Genetically modified yeast and fermentation method for xylitol production

Abstract

This specification discloses genetically modified yeast cells characterized by genetic modifications that result in the overexpression of a native enzyme capable of producing xylitol and having xylitol-5-phosphate phosphatase (X5PP) activity, and / or an exogenous polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity. The genetically modified yeast cells may be further genetically modified to overexpress a native RPE enzyme, to express an exogenous XPDH enzyme, to express an exogenous XKS enzyme, and / or to express an exogenous XDH enzyme.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 499,992, filed on 4 May 2023, the contents of which said Provisional Application are incorporated herein by reference in their entirety.

[0002] (Reference to sequence listing submitted via the Patent Center) The contents of the array list XML file "PT-1486-WO-PCT.xml", which was created on April 29, 2024, and submitted electronically through the Patent Center together with this application, and which has a size of 1,030,007 bytes, are incorporated herein by reference in their entirety. [Background technology]

[0003] Xylitol is a low-calorie sweetener used as a food additive and sugar substitute. Commonly used in drugs, nutritional supplements, confectionery, and toothpaste compositions, xylitol has also been associated with anticariogenic properties when used in chewing gum. Conventional methods of xylitol production, including the chemically catalyzed hydrogenation of xylose hydrolyzed from biomass-extracted xylan, are financially and environmentally costly. These methods require high temperatures and pressures, large amounts of water, and metal catalysts that must be mined. In contrast, fermentation processes, which are used on a large scale and commercially to produce other organic molecules (e.g., ethanol, citric acid, lactic acid, etc.), may offer a cost-effective and sustainable alternative to conventional xylitol processing methods.

[0004] Therefore, this specification provides genetically modified yeast and a fermentation method for xylitol production. [Overview of the project]

[0005] This disclosure provides genetically modified yeast cells capable of producing xylitol, wherein the genetically modified yeast cells include a gene modification resulting in the overexpression of a native enzyme having xylitol-5-phosphate phosphatase (X5PP) activity; and / or an exogenous polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity. The yeast cells may be osmotically tolerant yeast cells. The yeast cells may be cells belonging to the subphylum Ustyragomycetes or Saccharomyces. The yeast cells may be selected from the group consisting of Trichosporonoides megachiliensis, Trichosporonoides oenocephalis, Trichosporonoides nigrecens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustyraginomyces, trichomeoblasts, Yarowia liporitica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. The yeast cells may also be those of the genus Moniliella.

[0006] The cells may be Moniliella polynis cells, which, through genetic modification, overexpress a native X5PP 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% sequence identity with at least one of sequence IDs 198, 199, 200, or 221. X5PP activity in genetically modified yeast cells may be higher than that in equivalent cells lacking genetic modification. When genetically modified cells are used in a fermentation process in the presence of dextrose, the xylitol yield may be increased compared to the xylitol yield in an equivalent fermentation process using equivalent cells lacking genetic modification. Genetic modification may include replacing the native X5PP gene promoter with a heterologous or artificial promoter. Heterogeneous or artificial promoters may be selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139).

[0007] Genetic modification in genetically engineered cells may involve the addition of an exogenous polynucleotide sequence encoding the native X5PP enzyme, resulting in the genetically engineered cells containing at least one additional copy of the sequence encoding the native X5PP enzyme. Yeast cells may contain 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% sequence identity with the exogenous polynucleotide sequence encoding the enzyme having X5PP activity, and at least one of Sequence IDs 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188. Yeast cells may contain 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% sequence identity with an exogenous polynucleotide sequence encoding an enzyme having X5PP activity, and at least one of SEQ ID NOs: 200, 202, 203, 204, 206, and 213.

[0008] The genetically modified cells described herein may further include genetic modifications resulting in the overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity. The yeast cells may be Moniliella polynis cells, and the native RPE enzyme includes at least one of at least SEQ ID NOs: 179 and 180 with 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% sequence identity. The genetic modification resulting in the overexpression of the native RPE enzyme includes replacing the native RPE gene promoter with a heterologous or artificial promoter. Heterogeneous or artificial promoters may be selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139). Genetic modification resulting in overexpression of the native RPE enzyme may involve adding an exogenous polynucleotide encoding the native RPE enzyme, thereby causing the genetically modified cell to contain at least one additional copy of the sequence encoding the RPE enzyme.

[0009] The cells may produce xylitol and contain an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme with 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% sequence identity with at least one of SEQ ID NOs: 12-15, 28-31, and 33, or at least one of SEQ ID NOs: 14, 15, 28, or 31. The XPDH enzyme may have at least 85% sequence identity with at least one of SEQ ID NOs: 12-15, 28-31, and 33, or at least one of SEQ ID NOs: 14, 15, 28, or 31. The XPDH enzyme may have 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% sequence identity with at least one of sequence numbers 14, 15, 28, or 31. The XPDH 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% identity with at least sequence number 14.

[0010] The cells may be able to produce xylitol and may contain an exogenous polynucleotide sequence encoding a xylokinase (XKS) enzyme with at least one of sequence numbers 188 and 189 and 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% sequence identity. The cells may further contain an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme with at least one of sequence numbers 190, 191, and 192 and 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% sequence identity.

[0011] The genetically modified yeast cells described herein include at least one of SEQ ID NOs: 12-15, 28-31, and 33, and an exogenous polynucleotide sequence encoding the XPDH enzyme which 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, and a. A gene modification resulting in overexpression of a natural RPE enzyme having at least one more copy than the parent cell, the X5PP 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% sequence identity with SEQ ID NO: 200, and 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% identity with SEQ ID NO: 180, or b. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 202, or c. An exogenous polynucleotide sequence encoding the X5PP enzyme which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203, and a gene modification resulting in overexpression of the native RPE enzyme which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180, or d. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 204, or e. An exogenous polynucleotide sequence encoding an X5PP enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 205, and a gene modification resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180, or f. An exogenous polynucleotide sequence encoding the X5PP enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 206, and a gene modification resulting in overexpression of the native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180, or g. An exogenous polynucleotide sequence encoding the X5PP enzyme which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 210, and a gene modification resulting in overexpression of the native RPE enzyme which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180, or h. Includes an exogenous polynucleotide sequence encoding an X5PP enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 213, and a gene modification resulting in the overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180.

[0012] Any of the exogenous polynucleotide sequences can be operably linked to a heterologous or artificial promoter. Heterogeneous or artificial promoters may be selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139). Any of the exogenous polynucleotide sequences can be incorporated into the yeast cell genome at a locus selected from the ER1, ER3, PDC1, pyrF, TRP3, gpdIIA, and gpdIIB loci.

[0013] This specification also discloses a method for producing xylitol using genetically modified cells described herein, the method comprising the step of contacting a substrate containing dextrose with the genetically modified cells described herein, and xylitol is produced by fermentation of the substrate by the genetically modified cells. The fermentation temperature may be 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C or in between. The volume oxygen uptake rate (OUR) may be 0.5 to 40, 1 to 35, 2 to 30, 3 to 25, 4 to 20, or 5 to 15 mmol O2 / (L·h). The amount of xylitol is at least 0.2, 0.3, 0.5, 0.75, or 1.0 g L -1 h -1 It can be produced at the following rates. When fermentation is carried out at 35°C for 96 hours, xylitol production may be at least 20, 30, 50, 75, or 100 g / L. The rate, titer, and / or yield of xylitol production may be increased compared to equivalent fermentation carried out using equivalent yeast cells that lack the genetic modification to overexpress the X5PP enzyme and the exogenous polynucleotide sequence encoding the exogenous X5PP enzyme. The dextrose concentration may be at least 100 g / L.

[0014] This disclosure further provides the use of genetically modified yeast cells described herein for producing xylitol. [Brief explanation of the drawing]

[0015] This patent or application includes at least one drawing made in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0016] The drawings generally illustrate various embodiments considered herein by means of example, but not by limiting means. [Figure 1] The natural pentose phosphate pathway (dotted line and arrow) and the natural glycolysis pathway (solid line and arrow) in Moniliella polynis are shown. [Figure 2]Shows diversity in the galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) sequence space. [Figure 3] Shows the structural features of the NAD or NADP binding pocket located at +23 amino acids from the characteristic GXGXXG motif (SEQ ID NO: 133) of the XPDH enzyme. [Figure 4] Shows diversity in the ribulose-5-phosphate reductase sequence space. [Figure 5] Shows the in vitro activities of the TarJ’ and XPDH enzymes outlined in Example 3. [Figure 6] Shows the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites in the 96-hour shake flask fermentation of strains 1-1, 1-13a-f, and 1-15a-f outlined in Example 5. The data label reports the concentration (g / L) of xylitol. [Figure 7] Shows the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites in the 96-hour shake flask fermentation of strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, and 1-39a-f outlined in Example 5. The data label reports the concentration (g / L) of xylitol. [Figure 8] Shows the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites in the 96-hour shake flask fermentation of strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e outlined in Example 6. The data label reports the concentration (g / L) of xylitol. [Figure 9] Shows the concentrations (g / L) of erythritol, ribitol, arabitol, and xylitol metabolites in the 96-hour shake flask fermentation of strains 1-13c, 1-12a-e, 1-14a-e, and 1-16a-e outlined in Example 8. The data label reports the concentration (g / L) of xylitol (strains 1-13c, 1-14a-e, and 1-16a-e) or arabitol (strains 12a-e). [Figure 10]The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites of strains 13c, 1-36a-e, and 1-40a-e outlined in Example 9 after 96 hours of shaking flask fermentation are shown. The data labels report the xylitol concentration (g / L). [Figure 11] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites of strains 1-30a-e, 1-31a-e, 1-32a-e, and 1-13c outlined in Example 10 after 96 hours of shaking flask fermentation are shown. The data labels report the xylitol concentration (g / L). [Figure 12] This shows a comparison of xylitol and ribitol concentrations (g / L) produced in strains with RPE2 overexpression (3-7a-f and 3-8a-f) and strains without RPE2 overexpression (1-13c and 1-15a). [Figure 13] The yields of xylitol, ribitol, glycerol, and erythritol for strains with and without RPE2 overexpression (3-8a-f) are shown. [Figure 14] The xylitol and ribitol titers (g / L) for the indicated strains are shown. [Figure 15] The yields of xylitol, ribitol, glycerol, and erythritol for the shown strains with various RPE overexpression configurations are shown. [Figure 16] The xylitol concentration (g / L) after 96 hours of shaking flask fermentation of the strain outlined in Example 15 is shown. [Figure 17] The xylitol concentration (g / L) after 96 hours of shaking flask fermentation of the strain outlined in Example 15 is shown. [Figure 18] The xylitol rate (g / (L·h)) between 48 hours and 96 hours of shaking flask fermentation of the strain outlined in Example 15 is shown. [Figure 19] The xylitol rate (g / (L·h)) between 48 hours and 96 hours of shaking flask fermentation of the strain outlined in Example 15 is shown. [Figure 20]The xylitol yield (%) between 48 hours and 96 hours of shaking flask fermentation of the strain outlined in Example 15 is shown. [Figure 21] The xylitol yield (%) between 48 hours and 96 hours of shaking flask fermentation of the strain outlined in Example 15 is shown. [Figure 22] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 18 are shown. The data label reports the xylitol concentration (g / L). [Figure 23] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 18 are shown. The data label reports the xylitol concentration (g / L). [Figure 24] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 22 are shown. The data label reports the xylitol concentration (g / L). [Figure 25] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 22 are shown. The data label reports the xylitol concentration (g / L). [Figure 26] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 23 are shown. The data label reports the xylitol concentration (g / L). [Figure 27] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 23 are shown. The data label reports the xylitol concentration (g / L). [Figure 28] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 24 are shown. The data label reports the xylitol concentration (g / L). [Figure 29]The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 24 are shown. The data label reports the xylitol concentration (g / L). [Figure 30] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 25 are shown. The data label reports the xylitol concentration (g / L). [Figure 31] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 27 are shown. The data label reports the xylitol concentration (g / L). [Figure 32] The concentrations (g / L) of erythritol, ribitol, and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 27 are shown. The data label reports the xylitol concentration (g / L). [Figure 33] The concentrations (g / L) of erythritol and xylitol metabolites after 72 hours of shaking flask fermentation of the strain outlined in Example 28 are shown. The data label reports the xylitol concentration (g / L). [Figure 34] The xylitol concentration in the shaking flask fermentation outlined in Example 30 is shown. [Figure 35] The concentrations (g / l) of erythritol, xylitol, and glycerol after 96 hours of shaking flask fermentation outlined in Example 34 are shown. The data label reports the xylitol concentration (g / L). [Figure 36] The concentrations (g / l) of erythritol, xylitol, and glycerol after 96 hours of shaking flask fermentation outlined in Example 35 are shown. The data label reports the xylitol concentration (g / L). [Modes for carrying out the invention]

[0017] Herein, certain aspects of the disclosed subject matter are referred to in detail, and examples thereof are partially illustrated in the accompanying drawings. The disclosed subject matter is described together with the enumerated claims, but it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0018] In this document, the terms “a,” “an,” or “the” are used to include one or more unless the context explicitly indicates otherwise. The term “or” is used to refer to a non-exclusive “or” unless otherwise indicated. All publications, patents, and patent documents referenced in this document are incorporated herein by reference in whole, as if they were incorporated individually by reference. Where there is inconsistency in usage between this document and the documents thus incorporated by reference, the usage in the incorporated references should be interpreted as supplementing the usage in this document. In the event of irreconcilable conflict, the usage in this document shall prevail.

[0019] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical limits explicitly listed as range limits, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly listed. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (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%). The notation "approximately X to Y" has the same meaning as "approximately X to about Y" unless otherwise specified. Similarly, the notation "approximately X, Y, or about Z" has the same meaning as "approximately X, about Y, or about Z" unless otherwise specified.

[0020] Unless otherwise stated, ppm (parts per million), percentages, and ratios are based on weight. Weight-based percentages are also referred to as weight % or %(weight) below.

[0021] This disclosure relates to various recombinant cells genetically engineered to produce xylitol. Generally, the recombinant cells described herein are capable of producing xylitol and are characterized by overexpression of the X5PP enzyme or inclusion of an exogenous polynucleotide sequence encoding the X5PP enzyme. Recombinant yeast may further be characterized by overexpression of the ribulose 5-phosphatase epimerase (RPE) enzyme and inclusion of an exogenous polynucleotide sequence encoding the xylitol phosphate dehydrogenase (XPDH) enzyme. The present invention further provides a fermentation method for producing xylitol from dextrose using the genetically engineered cells described herein.

[0022] Generally, the recombinant cells described herein are yeast cells. As used herein, “yeast” means a eukaryotic unicellular microorganism classified as a member of the Kingdom Mycology. Yeasts are unicellular organisms that evolved from multicellular ancestors, and some species retain multicellular characteristics, such as forming strings of linked budding cells known as pseudohyphae or false hyphae. Yeast cells may also be referred to as yeast-like cells in the art, and as used herein, “yeast cells” encompass both yeast and yeast-like cells. Suitable yeasts and yeast-like host cells for modification include Saccharomyces cerevisiae, Chomagataera, Cluiveromyces (e.g., Cluiveromyces lactis, Cluiveromyces marsianus), Yarowia liporitica, Isatakenchia orientalis, Pichia galeyformis, Pichia YB-4149 (NRRL designated), Pichia pastris, Candida (e.g., Candida magnoliae, Candida etanorica), Pichia decericola, Pichia membranous membrane, and Pichia f This may include, but is not limited to, *Trichoderma*, *Aspergillus*, *Trichoderma*, *Miserisola thermophylla*, *Moniliella* (e.g., *Moniliella polinis*), *Pfaffia*, *Yamadajima*, *Hansenula*, *Pitia kudryavzevii*, *Trichosporonoides* (e.g., *Trichosporonoides megachiliensis*, *Trichosporonoides odocephalis*, *Trichosporonoides nigressens*), *Pseudozyma tsukubaensis*, *Trigopsis variabilis*, *Penicillium*, and *Torula*. Those skilled in the art will understand the requirements for selecting suitable yeast cells, and the recombinant yeast cells of this disclosure are not limited to those expressly enumerated herein. Methods for genetically manipulating yeast cells are known and described in the art, and those skilled in the art will understand the methods necessary to transform and manipulate suitable yeast cells.

[0023] Suitable yeast cells may be from the Basidiomycota and Ustyraginomikothinae subphyla. Suitable yeasts from the Ustyraginomikothinae subphylum include Ustyrago (e.g., U. synodontis, U. meidis, U. sphaerogena, U. cordal, U. sitaminea, U. coisis, U. syntellismae, U. esculenta, U. neglecta, U. crus galli, Ustyrago avenae), Sporisolium (e.g., Sporisolium excertum), and Moniera (e.g., M. polinis, M. tomento). This includes, but is not limited to, M. acetabtance, M. foncécae, M. madida, M. megachiliensis, M. osedocephalis, M. nigrecens, Pseudozyma (e.g., Pseudozyma tsukubaensis), and Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trichosporonoides osedocephalis, Trichosporonoides nigrecens). Yeasts of the subphylum Ustyraginomycochina are known and described in the art as potential productive organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol, as well as other valuable biotechnology applications.For example, Geiser et al. (Prospecting the biodiversity of the fungal family Ustilaginacceae for the production of value-added chemicals ed chemicals,” Fungal Biol Biotechnol, 2014, 1:2), Feldbrugge et al., (“The biotechnological use and potential of plant pathogenic smut fungi,” Appl Microbiol Biotechnol,2013,97(8):3253-65), Guevarra et al.,(``Accumulation of itaconic,2-hydroxyparaconic,itatartaric,and malic acids by strains of the genus Ustilago,Agric.,1990,54(9),2353-2358), and Moon et al.,(``Biotechnological production of erythritol and Its applications,”Appl Microbiol See Biotechnol, 2010, 86:1017-1025.

[0024] A suitable yeast may be from the subphylum Saccharomycotina, such as Saccharomyces cerevisiae.

[0025] Suitable yeast cells have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein, the "active pentose phosphate pathway" refers to glucose-6-phosphate, NADP. +Alternatively, it refers to the expression of one or more functional enzymes that convert NAD+ and water together into NADPH or NADH, CO2, and ribulose-5-phosphate. If the non-oxidative step continues, this pathway may further produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway may produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, or combinations thereof. Active pentose phosphate pathways may be naturally present in yeast cells or can be introduced into yeast cells through genetic engineering.

[0026] Yeast cells may be osmotically tolerant yeast cells. As used herein, “osmotically tolerant” means yeast that can grow and regenerate under high osmotic conditions (e.g., at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v), or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), at least 15% (w / v) sodium chloride). Species and strains of osmotically tolerant yeast are known and described in the art and include many species of yeast used in industrial fermentation processes. Similarly, methods for assaying yeast osmotic tolerance are known and described in the art. For example, see 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).

[0027] Recombinant yeast cells may be recombinant Moniliella cells, such as Moniliella polynis cells. Figure 1 shows the predicted native pentose phosphate and glycolysis pathway in Moniliella polynis. Moniliella has been previously used in the fermentation production of erythritol, and methods for genetically modifying and fermenting Moniliella are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb Cell Fact, 16:168).

[0028] Various plasmids and methods for the transformation of Moniliella are also described in the following examples. For example, Moniliella can be transformed using a binary polynucleotide sequence in which the desired exogenous polynucleotide is incorporated into a specific locus after recombination, and a selection marker can be expressed intracellularly. Suitable selection markers are known and described in the art. Selective markers may include, but are not limited to, amdS (e.g., degraded into the 3' portion (sequence number 167) and the 5' portion (sequence number 174)), the G418 resistance gene (e.g., degraded into the 3' portion (sequence number 172) and the 5' portion (sequence number 175)), the zeosin resistance gene (e.g., degraded into the 3' portion (sequence number 168) and the 5' portion (sequence number 169)), nulceotricin N-acetyltransferase (NAT) (e.g., degraded into the 3' portion (sequence number 171) and the 5' portion (sequence number 170)), and the invertase gene (SUC2) (e.g., the 3' portion of sequence number 173 and the 5' portion of sequence number 176).

[0029] The recombinant cells described herein comprise one or more exogenous polynucleotide sequences, which, when expressed, encode one or more polypeptides that improve glucose-to-xylitol fermentation by the recombinant cells.

[0030] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless expressly indicated otherwise.

[0031] As used herein, “exogenous” means genetic material or its expression products that originate from outside the host organism. For example, exogenous genetic material or its expression products may be a modified form of genetic material native to the host organism, may originate from another organism, may be a modified form of a component derived from another organism, or may be a synthetically derived component. For example, the K. lactis invertase gene becomes exogenous when introduced into S. cerevisiae.

[0032] As used herein, “natural” means genetic material or its expression products found within the genome of wild-type host cells, apart from inter-individual variations that do not affect function or expression. For the purposes of this application, Moniliella polynis cells “Moniliella tomentosa var polynis TCV364”, described in U.S. Patent No. 6,440,712, which is incorporated herein by reference in its entirety, and deposited on March 28, 1997, under the Budapest Convention with BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de l'Universite Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) under the number MUCL40385, are considered wild-type Moniliella polynis cells.

[0033] As used herein, the terms “polypeptide” and “peptide” refer to the collective primary, secondary, tertiary, and quaternary amino acid sequences and structures necessary to impart their function and properties to the listed polymers, and which are used interchangeably. As used herein, “enzyme” or “biosynthetic pathway enzyme” refers to a protein that catalyzes a chemical reaction. Any enumeration of a particular enzyme is understood to include cofactors, coenzymes, and metals necessary for the enzyme to function properly, either independently or as part of a biosynthetic pathway. Table 1 provides an overview of amino acids as understood in the art, along with their three-letter and one-letter symbols. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0034] [Table 1]

[0035] Mutants or sequences having substantial identity or homology to the polypeptides described herein may be used in the implementation of the disclosed recombinant cells, compositions, and methods. Such sequences may be called variants or modified sequences. That is, polypeptide sequences may be modified while still retaining the ability to exhibit the desired activity. Generally, mutants or modified sequences may have more than 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild type, naturally occurring polypeptide sequences, or mutant polypeptides described herein.

[0036] Where used herein, the phrases “% sequence identity,” “% identity,” and “percent identity” are used interchangeably and refer to the percentage of residue matching between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods for amino acid and nucleic acid sequence alignment are well known. Sequence alignment and sequence identity generation include global and local alignments, which typically use computational approaches. Alignment can be performed using the BLAST (National Center for Biological Information, NCBI) Basic Local Alignment Search Tool version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters. Maximum target sequences: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word size: 6; Maximum match in query range: 0; Matrix: BLOSUM62; Gap cost: (Present: 11, Extended: 1); Composition adjustment: Conditional composition score matrix adjustment; Filter: Not selected; Mask: Not selected. Nucleic acid sequence identity % between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequences: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word size: 28; Maximum match in query range: 0; Match / mismatch score: 1, -2; Gap cost: Linear; Filter: Low complexity region; Mask: Mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to have at least XX% identity to the reference sequence, or equivalently XX% sequence identity.

[0037] Polypeptide or polynucleotide sequence identity may be measured over the entire length of a defined polypeptide sequence, for example, as defined by a specific sequence number, or over a shorter length, for example, over the length of a fragment taken from a larger defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are merely illustrative, and it is understood that any fragment length supported by the sequences shown herein, in tables, figures, or sequence listings may be used to describe the length over which the identity percentage can be measured.

[0038] Polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein, the term “wild type” is a term of the art as understood by those skilled in the art, and means a typical form of a polypeptide, such as that found in nature, which is distinguished from variant or mutant forms. As used herein, “mutant,” “mutant,” or “derivative” means a polypeptide molecule having an amino acid sequence different from that of a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of amino acid residues compared to the reference molecule.

[0039] The amino acid sequences of polypeptide variants, mutants, derivatives, or fragments intended herein may include conserved amino acid substitutions compared to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conserved amino acid substitutions compared to a reference molecule. A “conserved amino acid substitution” is a substitution of one amino acid to another amino acid which is predicted to interfere least with the properties of the reference polypeptide. In other words, a conserved amino acid substitution substantially preserves the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of substitution, e.g., a beta-sheet or alpha-helix conformation, (b) the molecular charge and / or hydrophobicity at the site of substitution, and / or (c) the bulkiness of the side chain.

[0040] As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,” and “nucleic acid” are interchangeable and refer to a sequence of nucleotides or any fragment thereof. These phrases further refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. A DNA polynucleotide may be cDNA (e.g., coding DNA) or a genomic DNA sequence (e.g., including both introns and exons).

[0041] A polynucleotide is said to encode a polypeptide if, in its natural state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce a polypeptide or fragment thereof. The antisense strand of such a polynucleotide is also said to encode a sequence.

[0042] Those skilled in the art will understand the degeneracy of the genetic code and that various polynucleotides can code for the same polypeptide. In some embodiments, a polynucleotide (e.g., a polynucleotide encoding the X5PP polypeptide) can be codon-optimized for expression in specific cells, including but not limited to plant cells, bacterial cells, fungal cells, or animal cells. Polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, but any polynucleotide sequence encoding a desired form of the polypeptide described herein may be used. Thus, sequences not found in nature can be used. These may be desired, for example, to enhance the expression of polypeptides or proteins in heterologous expression systems. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and human hands can also be used to generate degenerate coding sequences.

[0043] Recombinant cells described herein may include deletions or disruptions in one or more native genes. The term “deletion or disruption” means a state of a native gene in a recombinant cell having either a completely removed coding region (deletion) or an alteration (by deletion, insertion, or mutation, etc.) of the gene, its promoter, or its terminator, resulting in the gene no longer producing an active expression product, a significant reduction in the amount of expression product (e.g., at least 75% or at least 90%), or a significantly reduced activity (e.g., at least 75% or at least 90%) of the expression product. Deletions or disruptions can be achieved by genetic engineering, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. The native gene to be deleted or disrupted may be replaced with an exogenous nucleic acid of interest for the expression of an exogenous gene product (e.g., polypeptide, enzyme, etc.).

[0044] Recombinant cells described herein may include one or more genetic modifications in which an exogenous nucleic acid is incorporated into the genome of a host cell. Those skilled in the art know how to select appropriate loci in the yeast genome for the incorporation of an exogenous nucleic acid. Suitable incorporation 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 M. polynis host cells, suitable interacting loci may include, but are not limited to, the ER1 locus (defined as the locus adjacent to SEQ ID NOs. 85 and 162), the ER3 locus (defined as the locus adjacent to SEQ ID NOs. 155 and 165), the PDC1 locus (defined as the locus adjacent to SEQ ID NOs. 152 and 164), the pyrF locus (defined as the locus adjacent to SEQ ID NOs. 153 and 163), the TRP3 locus (defined as the locus adjacent to SEQ ID NOs. 156 and 159), the gpdIIA locus (defined as the locus adjacent to SEQ ID NOs. 157 and 161); and the gpdIIB locus (defined as the locus adjacent to SEQ ID NOs. 158 and 166). Exogenous nucleic acids may further be incorporated into intergeneric regions or other locations in the host cell genome that are not specifically identified herein. Other suitable integration loci may be determined by those skilled in the art. Furthermore, those skilled in the art will recognize how to use sequences to design primers for verifying precise gene integration at selected loci.

[0045] Recombinant cells may have one or more copies of a given exogenous nucleic acid sequence that is integrated into a host chromosome and replicated together with the chromosome into which it is integrated. For example, yeast cells may 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 contain 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 that are integrated into the genome. The multiple copies of the polynucleotide sequence may all be integrated into a single locus, or they may be integrated into multiple loci.

[0046] The final step in the xylitol pathway from xylitol 5-phosphate to xylitol requires a phosphatase enzyme. The Saccharomyces cerevisiae PYP1 (polyolp 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 sugar alcohol phosphatase," ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova, et a., "Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae," J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the sorbitol-6-phosphatase enzyme class (Enzyme Commission (EC) 3.1.3.50). Since xylitol 5-phosphate is a molecule similar to a known substrate of PYP1, it is demonstrated herein that one or more PYP-like enzymes or PYP orthologues possess xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in recombinant cells as described herein. Escherichia coli HxpA (hexitol phosphatase A) is a HAD-like enzyme belonging to EC 3.1.3.50 that 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, it is also demonstrated herein that one or more HAD-like hydrolase enzymes or HAD-like hydrolase orthologues have xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in recombinant cells as described herein.

[0047] The recombinant cells described herein are capable of producing xylitol and are characterized by overexpression of a native enzyme having xylitol-5-phosphate phosphatase (X5PP) activity, and / or contain an exogenous polynucleotide sequence encoding a native or exogenous enzyme having xylitol-5-phosphate phosphatase (X5PP) activity. Generally, recombinant cells overexpressing the X5PP enzyme, or recombinant cells expressing the exogenous X5PP enzyme, produce more xylitol than equivalent cells lacking the exogenous X5PP enzyme or not overexpressing the X5PP enzyme. The enzyme can be any suitable enzyme having X5PP activity. As used herein, "X5PP enzyme" and "X5PP" are interchangeable and refer to an enzyme having X5PP activity. In this paper, "xylitol-5-phosphate phosphatase activity" and "X5PP activity" are used synonymously and refer to the catalytic ability to convert xylitol-5-phosphate to xylitol and phosphate. Suitable X5PP enzymes are divalent metal cations, e.g., Mg 2+ Mn 2+ , or Cole 2+This may include. Suitable enzymes having X5PP activity include, but are not limited to, those classified under EC 3.1.3.50, such as sugar alcohol phosphatases and HAD-like hydrolases. The polynucleotide encoding the X5PP enzyme can be derived from any suitable source. For example, the polynucleotide encoding the X5PP enzyme may be derived from Moniliella polinis, Saccharomyces cerevisiae, Lachancea dasiensis, Tetrapispira blattae, Saccharomyces pastorianus, Kazakhstania africana, Podospora comata, Geotrichum candidam, Ogataea haglelorum, Devariomyces fabri, Monilinia fructicola, Nadsonia fulvecens var. elongata DSM 6958, Escherichia coli, Wickerhamomyces ciferi, Bacillus amyloriquefaciens, etc. The X5PP enzyme may be a polypeptide having an amino acid sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% identical to at least one amino acid sequence from among SEQ ID NOs: 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188. The X5PP enzyme may be a polypeptide having an amino acid sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% identical to at least one amino acid sequence from sequence numbers 200, 202, 203, 204, 206, and 213. The X5PP enzyme may be a polypeptide having an amino acid sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% identical to at least one amino acid sequence from sequence numbers 200, 203, 204, 206, and 213.

[0048] The recombinant cells described herein may include a Moniliella polynis gene encoding the amino acid sequence of SEQ ID NO: 200, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0049] The recombinant cells described herein may include a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO: 201, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0050] The recombinant cells described herein may include the Lachansea dasiensis gene encoding the amino acid sequence of SEQ ID NO: 202, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0051] The recombinant cells described herein may include a tetrapisispora blattae gene encoding the amino acid sequence of SEQ ID NO: 203, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0052] The recombinant cells described herein may include a Saccharomyces pastorianus gene encoding the amino acid sequence of SEQ ID NO: 204, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0053] The recombinant cells described herein may include a Kazakhstani Africana gene encoding the amino acid sequence of SEQ ID NO: 205, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0054] The recombinant cells described herein may include a Podospora komata gene encoding the amino acid sequence of SEQ ID NO: 206, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0055] The recombinant cells described herein may include a Geotrichum candidam gene encoding the amino acid sequence of SEQ ID NO: 207, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0056] The recombinant cells described herein may include an ogattea hagleloram gene encoding the amino acid sequence of SEQ ID NO: 208, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0057] The recombinant cells described herein may include a devariomyces fabri gene encoding the amino acid sequence of SEQ ID NO: 209, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0058] The recombinant cells described herein may include a Monilina fructicola gene encoding the amino acid sequence of SEQ ID NO: 210, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0059] The recombinant cells described herein may contain the Nadsonia fulvecens variety elongata DSM 6958 gene encoding the amino acid sequence of SEQ ID NO: 211, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0060] The recombinant cells described herein may include an E. coli gene encoding the amino acid sequence of SEQ ID NO: 213, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0061] The recombinant cells described herein may include the Wickerhammyces ciferi gene encoding the amino acid sequence of SEQ ID NO: 214, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0062] The recombinant cells described herein may include a Moniliella polynis gene encoding the amino acid sequence of SEQ ID NO: 221, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0063] The recombinant cells described herein may include a Bacillus amyloricephasiens gene encoding the amino acid sequence of SEQ ID NO: 222, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0064] The recombinant cells described herein may include the Saccharomyces cerevisiae DOG2 gene encoding the amino acid sequence of SEQ ID NO: 189, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0065] The recombinant cells described herein may include the Saccharomyces cerevisiae DOG1 gene encoding the amino acid sequence of SEQ ID NO: 188, or an exogenous polynucleotide that may be derived therefrom. The exogenous polynucleotide 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.

[0066] Enzymes possessing X5PP activity may be native to host cells. For example, if the host organism is M. polynis, the X5PP enzyme may be an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with at least one of SEQ ID NOs: 198, 199, 200, or 221. Recombinant cells may contain an exogenous polynucleotide encoding an X5PP enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with at least one of SEQ ID NOs: 198, 199, 200, or 221. Recombinant cells may include, but are not limited to, the insertion of an additional copy of the nucleic acid encoding the native X5PP enzyme into a host cell (e.g., an additional copy of the polynucleotide encoding X5PP into a non-specific locus within the cell), the insertion of a constitutive promoter upstream of the coding region of the gene encoding the native X5PP enzyme in the host cell genome, and / or the modification of an existing promoter located upstream of the coding region of the gene encoding the native X5PP enzyme in the host cell genome. Those skilled in the art will recognize that there are many ways to increase the expression of the gene encoding the native X5PP enzyme, and will be able to select and apply an appropriate method.

[0067] As used herein, “overexpression” means a level of expression of a polypeptide in equivalent cells that is higher than the level of expression of the same polypeptide in the absence of the genetic modification or the exogenous polynucleotide encoding the polypeptide.

[0068] Recombinant cells described herein, which are capable of producing xylitol, contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, may further be characterized by overexpression of ribulose 5-phosphate epimerase (RPE enzyme). Generally, recombinant cells that overexpress RPE enzyme produce more xylitol than cells lacking RPE enzyme or equivalent cells that do not overexpress RPE enzyme.

[0069] The recombinant cells described herein may be capable of producing xylitol and may contain an exogenous polynucleotide sequence encoding an X5PP enzyme and / or may overexpress a native X5PP enzyme and contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have a genetic modification resulting in the overexpression of a native RPE enzyme. The RPE enzyme may be any suitable enzyme having 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 having RPE activity may be native to the host cell, or the RPE enzyme may be an exogenous RPE enzyme. For example, if the host organism is M. polynis, the RPE enzyme may be an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with at least one of SEQ ID NOs: 179 and 180. Recombinant cells may contain an exogenous polynucleotide encoding an RPE enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with at least one of SEQ ID NOs: 179 and 180. Recombinant cells may include at least one of sequence numbers 179 and 180 and gene modifications that increase the expression of the RPE enzyme with 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% identity. Gene modifications may include, but are not limited to, insertion of a further copy of the nucleic acid encoding the native RPE into the cell, insertion of a constitutive promoter upstream of the coding region of the native RPE gene in the host cell genome, and / or modification of a promoter located upstream of the coding region of the native RPE gene in the host cell genome.Those skilled in the art will recognize that the expression of the natural RPE gene can be increased by many methods known in the art, and will be able to appropriately select and apply such methods.

[0070] Recombinant cells described herein are capable of producing xylitol and may contain an exogenous polynucleotide sequence encoding the X5PP enzyme and / or overexpressing the native X5PP enzyme and an exogenous polynucleotide sequence encoding the xylitol-phosphate dehydrogenase (XPDH) enzyme. The exogenous polynucleotide sequence may be the exogenous xylitol-phosphate dehydrogenase (XPDH) gene. Recombinant cells described herein that are capable of producing xylitol and contain an exogenous polynucleotide sequence encoding the X5PP enzyme and / or overexpressing the native X5PP enzyme and an exogenous polynucleotide sequence encoding the XPDH enzyme may further contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have a genetic modification resulting in overexpression of the native RPE enzyme as described herein.

[0071] "Xylitol-phosphate dehydrogenase gene" and "XPDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylitol-phosphate dehydrogenase activity. As used herein, "xylitol-phosphate dehydrogenase activity" refers to the ability to catalyze the reaction that converts xylose-5-phosphate and NADPH or NADH(NAD(P)H) to xylitol-5-phosphate and NADP+ or NAD+(NAD(P)+). The XPDH gene may be derived from any suitable source. For example, the XPDH gene may be derived from Clostridium difficile, Lactobacillus rhamnosus, Bacillus halodurans, Alkalihalobacillus ligniniphyllus, Geotogalibacillus soli, Heindrixia sporothermdurans, Clostridium fungisorbens, or Neobacillus cucumis. The XPDH gene may encode amino acids having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with at least one amino acid sequence of SEQ ID NOs. 12-15, 28-32, or 33. The XPDH gene may encode amino acids having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with at least one amino acid sequence of SEQ ID NOs. 14, 15, 28, or 31.

[0072] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Clostridium difficile gene encoding the amino acids of SEQ ID NO: 12. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 12.

[0073] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain a Clostridium difficile-derived gene encoding the amino acids of SEQ ID NO: 13, or an exogenous polynucleotide that can be derived therefrom. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 13.

[0074] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Lactobacillus rhamnosus gene encoding the amino acids of SEQ ID NO: 14. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 14.

[0075] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Bacillus halodurans gene encoding the amino acids of SEQ ID NO: 15. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 15.

[0076] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Alkalihalobacillus ligniniphyllus gene encoding the amino acids of SEQ ID NO: 28. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 28.

[0077] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Geotogalibacillus soli gene encoding the amino acids of SEQ ID NO: 29. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 29.

[0078] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Heindrixia sporothermodurance gene encoding the amino acids of SEQ ID NO: 30. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 30.

[0079] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain a Clostridium fungisorbens gene encoding the amino acids of SEQ ID NO: 31, or an exogenous polynucleotide that can be derived therefrom. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 31.

[0080] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Neobacillus cucumis gene encoding the amino acids of SEQ ID NO: 33. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 33.

[0081] Recombinant cells described herein are capable of producing xylitol and may contain an exogenous polynucleotide sequence encoding the X5PP enzyme and / or overexpressing the native X5PP enzyme and an exogenous polynucleotide sequence encoding the xylulokinase (XKS) enzyme. Recombinant cells described herein that are capable of producing xylitol and contain an exogenous polynucleotide sequence encoding the X5PP enzyme and / or overexpressing the native X5PP enzyme and an exogenous polynucleotide sequence encoding the XKS enzyme may also contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have a genetic modification resulting in overexpression of the native RPE enzyme, as described herein. The exogenous polynucleotide sequence may be the exogenous xylulose glycophosphatase (XKS) gene.

[0082] "Xylurokinase gene" and "XKS gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylurokinase activity. As used herein, "xylurokinase activity" refers to the ability to catalyze the conversion of xylulose-5-phosphate and ADP to xylulose and ATP. The XKS gene may be derived from any suitable source. For example, the XKS gene may be derived from Saccharomyces cerevisiae. The XKS gene may encode amino acids having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to at least one amino acid sequence of SEQ ID NOs. 188 and 189. A further description of recombinant cells comprising polypeptides capable of producing xylitol and possessing xyllokinase activity is provided in U.S. Provisional Patent Application No. 63 / 364,363, filed on 9 May 2022, which is incorporated herein by reference in its entirety.

[0083] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from, or potentially derived from, the DOG1 sugar phosphatase gene of Saccharomyces cerevisiae, encoding the amino acids of SEQ ID NO: 188. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 188.

[0084] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from, or potentially derived from, the Saccharomyces cerevisiae DOG2 sugar phosphatase gene encoding the amino acids of SEQ ID NO: 189. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 189.

[0085] Recombinant cells described herein are capable of producing xylitol and may include an exogenous polynucleotide sequence encoding the X5PP enzyme and / or overexpressing the native X5PP enzyme, and may include an exogenous polynucleotide sequence encoding the XKS enzyme and an exogenous polynucleotide sequence encoding the xylitol dehydrogenase (XDH) enzyme. Recombinant cells described herein that are capable of producing xylitol and include an exogenous polynucleotide sequence encoding the X5PP enzyme and / or overexpressing the native X5PP enzyme, and may include an exogenous polynucleotide sequence encoding the XKS enzyme and an exogenous polynucleotide sequence encoding the XHD enzyme may further include an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have a genetic modification resulting in overexpression of the native RPE enzyme as described herein. The exogenous polynucleotide sequence may be the exogenous xylitol dehydrogenase (XDH) gene.

[0086] "Xylitol dehydrogenase gene" and "XDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylitol dehydrogenase activity. As used herein, "xylitol dehydrogenase activity" refers to xylitol and NAD from xylulose and NADH or NADPH. + or NADP +This refers to the ability to catalyze the conversion to xylitol. The XDH gene may be derived from any suitable source. For example, the XDH gene may be derived from Pichia stipitis, Rhodobacteriaceae bacteria, or Bemisia argentofoli. The XDH gene may encode amino acids with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to at least one amino acid sequence of SEQ ID NOs. 190, 191, or 192. A further description of recombinant cells comprising polypeptides capable of producing xylitol and having xyllokinase activity and polypeptides having xylitol dehydrogenase activity is provided in U.S. Provisional Patent Application No. 63 / 364,363, filed 9 May 2022, which is incorporated herein by reference in its entirety.

[0087] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide derived from or potentially derived from the Pichia stipitis-derived XDH gene (encoding the amino acids of SEQ ID NO: 190) with switched cofactors. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 190.

[0088] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide encoding the amino acids of SEQ ID NO: 191, which is derived from or may be derived from the Rhodobacteraceae SDR family oxidoreductase gene. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 191.

[0089] Recombinant cells may contain an exogenous polynucleotide sequence encoding the X5PP enzyme, and / or overexpress the native X5PP enzyme, and may further contain an exogenous polynucleotide encoding the amino acids of SEQ ID NO: 192, which is derived from or can be derived from a ketose reductase (sorbitol dehydrogenase) gene of a Rhodobacteriaceae bacterium. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 192.

[0090] Exogenous polynucleotides in recombinant cells described herein may be under the control of a promoter. For example, exogenous nucleic acids may be operably linked to heterologous or artificial promoters. Suitable promoters are well known and described in the art. Examples of promoters include pyruvate decarboxylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86); 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132); translation elongation factor This may include, but is not limited to, the following: 1 promoter (TEFp; SEQ ID NO: 133); modified TEFp (SEQ ID NO: 131); phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135); enolase promoter (ENO1p; SEQ ID NO: 136); asparagine synthetase promoter (ASNSp; SEQ ID NO: 137); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138); and RPL16B (SEQ ID NO: 139).

[0091] Exogenous nucleic acids in recombinant cells described herein may be under the control of terminators. For example, exogenous nucleic acids may be operably linked to heterologous or artificial terminators. Suitable terminators are well known and described in the art. Terminators include, but are not limited to, GAL10 terminators, PDC terminators, transaldolase terminators (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); and PGM1 terminator (PGM1t; SEQ ID NO: 144). This may include, but is not limited to, the following: 145); PYK1 terminator (PYK1t; SEQ ID NO: 146); RPLA terminator (RPLAt; SEQ ID NO: 147); transaldolase 1 terminator (TAL1t; SEQ ID NO: 148); TDH3 terminator (TDH3t; SEQ ID NO: 149); translation elongation factor 2 terminator (TEF2t; SEQ ID NO: 150); triose phosphate isomerase 1 terminator (TPI1t; SEQ ID NO: 151); and MpTEF1 (SEQ ID NO: 289).

[0092] A promoter or terminator is "operably ligated" to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to the polynucleotide is such that the promoter or terminator may, in some cases, perform its transcriptional regulatory function.

[0093] Polypeptides described herein may be provided as part of a construct. As used herein, the term “construct” means a recombinant polynucleotide (including, but not limited to, DNA and RNA), which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods comprising polynucleotide sequences derived from at least two different natural sources, or they may be synthesized. Thus, constructs may include, for example, novel modifications to endogenous genes introduced by genome editing techniques. Constructs may further include recombinant polynucleotides prepared, for example, using recombinant DNA methodologies. A construct may be a vector comprising a promoter operably ligated to a polynucleotide encoding a polypeptide described herein. As used herein, the term “vector” means a polynucleotide capable of transporting another polynucleotide to which it is ligated. A vector may be a plasmid, which means a circular double-stranded DNA loop into which further DNA segments may be incorporated.

[0094] The present invention further provides a fermentation method for producing xylitol using recombinant cells as described herein. The fermentation method includes the step of fermenting a substrate using genetically modified yeast as described herein to produce xylthiol. The fermentation method may include additional steps as will be understood by those skilled in the art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and isolating ethanol from the fermentation broth. The fermentation process may be a fully aerobic or partially aerobic process.

[0095] The fermentation method can be carried out using a suitable fermentation substrate. Substrates for the fermentation method 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.

[0096] The fermentation process can be carried out under a variety of conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, may be the ambient temperature. Alternatively, the fermentation temperature may be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably around 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to the specific range or temperature described herein and may be changed as needed.

[0097] The fermentation process can be carried out within a specific oxygen uptake rate (OUR) range. The volume OUR of the fermentation process may be in the range of 0.5–40, 1–35, 2–30, 3–25, 4–20, or 5–15 mmol O2 / (L·h). In some embodiments, the ratio OUR may be in the range of 0.05–10, 0.1–8, 0.15–5, 0.2–1, or 0.3–0.75 mmol O2 / (g cell dry weight·h). However, the volume measurement OUR or ratio OUR of the fermentation process is not limited to any specific rate or range listed herein.

[0098] The fermentation process can be carried out at various cell concentrations. In some embodiments, the dry cell weight at the end of fermentation can be 5-40, 8-30, or 10-20 g / L. Furthermore, the pitch density or pitting rate of the fermentation process can be varied. In some embodiments, the pitch density can be 0.05-11, 0.1-10, or 0.25-8 g / L.

[0099] The initial dextrose concentration of the fermentation can be at least 100, 200, 250, 300, 350, or at least 400 g / L of dextrose. The initial dextrose concentration may be 100 - 400, 150 - 350, or 250 - 325 g / L.

[0100] The fermentation process can be associated with various characteristics such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of the yeast used in the fermentation process and / or genetic modification of the yeast. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be regulated through a combination of yeast selection or modification and selection of fermentation process conditions.

[0101] The xylitol production rate of this process can be at least at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L -1 h -1 It may be. The polyol mass yield of this process can be at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 50 percent, at least 55 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, or at least 85 percent. The final polyol titer of this process can be at least 5, 10, 20, 30, 50, 75, or 100 g / L.

[0102] The fermentation process can be carried out as a dextrose supply batch. Further, the fermentation process can be a batch process, a continuous process, or a semi - continuous process, as understood by those skilled in the art.

Examples

[0103] The present invention will be described in more detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Therefore, the present invention should not be construed as being limited in any way to the following examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0104] Throughout the examples, strain numbering and sequence identification numbers are used consistently. For example, strain 1-1 in Example 4 is the same as strain 1-1 in Example 5.

[0105] Example 1 - Diversity of xylitol-phosphate dehydrogenase Approximately 3000 galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) enzyme sequences were obtained from Uniprot and analyzed. Figure 2 shows the native sequence diversity for this set of sequences. This set is diverse, with about 25% of the enzymes lacking homologs with more than 75% identity. Since these enzymes tend to prefer NAD over NADP as a cofactor, the cofactor binding preference of the homologs was evaluated using a method similar to that described by Duax et al., ("Rational proteomics I. Fingerprinting identification and cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme family," Proteins, 2003, 53(4):931-943). Cofactor binding pockets were identified based on their proximity to the Rossman fold (+23 to +30 amino acids derived from the GXGXXG motif (SEQ ID NO: 129)) and scored based on the total charge within an 8-residue window. The top 8 candidates predicted using NADP were selected for further characterization, along with 4 candidates and 3 controls predicted using NAD.

[0106] Further examination of the predicted structural features of the binding pocket for factors that may influence cofactor preference led to the identification of important aspartic acid residues. See Figure 3. Using the polypeptide of SEQ ID NO: 34 and its substitutions, a structural homology model was constructed to predict the confirmation of the cofactor binding pocket. Figure 3 shows the C-terminus of the second to last β-chain outside the Rossmann fold domain. While we do not wish to be bound by any particular theory, enzymes in which the first residue in this region (residue 198 for SEQ ID NO: 34) is aspartic acid and the second residue (residue 199 for SEQ ID NO: 34) is a large hydrophobic amino acid (e.g., isoleucine) are predicted to prefer the NAD cofactor due to the hydrogen bonding of aspartic acid to the hydroxyl group of NAD-ribose. However, enzymes in which the first residue (residue 198 for SEQ ID NO: 34) is alanine, glycine, or serine, and the second residue (residue 199 for SEQ ID NO: 34) is lysine or arginine, prefer the NADP cofactor because the positive charge on the lysine or arginine residue interacts with the negative charge of the phosphate of NADP, and the smaller residue at the first position allows space in the binding pocket for the phosphate. Based on this analysis, 12 further enzymes were selected for their predicted preference for NADP. Finally, six further enzymes with sequence similarity to the active XPDH enzyme were selected for testing.

[0107] Example 2 - Diversity of TarJ Approximately 800 ribulose 5-phosphate reductase sequences were obtained from Uniprot and analyzed. Figure 3 shows the native sequence diversity for this set of sequences. Overall, the diversity in this set is low, with only 10% of the enzymes lacking sequence similarity of more than 75% identity. Since these enzymes tend to prefer NADP over NAD as a cofactor, no scoring was performed, and the sequences were simply aligned in Geneious (ClustalW, default settings). Eight enzymes were selected for further analysis based on sequence similarity.

[0108] Example 3 - In vitro enzyme assay Polynucleotides encoding suspected XPDH homologs (Table 2) or TarJ' homologs (Table 3) were cloned into a vector (New England Biolabs, PURExpress® In Vitro Protein Synthesis) containing a T7 promoter and terminator for cell-free protein expression. Cell-free synthesized proteins were analyzed for activity against four substrates (ribulose 5-phosphate, xylulose 5-phosphate, ribulose, and xylulose) having either NADP or NAD cofactor. Seven enzymes (XPDH of SEQ ID NOs. 12 and 34, and TarJ' of SEQ ID NOs. 36, 37, 38, 40, and 42) were able to catalyze the reduction of either ribulose 5-phosphate or xylulose 5-phosphate (Figure 5), but were unable to catalyze the reduction of either xylulose or ribulose (data not shown).

[0109] [Table 2]

[0110] [Table 3]

[0111] Example 4 - Genetically modified Moniliella polynis strain Strain 1-1 is the Moniliella polinis host strain “Moniliella tomentosa varietal polinis TCV364,” described in U.S. Patent No. 6,440,712, which is incorporated herein by reference in its entirety, and was deposited under the Budapest Convention with BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de l'Universiche Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) under number MUCL40385 on March 28, 1997. Table 4 below lists various Moniliella polinis strains, including information on the parent strain, the sequence into which the parent strain was transformed, and characterization of the expression cassette contained in the transformed sequence. Each "XPDH / TarJ' homolog expression cassette" contained, in order, a 5'ER1 flanking sequence (SEQ ID NO: 85), an MpPYK1 promoter (SEQ ID NO: 86), a gene encoding the indicated XPDH or TarJ' homolog (one of SEQ ID NOs: 87-128), an Mp6PGD terminator (SEQ ID NO: 140), and the 5' portion of the G418 resistance gene expression cassette (SEQ ID NO: 175). Each "selective marker cassette" contained, in order, 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 flanking sequence (SEQ ID NO: 160). During bipartite transformation with both the XPDH / TarJ' homolog expression cassette and the selective marker cassette, the two cassettes are recombined to incorporate nucleotide sequences encoding both the XPDH or TarJ' homolog and the G418 resistance marker at the ER1 locus.

[0112] The indicated Moniliella polinis parent strain was transformed with the indicated sequence by first protoplastizing the parent strain by adding an enzyme mixture containing 0.6 M MgSO4, 7.5 g / L dilyselase, and 12.5 g / L Trichoderma harzianum lysase to the mycelial pellet of the parent strain. The protoplasts were then pelletized, 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 1.5–5 μg each of 5' and 3' DNA transformation fragments (3–10 μg total; see Table 4 for a list of fragments) were added to approximately 200 μL of the protoplast mixture (10 8 The mixture was prepared by adding it to cells / mL. 50% PEG in 1 mL of STC medium was then added to a mixture of salmon sperm DNA, transformed DNA, and protoplasts, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, the recovered broth (0.4 M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L glucose, pH 4.5) was added to the mixture and incubated at 27°C and 100 rpm for 16-24 hours. After incubation, the protoplasts were pelletized by centrifugation and resuspended in 1 mL of PBS.

[0113] The resuspended protoplasts were seeded onto PDA+250 mg / L Geneticin (G418) select plates and incubated at 30–35°C for at least 2–4 days until transformants proliferated. The resulting transformants were evaluated by colony PCR for the incorporation of the indicated sequences. The PCR-validated isolates were then designated as the indicated strain numbers. In some cases, one or more PCR-validated isolates, e.g., "sister" isolates, are indicated by letters following the strain number. For example, strain 1-2 has five sister isolates: strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.

[0114] 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 target 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 XPDH homolog of SEQ ID NO: 87, which encodes 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 target 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 Geneticin (G418) selective plate and incubated at 30-35°C for at least 2 days until the transformants proliferated. The resulting transformants were streaked onto PDA+geneticin (G418) plates for single-colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of the indicated sequence. The PCR-validated isolates were designated strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.

[0115] [Table 4]

[0116] Example 5 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, 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 (scheduled in Table 4 above) were run in shaking flasks.

[0117] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0118] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 72 and 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 6 and Figures 6 and 7.

[0119] [Table 5]

[0120] PCR validation showed the presence of the transformed polynucleotide sequence in the indicated strains, but further analysis revealed that in some strains, the sequence was not correctly integrated into the ER1 locus. Further analysis showed 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 were not integrated into the ER1 locus.

[0121] [Table 6]

[0122] Example 6 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e (scheduled in Table 4 above) were run in shaking flasks.

[0123] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0124] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 7 and Figure 8.

[0125] As shown in Figure 8, sister strains 1-34c and 1-34d produced 15.8 and 18.6 g / L of xylitol, respectively, while strains 1-34a, 1-34b, and 1-34e did not produce significantly more xylitol than the wild type (strain 1-1, Figure 6). Strains 1-34a, 1-34b, and 1-34e were initially PCR validated, and it was subsequently determined that the frameshift mutation contained an integrated polynucleotide that should encode a N. cucumber XPDH homolog, and therefore functional XPDH was not expressed. Thus, although the results appear variable, they are actually consistent given that strains 1-34a, 1-34b, and 1-34e do not contain the polynucleotide encoding functional XPDH.

[0126] [Table 7]

[0127] Example 7 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, 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 (scheduled in Table 4 above) were run in shaking flasks.

[0128] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0129] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 8.

[0130] [Table 8]

[0131] Example 8 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, 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 (scheduled in Table 4 above) were run in shaking flasks.

[0132] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0133] A 0.8 mL seed culture was inoculated into a 250 mL baffle-free flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 72 and 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 9 and Figure 9.

[0134] PCR validation showed that the transformed polynucleotide sequence was present in the indicated strains, but further analysis showed that in some strains, the sequence was not correctly integrated into the ER1 locus. Further analysis showed that strains 1-16b-e contained the transformed polynucleotide sequence, but it was not located at the ER1 locus. Further analysis was inconclusive regarding the integration site in strains 1-2c and 1-2d.

[0135] [Table 9]

[0136] Example 9 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-13c, 1-8a-d, 1-26a-e, 1-36a-e, 1-41a-e, 1-40a-e, and 1-20a-e (scheduled in Table 4 above) were run in shaking flasks.

[0137] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0138] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 10 and Figure 10.

[0139] PCR validation showed the presence of the transformed polynucleotide sequence in the indicated strains, but further analysis showed that in some strains, the sequence was not correctly integrated into the ER1 locus. Further analysis showed that strains 1-8c, 1-8d, and 1-41c contained the transformed polynucleotide sequence, but it was not integrated into the ER1 locus. Further analysis was inconclusive regarding the integration locus in strains 1-36a, 1-41b, 1-41e, and 1-20a-e.

[0140] [Table 10]

[0141] Example 10 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, 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 (scheduled in Table 4 above) were run in shaking flasks.

[0142] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0143] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 72 and 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 11 and Figure 11.

[0144] PCR validation showed the presence of the transformed polynucleotide sequence in the indicated strains, but further analysis showed that in some strains, the sequence was not correctly integrated into the ER1 locus. Further analysis showed that strains 1-30c and 1-30d contained the transformed polynucleotide sequence, but it was not integrated into the ER1 locus. Further analysis regarding the integrated locus in strain 1-6c was not conclusive.

[0145] [Table 11]

[0146] Example 11-2 Copy XPDH modified Moniliella polynis strain Strain 1-1 was transformed using SEQ ID NO: 55 and SEQ ID NO: 177 as described in Example 4. SEQ ID NO: 177 contains (i) the 3' portion of a G418 selectable marker (SEQ ID NO: 172); ii) an expression cassette of a Clostridium difficile-derived XPDH homolog 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 (SEQ ID NO: 98); and (iii) 3' flanking DNA for target chromosome integration into the ER1 locus (SEQ ID NO: 162). SEQ ID NO: 55 contains (i) 5' flanking DNA for target chromosome integration into the ER1 locus (SEQ ID NO: 85); ii) an expression cassette of a Clostridium difficile-derived XPDH homolog of 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 Geneticin (G418) selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA+Geneticin (G418) plate for single colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of two copies of the C. difficile XPDH sequence. The PCR-validated isolates were designated strains 2-1a, 2-1b, 2-1c, 2-1d, and 2-1e.

[0147] Strain 1-1 was transformed using SEQ ID NO: 57 and SEQ ID NO: 178 as described in Example 4. SEQ ID NO: 178 contains (i) the 3' portion of a G418 selectable marker (SEQ ID NO: 172); ii) an expression cassette of a Lactobacillus rhamnosus-derived XPDH homolog (SEQ ID NO: 100) encoding the amino acid sequence of SEQ ID NO: 14, under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) 3' flanking DNA (SEQ ID NO: 162) for target chromosome integration into the ER1 locus. SEQ ID NO: 57 contains (i) 5' flanking DNA (SEQ ID NO: 85) for target chromosome integration into the ER1 locus; (ii) an expression cassette of a Lactobacillus rhamnosus-derived XPDH homolog of SEQ ID NO: 100 encoding the amino acid sequence of SEQ ID NO: 14, under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) 5' portion of a G418 selectable marker (SEQ ID NO: 175). Transformants were selected on a PDA+250 mg / L Geneticin (G418) selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA+Geneticin (G418) plate for single colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of two copies of the L. rhamnosus XPDH sequence. The PCR-validated isolates were designated strains 2-2a, 2-2b, 2-2c, 2-2d, and 2-2e.

[0148] To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-13c, 2-1a-e, and 2-2a-e were run in shaking flasks.

[0149] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0150] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. After 72 and 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 12.

[0151] PCR validation showed the presence of the transformed polynucleotide sequence in the indicated strains, but further analysis showed that in some strains, the sequence did not precisely target the ER1 locus. Further analysis showed that strain 2-2e contained the transformed polynucleotide sequence but did not target the ER1 locus.

[0152] [Table 12]

[0153] Example 12 - Genetically modified Moniliella polynis strain Strain 1-1 was transformed with SEQ ID NOs: 186 and 187 as described in Example 4. SEQ ID NOs: 186 contained (i) 5' flanking DNA for target chromosome integration into the ER1 locus (SEQ ID NOs: 85), (ii) MpPYK1 promoter (SEQ ID NOs: 86), (iii) gene encoding Staphylococcus aureus xylitol dehydrogenase (SEQ ID NOs: 34), (iv) Mp6PGD terminator (SEQ ID NOs: 140), and (v) 5' portion of a G418 resistance gene expression cassette (SEQ ID NOs: 175). Sequence ID 187 contained (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 Saccharomyces cerevisiae DOG1 sugar phosphatase (SEQ ID NO: 188), (v) the MpENO1 terminator (SEQ ID NO: 142), and (vi) the 3' flanking gene for target chromosome integration into the ER1 locus (SEQ ID NO: 162). Transformants were selected on PDA + 250 mg / L Geneticin (G418) selection plates and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA + Geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the indicated sequences. The PCR-validated isolate was designated strain 3-1.

[0154] Table 13 below lists various Moniliella polinis strains, including information on the parent strain, the sequence into which the parent strain was transformed, and characterization of the expression cassette contained in the transformed sequence. The transformed fragment of SEQ ID NO: 181 contained, in order, a 5'ER3 flanking sequence (SEQ ID NO: 155), an MpPYK1 promoter (SEQ ID NO: 86), the gene encoding the M. polinis RPE2 polypeptide of SEQ ID NO: 180, an MpPYK terminator (SEQ ID NO: 146), and the 5' portion of the zeosin resistance gene expression cassette (SEQ ID NO: 169). The transformed fragment of SEQ ID NO: 182 contained, in order, the 3' portion of the zeosin 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 contained, in order, the 3' portion of the zeosin resistance gene expression cassette (SEQ ID NO: 168), the Mp6PGD promoter (SEQ ID NO: 130), the gene encoding the M. polynis RPE1 polypeptide (SEQ ID NO: 179), the Mp6PGD terminator (SEQ ID NO: 140), and the 3'ER3 flanking sequence (SEQ ID NO: 165). The transformed fragment of SEQ ID NO: 184 contained, in order, the 3' portion of the zeosin resistance gene expression cassette (SEQ ID NO: 168), the Mp6PGD promoter (SEQ ID NO: 130), the gene encoding the M. polynis RPE2 polypeptide (SEQ ID NO: 180), the Mp6PDG terminator (SEQ ID NO: 140), and the 3'ER3 flanking sequence (SEQ ID NO: 165). The transformed fragment of SEQ ID NO: 185 contained, in order, a 5'ER3 flanking sequence (SEQ ID NO: 155), an MpTEF1 promoter (SEQ ID NO: 133), and the 5' portion of a zeosin resistance gene expression cassette (SEQ ID NO: 169).

[0155] The indicated Moniliella polinis parent strain was transformed with the indicated sequence as described in Example 4 using either zeosin or G418 selection corresponding to the indicated selection marker. The resulting transformants were evaluated for incorporation of the indicated sequence by colony PCR. The PCR-validated isolates were then designated as the indicated strain numbers. In some cases, one or more PCR-validated isolates, e.g., "sister" isolates, are indicated by letters following the strain number. For example, strain 3-2 has four sister isolates, strains 3-2a, 3-2b, 3-2c, and 3-2d (collectively 3-2a-d).

[0156] [Table 13] na-non-applicable, second cassette used only for recombinant and select markers.

[0157] Example 13 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-1, 3-1, 3-2a-c, 3-3a-c, 3-4a-c, 3-5a-c, and 3-6a-c were run in shaking flasks.

[0158] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0159] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 48, 72, and 96 hours after incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 14 and Figures 14 and 15.

[0160] [Table 14]

[0161] Example 14 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, erythritol, and ethanol, strains 1-1, 1-13c, 1-15a, 3-7a-f, and 3-8a-f were run in shaking flasks.

[0162] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0163] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 48, 72, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 15 and Figures 12 and 13.

[0164] [Table 15]

[0165] Example 15 - Shaking Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, erythritol, and ethanol, 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 shaking flasks.

[0166] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0167] A 0.8 mL seed culture was inoculated into a 250 mL baffle-less flask containing the production medium (Table 5) to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 48, 72, and 96 hours after incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 16-18 and Figures 16-21.

[0168] The yield was calculated as the ratio of the product (i.e., xylitol) to the substrate consumed (i.e., the difference between one time point and the second time point) and expressed as a percentage.

[0169] [Table 16-1]

[0170] [Table 16-2]

[0171] [Table 17-1]

[0172] [Table 17-2]

[0173] [Table 18-1]

[0174] [Table 18-2]

[0175] [Table 18-3]

[0176] [Table 18-4]

[0177] [Table 18-5]

[0178] Example 16 - Diversity of phosphatases The final step in the xylitol pathway from xylitol 5-phosphate to xylitol requires a phosphatase enzyme. The Saccharomyces cerevisiae PYP1 polyolp 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 sugar alcohol phosphatase," ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova, et a., "Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae," J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the enzyme class of sorbitol-6-phosphatases (EC 3.1.3.50). Since xylitol 5-phosphate is a molecule similar to a known substrate of PYP1, orthologues of PYP1 in Moniliella are likely to be involved in the final catalytic step in the pathway for xylitol production. Queries of the Moniliella polynis genome of strain 1-1 found two genes with high homology to PYP1: RCSR00371 and RCSR15215. These two genes are 40% and 38% identical to ScPYP1, respectively, and 89% identical to each other.

[0179] E. 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, the M. polynis genome was searched for HxpA orthologues as alternative phosphatase candidates. RCSR21016 was identified as having 38% identity with HxpA and was uniquely identified as a sugar phosphatase with sequence similarity to DOG1 and DOG2 of S. cerevisiae. As shown in Examples 12-15, the expression of DOG1 and DOG2 in S. cerevisiae increases xylitol production in M. polinis.

[0180] Based on enzyme classification and / or sequence identity, 26 additional phosphatase candidates, including M. polinis RCSR00371, RCSR15215, and RCSR21016 phosphatases and S. cerevisiae PYP1 phosphatase, were selected for further analysis, as outlined in Table 19.

[0181] [Table 19]

[0182] Example 17 - Genetically modified Moniliella polynis strain The Moniliella polinis parent strains shown were transformed with the double transformation fragments shown in Table 20 using the transformation method outlined in Example 4. The resulting transformants were evaluated by colony PCR for the incorporation of the indicated sequences. The PCR-validated isolates were then designated as strain numbers shown in Table 20. In some cases, one or more PCR-validated isolates, e.g., "sister" isolates, are indicated by letters 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.

[0183] For example, strain 2-2b was transformed using SEQ ID NO: 223 and SEQ ID NO: 181. SEQ ID NO: 181 is described in Example 12 and includes, in this order, a 5'ER3 flanking sequence (SEQ ID NO: 155), an MpPYK1 promoter (SEQ ID NO: 86), the gene encoding the M. polynis RPE2 polypeptide (SEQ ID NO: 180), an MpPYK terminator (SEQ ID NO: 146), and the 5' portion of a zeosin resistance gene expression cassette (SEQ ID NO: 169). SEQ ID NO: 223 includes the 3' portion of a zeosin resistance gene expression cassette (SEQ ID NO: 168), an MpPGK1 promoter (SEQ ID NO: 135), the gene encoding the S. cerevisiae PYP1 polypeptide (SEQ ID NO: 224), an Mp6PGD terminator (SEQ ID NO: 140), and a 3'ER3 flanking sequence (SEQ ID NO: 165). Transformants were selected on a PDA+ zeosin select plate and incubated at 35°C for at least 2 days until the transformants proliferated. The obtained transformants were streaked onto PDA+zeosin plates for single-colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of the indicated sequence. The PCR-validated isolates were designated strains 4-1a, 4-1b, 4-1c, 4-1d, and 4-1e.

[0184] The phosphatase homolog transformation cassette outlined in Table 20 has the same components as Sequence ID No. 223 described above, but contains the indicated nucleotide sequence encoding the indicated polypeptide sequence.

[0185] [Table 20] na-non-applicable, second cassette used only for recombinant and select markers.

[0186] Example 18 - Shaking Flask Fermentation Assay 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 shaking flasks, and glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol were evaluated.

[0187] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0188] A product culture was formed by inoculating 0.4 mL of seed culture into a 250 mL baffle-free flask containing 20 mL of product medium (Table 5). The product culture was incubated at 35°C and 250 rpm. Samples were taken from the product culture at 48, 72, and 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. Fermentation results are reported in Table 21 and Figures 22 and 23. Results from 96 hours onward are not shown because part of the reaction consumed the entire glycerol feedstock, resulting in an inaccurate estimate of the reaction rate. The results demonstrate that overexpression of the X5PP enzyme of SEQ ID NOs. 198, 199, and 200 increases xylitol titer compared to the parent strain 2-2b and control strains (strains 3-12c and 3-12d) that express RPE and XPDH but lack X5PP. The results further indicate that expression of the exogenous X5PP enzyme from sequence number 201 also increased xylitol titer compared to strains 2-2b, 3-12c, and 3-12d.

[0189] [Table 21-1]

[0190] [Table 21-2]

[0191] The best xylitol-producing strains for each phosphatase enzyme were sequenced. All strains contained two copies of L. rhamnosus XPDH, as expected; however, there was variability in the gene copy numbers for phosphatases and RPE2, as outlined in Table 22.

[0192] [Table 22]

[0193] Example 19 - Shaking Flask Fermentation Assay Strains 1-1, 5-1a-e, 5-2a-e, 5-3a-e, and 5-4a-e were run in shaking flasks, and glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol were evaluated.

[0194] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0195] A product culture was formed by inoculating 0.4 mL of seed culture into a 250 mL baffle-free flask containing 20 mL of product medium (Table 5). The product culture was incubated at 35°C and 250 rpm. Samples were taken from the product culture at 24, 48, 72, and 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. Fermentation results at the final 96-hour point are reported in Tables 23 and 24. Results from 24, 48, and 72 hours are not shown. The results demonstrate that the phosphatases of SEQ ID NOs. 198-201 do not have the same effect on erythritol production as they do on xylitol production.

[0196] [Table 23]

[0197] [Table 24]

[0198] Example 20 - Shaking Flask Fermentation Assay 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 shaking flasks, and glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol were evaluated.

[0199] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0200] A 250 mL baffle-free flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results at the final 96-hour mark are reported in Table 25. Results from 24, 48, and 72 hours are not shown.

[0201] [Table 25]

[0202] [Table 26]

[0203] This example demonstrates the results of a shaking flask comparison of two X5PP enzymes (SEQ ID NOs: 200 and 201) with and without expression of RPE (SEQ ID NO: 180). The mean xylitol yields in strains containing overexpressed X5PP but not overexpressed RPE were 14% and 18.3% for phosphatases SEQ ID NOs: 200 and 201, respectively, compared to 11.7% for the control strain (see Table 26). These yields were significantly lower than the 33.7% and 37.7% yields observed in strains containing both X5PP and RPE expression.

[0204] Example 21 - Shaking Flask Fermentation Assay 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 shaking flasks, and glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol were evaluated.

[0205] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0206] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results at the final 96-hour mark are reported in Tables 27 and 28. Results from 24, 48, and 72 hours are not shown.

[0207] [Table 27]

[0208] [Table 28] * Values ​​in italics are outliers and are not used in the calculation of the average.

[0209] This shake flask fermentation assay examines the combination of the phosphatase of SEQ ID NO: 200, overexpression of RPE2 (SEQ ID NO: 180), and five different XPDH enzymes. Table Z shows the xylitol yields for the strains tested. For all of the XPDH candidates tested in combination with overexpression of RPE2 and the X5PP enzyme of SEQ ID NO: 200, a significant increase in yield was apparent. Strains with XPDHs of SEQ ID NOs: 14 and 15 had the highest 96-hour xylitol yields of 27% and 29.5%, respectively. All of the strains tested in this example have one copy of the XPDH shown, but strain 2-2b and its progeny have two copies of the sequence encoding the XPDH of SEQ ID NO: 14, potentially explaining why strains 4-4b and 4-4c have an average yield of 37.7% (see Table 26).

[0210] Example 22 - Shake Flask Fermentation Assay 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 evaluate glucose consumption and ribitol, xylitol, erythritol, glycerol, and ethanol production.

[0211] Strains were streaked onto YPD plates (bacterial peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, and agar 15 g / L) for biomass growth and incubated at 30 °C for 48 - 72 hours. Cells from the incubated YPD plates were scraped into 40 mL of rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffled flask. The cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15 - 20 to form a seed culture. The optical density was measured at a wavelength of 600 nm in a 1 cm path length cuvette using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 of 15 - 20 in about 32 - 50 hours.

[0212] A 250 mL baffle-free flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. Fermentation results at 48 and 72 hours are reported in Table 29. Results from 24 and 96 hours are not shown. The titer at 24 hours was low, and at 96 hours, most of the culture contained low concentrations of glucose or none at all. Due to an error in the shaking flask procedure for strain 5-15d, results for this strain are not reported. Results at 72 hours are also shown in Figures 24 and 25.

[0213] [Table 29-1]

[0214] [Table 29-2]

[0215] As shown in Figures 24 and 25, and as shown in Table 29, strains expressing the X5PP enzymes of SEQ ID NOs. 201–209 demonstrated increased xylitol titer compared to control strains 2-2b and RPE(3-12d). Strains expressing the X5PP enzymes of SEQ ID NOs. 201–207 further had at least one sister isolate with xylitol titer equivalent to or exceeding that of strain 4-4c expressing the X5PP of SEQ ID NO. 200. Some of the assayed strains also exhibited sister-to-sister variability. As demonstrated in some of the above examples, strains are verified by PCR to contain the desired polynucleotide sequence, although the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have made individual sisters different from each other. While the results here suggest that similar transformations may occur in these sisters, the results demonstrate the efficacy of the X5PP enzymes shown.

[0216] For example, PCR validation showed that the transformed polynucleotide sequence was present in the indicated strains, but further whole-genome sequencing analysis showed that in strains 5-12a-e, 5-15a-c, and 5-15e, the sequence was incorporated in one more copy and / or not precisely incorporated at the ER3 locus. The results of whole-genome sequencing for these strains are reported in Table 30 below. The results suggest that the variability between sister strains is likely due to differences in gene copy number and integration locus (of the XPDH, RPE2, and / or phosphatase genes), but that despite the variability in copy number and integration, the efficacy of the indicated X5PP enzyme is consistent.

[0217] [Table 30]

[0218] Example 23 - Shaking Flask Fermentation Assay 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 shaking flasks to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0219] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0220] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. High-performance liquid chromatography with a refractive index detector was used to analyze the samples for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol. Fermentation results at 48 and 72 hours are reported in Table 31. Results from 24 and 96 hours are not shown. The titer at 24 hours was low, and at 96 hours, most of the culture contained low concentrations of glucose or none at all. Results from 72 hours are also shown in Figures 26 and 27.

[0221] [Table 31-1]

[0222] [Table 31-2]

[0223] As shown in Figures 26 and 27, and as shown in Table 31, strains expressing the X5PP enzymes of SEQ ID NOs. 210, 211, 213, 214, and 189 demonstrated increased xylitol titers compared to the control strain 2-2b. The strains expressing the X5PP enzymes of SEQ ID NOs. 210 and 213 further had at least one sister isolate with xylitol titers equivalent to or exceeding those of strains 4-4b and 4-4c expressing the X5PP enzyme of SEQ ID NO. 200. Some of the assayed strains also exhibited sister-to-sister variability. As demonstrated in some of the above examples, strains are verified by PCR to contain the desired polynucleotide sequence, although the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or frameshifts or other mutations may have made individual sisters different from each other. While the results here suggest that similar transformations may occur in these sisters, the results demonstrate the efficacy of the X5PP enzymes shown.

[0224] Example 24 - Shaking Flask Fermentation Assay 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 shaking flasks to evaluate glucose consumption and the production of ribitol, erythritol, xylitol, glycerol, and ethanol.

[0225] The strain was streaked onto YPD plates (20 g / L bacteriological 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 40 mL of rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffled flask. The cells were incubated at 30 °C and 250 rpm until the optical density (OD600) reached 15 - 20 to form a seed culture. The optical density was measured at a wavelength of 600 nm in a 1 cm pathlength cuvette using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 of 15 - 20 in about 32 - 50 hours.

[0226] A 250 mL baffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of the seed culture to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture at 24 hours, 48 hours, 72 hours, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography equipped with a refractive index detector. The fermentation results at the 48 - hour and 72 - hour time points are reported in Table 32. The results from the 24 - hour and 96 - hour time points are not shown. The titer at the 24 - hour time point was low, and after 96 hours, many of the cultures contained low concentrations of glucose or none at all. The results from the 72 - hour time point are also shown in Figures 28 and 29.

[0227]

Table 32 - 1

[0228]

Table 32 - 2

[0229] As shown in Figures 28 and 29, and Table 32, strains expressing the X5PP enzymes of SEQ ID NOs. 221, 222, and 188 demonstrated increased xylitol titers compared to the control strain 2-2b. None of the tested strains had xylitol titers equivalent to or exceeding those of strains 4-4b and 4-4c expressing the X5PP of SEQ ID NO. 200. Some of the assayed strains also exhibited sister-to-sister variability. As demonstrated in some of the above examples, strains are verified by PCR to contain the desired polynucleotide sequence, but the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or a frameshift or other mutation may have made individual sisters different from each other. The results here suggest that similar transformations may occur in these sisters, but the results demonstrate the efficacy of the X5PP enzyme shown.

[0230] Example 25 - Shaking Flask Fermentation Assay 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 a shaking flask to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0231] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0232] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. High-performance liquid chromatography with a refractive index detector was used to analyze the samples for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol. Fermentation results at 48 and 72 hours are reported in Table 33. Results from 24 and 96 hours are not shown. The titer at 24 hours was low, and at 96 hours, most of the culture contained low concentrations of glucose or none at all. The results at 72 hours are also shown in Figure 30.

[0233] [Table 33]

[0234] The results of this example are consistent with those of the previous examples, demonstrating that strains expressing the X5PP enzyme of SEQ ID NOs. 202, 203, 204, 205, 206, 210, and 213 produce xylitol titers that are approximately equivalent to or greater than those produced by the control strain expressing the X5PP of SEQ ID NO. 200. As demonstrated in some of the above examples, strains are verified by PCR to contain the desired polynucleotide sequence, although the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or a frameshift or other mutation may have made individual sisters different from each other. The results here suggest that similar transformations may occur in these sisters, but the results demonstrate the effectiveness of the X5PP enzyme shown.

[0235] Example 26 - Genetically modified Moniliella polynis strain UV mutagenesis (Hoefer UV Crosslinker at 360 μJ / cm²) 3 Using the energy (used in the 3-12d sample) and selection of strain 3-12d, Moniliella polinis strains with reduced foaming during shaking flask fermentation were produced. Strains with a low-foaming phenotype were selected based on a visual evaluation of foaming during shaking flask fermentation compared to foaming in the parent strain 3-12d. The resulting low-foaming strain, containing two copies of the exogenous polynucleotide sequence encoding XPDH of SEQ ID NO: 14 integrated into the ER1 locus and one copy of the polynucleotide sequence encoding RPE of SEQ ID NO: 180 integrated into the ER3 locus, was designated 6-1.

[0236] Strain 6-1 was transformed with the Cre recombinase plasmid of SEQ ID NO: 288 using the transformation method outlined in Example 4. The resulting transformants were evaluated by colony PCR for the removal of G418 and the zeosin resistance selection marker. The PCR-validated isolate was designated as strain 6-2.

[0237] Strain 6-2 was non-selectively propagated on a YPD plate to induce plasmid loss of SEQ ID NO: 288. A single colony was attacked with biomass, and plasmid loss was confirmed by PCR. The PCR-validated isolate was designated strain 6-3.

[0238] The parental strain of Moniliella polinis shown, 6-3, was transformed with the double transformation fragments shown in Table 34 using the transformation method outlined in Example 4. The resulting transformants were evaluated by colony PCR for the incorporation of the shown sequences. The PCR-validated isolates were then designated as the indicated strain numbers shown in Table 34. In some cases, one or more PCR-validated isolates, e.g., "sister" isolates, are indicated by letters 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.

[0239] For example, strain 6-3 was transformed with SEQ ID NO: 280 and SEQ ID NO: 279. SEQ ID NO: 279 contained, in order, 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 contained the 5' gpdIIB flanking sequence (SEQ ID NO: 158), the PGK1 promoter (SEQ ID NO: 135), the gene encoding the Moniliella polynis 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 selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked onto a PDA+G418 plate to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of the indicated sequences. The PCR-validated isolates were designated strains 6-4a, 6-4b, 6-4c, 6-4d, and 6-4e.

[0240] The transformation cassettes for the phosphatase homologs outlined in Table 34, SEQ ID NOs. 281-287, have the same components as SEQ ID NOs. 280 but contain the indicated nucleotide sequence encoding the indicated polypeptide sequence. SEQ ID NOs. 181 is described in Example 12 and, in order, contained the 5'ER3 flanking sequence (SEQ ID NOs. 155), the MpPYK1 promoter (SEQ ID NOs. 86), the gene encoding the M. polynis RPE2 polypeptide of SEQ ID NOs. 180, the MpPYK terminator (SEQ ID NOs. 146), and the 5' portion of the zeosin resistance gene expression cassette (SEQ ID NOs. 169).

[0241] [Table 34]

[0242] Sequencing was performed on strains 6-12a-c to determine which sites the polynucleotides indicated were incorporated into and what the final copy number of a given gene was. Parent strain 6-3 contained one copy of the polynucleotide encoding RPE of SEQ ID NO: 180, incorporated into the ER3 locus on a single allele. In strain 6-12b, a second copy of the sequence encoding RPE was incorporated into the ER3 locus on a second allele, resulting in complete ER3 knockout. Strain 6-12b was further confirmed to have two copies of SEQ ID NO: 230, which encodes the X5PP enzyme of SEQ ID NO: 200. However, in strains 6-12a and 6-12c, transformation resulted in the substitution of previously incorporated sequences with new sequences encoding both RPE of SEQ ID NO: 180 and X5PP of SEQ ID NO: 200.

[0243] Example 27 - Shaking Flask Fermentation Assay 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 shaking flasks, and glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol were evaluated.

[0244] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0245] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. High-performance liquid chromatography with a refractive index detector was used to analyze the samples for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol. Fermentation results at 48 and 72 hours are reported in Table 35. Results from 24 and 96 hours are not shown. The titer at 24 hours was low, and at 96 hours, most of the culture contained low concentrations of glucose or none at all. The results at 72 hours are also shown in Figures 31 and 32.

[0246] In the case of strain 6-7b, there was an error during the shaking flask fermentation process. The results show little glucose consumption and low metabolite production, but this is likely due to an error in the fermentation setup and not a defect in the strain itself.

[0247] [Table 35-1]

[0248] [Table 35-2]

[0249] The results demonstrate that the same increase in xylitol titer and yield can be achieved when the sequence encoding X5PP is integrated into the gpdIIB locus, as seen in the previous examples where the sequence was integrated into the ER3 locus. As demonstrated in some of the examples above, strains are verified by PCR to contain the desired polynucleotide sequence, but the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or a frameshift or other mutation may have made individual sisters different from each other. The results here suggest that similar transformations may occur in these sisters, but the results demonstrate the efficacy of the X5PP enzyme shown.

[0250] Example 28 - Shaking Flask Fermentation Assay 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 a shaking flask to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0251] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0252] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. High-performance liquid chromatography with a refractive index detector was used to analyze the samples for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol. Fermentation results at 48 and 72 hours are reported in Table 36. Results from 24 and 96 hours are not shown. The titer at 24 hours was low, and at 96 hours, most of the culture contained low concentrations of glucose or none at all. The results at 72 hours are also shown in Figure 33.

[0253] In the case of strain 6-7b, there was an error during the shaking flask fermentation process. The results show little glucose consumption and low metabolite production, but this is likely due to an error in the fermentation setup and not a defect in the strain itself.

[0254] [Table 36]

[0255] Example 29 - Genetically modified Saccharomyces cerevisiae strain As demonstrated herein, the pathway for producing xylitol from glucose in Moniliella polinis utilizes two enzymes. First, xylitol 5-phosphate dehydrogenase (XPDH) extracts a carbon from the pentose phosphate pathway by converting xylulose 5-phosphate to xylitol 5-phosphate. Second, xylitol 5-phosphate decoplastyase (X5PP) removes phosphate from xylitol-5-phosphate to produce xylitol. To test the feasibility of this pathway in Saccharomyces cerevisiae, the following strains were constructed and tested.

[0256] Stock 7-1 Strain 7-1 is the yeast strain Saccharomyces cerevisiae CEN.PK 113-7D (MATaHIS 3 LEU2 TRP1 MAL2-8 SUC2; classification ID: NCBI: txid889517).

[0257] Dyeing 7-2 Strain 7-2 is a uracil nutrient derivative of strain 7-1, in which the Aspergillus nidurans amdS gene is inserted into the URA3 locus.

[0258] Stock 7-3 Strain 7-2 was transformed using the Li-acetate protocol (Gietz, RD, et al., "Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method," Methods Enzymol. 350, 87-96, 2002) with a DNA fragment containing a constitutively expressed synthetic transcription factor (sTF; nucleotide sequence (SEQ ID NO: 290; polypeptide SEQ ID NO: 291) and a sequence encoding the URA+ marker) to create strains 1-3. The DNA fragment was then processed using restriction endonuclease NotI to obtain plasmid B6622 (pKlURA3_TDH3cp-BM3R1-VP16; Rantasalo A., et al., "Synthetic toolkit for complex genetic circuit engineering in Saccharomyces cerevisiae," ACS). It was generated by digestion of Synth. Biol., 2018, 7, 6, 1573-1587). Transformants were selected on uracil-free SDA medium, and colony PCR was used to verify sTF insertion, yielding strain 7-3.

[0259] Stock 7-4 To generate G418-resistant uracil trophicer strain 7-4, the uracil marker in strain 7-3 was replaced with a marker conferring resistance to the antibiotic G418, which is bound to the LoxP site.

[0260] Stock 7-5 To generate strain 7-5, the G418 resistance cassette was removed from strain 7-4 by transforming it with a plasmid expressing Cre recombinase. The resulting strain 7-5 was ura3 - It includes the synthetic transcription factor of SEQ ID NO: 291, which is under the control of the TDH3cp promoter.

[0261] Stock 7-6-7-20 Strains 7-5 were separately transformed with transformation fragments of SEQ ID NOs. 292-307, as outlined in Table 37. SEQ ID NOs. 292-297 contain the Saccharomyces TDH3 promoter, a single xylitol 5-phosphate dehydrogenase (XPDH) or xylitol 5-phosphate phosphatase gene as described in Table 37, the Saccharomyces cerevisiae CYC1 terminator, the Saccharomyces URA3 expression cassette, an ampicillin resistance marker, and the Saccharomyces cerevisiae 2 micron origin of replication. Sequence IDs 298-306 contain the Saccharomyces cerevisiae TDH3 promoter, a single xylitol 5-phosphate phosphatase gene outlined in Table 37, the Saccharomyces cerevisiae CYC1 terminator, eight copies of the Bm3R1 transcription factor binding site, the Saccharomyces cerevisiae ENO1 promoter sequence, the xylitol 5-phosphate dehydrogenase gene, the Saccharomyces cerevisiae PDC5 terminator sequence, the Saccharomyces cerevisiae URA3 expression cassette, an ampicillin resistance marker, and the Saccharomyces cerevisiae 2-micron origin of replication. Sequence ID 307 contains the Saccharomyces TDH3 promoter, Saccharomyces CYC1 terminator, Saccharomyces cerevisiae URA3 expression cassette, ampicillin resistance marker, and Saccharomyces cerevisiae 2-micron origin of replication. Transformants were streaked onto ScD-Ura plates, and individual colonies were isolated. Five isolates from each transformation were designated as strain ae, as outlined in Table 37.

[0262] [Table 37]

[0263] Example 30 - Shaking Flask Fermentation Assay Cells 1-6a-e to 1-21a-e were inoculated into ScD-Ura plates (6.7 g / L yeast nitrogen base without amino acids, 1.9 g / L synthetic complete amino acid mix, 20 g / L glucose, and 20 g / L agar) and incubated at 30°C for 2 days. Cell slurries were prepared by inoculating small biomass patches into shallow 96-well microtiter plates containing 200 microliters of sterile DI water. 20 microliters of cell slurry were transferred to deep 96-well microtiter plates containing 500 microliters of buffered ScD-Ura medium (6.7 g / L yeast nitrogen base without amino acids, 1.9 g / L synthetic complete amino acid mix, 100 g / L glucose, 19.5 g / L MES buffer, pH adjusted to 6.0) and incubated in an orbital shaker at 30°C and 800 rpm for 24 hours. 25 microliters from the overnight culture were transferred to a 48-well flower plate containing 750 microliters of buffered ScD-Ura and incubated in an orbital shaker at 30°C and 800 rpm for 64 hours. Samples were taken for HPLC analysis to determine xylitol titer (25' Dulce method). The results in Table 38 show that the blank vector control strain and the X5PP-containing strain did not produce xylitol. Strains containing XPDH alone produced xylitol ranging from an average of 0.207+ / -0.025 to -0.304+ / -0.181 g / L, while strains containing both XPDH and X5PP produced xylitol ranging from 0.459+ / -0.018 to 0.633+ / -0.018. The results in Figure 34 show that XPDH alone, with the help of a natural phosphatase, can enable xylitol production in Saccharomyces cerevisiae. Xylitol production is improved when X5PP is introduced together with XPDH.

[0264] [Table 38-1]

[0265] [Table 38-2] * nd indicates that the value was not measured or was below the instrument's detection limit.

[0266] Example 31 - Genetically modified Yarowia liporitica strain As demonstrated herein, the pathway for producing xylitol from glucose in Moniliella polinis utilizes two enzymes. First, xylitol 5-phosphate dehydrogenase (XPDH) extracts a carbon from the pentose phosphate pathway by converting xylulose 5-phosphate to xylitol 5-phosphate. Second, xylitol 5-phosphate phosphatase (X5PP) removes a phosphate from xylitol-5-phosphate to produce xylitol. To test the feasibility of this pathway in Yarowia liporitica, the following strains were constructed and tested.

[0267] Stock 8-1 Strain 8-1 is the wild-type Yarrowia liporitica yeast strain (NRRL Y-63746).

[0268] Stocks 8-2 to 8-16 Strain 8-1 was transformed separately with the transformation fragments of SEQ ID NOs. 308-316. SEQ ID NOs. 208-313 contain, as shown in Table 39, a 5'ANT1 flanking sequence, eight copies of a synthetic transcription factor binding site, a core promoter, a single xylitol 5-phosphate dehydrogenase (XPDH) or xylitol 5-phosphate phosphatase gene, an ADH1 terminator, a core promoter, a synthetic transcription factor, a TEF1 terminator, a PGK promoter, a nulceotricin resistance gene, a TEF1 terminator, and a 3'ANT1 flanking sequence. Sequence IDs 314-322 contain, as described in Table 39, a 5'ANT1 flanking sequence, an ADH1 terminator, a xylitol 5-phosphate phosphatase gene, a core promoter, eight copies of a synthetic transcription factor binding site, a core promoter, xylitol 5-phosphate dehydrogenase, an ADH1 terminator, a core promoter, a synthetic transcription factor, a TEF1 terminator, a PGK promoter, a nurseotricin resistance gene, a TEF1 terminator, and a 3'ANT1 flanking sequence. Transformants were selected on YPD + 250 mg / L noseotricin plates, and individual colonies were evaluated for the incorporation of the indicated sequences by colony PCR. The isolates validated by five PCRs were designated as strain ae, as outlined in Table 39.

[0269] [Table 39]

[0270] Example 32 - Shaking Flask Fermentation Assay Strains 8-1 and 8-2a-e through 8-16a-e were seeded onto YPD plates (10 g / L yeast extract, 20 g / L yeast peptone, 20 g / L glucose, 20 g / L agar) and incubated at 30°C for 2 days. A cell slurry was prepared by inoculating small biomass patches into shallow 96-well microtiter plates containing 200 microliters of sterile DI water. 20 microliters of the cell slurry was transferred to deep 96-well microtiter plates containing 500 microliters of YP100D containing 10 g / L yeast extract, 20 g / L yeast peptone, and 100 g / L glucose, and incubated in an orbital shaker at 30°C and 800 rpm for 24 hours. Transfer 25 microliters from the overnight culture to a 48-well flower plate containing 750 microliters of YP100D and incubate in an orbital shaker at 30°C and 800 rpm for up to 96 hours. To determine the xylitol titer, take a sample for HPLC analysis.

[0271] Example 33 - Genetically modified Moniliella polynis strain Stock 9-1 To remove the zeosin resistance selection marker, strain 6-12b was transformed with the Cre recombinase plasmid of SEQ ID NO: 288 using the transformation method outlined in Example 4. The resulting transformants were evaluated for removal of the zeosin resistance selection marker by colony PCR. The PCR-validated isolate was designated strain 9-1.

[0272] Stock 9-2a-l Strain 9-1 was transformed with SEQ ID NOs: 279 and 280 using the transformation method outlined in Example 4. SEQ ID NOs: 279 contained the 3' portion of the G418 resistance gene expression cassette, the MpTEF1 terminator (SEQ ID NOs: 289), and the 3' gpdIIB flanking sequence (SEQ ID NOs: 166) in that order. SEQ ID NOs: 280 contained the 5' gpdIIB flanking sequence (SEQ ID NOs: 158), the PGK1 promoter, the gene encoding the Moniliella polynis phosphatase polypeptide of SEQ ID NOs: 210 (SEQ ID NOs: 230), the TDH3 terminator (SEQ ID NOs: 149), and the 5' portion of the G418 resistance gene expression cassette. Transformants were selected on a PDA+G418 selection plate and incubated at 35°C for at least 2 days until the transformants proliferated. The resulting transformants were streaked onto a PDA+G418 plate to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of the indicated sequences. The PCR-validated sister isolates were designated as strains 9-2a, 9-2b, 9-2c, 9-2e, 9-2f, 9-2g, 9-2h, 9-2i, 9-2j, 9-2k, and 9-2l.

[0273] Stock 9-3 Strain 6-12b was transformed with SEQ ID NOs: 323 and 324 using the transformation method outlined in Example 4. SEQ ID NOs: 323 contains a 5'ER1 flanking sequence, an MpPYK1 promoter, a polynucleotide sequence encoding B. halo of SEQ ID NOs: 14, an Mp6PGD terminator, and the 5' portion of the G418 resistance gene selection marker. SEQ ID NOs: 324 contains the 3' portion of the G418 resistance gene selection marker, an MPPYK1 promoter, a polynucleotide encoding XPDH of SEQ ID NOs: 14, an Mp6PGD terminator, and a 3'ER1 flanking sequence. Transformants were selected on a PDA+G418 selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked onto a PDA+G418 plate to isolate single colonies, and single colonies were selected. The selected colonies were evaluated by colony PCR for the incorporation of the indicated sequences. The sister isolates verified by PCR were designated as strains 9-3a, 9-3b, 9-3c, 9-3e, 9-3f, 9-3g, 9-3h, 9-3i, and 9-3j.

[0274] Example 34 - Shaking Flask Fermentation Assay Strains 6-12b, 9-1, and 9-2a-l were run in shaking flasks, and glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol were evaluated.

[0275] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0276] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 40. The results at 96 hours are also shown in Figure 35.

[0277] During the transformation of strain 6-12b with Cre recombinant plasmid, not only was the zeosin resistance marker gene removed, but the resulting strain 9-1 had only two copies of the X5PP gene encoding SEQ ID NO: 200, compared to strain 6-12b which had three copies. Transformation of strain 9-1 with an additional copy of the X5PP gene encoding SEQ ID NO: 200. As most notably observed at 72 hours, strain 9-1 had lower xylitol percentages and titers, but strain 9-2a-1, which recovered three copies of the X5PP gene, was closer to strain 6-12b. As demonstrated in some of the examples above, strains are verified by PCR to contain the desired polynucleotide sequence, but the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or a frameshift or other mutation may have made individual sisters different from each other. The results here suggest that similar transformations may occur in these sisters, but the results demonstrate the efficacy of the X5PP enzyme shown.

[0278] [Table 40-1]

[0279] [Table 40-2]

[0280] Example 35 - Shaking Flask Fermentation Assay Strains 6-12b and 9-3a-j were run in shaking flasks to evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol.

[0281] The strains were 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 propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm with a path length of 1 cm using a Genesys 20 spectrophotometer (Thermo Scientific). Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0282] A 250 mL unbaffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of seed culture to form the production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture at 24, 48, 72, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The fermentation results are reported in Table 41. The results at 96 hours are also shown in Figure 36.

[0283] As demonstrated in some of the examples above, strains are verified by PCR to contain the desired polynucleotide sequence, but the sequence may not be integrated into the exact locus, and may have multiple copies of the sequence integrated into its genome, or a frameshift or other mutation may have made individual sisters different from each other. The results here suggest that similar transformations occur in these sisters, but the results demonstrate the effectiveness of the X5PP enzyme shown. Further characterization demonstrated that strains 9-3a, 9-3b, and 9-3c correctly target the B. halo XPDH gene at the ER1 locus, replacing the L. rhamnosus XPDH gene. However, in strains 9-3d and 9-3e, the L. rhamnosus gene was removed from the ER1 locus, and the introduced B. halo XPDH gene was confirmed by PCR, but only partially targeted the ER1 locus. In strains 9-3f, 9-3g, 9-3h, 9-3i, and 9-3j, the presence of the B. haloXPDH gene was confirmed by PCR, but it was not targeted to the ER1 locus, while the L. rhamnosusXPDH gene remained present at the same locus. Overall, the addition of the B. haloXPDH gene to strain 6-12b resulted in similar variable xylitol production, which can be explained by accurate or inaccurate targeting.

[0284] [Table 41]

Claims

1. Genetically modified yeast cells capable of producing xylitol, wherein the genetically modified yeast cells are Genetic modification resulting in the overexpression of a natural enzyme having xylitol-5-phosphate phosphatase (X5PP) activity, and / or Genetically modified yeast cells containing an exogenous polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity.

2. The yeast cell according to claim 1, wherein the yeast cell is an osmotic pressure-tolerant yeast cell.

3. The yeast cell according to claim 1 or claim 2, wherein the yeast cell is a cell belonging to the subphylum Ustylagomycetes or the subphylum Saccharomyces.

4. The yeast cell according to any one of claims 1 to 3, wherein the yeast cell is selected from the group consisting of Trichosporonoides megachiriensis, Trichosporonoides oeducephalis, Trichosporonoides nigrescens, Pseudozaima tsukubaensis, Trigonopsis variabilis, Moniliella, Ustyraginomisetes, Trichosporon, Yarowia alipolitica, Saccharomyces cerevisiae, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium.

5. The yeast cell according to any one of claims 1 to 4, wherein the cell is a Moniliella polynis cell, and the gene modification overexpresses at least one of sequence IDs 198, 199, 200, or 221 and a natural X5PP 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% sequence identity.

6. The yeast cell according to any one of claims 1 to 5, wherein the gene modification comprises replacing the natural X5PP gene promoter with a heterologous or artificial promoter.

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

8. The yeast cell according to any one of claims 1 to 7, wherein the gene modification comprises adding an exogenous polynucleotide sequence encoding the natural X5PP enzyme, thereby the genetically modified cell comprising at least one additional copy of the sequence encoding the natural X5PP enzyme.

9. The yeast cell according to any one of claims 1 to 8, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having X5PP activity, at least one of SEQ ID NOs: 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 221, 222, 189, and 188, and sequence identity of 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%.

10. The yeast cell according to any one of claims 1 to 9, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having X5PP activity, at least one of SEQ ID NOs: 200, 202, 203, 204, 205, 206, 210, and 213, and sequence identity of 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%.

11. The yeast cell according to any one of claims 1 to 10, wherein the yeast cell comprises an exogenous polynucleotide sequence encoding an enzyme having X5PP activity, at least one of SEQ ID NOs: 200, 203, 204, 206, and 213, and sequence identity of 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%.

12. The yeast cell according to any one of claims 1 to 11, wherein the X5PP activity in the genetically modified yeast cell is higher than the X5PP activity in equivalent cells lacking the genetic modification or exogenous polynucleotide sequence.

13. The yeast cell according to any one of claims 1 to 12, wherein when the genetically modified cell is used in a fermentation process in the presence of dextrose, the titer and / or yield of xylitol is increased compared to the titer and / or yield of xylitol in an equivalent fermentation process using equivalent cells lacking the genetic modification or exogenous polynucleotide sequence.

14. The yeast cell according to any one of claims 1 to 13, further comprising a gene modification that results in the overexpression of a natural enzyme having ribulose-5-phosphate epimerase (RPE) activity.

15. The yeast cell according to claim 14, wherein the cell is a Moniliella polynis cell, and the native RPE enzyme comprises at least one of Sequence ID No. 179 and 180 with 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% sequence identity.

16. The yeast cell according to claim 14 or claim 15, wherein the gene modification resulting in overexpression of the natural RPE enzyme includes 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 or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139).

18. The yeast cell according to any one of claims 14 to 17, wherein the gene modification resulting in overexpression of the natural RPE enzyme includes the addition of an exogenous polynucleotide encoding the natural RPE enzyme, and as a result the genetically modified cell includes at least one additional copy of the sequence encoding the RPE enzyme.

19. The yeast cell according to any one of claims 1 to 18, wherein the cell further comprises at least one of sequence numbers 12-15, 28-31, and 33, and an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XPDH) 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% sequence identity.

20. The yeast cell according to claim 19, wherein the XPDH enzyme has at least 85% sequence identity with at least one of SEQ ID NOs: 12-15, 28-31, and 33, or at least one of SEQ ID NOs: 14, 15, 28, or 31.

21. The yeast cell according to claim 19 or claim 20, wherein the XPDH enzyme has at least 90% sequence identity with at least one of SEQ ID NOs: 12-15, 28-31, and 33, or at least one of SEQ ID NOs: 14, 15, 28, or 31.

22. The yeast cell according to any one of claims 1 to 21, wherein the cell further comprises at least one of sequence numbers 188 and 189 and an exogenous polynucleotide sequence encoding a xylokinase (XKS) 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% sequence identity.

23. The yeast cell according to claim 22, wherein the cell further comprises at least one of sequence numbers 190, 191, and 192 and an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) 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% sequence identity.

24. The yeast cells comprise at least one of sequence numbers 12-15, 28-31, and 33, and an exogenous polynucleotide sequence encoding the XPDH enzyme which 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. a. A polynucleotide sequence encoding an enzyme having X5PP activity, which is at least one copy more than that of the parent cell, wherein the X5PP enzyme has 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% sequence identity with SEQ ID NO: 200, and a gene modification resulting in overexpression of a natural RPE 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% sequence identity with SEQ ID NO: 180, or b. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 202, or c. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203, or d. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 204, or e. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 205, or f. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 206, or g. Genetic modifications resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 210, or h. A yeast cell according to any one of claims 1 to 13, comprising: an exogenous polynucleotide sequence encoding an X5PP enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 213; and a gene modification resulting in overexpression of a native RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

180.

25. The yeast cell according to claim 24, wherein the XPDH 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 sequence numbers 14, 15, 28, or 31.

26. The yeast cell according to claim 25, wherein the XPDH 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 SEQ ID NO:

14.

27. The yeast cell according to any one of claims 1 to 26, wherein one or more of the exogenous polynucleotide sequences are operably linked to a heterologous promoter or an artificial promoter.

28. The yeast cell according to claim 27, wherein the promoter is a constitutive promoter.

29. The yeast cell according to claim 27 or 28, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139).

30. The yeast cell according to any one of claims 1 to 29, wherein one or more of the exogenous polynucleotide sequences are incorporated into the genome of the yeast cell at a locus selected from the ER1 locus, ER3 locus, PDC1 locus, pyrF locus, TRP3 locus, gpdIIA locus, and gpdIIB locus.

31. A method for producing xylitol, A method comprising contacting a dextrose-containing substrate with genetically modified yeast cells according to any one of claims 1 to 30, wherein xylitol is produced by fermentation of the substrate by the genetically modified cells.

32. The fermentation temperature is 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C or somewhere in between, and the volumetric oxygen uptake rate (OUR) is 0.5 to 40, 1 to 35, 2 to 30, 3 to 25, 4 to 20, or 5 to 15 mmol O ２ The method according to claim 31, wherein / (L・h).

33. The xylitol is at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L －１ h －１ The method according to claim 31 or claim 32, which is generated at the speed of [speed].

34. The method according to any one of claims 31 to 33, wherein when the fermentation is carried out at 35°C for 96 hours, the xylitol production is at least 20, 30, 50, 75, or 100 g / L.

35. The method according to any one of claims 31 to 34, wherein the rate, titer, and / or yield of xylitol production are increased compared to equivalent fermentation performed using equivalent yeast cells lacking the gene modification for overexpressing the X5PP enzyme and lacking the exogenous polynucleotide sequence encoding the exogenous X5PP enzyme.

36. The method according to any one of claims 31 to 35, wherein the dextrose concentration is at least 100 g / L.

37. Use of genetically modified cells according to any one of claims 1 to 30 for producing xylitol.

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