Genetically Modified Yeast and Fermentation Method for the Production of Xylitol

Abstract

This specification discloses genetically engineered yeast cells capable of producing xylitol. The engineered yeast cells may contain an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 364,375, filed May 9, 2022, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing Filed via Patent Center The contents of the sequence list XML file "PT - 1349 - WO - PCT.xml", sized 448,927 bytes, created on May 4, 2023, and electronically filed with this application through the Patent Center, are hereby incorporated by reference in their entirety.

Background Art

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

[0004] Accordingly, provided herein are genetically modified yeasts and fermentation methods for producing xylitol while reducing the production of erythritol.

Summary of the Invention

[0005] The present disclosure provides a genetically engineered yeast cell capable of producing xylitol, wherein the engineered yeast cell comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme. The XPDH enzyme may comprise a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33. The yeast cell may be an osmotolerant yeast cell. The yeast cell may be a cell of the subphylum Ustilaginomycotina. The yeast cell may be selected from the group consisting of Trichosporonoides megachiliensis, Trichosporonoides oenocephalus, Trichosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomyces, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. The yeast cell may be a yeast cell of the genus.

[0006] The present disclosure also provides a genetically engineered Moniliella cell capable of producing xylitol, wherein the engineered Moniliella cell comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme that comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33.

[0007] The XPDH enzyme may have a sequence that is at least 85% identical to 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 a sequence that is at least 90% identical to 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.

[0008] The engineered cells described herein can be Moniliella polnis cells. Yeast cells can be capable of producing xylitol at a titer of at least 20, 30, 50, 75, or 100 g / L when used in a fermentation process at 35 °C for 96 hours in the presence of dextrose. Erythritol production by yeast cells can be reduced compared to erythritol production in equivalent yeast cells lacking an exogenous polynucleotide sequence. The exogenous polynucleotide sequence can be integrated into the genome of yeast cells at a locus selected from the ER1 locus, ER3 locus, PDC1 locus, pyrF locus, TRP3 locus, gpdIIA locus, and gpdIIB locus. The exogenous polynucleotide sequence may be operably linked to a heterologous promoter or an artificial promoter. The promoter may be a constitutive promoter. The promoter can be selected from the group consisting of the 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 (ASNS p; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139).

[0009] The present disclosure provides a method for producing xylitol, the method comprising contacting a substrate containing dextrose with an engineered yeast cell as described herein, and xylitol is produced by fermentation of the substrate by the engineered yeast. The present disclosure also provides a method for producing xylitol, the method comprising contacting a substrate containing dextrose with an engineered yeast cell comprising an exogenous polynucleotide sequence encoding a xylitol phosphate dehydrogenase (XPDH) enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33, and xylitol is produced by fermentation of the substrate by the engineered yeast. 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 volumetric 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 O 2- (L·h). Xylitol can be produced at a rate of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, or at least 1.0 g / L -1 h -1 . Xylitol production can be at least 10, 20, 30, 40, 50, 75, or 100 g / L when the fermentation is carried out at 35°C for 96 hours. Erythritol production can be reduced compared to an equivalent fermentation run using equivalent yeast cells lacking the exogenous polynucleotide sequence. Erythritol production can be less than 50, 40, 30, or 20 g / L when the fermentation is carried out at 35°C for 96 hours. Glycerol production can be reduced compared to an equivalent fermentation run using equivalent yeast cells lacking the exogenous polynucleotide sequence. Ethanol production can be reduced compared to an equivalent fermentation run using equivalent yeast cells lacking the exogenous polynucleotide sequence.

Brief Description of the Drawings

[0010] This patent or application includes at least one drawing created in color. Copies of this patent or patent application publication that include color drawings will be provided by the Patent Office upon request and payment of the required fees.

[0011] The drawings generally, but not by way of limitation, and by way of example, illustrate the various aspects discussed herein.

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Mode for Carrying Out the Invention

[0012] Here, certain aspects of the disclosed subject matter are specifically referenced, and examples thereof are illustrated, in part, in the accompanying drawings. It will be understood that the disclosed subject matter is described in conjunction with the recited claims, but the illustrated subject matter is not intended to limit the disclosed subject matter to the recited claims.

[0013] In this document, the terms "a", "an", or "the" are used to include one or more, unless the context clearly dictates otherwise. The term "or" is used to refer to non-exclusive "or", unless otherwise indicated. All publications, patents, and patent documents referred to in this document are hereby incorporated by reference in their entirety, as if each were individually incorporated by reference. In the event of inconsistent usage between this document and those documents incorporated by reference in this manner, the usage in the incorporated reference is to be construed as supplementing that of this document. In case of incompatible contradictions, the usage in this document prevails.

[0014] Values expressed in a range format are to be construed flexibly so as to include not only the numerical values explicitly listed as the limits of the range, but also all individual numerical values or sub-ranges subsumed within that range as if each were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" is to be construed as including not only about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The description "about X to Y" has the same meaning as "about X to about Y", unless otherwise indicated. Similarly, the description "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z", unless otherwise indicated.

[0015] Unless otherwise explicitly stated, ppm (parts per million) is on a percentage basis, and ratios are on a weight basis. Percentage by weight is also referred to hereinafter as weight% or % (weight).

[0016] The present disclosure relates to various recombinant cells engineered to produce xylitol. Generally, the recombinant cells described herein have an active pentose phosphate pathway and are characterized by the expression of an exogenous xylitol-phosphate dehydrogenase (XPDH) enzyme. The invention further provides a fermentation method for producing xylitol from dextrose using the genetically engineered cells described herein.

[0017] Generally, 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 are unicellular organisms that evolved from multicellular ancestors, and some species retain multicellular characteristics such as forming strings of connected budding cells known as pseudohyphae or false hyphae. Yeast cells may also be referred to in the art as yeast-like cells, and as used herein, "yeast cells" encompasses both yeast and yeast-like cells. Suitable yeasts and yeast-like host cells for modification include Saccharomyces cerevisiae, Komagataella, Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Yarrowia lipolytica, Issatchenkia orientalis, Pichia guilliermondii, Pichia genus YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnoliae, Candida ethanolica), Pichia desiccans, Pichia membranifaciens, Pichia fermentans, Aspergillus, Trichoderma, Myceliophthora thermophila, Moniliella (e.g., Moniliella polnis), Paffia, Yamadazyma, Hansenula, Pichia kudriavzevii, Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trichosporonoides odsephalis, Trichosporonoides nigrescens), Pseudozyma tsukubaensis, Trigonopsis variabilis, Penicillium, and Torula, but are not limited thereto. Those skilled in the art will understand the requirements for the selection of suitable yeast cells, and the recombinant yeast cells of the present disclosure are not limited to those expressly listed herein. Methods for genetic manipulation of 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.

[0018] Suitable yeast cells may be cells of the Basidiomycota and Ustilaginomycotina subphylum. Suitable yeast fungi of the Ustilaginomycotina subphylum include Ustilago (e.g., U. cynodontis, U. maydis, U. sphaerogena, U. corda, U. citrina, U. coicis, U. syntherismae, U. esculenta, U. neglecta, U. crus-galli, Ustilago avenae), Sporisorium (e.g., Sporisorium exsertum), Monographella (e.g., M. polonicum, M. tomentosa, M. acetabutans, M. fonsecae, M. madida, M. megachiliensis, M. oseccephalus, M. nigrescens), Pseudozyma (e.g., Pseudozyma tsukubaensis), and Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trichosporonoides oseccephalus, Trichosporonoides nigrescens), but are not limited thereto. Yeast fungi of the Ustilaginomycotina subphylum are known and described in the art as potential producers for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol as well as for other valuable biotechnology applications.For example, see 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 Biotechnol, 2010, 86:1017-1025).

[0019] Suitable yeast cells 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 convert glucose-6-phosphate, NADP + or NAD +, and water together into NADPH or NADH, CO2, and ribulose-5-phosphate. Continuing with the non-oxidative phase, this pathway can also produce other pentose (i.e., five-carbon) sugars. For example, the pentose phosphate pathway can produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose 6-phosphate, combinations thereof, etc., depending on the enzyme activities present. The active pentose phosphate pathway can be native to the yeast cells or introduced into the yeast cells by genetic engineering.

[0020] The yeast cell can be an osmotolerant yeast cell. As used herein, "osmotolerant" refers to a yeast capable of growth and regeneration under conditions of high osmotic pressure (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 osmotolerant yeasts are known and described in the art and include many species of yeasts used in industrial fermentation processes. Similarly, methods for assaying yeast osmotolerance are known and described in the art. See, for example, Tiwari, S. et al., ("Nectar yeast community of tropical flowering plants and assessment of their osmotolerance and xylitol-producing potential," Current Microbiology, 2022, 79:28).

[0021] The recombinant yeast cell may be a recombinant Moniliella cell, e.g., a Moniliella polnis cell. Figure 1 shows the predicted native pentose phosphate and glycolysis pathways in Moniliella polnis. Moniliella has previously been used in the fermentative 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).

[0022] Various plasmids and methods for the transformation of Moniliella are also described in the following examples. For example, Moniliella can be transformed using a bipartite polynucleotide sequence, in which case, after recombination, the exogenous polynucleotide of interest is integrated into the designated locus and the selectable marker becomes expressible in the cell. Suitable selectable markers are known and described in the art. Examples of selectable markers include amdS (e.g., degraded into a 3' portion (SEQ ID NO: 167) and a 5' portion (SEQ ID NO: 174)), the G418 resistance gene (e.g., degraded into a 3' portion (SEQ ID NO: 172) and a 5' portion (SEQ ID NO: 175)), the zeocin resistance gene (e.g., degraded into a 3' portion (SEQ ID NO: 168) and a 5' portion (SEQ ID NO: 169)), the nourseothricin N-acetyltransferase (NAT) (e.g., degraded into a 3' portion (SEQ ID NO: 171) and a 5' portion (SEQ ID NO: 170)), and the invertase gene (SUC2) (e.g., the 3' portion of SEQ ID NO: 173 and the 5' portion of SEQ ID NO: 176), but are not limited thereto.

[0023] The recombinant cells described herein, when expressed, contain one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that improve the fermentation of glucose to ribitol by the recombinant cells.

[0024] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless explicitly indicated otherwise.

[0025] As used herein, "exogenous" refers to genetic material or its expression product that is derived 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, can be derived from another organism, can be a modified form of a component derived from another organism, or can be a synthetically derived component. For example, the aK.S. lactis invertase gene becomes exogenous when introduced into S. cerevisiae.

[0026] As used herein, "natural" refers to genetic material or its expression product found within the genome of wild-type cells of a host cell, apart from inter-individual variations that do not affect function or expression. For the purposes of this application, the Monilia polinis cell "Monilia tomentosa var. polinis TCV364", described in U.S. Patent No. 6,440,712 and incorporated herein by reference in its entirety, and deposited under the Budapest Treaty on March 28, 1997 with the 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 accession number MUCL40385, is considered a wild-type Monilia polinis cell.

[0027] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequences and structures necessary to confer the function and characteristics to the polymers listed. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. Any listing 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. An overview of the amino acids and their three-letter and one-letter symbols understood in the art is shown in Table 1. The amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0028]

Table 1

[0029] Variants or sequences having substantial identity or homology to the polypeptides described herein can be utilized in the practice of the disclosed recombinant cells, compositions, and methods. Such sequences can be referred to as variant or modified sequences. That is, the polypeptide sequences can be modified while still retaining the ability to exhibit the desired activity. Generally, variant or modified sequences can include sequence identity of about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 95% with the wild-type, naturally occurring polypeptide sequence, or the variant polypeptides described herein.

[0030] 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 for amino acid and nucleic acid sequence alignment are well known. Methods for sequence alignment and generation of sequence identity include global alignment and local alignment, which typically use computational approaches. In some embodiments, the alignment can be performed using the default parameters with the BLAST (Basic Local Alignment Search Tool version 2.2.31 software of the National Center for Biological Information (NCBI)) local alignment search tool. The 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 adjusts the parameters of the short input sequence; expect threshold: 10; word size: 6; maximum matches in query range: 0; matrix: BLOSUM62; gap costs: (existence: 11, extension: 1); composition adjustment: conditional compositional 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 sequences: 100; short query: automatically adjusts the parameters of the short input sequence; expect threshold: 10; word size: 28; maximum matches in query range: 0; match / mismatch scores: 1, -2; gap costs: linear; filter: low complexity regions; mask: mask only for lookup tables. A sequence having an identity score of XX% (e.g., 80%) to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical to the reference sequence or equivalently to have XX% sequence identity.

[0031] Polypeptide or polynucleotide sequence identity may be measured over the length of the entire defined polypeptide sequence, as defined, for example, by a particular SEQ ID NO, or over a shorter length, for example, a fragment obtained from a larger defined polypeptide sequence, for example, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are merely exemplary, and it is understood that any fragment length supported by the sequences shown in the specification, tables, figures or sequence listing may be used to describe the length over which the percent identity is measured.

[0032] The polypeptides disclosed herein may include "variant" polypeptides, "mutants", and "derivatives" thereof. As used herein, the term "wild-type" is a term of the art understood by one of ordinary skill in the art and means the typical form of a naturally occurring polypeptide, as distinguished from a mutant form or a mutant form. As used herein, "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence different from the reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of amino acid residues as compared to the reference molecule.

[0033] The amino acid sequences of polypeptide variants, mutants, derivatives, or fragments contemplated herein may include conservative amino acid substitutions as compared to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions as compared to a reference molecule. A "conservative amino acid substitution" is a substitution of one amino acid for a different amino acid that is predicted to interfere the least with the properties of the reference polypeptide. In other words, a conservative 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 the substitution (e.g., as a β-sheet or α-helix conformation), (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain.

[0034] As used herein, the terms "polynucleotide", "polynucleotide sequence", and "nucleic acid sequence", and "nucleic acid" are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. These terms also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand. DNA polynucleotides can be cDNA (e.g., coding DNA) or genomic DNA sequences (e.g., including both introns and exons).

[0035] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those of skill in the art, it can be transcribed and / or translated to produce the polypeptide or a fragment thereof. The antisense strand of such a polynucleotide is also said to encode the sequence.

[0036] One skilled in the art understands the degeneracy of the genetic code and that various polynucleotides can encode the same polypeptide. In some embodiments, a polynucleotide (e.g., a polynucleotide encoding an XPDH 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 the desired form of the polypeptides described herein can be used. Thus, non-naturally occurring sequences can be used. These may be desired, for example, to enhance expression in a heterologous expression system of a polypeptide or protein. Computer programs for generating degenerate codon sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and human hands can also be used to generate degenerate codon sequences.

[0037] The recombinant cells described herein can include deletions or disruptions in one or more native genes. The term "deletion or disruption" has either a completely removed coding region (deletion), or a modification (such as by deletion, insertion, or mutation) of a gene, its promoter, or its terminator, such that the gene no longer produces an active expression product, the amount of the expression product is significantly reduced (e.g., at least 75% reduction or at least 90% reduction), or produces an expression product with significantly reduced activity (e.g., at least 75% reduction or at least 90% reduction), referring to the state of a native gene in a recombinant cell. Deletions or disruptions can be achieved by genetic engineering methods, directed evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. The native gene that is deleted or disrupted can be replaced with an exogenous nucleic acid of interest for the expression of an exogenous gene product (e.g., a polypeptide, an enzyme, etc.).

[0038] The recombinant cells described herein may include one or more genetic modifications in which exogenous nucleic acid is integrated into the genome of the host cell. Those skilled in the art know how to select appropriate loci in the yeast genome for integration of exogenous nucleic acid. Appropriate integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in M. polnis host cells, suitable interaction loci include, but are not limited to, the ER1 locus (defined as the locus adjacent to SEQ ID NO: 85 and SEQ ID NO: 162), the ER3 locus (defined as the locus adjacent to SEQ ID NO: 155 and SEQ ID NO: 165), the PDC1 locus (defined as the locus adjacent to SEQ ID NO: 152 and SEQ ID NO: 164), the pyrF locus (defined as the locus adjacent to SEQ ID NO: 153 and SEQ ID NO: 163), the TRP3 locus (defined as the locus adjacent to SEQ ID NO: 156 and SEQ ID NO: 159), the gpdIIA locus (defined as the locus adjacent to SEQ ID NO: 157 and SEQ ID NO: 161); and the gpdIIB locus (defined as the locus flanked by SEQ ID NO: 158 and SEQ ID NO: 166), but are not limited thereto. The exogenous nucleic acid may also be integrated into intergenic regions or other positions in the host cell genome not specifically identified herein. Other suitable integration loci may be determined by those skilled in the art. Further, those skilled in the art will recognize how to use the sequences to design primers to verify accurate gene integration at the selected locus.

[0039] The recombinant cell may be integrated into the host chromosome and may have one or more copies of a given exogenous nucleic acid sequence that is replicated along with the chromosome into which it is integrated. For example, a yeast cell 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. The recombinant cell may contain multiple (two or more) copies of a given polynucleotide sequence encoding a polypeptide described herein. The recombinant cell may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a polynucleotide sequence encoding a polypeptide described herein that is integrated into the genome. The multiple copies of the polynucleotide sequence may all be integrated at a single locus or may be integrated at multiple loci.

[0040] The recombinant cells described herein can produce xylitol and contain an exogenous polynucleotide sequence encoding the xylitol phosphate dehydrogenase (XPDH) enzyme. The exogenous polynucleotide sequence can be an exogenous xylitol-phosphate dehydrogenase (XPDH) gene. After the conversion of xylulose 5-phosphate to xylitol 5-phosphate, it is thought that the native phosphatase enzyme removes the phosphate to produce xylitol.

[0041] The "xylitol-phosphate dehydrogenase gene" and the "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" means xylulose-5-phosphate and NADPH or NADH to xylitol 5-phosphate and NADP + or NAD +Refers to the ability to catalyze the conversion to. The XPDH gene can be derived from any suitable source. For example, the XPDH gene can be derived from Clostridium difficile, Lactobacillus rhamnosus, Bacillus halodurans, Alkalihalobacillus ligniniphilus, Geotoga ribovars soli, Hendrixia sporothermodurans, Clostridium fungisolvens, or Neobacillus coagulans. The XPDH 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 to at least one amino acid sequence of SEQ ID NOs: 12-15, 28-32, or 33. The XPDH 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 to at least one amino acid sequence of SEQ ID NOs: 14, 15, 28, or 31.

[0042] The recombinant cell can contain an exogenous polynucleotide that is or is derived from the Clostridium difficile gene encoding the amino acid of SEQ ID NO: 12. The exogenous polynucleotide can encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0043] The recombinant cell may comprise a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or can be derived from a Clostridium difficile gene encoding the amino acids 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.

[0044] The recombinant cell may comprise an exogenous polynucleotide that is or can be derived from a 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 to the amino acid sequence of SEQ ID NO: 14.

[0045] The recombinant cell may comprise an exogenous polynucleotide that is or can be derived from a 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 to the amino acid sequence of SEQ ID NO: 15.

[0046] The recombinant cell may comprise an exogenous polynucleotide that is or can be derived from an Alkalihalobacillus ligniniphilus 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 to the amino acid sequence of SEQ ID NO: 28.

[0047] The recombinant cell may contain an exogenous polynucleotide that is or is derived from a Bacillus subtilis 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 to the amino acid sequence of SEQ ID NO: 29.

[0048] The recombinant cell may contain an exogenous polynucleotide that is or is derived from a Henneguya sp. 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 to the amino acid sequence of SEQ ID NO: 30.

[0049] The recombinant cell may contain an exogenous polynucleotide that is or is derived from a Clostridium fungisolvens gene encoding the amino acids of SEQ ID NO: 31. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0050] The recombinant cell may contain an exogenous polynucleotide that is or is derived from a 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 to the amino acid sequence of SEQ ID NO: 33.

[0051] The exogenous polynucleotide in the recombinant cell described in this specification may be under the control of a promoter. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial promoter. Suitable promoters are known and described in the art. Examples of promoters include the pyruvate decarboxylase promoter (PDC), the translation elongation factor 2 promoter (TEF2), SED1, the alcohol dehydrogenase 1A promoter (ADH1), the hexokinase 2 promoter (HXK2), the FLO5 promoter, the pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86); the 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130); the glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132); the translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133); the modified TEFp (SEQ ID NO: 131); the phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134); the 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135); the enolase promoter (ENO1p; SEQ ID NO: 136); the asparagine synthetase promoter (ASNSp; SEQ ID NO: 137); the 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138); and RPL16B (SEQ ID NO: 139), but are not limited thereto.

[0052] The exogenous nucleic acid in the recombinant cell described in this specification may 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. Terminators include, but are not limited to, GAL10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt; SEQ ID NO: 140); ASNS terminator (ASNSt; SEQ ID NO: 141); ENO1 terminator (ENO1t; SEQ ID NO: 142); hexokinase 1 terminator (HXK1t; SEQ ID NO: 143); PGK1 terminator (PGK1t; SEQ ID NO: 144); PGM1 terminator (PGM1t; SEQ ID NO: 145); PYK1 terminator (PYK1t; SEQ ID NO: 146); RPLA terminator (RPLAt: SEQ ID NO: 147); transaldolase 1 terminator (TAL1t; SEQ ID NO: 148); TDH3 terminator (TDH3t; SEQ ID NO: 149); translation elongation factor 2 terminator (TEF2t; SEQ ID NO: 150); triosephosphate isomerase 1 terminator (TPI1t; SEQ ID NO: 151).

[0053] A promoter or terminator is "operably linked" to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to the polynucleotide is such that the promoter or terminator, in some cases, performs its transcriptional regulatory function.

[0054] 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 can be single-stranded or double-stranded and can represent a sense strand or an antisense strand. A recombinant polynucleotide is a polynucleotide formed by laboratory methods that includes polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Thus, a construct can include, for example, new modifications to an endogenous gene introduced by genome editing techniques. A construct can also include, for example, a recombinant polynucleotide made using recombinant DNA methodology. A construct may be a vector that includes a promoter operably linked to a polynucleotide encoding a polypeptide described herein. As used herein, the term "vector" refers to a polynucleotide that can transport another polynucleotide to which it is ligated. A vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be incorporated.

[0055] The present invention also provides a fermentation method for producing xylitol using the recombinant cells described herein. The fermentation method includes a step of fermenting a substrate using the genetically engineered yeast described herein to produce xylitol. The fermentation method can 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 xylitol from the fermentation broth. The fermentation process may be a completely aerobic process.

[0056] The fermentation method can be carried out using a suitable fermentation substrate. The substrate for the fermentation method can include glucose, sucrose, galactose, mannose, molasses, xylose, fructose, a hydrolyzate of starch, a lignocellulose hydrolyzate, or a combination thereof. Those skilled in the art will recognize which fermentation substrate is suitable for a given fermenting organism and system.

[0057] The fermentation process can be carried out under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during the process, may be the ambient temperature. Alternatively, or additionally, 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 at about 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to the specific ranges or temperatures described herein and can be changed as needed.

[0058] The fermentation process can be carried out within a specific oxygen uptake rate (OUR) range. The volumetric OUR of the fermentation process can be in the range of 0.5 to 40, 1 to 35, 2 to 30, 3 to 25, 4 to 20, or 5 to 15 mmol O 2- (L·h). In some embodiments, the specific OUR can be in the range of 0.05 to 10, 0.1 to 8, 0.15 to 5, 0.2 to 1, or 0.3 to 0.75 mmol O 2- (g cell dry weight·h). However, the volumetric measured OUR or specific OUR of the fermentation process is not limited to any specific rate or range listed herein.

[0059] The fermentation process can be carried out at various cell concentrations. In some embodiments, the cell dry weight at the end of fermentation can be 5 to 40, 8 to 30, or 10 to 20 g cell dry weight / L. Further, the pitch density or pitching rate of the fermentation process can vary. In some embodiments, the pitch density can be 0.05 to 11, 0.1 to 10, or 0.25 to 8 g cell dry weight / L.

[0060] 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.

[0061] The fermentation process can be associated with various characteristics, although not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of 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 the selection of fermentation process conditions.

[0062] The xylitol production rate of this process can be at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g / L -1 h -1 and may be so. The xylitol mass yield of this process can be 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 xylitol titer of this process can be at least 20, 30, 50, 75, or 100 g / L.

[0063] 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

[0064] The present invention will be described in more detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0065] 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 approximately 25% of the enzymes having no 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 in a manner similar to that described by Duax et al., ("Rational proteomics I. Fingerprinting identification and cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme family," Proteins, 2003, 53(4):931-943). The cofactor binding pocket was identified by proximity to the Rossman fold (+23 to +30 amino acids from the GXGXXG motif (SEQ ID NO: 129)) and scored based on the total charge in an 8-residue window. The top 8 candidates predicted to use NADP, along with 4 candidates predicted to use NAD and 3 controls, were selected for further characterization.

[0066] Further examination of the structural features of the predicted binding pockets for factors that can affect cofactor preference identified important aspartic acid residues. See Figure 3. Using the polypeptide of SEQ ID NO: 34 and its variants, a structural homology model was constructed to predict the identification of the cofactor binding pocket. Figure 3 shows the C-terminus of the penultimate β-strand outside the Rossmann fold domain. Without wishing to be bound by any particular theory, enzymes in which the first residue in this region (residue 198 relative to SEQ ID NO: 34) is aspartic acid and the second residue (residue 199 relative to SEQ ID NO: 34) is a large hydrophobic amino acid (e.g., isoleucine) are predicted to prefer the NAD cofactor by hydrogen bonding of the aspartic acid to the hydroxyl group of the NAD ribose. However, enzymes in which the first residue (residue 198 relative to SEQ ID NO: 34) is alanine, glycine, or serine and the second residue (residue 199 relative to 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 within the binding pocket for said phosphate. Based on this analysis, 12 additional enzymes were selected for their predicted preference for NADP. Finally, 6 additional enzymes with sequence similarity to the active XPDH enzyme were selected for testing.

[0067] Example 2 - Diversity of TarJ Approximately 800 ribulose 5-phosphate reductase sequences were obtained from Uniprot and analyzed. Figure 3 shows the natural sequence diversity for this set of sequences. Overall, the diversity in this set is low, with only 10% of the enzymes having sequence similarity with an identity greater than 75%. 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.

[0068] Example 3 - In Vitro Enzyme Assay Polynucleotides encoding putative XPDH homologs (Table 2) or TarJ' homologs (Table 3) were cloned into a vector containing the T7 promoter and terminator for cell-free protein expression (New England Biolabs, PURExpress® In Vitro Protein Synthesis). Cell-free synthesized proteins were analyzed for activity against four substrates (ribulose 5-phosphate, xylulose 5-phosphate, ribulose, and xylulose) with either NADP or NAD cofactor. Seven enzymes (XPDH of SEQ ID NOs: 12 and 34, TarJ' of SEQ ID NOs: 36, 37, 38, 40, and 42) were able to catalyze the reduction of either ribulose 5-phosphate or xylulose 5-phosphate (Figure 5), but were unable to catalyze the reduction of xylulose or ribulose (data not shown).

[0069] [Table 2]

[0070] [Table 3]

[0071] Example 4 - Genetically Modified Moniliera polnis Strains Strain 1-1 is the Moniliella polnis host strain "Moniliella tomentosa var. polnis TCV364" described in U.S. Patent No. 6,440,712, which is incorporated herein by reference in its entirety, and was deposited on March 28, 1997, under the Budapest Treaty at 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 the accession number MUCL40385. Table 4 below lists various Moniliella polnis strains, including information on the parent strain, the sequences into which the parent strain was transformed, and the characterization of the expression cassettes contained in the transformed sequences. 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 a 5' portion of the G418 resistance gene expression cassette (SEQ ID NO: 175). Each "selectable marker cassette" contained, in order, a 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). Upon bipartite transformation with both the XPDH / TarJ' homolog expression cassette and the selectable marker cassette, the two cassettes were recombined for the incorporation of the nucleotide sequences encoding both the XPDH or TarJ' homolog and the G418 resistance marker at the ER1 locus.

[0072] The indicated Moniliera polinis parent strain was transformed with the indicated sequence by first protoplasting the parent strain by adding an enzyme mixture containing 0.6 M MgSO4, 7.5 g / L driselase, and 12.5 g / L Trichoderma harzianum lysing enzyme to the mycelial pellets of the parent strain. The protoplasts were then pelleted, 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 the 5’ and 3’ DNA transformation fragments (total 3 - 10 μg; see Table 4 for a list of fragments) were added to approximately 200 μL of the protoplast mixture (10 8 cells / mL) prepared. 50% PEG in 1 mL of STC medium was then added to the salmon sperm DNA, transformation DNA, and protoplast mixture, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, recovery 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 it was incubated at 27°C, 100 rpm for 16 - 24 hours. After incubation, the protoplasts were pelleted by centrifugation and resuspended in 1 mL of PBS.

[0073] The resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30 - 35°C for at least 2 - 4 days until transformants grew. The resulting transformants were evaluated by colony PCR for integration of the indicated sequence. The PCR-verified isolates were then designated as the indicated strain numbers. In some cases, one or more PCR-verified 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.

[0074] For example, strain 1-1 was transformed with SEQ ID NO: 43 and SEQ ID NO: 44. SEQ ID NO: 43 contains (i) a 3'-adjacent DNA (SEQ ID NO: 162) for targeted chromosomal integration into the ER1 locus, and (ii) the 3'-portion of a G418 resistance gene selectable marker (SEQ ID NO: 172). SEQ ID NO: 44 contains (i) an expression cassette of an XPDH homolog from M. sediminis of SEQ ID NO: 87 encoding the amino acid sequence of SEQ ID NO: 1 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; (ii) a 5'-adjacent DNA (SEQ ID NO: 85) for targeted chromosomal integration into the ER1 locus; (iii) the 5'-portion of a G418 resistance gene selection marker (SEQ ID NO: 175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30 - 35 °C for at least 2 days until the transformants grew. The obtained 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 the integration of the indicated sequences. The PCR-verified isolates were named strains 1-2a, 1-2b, 1-2c, 1-2d, and 1-2e.

[0075]

Table 4

[0076] Example 5 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, 1-39a-f, 1-42a-f, 1-13a-f, and 1-15a-f (summarized in Table 4 above) were run in shake flasks.

[0077] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30 °C for 48 - 72 hours. Cells from the incubated YPD plates were scraped into 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.

[0078] 0.8 mL of the seed culture was inoculated into a 250 mL baffled flask containing the production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 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 are reported in Table 6 and Figures 6 and 7.

[0079]

Table 5

[0080] PCR verification 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 - 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.

[0081]

Table 6

[0082] Example 6 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1 - 13c, 1 - 29a - e, 1 - 33a - e, and 1 - 34a - e (summarized in Table 4 above) were run in shake flasks.

[0083] The strains were 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 h. 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 approximately 32 - 50 h.

[0084] A 250 mL baffled flask containing the production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35 °C and 250 rpm. After 96 h of incubation, samples were taken from the production culture. 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 are reported in Table 7 and Figure 8.

[0085] 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-verified, but it was then determined that the integrated polynucleotide that should encode the N. cucumber XPDH homolog contained a frameshift mutation and no functional XPDH was expressed. Thus, although the results appear variable, they are actually consistent considering that strains 1-34a, 1-34b, and 1-34e do not contain the polynucleotide encoding functional XPDH.

[0086]

Table 7

[0087] Example 7 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1-13c, 1-17a-e, 1-18a-e, 19a-e, 1-21a-e, 1-22a-e, 1-23a-e, 1-24a-e, 1-25a-e, and 1-27a-d (summarized in Table 4 above) were run in shake flasks.

[0088] 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 path length cuvette using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 of 15 - 20 in about 32 - 50 hours.

[0089] 0.8 mL of the seed culture was inoculated into a 250 mL baffled flask containing the production medium (Table 5) to form a 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 equipped with a refractive index detector. The results are shown in Table 8.

[0090]

Table 8

[0091] Example 8 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1 - 13c, 1 - 3a - e, 1 - 10a - e, 1 - 11a - e, 1 - 12a - e, 1 - 14a - e, 1 - 16a - e, 1 - 28a - e, and 1 - 2a - e (summarized in Table 4 above) were run in shake flasks.

[0092] The strain was streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass growth and incubated at 30 °C for 48 - 72 hours. Cells from the incubated YPD plates were scraped into 40 mL of rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffled - free 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.

[0093] 0.8 mL of the seed culture was inoculated into a 250 mL baffled - free flask containing the production medium (Table 5) to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 - hour and 96 - hour incubations. 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 are reported in Table 9 and Figure 9.

[0094] PCR verification 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 in the ER1 locus. Further analysis was inconclusive about the integration positions in strains 1 - 2c and 1 - 2d.

[0095] [Table 9]

[0096] Example 9 - Shake - flask fermentation assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1-13c, 1-8a-d, 1-26a-e, 1-36a-e, 1-41a-e, 1-40a-e, and 1-20a-e (summarized in Table 4 above) were run in shake flasks.

[0097] The strains were 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 path length cuvette using a Genesys 20 spectrophotometer (Thermo Scientific). The seed culture reached an OD600 of 15 - 20 in approximately 32 - 50 hours.

[0098] 0.8 mL of the seed culture was inoculated into a 250 mL baffled flask containing the production medium (Table 5) to form a 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. 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 are reported in Tables 10 and 10.

[0099] PCR verification 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 1-20 a-e.

[0100]

Table 10

[0101] Example 10 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1-13c, 1-30a-e, 1-31a-e, 1-32a-e, 1-4a-e, 1-5a-e, 1-6a-e, 1-7a-e, and 1-9a-e (summarized in Table 4 above) were run in shake flasks.

[0102] Strains were 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 250 mL baffled flasks. 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 approximately 32 - 50 hours.

[0103] A 250 mL baffled flask containing the production medium (Table 5) was inoculated with 0.8 mL of the seed culture to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture after 72 hours and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography equipped with a refractive index detector. The fermentation results are reported in Tables 11 and 11.

[0104] PCR verification 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 at the ER1 locus. Further analysis showed that strains 1-30c and 1-30d contained the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integration locus in strain 1-6c was inconclusive.

[0105]

Table 11

Claims

1. A genetically engineered yeast cell capable of producing xylitol, wherein the engineered yeast cell contains an exogenous polynucleotide sequence encoding xylitol-phosphate dehydrogenase (XPDH) enzyme, a yeast cell.

2. The yeast cell according to claim 1, wherein the XPDH enzyme contains a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33.

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

4. The yeast cell according to any one of claims 1-3, wherein the yeast cell is a cell of the subphylum Ustilaginomycotina.

5. The yeast cell according to any one of claims 1-4, wherein the yeast cell is selected from the group consisting of Trichosporonoides megachiliensis, Trichosporonoides oenocephalus, Trichosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomyces, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium.

6. The yeast cell according to any one of claims 1-5, wherein the yeast cell is a yeast cell of the genus Moniliella.

7. A genetically engineered Moniliella cell capable of producing xylitol, wherein the engineered Moniliella cell A Moniliella cell comprising an exogenous polynucleotide sequence encoding a xylitol - phosphate dehydrogenase (XPDH) enzyme, wherein the sequence is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12 - 15, 28 - 31, and 33. **Claim 8** The yeast cell according to any one of claims 1 - 7, wherein the cell is a Moniliella polnis cell. **Claim 9** The yeast cell according to any one of claims 1 - 8, wherein when the yeast cell is used in a fermentation process at 35°C for 96 hours in the presence of dextrose, it can produce xylitol at a titer of at least 20, 30, 50, 75, or 100 g / L. **Claim 10** The yeast cell according to any one of claims 1 - 9, wherein the erythritol production by the yeast cell is reduced as compared to erythritol production in an equivalent yeast cell lacking the exogenous polynucleotide sequence. **Claim 11** The yeast cell according to any one of claims 1 - 10, wherein the exogenous polynucleotide sequence is integrated 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. **Claim 12** The yeast cell according to any one of claims 1 - 11, wherein the exogenous polynucleotide sequence is operably linked to a heterologous promoter or an artificial promoter. **Claim 13** The yeast cell according to claim 12, wherein the promoter is a constitutive promoter. **Claim 14** The yeast cell according to claim 12 or 13, wherein the constitutive 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).

15. The yeast cell according to any one of claims 1 to 14, wherein the XPDH enzyme has at least one of SEQ ID NOs: 12 to 15, 28 to 31, and 33, or a sequence that is at least 85% identical to at least one of SEQ ID NOs: 14, 15, 28, or 31.

16. The yeast cell according to any one of claims 1 to 15, wherein the XPDH enzyme has at least one of SEQ ID NOs: 12 to 15, 28 to 31, and 33, or a sequence that is at least 90% identical to at least one of SEQ ID NOs: 14, 15, 28, or 31.

17. A method for producing xylitol, the method comprising: contacting a substrate containing dextrose with the engineered yeast cell according to any one of claims 1 to 16, and producing xylitol by fermentation of the substrate by the engineered yeast.

18. A method for producing xylitol, the method comprising: A method comprising contacting a substrate containing dextrose with an engineered yeast cell containing an exogenous polynucleotide sequence encoding a xylitol phosphate dehydrogenase (XPDH) enzyme having a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33, wherein xylitol is produced by fermentation of the substrate by the engineered yeast.

19. The method according to claim 18, wherein the engineered yeast cell is a Moniera polnis cell.

20. The fermentation temperature is 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C or 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－ (L·h), the method according to any one of claims 17-19.

21. Xylitol is produced at a rate of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, or at least 1.0 gL －１ h －１ The method according to any one of claims 17-20.

22. When the fermentation is carried out at 35°C for 96 hours, the xylitol production is at least 10, 20, 30, 40, 50, 75, or 100 g / L, the method according to any one of claims 17-21.

23. The method according to any one of claims 17-22, wherein the production of erythritol is reduced as compared to an equivalent fermentation run using equivalent yeast cells lacking the exogenous polynucleotide sequence.

24. The method according to any one of claims 17 to 23, wherein when the fermentation is carried out at 35°C for 96 hours, the production of erythritol is less than 50 g / L, less than 40 g / L, less than 30 g / L, or less than 20 g / L. **Claim 25** The method according to any one of claims 17 to 24, wherein glycerol production is reduced as compared to an equivalent fermentation run using equivalent yeast cells lacking the exogenous polynucleotide sequence. **Claim 26** The method according to any one of claims 17 to 25, wherein ethanol production is reduced as compared to an equivalent fermentation run using equivalent yeast cells lacking the exogenous polynucleotide sequence.

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