).

[0050] Various plasmids and methods for transforming Mycelium are also described in the examples below. For example, Mycelium can be transformed using a bipartite polynucleotide sequence wherein, after recombination, the exogenous polynucleotide of interest is integrated at a specific locus and a selection marker is expressible in the cell. Suitable selection markers are known and used in the art. Selectable markers may include, but are not limited to, amdS (e.g., split into a 3' portion of SEQ ID NO: 167 and a 5' portion of SEQ ID NO: 174), a G418 resistance gene (e.g., split into a 3' portion of SEQ ID NO: 172 and a 5' portion of SEQ ID NO: 175), a zeocin resistance gene (e.g., split into a 3' portion of SEQ ID NO: 168 and a 5' portion of SEQ ID NO: 169), nourseothricin N-acetyltransferase (NAT) (e.g., split into a 3' portion of SEQ ID NO: 171 and a 5' portion of SEQ ID NO: 170), and an invertase gene (SUC2) (e.g., a 3' portion of SEQ ID NO: 173 and a 5' portion of SEQ ID NO: 176).

[0051] The recombinant cells described herein comprise one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve the fermentation of glucose to ribitol by the recombinant cell.

[0052] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless expressly stated otherwise.

[0053] As used herein, "exogenous" refers to genetic material or its expression product that originates from outside the host organism. For example, the exogenous genetic material or its expression product 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, when introduced into S. cerevisiae, the K. lactis invertase gene is exogenous.

[0054] As used herein, "native" refers to genetic material or its expression products found within the genome of a wild-type cell of a host cell, except for inter-individual mutations that do not affect function or expression. For the purposes of this application, the Moniliella tomentosa var pollinis TCV364 yeast cell described in US 6,440,712 is considered to be a wild-type Moniliella tomentosa yeast cell, which is incorporated herein by reference in its entirety and deposited 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) under the Budapest Treaty on March 28, 1997 under the number MUCL40385.

[0055] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the overall primary, secondary, tertiary and quaternary amino acid sequences and structures necessary to give the macromolecule its function and properties. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (either independently or as part of a biosynthetic pathway) is understood to include the cofactors, coenzymes and metals necessary for the enzyme to function properly. An overview of amino acids and their three letter and one letter symbols as understood in the art is provided in Table 1. The amino acid names, three letter symbols and one letter symbol are used interchangeably herein.

[0056] Table 1: Amino Acid Three-Letter and One-Letter Symbols

[0057]

[0058]

[0059] Variants or sequences having substantial identity or homology to the polypeptides described herein can be used to implement the disclosed recombinant cells, compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, the polypeptide sequence can be modified but still retains the ability to exhibit the desired activity. Typically, the variant or modified sequence can include greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the wild-type, naturally occurring polypeptide sequence or to a variant polypeptide as described herein.

[0060] 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. The generation of sequence alignments and sequence identities includes global alignments and local alignments performed using computational methods. Alignments can be performed using BLAST (National Center for Biological Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using standard protein BLAST with the following default parameters: maximum target sequence: 100; short query: automatically adjust parameters for short input sequences; expected threshold: 10; word length: 6; maximum number of matches within the query range: 0; matrix: BLOSUM62; gap penalty: (existence: 11, extension: 1); component adjustment: conditional component score matrix adjustment; filter: not selected; mask: not selected. The % nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within query range: 0; Match / mismatch score: 1, -2; Gap penalty: linear; Filter: Low complexity region; Mask: Mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently, to have XX% sequence identity with the reference sequence.

[0061] Polypeptide or polynucleotide sequence identity can be measured over the length of an entire defined polypeptide sequence (e.g., as defined by a particular SEQ ID number), or can be measured over a shorter length, for example, over the length of a fragment (e.g., 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 consecutive residues) taken from a larger defined polypeptide sequence. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown in this article, table, figure, or sequence listing can be used to describe the length over which a percentage identity can be measured.

[0062] The polypeptides disclosed herein may include "variant" polypeptides, "mutants" and "derivatives thereof". As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of the polypeptide in nature, as distinguished from a variant or mutant form. As used herein, a "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence that is different from a reference protein or polypeptide molecule. A variant or mutant may have an insertion, deletion or substitution of one or more amino acid residues relative to a reference molecule.

[0063] The amino acid sequences of polypeptide variants, mutants, derivatives or fragments 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 in which the amino acid is substituted with a different amino acid, wherein the substitution is predicted to interfere minimally with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially retain the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of the substitution, such 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 volume of the side chain.

[0064] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" and "nucleic acid" are used interchangeably and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. The DNA polynucleotide may be a cDNA (e.g., coding DNA) or a genomic DNA sequence (e.g., including introns and exons).

[0065] If in its natural state or when operated by methods known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof, then the polynucleotide is said to encode a polypeptide. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0066] Those skilled in the art understand that the degeneracy of genetic code and multiple polynucleotides can encode identical polypeptides.In some aspects, polynucleotides (for example, polynucleotides encoding RPE polypeptides) can be codon optimized to express in specific cells, and this specific cell includes but is not limited to plant cells, bacterial cells, fungal cells or animal cells.Although disclosed herein is a polypeptide encoded by a polynucleotide sequence found in various species, any polynucleotide sequence encoding the desired form of polypeptides described herein can be used.Therefore, non-naturally occurring sequences can be used.These sequences may be desirable, for example, to enhance the expression in the heterologous expression system of polypeptide or protein.Computer programs for the generation of degenerate coding sequences are available and can be used for this purpose.Pencil, paper, genetic code and hand can also be used to generate degenerate coding sequences.

[0067] The recombinant cells described herein may include deletions or destructions in one or more natural genes. The phrase "deletion or destruction" refers to the state of natural genes in recombinant cells, which have a completely eliminated coding region (deletion) or gene, its promoter or its terminator modification (such as by deletion, insertion or mutation), so that the gene no longer produces an active expression product, produces a severely reduced amount of expression product (e.g., reduced by at least 75% or reduced by at least 90%), or produces an expression product with severely reduced activity (e.g., reduced by at least 75% or reduced by at least 90%). Deletion or destruction can be achieved by genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi) and / or selection and screening. The natural gene to be deleted or destroyed can be replaced with an exogenous nucleic acid of interest for expressing an exogenous gene product (e.g., polypeptide, enzyme, etc.).

[0068] Recombinant cells as described herein may comprise one or more genetic modifications, wherein exogenous nucleic acids are integrated into the genome of the host cell. Those skilled in the art will know how to select suitable loci in the yeast genome for integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, 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 spore yeast host cell, suitable interaction sites may include, but are not limited to, the ER1 locus (defined as a locus flanked by SEQ ID NO: 85 and SEQ ID NO: 162), the ER3 locus (defined as a locus flanked by SEQ ID NO: 155 and SEQ ID NO: 165), the PDC1 locus (defined as a locus flanked by SEQ ID NO: 152 and SEQ ID NO: 164), the pyrF locus (defined as a locus flanked by SEQ ID NO: 153 and SEQ ID NO: 163), the TRP3 locus (defined as a locus flanked by SEQ ID NO: 156 and SEQ ID NO: 159), the gpdIIA locus (defined as a locus flanked by SEQ ID NO: 157 and SEQ ID NO: 161), and the gpdIIB locus (defined as a 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 can be determined by one skilled in the art. Furthermore, one skilled in the art will recognize how to use the sequences to design primers to verify correct gene integration at the selected locus.

[0069] Recombinant cells can have one or more copies of a given exogenous nucleic acid sequence, which is integrated into the host chromosome and replicated with the chromosome into which it is integrated. For example, yeast cells can be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide as described herein, and the polynucleotide sequence encoding the polypeptide can be integrated into the host chromosome with one or more copies. Recombinant cells may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide as described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten or more copies of a polynucleotide sequence encoding a polypeptide as described herein that is integrated into the genome. Multiple copies of the polynucleotide sequence can all be incorporated into a single locus or can be incorporated into multiple loci.

[0070] Recombinant cell as described herein can produce one or more polyols, and is characterized in that the overexpression of ribulose 5-phosphate epimerase (RPE enzyme).Generally, the recombinant cell comprising the overexpression of RPE enzyme produces more polyols than the equivalent cells lacking RPE enzyme or lacking the overexpression of RPE enzyme.The recombinant cell comprising the overexpression of RPE enzyme can produce arabitol, ribitol, xylitol or their combination.

[0071] Recombinant cells as described herein include exogenous polynucleotides encoding natural or exogenous RPE enzymes, or may have a genetic modification causing the overexpression of natural RPE enzymes. RPE enzymes 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 of catalyzing ribulose-5-phosphate to be converted into xylulose-5-phosphate. Enzymes with RPE activity may be natural to host cells, or RPE enzymes may be exogenous RPE enzymes. For example, when the host organism is spore yeast, RPE enzymes may be enzymes with at least one of SEQ ID NO:179 and 180 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 sequences. The recombinant cell may comprise an exogenous polynucleotide encoding an RPE enzyme having 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 comprise a genetic modification that increases the expression of an RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 179 and 180. The genetic modification may include, but is not limited to, inserting additional copies of nucleotides encoding a native RPE enzyme into the cell, inserting a constitutive promoter upstream of the coding region of a native RPE enzyme gene in the genome of the host cell, and / or modifying an existing promoter upstream of the coding region of a native RPE enzyme gene in the genome of the host cell. Those skilled in the art will recognize that expression of native RPE enzyme genes can be increased by a variety of methods known in the art, and will be able to select and apply these methods appropriately.

[0072] As used herein, "overexpression" refers to expression of a polypeptide at a level that is higher than the level of expression of the same polypeptide in an equivalent cell in the absence of a genetic modification or an exogenous polynucleotide encoding the polypeptide.

[0073] Recombinant cell as described herein can produce xylitol, and comprises the exogenous polynucleotide sequence of encoding xylitol-phosphate dehydrogenase (XPDH) and the genetic modification of the overexpression causing RPE enzyme.The exogenous polynucleotide sequence can be an exogenous xylitol-phosphate dehydrogenase (XPDH) gene.After xylulose 5-phosphate is converted into xylitol 5-phosphate, it is believed that natural phosphatase removes phosphoric acid to produce xylitol.

[0074] "Xylitol-phosphate dehydrogenase gene" and "XPDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylitol-phosphate dehydrogenase activity. As used herein, "xylitol-phosphate dehydrogenase activity" refers to catalyzing the conversion of xylulose-5-phosphate and NADPH or NADH into xylitol 5-phosphate and NADP + or NAD + The XPDH gene can be derived from any suitable source. For example, the XPDH gene can be derived from Clostridium difficile, Lactobacillus rhamnosus, Bacillushalodurans, Alkalihalobacillus ligniniphilus, Jeotgalibacillus soli, Heyndrickxiasporothermodurans, Clostridium fungisolvens and Neobacillus cucumis. The XPDH gene may encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs: 12-15, 28-32, or 33. The XPDH gene may encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs: 14, 15, 28, or 31.

[0075] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Clostridium difficile gene encoding the amino acid of SEQ ID NO: 12. The exogenous polynucleotide may encode an amino acid sequence having 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: 12.

[0076] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Clostridium difficile gene encoding the amino acid of SEQ ID NO: 13. The exogenous polynucleotide may encode an amino acid sequence having 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: 13.

[0077] The recombinant cell may include a genetic modification resulting in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Lactobacillus rhamnosus gene encoding the amino acid of SEQ ID NO: 14. The exogenous polynucleotide may encode an amino acid sequence having 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: 14.

[0078] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a halodurogenic Bacillus gene encoding the amino acid of SEQ ID NO: 15. The exogenous polynucleotide may encode an amino acid sequence having 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: 15.

[0079] The recombinant cell may include a genetic modification that results in overexpression of an RPE enzyme and an exogenous polynucleotide that is or may be derived from a ligninophilic halobacillus gene encoding the amino acid of SEQ ID NO: 28. The exogenous polynucleotide may encode an amino acid sequence having 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: 28.

[0080] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a soil saltwater marine bacillus gene encoding the amino acid of SEQ ID NO: 29. The exogenous polynucleotide may encode an amino acid sequence having 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: 29.

[0081] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from the Hyndry thermospore gene encoding the amino acid of SEQ ID NO: 30. The exogenous polynucleotide may encode an amino acid sequence having 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: 30.

[0082] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Clostridium fibrinolyticum gene encoding the amino acid of SEQ ID NO: 31. The exogenous polynucleotide may encode an amino acid sequence having 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: 31.

[0083] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Bacillus cucumeris gene encoding the amino acid of SEQ ID NO: 33. The exogenous polynucleotide may encode an amino acid sequence having 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: 33.

[0084] The recombinant cells described herein are capable of producing xylitol and include an exogenous polynucleotide sequence encoding a xylulokinase (XKS) and a genetic modification resulting in overexpression of the RPE enzyme. The exogenous polynucleotide sequence may be an exogenous xylulose phosphatase (XKS) gene.

[0085] "Xylulose kinase gene" and "XKS gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylulose kinase activity. As used herein, "xylulose kinase activity" refers to the ability to catalyze the conversion of xylulose-5-phosphate and ADP into xylulose and ATP. The XKS gene may be derived from any suitable source. For example, the XKS gene may be derived from Saccharomyces cerevisiae. The XPDH gene may encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with the amino acid sequence of at least one of SEQ ID NOs: 188 and 189. Additional descriptions of recombinant cells capable of producing xylitol and comprising a polypeptide having xylulose kinase activity are provided in U.S. Provisional Application No. 63 / 364363, filed May 9, 2022, which is incorporated herein by reference in its entirety.

[0086] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from the Saccharomyces cerevisiae DOG1 sugar phosphatase gene encoding the amino acid of SEQ ID NO: 188. The exogenous polynucleotide may encode an amino acid sequence having 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: 188.

[0087] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Saccharomyces cerevisiae DOG2 sugar phosphatase gene encoding the amino acid of SEQ ID NO: 189. The exogenous polynucleotide may encode an amino acid sequence having 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: 189.

[0088] The recombinant cells described herein may include an exogenous polynucleotide sequence encoding a xylulokinase (XKS), an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH), and a genetic modification resulting in overexpression of an RPE enzyme. The exogenous polynucleotide sequence may be an exogenous xylitol dehydrogenase (XDH) gene.

[0089] "Xylitol dehydrogenase gene" and "XDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylitol dehydrogenase activity. As used herein, "xylitol dehydrogenase activity" refers to a gene or polynucleotide that catalyzes the conversion of xylulose and NADH or NADPH into xylitol and NADPH. + or NADP+ The ability of the XDH gene to produce xylitol. The XDH gene can be derived from any suitable source. For example, the XDH gene can be derived from Pichia stipitis, Rhodobacteraceae bacterium, and Bemisia argentofolii. The XDH gene can encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with the amino acid sequence of at least one of SEQ ID NO: 190, 191 or 192. Additional descriptions of recombinant cells capable of producing xylitol and including a polypeptide having xylulokinase activity and a polypeptide having xylitol dehydrogenase activity are provided in U.S. Provisional Application No. 63 / 364363, filed May 9, 2022, which is incorporated herein by reference in its entirety.

[0090] The recombinant cell may include a genetic modification resulting in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Pichia stipitis XDH gene encoding a cofactor conversion of the amino acid of SEQ ID NO: 190. The exogenous polynucleotide may encode an amino acid sequence having 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: 190.

[0091] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Rhodobacteraceae SDR family oxidoreductase gene encoding the amino acid of SEQ ID NO: 191. The exogenous polynucleotide may encode an amino acid sequence having 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: 191.

[0092] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from the Bemisia argentofolii (silverleaf whitefly) ketose reductase (sorbitol dehydrogenase) gene encoding the amino acids of SEQ ID NO: 192. The exogenous polynucleotide may encode an amino acid sequence having 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: 192.

[0093] The recombinant cells described herein are capable of producing arabitol and include an exogenous polynucleotide sequence encoding arabitol 2-dehydrogenase (ARD2DH) and a genetic modification resulting in overexpression of the RPE enzyme. The exogenous polynucleotide sequence may be an exogenous ARD2DH gene.

[0094] "Arbitol 2-dehydrogenase" and "ARD2DH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having arabitol 2-dehydrogenase activity. As used herein, "arabitol 2-dehydrogenase activity" refers to the catalysis of the conversion of D-ribulose and NADH or NADPH into D-arabitol and NADPH. + or NADP + The enzyme having arabitol 2-dehydrogenase can be characterized under enzyme classification 1.1.1.250. The ARD2DH gene can be derived from any suitable source. For example, the ARD2DH gene can be derived from Beauveria bassiana, Pichia stipitis, Candida albicans, Kwoniella heveanensis, Candida maltosa. The ARD2DH gene can encode a polypeptide having 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 NO: 193, 194, 195, 196 or 197. The ARD2DH gene can encode a polypeptide having 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 descriptions of recombinant cells capable of producing arabitol and including a polypeptide having arabitol 2-dehydrogenase activity are provided in U.S. Provisional Application No. 63 / 364,359, filed May 9, 2022, which is incorporated herein by reference in its entirety.

[0095] The recombinant cell may include a genetic modification that results in overexpression of the 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 having 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.

[0096] The recombinant cell may include a genetic modification that results in overexpression of the 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 having 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.

[0097] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Candida albicans ARD2DH gene encoding the amino acid of SEQ ID NO: 195. The exogenous polynucleotide may encode an amino acid sequence having 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.

[0098] The recombinant cell may include a genetic modification that results in overexpression of the 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 having 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.

[0099] The recombinant cell may include a genetic modification that results in overexpression of the 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 having 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.

[0100] Recombinant cell as described herein can produce arabitol, and comprises the exogenous polynucleotide sequence of encoding arabitol-phosphate dehydrogenase (APDH) and the genetic modification causing the overexpression of RPE enzyme.The exogenous polynucleotide sequence can be an exogenous arabitol-phosphate dehydrogenase gene.After producing arabitol 5-phosphate or arabitol 1-phosphate, it is believed that natural phosphatase removes phosphoric acid to produce arabitol.

[0101] "Arabitol-phosphate dehydrogenase gene" and "APDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having arabitol-phosphate dehydrogenase activity. As used herein, "arabitol-phosphate dehydrogenase activity" refers to catalyzing (i) the conversion of xylulose 5-phosphate and NADPH or NADH into arabitol-1-phosphate and NADP + or NAD + and / or (ii) conversion of ribulose-5-phosphate and NADPH or NADH into arabitol-5-phosphate and NADP + or NAD + The APDH gene can be derived from any suitable source. For example, the ARDH gene can be derived from Lactobacillus salivarius cp400.

[0102] The recombinant cell may include a genetic modification that results in overexpression of the 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 having 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.

[0103] Recombinant cells as described herein can be capable of producing ribosol, and include exogenous polynucleotide sequences encoding ribulose-5-phosphate reductase (TarJ') and genetic modifications causing the overexpression of RPE enzymes. Exogenous polynucleotide sequences can be exogenous ribulose-5-phosphate reductase genes. After ribulose-5-phosphate is converted into ribosol 5-phosphate, it is believed that natural phosphatases remove phosphoric acid to produce ribosol.

[0104] "Ribulose-5-phosphate reductase gene" and "TarJ' gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having ribulose-5-phosphate reductase activity. As used herein, "ribulose-5-phosphate reductase activity" refers to an activity that catalyzes the conversion of ribulose-5-phosphate and NADPH or NADH into ribosol 5-phosphate and NADP + or NAD +The TarJ' gene may be derived from any suitable source. For example, the TarJ' gene may be derived from Clostridium difficile, Staphylococcus aureus, Staphylococcus aureus subsp. aureus 71193, Eubacterium ventriosum, Pradoshiasp. D12, Lactobacillus plantarum EGD-AQ4, or uncultured Ruminococcus sp. The TarJ' gene may encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs: 13, 34, 35, 36, 37, 38, or 39.

[0105] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Clostridium difficile gene encoding the amino acid of SEQ ID NO: 13. The exogenous polynucleotide may encode an amino acid sequence having 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: 13.

[0106] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a S. aureus gene encoding the amino acid of SEQ ID NO: 34. The exogenous polynucleotide may encode an amino acid sequence having 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: 34.

[0107] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Staphylococcus aureus subspecies aureus 71193 gene encoding the amino acid sequence of SEQ ID NO: 35. The exogenous polynucleotide may encode an amino acid sequence having 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: 35.

[0108] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a gene of Eubacterium convexoside that encodes the amino acid sequence of SEQ ID NO: 36. The exogenous polynucleotide may encode an amino acid sequence having 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: 36.

[0109] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Pradoshiella D12 gene encoding the amino acid sequence of SEQ ID NO: 37. The exogenous polynucleotide may encode an amino acid sequence having 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: 37.

[0110] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from a Lactobacillus plantarum EGD-AQ4 gene encoding the amino acid sequence of SEQ ID NO: 38. The exogenous polynucleotide may encode an amino acid sequence having 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: 38.

[0111] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or may be derived from an uncultured Ruminococcus gene encoding the amino acid sequence of SEQ ID NO: 39. The exogenous polynucleotide may encode an amino acid sequence having 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: 39.

[0112] The exogenous polynucleotides in the recombinant cells described herein can be under the control of a promoter. For example, the exogenous nucleic acid can be operably linked to a heterologous promoter or an artificial promoter. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarbonatase 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); translation 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); 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).

[0113] The exogenous nucleic acid in the recombinant cells described herein can be under the control of a terminator. For example, the exogenous nucleic acid can be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. The terminator may include, but is not limited to, a GAL10 terminator, a PDC terminator, a transaldolase terminator (TAL), a 6PGD terminator (6PGDt; SEQ ID NO: 140); an ASNS terminator (ASNSt; SEQ ID NO: 141); an ENO1 terminator (ENO1t; SEQ ID NO: 142); a hexokinase 1 terminator (HXK1t; SEQ ID NO: 143); a PGK1 terminator (PGK1t; SEQ ID NO: 144); a PGM1 terminator (PGM1t; SEQ ID NO: 145); a PYK1 terminator (PYK1t; SEQ ID NO: 146); a RPLA terminator (RPLAt; SEQ ID NO: 147); a transaldolase 1 terminator (TAL1t; SEQ ID NO: 148); a TDH3 terminator (TDH3t; SEQ ID NO: 149); a translation elongation factor 2 terminator (TEF2t; SEQ ID NO: 150). NO: 150); and triosephosphate isomerase 1 terminator (TPI1t; SEQ ID NO: 151);

[0114] A promoter or terminator is "operably linked" to a given polynucleotide (eg, a gene) if its position relative to the polynucleotide in the genome or expression cassette is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0115] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. A recombinant polynucleotide is a polynucleotide formed by a laboratory method, which includes a polynucleotide sequence derived from at least two different natural sources, or they may be synthetic. Therefore, a construct may include a new modification to an endogenous gene introduced by, for example, a genome editing technique. A construct may also include a recombinant polynucleotide produced using, for example, a recombinant DNA method. A construct may be a vector comprising a promoter operably connected to a polynucleotide encoding a polypeptide described herein. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide connected thereto. A vector may be a plasmid, which refers to a circular double-stranded DNA loop, into which an additional DNA fragment may be integrated.

[0116] The present disclosure also provides a fermentation method for producing polyols (e.g., xylitol, ribitol, arabitol) using the recombinant cells described herein. The fermentation method includes a step of using a genetically engineered yeast fermentation substrate as described herein to produce polyols (e.g., xylitol, ribitol, arabitol). As will be appreciated by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and separating polyols from the fermentation broth. The fermentation process may be a fully aerobic or partially aerobic process.

[0117] The fermentation process can be run using a suitable fermentation substrate. The substrate of the fermentation process can include hydrolyzates of glucose, sucrose, galactose, mannose, molasses, xylose, fructose, starch, lignocellulose hydrolyzates, or a combination thereof. Those skilled in the art will recognize which fermentation substrate is suitable for a given fermenting organism and system.

[0118] The fermentation process can be run under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained within a range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably about 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to any specific range or temperature described herein and can be modified as appropriate.

[0119] The fermentation process can be operated within a range of oxygen uptake rates (OUR). The volumetric OUR of the fermentation process can be in the range of 0.5 mmol O2 / (L·h) to 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) to 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) to 30 mmol O2 / (L·h), 3 mmol O2 / (L·h) to 25 mmol O2 / (L·h), 4 mmol O2 / (L·h) to 20 mmol O2 / (L·h), or 5 mmol O2 / (L·h) to 15 mmol O2 / (L·h). In some embodiments, the specific OUR can be in the range of 0.05 mmol O2 / (g cell dry weight h) to 10 mmol O2 / (g cell dry weight h), 0.1 mmol O2 / (g cell dry weight h) to 8 mmol O2 / (g cell dry weight h), 0.15 mmol O2 / (g cell dry weight h) to 5 mmol O2 / (g cell dry weight h), 0.2 mmol O2 / (g cell dry weight h) to 1 mmol O2 / (g cell dry weight h), or 0.3 mmol O2 / (g cell dry weight h) to 0.75 mmol O2 / (g cell dry weight h). However, the volume or specific OUR of the fermentation process is not limited to any specific rate or range described herein.

[0120] Fermentation process can be operated under various cell concentrations. In some embodiments, the dry cell weight at the end of fermentation can be 5g dry cell weight / L to 40g dry cell weight / L, 8g dry cell weight / L to 30g dry cell weight / L or 10g dry cell weight / L to 20g dry cell weight / L. In addition, the input density (pitch density) or input rate (pitchingrate) of fermentation process can change. In some embodiments, the input density can be 0.05g dry cell weight / L to 11g dry cell weight / L, 0.1g dry cell weight / L to 10g dry cell weight / L or 0.25g dry cell weight / L to 8g dry cell weight / L.

[0121] The initial dextrose concentration of the fermentation can be at least 100 g / L dextrose, 200 g / L dextrose, 250 g / L dextrose, 300 g / L dextrose, 350 g / L dextrose, or at least 400 g / L dextrose. The initial dextrose concentration can be between 100 g / L and 400 g / L, 150 g / L and 350 g / L, or 250 g / L and 325 g / L.

[0122] Fermentation processes can be associated with various characteristics, such as, but not limited to, fermentation production rate, approach fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of yeast and / or the genetic modification of yeast used in the fermentation process. These characteristics can be affected by regulating the fermentation process conditions. These characteristics can be regulated via a combination of yeast selection or modification and the selection of fermentation process conditions.

[0123] The polyol production rate of the process can be at least 0.2 g L -1 h -1 , 0.3 g L -1 h -1 , 0.5gL -1 h -1 , 0.75 g L -1 h -1 or at least 1.0 g L -1 h -1 The polyol mass yield of the process can be at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. The final polyol titer of the process can be at least 5 g / L, 10 g / L, 20 g / L, 30 g / L, 50 g / L, 75 g / L, or 100 g / L.

[0124] The fermentation process can be run as a dextrose fed batch.In addition, the fermentation process can be a batch process, a continuous process or a semi-continuous process, as understood by those skilled in the art.

[0125] Example

[0126] The present invention is further described in detail by reference to the following experimental examples. Unless otherwise stated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0127] Example 1 - Xylitol-phosphate dehydrogenase diversity

[0128] About three thousand galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) sequences were obtained from Uniprot and analyzed. Figure 2The natural sequence diversity of this group of sequences is shown. The group is diverse, with about 25% of the enzymes not having more than 75% identical homology. Since these enzymes tend to prefer NAD as a cofactor rather than NADP as a cofactor, the cofactor binding preference of homologs was evaluated 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). The cofactor binding pocket was identified by approaching the Rossman fold (from +23 to +30 amino acids of the GXGXXG motif (SEQ ID NO: 129)) and scored based on the total charge in an 8-amino acid residue window. The top 8 candidates predicted to use NADP, as well as 4 candidates predicted to use NAD and 3 controls were selected for further characterization.

[0129] Upon further examination of the structural features of the predicted binding pocket, important aspartate residues were identified that could influence cofactor preference. Figure 3 The polypeptide of SEQ ID NO: 34 and its substitutions were used to construct a structural homology model for predicting the conformation of the cofactor binding pocket. Figure 3 Shown is the C-terminal of the penultimate β-strand outside the Rossman fold domain.Do not wish to be bound by any particular theory, it is estimated that the enzyme wherein the first residue in this region (relative to residue 198 of SEQ ID NO:34) is aspartic acid and the second residue (relative to residue 199 of SEQ ID NO:34) is a large hydrophobic amino acid (e.g., isoleucine) will preferably NAD cofactor, this is due to the hydrogen bonding of the hydroxyl of aspartic acid and NAD ribose. However, the enzyme wherein the first residue (relative to residue 198 of SEQ ID NO:34) is alanine, glycine or serine and the second residue (relative to residue 199 of SEQ ID NO:34) is lysine or arginine will preferably NADP cofactor, because the positive charge on lysine or arginine residue will interact with the negative charge of the phosphoric acid of NADP and the smaller residue in the first position allows the space of the phosphoric acid in the binding pocket.Based on this analysis, according to the prediction preference to NADP, other 12 kinds of enzymes were selected.Finally, 6 other enzymes with sequence similarity to the active XPDH enzyme were selected for testing.

[0130] Example 2 - TarJ' diversity

[0131] Approximately eight hundred ribulose 5-phosphate reductase sequences were obtained from Uniprot and analyzed. Figure 3 The natural sequence diversity of this group of sequences is shown. Overall, the diversity in this group is low, because only 10% of the enzymes do not have more than 75% identical sequence similarity. Since these enzymes tend to prefer NADP rather than NAD as cofactors, no scoring is performed, and sequences are simply aligned in Geneous (ClustalW, default settings). Eight enzymes were selected for further analysis based on sequence similarity.

[0132] Example 3 - In vitro enzyme assay

[0133] The 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, InVitro Protein Synthesis). The activity of cell-free synthesized proteins on four substrates (ribulose 5-phosphate, xylulose 5-phosphate, ribulose, and xylulose) was analyzed with 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 ribulose 5-phosphate or xylulose 5-phosphate ( Figure 5 ), but could not catalyze the reduction of xylulose or ribulose (results not shown).

[0134] Table 2: XPDH homologs

[0135]

[0136] Table 3: TarJ' homologs

[0137]

[0138] Example 4 - Genetically modified Saccharomyces cerevisiae strains

[0139] Strain 1-1 is a Moniliella tomentosa host strain "Moniliellatomentosa var pollinis TCV364" described in US 6,440,712, which is incorporated herein by reference in its entirety and deposited under the Budapest Treaty on March 28, 1997 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). Table 4 below lists various Moniliella tomentosa strains, including information on the parent strain, the sequence of the transformed parent strain, and the characterization of the expression cassette contained in the transformed sequence. Each "XPDH / TarJ' homolog expression cassette" contains, 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), an 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" contains, 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). Following binary transformation with the XPDH / TarJ' homolog expression cassette and the selectable marker cassette, the two cassettes recombined to integrate the nucleotide sequence encoding the XPDH or TarJ' homolog and the G418 resistance marker at the ER1 locus.

[0140] The indicated Saccharomyces parent strains were transformed with the indicated sequences by first protoplasting the parent strains by adding an enzyme mixture containing 0.6M MgSO4, 7.5g / L collapsing enzyme, and 12.5g / L Trichoderma harzianum lyase to the mycelial pellet of the parent strains. The protoplasts were then pelleted, washed with 0.6M MgSO4, and resuspended in STC medium (0.6M sucrose, 50mM CaCl2, 10mM Tris-HCl, pH 7.5). The protoplasts were then precipitated with 100μg of single-stranded salmon sperm DNA and 1.5μg to 5μg of each 5'DNA and 3'DNA conversion fragments (3μg-10μg in total; see Table 4 for a list of fragments) to approximately 200μL of the protoplast mixture (10 8 Cells / mL) are added to prepare binary transformation. Then 1mL of STC culture medium containing 50% PEG is added to salmon sperm DNA, conversion DNA and protoplast mixture, and the resulting combination is incubated at room temperature for 15 minutes. After incubation, the recovery culture solution (0.4M sucrose, 1g / L yeast extract, 1g / L malt extract, 10g / L sugar, pH 4.5) is added to the mixture, and incubated at 27°C, 100rpm for 16 hours to 24 hours. After incubation, the protoplasts are precipitated and resuspended in 1mL PBS by centrifugation.

[0141] The resuspended protoplasts are spread on PDA+250mg / L geneticin (geneticin) (G418) selection plates and incubated at least 2 days-4 days at 30°C-35°C until transformant growth. The integration of the indicated sequence of the obtained transformant is evaluated by colony PCR. The isolate verified by PCR is then designated as the indicated strain number. In some cases, more than one isolate verified by PCR (e.g., "sister" isolate) is indicated by the letter after the strain number. For example, strain 1-strain 2 has 5 sister isolates: strains 1-2a, 1-2b, 1-2c, 1-2d and 1-2e.

[0142] For example, strain 1-1 is 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) the 3' portion of the G418 resistance gene selectable marker (SEQ ID NO:172). SEQ ID NO:44 contains (i) an expression cassette for an XPDH homolog from M. sediminis 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 (SEQ ID NO:87); (ii) 5' flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:85). And (iii) the 5' portion of the G418 resistance gene selectable marker (SEQ ID NO:175). Transformants were selected on PDA+250mg / L Geneticin (G418) selection plates and incubated at least 2 days at 30°C to 35°C until transformant growth. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and selection of single colonies. The integration of the indicated sequences of the selected colonies was evaluated by colony PCR. The isolates verified by PCR were named as strains 1-2a, 1-2b, 1-2c, 1-2d and 1-2e.

[0143] Table 4 .

[0144]

[0145]

[0146] Example 5 - Shake flask fermentation assay

[0147] 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 (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0148] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0149] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 72 hours and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 6 and Figure 6 and Figure 7 middle.

[0150] Table 5: Production medium

[0151]

[0152]

[0153] Although 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 1-35a, 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 included the transformed polynucleotide sequence, but it was not integrated at the ER1 locus.

[0154] Table 6: 96-hour shake flask results

[0155]

[0156]

[0157] Example 6 - Shake flask fermentation assay

[0158] Strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0159] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0160] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a 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. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 7 and Figure 8 middle.

[0161] like Figure 8 As shown in , although sister strains 1-34c and 1-34d produced 15.8 g / L and 18.6 g / L xylitol, respectively, strains 1-34a, 1-34b, and 1-34e did not produce more xylitol than the wild type (strains 1-1, Figure 6 ) significantly more xylitol. When strains 1-34a, 1-34b, and 1-34e were initially verified by PCR, it was later determined that the integrated polynucleotide that should encode the Neobacillus cucumeris XPDH homolog contained a frameshift mutation and did not express a functional XPDH. Therefore, although the results appear different, they are actually consistent considering that strains 1-34a, 1-34b, and 1-34e do not contain a polynucleotide encoding a functional XPDH.

[0162] Table 7: 96-hour shake flask results

[0163]

[0164] Example 7 - Shake flask fermentation assay

[0165] 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 (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol and ethanol production.

[0166] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0167] The 250ml non-baffled flask containing the production medium (Table 5) was inoculated with 0.8mL of seed culture to form a production culture. The production culture was incubated at 35°C and 250rpm. Samples were taken from the production culture after 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol and ethanol were analyzed by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 8.

[0168] Table 8: 96-hour shake flask results

[0169]

[0170]

[0171] Example 8 - Shake flask fermentation assay

[0172] 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 (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol and ethanol production.

[0173] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0174] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 72 hours and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 9 and Fig. 9 middle.

[0175] Although 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 1-16b-e included the transformed polynucleotide sequence, but it was not at the ER1 locus. Further analysis of the integration position in strains 1-2c and 1-2d was inconclusive.

[0176] Table 9: 96-hour shake flask results

[0177]

[0178]

[0179] Example 9 - Shake flask fermentation assay

[0180] Strains 1-13c, 1-8a-d, 1-26a-e, 1-36a-e, 1-41a-e, 1-40a-e, and 1-20a-e (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0181] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0182] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a 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. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 10 and Fig.10 middle.

[0183] Although 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 1-8c, 1-8d, and 1-41c included the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integration loci in strains 1-36a, 1-41b, 1-41e, and 1-20a-e was inconclusive.

[0184] Table 10: 96-hour shake flask results

[0185]

[0186] Example 10 - Shake flask fermentation assay

[0187] 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 (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0188] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0189] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 72 hours and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 11 and Fig.11 middle.

[0190] Although 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 included the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integration locus in strain 1-6c was inconclusive.

[0191] Table 11: 96-hour shake flask results

[0192]

[0193]

[0194] Example 11 - Two copies of XPDH modified Saccharomyces cerevisiae strain

[0195] Strain 1-1 was transformed with SEQ ID NO:55 and SEQ ID NO:177 as described in Example 4. SEQ ID NO:177 contains (i) the 3' portion of the G418 selectable marker (SEQ ID NO:172); (ii) an expression cassette (SEQ ID NO:98) encoding the XPDH homolog from C. difficile of 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) 3' flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:162). SEQ ID NO:55 contains (i) 5' flanking DNA (SEQ ID NO:85) for targeted chromosomal integration into the ER1 locus, (ii) an expression cassette (SEQ ID NO:98) encoding an XPDH homolog from Clostridium difficile 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) the 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°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and single colonies were selected. The selected colonies were evaluated for integration of two copies of the Clostridium difficile XPDH sequence by colony PCR. The PCR-confirmed isolates were designated strains 2-1a, 2-1b, 2-1c, 2-1d, and 2-1e.

[0196] Strain 1-1 was transformed with SEQ ID NO:57 and SEQ ID NO:178 as described in Example 4. SEQ ID NO:178 contains (i) the 3' portion of the G418 selectable marker (SEQ ID NO:172); (ii) an expression cassette (SEQ ID NO:100) encoding the XPDH homolog from Lactobacillus rhamnosus of 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) 3' flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:162). SEQ ID NO:57 contains (i) 5' flanking DNA (SEQ ID NO:85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette (SEQ ID NO:100) encoding the amino acid sequence of SEQ ID NO:14 from Lactobacillus rhamnosus under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (ii) the 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°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of two copies of the Lactobacillus rhamnosus XPDH sequence. The PCR-confirmed isolates were designated strains 2-2a, 2-2b, 2-2c, 2-2d, and 2-2e.

[0197] Strains 1-13c, 2-1a-e, and 2-2a-e were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0198] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0199] The 250ml non-baffled flask containing the production medium (Table 5) was inoculated with 0.8mL of seed culture to form a production culture. The production culture was incubated at 35°C and 250rpm. Samples were taken from the production culture after 72 hours and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol and ethanol were analyzed by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 12.

[0200] Although 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 targeted to the ER1 locus. Further analysis indicated that strain 2-2e included the transformed polynucleotide sequence, but it did not target the ER1 locus.

[0201] Table 12: 96 hour shake flask results

[0202]

[0203] Example 12 - Genetically modified Saccharomyces cerevisiae strains

[0204] Strain 1-1 was transformed with SEQ ID NO: 186 and SEQ ID NO: 187 as described in Example 4. SEQ ID NO: 186 contains (i) 5' flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO: 85), (ii) the MpPYK1 promoter (SEQ ID NO: 86), (iii) the gene encoding the S. aureus xylitol dehydrogenase of SEQ ID NO: 34, (iv) the Mp6PGD terminator (SEQ ID NO: 140), and (v) the 5' portion of the G418 resistance gene expression cassette (SEQ ID NO: 175). SEQ ID NO: 187 contains (i) the 3' portion of the G418 resistance gene expression cassette (SEQ ID No: 172), (ii) the MpTEF2 terminator (SEQ ID NO: 150), (iii) the MpPGK1 promoter (SEQ ID NO: 135), (iv) the gene encoding the Saccharomyces cerevisiae DOG1 sugar phosphatase of SEQ ID NO: 188, (v) the 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 ° C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA + Geneticin (G418) plates for single colony isolation and single colonies were selected. The integration of the indicated sequences of the selected colonies was evaluated by colony PCR. The isolate verified by PCR was called strain 3-1.

[0205] Table 13 below lists various strains of S. cerevisiae, including information about the parent strain, the sequence of the transformed parent strain, and the characterization of the expression cassette contained in the transformed sequence. The transformed fragment of SEQ ID NO: 181 contains, in order, 5'ER3 flanking sequences (SEQ ID NO: 155), MpPYK1 promoter (SEQ ID NO: 86), the gene encoding the S. cerevisiae RPE2 polypeptide of SEQ ID NO: 180, MpPYK terminator (SEQ ID NO: 146), and the 5' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 169). The transformed fragment of SEQ ID NO: 182 contains, in order, the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 168), MpTEF2 terminator (SEQ ID NO: 150), and 3'ER3 flanking sequences (SEQ ID NO: 165). The transformation fragment of SEQ ID NO: 183 contains the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 168), the Mp6PGD promoter (SEQ ID NO: 130), the gene encoding the RPE1 polypeptide of SEQ ID NO: 179, the Mp6PGD terminator (SEQ ID NO: 140), and the 3'ER3 flanking sequence (SEQ ID NO: 165) in order. The transformation fragment of SEQ ID NO: 184 contains the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 168), the Mp6PGD promoter (SEQ ID NO: 130), the gene encoding the RPE2 polypeptide of SEQ ID NO: 180, the Mp6PDG terminator (SEQ ID NO: 140), and the 3'ER3 flanking sequence (SEQ ID NO: 165) in order. The transformation fragment of SEQ ID NO: 185 contains, in order, the 5' ER3 flanking sequence (SEQ ID NO: 155), the MpTEF1 promoter (SEQ ID NO: 133), and the 5' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 169).

[0206] As described in Example 4, the indicated parent strain of the yeast of the spore spore was transformed with the indicated sequence using bleomycin or G418 selection corresponding to the indicated selection marker. The integration of the indicated sequence of the resulting transformant was evaluated by colony PCR. The isolates verified by PCR were then assigned to the indicated strain numbers. In some cases, more than one isolate verified by PCR (e.g., "sister" isolates) are indicated by the letter after the strain number. For example, strain 3-2 has 4 sister isolates: strains 3-2a, 3-2b, 3-2c and 3-2d (collectively referred to as 3-2a-d).

[0207] Table 13

[0208]

[0209] na - not applicable, the second cassette is only used for recombination and selection markers

[0210] Example 13 - Shake flask fermentation assay

[0211] 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 and ribitol, xylitol, glycerol, and ethanol production.

[0212] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0213] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 48 hours, 72 hours, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 14 and Fig.14 and Fig.15 middle.

[0214] Table 14: 96 hour shake flask results

[0215]

[0216] Example 14 - Shake flask fermentation assay

[0217] Strains 1-1, 1-13c, 1-15a, 3-7a-f, and 3-8a-f were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, erythritol, and ethanol production.

[0218] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0219] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 48 hours, 72 hours, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 15 and Fig.12 and Fig.13 middle.

[0220] Table 15: 96 hour shake flask results

[0221] strain glucose Ribitol Xylitol Erythritol glycerin Ethanol 3-7a 137.81 0.35 26.02 11.69 23.16 21.44 3-7b 59.81 2.78 30.09 24.53 21.77 37.56 3-7c 61.19 2.23 38.98 21.32 16.97 36.23 3-7d 81.93 1.70 36.50 19.87 20.79 24.54 3-7e 60.27 4.31 31.94 23.23 16.40 35.79 3-7f 73.27 3.77 27.98 32.10 17.63 28.82 3-8a 66.88 3.01 44.57 15.77 18.28 33.11 3-8b 77.84 1.03 48.40 9.95 20.58 30.25 3-8c 97.96 0.93 39.53 14.00 17.55 26.02 3-8d 73.16 0.84 48.83 12.32 17.96 34.21 3-8e 116.78 0.65 28.41 7.72 28.04 24.53 3-8f 81.82 1.01 46.42 11.13 19.83 31.73 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 1-15a 55.73 9.23 36.08 24.34 16.69 35.93 1-15a 60.66 8.93 35.24 22.91 15.91 37.51 1-1 55.56 0.63 0.33 59.10 15.20 43.83 1-1 57.65 0.59 0.09 58.06 15.22 44.85

[0222] Example 15 - Shake flask fermentation assay

[0223] 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 and ribitol, xylitol, glycerol, erythritol, and ethanol production.

[0224] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0225] A 250 ml unbaffled flask containing production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35° C. and 250 rpm. Samples were taken from the production culture after 48 hours, 72 hours, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 16, 17, and 18 and Figures 16 to 21 middle.

[0226] Yield was calculated as the ratio of product (ie, xylitol) expressed as a percentage to substrate consumed (ie, the difference between one time point and a second time point).

[0227] Table 16: Shake flask yield (48 hours - 96 hours)

[0228]

[0229]

[0230] Table 17: Shake flask rate (48 hours - 96 hours)

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238] Example 16 - Genetically Engineered Saccharomyces cerevisiae Strain

[0239] strain 4-1a-e

[0240] To test the activity of the RPE enzyme in a wild-type S. fascicularis background, strain 1-1 was transformed with SEQ ID NO: 181 and SEQ ID NO: 182 using the transformation method outlined in Example 4. The transformation fragment of SEQ ID NO: 181 contained, in order, the 5' ER3 flanking sequence (SEQ ID NO: 155), the MpPYK1 promoter (SEQ ID NO: 86), the gene encoding the S. fascicularis RPE2 polypeptide of SEQ ID NO: 180, the MpPYK terminator (SEQ ID NO: 146), and the 5' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 169). The transformation fragment of SEQ ID NO: 182 contained, in order, the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO: 168), the MpTEF2 terminator (SEQ ID NO: 150), and the 3' ER3 flanking sequence (SEQ ID NO: 165). Transformants were selected on PDA + bleomycin selection plates and incubated at 35 ° C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA + bleomycin plates for single colony isolation and single colonies were selected. The integration of the RPE2 gene sequence of the selected colonies was evaluated by colony PCR. The sister isolates verified by PCR were named strains 4-1a, 4-1b, 4-1c and 4-1d.

[0241] strain 4-2

[0242] Strain 1-1 was transformed with SEQ ID NO:57 and SEQ ID NO: 199 using the transformation method outlined in Example 4. SEQ ID NO: 199 contains the 3' portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the polypeptide encoding the RPE of SEQ ID NO: 180, the MpPYK1 terminator (SEQ ID NO: 86), and the 3' ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO:57 contains (i) 5' flanking DNA (SEQ ID NO:85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette (SEQ ID NO:100) encoding the amino acid sequence of SEQ ID NO:14 from Lactobacillus rhamnosus under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (ii) the 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°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the Lactobacillus rhamnosus XPDH sequence and the Saccharomyces cerevisiae RPE2 sequence. The PCR-verified isolate was named strain 4-2.

[0243] strain 4-3

[0244] Strain 1-1 was transformed with SEQ ID NO:57 and SEQ ID NO: 199 using the transformation method outlined in Example 4. SEQ ID NO: 199 contains the 3' portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the polypeptide encoding the RPE of SEQ ID NO: 180, the MpPYK1 terminator (SEQ ID NO: 86), and the 3' ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO:57 contains (i) 5' flanking DNA (SEQ ID NO:85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette (SEQ ID NO:100) encoding the amino acid sequence of SEQ ID NO:14 from Lactobacillus rhamnosus under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (ii) the 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°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the Lactobacillus rhamnosus XPDH sequence and the Saccharomyces cerevisiae RPE2 sequence. The PCR-verified isolate was named strain 4-2.

[0245] Strain 4-4a-e

[0246] Strain 1-1 was transformed with SEQ ID NO:57 and SEQ ID NO:200 using the transformation method outlined in Example 4. SEQ ID NO:200 contains the 3' portion of the G418 resistance marker (SEQ ID NO:172), the MpPGK1 promoter (SEQ ID NO:135), the gene encoding the R. paniculata RPE enzyme of SEQ ID NO:179, the MpPYK1 terminator (SEQ ID NO:86), and the 3' ER1 flanking sequence (SEQ ID NO:162). SEQ ID NO:57 contains (i) 5' flanking DNA (SEQ ID NO:85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette (SEQ ID NO:100) encoding the amino acid sequence of SEQ ID NO:14 from Lactobacillus rhamnosus under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (ii) the 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°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the Lactobacillus rhamnosus XPDH sequence and the Saccharomyces cerevisiae RPE2 sequence. The PCR-verified isolate was named strain 4-2.

[0247] Strains 4-5a-d

[0248] Strain 1-1 was transformed with SEQ ID NO:57 and SEQ ID NO: 199 using the transformation method outlined in Example 4. SEQ ID NO: 199 contains the 3' portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the polypeptide encoding the RPE of SEQ ID NO: 180, the MpPYK1 terminator (SEQ ID NO: 86), and the 3' ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO:57 contains (i) 5' flanking DNA (SEQ ID NO:85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette (SEQ ID NO:100) encoding the amino acid sequence of SEQ ID NO:14 from Lactobacillus rhamnosus under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (ii) the 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°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the Lactobacillus rhamnosus XPDH sequence and the Saccharomyces cerevisiae RPE2 sequence. The PCR-verified isolate was named strain 4-3.

[0249] Although strains 4-2, 4-3, and 4-5a-d are all transformation products of strain 1-1 having SEQ ID Nos: 57 and 199, they are the result of three different transformation reactions. Although they are all PCR verified, they are not sister isolates of a single transformation reaction and therefore have different strain numbers.

[0250] Example 17 - Shake flask fermentation assay

[0251] Strains 1-1 and 1-4a-d were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0252] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0253] A 250 ml unbaffled flask containing 20 ml of production medium (Table 5) was inoculated with 0.4 ml of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 48 hours, 72 hours, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 19 and Fig. 22 middle.

[0254] The results showed that overexpression of RPE2 in wild-type Rhizoctonia solani resulted in a reduction in erythritol production. The final erythritol titer was reduced by 25%-50%. The erythritol yield dropped from 32% to 20%-25%. The glucose consumption rate was also reduced.

[0255] Table 19 .

[0256]

[0257] na indicates that the metabolite is absent or below the detection limit of the instrument.

[0258] Example 18 - Shake flask fermentation assay

[0259] Strains 1-1, 2-2b, 4-2, 4-3, 4-4a-e, and 4-5a-d were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0260] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0261] A 250 ml unbaffled flask containing 20 ml of production medium (Table 5) was inoculated with 0.4 ml of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 48 hours, 72 hours and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 20 and 21 and Fig.23 middle.

[0262] The results showed that both RPE1 (SEQ ID NO: 179) and REP2 (SEQ ID NO: 180) from Rhizoctonia solani increased the titer and yield of xylitol production. Overexpression of the enzymes resulted in 20.9% and 18.4% xylitol yields, respectively, compared to 10.8% in strain 2-2b (which lacks RPE overexpression) and 0% in wild-type strain 1-1.

[0263] Table 20 .

[0264]

[0265]

[0266] Table 21 .

[0267]

Claims

1. A genetically engineered yeast cell capable of producing a polyol, the engineered yeast cell comprising: Genetic modification resulting in overexpression of a native enzyme with ribulose-5-phosphate epimerase (RPE) activity. The yeast cell according to claim 1 , wherein the yeast cell is an osmotolerant yeast cell.

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

4. The yeast cell according to any one of claims 1 to 3, wherein the yeast cell is selected from the group consisting of: giant Trichosporon, Trichosporon sphaerocephala, Trichosporon nigromatis, Pseudomonas tsukubaensis, Trigonella variabilis, Pseudomonas fasciatus, Ustilago, Trichosporon, Oleaginous yeast, Penicillium, Torula yeast, Pichia pastoris, Candida, Candida magnolii and Aureobasidium pullulans.

5. The yeast cell of any one of claims 1 to 4, wherein the cell is a Saccharomyces cerevisiae cell and the native RPE enzyme comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 179 and 180.

6. according to the yeast cell described in any one in claim 1 to 5, the RPE activity in the wherein said engineered yeast cell is higher than the RPE activity in the equivalent cell lacking said genetic modification.

7. The yeast cell according to any one of claims 1 to 6, wherein When the engineered cells are used in a fermentation process in the presence of dextrose, the yield of the polyol is increased relative to the yield of the polyol in an equivalent fermentation process using an equivalent cell lacking the genetic modification.

8. according to the yeast cell described in any one in claim 1 to 7, wherein said genetic modification comprises replacing the natural RPE gene promoter with a heterologous promoter or an artificial promoter.

9. The yeast cell according to claim 8, wherein the heterologous promoter 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), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 134), 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), NO:138) and RPL16B (SEQ ID NO:139).

10. The yeast cell of any one of claims 1 to 9, wherein the genetic modification comprises adding 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.

11. The yeast cell of any one of claims 1 to 10, wherein the cell is capable of producing ribitol and comprises an exogenous polynucleotide sequence encoding an enzyme having ribulose-5-phosphate reductase activity, the enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 13, 34, 35, 36, 37, 38 and 39.

12. The yeast cell of any one of claims 1 to 10, wherein the cell is capable of producing xylitol and comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH), the XPDH enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31 and 33.

13. The yeast cell of any one of claims 1 to 10, wherein the cell is capable of producing xylitol and comprises an exogenous polynucleotide sequence encoding a sugar phosphatase comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 188 and 189.

14. The yeast cell of claim 11, wherein the cell further comprises an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH), the XDH enzyme comprising A sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of ID NOs: 190, 191 and 192.

15. The yeast cell of any one of claims 1 to 10, wherein the cell is capable of producing arabitol and comprises (i) an exogenous polynucleotide sequence encoding an arabitol-phosphate dehydrogenase (APDH) comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11, and / or (ii) an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 193-197.

16. The yeast cell according to any one of claims 1 to 15, wherein the exogenous polynucleotide sequence is operably linked to a heterologous promoter or an artificial promoter.

17. The yeast cell of claim 16, wherein the promoter is a constitutive promoter.

18. The yeast cell of claim 16 or claim 17, wherein the heterologous promoter 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), translation 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), NO:138) and RPL16B (SEQ ID NO:139).

19. The yeast cell according to any one of claims 1 to 18, wherein the exogenous polynucleotide sequence is integrated into the genome of the yeast cell at a locus selected from the group consisting of ER1 locus, ER3 locus, PDC1 locus, pyrF locus, TRP3 locus, gpdIIA locus and gpdIIB locus.

20. A method for producing a polyol, the method comprising: A substrate comprising dextrose is contacted with the engineered yeast cell of any one of claims 1 to 19, wherein fermentation of the substrate by the engineered cell produces the polyol.

21. A method for producing a polyol (e.g., xylitol, arabitol, ribitol), the method comprising: A substrate comprising dextrose is contacted with an engineered yeast cell comprising a genetic modification resulting in overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity.

22. The method of claim 21, wherein the cell is a Mycelium cell and the native RPE enzyme comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 179 and 180.

23. The method according to any one of claims 20 to 22, wherein the fermentation temperature is 25°C to 45°C, 30°C to 40°C or 32°C to 37°C or therebetween, and the volumetric oxygen uptake rate (OUR) is between 0.5 mmol O2 / (L·h) and 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) and 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) and 30 mmol O2 / (L·h), 3 mmol O2 / (L·h) and 25 mmol O2 / (L·h), 4 mmol O2 / (L·h) and 20 mmol O2 / (L·h) or 5 mmol O2 / (L·h) and 15 mmol O2 / (L·h).

24. The method according to any one of claims 20 to 23, wherein the polyol is present in an amount of at least 0.2 g L -1 h -1 , 0.3 g L -1 h -1 , 0.5 g L -1 h -1 , 0.75 g L -1 h -1 or at least 1.0 g L -1 h -1 rate of production.

25. The method of any one of claims 20 to 24, wherein the polyol production is at least 20 g / L, 30 g / L, 50 g / L, 75 g / L or 100 g / L when the fermentation is run at 35°C for 96 hours.

26. The method of any one of claims 20 to 25, wherein the rate and / or yield of the polyol is increased relative to an equivalent fermentation run with an equivalent yeast cell lacking the genetic modification.

27. The method of any one of claims 20 to 26, wherein the concentration of dextrose is at least 100 g / L.

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