Genetically modified yeast and fermentation process for production of arabitol

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 499,991, filed May 4, 2023, the entire contents of which are incorporated herein by reference.

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

[0004] The contents of the sequence list XML file named "PT-1755-WO-PCT.xml", which is 803,466 bytes in size and was created on April 29, 2024 and electronically submitted with this application via the Patent Centre, are incorporated herein by reference in their entirety. Background Technology

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

[0006] In the development of microbial-based fermentation strategies for xylitol production, the production of metabolic pathway intermediates and alternative fermentation products is an important consideration. For example, the metabolic pathways active in xylitol production may overlap with those used to produce aritol, erythritol, ribitol, etc. Intermediates and products have their own uses and markets, making their fermentation commercially relevant. Therefore, this paper presents genetically modified yeast and fermentation methods for aritol production, based on closely related pathways used in xylitol production. Summary of the Invention

[0007] This disclosure provides genetically engineered yeast cells capable of producing xylitol, the engineered yeast cells comprising genetic modifications leading to the overexpression of a native enzyme having arabinoyl-5-phosphate phosphatase (A5PP) activity; and / or exogenous polynucleotide sequences encoding an enzyme having arabinoyl-5-phosphate phosphatase (A5PP) activity. The yeast cells may be osmotically resistant yeast cells. The yeast cells may be cells belonging to the subphylum Ustilaginomycotina or Saccharomycotina. Yeast cells can be selected from the group consisting of: *Trichosporonoides megachiliensis*, *Trychosporonoides oedocephalis*, *Trychosporonoides nigrescens*, *Pseudozyma tsukubaensis*, *Trigonopsis variabilis*, *Moniliella*, *Ustilaginomycetes*, *Trichosporon*, *Yarrowia lipolytica*, *Saccharomyces cerevisiae*, *Penicillium*, *Torula*, *Pichia*, *Candida*, *Candidamagnoliae*, and *Aureobasidium*. Yeast cells can also be from the genus *Moniliella*.

[0008] The yeast cells may be Moniliella pollinis cells, and genetic modifications result in the overexpression of a natural A5PP enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 198, 199, 200, or 221.

[0009] Genetic modification may include replacing the natural A5PP gene promoter with a heterologous promoter or an artificial promoter. Heterologous or artificial promoters may be selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6-phosphogluconic acid 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), phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p; SEQ ID NO:136), asparagine synthase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139).

[0010] Genetic modification may include adding a foreign polynucleotide sequence encoding a native A5PP enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding a native A5PP enzyme. Yeast cells may contain a foreign polynucleotide sequence encoding an enzyme having A5PP activity, and a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188. Yeast cells may contain a foreign polynucleotide sequence encoding an enzyme having A5PP activity, and a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO:200, 202, 203, 204, 206, and 213.

[0011] Generally, A5PP activity in genetically engineered yeast cells is higher than that in equivalent cells lacking genetic modification or exogenous polynucleotide sequences. When engineered cells are used in fermentation processes in the presence of dextran, the titer and / or yield of aritol increases compared to equivalent fermentation processes using equivalent cells lacking genetic modification or exogenous polynucleotide sequences.

[0012] Engineered yeast cells may additionally contain genetic modifications that lead to the overexpression of a native enzyme with ribulose-5-phosphate epimerase (RPE) activity. The cells may be *Moniliella pollinis* cells, and the native RPE enzyme may contain at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences to at least one of SEQ ID NO: 179 and 180. Genetic modifications leading to the overexpression of the native RPE enzyme may include replacing the native RPE gene promoter with a heterologous promoter or an artificial promoter. Heterologous or artificial promoters may be selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6-phosphogluconic acid 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), phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p; SEQ ID NO:136), asparagine synthase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139). Genetic modifications that lead to overexpression of the natural RPE enzyme may include the addition of a foreign polynucleotide encoding the natural RPE enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the natural RPE enzyme.

[0013] Yeast cells may additionally contain an exogenous polynucleotide sequence encoding an arachidonic acid-phosphate dehydrogenase (APDH) enzyme, which contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the same sequence as SEQ ID NO:11.

[0014] The cell may additionally contain a foreign polynucleotide sequence encoding an ARD2DH enzyme having 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: 193, 194, 195, 196, or 197.

[0015] Any of the exogenous polynucleotide sequences can be operatively linked to a heterologous promoter or an artificial promoter. Heterologous or artificial promoters may be selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86), 6-phosphogluconic acid 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), phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO:134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135), enolase promoter (ENO1p; SEQ ID NO:136), asparagine synthase promoter (ASNSp; SEQ ID NO:137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138), and RPL16B (SEQ ID NO:139). Any of the exogenous polynucleotide sequences can be integrated into the genome of a yeast cell at a locus selected from the ER1, ER3, PDC1, pyrF, TRP3, gpdIIA, and gpdIIB loci.

[0016] This disclosure also provides a method for producing arbitrol using engineered cells described herein, the method comprising contacting a substrate containing dextran with the engineered cells described herein, wherein the engineered cells ferment the substrate to produce arbitrol. The fermentation temperature may be 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, or between these temperatures. The volumetric oxygen consumption rate (OUR) may be between 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). At least 0.2 g L of arbitrol may be produced. -1 h -1 0.3g L -1 h -1 0.5g L -1 h -1 0.75g L -1 h -1 Or at least 1.0 g L -1 h -1 The rate of aritol production is as follows. When fermentation is run at 35°C for 96 hours, the aritol yield 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 aritol production may be increased relative to equivalent fermentation run with equivalent yeast cells lacking genetic modification that overexpresses A5PP enzyme and lacking exogenous polynucleotide sequences encoding exogenous A5PP enzyme. The concentration of dextran can be at least 100 g / L.

[0017] This disclosure also provides the use of the engineered yeast described herein for the production of aritol. Attached Figure Description

[0018] This patent or application contains at least one color-drawn drawing. A copy of this patent or patent application publication with a color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0019] The accompanying figures illustrate, in a manner that is not restrictive, various aspects described herein.

[0020] Figure 1 The natural pentose phosphate pathway (dashed lines and arrows) and natural glycolysis pathway (solid lines and arrows) in *Saccharomyces cerevisiae* are shown.

[0021] Figure 2 The sequence space diversity of galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) was demonstrated.

[0022] Figure 3 The structural features of the NAD or NADP binding bag located at the characteristic GXGXXG motif (SEQ ID NO:133) of the XPDH enzyme are shown.

[0023] Figure 4 This demonstrates the spatial diversity of ribulose-5-phosphate reductase sequences.

[0024] Figure 5 The in vitro activities of TarJ' and XPDH enzymes as outlined in Example 3 were demonstrated.

[0025] Figure 6 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 96 hours of shake-flask fermentation of strains 1-1, 1-13a-f, and 1-15a-f as outlined in Example 5 are shown. Xylitol concentration (g / L) is reported using data labels.

[0026] Figure 7 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 96 hours of shake-flask fermentation of strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, and 1-39a-f as outlined in Example 5 are shown. Xylitol concentration (g / L) is reported using data labels.

[0027] Figure 8 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 96 hours of shake-flask fermentation of strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e as outlined in Example 6 are shown. Xylitol concentration (g / L) is reported using data labels.

[0028] Figure 9 The concentrations (g / L) of erythritol, ribitol, aritol, and xylitol metabolites during 96 hours of shake-flask fermentation of strains 1-13c, 1-12a-e, 1-14a-e, and 1-16a-e as outlined in Example 8 are shown. Data labels report the concentrations (g / L) of xylitol (strains 1-13c, 1-14a-e, and 1-16a-e) or aritol (strain 12a-e).

[0029] Figure 10The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 96 hours of shake-flask fermentation of strains-13c, 1-36a-e, and 1-40a-e as outlined in Example 9 are shown. Xylitol concentration (g / L) is reported using data labels.

[0030] Figure 11 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 96 hours of shake-flask fermentation of strains 1-30a-e, 1-31a-e, 1-32a-e, and 1-13c as outlined in Example 10 are shown. Xylitol concentration (g / L) is reported using data labels.

[0031] Figure 12 The comparison of xylitol and ribitol concentrations (g / L) produced in strains with (3-7a-f and 3-8a-f) and those without (1-13c and 1-15a) RPE2 overexpression is shown.

[0032] Figure 13 The corresponding yields of xylitol, ribitol, glycerol, and erythritol are shown for strains with (3-8a-f) and those without (1-15a) RPE2 overexpression.

[0033] Figure 14 The xylitol and ribitol titers (g / L) of the strain shown are displayed.

[0034] Figure 15 The yields of xylitol, ribitol, glycerol, and erythritol of the strains shown with different configurations of RPE overexpression are displayed.

[0035] Figure 16 The xylitol concentration (g / L) of the strains shown in Example 15 during 96 hours of shake-flask fermentation is displayed.

[0036] Figure 17 The xylitol concentration (g / L) of the strains shown in Example 15 during 96 hours of shake-flask fermentation is displayed.

[0037] Figure 18 The xylitol rate (g / (L•h)) between 48 hours and 96 hours of shake-flask fermentation of the strains shown in Example 15 is displayed.

[0038] Figure 19 The xylitol rate (g / (L•h)) between 48 hours and 96 hours of shake-flask fermentation of the strains shown in Example 15 is displayed.

[0039] Figure 20 The xylitol yield (%) between 48-hour and 96-hour shake-flask fermentation of the strains shown in Example 15 is displayed.

[0040] Figure 21 The xylitol yield (%) between 48-hour and 96-hour shake-flask fermentation of the strains shown in Example 15 is displayed.

[0041] Figure 22 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 18 are shown. The xylitol concentration (g / L) is reported using data labels.

[0042] Figure 23 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 18 are shown. The xylitol concentration (g / L) is reported using data labels.

[0043] Figure 24 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 22 are shown. The xylitol concentration (g / L) is reported using data labels.

[0044] Figure 25 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 22 are shown. The xylitol concentration (g / L) is reported using data labels.

[0045] Figure 26 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 23 are shown. The xylitol concentration (g / L) is reported using data labels.

[0046] Figure 27 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 23 are shown. The xylitol concentration (g / L) is reported using data labels.

[0047] Figure 28 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 24 are shown. The xylitol concentration (g / L) is reported using data labels.

[0048] Figure 29 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 24 are shown. The xylitol concentration (g / L) is reported using data labels.

[0049] Figure 30The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 25 are shown. The xylitol concentration (g / L) is reported using data labels.

[0050] Figure 31 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 27 are shown. The xylitol concentration (g / L) is reported using data labels.

[0051] Figure 32 The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 27 are shown. The xylitol concentration (g / L) is reported using data labels.

[0052] Figure 33 The concentrations (g / L) of erythritol and xylitol metabolites during 72-hour shake-flask fermentation of the strains as outlined in Example 28 are shown. The xylitol concentration (g / L) is reported using data labels.

[0053] Figure 34 The concentrations (g / L) of erythritol, glycerol, and aritol metabolites during 72-hour shake-flask fermentation of the strain as outlined in Example 30 are shown. The aritol concentration (g / L) is reported using data labels.

[0054] Figure 35 The concentrations (g / L) of xylitol and arbutin metabolites from the fermentation described in Example 32 are shown.

[0055] Figure 36 The concentrations (g / L) of xylitol and arbutin metabolites, as measured by HPIC, during the fermentation described in Example 32 are shown. Detailed Implementation

[0056] Reference will now be made specifically to certain aspects of the subject matter disclosed herein, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0057] In this document, unless the context clearly requires otherwise, the terms "an," "a," or "the" are used to include one or more. Unless otherwise indicated, the term "or" is used to mean a non-exclusive "or." All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety as if individually cited. In the event of any inconsistency between the usage in this document and those documents so incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of irreconcilable inconsistencies, the usage in this document shall prevail.

[0058] Values ​​expressed in range format should be interpreted flexibly to include not only the values ​​explicitly listed as limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise stated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise stated, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0059] Unless otherwise specified, ppm (parts per million), percentages, and ratios are based on weight. Percentages based on weight are also referred to below as weight% or (weight)%.

[0060] This disclosure relates to various recombinant cells engineered to produce aritol. Generally, the recombinant cells described herein are capable of producing aritol and are characterized by the overexpression of a phosphatase. Recombinant yeast may further be characterized by the overexpression of an X5PP enzyme or the inclusion of a foreign polynucleotide sequence encoding an X5PP enzyme. Recombinant yeast may further be characterized by the overexpression of a ribulose-5-phosphate epimerase (RPE) and the incorporation of a foreign polynucleotide sequence encoding aritol phosphate dehydrogenase (APDH) and / or ARD2DH enzymes. This disclosure also provides a fermentation method for producing aritol from dextran using the genetically engineered cells described herein.

[0061] Generally speaking, the recombinant cells described herein are yeast cells. As used herein, “yeast” refers to a eukaryotic unicellular microorganism classified as a member of the kingdom Fungi. Yeasts are unicellular organisms that evolved from multicellular ancestors, and some species retain multicellular characteristics, such as forming connected budding cell chains called pseudohyphae or false hyphae. Yeast cells are also referred to in the art as yeast-like cells, and as used herein, “yeast cell” encompasses both yeast and yeast-like cells. Suitable yeasts and yeast-like host cells used for modification may include, but are not limited to: *Saccharomyces cerevisiae*, *Komagataella* sp., *Kluyveromyces* (e.g., *Kluyveromyces lactis*, *Kluyveromyces marxianus*), *Yersinia lipolytica*, *Issatchenkia orientalis*, *Pichia galeiformis*, *Pichia* YB-4149 (NRRL name), *Pichia pastoris*, *Candida* (e.g., *Candida magnolia*, *Candida ethanolica*), *Pichia deserticola*, *Pichiamembranifadens*, *Pichia fermentans*, *Aspergillus flavus*, etc. Aspergillus, Trichoderma, Myceliphthora thermophila, Trichoderma (e.g., *Trichoderma*), Pfaffi, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., *Trichosporonoides macrocarpa*, *Trichoderma spp.*, *Trichoderma melanogaster*), *Trichosporonoides tsukuba*, *Trichoderma mutagenes*, Penicillium, and *Trichoderma spp.*. Those skilled in the art will understand that the requirements for selecting suitable yeast cells and the recombinant yeast cells of this disclosure are not limited to those explicitly listed herein. Methods for genetic engineering yeast cells are known and described in the art, and those skilled in the art will understand the methods necessary for transforming and engineering suitable yeast cells.

[0062] Suitable yeast cells can be Basidiomycota or Ustilago smut. Suitable yeasts of the Ustilago subphylum include, but are not limited to, *Ustilago* (e.g., *U. cynodontis*, *U. maydis*, *U. sphaerogena*, *U. cordal*, *U. scitaminea*, *U. coicis*, *U. syntherismae*, *U. esculenta*, *U. neglecta*, *U. crus-galli*, *Ustilago avenae*), *Sporisorium* (e.g., *Sporisorium exsertum*), and *M. tomentosa* (e.g., *M. acetoabutans*, *M. fonsecae*, *M.*). Madida, *M. megachiliensis*, *M. ocedocephalis*, *M. nigrescens*, and *Pseudozyma* (e.g., *Pseudozyma tsukuba*) and *Pseudozyma* species (e.g., *Pseudozyma megachiliensis*, *Pseudozyma ocedocephalis*, *Pseudozyma nigrescens*). Yeasts of the subphylum Ustilagoeum are known and described in the art as potential production organisms for valuable chemicals such as itaconic acid esters, malate esters, succinate esters, mannitol, and erythritol, as well as other valuable biotechnological applications.See, e.g., Geiser et al. 97(8):3253-65), Guevarra et al. ("Accumulation of itaconic, 2-hydroxyparaconic, itatartaric, and malic acids by strains of thegenus Ustilago, Agric.Biol.Chem., 1990, 54(9), 2353-2358) and Moon et al. ("Biotechnological production of erythritol and its applications,” ApplMicrobiol Biotechnol, 2010, 86:1017-1025).

[0063] Suitable yeast cells will possess an active pentose phosphate pathway for the production of ribulose-5-phosphate. As used herein, the "active pentose phosphate pathway" refers to the expression of one or more functional enzymes that together produce glucose-6-phosphate, NADP... + Alternatively, NAD+ and water are converted into NADPH or NADH, CO2, and ribulose-5-phosphate. Continuing in the non-oxidative phase, this pathway can also produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway can produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, and combinations thereof. The active pentose phosphate pathway can be native to yeast cells or can be introduced into yeast cells through genetic engineering.

[0064] Yeast cells can be osmolyzed yeast cells. As used herein, “osmolyzed” means yeast capable of growing and reproducing under conditions of high molar osmolar concentrations, such as at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v), or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), or at least 15% (w / v) sodium chloride. Species and strains of osmolyzed yeast are known and described in the art, including many species of yeast used in industrial fermentation processes. Similarly, methods for determining yeast osmolyzability are known and described in the art. See, for example, Tiwari, S. et al. (“Nectar yeast community of tropical flowering plants and assessment of their osmotolerance and xylitol-producing potential,” Current Microbiology, 2022, 79:28).

[0065] Recombinant yeast cells can be recombinant moniliform cells, such as moniliform yeast cells. Figure 1 The predicted natural pentose phosphate and glycolysis pathways in *Fragaria filamentous fungi* are shown. *Fragaria filamentous fungi* has been previously used in the fermentation production of erythritol, and methods for genetic modification and fermentation of *Fragaria filamentous fungi* are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb CellFact, 16:168).

[0066] Various plasmids and methods for transforming *Strombophytum comosum* are also described in the following examples. For example, *Strombophytum comosum* can be transformed using a dinucleotide sequence, wherein, after recombination, the exogenous polynucleotide of interest is integrated at a specific locus and a selection marker is expressed intracellularly. Suitable selection markers are known and used in the art. Optional markers may include, but are not limited to, amdS (e.g., broken into 3' parts SEQ ID NO:167 and 5' parts SEQ ID NO:174), G418 resistance gene (e.g., broken into 3' parts SEQ ID NO:172 and 5' parts SEQ ID NO:175), bleomycin (zeocin) resistance gene (e.g., broken into 3' parts SEQ ID NO:168 and 5' parts SEQ ID NO:169), nourseothricin N-acetyltransferase (NAT) (e.g., broken into 3' parts SEQ ID NO:171 and 5' parts SEQ ID NO:170), and invertase gene (SUC2) (e.g., the 3' part of SEQ ID NO:173 and the 5' part of SEQ ID NO:176).

[0067] The recombinant cells described herein contain one or more polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve the recombinant cells' fermentation of glucose into araitol.

[0068] The terms “glucose” and “dextrose” are used interchangeably in this document and refer to D-glucose unless otherwise explicitly stated.

[0069] As used herein, “exogenous” refers to genetic material or its expression product originating outside the host organism. For example, exogenous genetic material or its expression product can be a modified form of the host organism’s natural genetic material, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component. For example, when introduced into Saccharomyces cerevisiae, the Kluyveromyces lactis invertase gene is exogenous.

[0070] As used herein, “natural” means genetic material or its expression products found in the genome of wild-type cells of a host cell, except for inter-individual mutations that do not affect function or expression. For the purposes of this application, the Moniliella tomentosa var pollinis TCV364 cells described in US 6,440,712 are considered wild-type Moniliella tomentosa cells, which are incorporated herein by reference in their entirety and were deposited on March 28, 1997, under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800Vilvoorde).

[0071] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the total primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the macromolecule its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (independently or as part of a biosynthetic pathway) should be understood to include cofactors, coenzymes, and metals necessary for the enzyme to function properly. An overview of amino acids and their three-letter and one-letter symbols as understood in the art is provided in Table 1. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0072] Table 1: Three-letter and one-letter symbols for amino acids

[0073]

[0074] Variants or sequences having substantially the same identity or homology with the polypeptides described herein can be used to implement the disclosed recombinant cells, compositions, and methods. Such sequences may be referred to as variants or modified sequences. That is, the polypeptide sequence can be modified but still retains the ability to exhibit the desired activity. Typically, variants or modified sequences may include greater than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild-type, naturally occurring polypeptide sequence or with the variant polypeptides described herein.

[0075] As used herein, the phrases “sequence identity %”, “identity %”, and “identity percentage” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a normalized algorithm. Methods for amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and sequence identity generation involve both global and local alignments performed using computational methods. Alignment can be performed using BLAST (National Center for Biotechnology Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using a standard protein BLAST with the following default parameters: Maximum target sequence: 100; Short query: Parameters automatically adjusted for short input sequences; Expected threshold: 10; Word length: 6; Maximum number of matches within the query range: 0; Matrix: BLOSUM62; Empty penalty: (Existence: 11, Expansion: 1); Component adjustment: Conditional component score matrix adjustment; Filter: Not selected; Mask: Not selected. The standard nucleotide BLAST algorithm with the following default parameters can be used to determine the percentage of nucleic acid sequence identity between nucleic acid sequences: Maximum target sequence: 100; Short query: Parameters are automatically adjusted for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within the query range: 0; Match / mismatch score: 1, -2; Gaps penalty: Linear; Filter: Low complexity region; Mask: Mask only for lookup tables. Sequences that have an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently have XX% sequence identity with the reference sequence.

[0076] Peptide or polynucleotide sequence identity can be measured over the entire length of a defined peptide sequence (e.g., as defined by a specific SEQ ID number) or over a shorter length, such as over a fragment taken from a larger defined peptide sequence (e.g., a fragment of at least 15, 20, 30, 40, 50, 70, or 150 consecutive residues). Such lengths are merely exemplary and it should be understood that any fragment length supported by the sequences shown in this document, tables, figures, or sequence listings can be used to describe the length of measurable percentage identity.

[0077] The polypeptides disclosed herein may include “variant” polypeptides, “mutants”, and “derivatives thereof.” As used herein, the term “wild-type” is a term understood by those skilled in the art and refers to the typical form of the polypeptide in nature, as distinct from variant or mutant forms. As used herein, “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence different from that of a reference protein or polypeptide molecule. Variants or mutants may have insertions, deletions, or substitutions of one or more amino acid residues relative to the reference molecule.

[0078] The amino acid sequences of peptide variants, mutants, derivatives, or fragments considered herein may include conserved amino acid substitutions relative to a reference amino acid sequence. For example, variant, mutant, derivative, or fragment peptides may include conserved amino acid substitutions relative to a reference molecule. A “conserved amino acid substitution” is one in which an amino acid is substituted to a different amino acid that is predicted to have the least interference with the properties of the reference peptide. In other words, a conserved amino acid substitution essentially preserves the structure and function of the reference peptide. Conserved amino acid substitutions typically maintain (a) the structure of the peptide backbone in the substituted region, such as a β-sheet or α-helical conformation, (b) the charge and / or hydrophobicity of the molecule at the substitution site, and / or (c) the volume of the side chains.

[0079] As used herein, the terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence” are used interchangeably with “nucleic acid” and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may indicate a sense strand or antisense strand. DNA polynucleotides may be cDNA (e.g., encoding DNA) or genomic DNA sequences (e.g., including introns and exons).

[0080] A polynucleotide is said to encode a polypeptide if, in its natural state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce a polypeptide or a fragment thereof. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0081] Those skilled in the art understand the degeneracy of the genetic code and that multiple polynucleotides can encode the same polypeptide. In some aspects, polynucleotides (e.g., polynucleotides encoding the A5PP polypeptide) can be codon-optimized for expression in specific cells, including but not limited to plant cells, bacterial cells, fungal cells, or animal cells. While polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptide described herein may be used. Therefore, sequences that are not naturally occurring may be used. These sequences may be desired, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and the human hand can also be used to generate degenerate coding sequences.

[0082] The recombinant cells described herein may contain deletions or disruptions in one or more natural genes. The phrase "deletion or disruption" refers to the state of a natural gene in a recombinant cell, having a completely eliminated coding region (deletion) or a modification of the gene, its promoter, or its terminator (such as by deletion, insertion, or mutation) such that the gene no longer produces an active expression product, produces a severely reduced amount of expression product (e.g., reduced by at least 75% or at least 90%), or produces an expression product with severely reduced activity (e.g., reduced by at least 75% or at least 90%). Deletions or disruptions can be achieved through genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. The natural gene to be deleted or disrupted can be replaced with a foreign nucleic acid of interest for the expression of a foreign gene product (e.g., peptides, enzymes, etc.).

[0083] The recombinant cells described herein may contain one or more genetic modifications, wherein exogenous nucleic acids are integrated into the genome of the host cell. Those skilled in the art know how to select suitable loci in the yeast genome for the integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in *Saccharomyces cerevisiae* host cells, suitable interaction sites may include, but are not limited to, the ER1 locus (defined as a locus flanking SEQ ID NO:85 and SEQ ID NO:162), the ER3 locus (defined as a locus flanking SEQ ID NO:155 and SEQ ID NO:165), the PDC1 locus (defined as a locus flanking SEQ ID NO:152 and SEQ ID NO:164), the pyrF locus (defined as a locus flanking SEQ ID NO:153 and SEQ ID NO:163), the TRP3 locus (defined as a locus flanking SEQ ID NO:156 and SEQ ID NO:159), the gpdIIA locus (defined as a locus flanking SEQ ID NO:157 and SEQ ID NO:161), and the gpdIIB locus (defined as a locus flanking SEQ ID NO:158 and SEQ ID NO:166). Exogenous nucleic acids may also be integrated into intergenic regions or other locations in the host cell genome not specifically specified herein. Those skilled in the art can identify other suitable integration loci. Furthermore, those skilled in the art will recognize how sequences can be used to design primers to verify correct gene integration at the selected locus.

[0084] Recombinant cells may have one or more copies of a given exogenous nucleic acid sequence, which is integrated into the host chromosome and replicates along with the chromosome into which it is integrated. For example, yeast cells can be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide described herein, and the polynucleotide sequence encoding the polypeptide may be integrated into the host chromosome in one or more copies. Recombinant cells may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten or more copies of the polynucleotide sequence encoding a polypeptide described herein integrated into the genome. The multiple copies of the polynucleotide sequence may be incorporated entirely into a single locus or may be incorporated into multiple loci.

[0085] The final step in the araitol pathway, from araitol 5-phosphate to araitol, requires a phosphatase. Saccharomyces cerevisiae PYP1 (polyol phosphatase) is an example. 1 The gene encodes a sugar alcohol phosphatase that hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (D)-glycerol-3-phosphate (Xu et al., “Discovery and functional characterization of a yeast sugaralcohol phosphatase,” ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova et al., “Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae,” J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the sorbitol-6-phosphatase class (Enzyme Commission (EC) 3.1.3.50). Since araitol 5-phosphate and xylitol 5-phosphate are molecules with known substrate similarities to PYP1, this paper demonstrates that one or more PYP-like enzymes or PYP orthologs possess xylitol-5-phosphate phosphatase activity and / or araitol-5-phosphate phosphatase activity and can be used to increase xylitol or araitol production in the recombinant cells described herein. *Escherichia coli* HxpA (hexitol phosphatase A) is a HAD-like enzyme belonging to EC 3.1.3.50, which has a substrate profile similar to PYP1 (Kuznetsova et al., “Genome wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphate family,” 2006, J. Biol. Chem., 281, 36149-36161). Therefore, this paper also demonstrates that one or more HAD-like hydrolases or HAD-like hydrolases or their orthologs possess xylitol-5-phosphate phosphatase activity and / or aritol-5-phosphate phosphatase activity, and can be used to increase xylitol or aritol production in the recombinant cells described herein.

[0086] The recombinant cells described herein are capable of producing araitol and are characterized by overexpression of a natural enzyme with araitol-5-phosphate phosphatase (A5PP) activity and / or inclusion of a foreign polynucleotide sequence encoding a natural or foreign enzyme with araitol-5-phosphate phosphatase (A5PP) activity. Generally, recombinant cells including overexpression of A5PP enzyme or expression of foreign A5PP enzyme produce more araitol compared to equivalent cells lacking foreign A5PP enzyme or lacking overexpression of A5PP enzyme. The enzyme can be any suitable enzyme with A5PP activity. As used herein, “A5PP enzyme” and “A5PP” are interchangeable and refer to an enzyme with A5PP activity. In this document, “araitol-5-phosphate phosphatase activity” and “A5PP activity” are used interchangeably and refer to the ability to catalyze the conversion of araitol-5-phosphate to araitol and phosphate. Suitable A5PP enzymes may include divalent metal cations, such as Mg2+. 2+ Mn 2+ or Co 2+Suitable enzymes with A5PP activity may include, but are not limited to, those classified as EC3.1.3.50, such as sugar alcohol phosphatases and HAD-like hydrolases. The polynucleotide encoding an A5PP enzyme may be derived from any suitable source. For example, polynucleotides encoding the A5PP enzyme can be derived from *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Lachancea dasiensis*, *Tetrapisispora blattae*, *Saccharomyces pastorianus*, *Kazachstania Africana*, *Podospora comata*, *Geotrichum candidum*, *Ogattaea haglerorum*, *Debaryomyces fabryi*, *Monilinia fructicola*, *Nadsonia fulvescens* longleaf variant DSM 6958, *Escherichia coli*, *Wickerhamomyces ciferrii*, and *Bacillus amyloliquefaciens*. A5PP enzyme can be a polypeptide having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NO: 200, 201, 202, 203, 204, 205, 206, 210, and 213. A5PP enzyme can be a polypeptide having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NO: 200, 203, 204, 206, and 213.

[0087] The A5PP enzyme described herein also exhibits activity towards xylitol-5-phosphate and can be described as a xylitol-5-phosphate phosphatase with xylitol-5-phosphate phosphatase activity. As used herein, "xylitol-5-phosphate phosphatase activity" and "X5PP activity" are used interchangeably and refer to the ability to catalyze the conversion of xylitol-5-phosphate to xylitol and phosphate. Therefore, the polypeptide described herein can be both an A5PP enzyme and an X5PP enzyme, as a given polypeptide possesses both A5PP and X5PP activities. Describing an enzyme as having X5PP activity or as an X5PP enzyme herein does not imply that the enzyme is not an A5PP enzyme.

[0088] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a *Saccharomyces cerevisiae* gene encoding the amino acid sequence of SEQ ID NO:200. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:200.

[0089] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO:201. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:201.

[0090] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a Darcylla cylindrica gene encoding the amino acid sequence of SEQ ID NO:202. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:202.

[0091] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a Tetradisporum roxburghii gene encoding the amino acid sequence of SEQ ID NO:203. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:203.

[0092] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a Pasteurella gene encoding the amino acid sequence of SEQ ID NO:204. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:204.

[0093] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from an African Kazakhstani yeast gene encoding the amino acid sequence of SEQ ID NO:205. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:205.

[0094] The recombinant cells may contain a foreign polynucleotide sequence that is or may be derived from a *Trichoderma longicornis* gene encoding the amino acid sequence of SEQ ID NO:206. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:206.

[0095] The recombinant cells may contain a foreign polynucleotide sequence that is or may be derived from a Geotrichum candidum gene encoding the amino acid sequence of SEQ ID NO:207. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:207.

[0096] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a Hagler's Ogata yeast gene encoding the amino acid sequence of SEQ ID NO:208. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:208.

[0097] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a *Fabried barley* gene encoding the amino acid sequence of SEQ ID NO:209. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:209.

[0098] The recombinant cells may contain a foreign polynucleotide sequence that is or may be derived from a *Sclerotinia sclerotiorum* gene encoding the amino acid sequence of SEQ ID NO:210. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:210.

[0099] The recombinant cells may contain a foreign polynucleotide sequence that is, or may be derived from, the gene encoding the amino acid sequence of SEQ ID NO:211, of the *Rhodotorula rubiginii* longleaf variety DSM 6958. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:211.

[0100] The recombinant cells may contain a foreign polynucleotide sequence that is or may be derived from an E. coli gene encoding the amino acid sequence of SEQ ID NO:213. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:213.

[0101] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from the *Wickhamia spp.* gene encoding the amino acid sequence of SEQ ID NO:214. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:214.

[0102] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from a *Saccharomyces cerevisiae* gene encoding the amino acid sequence of SEQ ID NO:221. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:221.

[0103] The recombinant cells may contain a foreign polynucleotide sequence that is or may be derived from a Bacillus amyloliquefaciens gene encoding the amino acid sequence of SEQ ID NO:222. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:222.

[0104] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* DOG2 gene encoding the amino acid sequence of SEQ ID NO:189. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:189.

[0105] The recombinant cell may contain a foreign polynucleotide sequence that is or may be derived from the *Saccharomyces cerevisiae* DOG1 gene encoding the amino acid sequence of SEQ ID NO:188. The foreign polynucleotide sequence may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:188.

[0106] Enzymes with A5PP activity can be native to the host cell. For example, when the host organism is *Saccharomyces cerevisiae*, the A5PP enzyme can be an enzyme having a sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% with at least one of SEQ ID NO: 198, 199, 200, or 221. Recombinant cells may contain exogenous polynucleotides encoding an A5PP enzyme having a sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% with at least one of SEQ ID NO: 198, 199, 200, or 221. The recombinant cell may contain genetic modifications that increase the expression of an A5PP enzyme, wherein the A5PP enzyme is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 198, 199, 200, or 221. Genetic modifications may include, but are not limited to, inserting an additional copy of the nucleotide encoding the natural A5PP enzyme into the cell (e.g., integrating an additional copy of the A5PP-encoding polynucleotide into a non-natural locus in the cell), inserting a constitutive promoter upstream of the coding region of the natural A5PP enzyme-encoding gene in the host cell's genome, and / or modifying an existing promoter upstream of the coding region of the natural A5PP enzyme-encoding gene in the host cell's genome. Those skilled in the art will recognize that the expression of the natural A5PP enzyme-encoding gene can be increased by a variety of methods known in the art, and will be able to appropriately select and apply these methods.

[0107] As used herein, “overexpression” means that the expression level of a polypeptide is higher than that of the same polypeptide in equivalent cells in the absence of genetic modification or exogenous polynucleotides encoding the polypeptide.

[0108] The recombinant cells described herein, capable of producing aritol and including overexpression of exogenous polynucleotide sequences encoding A5PP enzymes and / or native A5PP enzymes, are further characterized by overexpression of ribulose-5-phosphate epimerase (RPE enzyme). Generally, recombinant cells including RPE enzyme overexpression produce more aritol compared to equivalent cells lacking RPE enzyme or lacking RPE enzyme overexpression.

[0109] The recombinant cells described herein are capable of producing aritol, contain an exogenous polynucleotide sequence encoding an A5PP enzyme and / or overexpress a native A5PP enzyme, and may contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have genetic modifications leading to the overexpression of a native RPE enzyme. The RPE enzyme can be any suitable enzyme with ribulose-5-phosphate epimerase activity. As used herein, “ribulose-5-phosphate epimerase activity” and “RPE activity” are used interchangeably and refer to the ability to catalyze the conversion of ribulose-5-phosphate to xylulose-5-phosphate. The enzyme with RPE activity can be native to the host cell, or the RPE enzyme can be an exogenous RPE enzyme. For example, when the host organism is *Saccharomyces cerevisiae*, the RPE enzyme can be an enzyme with a sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to at least one of SEQ ID NO: 179 and 180. The recombinant cell may contain an exogenous polynucleotide encoding an RPE enzyme having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with at least one of SEQ ID NO: 179 and 180. The recombinant cell may contain genetic modifications that increase the expression of the RPE enzyme, having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with at least one of SEQ ID NO: 179 and 180. Genetic modifications may include, but are not limited to, inserting an additional copy of the nucleotide encoding the natural RPE enzyme into the cell, inserting a constitutive promoter upstream of the coding region of the natural RPE enzyme gene in the host cell's genome, and / or modifying an existing promoter upstream of the coding region of the natural RPE enzyme gene in the host cell's genome. Those skilled in the art will recognize that the expression of the natural RPE enzyme gene can be increased by a variety of methods known in the art, and will be able to appropriately select and apply these methods.

[0110] The recombinant cells described herein are capable of producing aritol, containing exogenous polynucleotides encoding the A5PP enzyme and / or overexpressing the native A5PP enzyme, and may contain exogenous polynucleotide sequences encoding aritol 2-dehydrogenase (ARD2DH) enzyme. The recombinant cells described herein are capable of producing aritol, containing exogenous polynucleotides encoding the A5PP enzyme and / or overexpressing the native A5PP enzyme, and may contain exogenous polynucleotide sequences encoding the aritol 2-dehydrogenase (ARD2DH) enzyme, and may contain exogenous polynucleotides encoding the native or exogenous RPE enzyme, or may have genetic modifications leading to overexpression of the native RPE enzyme, as described herein. The exogenous polynucleotide sequence may be an exogenous ARD2DH gene.

[0111] The terms "araitol 2-dehydrogenase" and "ARD2DH gene" are used interchangeably in this document and refer to any gene or polynucleotide encoding a polypeptide with araitol 2-dehydrogenase activity. As used herein, "araitol 2-dehydrogenase activity" refers to the catalytic conversion of D-ribulose and NADH or NADPH to D-araitol and NAD2DH. + or NADP + The ability to encode ARD2DH enzymes is described. Enzymes possessing ARD2DH 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 pastoris*, *Candida albicans*, *Kwoniella heveanensis*, or *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 with at least one of SEQ ID NO: 193, 194, 195, 196, or 197. The ARD2DH gene may 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 with at least one of SEQ ID NO: 194, 195, 196, or 197. Additional description of recombinant cells capable of producing aritol and comprising a polypeptide having aritol 2-dehydrogenase activity is provided in U.S. Provisional Application No. 63 / 364359, filed May 9, 2022, which is incorporated herein by reference in its entirety.

[0112] The recombinant cells may include genetic modifications leading to overexpression of the RPE enzyme and a foreign polynucleotide, which is or may be derived from the Beauveria bassiana ARD2DH gene encoding the amino acid sequence of SEQ ID NO:193. The foreign 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 with the amino acid sequence of SEQ ID NO:193.

[0113] The recombinant cell may include genetic modifications leading to overexpression of the RPE enzyme and a foreign polynucleotide, which is or may be derived from the Pichia pastoris ARD2DH gene encoding the amino acid sequence of SEQ ID NO:194. The foreign 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 with the amino acid sequence of SEQ ID NO:194.

[0114] The recombinant cell may include genetic modifications leading to overexpression of the RPE enzyme and a foreign polynucleotide, which is or may be derived from the Candida albicans ARD2DH gene encoding the amino acid sequence of SEQ ID NO:195. The foreign 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 with the amino acid sequence of SEQ ID NO:195.

[0115] The recombinant cells may include genetic modifications leading to overexpression of the RPE enzyme and a foreign polynucleotide, which is or may be derived from the Kwoniella heveanensis ARD2DH gene encoding the amino acid sequence of SEQ ID NO:196. The foreign 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 with the amino acid sequence of SEQ ID NO:196.

[0116] The recombinant cells may include genetic modifications leading to overexpression of the RPE enzyme and a foreign polynucleotide, which is or may be derived from the *Candida maltose* ARD2DH gene encoding the amino acid sequence of SEQ ID NO:197. The foreign 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 with the amino acid sequence of SEQ ID NO:197.

[0117] The recombinant cells described herein are capable of producing arabinoliter, containing a foreign polynucleotide sequence encoding an A5PP enzyme and / or overexpressing the natural A5PP enzyme, and may contain a foreign polynucleotide sequence encoding an arabinoliter phosphate dehydrogenase (APDH) enzyme. The foreign polynucleotide sequence may be the foreign arabinoliter-phosphate dehydrogenase (APDH) gene. The recombinant cells described herein, capable of producing arabinoliter, containing a foreign polynucleotide sequence encoding an A5PP enzyme and / or overexpressing the natural A5PP enzyme, and containing a foreign polynucleotide sequence encoding an APDH enzyme, may also contain a foreign polynucleotide encoding a natural or foreign RPE enzyme, or may have genetic modifications leading to the overexpression of the natural RPE enzyme, as described herein.

[0118] The terms "araitol-phosphate dehydrogenase gene" and "APDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having araitol-phosphate dehydrogenase activity. As used herein, "araitol-phosphate dehydrogenase activity" refers to the catalytic conversion of (i) xylulose 5-phosphate and NADPH or NADH to araitol-1-phosphate and NADP. + or NAD + and / or (ii) ribulose-5-phosphate and NADPH or NADH are converted to araitol-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.

[0119] The recombinant cell may include genetic modifications leading to overexpression of the RPE enzyme and a foreign polynucleotide, which is or may be derived from the *Lactobacillus salivarius* cp400 gene encoding the amino acid sequence of SEQ ID NO:11. The foreign 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 with the amino acid sequence of SEQ ID NO:11.

[0120] The exogenous polynucleotides in the recombinant cells described herein can be controlled by a promoter. For example, exogenous nucleic acids can be operatively linked to heterologous or artificial promoters. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarbonylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:86); 6-phosphogluconic acid 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); phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO:134); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:135); enolase promoter (ENO1p; SEQ ID NO:136); asparagine synthase promoter (ASNSp; SEQ ID NO:136). NO:137); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:138); and RPL16B (SEQ ID NO:139).

[0121] The exogenous nucleic acids in the recombinant cells described herein can be controlled by terminators. For example, the exogenous nucleic acids can be operatively linked to heterologous or artificial terminators. Suitable terminators are known and described in the art. Terminators may include, but are not limited to, GAL10 terminator, PDC terminator, transaldolase terminator (TAL), 6PGD terminator (6PGDt; SEQ ID NO:140), ASNS terminator (ASNSt; SEQ ID NO:141), ENO1 terminator (ENO1t; SEQ ID NO:142), hexokinase 1 terminator (HXK1t; SEQ ID NO:143), PGK1 terminator (PGK1t; SEQ ID NO:144), PGM1 terminator (PGM1t; SEQ ID NO:145), PYK1 terminator (PYK1t; SEQ ID NO:146), RPLA terminator (RPLAt; SEQ ID NO:147), transaldolase 1 terminator (TAL1t; SEQ ID NO:148), TDH3 terminator (TDH3t; SEQ ID NO:149), and translation elongation factor 2 terminator (TEF2t; SEQ ID NO:149). NO:150); triose phosphate isomerase 1 terminator (TPI1t; SEQ ID NO:151); and MpTEF1 (SEQ ID NO:289).

[0122] If the position of a promoter or terminator relative to the polynucleotide in the genome or expression cassette allows the promoter or terminator (as the case may be) to perform its transcriptional control function, then the promoter or terminator is “operably linked” to the given polynucleotide (e.g., a gene).

[0123] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent sense or antisense strands. Recombinant polynucleotides are polynucleotides formed by laboratory methods and may comprise polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Therefore, a construct may include novel modifications to an endogenous gene introduced, for example, by genome editing techniques. A construct may also include recombinant polynucleotides produced using, for example, recombinant DNA methods. A construct may be a vector containing a promoter operatively linked to a polynucleotide encoding the polypeptide described herein. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide linked thereto. A vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA fragments may be integrated.

[0124] This disclosure also provides a fermentation method for producing arabinol using the recombinant cells described herein. The fermentation method includes the step of using a genetically engineered yeast fermentation substrate described herein to produce arabinol. As will be understood by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and separating arabinol from the fermentation broth. The fermentation process may be a fully aerobic or partially aerobic process.

[0125] Fermentation methods can be operated using suitable fermentation substrates. Substrates for fermentation methods may include glucose, sucrose, galactose, mannose, molasses, xylose, fructose, starch hydrolysates, lignocellulose hydrolysates, or combinations thereof. Those skilled in the art will recognize which fermentation substrates are suitable for a given fermentation organism and system.

[0126] Fermentation processes can be operated under a variety of conditions. The fermentation temperature (i.e., the temperature of the fermentation broth during processing) can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably about 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to any particular range or temperature described herein and can be appropriately modified.

[0127] The fermentation process can operate within a certain oxygen uptake (OUR) range. 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 implementations, the ratio of OUR can range from 0.05 mmol O2 / (g cell dry weight • h) to 10 mmol O2 / (g cell dry weight • h), 0.1 mmol O2 / (g cell dry weight • h) to 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 of the fermentation process or the ratio of OUR is not limited to any specific rate or range described herein.

[0128] The fermentation process can be operated at various cell concentrations. In some embodiments, the cell dry weight at the end of fermentation can be from 5 g / L to 40 g / L, 8 g / L to 30 g / L, or 10 g / L to 20 g / L. Furthermore, the pitch density or pitting rate of the fermentation process can vary. In some embodiments, the pitch density can be from 0.05 g / L to 11 g / L, 0.1 g / L to 10 g / L, or 0.25 g / L to 8 g / L.

[0129] The initial dextrose concentration for 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.

[0130] Fermentation processes can be associated with a variety of characteristics, including, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be influenced by yeast selection and / or genetic modifications of the yeast used in the fermentation process. These characteristics can also be influenced by adjusting fermentation process conditions. These characteristics can be modulated through a combination of yeast selection or modification and the selection of fermentation process conditions.

[0131] The aritol production rate of this process can be at least 0.2 g / L. -1 h -1 0.3g L -1 h -1 0.5g L -1 h -1 0.75g L -1 h -1 Or at least 1.0 g L -1 h -1 The arbitrol yield of this process can be at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. The final arbitrol titer of this process can be at least 5 g / L, 10 g / L, 20 g / L, 30 g / L, 50 g / L, 75 g / L, or 100 g / L.

[0132] The fermentation process can be operated as a dextran feed batch. Furthermore, the fermentation process can be a batch process, a continuous process, or a semi-continuous process, as understood by those skilled in the art.

[0133] Example

[0134] The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

[0135] Throughout the embodiments, strain numbers and sequence identification numbers were used consistently. For example, strain 1-1 in Example 4 is the same as strain 1-1 in Example 5, etc.

[0136] Example 1: Diversity of xylitol-phosphate dehydrogenase

[0137] Approximately three thousand galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) enzyme sequences were obtained from Uniprot and analyzed. Figure 2The natural sequence diversity of this group of sequences is shown. The group is diverse, with approximately 25% of the enzymes not sharing more than 75% homology. Since these enzymes tend to prefer NAD over NADP as a cofactor, cofactor binding preference of homologs was assessed in a manner similar to that described by Duax et al. (“Rational proteomics I. Fingerprinting identification and cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme family,” Proteins, 2003, 53(4):931-943). Cofactor binding bags were identified by approximation of the Rossman fold (from +23 to +30 amino acids of the GXGXXG motif (SEQ ID NO:129)) and scored based on the total charge within an 8-amino acid residue window. The top 8 candidates predicted to use NADP, along with 4 candidates predicted to use NAD and 3 controls, were selected for further characterization.

[0138] Further examination of the predicted structural features of the binding bag revealed important aspartic acid residues that could potentially influence cofactor preference. See also Figure 3 The polypeptide of SEQ ID NO:34 and its substitutions were used to construct a structural homology model for predicting the cofactor-binding bag conformation. Figure 3 The C-terminus of the penultimate β-chain outside the Rossman fold domain is shown. Without being bound by any particular theory, it is expected that enzymes in which the first residue (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 preferentially be NAD cofactors due to the hydrogen bonding between aspartic acid and the hydroxyl group of NAD ribose. However, enzymes in which 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 preferentially be NADP cofactors because the positive charge on the lysine or arginine residue will interact with the negative charge of the phosphate of NADP, and the smaller residue in the first position allows space for the phosphate in the binding pouch. Based on this analysis, 12 additional enzymes were selected according to the predicted preference for NADP. Finally, 6 additional enzymes with sequence similarity to the active XPDH enzyme were selected for testing.

[0139] Example 2: TarJ' diversity

[0140] Approximately 800 ribulose 5-phosphoreductase 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, as only 10% of the enzymes do not have more than 75% identical sequence similarity. Since these enzymes tend to prefer NADP over NAD as a cofactor, no scoring was performed, and the sequences were simply aligned in Geneous (ClustalW, default setting). Eight enzymes were selected for further analysis based on sequence similarity.

[0141] Example 3: In vitro enzyme assay

[0142] Polynucleotides encoding suspected XPDH homologues (Table 2) or TarJ' homologues (Table 3) were cloned into vectors containing T7 promoters and terminators for cell-free protein expression (New England Biolabs, PURExpress). ® InVitro Protein Synthesis. The activities of cell-free synthesized proteins on four substrates (ribulose 5-phosphate, xylulose 5-phosphate, ribulose, and xylulose) were analyzed using NADP or NAD cofactors. Seven enzymes (XPDH of SEQ ID NO: 12 and 34, TarJ' of SEQ ID NO: 36, 37, 38, 40, and 42) were able to catalyze the reduction of ribulose 5-phosphate or xylulose 5-phosphate. Figure 5 However, it cannot catalyze the reduction of xylulose or ribulose (results not shown).

[0143] Table 2: XPDH homologs

[0144]

[0145]

[0146] Table 3: TarJ' homologues

[0147]

[0148] Example 4: Genetically modified *Saccharomyces cerevisiae* strain

[0149] Strain 1-1 is the host strain of *Moniliellatomentosa* “Moniliellatomentosa var pollinis TCV364” described in US 6,440,712, which is incorporated herein by reference in its entirety and was deposited on March 28, 1997, under the Budapest Treaty, with the accession number MUCL40385 at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde). Table 4 below lists various *Moniliellatomentosa* strains, including information on the parent strains, sequences of the transformed parent strains, and characterization of the expression cassettes contained in the transformed sequences. Each “XPDH / TarJ’ homologue expression cassette” contains, in sequence, a 5’ ER1 side sequence (SEQ ID NO:85), an MpPYK1 promoter (SEQ ID NO:86), a gene encoding the indicated XPDH or TarJ’ homologue (one of SEQ ID NO:87-128), an Mp6PGD terminator (SEQ ID NO:140), and the 5’ portion of the G418 resistance gene expression cassette (SEQ ID NO:175). Each “optional marker cassette” contains, in sequence, the 3’ portion of the G418 resistance gene expression cassette (SEQ ID NO:172), an MpTEF2 terminator (SEQ ID NO:150), and a 3’ ER1 side sequence (SEQ ID NO:160). After binary transformation using the XPDH / TarJ' homologue expression cassette and the optional marker cassette, the two cassettes were recombined to integrate the nucleotide sequence encoding XPDH or TarJ' homologue and the G418 resistance marker at the ER1 locus.

[0150] The parent strain was first protoplastized by adding an enzyme mixture containing 0.6 M MgSO4, 7.5 g / L lysis enzyme, and 12.5 g / L Trichoderma harzianum lyase to the mycelial pellet of the parent strain, thereby transforming the indicated *Trichoderma harzianum* parent strain with the indicated sequence. The protoplast pellet was then washed with 0.6 M MgSO4 and resuspended in STC medium (0.6 M sucrose, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). 100 µg of single-stranded salmon sperm DNA and 1.5 µg to 5 µg of 5' and 3' DNA transformation fragments (total 3 µg–10 µg; fragment list see Table 4) were added to approximately 200 μL of the protoplast mixture (10... 8 The bipartite transformation was prepared using a concentration of 1 cell / mL. Then, 1 mL of STC medium containing 50% PEG was added to the salmon sperm DNA, transformed DNA, and protoplast mixture, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, the recovered culture medium (0.4 M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L sugar, pH 4.5) was added to the mixture, and incubation was carried out at 27°C and 100 rpm for 16 to 24 hours. After incubation, the protoplasts were precipitated by centrifugation and resuspended in 1 mL of PBS.

[0151] Resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30-35°C for at least 2-4 days until transformants grew. Integration of the indicated sequence into the transformed organisms was evaluated by colony PCR. The PCR-validated isolates were then assigned the indicated strain number. In some cases, more than one PCR-validated isolate (e.g., "sister" isolates) were indicated by a letter following the strain number. For example, strains 1-2 had five sister isolates: strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.

[0152] For example, strain 1-1 was transformed with SEQ ID NO:43 and SEQ ID NO:44. SEQ ID NO:43 contains (i) 3' flanking DNA for targeting chromosome integration into the ER1 locus (SEQ ID NO:162), and (ii) the 3' portion of a G418 resistance gene selectable marker (SEQ ID NO:172). SEQ ID NO:44 contains (i) an expression cassette (SEQ ID NO:87) of an XPDH homologue of *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; (ii) 5' flanking DNA for targeting chromosome integration into the ER1 locus (SEQ ID NO:85); and (iii) the 5' portion of a G418 resistance gene selectable marker (SEQ ID NO:175). Transformants were selected on a PDA + 250 mg / L genimycin (G418) selection plate and incubated at 30-35°C for at least 2 days until growth. The resulting transformants were streaked onto a PDA + genimycin (G418) plate for single-colony isolation, and single colonies were selected. Integration of the indicated sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.

[0153] Table 4.

[0154]

[0155]

[0156] Example 5: Shake-flask fermentation determination

[0157] 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 the production of ribitol, xylitol, glycerol, and ethanol.

[0158] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0159] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 6 and... Figure 6 and Figure 7 middle.

[0160] Table 5: Production Culture Media

[0161]

[0162] Although PCR verification indicated the presence of the transformed polynucleotide sequence in the indicated strains, further analysis showed that in some strains, the sequence was not properly 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 contained the transformed polynucleotide sequence, but it was not integrated at the ER1 locus.

[0163] Table 6: Results of 96-hour shaking test

[0164]

[0165]

[0166] Example 6: Shake-flask fermentation assay

[0167] 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 the production of ribitol, xylitol, glycerol, and ethanol.

[0168] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0169] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 7 and... Figure 8 middle.

[0170] like Figure 8 As shown, 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 (strain 1-1). Figure 6 Significantly more xylitol. When strains 1-34a, 1-34b, and 1-34e were initially validated by PCR, the integrated polynucleotide that was later determined to encode the *Neocobacterium cucumberis* XPDH homologue contained a frameshift mutation and did not express 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 the polynucleotide encoding functional XPDH.

[0171] Table 7: Results of 96-hour shaking flask operation

[0172]

[0173]

[0174] Example 7: Shake-flask fermentation determination

[0175] 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 the production of ribitol, xylitol, glycerol, and ethanol.

[0176] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0177] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. The glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 8.

[0178] Table 8: Results of 96-hour shaking flask operation

[0179]

[0180]

[0181] Example 8: Shake-flask fermentation determination

[0182] 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 the production of ribitol, xylitol, glycerol, and ethanol.

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

[0184] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 9 and Figure 9 middle.

[0185] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the indicated strains, further analysis indicated that in some strains, the sequence was not properly integrated at the ER1 locus. Further analysis indicated that strains 1-16b-e contained the transformed polynucleotide sequence, but it was not at the ER1 locus. Further analysis of the integration site in strains 1-2c and 1-2d was inconclusive.

[0186] Table 9: Results of 96-hour shaking flask operation

[0187]

[0188]

[0189] Example 9: Shake-flask fermentation determination

[0190] 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 the production of ribitol, xylitol, glycerol, and ethanol.

[0191] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0192] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 10 and... Figure 10 middle.

[0193] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the indicated strains, further analysis indicated that in some strains, the sequence was not properly integrated at the ER1 locus. Further analysis indicated that strains 1-8c, 1-8d, and 1-41c contained the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integrated locus in strains 1-36a, 1-41b, 1-41e, and 1-20a-e was inconclusive.

[0194] Table 10: Results of 96-hour shaking flask operation

[0195]

[0196]

[0197] Example 10: Shake Flask Fermentation Determination

[0198] 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 the production of ribitol, xylitol, glycerol, and ethanol.

[0199] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0200] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 11 and... Figure 11 middle.

[0201] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the indicated strains, further analysis indicated that in some strains, the sequence was not properly integrated at the ER1 locus. Further analysis indicated that strains 1-30c and 1-30d contained the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integrated locus in strain 1-6c was inconclusive.

[0202] Table 11: Results of 96-hour shaking flask operation

[0203]

[0204]

[0205] Example 11: Two copies of XPDH-modified *Saccharomyces cerevisiae* strain

[0206] 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 amino acid sequence of SEQ ID NO: 12, controlled by the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) 3' flanking DNA for targeting chromosome integration into the ER1 locus (SEQ ID NO: 162). SEQ ID NO:55 contains (i) 5' flanking DNA for targeting chromosomal integration into the ER1 locus (SEQ ID NO:85), (ii) an expression cassette (SEQ ID NO:98) encoding the amino acid sequence of SEQ ID NO:12 under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140; and (iii) the 5' portion of a G418 selectable marker (SEQ ID NO:175). Transformants were selected on a PDA + 250 mg / L genimycin (G418) selection plate and incubated at 35°C for at least 2 days until growth. The resulting transformants were streaked onto PDA + genimycin (G418) plates for single-colony isolation and selection. Integration of two copies of the Clostridium difficile XPDH sequence into the selected colonies was evaluated by colony PCR. The isolates validated by PCR were named strains 2-1a, 2-1b, 2-1c, 2-1d, and 2-1e.

[0207] 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) of an XPDH homologue of Lactobacillus rhamnosus encoding the amino acid sequence of SEQ ID NO: 14 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) 3' flanking DNA for targeting chromosome integration into the ER1 locus (SEQ ID NO: 162). SEQ ID NO:57 contains (i) 5' flanking DNA for targeting chromosomal integration into the ER1 locus (SEQ ID NO:85); (ii) an expression cassette (SEQ ID NO:100) encoding the amino acid sequence of Lactobacillus rhamnosus XPDH homologue 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 a PDA + 250 mg / L genimycin (G418) selection plate and incubated at 35°C for at least 2 days until growth. The resulting transformants were streaked on a PDA + genimycin (G418) plate for single-colony isolation and single-colony selection. Integration of two copies of the Lactobacillus rhamnosus XPDH sequence into the selected colonies was evaluated by colony PCR. The isolates validated by PCR were named strains 2-2a, 2-2b, 2-2c, 2-2d, and 2-2e.

[0208] Strains 1-13c, 2-1a-e, and 2-2a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol.

[0209] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0210] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 and 96 hours of incubation. The glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 12.

[0211] Although PCR validation indicated the presence of the transformed polynucleotide sequence in the indicated strains, further analysis showed that in some strains, the sequence did not correctly target the ER1 locus. Further analysis indicated that strain 2-2e contained the transformed polynucleotide sequence, but it did not target the ER1 locus.

[0212] Table 12: Results of 96-hour shaking flask operation

[0213]

[0214] Example 12: Genetically modified *Saccharomyces cerevisiae* strain

[0215] 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 targeting chromosomal integration into the ER1 locus (SEQ ID NO:85), (ii) the MpPYK1 promoter (SEQ ID NO:86), (iii) the gene encoding Staphylococcus 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 SEQ ID NO:188, (v) the MpENO1 terminator (SEQ ID NO:142), and (vi) 3' flanking DNA for targeting chromosomal integration into the ER1 locus (SEQ ID NO:162). Transformants were selected on a PDA + 250 mg / L genimycin (G418) selection plate and incubated at 35°C for at least 2 days until growth. The resulting transformants were streaked on a PDA + genimycin (G418) plate for single-colony isolation and single-colony selection. Integration of the indicated sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were designated strain 3-1.

[0216] Table 13 below lists various *Bacillus subtilis* strains, including information on the parental strains, sequences of the transformed parental strains, and characterization of the expression cassettes contained in the transformed sequences. The transformed fragment of SEQ ID NO:181 sequentially contains a 5' ER3 flanking sequence (SEQ ID NO:155), an MpPYK1 promoter (SEQ ID NO:86), the gene encoding the *Bacillus subtilis* RPE2 polypeptide of SEQ ID NO:180, an 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 sequentially contains the 3' portion of the bleomycin resistance gene expression cassette (SEQ ID NO:168), an MpTEF2 terminator (SEQ ID NO:150), and a 3' ER3 flanking sequence (SEQ ID NO:165). The transformed fragment of SEQ ID NO:183 contains, in sequence, 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 *Saccharomyces cerevisiae* RPE1 polypeptide (SEQ ID NO:179), the Mp6PGD terminator (SEQ ID NO:140), and a 3' ER3 flanking sequence (SEQ ID NO:165). The transformed fragment of SEQ ID NO:184 contains, in sequence, 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 *Saccharomyces cerevisiae* RPE2 polypeptide (SEQ ID NO:180), the Mp6PDG terminator (SEQ ID NO:140), and a 3' ER3 flanking sequence (SEQ ID NO:165). The transformed fragment of SEQ ID NO:185 contains, in sequence, a 5'ER3 side-joint sequence (SEQ ID NO:155), an MpTEF1 promoter (SEQ ID NO:133), and the 5' portion of the bleomycin resistance gene expression cassette (SEQ ID NO:169).

[0217] As described in Example 4, the parent strain of *Synthia spp.* was transformed with the indicated sequence using bleomycin or G418 selection, corresponding to the indicated selection marker. Integration of the indicated sequence into the resulting transformants was evaluated by colony PCR. The PCR-validated isolates were then assigned the indicated strain number. In some cases, more than one PCR-validated isolate (e.g., "sister" isolates) is indicated by a letter following the strain number. For example, strain 3-2 has four sister isolates: strains 3-2a, 3-2b, 3-2c, and 3-2d (collectively referred to as 3-2a-d).

[0218] Table 13.

[0219]

[0220] na - Not applicable; the second box is only for recombination and marker selection.

[0221] Example 13: Shake-flask fermentation determination

[0222] 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 the production of ribitol, xylitol, glycerol, and ethanol.

[0223] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0224] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 14 and... Figure 14 and Figure 15 middle.

[0225] Table 14: Results of 96-hour shaking flask operation

[0226]

[0227] Example 14: Shake-flask fermentation assay

[0228] Strains 1-1, 1-13c, 1-15a, 3-7a-f, and 3-8a-f were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, glycerol, erythritol, and ethanol.

[0229] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0230] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 15 and... Figure 12 and Figure 13 middle.

[0231] Table 15: Results of 96-hour shake flask operation (g / L)

[0232]

[0233] Example 15: Shake-flask fermentation assay

[0234] 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 the production of ribitol, xylitol, glycerol, erythritol, and ethanol.

[0235] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0236] 0.8 mL of seed culture was inoculated into 250 mL baffle-less flasks containing production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Tables 16 to 18 and Figure 16 to Figure 21 middle.

[0237] Yield is calculated as a percentage of product (i.e., xylitol) to substrate consumed (i.e., the difference between one time point and the second time point).

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] Example 16: Phosphatase Diversity

[0246] The final step in the xylitol pathway, from xylitol 5-phosphate to xylitol, requires a phosphatase. The Saccharomyces cerevisiae PYP1 (polyol phosphatase 1) gene encodes a sugar alcohol phosphatase that hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (D)-glycerol-3-phosphate (Xu et al., “Discovery and functional characterization of a yeast sugaralcohol phosphatase,” ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova et al., “Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae,” J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the sorbitol-6-phosphatase class (EC 3.1.3.50). Since xylitol 5-phosphate is a molecule similar to a known substrate of PYP1, orthologs of PYP1 in *Saccharomyces cerevisiae* may be responsible for the final catalytic step in the xylitol production pathway. A genome search of *Saccharomyces cerevisiae* strain 1-1 revealed two genes with high homology to PYP1: RCSR00371 and RCSR15215. These two genes share 40% and 38% identity with ScPYP1, respectively, and 89% identity with each other.

[0247] Escherichia coli HxpA (hexitol phosphatase A) is a HAD-like enzyme belonging to EC 3.1.3.50, with a substrate profile similar to PYP1 (Kuznetsova et al., “Genome wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphate family,” 2006, J. Biol. Chem., 281, 36149-36161). HxpA and PYP1 share low sequence similarity, and therefore, a search was conducted in the *Saccharomyces cerevisiae* genome for HxpA orthologs as alternative phosphatase candidates. RCSR21016 was identified as 38% identical to HxpA and independently identified as a sugar phosphatase with sequence similarity to *Saccharomyces cerevisiae* DOG1 and DOG2. As shown in Examples 12-15, expression of *Saccharomyces cerevisiae* DOG1 and DOG2 increased xylitol production in *Saccharomyces cerevisiae*.

[0248] Based on enzyme classification and / or sequence identity with *Saccharomyces cerevisiae* RCSR00371, RCSR15215 and RCSR21016 phosphatases and *Saccharomyces cerevisiae* PYP1 phosphatases, 26 additional phosphatase candidates were selected for further analysis, as outlined in Table 19.

[0249] Table 19: Phosphatases

[0250]

[0251] Example 17: Genetically modified *Saccharomyces cerevisiae* strain

[0252] Using the transformation method outlined in Example 4, the indicated *Saccharomyces cerevisiae* parent strain was transformed with the bispecific transformation fragments indicated in Table 20. Integration of the indicated sequence into the resulting transformants was evaluated by colony PCR. The PCR-validated isolates were then designated with the strain numbers indicated in Table 20. In some cases, more than one PCR-validated isolate (e.g., "sister" isolates) is indicated by the letter following the strain number. For example, strain 4-1 has five sister isolates: strains 4-1a, 4-1b, 4-1c, 4-1d, and 4-1e.

[0253] For example, strain 2-2b was transformed with SEQ ID NO:223 and SEQ ID NO:181. SEQ ID NO:181 is described in Example 12 and contains, in sequence, a 5' ER3 side sequence (SEQ ID NO:155), an MpPYK1 promoter (SEQ ID NO:86), a gene encoding the *Saccharomyces cerevisiae* RPE2 polypeptide (SEQ ID NO:180), an MpPYK terminator (SEQ ID NO:146), and the 5' portion of a bleomycin resistance gene expression cassette (SEQ ID NO:169). SEQ ID NO:223 contains the 3' portion of a bleomycin resistance gene expression cassette (SEQ ID NO:168), an MpPGK1 promoter (SEQ ID NO:135), a gene encoding the *Saccharomyces cerevisiae* PYP1 polypeptide (SEQ ID NO:224), an Mp6PGD terminator (SEQ ID NO:140), and a 3' ER3 side sequence (SEQ ID NO:165). Transformants were selected on a PDA + bleomycin selection plate and incubated at 35°C for at least 2 days until growth. The resulting transformants were streaked onto a PDA + bleomycin plate for single-colony isolation, and single colonies were selected. Integration of the indicated sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 4-1a, 4-1b, 4-1c, 4-1d, and 4-1e.

[0254] The conversion cassettes of the phosphatase homologues outlined in Table 20 have the same components as SEQ ID NO:223 above, but contain the indicated nucleotide sequence encoding the indicated polypeptide sequence.

[0255] Table 20.

[0256]

[0257]

[0258] na - Not applicable; the second box is only for recombination and marker selection.

[0259] Example 18: Shake-flask fermentation assay

[0260] Strains 2-2b, 1-14d, 3-12c, 3-12d, 4-1a-e, 4-2a-e, 4-3a-e, 4-4a-e, 4-5a-e, 4-6a-b, 4-7a-e, and 4-8a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0261] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0262] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 21 and... Figure 22 and Figure 23Results at the 96-hour time point are not shown because some reactions consumed all the glycerol feedstock, leading to inaccurate estimations of the reaction rates. Results demonstrated that overexpression of the X5PP enzymes in SEQ ID NO:198, 199, and 200 increased xylitol titers compared to parental strain 2-2b and control strains expressing RPE and XPDH but lacking X5PP (strains 3-12c and 3-12d). Results also showed that expression of the exogenous X5PP enzyme in SEQ ID NO:201 increased xylitol titers compared to strains 2-2b, 3-12c, and 3-12d.

[0263]

[0264]

[0265]

[0266] The top xylitol-producing strains for each phosphatase were sequenced. All strains contained two copies of Lactobacillus rhamnosus XPDH, as expected; however, the copy number of the phosphatase and RPE2 genes was variable, as outlined in Table 22.

[0267] Table 22: Gene Copy Number

[0268]

[0269] Example 19: Shake-flask fermentation assay

[0270] Strains 1-1, 5-1a-e, 5-2a-e, 5-3a-e, and 5-4a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0271] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0272] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. The fermentation results at the final 96-hour time point are reported in Tables 23 and 24. Results at the 24, 48, and 72-hour time points are not shown. The results demonstrate that the phosphatases of SEQ ID NO:198-201 have a different effect on erythritol production than they have on xylitol production.

[0273] Table 23: Results of 96-hour shaking flask operation

[0274]

[0275] Table 24: Erythritol Yield (%)

[0276]

[0277] Example 20: Shake-flask fermentation determination

[0278] Strains 2-2b, 4-1a, 4-1c, 4-4b, 4-4c, 5-1a-e, 5-2a-e, 5-3a-e, and 5-4a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0279] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0280] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. The fermentation results at the final 96-hour time point are reported in Tables 25 and 26. Results at the 24, 48, and 72-hour time points are not shown.

[0281] Table 25: Results of 96-hour shaking flask operation

[0282]

[0283] Table 26: Yield of 96-hour shake flask

[0284]

[0285]

[0286] This example demonstrates shake-flask results comparing two X5PP enzymes (SEQ ID NO: 200 and 201) with and without RPE expression as shown in SEQ ID NO: 180. Compared to the 11.7% yield in the control strain, the average xylitol yield in the strain containing overexpressed X5PP but without overexpressed RPE was 14% and 18.3% with the phosphatases of SEQ ID NO: 200 and 201, respectively (see Table 26). These yields are significantly lower than the 33.7% and 37.7% yields observed in strains containing both X5PP and RPE expression.

[0287] Example 21: Shake Flask Fermentation Determination

[0288] Strains 1-13c, 1-15a, 1-29c, 1-32d, 1-16a, 3-7a, 3-8b, 3-9c, 3-15d, 3-17d, 5-7a-e, 5-8a-e, 5-9a-e, 5-10a-e, and 5-11a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0289] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0290] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography with a refractive index detector. The fermentation results at the final 96-hour time point are reported in Tables 27 and 28. Results at the 24, 48, and 72-hour time points are not shown.

[0291] Table 27: Results of 96-hour shake flask operation

[0292]

[0293]

[0294] Table 28: Xylitol Yield (%)

[0295]

[0296]

[0297] Italicized values ​​are outliers and are not used to calculate the average.

[0298] This shake-flask fermentation assay examined combinations of phosphatase with overexpression of SEQ ID NO:200 (RPE2, SEQ ID NO:180) and five different XPDH enzymes. Table 28 shows the xylitol yields of the tested strains. Significant yield increases were evident for all XPDH candidates tested in combination with overexpression of the X5PP enzyme with RPE2 and SEQ ID NO:200. Strains with XPDHs of SEQ ID NO:14 and 15 exhibited the highest 96-hour xylitol yields, at 27% and 29.5%, respectively. The strains tested in this example all had one copy of the indicated XPDH, while strain 2-2b and its progeny had two copies of the sequence encoding the XPDH of SEQ ID NO:14, which may explain why strains 4-4b and 4-4c had an average yield of 37.7% (see Table 26).

[0299] Example 22: Shake-flask fermentation assay

[0300] Strains 2-2b, 3-12d, 4-4c, 5-12a-e, 5-13a-d, 5-14a-e, 5-15a-e, 5-16a-e, 5-17a-e, 5-18a-e, 5-19a-d, and 5-20a-d were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0301] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0302] To form a production culture, 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5). The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at the 48 and 72 hour time points are reported in Table 29. Results at the 24 and 96 hour time points are not shown. Titers were low at the 24 hour time point, and after 96 hours, many cultures contained low or no glucose concentrations. An error occurred when running shake flasks with the strain from 5–15 days, therefore results for that strain are not reported. Results at the 72 hour time point are also shown. Figure 24 and Figure 25 middle.

[0303]

[0304]

[0305]

[0306] like Figure 24 and Figure 25 As shown in the figures and the results in Table 29, strains expressing the X5PP enzyme of SEQ ID NO:201-209 exhibited increased xylitol titers compared to control strains 2-2b and RPE-expressing strains (3-12d). Strains expressing the X5PP enzyme of SEQ ID NO:201-207 also had at least one sister isolate with xylitol titers equal to or higher than those of strains 4-4c expressing X5PP of SEQ ID NO:200. Sister-to-sister variability was also observed in several of the tested strains. As demonstrated in the examples above, although the strains were PCR-verified to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sisters to differ from others. The results here indicate that similar transformations occurred in these sisters, but the results do demonstrate the effectiveness of the indicated X5PP enzyme.

[0307] For example, although PCR validation indicated the presence of the transformed polynucleotide sequence in the indicated strains, further whole-genome sequencing analysis revealed that for strains 5-12a-e, 5-15a-c, and 5-15d, the sequence was integrated with more than one copy and / or not properly integrated at the ER3 locus. The whole-genome sequencing results for these strains are reported in Table 30 below. The results suggest that sister-to-sister variability may be due to differences in copy number and integration locus (of the XPDH, RPE2, and / or phosphatase genes), but even with copy number and integration variability, the effectiveness of the indicated X5PP enzyme remained consistent.

[0308] Table 30.

[0309]

[0310]

[0311] Example 23: Shake-flask fermentation assay

[0312] Strains 2-2b, 4-4b, 4-4c, 5-21a-c, 5-22, 5-23a-c, 5-24a-b, 5-25a-e, 5-26a-d, 5-27, 5-28a-d, 5-29, 5-30a-b, 5-31a-b, 5-32a-c, and 5-33 were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0313] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0314] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form production cultures. The production cultures were incubated at 35 °C and 250 rpm. Samples were taken from the production cultures after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at the 48 and 72 hour time points are reported in Table 31. Results at the 24 and 96 hour time points are not shown. Titers were low at the 24 hour time point, and after 96 hours, many cultures contained low or no glucose concentrations. Results from the 72 hour time point are also shown... Figure 26 and Figure 27 middle.

[0315]

[0316]

[0317] like Figure 26 and Figure 27 As shown in the figures and the results in Table 31, strains expressing the X5PP enzymes of SEQ ID NO: 210, 211, 213, 214, and 189 exhibited increased xylitol titers compared to control strain 2-2b. Strains expressing the X5PP enzymes of SEQ ID NO: 210 and 213 also had at least one sister isolate with a xylitol titer equal to or higher than that of strains 4-4b and 4-4c expressing the X5PP enzyme of SEQ ID NO: 200. Sister-to-sister variability was also observed in several of the tested strains. As demonstrated in the examples above, although the strain was PCR-verified to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sisters to differ from other sisters. The results here indicate that similar transformations occurred in these sisters, but the results do demonstrate the effectiveness of the indicated X5PP enzyme.

[0318] Example 24: Shake-flask fermentation assay

[0319] Strains 2-2b, 4-4b, 4-4c, 5-34a-c, 5-35a-c, 5-36a-b, 5-37a-c, 5-38, 5-39a-b, 5-40a-c, 5-41a-b, 5-42a-d, 5-43a-f, 5-44a-b, 5-45a-d, 5-46, and 5-47a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0320] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0321] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form production cultures. The production cultures were incubated at 35 °C and 250 rpm. Samples were taken from the production cultures after 24, 48, 72, and 96 hours of incubation. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at the 48 and 72 hour time points are reported in Table 32. Results at the 24 and 96 hour time points are not shown. Titers were low at the 24 hour time point, and after 96 hours, many cultures contained low or no glucose concentrations. Results from the 72 hour time point are also shown... Figure 28 and Figure 29 middle.

[0322]

[0323]

[0324]

[0325] like Figure 28 and Figure 29As shown in the figures and the results in Table 32, strains expressing the X5PP enzymes SEQ ID NO: 221, 222, and 188 exhibited increased xylitol titers compared to control strain 2-2b. None of the tested strains had xylitol titers equal to or higher than those of strains 4-4b and 4-4c expressing X5PP SEQ ID NO: 200. Sister-to-sister variability was also observed in several of the tested strains. As demonstrated in the examples above, although the strains were PCR-verified to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sisters to differ from others. The results here indicate that similar transformations occurred in these sisters, but the results do demonstrate the effectiveness of the indicated X5PP enzyme.

[0326] Example 25: Shake-flask fermentation determination

[0327] Strains 4-4b, 4-4c, 5-12d, 5-12e, 5-14b, 5-14c, 5-15c, 5-16d, 5-16e, 5-17e, 5-21a, 5-21b, 5-26b, and 5-26d were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0328] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0329] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at the 48 and 72 hour time points are reported in Table 33. Results at the 24 and 96 hour time points are not shown. Titers were low at the 24 hour time point, and after 96 hours, many cultures contained low or no glucose concentrations. Results at the 72 hour time point are also shown... Figure 30 middle.

[0330]

[0331] The results of this embodiment are consistent with those of the previous embodiments, demonstrating that the xylitol titers produced by strains expressing the X5PP enzymes of SEQ ID NO: 202, 203, 204, 205, 206, 210, and 213 are approximately equal to or greater than the xylitol titers produced by the control strain expressing X5PP of SEQ ID NO: 200. As demonstrated in the preceding embodiments, although the strains were PCR-verified to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, it may have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sisters to differ from other sisters. The results here indicate that similar transformations occur in these sisters, but the results do indeed demonstrate the effectiveness of the indicated X5PP enzyme.

[0332] Example 26: Genetically modified *Saccharomyces cerevisiae* strain

[0333] UV mutagenesis was used (using Hoefer UV crosslinking agent, energy 360 uJ / cm²). 3 The selection of strains 3-12d was used to generate *Saccharomyces cerevisiae* strains with reduced foaming during shake-flask fermentation. Based on visual evaluation of foaming during shake-flask fermentation compared to the parental 3-12d strain, strains with a low-foaming phenotype were selected. The resulting low-foaming strain was named 6-1, containing two copies of the exogenous polynucleotide sequence encoding XPDH (SEQ ID NO: 14) integrated at the ER1 locus and one copy of the polynucleotide sequence encoding RPE (SEQ ID NO: 180) integrated at the ER3 locus.

[0334] Using the transformation method outlined in Example 4, strain 6-1 was transformed with the Cre recombinase plasmid of SEQ ID NO:288. The removal of bleomycin resistance selection markers in the resulting transformants was evaluated by colony PCR. The PCR-validated isolate was named strain 6-2.

[0335] Strain 6-2 was nonselectively grown on YPD plates to allow for the loss of the plasmid in SEQ ID NO:288. The biomass was subjected to single-colony knockout and evaluated by PCR to confirm the plasmid loss. The PCR-validated isolate was named strain 6-3.

[0336] Using the transformation method outlined in Example 4, the indicated *Saccharomyces cerevisiae* parent strain 6-3 was transformed with the bispecific transformation fragments indicated in Table 34. Integration of the indicated sequence into the resulting transformants was evaluated by colony PCR. The PCR-validated isolates were then designated with the strain numbers indicated in Table 34. In some cases, more than one PCR-validated isolate (e.g., "sister" isolates) is indicated by the letter following the strain number. For example, strain 6-4 has five sister isolates: strains 6-4a, 6-4b, 6-4c, 6-4d, and 6-4e.

[0337] For example, strain 6-3 was transformed using SEQ ID NO:280 and SEQ ID NO:279. SEQ ID NO:279 contains, in sequence, the 3' portion of the G418 resistance gene expression cassette (SEQ ID NO:172), the MpTEF1 terminator (SEQ ID NO:289), and the 3' gpdIIB flanking sequence (SEQ ID NO:166). SEQ ID NO:280 contains the 5' gpdIIB flanking sequence (SEQ ID NO:158), the PGK1 promoter (SEQ ID NO:135), the gene encoding the *Saccharomyces cerevisiae* phosphatase polypeptide of SEQ ID NO:210 (SEQ ID NO:230), the TDH3 terminator (SEQ ID NO:149), and the 5' portion of the G418 resistance gene expression cassette (SEQ ID NO:175). Transformants were selected on a PDA + G418 selector plate and incubated at 35°C for at least 2 days until growth. The obtained transformants were streaked onto PDA + G418 plates for single-colony isolation, and single colonies were selected. The integration of the indicated sequence into the selected colonies was evaluated by colony PCR. The PCR-validated isolates were named strains 6-4a, 6-4b, 6-4c, 6-4d, and 6-4e.

[0338] The transformation cassettes of the phosphatase homologues SEQ ID NO:281-287 summarized in Table 34 have the same components as SEQ ID NO:280, but contain the indicated nucleotide sequence encoding the indicated polypeptide sequence. SEQ ID NO:181 is described in Example 12 and contains, in sequence, a 5' ER3 side-joint sequence (SEQ ID NO:155), an MpPYK1 promoter (SEQ ID NO:86), a gene encoding the *Saccharomyces cerevisiae* RPE2 polypeptide of SEQ ID NO:180, an MpPYK terminator (SEQ ID NO:146), and the 5' portion of a bleomycin resistance gene expression cassette (SEQ ID NO:169).

[0339] Table 34.

[0340]

[0341] Strains 6-12a-c were sequenced to determine the sites at which the indicated polynucleotides were integrated and the final copy number of the given gene. Parental strain 6-3 contained one copy of the polynucleotide encoding the RPE (SEQ ID NO:180) integrated at the ER3 locus on a single allele. In strain 6-12b, a second copy of the RPE-coding sequence was integrated at the ER3 locus on the second allele, resulting in complete ER3 knockout. Strain 6-12b was also confirmed to have two copies of the X5PP enzyme (SEQ ID NO:200) encoded by SEQ ID NO:230. However, in strains 6-12a and 6-12c, transformation resulted in the replacement of the previously integrated sequence with a new sequence encoding both the RPE (SEQ ID NO:180) and the X5PP enzyme (SEQ ID NO:200).

[0342] Example 27: Shake-flask fermentation determination

[0343] Strains 6-3, 6-4a-e, 6-5a-e, 6-6a-e, 6-7a-e, 6-8a-e, 6-9a-e, 6-10a-e, and 6-11a-e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0344] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0345] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form production cultures. The production cultures were incubated at 35 °C and 250 rpm. Samples were taken from the production cultures after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at the 48 and 72 hour time points are reported in Table 35. Results at the 24 and 96 hour time points are not shown. Titers were low at the 24 hour time point, and after 96 hours, many cultures contained low or no glucose concentrations. Results at the 72 hour time point are also shown... Figure 31 and Figure 32 middle.

[0346] For strain 6-7b, there was an error in the shake-flask fermentation run. Although the results showed almost no glucose consumption and low metabolite yield, this was likely due to an error in the fermentation setup rather than a defect in the strain.

[0347]

[0348]

[0349]

[0350] The results demonstrate that the same increase in xylitol titer and yield can be achieved when the X5PP coding sequence is integrated at the gpdIIB locus, as seen in previous examples where the sequence was integrated at the ER3 locus. As demonstrated in the preceding examples, although the strain was PCR-verified to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, it may already have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sisters to differ from others. The results here indicate that similar transformations occur in these sisters, but the results do indeed demonstrate the effectiveness of the indicated X5PP enzyme.

[0351] Example 28: Shake-flask fermentation determination

[0352] Strains 6-3, 6-12a, 6-12b, 6-12c, 6-4d, 6-4e, 6-7d, 6-8a, 6-8e, 6-9b, 6-9d, 6-11a, 6-11b, 6-11c, 6-11d, and 6-11e were run in shake flasks to assess glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0353] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0354] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, xylitol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at the 48 and 72 hour time points are reported in Table 36. Results at the 24 and 96 hour time points are not shown. Titers were low at the 24 hour time point, and after 96 hours, many cultures contained low or no glucose concentrations. Results at the 72 hour time point are also shown...Figure 33 middle.

[0355] For strain 6-7b, there was an error in the shake-flask fermentation run. Although the results showed almost no glucose consumption and low metabolite yield, this was likely due to an error in the fermentation setup rather than a defect in the strain.

[0356]

[0357] Example 29: Genetically modified *Saccharomyces cerevisiae* strain

[0358] Using the transformation method outlined in Example 4, the indicated *Saccharomyces cerevisiae* parent strains 1-12a were transformed with the bispecific transformation fragments indicated in Table 37. The transformation fragments of SEQ ID NO: 181, 182, 185, and 229 are described in the previous examples. Integration of the indicated sequence into the resulting transformants was evaluated by colony PCR. The PCR-validated isolates were then designated with the strain numbers indicated in Table 37. In some cases, more than one PCR-validated isolate (e.g., "sister" isolates) is indicated by the letter following the strain number. For example, strain 7-1 has five sister isolates: strains 7-1a, 7-1b, 7-1c, 7-1d, and 7-1e.

[0359] Table 37.

[0360]

[0361] Example 30: Shake-flask fermentation determination

[0362] Strains 1-12a, 7-1a-e, 7-2a-e, and 7-3a-e were run in shake flasks to assess glucose consumption and the production of arbitol, ribitol, erythritol, glycerol, and ethanol.

[0363] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0364] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after incubation at 24, 48, 72, and 96 hours. Glucose, ribitol, arabinol, erythritol, glycerol, and ethanol in the samples were analyzed by high-performance liquid chromatography with a refractive index detector. Fermentation results at all time points are reported in Table 38. Data on arabinol yield are provided in Table 39. Data at the 72-hour time point are also shown. Figure 34 middle.

[0365] For strain 7-1b, there was an error in the shake-flask fermentation run. Although the results showed almost no glucose consumption and low metabolite yield, this was likely due to an error in the fermentation setup rather than a defect in the strain.

[0366] The results demonstrated that, relative to the parent strain, overexpression of the native RPE enzyme and / or the native A5PP enzyme simultaneously increased both araitol titer and araitol yield. As demonstrated in the examples above, although the strain was PCR-verified to contain the desired polynucleotide sequence, the sequence may not have been integrated at the correct locus, it may already have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have caused individual sisters to differ from others. The results here indicate that similar transformations occur in these sisters, but the results do indeed demonstrate the effectiveness of the indicated RPE and A5PP enzymes.

[0367]

[0368]

[0369] Table 39: Aritol Yield

[0370]

[0371] Example 31: Genetically modified *Saccharomyces cerevisiae* strain

[0372] strain 8-1

[0373] Strain 6-12b was transformed with SEQ ID NO:290 using the transformation protocol outlined in Example 4, and positive transformants were selected using a norsyl selection plate. SEQ ID NO:290 contains a deletion construct for removing one copy of the RSCR18717 gene. The PCR-validated isolate in which one copy of RCSR18717 was knocked out was named strain 8-1.

[0374] strain 8-2

[0375] Using the transformation protocol outlined in Example 4, strain 8-1 was transformed with SEQ ID NO:291. SEQ ID NO:291 contains a construct for selecting markers of bleomycin and noresorcin resistance. The PCR-validated isolate in which the bleomycin and noresorcin resistance selection markers were removed was named strain 8-1.

[0376] strain 8-3

[0377] Strain 8-2 was transformed with SEQ ID NO:290 using the transformation protocol outlined in Example 4, and positive transformants were selected using a noroside selection plate. SEQ ID NO:299 contains a deletion construct for removing one copy of the RSCR18717 gene. The PCR-validated isolate in which both copies of the RSCR18717 gene were knocked out was named strain 8-3.

[0378] strain 8-4

[0379] Strain 8-3 was transformed with SEQ ID NO:292 and SEQ ID NO:279 using the transformation protocol outlined in Example 4, and positive transformants were selected on a geneticmycin (G418) selector. The PCR-validated isolate containing two copies of the gene encoding X5PP (SEQ ID NO:200) was named strain 8-4.

[0380] strain 8-5

[0381] Using the transformation protocol outlined in Example 4, strain 8-4 was transformed with SEQ ID NO:293. SEQ ID NO:293 contains a construct for selecting markers of resistance to cyclonorrhizin and genimycin (G418). The PCR-validated isolate in which both cyclonorrhizin and genimycin resistance selection markers were removed was named strain 8-5.

[0382] strain 8-6

[0383] UV mutagenesis was used (using Hoefer UV crosslinking agent, energy 360 uJ / cm²). 3The selection of strains 8-5 and 9-3 was used to generate *Saccharomyces cerevisiae* strains with enhanced xylitol production rates in shake-flask fermentation. Based on xylitol production in shake-flask fermentation (e.g., measured by HPLC) compared to that of parental strain 9-3, strains with high xylitol yields were selected. The resulting strain with enhanced xylitol yield was named strain 8-6, containing two copies of the gene encoding XPDH (SEQ ID NO: 14), two additional copies (total of three copies) of the gene encoding RPE (SEQ ID NO: 180), three additional copies (total of five copies) of the gene encoding X5PP (SEQ ID NO: 200), and two alleles of the RCSR18717 gene knocked out.

[0384] Strain 8-7

[0385] UV mutagenesis was used (using Hoefer UV crosslinking agent, energy 360 uJ / cm²). 3 The selection of strains 8-6 and 9-4 was used to generate *Saccharomyces cerevisiae* strains with enhanced xylitol production rates in shake-flask fermentation. Based on xylitol production in shake-flask fermentation (e.g., measured by HPLC) compared to that of parental strain 9-4, strains with high xylitol yields were selected. The resulting strain with enhanced xylitol yield was named strain 8-7, containing two copies of the gene encoding XPDH (SEQ ID NO: 14), two additional copies (total of three copies) of the gene encoding RPE (SEQ ID NO: 180), three additional copies (total of five copies) of the gene encoding X5PP (SEQ ID NO: 200), and two alleles of the RCSR18717 gene knocked out.

[0386] strains 8-10 and 8-11

[0387] To test erythritol production in xylitol-producing strains exhibiting reduced erythritol production and increased rates, the polynucleotide encoding XPDH at ER1 (SEQ ID NO:14) was replaced with the polynucleotide sequence encoding APDH (SEQ ID NO:11), as outlined in the following strains.

[0388] The parent strain was transformed using the transformation method outlined in Example 4, with the transformation fragments indicated in Table 40. The remaining transformation fragments are summarized below:

[0389] a. SEQ ID NO:294 contains a 5' ER1 side-joint sequence (SEQ ID NO:85), an MpPYK1 promoter (SEQ ID NO:86), a gene encoding APDH of SEQ ID NO:11, an Mp6PGD terminator (SEQ ID NO:140), and the 5' portion of the norsinosin resistance gene expression cassette (SEQ ID NO:170).

[0390] b. SEQ ID NO:295 contains the 3' portion of the norsulitis resistance gene expression cassette (SEQ ID NO:171), the MpPYK1 promoter (SEQ ID NO:86), the gene encoding APDH of SEQ ID NO:11, the Mp6PGD terminator (SEQ ID NO:140), and the 3' ER1 side sequence (SEQ ID NO:162).

[0391] c. SEQ ID NO:296 contains a 5' ER1 side-joint sequence (SEQ ID NO:85); an MpPYK1 promoter (SEQ ID NO:86); a nucleic acid sequence encoding APDH of SEQ ID NO:11; an Mp6PGD terminator (SEQ ID NO:140); and the 5' portion of the G418 resistance gene expression cassette (SEQ ID NO:175).

[0392] d.SEQ ID NO:298 contains the 3' portion (SEQ ID NO:172) of the G418 resistance gene expression cassette and the 3' ER1 side sequence (SEQ ID NO:162).

[0393] The integration of the indicated sequence into the transformed organisms was evaluated by colony PCR. The PCR-validated isolates were then assigned the strain numbers indicated in Table 37. In some cases, more than one PCR-validated isolate (e.g., "sister" isolates) was indicated by a letter following the strain number. For example, strains 8-9 had three sister isolates: strains 8-9a, 8-9b, and 8-9c.

[0394] Table 40.

[0395]

[0396] Example 32: Shake-flask fermentation determination

[0397] Strains 8-9a-j, 8-10a-j, 1-12a, 7-3a, and 8-7 were run in shake flasks to assess glucose consumption and the production of aritol, xylitol, erythritol, glycerol, and ethanol.

[0398] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 250 mL baffleless flasks containing 40 mL of enrichment medium (170 g / L glucose and 10 g / L yeast extract). Cells were incubated at 30°C and 250 rpm until the optical density (OD600) reached 15–20 to form a seed culture. Optical density was measured at 600 nm wavelength and 1 cm path length using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 between 15 and 20 within approximately 32–50 hours.

[0399] 0.4 mL of seed culture was inoculated into 250 mL baffle-less flasks containing 20 mL of production medium (Table 5) to form production cultures. The production cultures were incubated at 35 °C and 250 rpm. Samples were taken from the production cultures after 24, 48, 72, and 96 hours of incubation. Glucose, arabinol, xylitol, erythritol, glycerol, and ethanol were analyzed in the samples by high-performance liquid chromatography (HPLC) with a refractive index detector, and the results at the 96-hour time point are reported in Table 41. Figure 35 In addition, for strains 7-3a, 8-9h, 8-10b, 8-10c, and 8-10d, samples collected at 96 hours were analyzed by high-performance ion chromatography (HPIC) to resolve slight peak overlaps observed in the HPLC results, and the results are reported in Table 42 and... Figure 36 middle.

[0400] Table 41.

[0401]

[0402]

[0403] Table 42.

[0404]

[0405] The results showed that the substitution of the XPDH sequence at the ER1 locus encoding SEQ ID NO:14 was incomplete in most of the tested strains. Generally, transformation with two copies of the APDH sequence encoding SEQ ID NO:11 (strains 8-10a-j) enabled arabinol production, indicating at least a partial substitution of the XPDH coding sequence. Both strains 8-9h and 8-10a-j showed arabinol production, suggesting that APDH expression levels competed with XPDH, resulting in less xylitol production compared to the parental strain 8-7. As demonstrated by the HPIC results, strain 8-10c produced no xylitol and significantly more arabinol compared to the control strain 7-3a.

Claims

1. A genetically engineered yeast cell capable of producing araitol, said engineered yeast cell comprising: Genetic modifications that lead to overexpression of the native enzyme with arabino-5-phosphate phosphatase (A5PP) activity; and / or The exogenous polynucleotide sequence encoding an enzyme with araitol-5-phosphate phosphatase (A5PP) activity.

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

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

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

5. The yeast cell according to any of the preceding claims, wherein the cell is a *Saccharomyces cerevisiae* cell, and the genetic modification results in the overexpression of a natural A5PP enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 198, 199, 200, or 221.

6. The yeast cell according to any of the preceding claims, wherein the genetic modification comprises replacing the natural A5PP gene promoter with a heterologous promoter or an artificial promoter.

7. The yeast cell according to claim 6, wherein the heterologous promoter or artificial promoter is selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconic acid 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), phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 86). NO:139).

8. The yeast cell according to any of the preceding claims, wherein the genetic modification comprises adding an exogenous polynucleotide sequence encoding the natural A5PP enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the natural A5PP enzyme.

9. A yeast cell according to any of the preceding claims, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having A5PP activity, and a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188.

10. The yeast cell according to any of the preceding claims, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having A5PP activity, and a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 200, 202, 203, 204, 205, 206, 210, and 213.

11. The yeast cell according to any of the preceding claims, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having A5PP activity, and a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 200, 203, 204, 206, and 213.

12. The yeast cell according to any of the preceding claims, wherein the A5PP activity in the genetically engineered yeast cell is higher than that in an equivalent cell lacking the genetic modification or the exogenous polynucleotide sequence.

13. The yeast cell according to any of the preceding claims, wherein, When the engineered cells are used in a fermentation process in the presence of dextran, the titer and / or yield of aritol increases relative to the titer and / or yield of aritol in an equivalent fermentation process using equivalent cells lacking the genetic modification or exogenous polynucleotide sequence.

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

15. The yeast cell of claim 14, wherein the cell is a *Saccharomyces cerevisiae* 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% of the sequence of at least one of SEQ ID NO: 179 and 180.

16. The yeast cell of claim 14 or claim 15, wherein the genetic modification leading to overexpression of the natural RPE enzyme comprises replacing the natural RPE gene promoter with a heterologous promoter or an artificial promoter.

17. The yeast cell according to any one of claims 14 to 16, wherein the heterologous promoter or artificial promoter is selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconic acid 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), phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 86). NO:138) and RPL16B (SEQ ID NO:139).

18. The yeast cell according to any one of claims 14 to 17, wherein the genetic modification leading to overexpression of the natural RPE enzyme comprises adding an exogenous polynucleotide encoding the natural RPE enzyme, such that the genetically engineered cell contains at least one additional copy of the sequence encoding the RPE enzyme.

19. A yeast cell according to any of the preceding claims, wherein the cell further comprises an exogenous polynucleotide sequence encoding an arabinophosphate dehydrogenase (APDH) enzyme, the arabinophosphate dehydrogenase (APDH) enzyme comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

11.

20. A yeast cell according to any of the preceding claims, wherein the cell further comprises an exogenous polynucleotide sequence encoding an aritol 2-dehydrogenase (ARD2DH) enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 193, 194, 195, 196, or 197.

21. A yeast cell according to any of the preceding claims, wherein the cell further comprises an exogenous polynucleotide sequence encoding an aritol 2-dehydrogenase (ARD2DH) enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 194, 195, 196, or 197.

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

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

24. The yeast cell according to claim 22 or 23, wherein the heterologous promoter or artificial promoter is selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconic acid 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), phosphoglucosidase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 86). NO:138) and RPL16B (SEQ ID NO:139).

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

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

27. The method of claim 26, wherein the fermentation temperature is 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, or between therewith, and the volumetric oxygen consumption rate (OUR) is between 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).

28. The method according to claim 26 or 27, wherein the arabinool is at least 0.2 g / L. -1 h -1 0.3g L -1 h -1 0.5g L -1 h -1 0.75g L -1 h -1 Or at least 1.0 g L -1 h -1 Production rate.

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

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

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

32. Use of the engineered yeast according to any one of claims 1 to 25 for the production of aritol.

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