Genetically modified yeast and fermentation process for production of ribitol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing ribitol. The genetically engineered yeast cell may comprise an exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NO: 13, 34, 35, 36, 37, 38, and 39.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 364,370, filed May 9, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listing submitted via Patent Center

[0004] The contents of the sequence listing XML file named "PT-1348-WO-PCT.xml", which was created on May 4, 2023 and electronically submitted via the Patent Center with this application, and is 448,927 bytes in size, are incorporated herein by reference in its entirety. Background Art

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

[0006] In the development of the fermentation strategy based on microorganisms for the production of xylitol, the production of metabolic pathway intermediates and alternative fermentation products is an important consideration. For example, metabolic pathways active in xylitol production may overlap with metabolic pathways for the production of arabitol, erythritol, ribitol, etc. Intermediates and products have their own uses and markets, which makes their fermentation commercially relevant. Therefore, this paper provides genetically modified yeast and fermentation methods for the production of ribitol. Summary of the invention

[0007] The present disclosure provides a genetically engineered yeast cell capable of producing ribitol, the engineered yeast cell comprising an exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase. The ribulose-5-phosphate reductase may comprise a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 13, 34, 35, 36, 37, 38 and 39. The yeast cell may be an osmotic yeast cell. The yeast cell may be a cell of the subdivision Ustilaginomycotina. The yeast cell may be selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium. The yeast cell may be a yeast cell of the genus Moniliella.

[0008] The present disclosure also provides a genetically engineered Mycospore cell capable of producing ribitol, the engineered Mycospore cell comprising an exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase, the 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: 13, 34, 35, 36, 37, 38 and 39.

[0009] The ribulose-5-phosphate reductase may have a sequence at least 85% identical to at least one of SEQ ID NOs: 34-39. The ribulose-5-phosphate reductase may have a sequence at least 90% identical to at least one of SEQ ID NOs: 34-39. The ribulose-5-phosphate reductase may comprise a sequence 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: 34, 36, 37, 38, and 39, and the yeast may be capable of producing at least 1 g / L, 2 g / L, 5 g / L, 7.5 g / L, or 10 g / L of ribitol.

[0010] Yeast cell can be Moniliella pollinis cell.When being used in fermentation process for at least 96 hours under the presence of dextrose, yeast cell can produce ribitol with a titer of at least 20g / L, 30g / L, 50g / L, 75g / L or 100g / L.The erythritol output of yeast cell can be reduced relative to the erythritol output in the equivalent yeast cell lacking exogenous polynucleotide sequence.Exogenous polynucleotide sequence can be integrated into the genome of yeast cell at the locus selected from ER1 locus, ER3 locus, PDC1 locus, pyrF locus, TRP3 locus, gpdIIA locus and gpdIIB locus.Exogenous polynucleotide sequence can be operably connected with heterologous promoter or artificial promoter.Promoter can be constitutive promoter. The 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).

[0011] The present disclosure also provides a method for producing ribitol 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 yeast produces ribitol. The present disclosure also provides a method for producing ribitol, the method comprising contacting a substrate comprising dextrose with an engineered yeast cell, the engineered yeast cell comprising an exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase, the ribulose-5-phosphate reductase 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: 34, 36, 37, 38 and 39, wherein fermentation of the substrate by the engineered yeast produces ribitol. The fermentation temperature may be 25°C to 45°C, 30°C to 40°C or 32°C to 37°C or between them. The volumetric oxygen uptake rate (OUR) may be between 0.5 mmol O2 / (L·h) and 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) and 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) and 30 mmol O2 / (L·h), 3 mmol O2 / (L·h) and 25 mmol O2 / (L·h), 4 mmol O2 / (L·h) and 20 mmol O2 / (L·h), or 5 mmol O2 / (L·h) and 15 mmol O2 / (L·h). Ribitol may be at least 0.2 g L -1 h -1 , 0.3 g L - 1 h -1 , 0.5 g L -1 h -1 , 0.75 g L -1 h -1 or at least 1.0 g L -1 h -1When the fermentation is run at 35°C for 96 hours, the ribitol production may be at least 20 g / L, 30 g / L, 50 g / L, 75 g / L or 100 g / L. The erythritol production may be reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking an exogenous polynucleotide sequence. When the fermentation is run at 35°C for 96 hours, the erythritol production may be less than 40 g / L, 30 g / L or less than 20 g / L. The glycerol production may be reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking an exogenous polynucleotide sequence. The ethanol production may be reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking an exogenous polynucleotide sequence. The ribulose-5-phosphate reductase may comprise a sequence 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: 34, 36, 37, 38, and 39, and the yeast produces at least 1 g / L, 2 g / L, 5 g / L, 7.5 g / L, or 10 g / L ribitol when the fermentation is run at 35°C for 96 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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 Office upon request and payment of the necessary fee.

[0013] The drawings illustrate generally by way of example and not by way of limitation various aspects described herein.

[0014] Figure 1 The native pentose phosphate pathway (dashed line and arrows) and the native glycolytic pathway (solid line and arrows) in the yeast Mycorrhizal sp. are shown.

[0015] Figure 2 Diversity in the galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) sequence space is shown.

[0016] Figure 3 Structural features of the NAD or NADP binding pocket located at the +23 amino acids from the characteristic GXGXXG motif of the XPDH enzyme (SEQ ID NO: 133) are shown.

[0017] Figure 4 The diversity in ribulose-5-phosphate reductase sequence space is shown.

[0018] Figure 5 The in vitro activities of TarJ' and XPDH enzymes as outlined in Example 3 are shown.

[0019] Figure 6Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-1, 1-13a-f, and 1-15a-f as outlined in Example 5. Data markers report the concentration of xylitol (g / L).

[0020] Figure 7 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, and 1-39a-f as outlined in Example 5. Data markers report the concentration of xylitol (g / L).

[0021] Figure 8 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e as outlined in Example 6. Data markers report the concentration of xylitol (g / L).

[0022] Fig. 9 Shown are the concentrations (g / L) of erythritol, ribitol, arabitol, and xylitol metabolites from 96 hour shake flask fermentations of strains 1-13c, 1-12a-e, 1-14a-e, and 1-16a-e as outlined in Example 8. Data markers report the concentration (g / L) of xylitol (strains 1-13c, 1-14a-e, and 1-16a-e) or arabitol (strains 12a-e).

[0023] Fig.10 Shown are the concentrations (g / L) of erythritol, ribitol, and xylitol metabolites from 96 hour shake flask fermentations of strains-13c, 1-36a-e, and 1-40a-e as outlined in Example 9. Data markers report the concentration of xylitol (g / L).

[0024] Fig.11 Shown are the concentrations of erythritol, ribitol, and xylitol metabolites (g / L) from 96 hour shake flask fermentations of strains 1-30a-e, 1-31a-e, 1-32a-e, and 1-13c as outlined in Example 10. Data markers report the concentration of xylitol (g / L). DETAILED DESCRIPTION

[0025] Reference will now be made in detail to certain aspects of the presently disclosed subject matter, examples of which are partially illustrated in the accompanying drawings.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0026] In this document, the terms "a", "an" or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of an inconsistency between the usages of this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0027] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

[0028] Unless expressly stated, ppm (parts per million), percentages and ratios are based on weight. Percentages based on weight are also referred to below as wt% or (wt)%.

[0029] The present disclosure relates to various recombinant cells engineered to produce ribitol. In general, the recombinant cells described herein have an active pentose phosphate pathway and are characterized by the expression of exogenous ribulose-5-phosphate reductase. The present disclosure also provides a fermentation method for producing ribitol from dextrose using the genetically engineered cells described herein.

[0030] In general, the recombinant cells described herein are yeast cells. As used herein, "yeast" refers to eukaryotic unicellular microorganisms classified as members of the kingdom Fungi. Yeast is a unicellular organism that evolved from a multicellular ancestor, some of which retain multicellular characteristics, such as forming connected strings of budding cells known as pseudohyphae or false hyphae. Yeast cells may also be referred to as yeast-like cells in the art, and as used herein, "yeast cells" encompass both yeast and yeast-like cells. Suitable yeast and yeast-like host cells for modification can include, but are not limited to, Saccharomyces cerevisiae, Komagataella sp., Kluyveromyces (e.g., Kluyveromyces lactis, Kluveromyces marxianus), oleaginous yeast, Issatchenkia orientalis, Pichia galeiformis, Pichia YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnolii, Candida ethanolica), Pichia deserticola, Pichia membranifadens, Pichia fermentans (e.g., fermentans), Aspergillus, Trichoderma, Myceliphthora thermophila, spores (e.g., spores yeast), Pfaffia, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., Trichosporonoides megafacies, Trichosporonoides globosum, Trichosporonoides nigrofacies), Pseudomonas tsukubaensis, Trichosporon variabilis, Penicillium, and Torula yeast. One of ordinary skill will appreciate that the requirements for selecting suitable yeast cells and recombinant yeast cells of the present disclosure are not limited to those explicitly listed herein. Methods for genetic engineering of yeast cells are known and described in the art, and one of ordinary skill will appreciate the methods necessary to transform and engineer suitable yeast cells.

[0031] Suitable yeast cells can be Basidiomycota and Ustilago. Suitable yeasts of the Ustilago include, but are not limited to, Ustilago (e.g., U. cynodontis, U. maydis, U. sphaerogena, U. cordal, U. scitaminea, U. coicis, U. syntherismae, U. esculenta, U. neglecta, U. crus-galli, Ustilago avenae), Sporisorium (e.g., Sporisorium spores), Sporisorium spores. exsertum), spores (e.g., spores yeast, M. tomentosa, M. acetoabutans, M. fonsecae, M. madida, M. megachiliensis, M. ocedocephalis, M. nigrescens), and Pseudozyma (e.g., Pseudozyma tsukubae), and Trichosporon (e.g., Trichosporon megatus, Trichosporon sphericalus, Trichosporon nigrescens). Yeasts of the subdivision Ustilagomyces are known and described in the art as potential production organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol, as well as other valuable biotechnological applications.See, for example, Geiser et al. Biotechnol, 2013, 97(8): 3253-65), Guevarra et al. ("Accumulation of itaconic, 2-hydroxyparaconic, itataric, and malic acids by strains of the genus Ustilago, Agric.Biol.Chem., 1990, 54(9), 2353-2358) and Moon et al. ("Biotechnological productionof erythritol and its applications,” Appl Microbiol Biotechnol, 2010,86:1017-1025).

[0032] Suitable yeast cells will have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein, "active pentose phosphate pathway" refers to the expression of one or more functional enzymes that together convert glucose-6-phosphate, NADP + Or NAD+ and water are converted into NADPH or NADH, CO2 and ribulose-5-phosphate. Continuing in the non-oxidative stage, the pathway can also produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway can produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, combinations thereof, etc. The active pentose phosphate pathway can be natural to yeast cells or can be introduced into yeast cells by genetic engineering.

[0033] Yeast cells can be osmotic yeast cells. As used herein, "osmotic resistance" refers to yeast that can grow and reproduce under conditions of high molar osmotic pressure concentrations, such as at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v) or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), at least 15% (w / v) sodium chloride. The species and strains of osmotic yeasts are known and described in the art, including many types of yeasts used in industrial fermentation processes. Similarly, methods for determining yeast osmotic resistance are known and described in the art. See, for example, Tiwari, S. et al. ("Nectaryeast community of tropical flowering plants and assessment of theirosmotolerance and xylitol-producing potential," Current Microbiology, 2022, 79: 28).

[0034] The recombinant yeast cell can be a recombinant Myristica spp. cell, such as a Myristica spp. yeast cell. Figure 1 The predicted native pentose phosphate and glycolytic pathways in the yeast of the spores are shown. The spores have been previously used in the fermentation production of erythritol, and methods for genetic modification and fermentation of the spores are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi," 2017, Microb Cell Fact, 16: 168).

[0035] Various plasmids and methods for transforming Mycelium are also described in the examples below. For example, Mycelium can be transformed using a bipartite polynucleotide sequence wherein, after recombination, the exogenous polynucleotide of interest is integrated at a specific locus and a selection marker is expressible in the cell. Suitable selection markers are known and used in the art. Selectable markers may include, but are not limited to, amdS (e.g., split into a 3' portion of SEQ ID NO: 167 and a 5' portion of SEQ ID NO: 174), a G418 resistance gene (e.g., split into a 3' portion of SEQ ID NO: 172 and a 5' portion of SEQ ID NO: 175), a zeocin resistance gene (e.g., split into a 3' portion of SEQ ID NO: 168 and a 5' portion of SEQ ID NO: 169), nourseothricin N-acetyltransferase (NAT) (e.g., split into a 3' portion of SEQ ID NO: 171 and a 5' portion of SEQ ID NO: 170), and an invertase gene (SUC2) (e.g., a 3' portion of SEQ ID NO: 173 and a 5' portion of SEQ ID NO: 176).

[0036] The recombinant cells described herein comprise one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve the fermentation of glucose to ribitol by the recombinant cell.

[0037] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless expressly stated otherwise.

[0038] As used herein, "exogenous" refers to genetic material or its expression product that originates from outside the host organism. For example, the exogenous genetic material or its expression product can be a modified form of genetic material native to the host organism, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component. For example, when introduced into S. cerevisiae, the K. lactis invertase gene is exogenous.

[0039] As used herein, "native" refers to genetic material or its expression products found within the genome of a wild-type cell of a host cell, except for inter-individual mutations that do not affect function or expression. For the purposes of this application, the Moniliella tomentosa var pollinis TCV364 yeast cell described in US 6,440,712 is considered to be a wild-type Moniliella tomentosa yeast cell, which is incorporated herein by reference in its entirety and deposited at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) under the Budapest Treaty on March 28, 1997 under the number MUCL40385.

[0040] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the overall primary, secondary, tertiary and quaternary amino acid sequences and structures necessary to give the macromolecule its function and properties. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (either independently or as part of a biosynthetic pathway) is understood to include the cofactors, coenzymes and metals necessary for the enzyme to function properly. An overview of amino acids and their three letter and one letter symbols as understood in the art is provided in Table 1. The amino acid names, three letter symbols and one letter symbol are used interchangeably herein.

[0041] Table 1: Amino Acid Three-Letter and One-Letter Symbols

[0042] Amino Acids Three letter symbols A letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0043] Variants or sequences having substantial identity or homology to the polypeptides described herein can be used to implement the disclosed recombinant cells, compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, the polypeptide sequence can be modified but still retains the ability to exhibit the desired activity. Typically, the variant or modified sequence can include greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the wild-type, naturally occurring polypeptide sequence or to a variant polypeptide as described herein.

[0044] As used herein, the phrases "% sequence identity", "% identity" and "percent identity" are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well known. The generation of sequence alignments and sequence identities includes global alignments and local alignments performed using computational methods. Alignments can be performed using BLAST (National Center for Biological Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using standard protein BLAST with the following default parameters: maximum target sequence: 100; short query: automatically adjust parameters for short input sequences; expected threshold: 10; word length: 6; maximum number of matches within the query range: 0; matrix: BLOSUM62; gap penalty: (existence: 11, extension: 1); component adjustment: conditional component score matrix adjustment; filter: not selected; mask: not selected. The % nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within query range: 0; Match / mismatch score: 1, -2; Gap penalty: linear; Filter: Low complexity region; Mask: Mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently, to have XX% sequence identity with the reference sequence.

[0045] Polypeptide or polynucleotide sequence identity can be measured over the length of an entire defined polypeptide sequence (e.g., as defined by a particular SEQ ID number), or can be measured over a shorter length, for example, over the length of a fragment (e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues) taken from a larger defined polypeptide sequence. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown in this article, table, figure, or sequence listing can be used to describe the length over which a percentage identity can be measured.

[0046] The polypeptides disclosed herein may include "variant" polypeptides, "mutants" and "derivatives thereof". As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of the polypeptide in nature, as distinguished from a variant or mutant form. As used herein, a "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence that is different from a reference protein or polypeptide molecule. A variant or mutant may have an insertion, deletion or substitution of one or more amino acid residues relative to a reference molecule.

[0047] The amino acid sequences of polypeptide variants, mutants, derivatives or fragments contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. "Conservative amino acid substitutions" are those substitutions in which the amino acid is substituted with a different amino acid, wherein the substitution is predicted to interfere minimally with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially retain the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of the substitution, such as a beta sheet or alpha helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the volume of the side chain.

[0048] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" and "nucleic acid" are used interchangeably and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. The DNA polynucleotide may be a cDNA (e.g., coding DNA) or a genomic DNA sequence (e.g., including introns and exons).

[0049] If in its natural state or when operated by methods known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof, then the polynucleotide is said to encode a polypeptide. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0050] Those skilled in the art understand that the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, polynucleotides (e.g., polynucleotides encoding TarJ' polypeptides) can be codon-optimized to be expressed in specific cells, and the specific cells include but are not limited to plant cells, bacterial cells, fungal cells, or animal cells. Although polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptides described herein can be used. Therefore, non-naturally occurring sequences can be used. These sequences may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, genetic codes, and hands can also be used to generate degenerate coding sequences.

[0051] The recombinant cells described herein may include deletions or destructions in one or more natural genes. The phrase "deletion or destruction" refers to the state of natural genes in recombinant cells, which have a completely eliminated coding region (deletion) or gene, its promoter or its terminator modification (such as by deletion, insertion or mutation), so that the gene no longer produces an active expression product, produces a severely reduced amount of expression product (e.g., reduced by at least 75% or reduced by at least 90%), or produces an expression product with severely reduced activity (e.g., reduced by at least 75% or reduced by at least 90%). Deletion or destruction can be achieved by genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi) and / or selection and screening. The natural gene to be deleted or destroyed can be replaced with an exogenous nucleic acid of interest for expressing an exogenous gene product (e.g., polypeptide, enzyme, etc.).

[0052] Recombinant cells as described herein may comprise one or more genetic modifications, wherein exogenous nucleic acids are integrated into the genome of the host cell. Those skilled in the art will know how to select suitable loci in the yeast genome for integration of exogenous nucleic acids. Suitable integration loci may include, but are not limited to, PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in a spore yeast host cell, suitable interaction sites may include, but are not limited to, the ER1 locus (defined as a locus flanked by SEQ ID NO: 85 and SEQ ID NO: 162), the ER3 locus (defined as a locus flanked by SEQ ID NO: 155 and SEQ ID NO: 165), the PDC1 locus (defined as a locus flanked by SEQ ID NO: 152 and SEQ ID NO: 164), the pyrF locus (defined as a locus flanked by SEQ ID NO: 153 and SEQ ID NO: 163), the TRP3 locus (defined as a locus flanked by SEQ ID NO: 156 and SEQ ID NO: 159), the gpdIIA locus (defined as a locus flanked by SEQ ID NO: 157 and SEQ ID NO: 161), and the gpdIIB locus (defined as a locus flanked by SEQ ID NO: 158 and SEQ ID NO: 166). The exogenous nucleic acid may also be integrated in an intergenic region or other location in the host cell genome not specifically specified herein. Other suitable integration loci can be determined by one skilled in the art. Furthermore, one skilled in the art will recognize how to use the sequences to design primers to verify correct gene integration at the selected locus.

[0053] The recombinant cells described herein are capable of producing ribitol and include an exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase (TarJ') enzyme. The exogenous polynucleotide sequence can be an exogenous ribulose-5-phosphate reductase gene. After ribulose 5-phosphate is converted into ribitol 5-phosphate, it is believed that natural phosphatases remove phosphate to produce ribitol.

[0054] Recombinant cells can have one or more copies of a given exogenous nucleic acid sequence, which is integrated into the host chromosome and replicated with the chromosome into which it is integrated. For example, yeast cells can be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide as described herein, and the polynucleotide sequence encoding the polypeptide can be integrated into the host chromosome with one or more copies. Recombinant cells may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide as described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten or more copies of a polynucleotide sequence encoding a polypeptide as described herein that is integrated into the genome. Multiple copies of the polynucleotide sequence can all be incorporated into a single locus or can be incorporated into multiple loci.

[0055] "Ribulose-5-phosphate reductase gene" and "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" refers to a gene that catalyzes the conversion of ribulose-5-phosphate and NADPH or NADH into ribitol 5-phosphate and NADP + or NAD + 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, Pradoshia sp. D12, Lactobacillus plantarum EGD-AQ4 or uncultured Ruminococcus sp. The TarJ' 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 the amino acid sequence of at least one of SEQ ID NO:13, 34, 35, 36, 37, 38 or 39.

[0056] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Clostridium difficile gene encoding the amino acid of SEQ ID NO: 13. The exogenous polynucleotide may encode an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0057] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a S. aureus gene encoding the amino acid of SEQ ID NO: 34. 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: 34.

[0058] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a 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.

[0059] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Eubacterium convexoside 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.

[0060] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Pradoshila D12 gene encoding the amino acid sequence 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.

[0061] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Lactobacillus plantarum EGD-AQ4 gene encoding the amino acid sequence 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.

[0062] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from an uncultured Ruminococcus 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.

[0063] The exogenous polynucleotides in the recombinant cells described herein can be under the control of a promoter. For example, the exogenous nucleic acid can be operably linked to a heterologous or artificial promoter. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarbonatase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p; SEQ ID NO: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); 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).

[0064] The exogenous nucleic acid in the recombinant cells described herein can be under the control of a terminator. For example, the exogenous nucleic acid can be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. The terminator may include, but is not limited to, a GAL10 terminator, a PDC terminator, a transaldolase terminator (TAL), a 6PGD terminator (6PGDt; SEQ ID NO: 140); an ASNS terminator (ASNSt; SEQ ID NO: 141); an ENO1 terminator (ENO1t; SEQ ID NO: 142); a hexokinase 1 terminator (HXK1t; SEQ ID NO: 143); a PGK1 terminator (PGK1t; SEQ ID NO: 144); a PGM1 terminator (PGM1t; SEQ ID NO: 145); a PYK1 terminator (PYK1t; SEQ ID NO: 146); a RPLA terminator (RPLAt; SEQ ID NO: 147); a transaldolase 1 terminator (TAL1t; SEQ ID NO: 148); a TDH3 terminator (TDH3t; SEQ ID NO: 149); a translation elongation factor 2 terminator (TEF2t; SEQ ID NO: 150). NO: 150); and triosephosphate isomerase 1 terminator (TPI1t; SEQ ID NO: 151);

[0065] A promoter or terminator is "operably linked" to a given polynucleotide (e.g., a gene) if its position relative to the polynucleotide in the genome or expression cassette is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0066] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. A recombinant polynucleotide is a polynucleotide formed by a laboratory method, which includes a polynucleotide sequence derived from at least two different natural sources, or they may be synthetic. Therefore, a construct may include a new modification to an endogenous gene introduced by, for example, genome editing technology. A construct may also include a recombinant polynucleotide produced using, for example, a recombinant DNA method. A construct may be a vector comprising a promoter operably connected to a polynucleotide encoding a polypeptide described herein. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide connected thereto. A vector may be a plasmid, which refers to a circular double-stranded DNA loop, into which an additional DNA fragment may be integrated.

[0067] The present disclosure also provides a fermentation method for producing ribitol 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 ribitol. As will be appreciated by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and separating ribitol from the fermentation broth. The fermentation process may be a fully or partially aerobic process.

[0068] The fermentation process can be run using a suitable fermentation substrate. The substrate of the fermentation process can include hydrolyzates of glucose, sucrose, galactose, mannose, molasses, xylose, fructose, starch, lignocellulose hydrolyzates, or a combination thereof. Those skilled in the art will recognize which fermentation substrate is suitable for a given fermenting organism and system.

[0069] The fermentation process can be run under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, can be ambient temperature. Alternatively or additionally, the fermentation temperature can be maintained within a predetermined range. For example, the fermentation temperature can be maintained within a range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably about 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to any specific range or temperature described herein and can be modified as appropriate.

[0070] The fermentation process can be operated within a range of oxygen uptake rates (OUR). The volumetric OUR of the fermentation process can be in the range of 0.5 mmol O2 / (L·h) to 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) to 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) to 30 mmol O2 / (L·h), 3 mmol O2 / (L·h) to 25 mmol O2 / (L·h), 4 mmol O2 / (L·h) to 20 mmol O2 / (L·h), or 5 mmol O2 / (L·h) to 15 mmol O2 / (L·h). In some embodiments, the specific OUR can be in the range of 0.05 mmol O2 / (g cell dry weight h) to 10 mmol O2 / (g cell dry weight h), 0.1 mmol O2 / (g cell dry weight h) to 8 mmol O2 / (g cell dry weight h), 0.15 mmol O2 / (g cell dry weight h) to 5 mmol O2 / (g cell dry weight h), 0.2 mmol O2 / (g cell dry weight h) to 1 mmol O2 / (g cell dry weight h), or 0.3 mmol O2 / (g cell dry weight h) to 0.75 mmol O2 / (g cell dry weight h). However, the volume or specific OUR of the fermentation process is not limited to any specific rate or range described herein.

[0071] Fermentation process can be operated under various cell concentrations. In some embodiments, the dry cell weight at the end of fermentation can be 5g dry cell weight / L to 40g dry cell weight / L, 8g dry cell weight / L to 30g dry cell weight / L or 10g dry cell weight / L to 20g dry cell weight / L. In addition, the input density (pitch density) or input rate (pitchingrate) of fermentation process can change. In some embodiments, the input density can be 0.05g dry cell weight / L to 11g dry cell weight / L, 0.1g dry cell weight / L to 10g dry cell weight / L or 0.25g dry cell weight / L to 8g dry cell weight / L.

[0072] The initial dextrose concentration of the fermentation can be at least 100 g / L dextrose, 200 g / L dextrose, 250 g / L dextrose, 300 g / L dextrose, 350 g / L dextrose, or at least 400 g / L dextrose. The initial dextrose concentration can be between 100 g / L and 400 g / L, 150 g / L and 350 g / L, or 250 g / L and 325 g / L.

[0073] Fermentation processes can be associated with various characteristics, such as, but not limited to, fermentation production rate, approach fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of yeast and / or the genetic modification of yeast used in the fermentation process. These characteristics can be affected by regulating the fermentation process conditions. These characteristics can be regulated via a combination of yeast selection or modification and the selection of fermentation process conditions.

[0074] The ribitol production rate of the process can be at least 0.2 g L -1 h -1 , 0.3 g L -1 h -1 , 0.5 g L -1 h -1 , 0.75 g L -1 h -1 or at least 1.0 g L -1 h -1 The mass yield of ribitol of the process may be at least 55%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85%. The final ribitol titer of the process may be at least 20 g / L, 30 g / L, 50 g / L, 75 g / L or 100 g / L.

[0075] The fermentation process can be run as a dextrose fed batch.In addition, the fermentation process can be a batch process, a continuous process or a semi-continuous process, as understood by those skilled in the art.

[0076] Example

[0077] The present invention is further described in detail by reference to the following experimental examples. Unless otherwise stated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0078] Example 1 - Xylitol-phosphate dehydrogenase diversity

[0079] About three thousand galactitol-1-phosphate-5-dehydrogenase (G1PDH) / xylitol-phosphate dehydrogenase (XPDH) enzyme sequences were obtained from Uniprot and analyzed. Figure 2 The natural sequence diversity of this group of sequences is shown. The group is diverse, with about 25% of the enzymes not having more than 75% identical homology. Since these enzymes tend to prefer NAD as a cofactor rather than NADP as a cofactor, the cofactor binding preference of 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 approaching the Rossman fold (from +23 to +30 amino acids of the GXGXXG motif (SEQ ID NO: 129)) and scored based on the total charge in an 8-amino acid residue window. The top 8 candidates predicted to use NADP, as well as 4 candidates predicted to use NAD and 3 controls were selected for further characterization.

[0080] Upon further examination of the structural features of the predicted binding pocket, important aspartate residues were identified that could influence cofactor preference. Figure 3 The polypeptide of SEQ ID NO: 34 and its substitutions were used to construct a structural homology model for predicting the conformation of the cofactor binding pocket. Figure 3Shown is the C-terminal of the penultimate β-strand outside the Rossman fold domain.Do not wish to be bound by any particular theory, it is estimated that the enzyme wherein the first residue in this region (relative to residue 198 of SEQ ID NO:34) is aspartic acid and the second residue (relative to residue 199 of SEQ ID NO:34) is a large hydrophobic amino acid (e.g., isoleucine) will preferably NAD cofactor, this is due to the hydrogen bonding of the hydroxyl of aspartic acid and NAD ribose. However, the enzyme wherein the first residue (relative to residue 198 of SEQ ID NO:34) is alanine, glycine or serine and the second residue (relative to residue 199 of SEQ ID NO:34) is lysine or arginine will preferably NADP cofactor, because the positive charge on lysine or arginine residue will interact with the negative charge of the phosphoric acid of NADP and the smaller residue in the first position allows the space of the phosphoric acid in the binding pocket.Based on this analysis, according to the prediction preference to NADP, other 12 kinds of enzymes were selected.Finally, 6 other enzymes with sequence similarity to the active XPDH enzyme were selected for testing.

[0081] Example 2 - TarJ' diversity

[0082] Approximately eight hundred ribulose 5-phosphate reductase sequences were obtained from Uniprot and analyzed. Figure 3 The natural sequence diversity of this group of sequences is shown. Overall, the diversity in this group is low, because only 10% of the enzymes do not have more than 75% identical sequence similarity. Since these enzymes tend to prefer NADP rather than NAD as cofactors, no scoring is performed, and sequences are simply aligned in Geneous (ClustalW, default settings). Eight enzymes were selected for further analysis based on sequence similarity.

[0083] Example 3 - In vitro enzyme assay

[0084] The polynucleotides encoding suspected XPDH homologs (Table 2) or TarJ' homologs (Table 3) were cloned into a vector containing a T7 promoter and terminator for cell-free protein expression (New England Biolabs, In Vitro Protein Synthesis). The activity of cell-free synthesized proteins on four substrates (ribulose 5-phosphate, xylulose 5-phosphate, ribulose, and xylulose) was analyzed with NADP or NAD cofactors. Seven enzymes (XPDH of SEQ ID NOs: 12 and 34, TarJ' of SEQ ID NOs: 36, 37, 38, 40, and 42) were able to catalyze the reduction of ribulose 5-phosphate or xylulose 5-phosphate ( Figure 5 ), but could not catalyze the reduction of xylulose or ribulose (results not shown).

[0085] Table 2: XPDH homologs

[0086]

[0087] Table 3: TarJ' homologs

[0088]

[0089] Example 4 - Genetically modified Saccharomyces cerevisiae strains

[0090] Strain 1-1 is a Moniliellatomentosa host strain "Moniliellatomentosa var pollinis TCV364" described in US 6,440,712, which is incorporated herein by reference in its entirety, and deposited under the Budapest Treaty on March 28, 1997 at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycothèque de l'Université Catholique de Louvain by Eridania Béghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde). Table 4 below lists various Moniliellatomentosa yeast strains, including information on the parent strain, the sequence of the transformed parent strain, and the characteristics of the expression cassette contained in the transformed sequence. Each "XPDH / TarJ' homolog expression cassette" contains, in order, a 5'ER1 flanking sequence (SEQ ID NO: 85), a MpPYK1 promoter (SEQ ID NO: 86), a gene encoding the indicated XPDH or TarJ' homolog (one of SEQ ID NOs: 87-128), an Mp6PGD terminator (SEQ ID NO: 140), and a 5' portion of a G418 resistance gene expression cassette (SEQ ID NO: 175). Each "selectable marker cassette" contains, in order, a 3' portion of a 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). Following binary transformation with the XPDH / TarJ' homolog expression cassette and the selectable marker cassette, the two cassettes recombined to integrate the nucleotide sequence encoding the XPDH or TarJ' homolog and the G418 resistance marker at the ER1 locus.

[0091] The indicated Saccharomyces parent strains were transformed with the indicated sequences by first protoplasting the parent strains by adding an enzyme mixture containing 0.6M MgSO4, 7.5g / L collapsing enzyme, and 12.5g / L Trichoderma harzianum lyase to the mycelial pellet of the parent strains. The protoplasts were then pelleted, washed with 0.6M MgSO4, and resuspended in STC medium (0.6M sucrose, 50mM CaCl2, 10mM Tris-HCl, pH 7.5). The protoplasts were then precipitated with 100μg of single-stranded salmon sperm DNA and 1.5μg to 5μg of each 5'DNA and 3'DNA conversion fragments (3μg-10μg in total; see Table 4 for a list of fragments) to approximately 200μL of the protoplast mixture (10 8 Cells / mL) are added to prepare binary transformation. Then 1mL of STC culture medium containing 50% PEG is added to salmon sperm DNA, conversion DNA and protoplast mixture, and the resulting combination is incubated at room temperature for 15 minutes. After incubation, the recovery culture solution (0.4M sucrose, 1g / L yeast extract, 1g / L malt extract, 10g / L sugar, pH 4.5) is added to the mixture, and incubated at 27°C, 100rpm for 16 hours to 24 hours. After incubation, the protoplasts are precipitated and resuspended in 1mL PBS by centrifugation.

[0092] The resuspended protoplasts are spread on PDA+250mg / L geneticin (geneticin) (G418) selection plates and incubated at least 2 days-4 days at 30°C-35°C until transformant growth. The integration of the indicated sequence of the obtained transformant is evaluated by colony PCR. The isolate verified by PCR is then designated as the indicated strain number. In some cases, more than one isolate verified by PCR (e.g., "sister" isolate) is indicated by the letter after the strain number. For example, strain 1-strain 2 has 5 sister isolates: strains 1-2a, 1-2b, 1-2c, 1-2d and 1-2e.

[0093] For example, strain 1-1 is transformed with SEQ ID NO:43 and SEQ ID NO:44. SEQ ID NO:43 contains (i) 3' flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID No:162), and (ii) the 3' portion of the G418 resistance gene selectable marker (SEQ ID NO:172). SEQ ID NO:44 contains (i) an expression cassette for an XPDH homolog from M. sediminis encoding the amino acid sequence of SEQ ID NO:1 under the control of the PYK1 promoter of SEQ ID NO:86 and the PGD terminator of SEQ ID NO:140 (SEQ ID NO:87); (ii) 5' flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:85); and (iii) the 5' portion of the G418 resistance gene selectable marker (SEQ ID NO:175). Transformants were selected on PDA+250mg / L Geneticin (G418) selection plates and incubated at least 2 days at 30°C to 35°C until transformant growth. The resulting transformants were streaked on PDA+Geneticin (G418) plates for single colony isolation and selection of single colonies. The integration of the indicated sequences of the selected colonies was evaluated by colony PCR. The isolates verified by PCR were named as strains 1-2a, 1-2b, 1-2c, 1-2d and 1-2e.

[0094] Table 4

[0095]

[0096]

[0097] Example 5 - Shake flask fermentation assay

[0098] Strains 1-1, 1-35a-d, 1-37a-d, 1-38a-f, 1-39a-f, 1-42a-f, 1-13a-f, and 1-15a-f (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0099] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0100] A 250 ml unbaffled flask containing 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 with a refractive index detector. The fermentation results are reported in Table 6 and Figure 6 and Figure 7 middle.

[0101] Table 5: Production medium

[0102] Components Concentration (unit) glucose 300(g / L) KH2PO4 1.27(g / L) (NH4)2HPO4 0.13(g / L) (NH4)2SO4 1.80(g / L) Urea 2.85(g / L) Citric Acid 200.0(mg / L) MgSO4-7H2O 690.2 (mg / L) FeSO4-7H2O 32.35 (mg / L) ZnSO4-7H2O 4.40(mg / L) MnSO4-H2O 0.53 (mg / L) CuSO4-5H2O 0.45(mg / L) CaCl2-2H2O 91.60(mg / L) Na2SO4 154.50(mg / L) Thiamine-HCl 10(mg / L) Choline-Cl 100(mg / L) Defoaming agent CF-32 1.00(g / L)

[0103] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-35a, 1-37a-d, 1-38a-c, 1-39d-f, 1-42a-b, 1-42d, 1-13a-b, 1-13d-e, 1-15b-c, and 1-15e-f included the transformed polynucleotide sequence, but it was not integrated at the ER1 locus.

[0104] Table 6: 96-hour shake flask results

[0105]

[0106]

[0107] Example 6 - Shake flask fermentation assay

[0108] Strains 1-13c, 1-29a-e, 1-33a-e, and 1-34a-e (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0109] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0110] A 250 ml unbaffled flask containing 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 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 7 and Figure 8 middle.

[0111] like Figure 8 As shown in , although sister strains 1-34c and 1-34d produced 15.8 g / L and 18.6 g / L xylitol, respectively, strains 1-34a, 1-34b, and 1-34e did not produce more xylitol than the wild type (strains 1-1, Figure 6 ) significantly more xylitol. When strains 1-34a, 1-34b, and 1-34e were initially verified by PCR, it was later determined that the integrated polynucleotide that should encode the Neobacillus cucumeris XPDH homolog contained a frameshift mutation and did not express a functional XPDH. Therefore, although the results appear different, they are actually consistent considering that strains 1-34a, 1-34b, and 1-34e do not contain a polynucleotide encoding a functional XPDH.

[0112] Table 7: 96-hour shake flask results

[0113]

[0114] Example 7 - Shake flask fermentation assay

[0115] Strains 1-13c, 1-17a-e, 1-18a-e, 19a-e, 1-21a-e, 1-22a-e, 1-23a-e, 1-24a-e, 1-25a-e and 1-27a-d (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol and ethanol production.

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

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

[0118] Table 8: 96-hour shake flask results

[0119]

[0120]

[0121] Example 8 - Shake flask fermentation assay

[0122] Strains 1-13c, 1-3a-e, 1-10a-e, 1-11a-e, 1-12a-e, 1-14a-e, 1-16a-e, 1-28a-e and 1-2a-e (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol and ethanol production.

[0123] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0124] A 250 ml unbaffled flask containing 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 with a refractive index detector. The fermentation results are reported in Tables 9 and Fig. 9 middle.

[0125] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-16b-e included the transformed polynucleotide sequence, but it was not at the ER1 locus. Further analysis of the integration position in strains 1-2c and 1-2d was inconclusive.

[0126] Table 9: 96-hour shake flask results

[0127]

[0128]

[0129] Example 9 - Shake flask fermentation assay

[0130] Strains 1-13c, 1-8a-d, 1-26a-e, 1-36a-e, 1-41a-e, 1-40a-e, and 1-20a-e (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0131] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0132] A 250 ml unbaffled flask containing 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 96 hours of incubation. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high performance liquid chromatography with a refractive index detector. The fermentation results are reported in Tables 10 and Fig.10 middle.

[0133] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-8c, 1-8d, and 1-41c included the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integration loci in strains 1-36a, 1-41b, 1-41e, and 1-20a-e was inconclusive.

[0134] Table 10: 96-hour shake flask results

[0135]

[0136]

[0137] Example 10 - Shake flask fermentation assay

[0138] Strains 1-13c, 1-30a-e, 1-31a-e, 1-32a-e, 1-4a-e, 1-5a-e, 1-6a-e, 1-7a-e, and 1-9a-e (summarized in Table 4 above) were run in shake flasks to assess glucose consumption and ribitol, xylitol, glycerol, and ethanol production.

[0139] The strain was streaked on YPD plates (bacterial peptone 20g / L, yeast extract 10g / L, glucose 20g / L and agar 15g / L) for biomass growth and incubated at 30°C for 48 hours-72 hours. The cells from the incubated YPD plates were scraped into a 250mL baffle-free flask containing 40mL enriched medium (170g / L glucose, 10g / L yeast extract). Cells were incubated at 30°C and 250rpm until optical density (OD600) reached 15-20 to form seed cultures. The spectrophotometer (Thermo Scientific) of model Genesys20 was used to measure optical density under 600nm wavelength and 1cm pathlength cuvette. Seed cultures reached an OD600 between 15 and 20 in approximately 32 hours to 50 hours.

[0140] A 250 ml unbaffled flask containing 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 with a refractive index detector. The fermentation results are reported in Tables 11 and Fig.11 middle.

[0141] Although PCR verification indicated that the transformed polynucleotide sequence was present in the indicated strains, further analysis indicated that in some strains, the sequence was not correctly integrated at the ER1 locus. Further analysis indicated that strains 1-30c and 1-30d included the transformed polynucleotide sequence, but it was not integrated at the ER1 locus. Further analysis of the integration locus in strain 1-6c was inconclusive.

[0142] Table 11: 96-hour shake flask results

[0143]

[0144]

Claims

1. A genetically engineered yeast cell capable of producing ribitol, the engineered yeast cell comprising: An exogenous polynucleotide sequence encoding ribulose-5-phosphate reductase.

2. The yeast cell of claim 1, wherein the ribulose-5-phosphate reductase 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: 13, 34, 35, 36, 37, 38, and 39.

3. The yeast cell according to claim 1 or claim 2, wherein the yeast cell is an osmotolerant yeast cell. The yeast cell according to any one of claims 1 to 3, wherein the yeast cell is a cell of the subdivision Ustilagomycotina.

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

6. A genetically engineered Mycelium cell capable of producing ribitol, the engineered Mycelium cell comprising: An exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase 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: 13, 34, 35, 36, 37, 38, and 39.

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

8. The yeast cell of any one of claims 1 to 7, wherein the yeast cell is capable of producing ribitol at a titer of at least 20 g / L, 30 g / L, 50 g / L, 75 g / L or 100 g / L when used in a fermentation process in the presence of dextrose for at least 96 hours.

9. The yeast cell of any one of claims 1 to 8, wherein the erythritol production of the yeast cell is reduced relative to the erythritol production in an equivalent yeast cell lacking the exogenous polynucleotide sequence.

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

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

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

13. The yeast cell of claim 11 or claim 12, wherein the constitutive heterologous promoter 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), NO:138) and RPL16B (SEQ ID NO:139).

14. The yeast cell of any one of claims 1 to 13, wherein the ribulose-5-phosphate reductase has a sequence at least 85% identical to at least one of SEQ ID NOs: 34-39.

15. The yeast cell of any one of claims 1 to 14, wherein the ribulose-5-phosphate reductase has a sequence at least 90% identical to at least one of SEQ ID NOs: 34-39.

16. The yeast cell of any one of claims 1 to 15, wherein the ribulose-5-phosphate reductase comprises a sequence 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: 34, 36, 37, 38 and 39, and the yeast is capable of producing at least 1 g / L, 2 g / L, 5 g / L, 7.5 g / L or 10 g / L ribitol.

17. A method for producing ribitol, the method comprising: A substrate comprising dextrose is contacted with the engineered yeast cell of any one of claims 1 to 16, wherein fermentation of the substrate by the engineered yeast produces ribitol.

18. A method for producing ribitol, the method comprising: A substrate comprising dextrose is contacted with an engineered yeast cell comprising an exogenous polynucleotide sequence encoding a ribulose-5-phosphate reductase 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: 34, 36, 37, 38, and 39, wherein fermentation of the substrate by the engineered yeast produces ribitol.

19. The method of claim 18, wherein the engineered yeast cell is a Mycosporium sp.

20. The method according to any one of claims 17 to 19, wherein the fermentation temperature is 25°C to 45°C, 30°C to 40°C or 32°C to 37°C or therebetween, and the volumetric oxygen uptake rate (OUR) is between 0.5 mmol O2 / (L·h) and 40 mmol O2 / (L·h), 1 mmol O2 / (L·h) and 35 mmol O2 / (L·h), 2 mmol O2 / (L·h) and 30 mmol O2 / (L·h), Between 3mmol O2 / (L·h) and 25mmol O2 / (L·h), 4mmol O2 / (L·h) and 20mmol O2 / (L·h) or 5mmol O2 / (L·h) and 15mmol O2 / (L·h).

21. The method of any one of claims 17 to 20, wherein ribitol is present in an amount of at least 0.2 g / L -1 h -1 , 0.3 g L - 1 h -1 , 0.5 g L -1 h -1 , 0.75 g L -1 h -1 or at least 1.0 g L -1 h -1 rate of production.

22. The method of any one of claims 17 to 21, wherein ribitol production is at least 20 g / L, 30 g / L, 50 g / L, 75 g / L or 100 g / L when the fermentation is run at 35°C for 96 hours.

23. The method of any one of claims 17 to 22, wherein erythritol production is reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking the exogenous polynucleotide sequence.

24. The method of any one of claims 17 to 23, wherein erythritol production is less than 40 g / L, 30 g / L, or less than 20 g / L when the fermentation is run at 35°C for 96 hours.

25. The method of any one of claims 17 to 24, wherein the ribulose-5-phosphate reductase comprises a sequence 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: 34, 36, 37, 38, and 39, and the yeast produces at least 1 g / L, 2 g / L, 5 g / L, 7.5 g / L, or 10 g / L ribitol when the fermentation is run at 35°C for 96 hours.

26. The method of any one of claims 17 to 25, wherein glycerol production is reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking the exogenous polynucleotide sequence.

27. The method of any one of claims 17 to 26, wherein ethanol production is reduced relative to an equivalent fermentation run with an equivalent yeast cell lacking the exogenous polynucleotide sequence.

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