Genetically modified yeast and fermentation process for producing xylitol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing xylitol. The engineered yeast cell may comprise an exogenous polynucleotide sequence encoding a sugar phosphatase comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 8 and SEQ ID NO: 20.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 364,363, filed on May 9, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listing submitted via Patent Center

[0004] The contents of the sequence listing XML file named "PT-1346-WO-PCT.xml", which was created on April 13, 2023 and electronically submitted via Patent Center with this application, and is 246,439 bytes in size, are incorporated herein by reference in its entirety. Background Art

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

[0006] Thus, provided herein are genetically modified yeast and fermentation methods for producing xylitol while reducing erythritol production. Summary of the invention

[0007] The present disclosure provides a genetically engineered yeast cell capable of producing xylitol, the engineered yeast cell comprising an exogenous polynucleotide sequence encoding a xylulokinase (XKS), the XKS comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NO: 8 and SEQ ID NO: 20. The yeast cell may be an osmotic yeast cell. The yeast cell may be a cell of the subdivision Ustilaginomycotina. The yeast cell may be selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. The yeast cell may be a yeast cell of the genus Moniliella.

[0008] The present disclosure also provides a genetically engineered Mycelium cell capable of producing xylitol, the engineered Mycelium cell comprising an exogenous polynucleotide sequence encoding a xylulokinase (XKS), the XKS comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NO: 8 and SEQ ID NO: 20.

[0009] XKS may have a sequence that is at least 85% identical to at least one of SEQ ID NO: 8 and SEQ ID NO: 20. XKS may have a sequence that is at least 90% identical to at least one of SEQ ID NO: 8 and SEQ ID NO: 20.

[0010] The engineered cells described herein can be Moniliella pollinis cells. When used in a fermentation process for 96 hours in the presence of dextrose, the yeast cells described herein can produce xylitol at a titer of at least 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L or 2.5 g / L. The erythritol production of the engineered cells described herein can be reduced relative to the erythritol production in the equivalent yeast cells lacking exogenous polynucleotide sequences.

[0011] The exogenous polynucleotide sequence can be operably linked to a heterologous promoter or an artificial promoter. The promoter can be a constitutive promoter. The promoter may be selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:61), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:52), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:54), translation elongation factor 1 promoter (TEFp; SEQ ID NO:55), modified TEFp (SEQ ID NO:53), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:56), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:57), enolase promoter (ENO1p; SEQ ID NO:58), asparagine synthetase promoter (ASNSp; SEQ ID NO:59), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:60) and RPL16B (SEQ ID NO:62).

[0012] The exogenous polynucleotide sequence can be integrated into the genome of the yeast cell at a locus selected from the group consisting of the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdIIA locus, and the gpdIIB locus.

[0013] The yeast cell may further comprise an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH), the XDH comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 14. The XDH may have a sequence that is at least 85% identical to at least one of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 14. The XDH may have a sequence that is at least 90% identical to at least one of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 14.

[0014] The present disclosure also provides a method for producing xylitol using the engineered cells described herein, the method comprising contacting a substrate comprising dextrose with an engineered cell described herein, wherein fermentation of the substrate by the engineered yeast produces xylitol. The present disclosure also provides a method for producing xylitol, the method comprising contacting a substrate comprising dextrose with an engineered yeast cell, the engineered yeast cell comprising an exogenous polynucleotide sequence encoding a sugar phosphatase, the sugar phosphatase comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical sequence to at least one of SEQ ID NO: 8 and SEQ ID NO: 20, wherein fermentation of the substrate by the engineered yeast produces xylitol. The fermentation temperature may be 25°C to 45°C, 30°C to 40°C or 32°C to 37°C or therebetween. The volumetric oxygen consumption rate (OUR) may be between 0.5 mmol / L. 2 / (L·h) to 40mmol O 2 / (L·h), 1mmol O 2 / (L·h) to 35mmol O 2 / (L·h), 2mmol O 2 / (L·h) to 30mmol O 2 / (L·h), 3mmol O 2 / (L·h) to 25mmol O 2 / (L·h), 4mmol O 2 / (L·h) to 20mmol O 2 / (L·h) or 5mmol O 2 / (L·h) to 15mmol O 2 / (L·h). Erythritol production can be reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking an exogenous polynucleotide sequence. When the fermentation is run at 35°C for 96 hours, erythritol production can be less than 40g / L, 30g / L, or less than 20g / L. When the fermentation is run at 35°C for 96 hours, xylitol production can be at least 0.5g / L, 1.0g / L, 1.5g / L, 2.0g / L, 2.5g / L, or 3.0g / L. Glycerol production can be reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking an exogenous polynucleotide sequence. Ethanol production can be reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking an exogenous polynucleotide sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings illustrate generally by way of example and not by way of limitation various aspects described herein.

[0016] Figure 1 The native pentose phosphate pathway (dashed line and arrows) and the native glycolytic pathway (solid line and arrows) in the yeast Mycorrhizal sp. are shown.

[0017] Figure 2 Shown are the concentrations of glucose, erythritol, and xylitol metabolites (g / L) over 96 hour shake flask fermentations of strains 1-1, 1-2a-c, 1-3a-c, 1-4a-c, 1-5a-c, 1-6a-c, 1-7a-c, 1-8a-c, 1-9a-c, 1-10a-c, 1-11a-c, and 1-12a-c as outlined in Example 2.

[0018] Figure 3 Shown are xylitol, ribitol, and erythritol metabolite concentrations (g / L) from 96 hour shake flask fermentations of strains 1-13, 1-14a, 1-14c, 1-8a, 1-8b, and 1-8c with baffled flasks (BF) and without baffled flasks (no BF) as outlined in Example 3.

[0019] Figure 4 Shown are C6 polyol and xylitol metabolite concentrations (g / L) from 96 hour shake flask fermentations of strains 1-14c, 1-15a-d, 1-16a-d, 1-17a-d, 1-18a-e, and 1-19a-e as outlined in Example 4. Data markers report the concentration of xylitol (g / L).

[0020] Figure 5 Shown are C6 polyol and xylitol metabolite concentrations (g / L) from 96 hour shake flask fermentations of strains 1-14c, 1-20a-e, 1-21a-e, 1-23a-e, and 1-24a-e as outlined in Example 5. Data markers report the concentration of xylitol (g / L).

[0021] Figure 6 Shown are ribitol and xylitol metabolite concentrations (g / L) from 96 hour shake flask fermentations of strains 1-1, 1-20b, 1-24e, 1-25a-d, 1-26a-e, 1-27a-e, and 1-28a-c as outlined in Example 6. Data markers report the concentration of xylitol (g / L). DETAILED DESCRIPTION

[0022] Reference will now be made in detail to certain aspects of the presently disclosed subject matter, examples of which are partially illustrated in the accompanying drawings.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0023] In this document, the terms "a", "an" or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of an inconsistency between the usages of this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0024] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly recited as limits of the range, but also all individual values ​​or sub-ranges encompassed within the range, as if each value and sub-range were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and sub-ranges (e.g., 0.1% to 0.5%,

[0025] 1.1% to 2.2%, 3.3% to 4.4%). Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0026] Unless expressly stated, ppm (parts per million), percentages and ratios are based on weight. Percentages based on weight are also referred to below as wt% or (wt)%.

[0027] The present disclosure relates to various recombinant cells engineered to produce xylitol. Generally speaking, the recombinant cells described herein have an active pentose phosphate pathway and are characterized by the expression of an exogenous sugar phosphatase (XKS). The recombinant cells described herein may also optionally express an exogenous xylitol dehydrogenase (XDH). The present disclosure also provides a fermentation method for producing xylitol from dextrose using the genetically engineered cells described herein.

[0028] In general, the recombinant cells described herein are yeast cells. As used herein, "yeast" refers to eukaryotic unicellular microorganisms classified as members of the kingdom Fungi. Yeast is a unicellular organism that evolved from a multicellular ancestor, some of which retain multicellular characteristics, such as forming connected strings of budding cells known as pseudohyphae or false hyphae. Yeast cells may also be referred to as yeast-like cells in the art, and as used herein, "yeast cells" encompass both yeast and yeast-like cells. Suitable yeast and yeast-like host cells for modification can include, but are not limited to, Saccharomyces cerevisiae, Komagataella sp., Kluyveromyces (e.g., Kluyveromyces lactis, Kluveromyces marxianus), oleaginous yeast, Issatchenkia orientalis, Pichia galeiformis, Pichia YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnolii, Candida ethanolica), Pichia deserticola, Pichia membranifadens, Pichia fermentans (e.g., fermentans), Aspergillus, Trichoderma, Myceliphthora thermophila, spores (e.g., spores yeast), Pfaffia, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., Trichosporonoides megafacies, Trichosporonoides globosum, Trichosporonoides nigrofacies), Pseudomonas tsukubaensis, Trichosporon variabilis, Penicillium, and Torula yeast. One of ordinary skill will appreciate that the requirements for selecting suitable yeast cells and recombinant yeast cells of the present disclosure are not limited to those explicitly listed herein. Methods for genetic engineering of yeast cells are known and described in the art, and one of ordinary skill will appreciate the methods necessary to transform and engineer suitable yeast cells.

[0029] Suitable yeast cells can be Basidiomycota and Ustilago. Suitable yeasts of the Ustilago include, but are not limited to, Ustilago (e.g., U. cynodontis, U. maydis, U. sphaerogena, U. cordal, U. scitaminea, U. coicis, U. syntherismae, U. esculenta, U. neglecta, U. crus-galli, Ustilago avenae), Sporisorium (e.g., Sporisorium spores), Sporisorium spores. exsertum), spores (e.g., spores yeast, M. tomentosa, M. acetoabutans, M. fonsecae, M. madida, M. megachiliensis, M. ocedocephalis, M. nigrescens), and Pseudozyma (e.g., Pseudozyma tsukubae), and Trichosporon (e.g., Trichosporon megatus, Trichosporon sphericalus, Trichosporon nigrescens). Yeasts of the subdivision Ustilagomyces are known and described in the art as potential production organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol, as well as other valuable biotechnological applications.See, for example, Geiser et al. Biotechnol, 2013, 97(8): 3253-65), Guevarra et al. ("Accumulation of itaconic, 2-hydroxyparaconic, itataric, and malic acids by strains of the genus Ustilago, Agric.Biol.Chem., 1990, 54(9), 2353-2358) and Moon et al. ("Biotechnological productionof erythritol and its applications,” Appl Microbiol Biotechnol, 2010,86:1017-1025).

[0030] Suitable yeast cells will have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein, "active pentose phosphate pathway" refers to the expression of one or more functional enzymes that together convert glucose-6-phosphate, NADP + Or NAD+ and water are converted into NADPH or NADH, CO2 and ribulose-5-phosphate. Continuing in the non-oxidative stage, the pathway can also produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway can produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, combinations thereof, etc. The active pentose phosphate pathway can be natural to yeast cells, or can be introduced into yeast cells by genetic engineering.

[0031] Yeast cells can be osmotic yeast cells. As used herein, "osmotic resistance" refers to yeast that can grow and reproduce under conditions of high molar osmotic pressure concentrations, such as at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v) or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), at least 15% (w / v) sodium chloride. The species and strains of osmotic yeasts are known and described in the art, including many types of yeasts used in industrial fermentation processes. Similarly, methods for determining yeast osmotic resistance are known and described in the art. See, for example, Tiwari, S. et al. ("Nectaryeast community of tropical flowering plants and assessment of theirosmotolerance and xylitol-producing potential," Current Microbiology, 2022, 79: 28).

[0032] The recombinant yeast cell can be a recombinant Myristica spp. cell, such as a Myristica spp. yeast cell. Figure 1 The predicted native pentose phosphate and glycolytic pathways in the yeast of the spores are shown. The spores have been previously used in the fermentation production of erythritol, and methods for genetic modification and fermentation of the spores are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb Cell Fact, 16: 168).

[0033] Various plasmids and methods for transforming Myrtillus spp. are also described in the examples below. For example, Myrtillus spp. can be transformed using a bipartite polynucleotide sequence, wherein upon recombination, the exogenous polynucleotide of interest is integrated at a specific locus and a selectable marker is expressible in the cell. Suitable selectable markers are known and used in the art. Selectable markers may include, but are not limited to, amdS (e.g., broken into a 3' portion SEQ ID NO:91 and a 5' portion SEQ ID NO:92), a G418 resistance gene (e.g., broken into a 3' portion SEQ ID NO:97 and a 5' portion SEQ ID NO:98), a zeocin resistance gene (e.g., broken into a 3' portion SEQ ID NO:93 and a 5' portion SEQ ID NO:94), a nourseothricin N-acetyltransferase (NAT) (e.g., broken into a 3' portion SEQ ID NO:95 and a 5' portion SEQ ID NO:96), and an invertase gene (SUC2) (e.g., a 3' portion of SEQ ID NO:99 and a 5' portion of SEQ ID NO:100).

[0034] The recombinant cells described herein comprise one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve the fermentation of glucose to ribitol by the recombinant cell.

[0035] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless expressly stated otherwise.

[0036] As used herein, "exogenous" refers to genetic material or its expression product that originates from outside the host organism. For example, the exogenous genetic material or its expression product can be a modified form of genetic material native to the host organism, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component. For example, when introduced into S. cerevisiae, the K. lactis invertase gene is exogenous.

[0037] As used herein, "native" refers to genetic material or its expression products found within the genome of a wild-type cell of a host cell, except for inter-individual mutations that do not affect function or expression. For the purposes of this application, the Moniliella tomentosa var pollinis TCV364 yeast cell described in US 6,440,712 is considered to be a wild-type Moniliella tomentosa yeast cell, which is incorporated herein by reference in its entirety and deposited at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) under the Budapest Treaty on March 28, 1997 under the number MUCL40385.

[0038] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the overall primary, secondary, tertiary and quaternary amino acid sequences and structures necessary to give the macromolecule its function and properties. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (either independently or as part of a biosynthetic pathway) is understood to include the cofactors, coenzymes and metals necessary for the enzyme to function properly. An overview of amino acids and their three letter and one letter symbols as understood in the art is provided in Table 1. The amino acid names, three letter symbols and one letter symbol are used interchangeably herein.

[0039] Table 1: Amino Acid Three-Letter and One-Letter Symbols

[0040] Amino Acids Three letter symbols A letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0041] Variants or sequences having substantial identity or homology to the polypeptides described herein can be used to implement the disclosed recombinant cells, compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, the polypeptide sequence can be modified but still retains the ability to exhibit the desired activity. Typically, the variant or modified sequence can include a sequence identity greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% to a wild-type, naturally occurring polypeptide sequence or to a variant polypeptide as described herein.

[0042] As used herein, the phrases "% sequence identity", "% identity" and "percent identity" are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well known. The generation of sequence alignments and sequence identities includes global alignments and local alignments performed using computational methods. Alignments can be performed using BLAST (National Center for Biological Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using standard protein BLAST with the following default parameters: maximum target sequence: 100; short query: automatically adjust parameters for short input sequences; expected threshold: 10; word length: 6; maximum number of matches within the query range: 0; matrix: BLOSUM62; gap penalty: (existence: 11, extension: 1); component adjustment: conditional component score matrix adjustment; filter: not selected; mask: not selected. The % nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within query range: 0; Match / mismatch score: 1, -2; Gap penalty: Linear; Filter: Low complexity region; Mask: Mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently, to have XX% sequence identity with the reference sequence.

[0043] Polypeptide or polynucleotide sequence identity can be measured over the length of an entire defined polypeptide sequence (e.g., as defined by a particular SEQ ID number), or can be measured over a shorter length, for example, over the length of a fragment (e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues) taken from a larger defined polypeptide sequence. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown in this article, table, figure, or sequence listing can be used to describe the length over which a percentage identity can be measured.

[0044] The polypeptides disclosed herein may include "variant" polypeptides, "mutants" and "derivatives thereof". As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of the polypeptide in nature, as distinguished from a variant or mutant form. As used herein, a "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence that is different from a reference protein or polypeptide molecule. A variant or mutant may have an insertion, deletion or substitution of one or more amino acid residues relative to a reference molecule.

[0045] The amino acid sequences of polypeptide variants, mutants, derivatives or fragments contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. "Conservative amino acid substitutions" are those substitutions in which the amino acid is substituted with a different amino acid, wherein the substitution is predicted to interfere minimally with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially retain the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of the substitution, such as a beta sheet or alpha helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the volume of the side chain.

[0046] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" and "nucleic acid" are used interchangeably and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. The DNA polynucleotide may be a cDNA (e.g., coding DNA) or a genomic DNA sequence (e.g., including introns and exons).

[0047] If in its natural state or when operated by methods known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof, then the polynucleotide is said to encode a polypeptide. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0048] Those skilled in the art understand the degeneracy of the genetic code and that multiple polynucleotides can encode the same polypeptide. In some aspects, polynucleotides (e.g., polynucleotides encoding XKS or XDH polypeptides) can be codon-optimized for expression in specific cells, including but not limited to plant cells, bacterial cells, fungal cells, or animal cells. Although polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptides described herein can be used. Therefore, non-naturally occurring sequences can be used. These sequences may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, genetic codes, and human hands can also be used to generate degenerate coding sequences.

[0049] The recombinant cells described herein may include deletions or destructions in one or more natural genes. The phrase "deletion or destruction" refers to the state of natural genes in recombinant cells, which have a completely eliminated coding region (deletion) or gene, its promoter or its terminator modification (such as by deletion, insertion or mutation), so that the gene no longer produces an active expression product, produces a severely reduced amount of expression product (e.g., reduced by at least 75% or reduced by at least 90%), or produces an expression product with severely reduced activity (e.g., reduced by at least 75% or reduced by at least 90%). Deletion or destruction can be achieved by genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi) and / or selection and screening. The natural gene to be deleted or destroyed can be replaced with an exogenous nucleic acid of interest for expressing an exogenous gene product (e.g., polypeptide, enzyme, etc.).

[0050] Recombinant cells as described herein may comprise one or more genetic modifications, wherein exogenous nucleic acids are integrated into the genome of the host cell. Those skilled in the art will know how to select suitable loci in the yeast genome for integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in a spore yeast host cell, suitable interaction sites may include, but are not limited to, the ER1 locus (defined as the locus flanked by SEQ ID NO: 87 and SEQ ID NO: 88), the ER3 locus (defined as the locus flanked by SEQ ID NO: 81 and SEQ ID NO: 82), the PDC1 locus (defined as the locus flanked by SEQ ID NO: 75 and SEQ ID NO: 76), the pyrF locus (defined as the locus flanked by SEQ ID NO: 77 and SEQ ID NO: 78), the TRP3 locus (defined as the locus flanked by SEQ ID NO: 83 and SEQ ID NO: 84), the gpdIIA locus (defined as the locus flanked by SEQ ID NO: 85 and SEQ ID NO: 86), and the gpdIIB locus (defined as the locus flanked by SEQ ID NO: 89 and SEQ ID NO: 90). The exogenous nucleic acid may also be integrated in an intergenic region or other location in the host cell genome not specifically specified herein. Other suitable integration loci can be determined by one skilled in the art. Furthermore, one skilled in the art will recognize how to use the sequences to design primers to verify correct gene integration at the selected locus.

[0051] Recombinant cells can have one or more copies of a given exogenous nucleic acid sequence, which is integrated into the host chromosome and replicated with the chromosome into which it is integrated. For example, yeast cells can be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide as described herein, and the polynucleotide sequence encoding the polypeptide can be integrated into the host chromosome with one or more copies. Recombinant cells may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide as described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten or more copies of a polynucleotide sequence encoding a polypeptide as described herein that is integrated into the genome. Multiple copies of the polynucleotide sequence can all be incorporated into a single locus or can be incorporated into multiple loci.

[0052] The recombinant cells described herein are capable of producing xylitol and include an exogenous polynucleotide sequence encoding a xylulokinase (XKS). The exogenous polynucleotide sequence may be an exogenous xylulose phosphatase (XKS) gene.

[0053] "Xylulose kinase gene" and "XKS gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylulose kinase activity. As used herein, "xylulose kinase activity" refers to the ability to catalyze the conversion of xylulose-5-phosphate and ADP to xylulose and ATP. The XKS gene may be derived from any suitable source. For example, the XKS gene may be derived from Saccharomyces cerevisiae. The XKS gene may encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NO: 8 and SEQ ID NO: 20.

[0054] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from the Saccharomyces cerevisiae DOG1 sugar phosphatase gene encoding the amino acid of SEQ ID NO: 8. 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: 8.

[0055] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Saccharomyces cerevisiae DOG2 sugar phosphatase gene encoding the amino acid of SEQ ID NO: 20. 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: 20.

[0056] The recombinant cells described herein may further include an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH). The exogenous polynucleotide sequence may be an exogenous xylitol dehydrogenase (XDH) gene.

[0057] "Xylitol dehydrogenase gene" and "XDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having xylitol dehydrogenase activity. As used herein, "xylitol dehydrogenase activity" refers to a gene or polynucleotide that catalyzes the conversion of xylulose and NADH or NADPH into xylitol and NADPH. + or NADP +The XDH gene can be derived from any suitable source. For example, the XDH gene can be derived from Pichia stipitis, Rhodobacteraceae bacteria and Bemisia argentofolii. The XDH gene can encode an amino acid having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with the amino acid sequence of at least one of SEQ ID NO: 1, SEQ ID NO: 13 and SEQ ID NO: 14.

[0058] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a cofactor-switching Pichia stipitis XDH gene encoding the amino acid of SEQ ID NO: 1. 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: 1.

[0059] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Rhodobacteraceae SDR family oxidoreductase gene encoding the amino acid of SEQ ID NO: 13. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0060] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from the Bemisia argentofolii (silverleaf whitefly) ketose reductase (sorbitol dehydrogenase) gene encoding the amino acid of SEQ ID NO: 14. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0061] The exogenous polynucleotides in the recombinant cells described herein can be under the control of a promoter. For example, the exogenous nucleic acid can be operably linked to a heterologous promoter or an artificial promoter. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarbonatase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:61); 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:52); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:54); translation elongation factor 1 promoter (TEFp; SEQ ID NO:55); modified TEFp (SEQ ID NO:53); phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:56); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:57); enolase promoter (ENO1p; SEQ ID NO:58); asparagine synthetase promoter (ASNSp; SEQ ID NO:59); ID NO:59); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:61); RPL16B (SEQ ID NO:62).

[0062] The exogenous nucleic acid in the recombinant cells described herein can be under the control of a terminator. For example, the exogenous nucleic acid can be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. The terminator may include, but is not limited to, a GAL10 terminator, a PDC terminator, a transaldolase terminator (TAL), a 6PGD terminator (6PGDt; SEQ ID NO:63); an ASNS terminator (ASNSt; SEQ ID NO:64); an ENO1 terminator (ENO1t; SEQ ID NO:65); a hexokinase 1 terminator (HXK1t; SEQ ID NO:66); a PGK1 terminator (PGK1t; SEQ ID NO:67); a PGM1 terminator (PGM1t; SEQ ID NO:68); a PYK1 terminator (PYK1t; SEQ ID NO:69); a RPLA terminator (RPLAt; SEQ ID NO:70); a transaldolase 1 terminator (TAL1t; SEQ ID NO:71); a TDH3 terminator (TDH3t; SEQ ID NO:72); a translation elongation factor 2 terminator (TEF2t; SEQ ID NO:73); ID NO:73); triosephosphate isomerase 1 terminator (TPI1t; SEQ ID NO:74); and MpTEF1t (SEQ ID NO:101).

[0063] A promoter or terminator is "operably linked" to a given polynucleotide (eg, a gene) if its position relative to the polynucleotide in the genome or expression cassette is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0064] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. A recombinant polynucleotide is a polynucleotide formed by a laboratory method, which includes a polynucleotide sequence derived from at least two different natural sources, or they may be synthetic. Therefore, a construct may include a new modification to an endogenous gene introduced by, for example, genome editing technology. A construct may also include a recombinant polynucleotide produced using, for example, a recombinant DNA method. A construct may be a vector comprising a promoter operably connected to a polynucleotide encoding a polypeptide described herein. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide connected thereto. A vector may be a plasmid, which refers to a circular double-stranded DNA loop, into which an additional DNA fragment may be integrated.

[0065] The present disclosure also provides a fermentation method for producing xylitol using the recombinant cells described herein. The fermentation method includes the step of fermenting a substrate using the genetically engineered yeast described herein to produce xylitol. As will be appreciated by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and separating xylitol from the fermentation broth. The fermentation process may be a fully aerobic process or a partially aerobic process.

[0066] The fermentation process can be run using a suitable fermentation substrate. The substrate of the fermentation process can include hydrolyzates of glucose, sucrose, galactose, mannose, molasses, xylose, fructose, starch, lignocellulose hydrolyzates, or a combination thereof. Those skilled in the art will recognize which fermentation substrate is suitable for a given fermenting organism and system.

[0067] The fermentation process can be run under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained within a range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably about 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to any specific range or temperature described herein and can be modified as appropriate.

[0068] The fermentation process can be run within a certain range of oxygen uptake rate (OUR). The volume of the fermentation process can be 0.5 mmol O 2 / (L·h) to 40mmol O 2 / (L·h), 1mmol O 2 / (L·h) to 35mmol O 2 / (L·h), 2mmol O 2 / (L·h) to 30mmol O 2 / (L·h), 3mmol O 2 / (L·h) to 25mmol O 2 / (L·h), 4mmol O 2 / (L·h) to 20mmol O 2 / (L·h) or 5mmol O 2 / (L·h) to 15mmol O 2 / (L·h). In some embodiments, the specific OUR can be within the range of 0.05 mmol. 2 / (g cell dry weight·h) to 10mmol O 2 / (g cell dry weight·h), 0.1mmol O 2 / (g cell dry weight·h) to 8mmol O 2 / (g cell dry weight·h), 0.15mmol O 2 / (g cell dry weight·h) to 5mmol O 2 / (g cell dry weight·h), 0.2mmol O 2 / (g cell dry weight·h) to 1mmol O 2 / (g cell dry weight·h) or 0.3mmol O 2 / (g cell dry weight·h) to 0.75mmol O 2 / (g cell dry weight·h). However, the volume or specific OUR of the fermentation process is not limited to any specific rate or range described herein.

[0069] Fermentation process can be operated under various cell concentrations. In some embodiments, the dry cell weight at the end of fermentation can be 5g dry cell weight / L to 40g dry cell weight / L, 8g dry cell weight / L to 30g dry cell weight / L or 10g dry cell weight / L to 20g dry cell weight / L. In addition, the input density (pitch density) or input rate (pitchingrate) of fermentation process can change. In some embodiments, the input density can be 0.05g dry cell weight / L to 11g dry cell weight / L, 0.1g dry cell weight / L to 10g dry cell weight / L or 0.25g dry cell weight / L to 8g dry cell weight / L.

[0070] The initial dextrose concentration of the fermentation can be at least 100 g / L dextrose, 200 g / L dextrose, 250 g / L dextrose, 300 g / L dextrose, 350 g / L dextrose, or at least 400 g / L dextrose. The initial dextrose concentration can be between 100 g / L and 400 g / L, 150 g / L and 350 g / L, or 250 g / L and 325 g / L.

[0071] Fermentation processes can be associated with various characteristics, such as, but not limited to, fermentation production rate, approach fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of yeast and / or the genetic modification of yeast used in the fermentation process. These characteristics can be affected by regulating the fermentation process conditions. These characteristics can be regulated via a combination of yeast selection or modification and the selection of fermentation process conditions.

[0072] The xylitol production rate of the process can be at least 0.2 g L -1 h -1 , 0.3 g L -1 h -1 , 0.5 g L -1 h -1 , 0.75gL -1 h -1 or at least 1.0 g L -1 h -1 The final xylitol titer of the process may be at least 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, or 3.0 g / L.

[0073] The fermentation process can be run as a dextrose fed batch.In addition, the fermentation process can be a batch process, a continuous process or a semi-continuous process, as understood by those skilled in the art.

[0074] Example

[0075] The present invention is further described in detail by reference to the following experimental examples. Unless otherwise stated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0076] Example 1 - Genetically modified Saccharomyces cerevisiae strains

[0077] Strain 1-1 is a Moniliella tomentosa host strain "Moniliellatomentosa var pollinis TCV364" described in US 6,440,712, which is incorporated herein by reference in its entirety and deposited under the Budapest Treaty on March 28, 1997 at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde). Table 2 below lists various Moniliella tomentosa strains, including information on the parent strain, the sequence of the transformed parent strain, and the characterization of the expression cassette contained in the transformed sequence.

[0078] In Table 2, the following cassettes were used. Each "XHD homolog + 3'G418 expression cassette" contained, in order, a 5'ER1 flanking sequence (SEQ ID NO: 87), an MpPGM1 promoter (SEQ ID NO: 56), a gene encoding an indicated XDH enzyme homolog (one of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 14), an MpPGK1 terminator (SEQ ID NO: 67), and the 3' portion of the G418 resistance gene (SEQ ID NO: 97). Each "XKS / phosphatase homolog + 5'G418 expression cassette" contains, in order, the 5' portion of the G418 resistance gene (SEQ ID NO:98), the MpPGK promoter (SEQ ID NO:57), a gene encoding the indicated XKS / phosphatase homolog (SEQ ID NO:35 or one of SEQ ID NOs:41-50), the MpENO1 terminator (SEQ ID NO:65), and a 3'ER1 flanking sequence (SEQ ID NO:88).

[0079] In Table 3, the following cassettes were used. Each "XDH homolog expression cassette" contains, in order, a 5'ER1 flanking sequence (SEQ ID NO: 87), a MpPYK1 promoter (SEQ ID NO: 61), a gene encoding the indicated XDH homolog (one of SEQ ID NOs: 15-19), an Mp6PGD terminator (SEQ ID NO: 63), and a 5' portion of a bleomycin resistance gene (SEQ ID NO: 93). The "selection marker cassette" of SEQ ID NO: 38 contains, in order, a 3' portion of a bleomycin selection marker gene (SEQ ID NO: 93), an MpTEF2 terminator (SEQ ID NO: 73), and a 3'ER1 flanking sequence (SEQ ID NO: 88). Each "XKS / phosphatase expression cassette" contains, in order, a 5'ER1 flanking sequence (SEQ ID NO:87), a PGK1 promoter (SEQ ID NO:67), a gene encoding an indicated XKS / phosphatase homolog (SEQ ID NO:8 or one of SEQ ID NOs:20-24), an MpENO1 terminator (SEQ ID NO:65), a 3' portion of a G418 (SEQ ID NO:97) or bleomycin (SEQ ID NO:93) resistance gene, and an MpTEF2 terminator (SEQ ID NO:73). The "selectable marker cassette" of SEQ ID NO:37 contains, in order, a 5' portion of a bleomycin resistance gene and a 3'ER1 flanking sequence (SEQ ID NO:88). The "selectable marker cassette" of SEQ ID NO:36 contains, in order, a 5' portion of a G418 resistance gene and a 3'ER1 flanking sequence (SEQ ID NO:88).

[0080] Following binary transformation with the two cassettes indicated in Tables A and B, the two cassettes recombined to integrate the nucleotide sequences encoding the indicated XDH or XKS / phosphatase homologs and the indicated selectable marker at the ER1 locus.

[0081] The mycelial pellet of the parent strain was supplemented with 0.6 M MgSO 4 The indicated Saccharomyces parent strains were transformed with the indicated sequences by first protoplasting the parent strains with an enzyme mixture of 7.5 g / L lyase, 7.5 g / L lyase from Trichoderma harzianum, and 12.5 g / L lyase from Trichoderma harzianum. The protoplasts were then pelleted and washed with 0.6 M MgSO 4The cells were washed and resuspended in STC medium (0.6 M sucrose, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). 100 μg of single-stranded salmon sperm DNA and 1.5 μg to 5 μg of each 5'DNA and 3'DNA conversion fragment (3 μg-10 μg in total; see Table 4 for a list of fragments) were added to about 200 μL of the protoplast mixture (10 8 Cells / mL) are added to prepare binary transformation. Then 1mL of STC culture medium containing 50% PEG is added to salmon sperm DNA, conversion DNA and protoplast mixture, and the resulting combination is incubated at room temperature for 15 minutes. After incubation, the recovery culture solution (0.4M sucrose, 1g / L yeast extract, 1g / L malt extract, 10g / L sugar, pH 4.5) is added to the mixture, and incubated at 27°C, 100rpm for 16 hours to 24 hours. After incubation, the protoplasts are precipitated and resuspended in 1mL PBS by centrifugation.

[0082] The resuspended protoplasts are spread on PDA+250mg / L geneticin (geneticin) (G418) or PDA+100mg / L bleomycin selection plates and incubated at least 2 days-4 days at 30°C-35°C until transformant growth. The integration of the indicated sequence of the obtained transformant is evaluated by colony PCR. The isolate verified by PCR is then designated as the indicated strain number. In some cases, more than one isolate verified by PCR (e.g., "sister" isolate) is indicated by the letter after the strain number. For example, strain 1-strain 2 has 3 sister isolates: strain 1-2a, strain 1-2b and strain 1-2c.

[0083] For example, strain 1-1 is transformed with SEQ ID NO:39 and SEQ ID NO:41. SEQ ID NO:39 contains i) 5'ER1 flanking sequence (SEQ ID NO:87), ii) MpPGM1 promoter (SEQ ID NO:56), iii) expression cassette for cofactor switching XDH from Pichia stipitis of SEQ ID NO:1, iv) MpPGK1 terminator (SEQ ID NO:67), and v) 3' portion of G418 resistance gene (SEQ ID NO:97). SEQ ID NO:41 contains i) 5' portion of G418 resistance gene (SEQ ID NO:98), ii) MpPGK promoter (SEQ ID NO:57), iii) expression cassette encoding Hypsizygous marmoreus XKS1 of SEQ ID NO:2, iv) MpENO1 terminator (SEQ ID NO:65), and v) 3'ER1 flanking sequence (SEQ ID NO:88). Transformants were selected on PDA+250mg / L Geneticin (G418) selection plates and incubated at least 2 days at 35°C until transformant growth. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony separation and single colony selection. The integration of the indicated sequence of the selected colony was evaluated by colony PCR. The isolates verified by PCR were named as strain 1-2a, strain 1-2b and strain 1-2c.

[0084]

[0085]

[0086]

[0087] Example 2 - Shake flask fermentation assay

[0088] Strains 1-2a-c, 1-3a-c, 1-4a-c, 1-5a-c, 1-6a-c, 1-7a-c, 1-8a-c, 1-9a-c, 1-10a-c, 1-11a-c and 1-12a-c (summarized in Table 2 above) were run in shake flasks to evaluate xylitol, erythritol, ribitol, glycerol, arabitol and ethanol production and glucose consumption.

[0089] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0090] The seed culture was used to inoculate a 250 ml unbaffled flask containing production medium (Table C) to form a production culture (about 0.8 mL seed culture) with a starting OD600 of about 0.4. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, arabitol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 5 and Figure 2 Expression of both the Pichia stipitis cofactor-converting XDH (SEQ ID NO: 1) and the Saccharomyces cerevisiae DOG1 sugar phosphatase (SEQ ID NO: 8) in Saccharomyces cerevisiae resulted in the production of xylitol.

[0091] Table 4: Production medium

[0092]

[0093]

[0094] Table 5: 96-hour shake flask results

[0095]

[0096] Example 3 - Shake flask fermentation assay

[0097] Strains 1-8a-c, 1-13, 1-14a, and 1-14c (summarized in Table 2 above) were run in duplicate in shake flasks to assess xylitol, erythritol, ribitol, glycerol, and ethanol production and glucose consumption.

[0098] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0099] 0.8 mL of seed culture was used to inoculate a 250 ml non-baffled flask or a 250 ml baffled flask containing production medium (Table B) to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 48 hours, 72 hours and 96 hours of incubation. The samples were analyzed for glucose, arabitol, ribitol, xylitol, erythritol, glycerol and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 6 and Figure 2 As demonstrated by the results, expression of Saccharomyces cerevisiae DOG1 sugar phosphatase (SEQ ID NO:9) in combination with Pichia stipitis cofactor-converting XDH (SEQ ID NO:1), Rhodobacteraceae SDR family oxidoreductase R (SEQ ID NO:13), or Bemisia argentofolii (silverleaf whitefly) ketose reductase (sorbitol dehydrogenase) (SEQ ID NO:14) in Saccharomyces cerevisiae produced xylitol after only 48 hours of fermentation.

[0100] Although all strains tested produced xylitol, the production level could be altered by changing the aeration of the fermentation. Figure 3 As shown in , under high aeration (i.e., fermentation in baffled flasks), strain 1-14c produced approximately 0.5 g / L xylitol after 96 hours, whereas fermentation under lower aeration (i.e., in unbaffled flasks) resulted in xylitol production of over 2.5 g / L.

[0101] Although PCR validation indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence did not correctly target the ER1 locus or integrated multiple copies. Further analysis indicated that strains 1-14b and 1-13 included the transformed polynucleotide sequence, but it did not target the ER1 locus. Due to resource limitations, strain 1-14b was not run in this shake flask experiment. Further analysis also indicated that strain 1-14c included 8 copies of the polynucleotide encoding SEQ ID NO: 14 and 10 copies of the polynucleotide encoding SEQ ID NO: 8, but neither sequence correctly targeted the ER1 locus.

[0102] Table 6: Shake flask results

[0103]

[0104]

[0105] Example 4 - Shake flask fermentation assay

[0106] Strains 1-14c, 1-15a-d, 1-16a-d, 1-17a-e, 1-18a-e, and 1-19a-e (summarized in Tables A and B above) were run in shake flasks to evaluate xylitol, erythritol, ribitol, glycerol, and ethanol production and glucose consumption.

[0107] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0108] A 250 ml unbaffled flask containing production medium (Table C) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. The samples were analyzed for glucose, arabitol, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 7 and Figure 4 middle.

[0109] As summarized in Table 2, strain 1-14c was transformed with one of the XDH homolog candidates. Shake flask characterization of these strains confirmed that none of the tested XDH homolog candidates increased xylitol production above the production level of the 1-14c parent strain under the same conditions. In fact, some strains had reduced xylitol production relative to the parent strain.

[0110] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence did not correctly target the ER1 locus. Further analysis indicated that strains 1-15a-d, 1-16a-d, 1-17a-e, 1-18a-e, and 1-19a-e included the transformed polynucleotide sequence, but it did not target the ER1 locus.

[0111] Table 7: 96-hour shake flask results

[0112]

[0113]

[0114] Example 5 - Shake flask fermentation assay

[0115] Strains 1-14c, 1-20a-e, 1-21a-e, 1-23a-e, and 1-24a-e (summarized in Tables A and B above) were run in shake flasks to evaluate xylitol, erythritol, ribitol, glycerol, and ethanol production and glucose consumption.

[0116] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0117] A 250 ml unbaffled flask containing production medium (Table C) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. The samples were analyzed for glucose, arabitol, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 8 and Figure 5 middle.

[0118] As summarized in Table 2, strain 1-14c was transformed with one of the XKS / sugar phosphatase homolog candidates. Strains expressing S. cerevisiae DOG2 sugar phosphatase, bacterial Acidobacterium phosphatase homolog, or P. puerhi HAD-IA family hydrolase homolog increased the final titer of xylitol titer. The results for the Interrhizobium HAD family hydrolase homolog candidate were similar to those reported for the E. coli hexitol phosphatase gene (data not shown).

[0119] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence did not correctly target the ER1 locus. Further analysis indicated that strains 1-20a-e, 1-21a-e, 1-23a-e, and 1-24a-e included the transformed polynucleotide sequence, but it did not target the ER1 locus.

[0120] Table 8: 96-hour shake flask results

[0121]

[0122] Example 6 - Shake flask fermentation assay

[0123] Strains 1-1, 1-20b, 1-24e, 1-25a-d, 1-26a-e, 1-27a-e and 1-28a-c (summarized in Tables A and B above) were run in duplicate in shake flasks to evaluate xylitol, erythritol, ribitol, glycerol and ethanol production and glucose consumption.

[0124] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0125] A 250 ml unbaffled flask containing production medium (Table C) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. Samples were taken from the production culture after 96 hours of incubation. The samples were analyzed for glucose, arabitol, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 9 and Figure 6 middle.

[0126] The strains tested in this example included the phosphatase candidates that produced increased xylitol titers in Example 5, which were transformed into a wild-type background to observe metabolite production. Strains expressing S. cerevisiae DOG1 or S. cerevisiae DOG2 sugar phosphatases produced more xylitol than the wild-type parent background.

[0127] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly targeted to the ER1 locus. Further analysis indicated that strains 1-20b, 1-24e, 1-25a-d, 1-26a-c, 1-27a-c, and 1-28a-b included the transformed polynucleotide sequence, but it did not target the ER1 locus.

[0128] Table 9: Average 96-hour shake flask results

[0129]

[0130]

Claims

1. A genetically engineered yeast cell capable of producing xylitol, the engineered yeast cell comprising: An exogenous polynucleotide sequence encoding a xylulokinase (XKS), wherein the XKS comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NO: 8 and SEQ ID NO:

20. The yeast cell according to claim 1 , wherein the yeast cell is an osmotolerant yeast cell.

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

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

6. A genetically engineered Mycelium cell capable of producing xylitol, the engineered Mycelium cell comprising: An exogenous polynucleotide sequence encoding a xylulokinase (XKS), wherein the XKS comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NO: 8 and SEQ ID NO:

20.

7. The yeast cell according to any one of claims 1 to 6, wherein the cell is a Trichoderma sp. yeast cell.

8. The yeast cell of any one of claims 1 to 7, wherein the yeast cell is capable of producing xylitol at a titer of at least 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L or 2.5 g / L when used in a fermentation process in the presence of dextrose for 96 hours.

9. The yeast cell according to any one of claims 1 to 8, wherein the exogenous polynucleotide sequence is integrated into the genome of the yeast cell at a locus selected from the group consisting of ER1 locus, ER3 locus, PDC1 locus, pyrF locus, TRP3 locus, gpdIIA locus and gpdIIB locus.

10. The yeast cell according to any one of claims 1 to 9, wherein the exogenous polynucleotide sequence is operably linked to a heterologous promoter or an artificial promoter.

11. The yeast cell of claim 10, wherein the promoter is a constitutive promoter.

12. The yeast cell of claim 10 or claim 11, wherein the heterologous promoter or artificial promoter is selected from the group consisting of: pyruvate kinase 1 promoter (PYK1p; SEQ ID NO:61), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:52), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:54), translation elongation factor 1 promoter (TEFp; SEQ ID NO:55), modified TEFp (SEQ ID NO:53), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO:56), SEQ ID NO:56), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO:57), enolase promoter (ENO1p; SEQ ID NO:58), asparagine synthetase promoter (ASNSp; SEQ ID NO:59), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO:60), and RPL16B (SEQ ID NO:62).

13. The yeast cell according to any one of claims 1 to 12, wherein the yeast cell further comprises: An exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH), wherein the XDH comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO:

14.

14. The yeast cell of any one of claims 1 to 13, wherein the yeast cell is capable of producing xylitol at a titer of at least 1.0 g / L, 2.0 g / L, 2.5 g / L or 3.0 g / L when used in a fermentation process in the presence of dextrose for at least 96 hours.

15. The yeast cell of any one of claims 1 to 14, wherein the erythritol production of the yeast cell is reduced relative to the erythritol production in an equivalent yeast cell lacking the exogenous polynucleotide sequence encoding the sugar phosphatase.

16. The yeast cell of any one of claims 1 to 15, wherein the sugar phosphatase has a sequence that is at least 85% identical to at least one of SEQ ID NO: 8 and SEQ ID NO:

20.

17. The yeast cell of any one of claims 1 to 16, wherein the sugar phosphatase has a sequence that is at least 90% identical to at least one of SEQ ID NO: 8 and SEQ ID NO:

20.

18. The yeast cell of any one of claims 1 to 17, wherein the XHD has a sequence that is at least 85% identical to at least one of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO:

14.

19. The yeast cell of any one of claims 1 to 18, wherein the XDH has a sequence that is at least 90% identical to at least one of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO:

14.

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

21. A method for producing xylitol, the method comprising: A substrate comprising dextrose is contacted with an engineered yeast cell comprising an exogenous polynucleotide sequence encoding a sugar phosphatase comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 8 and SEQ ID NO: 20, wherein fermentation of the substrate by the engineered yeast produces xylitol.

22. The method of claim 21, wherein the engineered yeast cell is a Mycosporium yeast cell.

23. The method according to any one of claims 20 to 22, wherein the fermentation temperature is from 25°C to 45°C, from 30°C to 40°C or from 32°C to 37°C or therebetween.

24. The method of any one of claims 20 to 23, wherein the xylitol production is at least 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L or 3.0 g / L when the fermentation is run at 35°C for 96 hours.

25. The method of any one of claims 20 to 24, wherein erythritol production is reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking the exogenous polynucleotide sequence.

26. The method of any one of claims 20 to 25, wherein erythritol production is less than 40 g / L, 30 g / L, or less than 20 g / L when the fermentation is run at 35°C for 96 hours.

27. The method of any one of claims 20 to 26, wherein the volumetric oxygen uptake rate (OUR) is between 0.5 mmol O2 / (L·h) and 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) and 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) and 30 mmol O2 / (L·h), 3 mmol O2 / (L·h) and 25 mmol O2 / (L·h), 4 mmol O2 / (L·h) and 20 mmol O2 / (L·h), or 5 mmol O2 / (L·h) and 15 mmol O2 / (L·h).

28. The method of any one of claims 20 to 27, wherein glycerol production is reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking the exogenous polynucleotide sequence.

29. The method of any one of claims 20 to 28, wherein ethanol production is reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking the exogenous polynucleotide sequence.

30. Use of the engineered yeast cell according to any one of claims 1 to 19 for the production of xylitol.

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