Genetically Modified Yeast and Fermentation Method for the Production of Polyol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing polyols and characterized by overexpression of an enzyme having ribulose 5-phosphate epimerase (RPE) activity. For example, the cells may be Moniliella polnis cells, and the native RPE enzyme comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical sequence to at least one of SEQ ID NOs: 179 and 180.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 364,382, filed May 9, 2022, which is hereby incorporated by reference in its entirety.

[0002] (Reference to a Sequence Listing Submitted via the Patent Center) The contents of the sequence list XML file "PT - 1386 - WO - PCT.xml" created on May 4, 2023, and electronically submitted with this application through the Patent Center, which is 512,801 bytes in size, 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, dietary supplements, 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 used on a large scale commercially 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] In the development of microbial-based fermentation strategies for xylitol production, the production of metabolic pathway intermediates and alternative fermentation products are important considerations. For example, metabolic pathways active in xylitol production may overlap with those for the production of arabitol, erythritol, ribitol, etc. Intermediates and products have their own uses and markets that make their fermentation commercially relevant. Accordingly, provided herein are genetically modified yeasts and fermentation methods for the production of polyols including xylitol, arabitol, and ribitol.

Summary of the Invention

[0005] The present disclosure provides a genetically engineered yeast cell capable of producing a polyol, the genetically engineered yeast cell comprising a genetic modification that results in overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity. The yeast cell can 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 Moniliella. The yeast cell may be Moniliella polnis cells, and the native RPE enzyme comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 179 and 180. The RPE activity in the genetically engineered yeast cell may be higher than the RPE activity in an equivalent cell lacking the genetic modification. When the engineered cell is used in a fermentation process in the presence of dextrose, the yield of the polyol can be increased compared to the yield of the polyol in an equivalent fermentation process using an equivalent cell lacking the genetic modification.

[0006] The genetic modification may include substitution of the native RPE gene promoter with a heterologous or artificial promoter. The heterologous or artificial promoter may be selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139). The genetic modification may include addition of an exogenous polynucleotide encoding a native RPE enzyme such that the genetically engineered cell contains at least one additional copy of the sequence encoding the RPE enzyme.

[0007] The cell may be capable of producing ribitol and contains an exogenous polynucleotide sequence encoding an enzyme having ribulose-5-phosphate reductase activity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 13, 34, 35, 36, 37, 38, and 39.

[0008] The cell may be able to produce xylitol and contains an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 12-15, 28-31, and 33.

[0009] The cell may be able to produce xylitol and contains an exogenous polynucleotide sequence encoding a sugar phosphatase enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 188 and 189. The cell may further contain an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 190, 191, and 192.

[0010] The cell may be able to produce arabinitol and comprises (i) an exogenous polynucleotide sequence encoding an arabinitol-phosphate dehydrogenase (APDH) enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11, and / or (ii) an exogenous polynucleotide sequence encoding an arabinitol 2-dehydrogenase (ARD2DH) enzyme comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 193-197.

[0011] Any of the exogenous polynucleotide sequences may be operably linked to a heterologous or artificial promoter. The heterologous or artificial promoter may be selected from the group consisting of 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). Any of the exogenous polynucleotide sequences may be integrated into the genome of the yeast cell at a locus selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdIIA locus, and the gpdIIB locus.

[0012] The present disclosure also provides a method for producing a polyol using the engineered cells described herein, the method comprising contacting a substrate comprising dextrose with the engineered cells described herein, wherein fermentation of the substrate by the engineered cells produces the polyol. The present disclosure also provides a method for producing a polyol (e.g., xylitol, arabinitol, ribitol), the method comprising contacting a substrate comprising dextrose with an engineered yeast cell comprising a genetic modification that results in overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity. The cell may be a Moniliella cell, and the native RPE enzyme comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 179 and 180. The fermentation temperature may be 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C or thereabouts. The volumetric oxygen uptake rate (OUR) can 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). The polyol can be produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g / L -1 h -1 . When the fermentation is carried out at 35°C for 96 hours, the polyol production can be at least 20, 30, 50, 75, or 100 g / L. The rate and / or yield of the polyol can be increased compared to an equivalent fermentation run using equivalent yeast cells lacking the genetic modification. The concentration of dextrose can be at least 100 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0014] The drawings generally, but not by way of limitation, illustrate various aspects discussed herein by way of example.

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DETAILED DESCRIPTION OF THE INVENTION

[0015] Here, certain aspects of the disclosed subject matter are specifically referred to, examples of which are illustrated in part in the accompanying drawings. It will be understood that the disclosed subject matter is described with the appended claims, but the illustrated subject matter is not intended to limit the disclosed subject matter to the claims.

[0016] In this document, the terms "a", "an", or "the" are used to include one or more, unless the context clearly indicates otherwise. The term "or" is used to refer to a non-exclusive "or", unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated herein by reference in their entirety as if each were individually incorporated by reference. Where there is a lack of consistency in 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.

[0017] Values expressed in a range format are to be construed flexibly, including not only the explicitly recited numerical values as the limits of the range, but also all individual numerical values or sub-ranges included within that range as if each were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" is to be construed to include 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.

[0018] 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).

[0019] The present disclosure relates to various recombinant cells engineered to produce polyols including, but not limited to, xylitol, arabitol, and ribitol. Generally, the recombinant cells described herein are capable of producing a polyol (e.g., xylitol, arabitol, and / or ribitol) and are characterized by overexpression of the ribulose 5-phosphatase epimerase (RPE) enzyme. The present disclosure further provides a fermentation method for producing the polyol from dextrose using the genetically engineered cells described herein.

[0020] 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 deserticola, 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.

[0021] 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. sintherismae, U. esculenta, U. neglecta, U. crus-galli, Ustilago avenae), Sporisorium (e.g., Sporisorium exsertum), Monographella (e.g., M. polynis, M. tomentosa, M. acetabutans, M. fonsecae, M. madida, M. megachiliensis, M. oedocephalis, M. nigrescens), Pseudozyma (e.g., Pseudozyma tsukubaensis), and Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trichosporonoides oedocephalis, 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 biotechnological 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).

[0022] 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., 5-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.

[0023] The yeast cells can be osmotolerant yeast cells. As used herein, "osmotolerant" refers to yeast that can grow and regenerate 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). The species and strains of osmotolerant yeast are known and described in the art and include many species of yeast 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).

[0024] The recombinant yeast cells may be recombinant Moniliella cells, e.g., Moniliella pollinis cells. Figure 1 shows the predicted native pentose phosphate and glycolytic pathways in Moniliella pollinis. 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).

[0025] 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 the exogenous polynucleotide of interest is integrated into a specific locus after recombination and the selectable marker can be expressed intracellularly. 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 nucleotricin 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.

[0026] 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.

[0027] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless explicitly indicated otherwise.

[0028] 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 the 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 K.S. lactis invertase gene becomes exogenous when introduced into S. cerevisiae.

[0029] As used herein, "native" 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 Moniliella polnis cell "Moniliella tomentosa var. polnis TCV364", described in U.S. Patent No. 6,440,712, which is hereby incorporated by reference in its entirety, and deposited 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 on March 28, 1997, is considered a wild-type Moniliella polnis cell.

[0030] 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 recited macromolecule. 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 the 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.

[0031] [Table 1]

[0032] Variants or sequences having substantial identity or homology with 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 sequence 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.

[0033] 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 BLAST (Basic Local Alignment Search Tool) version 2.2.31 software of the National Center for Biological Information (NCBI) 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 sequences: 100; Short query: Automatically adjust parameters for short input sequences; 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 adjust parameters for short input sequences; 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.

[0034] Polypeptide or polynucleotide sequence identity may be measured over the length of the entire defined polypeptide sequence, such as defined by a particular SEQ ID NO., or over a shorter length, such as a fragment obtained from a larger defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 consecutive residues. Such lengths are merely exemplary, and it is understood that the length over which the percent identity is measured may be described using any fragment length supported by the sequences shown in the specification, tables, figures or sequence listing.

[0035] 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.

[0036] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments contemplated herein may include conservative amino acid substitutions compared to the reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions compared to the 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.

[0037] 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. A DNA polynucleotide may be a cDNA (e.g., coding DNA) or a genomic DNA sequence (e.g., including both introns and exons).

[0038] A polynucleotide is said to encode a polypeptide if, in its natural 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.

[0039] One of ordinary skill 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 RPE polypeptide) can be codon-optimized for expression in a particular cell, including but not limited to a plant cell, a bacterial cell, a fungal cell, or an animal cell. 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 coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and human hands can also be used to generate degenerate coding sequences.

[0040] The recombinant cells described herein can include deletions or disruptions in one or more native genes. The term "deletion or disruption" refers to a coding region that has been completely removed (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 the expression product produced has significantly reduced activity (e.g., at least 75% reduction or at least 90% reduction), 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.).

[0041] 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 the 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 the exact gene integration at the selected locus.

[0042] Recombinant cells can have one or more copies of a given exogenous nucleic acid sequence that is integrated into the host chromosome and replicated along with the chromosome into which it is integrated. For example, yeast cells may be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide described herein, and the polynucleotide sequence encoding the polypeptide may be integrated into the host chromosome in one or more copies. Recombinant cells can contain multiple (two or more) copies of a given polynucleotide sequence encoding a polypeptide described herein. Recombinant cells can 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. Multiple copies of the polynucleotide sequence may all be integrated at a single locus or may be integrated at multiple loci.

[0043] The recombinant cells described herein can produce one or more polyols and are characterized by overexpression of ribulose 5-phosphate epimerase (RPE enzyme). In general, recombinant cells comprising overexpression of the RPE enzyme produce more polyols than equivalent cells lacking the RPE enzyme or lacking overexpression of the RPE enzyme. Recombinant cells comprising overexpression of the RPE enzyme may be able to produce arabitol, ribitol, xylitol, or combinations thereof.

[0044] The recombinant cells described herein may contain an exogenous polynucleotide encoding a native or exogenous RPE enzyme, or may have a genetic modification that results in overexpression of the native RPE enzyme. The RPE enzyme can be any suitable enzyme having ribulose 5-phosphate epimerase activity. As used herein, "ribulose 5-phosphate epimerase activity" and "RPE activity" are used interchangeably and refer to the ability to catalyze the conversion of ribulose-5-phosphate to xylulose-5-phosphate. The enzyme having RPE activity can be native to the host cell, or the RPE enzyme can be an exogenous RPE enzyme. For example, when the host organism is M. pollinis, the RPE enzyme can be an enzyme having a sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to at least one of SEQ ID NOs: 179 and 180. The recombinant cell can contain an exogenous polynucleotide encoding an RPE enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of SEQ ID NOs: 179 and 180. The recombinant cell can contain a genetic modification that increases the expression of an RPE enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 179 and 180. Examples of genetic modifications can include, but are not limited to, insertion of additional copies of the nucleic acid encoding native RPE into the cell, insertion of a constitutive promoter upstream of the coding region of the native RPE gene in the genome of the host cell, and / or modification of the promoter present upstream of the coding region of the native RPE gene in the genome of the host cell. Those skilled in the art will recognize that the expression of the native RPE gene can be increased by many methods known in the art and can appropriately select and apply such methods.

[0045] As used herein, "overexpression" refers to the expression level of a polypeptide that is higher than the expression level of the same polypeptide in equivalent cells in the absence of a genetic modification or an exogenous polynucleotide encoding the polypeptide.

[0046] The recombinant cells described herein are capable of producing xylitol and contain an exogenous polynucleotide sequence encoding the xylitol-phosphate dehydrogenase (XPDH) enzyme and a genetic modification that results in overexpression of the RPE 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 believed that a native phosphatase enzyme removes the phosphate to produce xylitol.

[0047] 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 ribavtilis soli, Hendrixia sporothermodurans, Clostridium fungisolvens, or Neobacillus cucumis. 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.

[0048] The recombinant cell can include 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 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.

[0049] 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.

[0050] 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 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.

[0051] 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 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.

[0052] The recombinant cell may contain a genetic modification that results in overexpression of RPE, and an exogenous polynucleotide that is or can be derived from an Alkalihalobacillus ligniniphilus gene encoding the amino acid sequence 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.

[0053] The recombinant cell may contain a genetic modification that results in overexpression of RPE and an exogenous polynucleotide that is or can be derived from a Chogaliibacillus soli gene encoding the amino acid sequence 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.

[0054] The recombinant cell may contain a genetic modification that results in overexpression of RPE, and an exogenous polynucleotide that is or can be derived from a Henneguya sp. gene encoding the amino acid sequence 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.

[0055] The recombinant cell may contain a genetic modification that results in overexpression of RPE, and an exogenous polynucleotide that is or can be derived from a Clostridium fungisolvens gene encoding the amino acid sequence 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.

[0056] The recombinant cell may contain a genetic modification that results in overexpression of RPE, and an exogenous polynucleotide that is or can be derived from a Neobacillus kocurii gene encoding the amino acid sequence 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.

[0057] The recombinant cells described herein are capable of producing xylitol and contain an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme and a genetic modification that results in overexpression of the RPE enzyme. The exogenous polynucleotide sequence can be an exogenous xylulose sugar phosphatase (XKS) gene.

[0058] The "xylulokinase gene" and the "XKS gene" are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide having xylulokinase activity. As used herein, "xylulokinase activity" refers to the ability to catalyze the conversion of xylulose-5-phosphate and ADP to xylulose and ATP. The XKS gene can be derived from any suitable source. For example, the XKS gene may be derived from Saccharomyces cerevisiae. 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 of the amino acid sequences of SEQ ID NOs: 188 and 189. Further description of recombinant cells that can produce xylitol and contain a polypeptide having xylulokinase activity is provided in U.S. Patent Application No. 63 / 364,363, filed May 9, 2022, which is hereby incorporated by reference in its entirety.

[0059] Recombination can include genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or can be derived from the Saccharomyces cerevisiae DOG1 phosphoglycosidase gene encoding the amino acid sequence of SEQ ID NO: 188. 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: 188.

[0060] 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 the Saccharomyces cerevisiae DOG2 phosphoglycosidase gene encoding the amino acid of SEQ ID NO: 189. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 189.

[0061] The recombinant cells described herein may comprise an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme, an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme, and a genetic modification that results in overexpression of the RPE enzyme. The exogenous polynucleotide sequence may be an exogenous xylitol dehydrogenase (XDH) gene.

[0062] The "xylitol dehydrogenase gene" and the "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" means xylitol and NAD + or NADP +Refers to the ability to catalyze the conversion to. The XDH gene can be derived from any suitable source. For example, the XDH gene can be derived from Pichia stipitis, Rhodobacteraceae bacteria, or Bemisia argentifolii. The XDH gene can encode an amino acid with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to at least one of the amino acid sequences of SEQ ID NO: 190, 191, or 192. Further description of recombinant cells capable of producing xylitol and containing a polypeptide having xylulokinase activity and a polypeptide having xylitol dehydrogenase activity is provided in U.S. Provisional Patent Application No. 63 / 364,363, filed on May 9, 2022, which is hereby incorporated by reference in its entirety.

[0063] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or can be derived from the cofactor-switch Pichia stipitis XDH gene encoding the amino acids of SEQ ID NO: 190. 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: 190.

[0064] The recombinant cell may include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or can be derived from the Rhodobacteraceae bacterium SDR family oxidoreductase gene encoding the amino acids of SEQ ID NO: 191. 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: 191.

[0065] The recombinant cell may comprise a genetic modification that results in overexpression of RPE and an exogenous polynucleotide that is or can be derived from the Bemisia argentifolii (Silverleaf Whitefly) ketose reductase (sorbitol dehydrogenase) gene encoding the amino acid sequence of SEQ ID NO: 192. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 192.

[0066] The recombinant cells described herein can produce arabitol and comprise an exogenous polynucleotide sequence encoding an arabitol 2-dehydrogenase (ARD2DH) enzyme and a genetic modification that results in overexpression of the RPE enzyme. The exogenous polynucleotide sequence can be an exogenous ARD2DH gene.

[0067] The "arabitol 2-dehydrogenase gene" and the "ARD2DH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having arabitol 2-dehydrogenase activity. As used herein, "arabitol 2-dehydrogenase activity" means D-arabitol and NAD + or NADP +Refers to the ability to catalyze the conversion to. Enzymes having arabinitol 2-dehydrogenase can be characterized under Enzyme Classification 1.1.1.250. The ARD2DH gene can be derived from any suitable source. For example, the ARD2DH gene can be derived from Beauveria bassiana, Pichia stipitis, Candida albicans, Quambalaria heveanensis, Candida maltosa. The ARD2DH gene can encode a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of the amino acid sequences of SEQ ID NOs: 193, 194, 195, 196, or 197. The ARD2DH gene can encode a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of the amino acid sequences of SEQ ID NOs: 194, 195, 196, or 197. Further description of recombinant cells capable of producing arabinitol and containing a polypeptide having arabinitol 2-dehydrogenase activity is provided in U.S. Patent Application No. 63 / 364,359, filed May 9, 2022, which is hereby incorporated by reference in its entirety.

[0068] The recombinant cell can include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or can be derived from the Beauveria bassiana ARD2DH gene encoding the amino acids of SEQ ID NO: 193. 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: 193.

[0069] 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 the Pichia stipitis ARD2DH gene encoding the amino acids of SEQ ID NO: 194. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 194.

[0070] 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 the Candida albicans ARD2DH gene encoding the amino acids of SEQ ID NO: 195. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 195.

[0071] The recombinant cell may comprise a genetic modification that results in overexpression of the RPE and an exogenous polynucleotide that is or can be derived from the Quoniella heveanensis ARD2DH gene encoding the amino acid sequence of SEQ ID NO: 196. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 196.

[0072] 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 the Candida maltosa ARD2DH gene encoding the amino acid of SEQ ID NO: 197. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 197.

[0073] The recombinant cells described herein can produce arabitol and comprise an exogenous polynucleotide sequence encoding an arabitol-phosphate dehydrogenase (APDH) enzyme and a genetic modification that results in overexpression of the RPE enzyme. The exogenous polynucleotide sequence may be an exogenous arabitol phosphate dehydrogenase gene. Following the production of arabitol 5-phosphate or arabitol 1-phosphate, it is believed that the native phosphatase enzyme removes the phosphate to produce arabitol.

[0074] The "arabitol-phosphate dehydrogenase gene" and the "APDH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having arabitol-phosphate dehydrogenase activity. As used herein, "arabitol-phosphate dehydrogenase activity" means the ability to catalyze the conversion of (i) xylulose 5-phosphate and NADPH or NADH to arabitol-1-phosphate and NADP + or NAD + and / or (ii) ribulose-5-phosphate and NADPH or NADH to arabitol-5-phosphate and NADP + or NAD + The APDH gene can be derived from any suitable source. For example, the ARDH gene can be derived from Lactobacillus salivarius cp400.

[0075] 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 the Lactobacillus salivarius cp400 gene encoding the amino acid of SEQ ID NO: 11. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0076] The recombinant cells described herein may be capable of producing ribitol and comprise an exogenous polynucleotide sequence encoding the ribulose-5-phosphate reductase (TarJ’) enzyme and a genetic modification that results in overexpression of the RPE enzyme. The exogenous polynucleotide sequence may be an exogenous ribulose-5-phosphate reductase gene. After the conversion of ribulose-5-phosphate to ribitol 5-phosphate, it is believed that a native phosphatase enzyme removes the phosphate to produce ribitol.

[0077] The “ribulose-5-phosphate reductase gene” and the “TarJ’ gene” are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having ribulose-5-phosphate reductase activity. As used herein, “ribulose-5-phosphate reductase activity” means ribulose-5-phosphate and NADPH or NADH to ribitol 5-phosphate and NADP + or NAD +Refers to the ability to catalyze the conversion to. The TarJ' gene can be derived from any suitable source. For example, the TarJ' gene can be derived from Clostridium difficile, Staphylococcus aureus, Staphylococcus aureus subsp. aureus 71193, Eubacterium ventriosum, Prevotella sp. D12, Lactobacillus plantarum EGD-AQ4, or uncultured Lachnospiraceae. The TarJ' gene can encode an amino acid with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to at least one of the amino acid sequences of SEQ ID NO: 13, 34, 35, 36, 37, 38, or 39.

[0078] The recombinant cell can include 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 acid of SEQ ID NO: 13. 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: 13.

[0079] The recombinant cell can include a genetic modification that results in overexpression of the RPE enzyme and an exogenous polynucleotide that is or can be derived from a Staphylococcus aureus gene encoding the amino acid of SEQ ID NO: 34. 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: 34.

[0080] 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 the Staphylococcus aureus subsp. aureus 71193 gene encoding the amino acid sequence of SEQ ID NO: 35. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 35.

[0081] 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 the Eubacterium ventriosum gene encoding the amino acid sequence of SEQ ID NO: 36. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 36.

[0082] The recombination may comprise a genetic modification that results in overexpression of the RPE and an exogenous polynucleotide that is or can be derived from the Porphyromonas sp. D12 gene encoding the amino acid of SEQ ID NO: 37. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37.

[0083] 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 the Lactobacillus plantarum EGD-AQ4 gene encoding the amino acid of SEQ ID NO: 38. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0084] 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 the uncultured Luminococcus species gene encoding the amino acid sequence of SEQ ID NO: 39. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 39.

[0085] 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), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86); 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132); translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133); modified TEFp (SEQ ID NO: 131); phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134); 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135); enolase promoter (ENO1p; SEQ ID NO: 136); asparagine synthetase promoter (ASNSp; SEQ ID NO: 137); 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138); and RPL16B (SEQ ID NO: 139), but are not limited thereto.

[0086] 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 may 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).

[0087] 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.

[0088] 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 linked. A vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be incorporated.

[0089] The present disclosure also provides a fermentation method for producing polyols (e.g., xylitol, ribitol, arabinitol) 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 a polyol (e.g., xylitol, ribitol, arabinitol). 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 the polyol from the fermentation broth. The fermentation process may be a fully aerobic or partially aerobic process.

[0090] 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.

[0091] 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.

[0092] 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 of the specific rates or ranges listed herein.

[0093] 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.

[0094] 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.

[0095] The fermentation process can be associated with various characteristics such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of the yeast used in the fermentation process and / or genetic modification of the yeast. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be regulated through a combination of yeast selection or modification and selection of fermentation process conditions.

[0096] The polyol production rate of the process can be at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 gL -1 h -1 It may be. The polyol mass yield of the method can be at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 50 percent, at least 55 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, or at least 85 percent. The final polyol titer of the method can be at least 5, 10, 20, 30, 50, 75, or 100 g / L.

[0097] 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

[0098] 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.

[0099] 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.

[0100] 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 second to last β-strand outside the Rossmann fold domain. Without wishing to be bound by any particular theory, an enzyme 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) is predicted to prefer the NAD cofactor by hydrogen bonding of aspartic acid to the hydroxyl group of NAD ribose. However, an enzyme 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 prefers 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 the 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.

[0101] 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 identities 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.

[0102] 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 NO: 12 and 34, TarJ' of SEQ ID NO: 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).

[0103] [Table 2]

[0104] [Table 3]

[0105] 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’Universite 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 to 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.

[0106] The indicated Moniliella polnis parent strain was treated with 0.6M MgSO 4The parent strain was first protoplasted by adding an enzyme mixture containing 7.5 g / L zymolase and 12.5 g / L Trichoderma harzianum lytic enzyme to the mycelial pellets of the parent strain, and then transformed with the indicated sequences. The protoplasts were then pelleted, washed with 0.6 M MgSO 4 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) to prepare. 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 the mixture 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.

[0107] The resuspended protoplasts were seeded onto 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 the integration of the indicated sequences. 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.

[0108] 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 derived 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.

[0109]

Table 4-1

[0110]

Table 4-2

[0111] Example 5 - Shake Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, 1-39a-f, 1-42a-f, 1-13a-f, and 1-15a-f (summarized in Table 4 above) were run in shake flasks.

[0112] 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.

[0113] 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.

[0114]

Table 5

[0115] 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 contain the transformed polynucleotide sequence but were not integrated into the ER1 locus.

[0116]

Table 6

[0117] 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.

[0118] 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 - 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 approximately 32 - 50 hours.

[0119] A 250 - mL baffled - free 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 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 fermentation results are reported in Table 7 and Figure 8.

[0120] As shown in Figure 8, sister strains 1-34c and 1-34d produced 15.8 and 18.6 g / L 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.

[0121]

Table 7

[0122] 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.

[0123] 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.

[0124] 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.

[0125]

Table 8 - 1

[0126]

Table 8 - 2

[0127] 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.

[0128] 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 approximately 32 - 50 hours.

[0129] 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 9 and Figure 9.

[0130] 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 sites in strains 1 - 2c and 1 - 2d.

[0131]

Table 9 - 1

[0132]

Table 9-2

[0133] 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.

[0134] 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.

[0135] 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 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.

[0136] PCR verification showed that the transformed polynucleotide sequence was present in the strains tested, but further analysis showed that in some strains, the sequence was not correctly integrated into the ER1 locus. Further analysis showed that strains 1-8c, 1-8d, and 1-41c contained the transformed polynucleotide sequence, but it was not integrated into the ER1 locus. Further analysis was inconclusive regarding the integration locus in strains 1-36a, 1-41b, 1-41e, and 1-20a-e.

[0137]

Table 10

[0138] 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.

[0139] 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 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 path length cuvette using a Genesys 20 spectrophotometer (Thermo Scientific). The seed cultures reached an OD600 of 15 - 20 in approximately 32 - 50 h.

[0140] 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.

[0141] 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 1-30c and 1-30d contained the transformed polynucleotide sequence, but that it was not integrated into the ER1 locus. Further analysis of the integration locus in strain 1-6c was inconclusive.

[0142]

Table 11-1

[0143]

Table 11-2

[0144] Example 11-2 Copy XPDH-Modified Moniliella polnis Strains Strain 1-1 was transformed as described in Example 4 using SEQ ID NO: 55 and SEQ ID NO: 177. SEQ ID NO: 177 contains (i) the 3' portion of the G418 selectable marker enabling marker (SEQ ID NO: 172); (ii) an expression cassette of an XPDH homolog from Clostridium difficile encoding the amino acid sequence of SEQ ID NO: 12 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140 (SEQ ID NO: 98); and (iii) the 3' flanking DNA (SEQ ID NO: 162) for targeted chromosomal integration into the ER1 locus. SEQ ID NO: 55 contains (i) the 5' flanking DNA (SEQ ID NO: 85) for targeted chromosomal integration into the ER1 locus, (ii) the expression cassette of SEQ ID NO: 98 of an XPDH homolog from Clostridium difficile encoding the amino acid sequence of SEQ ID NO: 12 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 5' portion of the G418 selectable marker (SEQ ID NO: 175). The transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the integration of two copies of the C. difficile XPDH sequence by colony PCR. The PCR-verified isolates were named strains 2-1a, 2-1b, 2-1c, 2-1d, and 2-1e.

[0145] Strain 1-1 was transformed as described in Example 4 using SEQ ID NO: 57 and SEQ ID NO: 178. SEQ ID NO: 178 contains (i) the 3' portion of the G418 selectable marker (SEQ ID NO: 172); (ii) an expression cassette of an XPDH homolog from Lactobacillus rhamnosus (SEQ ID NO: 100) encoding the amino acid sequence of SEQ ID NO: 14 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 3' flanking DNA (SEQ ID NO: 162) for targeted chromosomal integration into the ER1 locus. SEQ ID NO: 57 contains (i) the 5' flanking DNA (SEQ ID NO: 85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette of the XPDH homolog from Lactobacillus rhamnosus of SEQ ID NO: 100 encoding the amino acid sequence of SEQ ID NO: 14 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 5' portion of the G418 selectable marker (SEQ ID NO: 175). The transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35°C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the integration of two copies of the L. rhamnosus XPDH sequence by colony PCR. The PCR-verified isolates were named strains 2-2a, 2-2b, 2-2c, 2-2d, and 2-2e.

[0146] To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1-13c, 2-1a-e, and 2-2a-e were run in shake flasks.

[0147] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass 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.

[0148] 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 shown in Table 12.

[0149] 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 accurately targeted to the ER1 locus. Further analysis showed that strain 2 - 2e contains the transformed polynucleotide sequence but does not target the ER1 locus.

[0150]

Table 12

[0151] Example 12 - Genetically Modified Moniliella polnis Strains Strain 1-1 was transformed with SEQ ID NO: 186 and SEQ ID NO: 187 as described in Example 4. SEQ ID NO: 186 contained (i) the 5'-flanking DNA (SEQ ID NO: 85) for targeted chromosomal integration into the ER1 locus, (ii) the MpPYK1 promoter (SEQ ID NO: 86), (iii) the gene encoding the Staphylococcus aureus xylitol dehydrogenase of SEQ ID NO: 34, (iv) the Mp6PGD terminator (SEQ ID NO: 140), and (v) the 5' portion of the G418 resistance gene expression cassette (SEQ ID NO: 175). SEQ ID NO: 187 contained (i) the 3' portion of the G418 resistance gene expression cassette (SEQ ID NO: 172), (ii) the MpTEF2 terminator (SEQ ID NO: 150), (iii) the MpPGK1 promoter (SEQ ID NO: 135), (iv) the gene encoding the Saccharomyces cerevisiae DOG1 phosphatase of SEQ ID NO: 188, (v) the MpENO1 terminator (SEQ ID NO: 142), and (vi) the 3'-flanking gene (SEQ ID NO: 162) for targeted chromosomal integration into the ER1 locus. The transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35°C for at least 2 days until the transformants grew. The 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 isolate was named strain 3-1.

[0152] Table 13 below lists various Moniliera polnis strains, including information on the parental strain, the sequences into which the parental strain was transformed, and the characterization of the expression cassettes contained in the transformed sequences. The transformation fragment of SEQ ID NO: 181 contained, in order, the 5' ER3 flanking sequence (SEQ ID NO: 155), the MpPYK1 promoter (SEQ ID NO: 86), the gene encoding the M. polnis RPE2 polypeptide of SEQ ID NO: 180, the MpPYK terminator (SEQ ID NO: 146), and the 5' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 169). The transformation fragment of SEQ ID NO: 182 contained, in order, the 3' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 168), the MpTEF2 terminator (SEQ ID NO: 150), and the 3' ER3 flanking sequence (SEQ ID NO: 165). The transformation fragment of SEQ ID NO: 183 contained, in order, the 3' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 168), the Mp6PGD promoter (SEQ ID NO: 130), the gene encoding the M. pollinis RPE1 polypeptide of SEQ ID NO: 179, the Mp6PGD terminator (SEQ ID NO: 140), and the 3' ER3 flanking sequence (SEQ ID NO: 165). The transformation fragment of SEQ ID NO: 184 contained, in order, the 3' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 168), the Mp6PGD promoter (SEQ ID NO: 130), the gene encoding the M. pollinis RPE2 polypeptide (SEQ ID NO: 180), the Mp6PDG terminator (SEQ ID NO: 140), and the 3' ER3 flanking sequence (SEQ ID NO: 165). The transformation fragment of SEQ ID NO: 185 contained, in order, the 5' ER3 flanking sequence (SEQ ID NO: 155), the MpTEF1 promoter (SEQ ID NO: 133), and the 5' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 169).

[0153] The Moniliella polnis parental strain shown was transformed with the sequence shown as described in Example 4 using either zeocin or G418 selection corresponding to the selection marker shown. The resulting transformants were evaluated by colony PCR for the integration of the sequence shown. The PCR-verified isolates were then designated as the strain numbers shown. In some cases, one or more PCR-verified isolates, such as "sister" isolates, are indicated by letters following the strain number. For example, strain 3-2 has four sister isolates, strains 3-2a, 3-2b, 3-2c, and 3-2d (collectively 3-2a-d).

[0154]

Table 13

[0155] Example 13 - Shake flask fermentation assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-1, 3-1, 3-2a-c, 3-3a-c, 3-4a-c, 3-5a-c, and 3-6a-c were run in shake flasks.

[0156] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass 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.

[0157] 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 at 48, 72, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography equipped with a refractive index detector. The fermentation results are reported in Table 14 as well as Figures 14 and 15.

[0158]

Table 14

[0159] Example 14 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, erythritol, and ethanol production, strains 1-1, 1-13c, 1-15a, 3-7a-f, and 3-8a-f were run in shake flasks.

[0160] 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.

[0161] 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 at 48, 72, and 96 hours of incubation. 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 15 and Figures 12 and 13.

[0162]

Table 15

[0163] Example 15 - Shake Flask Fermentation Assay To evaluate glucose consumption and ribitol, xylitol, glycerol, erythritol, and ethanol production, strains 1-13c, 1-29c, 1-34d, 1-30b, 1-31d, 1-32d, 1-14d, 1-16a, 2-1c, 2-2b, 3-8b, 3-9a-d, 3-10a-e, 3-11a-e, 3-12a-e, 3-13a-e, 3-14a-e, 3-15a-e, 3-16a-e, and 3-17a-e were run in shake flasks.

[0164] 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. 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.

[0165] 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 at 48, 72, and 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography equipped with a refractive index detector. The fermentation results are reported in Tables 16, 17, and 18 and Figures 16 - 21.

[0166] The yield was calculated as the ratio of the product (i.e., xylitol) to the consumed substrate (i.e., the difference between one time point and a second time point) and expressed as a percentage.

[0167]

Table 16

[0168]

Table 17

[0169]

Table 18 - 1

[0170]

Table 18 - 2

[0171]

Table 18 - 3

[0172]

Table 18 - 4

[0173]

Table 18-5

[0174]

Table 18-6

[0175] Example 16 - Genetically Engineered Monilinia Polynes Strain Strain 4-1a-e To test the activity of the RPE enzyme in the wild-type Monilinia Polynes background, strain 1-1 was transformed with SEQ ID NO: 181 and SEQ ID NO: 182 using the transformation method outlined in Example 4. The transformation fragment of SEQ ID NO: 181 contained, in order, the 5' ER3 flanking sequence (SEQ ID NO: 155), the MpPYK1 promoter (SEQ ID NO: 86), the gene encoding the M. Polynes RPE2 polypeptide of SEQ ID NO: 180, the MpPYK terminator (SEQ ID NO: 146), and the 5' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 169). The transformation fragment of SEQ ID NO: 182 contained, in order, the 3' portion of the zeocin resistance gene expression cassette (SEQ ID NO: 168), the MpTEF2 terminator (SEQ ID NO: 150), and the 3' ER3 flanking sequence (SEQ ID NO: 165). Transformants were selected on PDA + zeocin selection plates and incubated at 35°C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + zeocin plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for the integration of the RPE2 gene sequence. Sister isolates verified by PCR were named strains 4-1a, 4-1b, 4-1c, and 4-1d.

[0176] Strain 4-2 Using the transformation method outlined in Example 4, strain 1-1 was transformed with SEQ ID NO: 57 and SEQ ID NO: 199. SEQ ID NO: 199 contains the 3’ portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the polynucleotide encoding RPE of SEQ ID NO: 180, the MpPYK1 terminator (SEQ ID NO: 86), and the 3’ ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO: 57 contains (i) the 5’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO: 85); (ii) an expression cassette encoding the amino acid sequence of SEQ ID NO: 14 from Lactobacillus rhamnosus XPDH homolog under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 5’ portion of the G418 selection marker (SEQ ID NO: 175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the integration of the L. rhamnosus XPDH sequence and the M. polnis RPE2 sequence by colony PCR. The PCR-verified isolates were designated as strain 4-2.

[0177] Strain 4-3 Using the transformation method outlined in Example 4, strain 1-1 was transformed with SEQ ID NO: 57 and SEQ ID NO: 199. SEQ ID NO: 199 contains the 3’ portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the polynucleotide encoding RPE of SEQ ID NO: 180, the MpPYK1 terminator (SEQ ID NO: 86), and the 3’ ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO: 57 contains (i) the 5’ flanking DNA (SEQ ID NO: 85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette of an XPDH homolog derived from Lactobacillus rhamnosus of SEQ ID NO: 100, encoding the amino acid sequence of SEQ ID NO: 14, under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 5’ portion of the G418 selection marker (SEQ ID NO: 175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the integration of the L. rhamnosus XPDH sequence and the M. polnis RPE2 sequence by colony PCR. The PCR-verified isolates were designated as strain 4-2.

[0178] Strain 4-4a-e Using the transformation method outlined in Example 4, strain 1-1 was transformed with SEQ ID NO: 57 and SEQ ID NO: 200. SEQ ID NO: 200 contains the 3’ portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the gene encoding M. polnis RPE of SEQ ID NO: 179, the MpPYK1 terminator (SEQ ID NO: 86), and the 3’ ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO: 57 contains (i) the 5’ flanking DNA (SEQ ID NO: 85) for targeted chromosomal integration into the ER1 locus; (ii) an expression cassette of an XPDH homolog derived from Lactobacillus rhamnosus of SEQ ID NO: 100 encoding the amino acid sequence of SEQ ID NO: 14 under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 5’ portion of the G418 selection marker (SEQ ID NO: 175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the integration of the L. rhamnosus XPDH sequence and the M. polnis RPE2 sequence by colony PCR. The PCR-verified isolates were designated as strain 4-2.

[0179] Strain 4-5a-d Using the transformation method outlined in Example 4, strain 1-1 was transformed with SEQ ID NO: 57 and SEQ ID NO: 199. SEQ ID NO: 199 contains the 3’ portion of the G418 resistance marker (SEQ ID NO: 172), the MpPGK1 promoter (SEQ ID NO: 135), the polynucleotide encoding RPE of SEQ ID NO: 180, the MpPYK1 terminator (SEQ ID NO: 86), and the 3’ ER1 flanking sequence (SEQ ID NO: 162). SEQ ID NO: 57 contains (i) the 5’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO: 85); (ii) an expression cassette encoding the amino acid sequence of SEQ ID NO: 14 from Lactobacillus rhamnosus XPDH homolog under the control of the PYK1 promoter of SEQ ID NO: 86 and the PGD terminator of SEQ ID NO: 140; and (iii) the 5’ portion of the G418 selection marker (SEQ ID NO: 175). Transformants were selected on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 35 °C for at least 2 days until the transformants grew. The obtained transformants were streaked on PDA + geneticin (G418) plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the integration of the L. rhamnosus XPDH sequence and the M. polnis RPE2 sequence by colony PCR. The PCR-verified isolates were named strain 4-3.

[0180] Strains 4-2, 4-3, and 4-5a-d were all products of the transformation of strain 1-1 with SEQ ID NO: 57 and 199, but these were the results of three different transformation reactions. They were all PCR-verified, but they were not sister isolates for a single transformation reaction and thus had different strain numbers.

[0181] Example 17 - Shake Flask Fermentation Assay To evaluate glucose consumption and the production of ribitol, xylitol, glycerol, and ethanol, strains 1-1 and 1-4a-d were run in shake flasks.

[0182] 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 approximately 32 - 50 hours.

[0183] A 250 mL baffled flask containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of the seed culture to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture at 48, 72, 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 19 and Figure 22.

[0184] The results demonstrate that overexpression of the RPE2 enzyme in wild - type Moniliella polnis leads to a decrease in erythritol production. The final erythritol titer decreased by 25 - 50%. The erythritol yield decreased from 32% to 20 - 25%. The glucose consumption rate also decreased.

[0185]

Table 19

[0186] Example 18 - Shake - flask fermentation assay To evaluate glucose consumption and ribitol, xylitol, glycerol, and ethanol production, strains 1-1, 2-2b, 4-2, 4-3, 4-4a-e, and 4-5a-d were run in shake flasks.

[0187] 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 250 mL baffled flasks without baffles. 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 h.

[0188] A 250 mL baffled flask without baffles containing 20 mL of production medium (Table 5) was inoculated with 0.4 mL of the seed culture to form a production culture. The production culture was incubated at 35 °C and 250 rpm. Samples were taken from the production culture at 48, 72, and 96 h of incubation. 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 20 and 21 and Figure 23.

[0189] The results show that both RPE1 (SEQ ID NO: 179) and REP2 (SEQ ID NO: 180) from M. polnis increase the titer and yield of xylitol production. Overexpression of the enzyme resulted in xylitol yields of 20.9% and 18.4%, respectively, compared to a yield of 10.8% in strain 2-2b lacking RPE overexpression and a yield of 0% in wild-type strain 1-1.

[0190]

Table 20-1

[0191]

Table 20-2

[0192]

Table 21

Claims

1. A genetically engineered yeast cell capable of producing a polyol, wherein the genetically engineered yeast cell comprises a genetic modification that results in overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity, a genetically engineered yeast cell capable of producing a polyol.

2. 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 subphylum Ustilaginomycotina.

4. The yeast cell according to any one of claims 1 to 3, wherein the yeast cell is selected from the group consisting of Trichosporonoides megachiliensis, Trichosporonoides oenocephalus, Trichosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomyces, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium.

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

6. The yeast cell according to any one of claims 1 to 5, wherein the RPE activity in the genetically engineered yeast cell is higher than the RPE activity in an equivalent cell lacking the genetic modification.

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

8. The yeast cell according to any one of claims 1 to 7, wherein the genetic modification comprises substitution of the native RPE gene promoter with a heterologous or artificial promoter.

9. The yeast cell according to claim 8, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139).

10. The yeast cell according to any one of claims 1 to 9, wherein the genetic modification comprises the addition of an exogenous polynucleotide encoding the native RPE enzyme such that the genetically engineered cell contains at least one additional copy of the sequence encoding the RPE enzyme.

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

12. The yeast cell according to any one of claims 1 to 10, wherein the cell is capable of producing xylitol and comprises an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) 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.

13. The yeast cell according to any one of claims 1 to 10, wherein the cell is capable of producing xylitol and comprises an exogenous polynucleotide sequence encoding a sugar phosphatase 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: 188 and 189.

14. The yeast cell according to claim 11, wherein the cell further comprises an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) 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: 190, 191, and 192.

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

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

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

18. The yeast cell according to claim 16 or 17, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: 86), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 130), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p; SEQ ID NO: 132), translation elongation factor 1 promoter (TEFp; SEQ ID NO: 133), modified TEFp (SEQ ID NO: 131), phosphoglucomutase 1 promoter (PGM1p; SEQ ID NO: 134), 3-phosphoglycerate kinase promoter (PGK1p; SEQ ID NO: 135), enolase promoter (ENO1p; SEQ ID NO: 136), asparagine synthetase promoter (ASNSp; SEQ ID NO: 137), 50S ribosomal protein L1 promoter (RPLAp; SEQ ID NO: 138), and RPL16B (SEQ ID NO: 139).

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

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

21. A method for producing a polyol (e.g., xylitol, arabinitol, ribitol), the method comprising contacting a substrate containing dextrose with a genetically engineered yeast cell comprising a genetic modification that results in overexpression of a native enzyme having ribulose-5-phosphate epimerase (RPE) activity.

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

23. The 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), and the method according to any one of claims 20 to 22.

24. The method according to any one of claims 20 to 23, wherein the polyol is produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g / L －１ h －１ ​ **Claim 25** The method according to any one of claims 20 to 24, wherein when the fermentation is carried out at 35 °C for 96 hours, the polyol production is at least 20, 30, 50, 75, or 100 g / L. **Claim 26** The method according to any one of claims 20 to 25, wherein the rate and / or yield of the polyol is increased as compared to an equivalent fermentation run using equivalent yeast cells lacking the genetic modification. **Claim 27** The method according to any one of claims 20 to 26, wherein the concentration of dextrose is at least 100 g / L.

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