Genetically modified microorganism and fermentation process for the production of mannitol

Abstract

Disclosed herein are genetically engineered cells capable of producing D-mannitol. The genetically engineered cells comprise an exogenous polynucleotide sequence encoding a mannitol-1-phosphate dehydrogenase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:16 and an exogenous polynucleotide sequence encoding a mannitol-1-phosphate phosphatase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:18.

Description

PT-2221-WO-PCTGENETICALLY MODIFIED MICROORGANISM AND FERMENTATION PROCESS FOR THE PRODUCTION OF MANNITOLCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 749,095, filed January 24, 2025, which is incorporated by reference herein in its entirety.REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA PATENT CENTER

[0002] The content of the Sequence Listing XML file of the sequence listing named “PT-2221- WO-PCT.xml” which is 87,113 bytes in size created on January 23, 2026, and electronically submitted via Patent Center herewith the application is incorporated by reference in its entirety.BACKGROUND

[0003] Mannitol is a naturally occurring six-carbon polyol with a variety of uses in the food and pharmaceutical industries. Current industrial methods for the production of mannitol involve hydrogenation of other sugars (e.g., mannose and fructose), which is costly and inefficient. In contrast, fermentation processes have been used commercially at large scale to produce other organic molecules, such as ethanol, citric acid, lactic acid, and the like, and may offer a cost effective and sustainable alternative to current mannitol processing methods. Accordingly, provided herein are genetically modified microorganisms and fermentation methods for the production of mannitol.SUMMARY

[0004] The present disclosure provides a genetically engineered cell capable of producing D-mannitol, the engineered cell comprising an exogenous polynucleotide sequence encoding a mannitol- 1 -phosphate dehydrogenase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16; and an exogenous polynucleotide sequence encoding a mannitol -1 -phosphate phosphatase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 18. The yeast cell may be selected from the group consisting of Kluyveromyces marxianus. Kluyveromyces lactis, Komagataella phaffii, Saccharomyces cerevisiae. Yarrowia lipolytica, and Moniliella pollinis.PT-2221-WO-PCT

[0005] The genetically engineered cell may additionally comprise a deletion or disruption in a native PFK1 gene. When the cell is a Kluyveromyces marxianus cell, the native PFK1 gene may encodes PFK1 enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 19. When the cell is a Kluyveromyces marxianus cell the native PFK1 gene may be at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:20.

[0006] The mannitol -1 -phosphate dehydrogenase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to of SEQ ID NO: 16; the mannitol -1 -phosphate phosphatase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NOs:18; and the cell is capable of producing at least 25 g / L D-mannitol.

[0007] The mannitol -1 -phosphate dehydrogenase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to of SEQ ID NO: 16; the mannitol -1 -phosphate phosphatase may be at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NOs:18; the cell may comprise a deletion or disruption of a native PFK1 gene, and the cell is capable of producing at least 70 g / L D-mannitol.

[0008] In the genetically engineered cells described herein, one or more of the exogenous polynucleotide sequences is operably linked to a heterologous promoter and / or a heterologous terminator. The promoter may be selected from the group consisting of pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoter (ADHlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFlp); phosphoglucomutase 1 promoter (PGMlp); 3 -phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B; and PDC1 promoter. The terminator may be selected from the group consisting of GAL10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TAL It); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); fructose-bisphosphate aldolase terminator (FBAlt); TEF1; i so- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator.PT-2221-WO-PCT

[0009] The disclosure also provides a method for producing D-mannitol, the method comprising contacting a substrate with the genetically engineered cells described herein. The engineered cells may produce at least 20 g / L, at least 25 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, or at least 70 g / L mannitol after 120 hours. The substrate may comprise starch, glucose, sucrose, cellulosic biomass, or combinations thereof.

[0010] The disclosure further provides a use of the engineered cell(s) as described herein for the production of D-mannitol.BRIEF DESCRIPTION OF THE FIGURES

[0011] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and the payment of the necessary fee.

[0012] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0013] FIG. 1 shows a proposed pathway for the production of D-mannitol from sucrose, starch, and / or glucose.DETAILED DESCRIPTION

[0014] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0015] 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 nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0016] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include allPT-2221-WO-PCTthe individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0017] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis is also referred to as wt% or % (wt) below.

[0018] This disclosure relates to various recombinant cells engineered to produce D-mannitol. In general, the recombinant cells described herein include a heterologous polynucleotide encoding a mannitol- 1 -phosphate dehydrogenase, for example the mannitol- 1 -phosphate dehydrogenase enzyme of SEQ ID NO: 16, or sequences at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The recombinant cell additionally includes a heterologous nucleic acid encoding a mannitol- 1 -phosphate phosphatase, for example the mannitol -1 -phosphate phosphatase enzyme of SEQ ID NO: 18 or sequences at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The recombinant cell may additionally include a deletion or disruption in a native PFK1 gene. The disclosure further provides fermentation methods for the production of D-mannitol using the genetically engineered cells described herein.

[0019] In general, recombinant cells described herein are yeast cells. As used herein, “yeast” refers to eukaryotic single celled microorganisms classified as members of the fungus kingdom. Yeast are unicellular organisms which evolved from multicellular ancestors with some species retaining multicellular characteristics such as forming strings of connected budding cells known as pseudo hyphae or false hyphae. Yeast cells may also be referred to in the art as yeast-like cells, and as used herein “yeast cell” encompasses both yeast and yeast-like cells. Suitable yeast and yeast-like host cells for modification may include, but are not limited to, Saccharomyces cerevisiae. Komagataella sp., Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus). Yarrow ia lipolytica, Issatchenkia orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnolias, Candida elhanoUca), Pichia deserticola, Pichia membranifadens, Pichia fermentans, Aspergillus, Trichoderma,PT-2221-WO-PCTMyceliphthora thermophila, Moniliella (e.g., Moniliella pollinis), Pfaffia, Yamadazyma, Hansenula, Pichia kudriavzevvi, Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens), Pseudozyma tsukubaensis, Trigonopsis variabilis, Penicillium, and Torula. An ordinarily skilled artisan would understand the requirements for selection of a suitable yeast cell, and recombinant yeast cells of the present disclosure are not limited to those expressly recited herein. Methods for genetic engineering of yeast cells are known and described in the art and a skilled artisan would understand the methods necessary to transform and engineer a suitable yeast cell.

[0020] The terms “glucose” and “dextrose” are used interchangeably herein and refer to D-glucose except where expressly indicated otherwise.

[0021] The recombinant cells described herein include one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, enable the production of D-mannitol by the recombinant cells.

[0022] As used herein, “exogenous” refers to genetic material or an expression product thereof that originates from outside of the host organism. For example, the exogenous genetic material or expression product thereof 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, a Lactobacillus helveticus lactate dehydrogenase gene is exogenous when introduced into S. cerevisiae.

[0023] As used herein, “native” refers to genetic material or an expression product thereof that is found, apart from individual-to-individual mutations which do not affect function or expression, within the genome of wild-type cells of the host cell.

[0024] For the purposes of this application, the Kluyveromyces marxianus cell of Kluyveromyces marxianus CD21, deposited under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms (BCCM) / Mycotheque de 1'Universite Catholique de Louvain (MUCL), Croix du Sud, box L7.05.06, B-1348 Louvain-la-Neuve, Belgium) on October 18, 2024 under accession number 58456, is considered the wild-type Kluyveromyces marxianus cell.

[0025] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the recited macromolecule its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathwayPT-2221-WO-PCTis understood to include the co-f actors, co-enzymes, and metals necessary for the enzyme to properly function. A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.Table 1 : Amino Acid three and one letter symbols

[0026] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include greater than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild-type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.

[0027] As used herein, the phrases “percent 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. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool)PT-2221-WO-PCTversion 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0028] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0029] 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 skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.

[0030] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as 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.PT-2221-WO-PCT“Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, 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 bulk of the side chain.

[0031] As used herein, 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 phrases 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 the antisense strand. The DNA polynucleotides may be a cDNA or a genomic DNA sequence.

[0032] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

[0033] Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, the polynucleotides may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in various organisms are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These 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. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.

[0034] The recombinant cells described herein may include deletions or disruptions in one or more native genes. The phase “deletion or disruption” refers to the status of a native gene in the recombinant cell that has either a completely eliminated coding region (deletion) or a modification of the gene, its promoter, or its terminator (such as by a deletion, insertion, or mutation) so that the gene no longer produces an active expression product, produces severely reduced quantities of the expression product (e.g., at least a 75% reduction or at least a 90% reduction) or producesPT-2221-WO-PCTan expression product with severely reduced activity (e.g., at least 75% reduced or at least 90% reduced). The deletion or disruption can be achieved by genetic engineering methods, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. Deletion or disruption of a native host cell gene can be coupled to the incorporation of one or more polynucleotide sequences (e.g., an exogenous or native polynucleotide sequence) into the host cell at the locus of the host cell gene to be deleted or disrupted. The polynucleotide sequence to be inserted may be designed to replace all or a portion of the host cell gene to be deleted or disrupted. The polynucleotide sequence may encode for a gene product of interest, for example, a polypeptide, an enzyme, and the like. The deletion or disruption can also be accomplished using a deletion construct that does not contain a polynucleotide sequence to be integrated. Other methods for gene disruption or deletion are known and described in the art.

[0035] The recombinant cells described herein may include a deletion or disruption of a native phosphofructokinase- 1 (PFK1) gene. The native PFK1 gene encodes an enzyme that has phosphofructokinase- 1 activity. As used herein “phosphofructokinase- 1 activity” and “PFK1 activity” are used interchangeably and refer to enzymes that catalyze the irreversible conversion of beta-D-fructose 6-phosphate, adenosine triphosphate (ATP) to beta-D-fructose 1,6-bisphosphate, adenosine diphosphate (ADP) and water. PFK1 is an enzyme in the glycolytic pathway and is the first irreversible reaction unique to the glycolytic pathway. Without being bound by any particular theory, method, or mode of action, when activity of PFK1 is reduced or eliminated more cellular fructose-6-phosphate would be available to produce D-mannitol. When the host cell contains multiple PFK1 genes, it is preferred to delete or disrupt at least one of them. When the host cell contains, multiple alleles of a given PKF1 gene, it is preferred to delete or disrupt one, both, or all alleles of a given PFK1 gene.

[0036] When the recombinant cell is a Kluyveromyces marxianus cell, the recombinant cell may comprise a deletion or disruption of a PFK1 gene encoding an amino acid sequence 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: 19. When the recombinant cell is a Kluyveromyces marxianus cell, the recombinant cell may comprise a deletion or disruption of a PFK1 gene with a nucleotide sequence 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:20. When the recombinant cell is a Kluyveromyces marxianus cell, the PFK1 gene may be deleted or disrupted by insertion of an exogenous or native nucleic acid sequence at the PFK1 locus to replace the PFK1 gene. For example, in Kluyveromyces marxianus the PFK1 locus is flanked by SEQ ID NOs: 11 and 12.PT-2221-WO-PCT

[0037] The recombinant cells described herein may include one or more genetic modifications in which an exogenous nucleic acid is integrated into the genome of the host cell. One of skill in the art know how to select suitable loci in a yeast genome for integration of the exogenous nucleic acid. Suitable integration loci may include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adhl202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdllA, and gpdllB loci. For example, in af. marxianus host cell, suitable integration loci may include, but are not limited to, the CYB2 locus (defined as the locus flanked by SEQ ID NO:3 and SEQ ID NO:4), the PFK1 locus (defined as the locus flanked by SEQ ID NO: 11 and 12), and the GPD1 locus (defined as the locus flanked by SEQ ID NO:29 and 30). 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 may be determined by one of skill in the art. Furthermore, one of skill in the art would recognize how to use sequences to design primers to verify correct gene integration at the chosen locus.

[0038] The recombinant cell may have one or more copies of a given exogenous nucleic acid sequence integrated in a host chromosome(s) and replicated together with the chromosome(s) into which it has been integrated. For example, the yeast cell may be transformed with nucleic acid construct including a polynucleotide sequence encoding for a polypeptide described herein and the polynucleotide sequence encoding for the polypeptide may be integrated in one or more copies in a host chromosome(s). The recombinant cell may include multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. The recombinant cell may have one, two, three, four, five, six, seven, eight, nine, ten, or more copies of a polynucleotide sequence encoding a polypeptide described herein integrated into the genome. The multiple copies of said polynucleotide sequence may all be incorporated at a single locus or may be incorporated at multiple loci. The recombinant cell may include one or more copies of a given exogenous nucleic acid sequence including on a plasmid within the recombinant cell.

[0039] The recombinant cells described herein are capable of producing D-mannitol and include an exogenous polynucleotide sequence encoding a mannitol- 1 -phosphate dehydrogenase enzyme. The enzyme may be any suitable enzyme with mannitol -1 -phosphate dehydrogenase activity. The exogenous polynucleotide sequence may be an exogenous mannitol -1 -phosphate dehydrogenase gene.

[0040] As used herein “mannitol -1 -phosphate dehydrogenase gene” refers to any gene or polynucleotide sequence that encodes a mannitol- 1 -phosphate dehydrogenase enzyme. As used herein “mannitol -1 -phosphate dehydrogenase enzyme” refers to a polypeptide with mannitol-1-PT-2221-WO-PCTphosphate dehydrogenase activity. As used herein “mannitol- 1 -phosphate dehydrogenase activity” refers to the ability to catalyze the conversion of D-fructose-6-phosphate, NAD(P)H, and a proton (H+) to D-mannitol-1 -phosphate and NAD(P)+. The mannitol- 1 -phosphate dehydrogenase enzyme can be from any suitable organism or may be synthetic. Suitable mannitol -1 -phosphate dehydrogenase enzymes may include, but are not limited to, enzymes categorized under Enzyme Commission (EC) number 1.1.1.17. Suitable enzymes include the mannitol-1-phosphate dehydrogenase enzyme from Aspergillus niger. The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from an Aspergillus niger gene encoding the amino acid sequence of SEQ ID NO: 16. The mannitol- 1 -phosphate dehydrogenase gene may encode an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16.

[0041] As used herein, “NAD(P)H” refers to nicotinamide adenine dinucleotide (phosphate) hydrogen and is inclusive of both NADH and NADPH. As is understood in the art, inclusion of the phosphate (or “P” abbreviation) in parentheses indicates that the phosphate may be absent or present and the name and abbreviation are inclusive of both. Similarly, “NAD(H)” or “NADP(H)” refers to both the reduced and oxidized forms of the cofactor.

[0042] The recombinant cells described herein are capable of producing D-mannitol and include an exogenous polynucleotide sequence encoding a mannitol- 1 -phosphate phosphatase enzyme. The enzyme may be any suitable enzyme with mannitol -1 -phosphate phosphatase activity. The exogenous polynucleotide sequence may be an exogenous mannitol -1 -phosphate phosphatase gene.

[0043] As used herein “mannitol- 1 -phosphate phosphatase gene” refers to any gene or polynucleotide sequence that encodes a mannitol- 1 -phosphate phosphatase enzyme. As used herein “mannitol -1 -phosphate phosphatase enzyme” refers to a polypeptide with mannitol-1-phosphate phosphatase activity. As used herein “mannitol- 1 -phosphate phosphatase activity” refers to the ability to catalyze the conversion of D-mannitol-1 -phosphate and water to D-mannitol and inorganic phosphate. The mannitol- 1 -phosphate phosphatase enzyme can be from any suitable organism or may be synthetic. Suitable mannitol -1 -phosphate phosphatase enzymes may include, but are not limited to, enzymes categorized under Enzyme Commission (EC) number 3.1.3.22. Suitable enzymes include the mannitol- 1 -phosphate phosphatase enzyme from Eimeria tenella. The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from an Eimeria tenella gene encoding the amino acid sequence of SEQ ID NO: 18. The mannitol- 1 -phosphate phosphatase gene may encode an amino acid sequence at least 70%, at leastPT-2221-WO-PCT80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 18.

[0044] The exogenous nucleic acids in the recombinant cells described herein 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. Promoters may include, but are not limited to, 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 (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFp); phosphoglucomutase 1 promoter (PGMlp); 3 -phosphoglycerate kinase promoter (PGKlp; e.g., SEQ ID NO:22); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B (SEQ ID NO:23); ScPDClp (SEQ ID NO: 17); KmTDH3p (SEQ ID NO:21); KmPDClp (SEQ ID NO:24).

[0045] The exogenous nucleic acids in the recombinant cells described herein 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 may include, but are not limited to, GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt; SEQ ID NO:288); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); fructose-bisphosphate aldolase (FBAlt; SEQ ID NO:28); TEF1; i so- 1 -cytochrome c (CYC1); KmHXK2t (SEQ ID NO:27); KmGPMlt (SEQ ID NO:25); KmPGKlt (SEQ ID NO:26); ScURA3t (SEQ ID NO:8); URA3; ADH1; and ScGALlO.

[0046] 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 said polynucleotide is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0047] The polynucleotides described herein may be provided as part of a construct. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratoryPT-2221-WO-PCTmethods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. The construct may be a vector including a promoter operably linked to the polynucleotide encoding the mannitol -1 -phosphate dehydrogenase or the mannitol- 1 -phosphate phosphatase. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. The vector may be a plasmid, which refers to a circular doublestranded DNA loop into which additional DNA segments may be integrated.

[0048] The disclosure also provides fermentation methods for the production of D-mannitol using the recombinant cells described herein. The fermentation methods include the step of fermenting a substrate using the genetically engineered yeast cells or the genetically engineered bacterial cells described herein to produce D-mannitol. The fermentation method can include additional steps, as would be understood by a person 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 ethanol from the fermentation broth.

[0049] The fermentation substrate can comprise a starch. Starch can be obtained from a natural source, such as a plant source. Starch can also be obtained from a feedstock with high starch or sugar content, including, but not limited to corn, sweet sorghum, fruits, sweet potato, rice, barley, sugar cane, sugarbeets, wheat, cassava, potato, tapioca, arrowroot, peas, or sago. The fermentation substrate may be from lignocellulosic biomass such as wood, straw, grasses, or algal biomass, such as microalgae and macroalgae. The fermentation substrate may include cellulosic or lignocellulosic biomass. The fermentation substrate may be from grasses, trees, or agricultural and forestry residues, such as corn cobs and stalks, rice straw, sawdust, and wood chips. The fermentation substrate can also comprise a sugar, such as glucose (dextrose) or sucrose, and / or a polysaccharide, such as maltodextrin. The fermentation substrate may be physically (e.g., heat, pressure, and the like) or chemically (e.g., acid, hydrolysis, enzyme treatment, such as glucoamylase, and the like) pretreated prior to or during the fermentation process.

[0050] Media for fermentation of the engineered cells described herein can be supplemented with various components. For example, media for fermentation of the engineered cells described herein can be supplemented with a glucoamylase, e.g., the glucoamylase Spirizyme™PT-2221-WO-PCT(Novozymes, Bagsvaerd, Denmark) and / or the amyloglucosidase from Aspergillus niger sold under the trade name AMG 300L™ by Sigma- Aldrich.

[0051] The fermentation process can be run under various conditions. The fermentation temperature, i.e., the temperature of the fermentation broth during processing, may be 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 50 °C, 26 °C to 45 °C, 27 °C to 40 °C, 280to 35 °C, or 29 °C to 32 °C. The fermentation temperature may be maintained at a temperature of, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50°C, or any value in between or range thereof. However, a skilled artisan will recognize that the fermentation temperature is not limited to any specific range recited herein and may be modified as appropriate.

[0052] The pH of a culture medium described herein may be controlled for optimal D-mannitol production. The pH of the culture or a fermentation mixture of an engineered cell described herein may be in the range of between 3.0 and 7.5. The pH may be maintained for at least part of the incubation at 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.8, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and / or 7.5. The pH may be maintained at a range between 6.0 and 7.0, between 6.2 and 6.7, or between 6.3 to 6.6.

[0053] The engineered cell (yeast and / or bacteria) may be cultured for approximately 24-120+ hours. For example, the engineered cell may be cultured for approximately 12, 18, 24, 32, 48, 72, 96 hours, 114 hours, 120 hours, or more than 120 hours. The engineered cell described herein may be cultured for approximately 48 to 120 hours. A culture (fermentation) time of about 48 hours or 120 hours can be a representative time for similar commercial-scale fermentation processes. Accordingly, a 48-hour or 120-hour time point can be used to compare the fermentation performance of different genetically engineered cell strains.

[0054] Reaction parameters can be measured or adjusted during the production of D-mannitol. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox- potential, concentration of reaction substrate and / or product, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, ethanol concentration, fermentation substrate concentration, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of anPT-2221-WO-PCTion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products), physical / mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as thermodynamic parameters, such as temperature, light intensity / quality, etc.). Sensors to measure the parameters described herein are well known to one of ordinary skill in the art.

[0055] 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 cell (yeast and / or bacteria) and / or genetic modification of the cell used in the fermentation process. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be adjusted via a combination of cell selection or modification and the selection of fermentation process conditions.

[0056] The final D-mannitol titer may be at least at least 20 g / L, at least 25 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, or at least 70 g / L.EXAMPLES

[0057] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0058] Strain numbering is consistent throughout the Examples. For example, strain 1.3 in Example 1 is the same strain as strain 1.3 in Examples 2.Example 1 - Kluyveromyces marxianus Strain Engineering

[0059] The proposed pathway to produce D-mannitol from sucrose, starch, and / or glucose is shown in FIG. 1. In this pathway, glucose and / or fructose are converted to fructose-6-phosphate. The D-fructose-6-phosphate is converted to D-mannitol- 1 -phosphate by a hexose phosphate dehydrogenase enzyme, for example mannitol -1 -phosphate dehydrogenase. Finally, the D-mannitol-1 -phosphate is converted to D-mannitol by a hexose phosphate phosphatase enzyme, for example, mannitol- 1 -phosphate phosphatase. To test this pathway and demonstrate the productionPT-2221-WO-PCTof D-mannitol in a genetically engineered yeast (K. marxianus), the strains described in this, and subsequent examples, were built and tested.Strain 1.1

[0060] Strain 1.1 is a yeast cell Kluyveromyces marxianus CD21, deposited under Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain (BCCM MUCL) designation 58456.Stain 1.2

[0061] Strain 1.2 is an uracil auxotroph derivative of strain 1.1 with a deletion of the URA3 locus.Strain 1.3

[0062] Strain 1.2 was transformed with SEQ ID NO:1 and SEQ ID NO:2. SEQ ID NO:1 contained a 5’ CYB2 flanking sequence (SEQ ID NO:3), the ScURA3 promoter (SEQ ID NO:5), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:6). SEQ ID NO:2 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:7), the ScURA3 terminator (SEQ ID NO: 8), and a 3’ CYB2 flanking sequence (SEQ ID NO: 4). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for deletion of the gene at the CYB2 locus. A PCR verified isolate was designated strain 1.3.Strain 1.4

[0063] Strain 1.2 was transformed with SEQ ID NO:9 and SEQ ID NO: 10. SEQ ID NO:9 contained a 5’ PFK1 flanking sequence (SEQ ID NO: 11), the ScURA3 promoter (SEQ ID NO: 5), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:6). SEQ ID NO: 10 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:7), the ScURA3 terminator (SEQ ID NO:8), and a 3’ PFK1 flanking sequence (SEQ ID NO: 12). Resulting transformants were streaked for single colony isolation on ScD-ura plates and a single colony was selected. The selected colony was evaluated by colony PCR for knockout of the PKF1 gene and designated strain 1.4.Strain 1.5a-dPT-2221-WO-PCT

[0064] Strain 1.2 was transformed with SEQ ID NO: 13 and SEQ ID NO: 14. SEQ ID NO: 13 contained a 5’ CYB2 flanking sequence (SEQ ID NO:3), the KmENOl terminator (SEQ ID NO: 15), a polynucleotide sequence encoding the mannitol- 1 -phosphate dehydrogenase of SEQ ID NO: 16, the ScPDCl promoter (SEQ ID NO: 17), the ScURA3 promoter (SEQ ID NO: 5), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:6). SEQ ID NO: 14 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:7), the ScURA3 terminator (SEQ ID NO:8), the ScPDCl promoter (SEQ ID NO: 17), a polynucleotide sequence encoding the mannitol- 1 -phosphate phosphatase of SEQ ID NO: 18, the KmENOl terminator (SEQ ID NO: 15), and a 3’ CYB2 flanking sequence (SEQ ID NO:4). Resulting transformants were streaked for single colony isolation on ScD-ura places and single colonies were selected. The selected colonies were evaluated by colony PCR for incorporation of the indicated coding sequences. PCR verified sister strains were designated strains 1.5a, 1.5b, 1.5c, and 1.5d.Strain 1.6

[0065] Strain 1.4 was transformed with the Cre recombinase plasmid of SEQ ID NO:292. Removal of the ScURA3 selection marker gene was confirmed by lack of growth on ScD-ura plates. The resulting transformants were evaluated by colony PCR for removal of the ScURA3 selection marker gene. A PCR verified isolate was designated strain 1.6.Strain 1.7a-d

[0066] Strain 1.6 was transformed with SEQ ID NO: 13 and SEQ ID NO: 14. Resulting transformants were streaked for single colony isolation on ScD-ura places and single colonies were selected. The selected colonies were evaluated by colony PCR for incorporation of the indicated coding sequences. PCR verified sister strains were designated strains 1.7a, 1.7b, 1.7c, and 1.7d.Strain 1.8a-c

[0067] Strain 1.2 was transformed with SEQ ID NO:31 and SEQ ID NO:32. SEQ ID NO:31 contained a 5’ GPD1 flanking sequence (SEQ ID NO:29), the KmENOl terminator (SEQ ID NO: 15), a polynucleotide sequence encoding the mannitol -1 -phosphate dehydrogenase of SEQ ID NO: 16, the ScPDCl promoter (SEQ ID NO: 17), the ScURA3 promoter (SEQ ID NO:5), and a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:6). SEQ ID NO:32 contained a 3’ portion of the ScURA3 selection marker gene (SEQ ID NO:7), the ScURA3 terminator (SEQPT-2221-WO-PCTID N0:8), the ScPDCl promoter (SEQ ID NO: 17), a polynucleotide sequence encoding the mannitol- 1 -phosphate phosphatase of SEQ ID NO: 18, the KmENOl terminator (SEQ ID NO: 15), and a 3’ GPD1 flanking sequence (SEQ ID NO:30). Resulting transformants were streaked for single colony isolation on ScD-ura places and single colonies were selected. The selected colonies were evaluated by colony PCR for incorporation of the indicated coding sequences. PCR verified sister strains were designated strains 1.8a, 1.8b, and 1.8c.Example 2 - Microtiter Plate Fermentation Assay

[0068] Strains 1.3, 1.4, 1 ,5a-d, and 1 ,7a-d were fermented in flower plates to assay production of D-mannitol and ethanol and consumption of glucose.

[0069] Strains were struck on a ScD-ura plates and grown until single colonies formed (1-3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a 96 deep 48-well microtiter flower plate containing 1000 microliters of ScD-Ura media (6.7g / L yeast nitrogen base without amino acids, 2.0 g / L Synthetic Complete amino acid mix, 20 g / L glucose), and incubated for 24 hours at 30°C and 900 RPM in an orbital shaker. The overnight cultures were transferred to a 48 well flower plate containing 1000 microliters of 200 g / L dextrose DMlu media (Table 2) to an OD600 = 0.2, incubated at 30°C, 80% relative humidity, and 900 RPM in an orbital shaker. Samples were taken at 48 hours (strains 1.3 and 1.5a-d) or 120 hours (strains 1.4 and 1 ,7a-d) for analysis by HPLC to determine D-mannitol, ethanol, and glucose titers. Strains including the PFK knockout were allowed to go longer prior to sampling due to the slower growth rate. Results are shown in Table 5.Table 2: DMlu Production Medium - DextrosePT-2221-WO-PCTTable 3: lOOOx trace elementsTable 4: 1OOOX DM1 vitamin solutionTable 5: Flower Plate HPLC ResultsPT-2221-WO-PCTnd = not detected

[0070] Strains 1.5a-d, which express the mannitol- 1 -phosphate dehydrogenase of SEQ ID NO: 16 and the mannitol -1 -phosphate phosphatase of SEQ ID NO: 18 produced between 33-46 g / L of mannitol, but also produced about 10 g / L ethanol. Similarly, the equivalent strain (1.3, lacking the mannitol -1-phosphase dehydrogenase and mannitol -1 -phosphate phosphatase expression but including the loss of the native gene at the CYB2 locus) produced a significant amount of ethanol, but did not produce mannitol.

[0071] Strains 1.7a-d, which express the mannitol- 1 -phosphate dehydrogenase of SEQ ID NO: 16 and the mannitol- 1 -phosphate phosphatase of SEQ ID NO: 18 and included a deletion of the PFK1 gene, produced approximately 80 g / L mannitol but did not produce any ethanol. Similarly, the equivalent strain (1.4, lacking the mannitol- 1-phosphase dehydrogenase and mannitol- 1 -phosphate phosphatase expression but with the PFK1 deletion) did not produce ethanol or mannitol. These results indicate that the deletion of the native PFK1 gene reduces ethanol production and increases mannitol production. However, based on the results see for strains 1.5a-d, the PFK1 deletion is not required for mannitol production.

[0072] While a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct locus, it may have had multiple copies of thePT-2221-WO-PCTsequence integrated into its genome, or a frameshift or other mutation caused an individual sister(s) to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters, for example strain 1.7d. However, the results still demonstrate that the engineering of yeast cells to produce D-mannitol.Example 3: Shake Flask Fermentation Assay

[0073] Strains 1.5d and 1.7a were assayed to evaluate production of D-mannitol and ethanol and consumption of glucose.

[0074] Strains were struck on a ScD-ura plates and grown until single colonies formed (overnight at 30 °C or 2-3 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 25 ml of ScD-ura medium (6.7g / L yeast nitrogen base without amino acids, 2.0 g / L Synthetic Complete amino acid mix, 20 g / L glucose) and incubated overnight at 30°C, 250 rpm with 70% humidity. The overnight cultures were diluted into 40 ml DMlu medium (Table 2), with either 300 g / L or 250 g / L dextrose (in place of the 200 g / L indicated in Table 2) as indicated in Table 6, in 250 ml baffled Erlenmeyer flasks to achieve an initial cell density of OD600 = 0.1 or 0.4 as indicated in Table 6, and incubated at 30°C, 250 rpm with 70% humidity. Samples were withdrawn at 68, 95, 142, and 166 hours into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, and stored at -20°C until analysis. Samples were analyzed for D-mannitol, ethanol, and glucose by HPLC. Results are shown in Table 6.

[0075] Results show that strain 1.7a produced approximately 125 g / L mannitol in shake flask fermentation with low residual glucose and ethanol when provided 250 g / L dextrose. Increasing initial dextrose to 300 g / L led to incomplete dextrose consumption at the final 166-hour timepoint with mannitol titer of approximately 50 g / L. Increasing both initial dextrose to 300 g / L and initial OD to 0.4 increased dextrose consumption but did not increase mannitol titer. Strain 1.5d was able to consume 250 g / L initial dextrose within 68 hours and produced approximately 24 g / L mannitol with approximately 48 g / L ethanol. Increasing initial dextrose to 300 g / L slowed dextrose consumption and increased ethanol titer but did not increase mannitol titer.Table 6:PT-2221-WO-PCTnd is not detected, below instrument detection limitExample 4: Microtiter Plate Fermentation Assay

[0076] Strains 1.7a and 1.8a-c were fermented in flower plates to assay production of D-mannitol and ethanol and consumption of glucose.

[0077] Strain 1.7a was restruck on ScD-ura plates and seven (7) individual colonies were chosen for the flower plate fermentation assay. Strains were struck on a ScD-ura plates and grown until single colonies formed (1-3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). A small patch of biomass was used to inoculate a 96 deep 48-well microtiter flower plate containingPT-2221-WO-PCT1000 microliters of ScD-Ura media (6.7g / L yeast nitrogen base without amino acids, 2.0 g / L Synthetic Complete amino acid mix, 20 g / L glucose), and incubated for 24 hours at 30°C and 900 RPM in an orbital shaker. The overnight cultures were transferred to a 48 well flower plate containing 1000 microliters of 200 g / L dextrose DMlu media (Table 2) to an OD600 = 0.2, incubated at 30°C, 80% relative humidity, and 900 RPM in an orbital shaker. Samples were taken at 88 hours for analysis by HPLC to determine D-mannitol, ethanol, and glucose titers. Results are shown in Table 7.Table 7:nd is not detected, below instrument detection limit

[0078] Results show that strains including the genes encoding the mannitol- 1 -phosphate dehydrogenase of SEQ ID NO: 16 and the mannitol -1 -phosphate phosphatase of SEQ ID NO: 18 integrated at the GPD1 locus (Strains 1.8a-c) were able to produce at least 17.0 g / L D-mannitol. Based on these results, its likely that deletion of the PFK gene, which is deleted in the 1.7a strain, would further improve the production of D-mannitol in these strains.

[0079] While a strain is PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct locus, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister(s) to vary from the others. The results here suggest that similar transformation occurrences are present in the sisters of strain 1.8. Additionally, following the assay, it was discovered thatPT-2221-WO-PCTsome ScD-ura plates used for seeding were improperly prepared, which accounts for the high residual glucose and ethanol production seen in the cultures of strains 1.7a(l), 1.7a(5), and 1.7a(6) However, the results still demonstrate the engineering of yeast cells to produce D-mannitol.Example 5: Microtiter Plate Fermentation Assay

[0080] Strain 1.7a was fermented using various carbon feedstock to assay production of mannitol. Strain 1.7a was struck on an ScD-ura plate and grown until single colonies formed (1-3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 40 ml of ScD-ura medium (6.7g / L yeast nitrogen base without amino acids, 2.0 g / L Synthetic Complete amino acid mix, 20 g / L glucose) and incubated overnight at 30°C, 250 rpm with 70% humidity. Microtiter flower plates containing 800 microliters of the DMul medium indicated in Table 8 (DMul base medium of Table 2, where the 200 g / L dextrose was replaced with the indicated carbon source in Table 8) were inoculated to an OD600 of 0.2 from the overnight culture and incubated for 48 hours at 30°C, 80% relative humidity, and 900 RPM in an orbital shaker. Samples were analyzed for D-mannitol, ethanol, glycerol, and glucose by HPLC. Results are shown in Table 8.Table 8.PT-2221-WO-PCT

[0081] Results show that strain 1 ,7a was able to product mannitol from dextrose, fructose, and sucrose carbon sources as well as the combination of dextrose and fructose.Example 6: Shake Flask Fermentation Assays

[0082] Strain 1.7a was fermented using various carbon feedstock to assay production of mannitol. Strain 1.7a was struck on struck on an ScD-ura plate and grown until single colonies formed (1-3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scraped into sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 40 ml of ScD-ura medium (6.7g / L yeast nitrogen base without amino acids, 2.0 g / L Synthetic Complete amino acid mix, 20 g / L glucose) and incubated overnight at 30°C, 250 rpm with 70% humidity. Baffled Erlenmeyer shake flasks containing 40 mL of the medium indicated in Table 9 were inoculated from the overnight cultures to an OD600 of 0.2 and incubated at 30°C, 250 rpm, and 70% humidity. Samples were withdrawn after 144 hours of incubation into microcentrifuge tubes, centrifuged at 14,000 x g for 2 min, and stored at -20°C until analysis. Samples were analyzed for D-mannitol, ethanol, glycerol, and glucose by HPLC. In Table 9, the media is the DMul medium of Table 2. In some shake flasks, the 200 g / L dextrose of the DMul medium is replaced with either 200 g / L sucrose, or 200g / L maltodextrin. Media using maltodextrin as a carbon source also included added glucoamylase (GA) enzyme at the concentration listed in Table 9. Results are shown in Table 9.PT-2221-WO-PCTTable 9.

[0083] Results show that strain 1 ,7a was able to produce mannitol from a maltodextrin carbon source when a glucoamylase enzymes was also present.PT-2221-WO-PCTExample 7: Genetically Modified Kluyveromyces lactis Strains

[0084] The proposed pathway to produce D-mannitol from sucrose, starch, and / or glucose is shown in FIG. 1. In this pathway, glucose and / or fructose are converted to fructose-6-phosphate. The fructose-6-phosphate is converted to D-mannitol- 1 -phosphate by a mannitol- 1 -phosphate dehydrogenase enzyme. Finally, the D-mannitol- 1 -phosphate is converted to D-mannitol by a mannitol- 1 -phosphate phosphatase. To test this pathway and demonstrate the production of D-mannitol in a genetically engineered yeast (K lactis), the strains described in this, and subsequent examples were built and tested.Strain 2.1

[0085] Strain 2.1 is a yeast cell Kluyveromyces lactis CD684, which is an uracil auxotroph with a deletion of the URA3 locus. The K lactis CD684 cell is equivalent to the Kluyveromyces lactis (Dombrowski) van der Walt MYA-2288™, deposited with the American Type Culture Collection (ATCC) and available under the strain designation 22A295-1.Strain 2.2

[0086] Strain 2.1 was transformed with SEQ ID NO:33 and SEQ ID NO:34. SEQ ID NO:33 contained a 5’ K lactis PFK1 flanking sequence (SEQ ID NO:35), the KmENOl terminator (SEQ ID NO: 15), a polynucleotide sequence encoding the mannitol -1 -phosphate dehydrogenase of SEQ ID NO: 16, the ScPDCl promoter (SEQ ID NO: 17), the ScURA3 promoter (SEQ ID NO:5), and a 5’ portion of the ScURA3 selection marker gene (SEQ ID NO:6). SEQ ID NO:34 contained 3’ portion of the ScURA3 selection marker gene (SEQ ID NO: 7), the ScURA3 terminator (SEQ ID NO:8), the ScPDCl promoter (SEQ ID NO: 17), a polynucleotide sequence encoding the mannitol-1-phosphate phosphatase of SEQ ID NO: 18, the KmENOl terminator (SEQ ID NO: 15), and a 3’ K. lactis PFK1 flanking sequence (SEQ ID NO:36). Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the indicated sequence. PCR verified isolates were designated strains 2.2a-d.Example 8: Fermentation Assay

[0087] Strains 2.2a-d were fermented in a microtiter plate to assay production of mannitol. Strains 2.2a-d were struck on ScD-ura plates and grown until single colonies formed (1-3 days at 30 °C or 2-5 days at room temperature (about 25 °C)). Cells from the ScD-ura plate were scrapedPT-2221-WO-PCTinto sterile seed vessels (250 ml baffled Erlenmeyer flasks) containing 40 ml of ScD-ura medium (6.7g / L yeast nitrogen base without amino acids, 2.0 g / L Synthetic Complete amino acid mix, 20 g / L glucose) and incubated overnight at 30°C, 250 rpm with 70% humidity. Microtiter flower plates containing 800 microliters of the DMul medium (Table 2, except the 200 g / L dextrose was replaced with 100 g / L dextrose) were inoculated to an OD600 of 0.2 from the overnight culture and incubated for 96 hours at 30°C, 80% relative humidity, and 800 RPM in an orbital shaker. Samples were analyzed for D-mannitol and dextrose by HPLC. Results are shown in Table 10.Table 10.

[0088] Result show that K. lactis strains expressing the mannitol- 1 -phosphate dehydrogenase of SEQ ID NO: 16 and the mannitol- 1 -phosphate phosphatase of SEQ ID NO: 18 were able to produce D-mannitol and consumed all available dextrose in the 96 hour fermentation relaxation.

Claims

PT-2221-WO-PCTCLAIMSWhat is claimed is:

1. A genetically engineered yeast cell capable of producing D-mannitol, the engineered cell comprisingan exogenous polynucleotide sequence encoding a mannitol- 1 -phosphate dehydrogenase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16; andan exogenous polynucleotide sequence encoding a mannitol- 1 -phosphate phosphatase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 18.

2. The engineered cell of claim 1, wherein the yeast cell is selected from the group consisting of Kluyveromyces marxianus. Kluyveromyces lactis, Komagataella phaffii, Saccharomyces cerevisiae. Yarrowia lipolytica, and Moniliella pollinis.

3. The engineered cell of claim 1 or claim 2, wherein the mannitol- 1 -phosphate dehydrogenase is at least 80% identical to SEQ ID NO: 16; and the mannitol- 1 -phosphate phosphatase is at least 80% identical to SEQ ID NO: 18.

4. The engineered cell of any preceding claim, wherein the mannitol- 1 -phosphate dehydrogenase is at least 90% identical to SEQ ID NO: 16; and the mannitol- 1 -phosphate phosphatase is at least 90% identical to SEQ ID NO: 18.

5. The engineered cell of claim 1, wherein the engineered cell additionally comprises a deletion or disruption in a native phosphofructokinase- 1 (PFK1) gene.

6. The engineered cell of any one of claims 2-5, wherein, when the cell is a Kluyveromyces marxianus cell, the native PFK1 gene encodes a PFK1 enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 19.PT-2221-WO-PCT7. The engineered cell of any one of claims 2-6, wherein, when the cell is a Kluyveromyces marxianus cell, the native PFK1 gene is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:20.

8. The engineered cell of any preceding claim, wherein the mannitol- 1 -phosphate dehydrogenase is at least 80%, at least 85%, at least 90%, or at least 95% identical to of SEQ ID NO: 16; wherein the mannitol- 1 -phosphate phosphatase is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NOs: 18; and wherein the cell is capable of producing at least 25 g / L D-mannitol.

9. The engineered cell of any preceding claim, wherein the mannitol- 1 -phosphate dehydrogenase is at least 80%, at least 85%, at least 90%, or at least 95% identical to of SEQ ID NO: 16; wherein the mannitol- 1 -phosphate phosphatase is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NOs: 18; wherein the cell comprises a deletion or disruption in a native PFK1 gene encoding a polypeptide at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NOs: 19, and wherein the cell is capable of producing at least 70 g / L D-mannitol.

10. The engineered cell of any proceeding claim, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous promoter and / or a heterologous terminator.

11. The engineered cell of claim 10, wherein the promoter is selected from the group consisting of pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1 A promoter (ADHlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFlp); phosphoglucomutase 1 promoter (PGMlp); 3 -phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B; and PDC1 promoter.PT-2221-WO-PCT12. The engineered cell of claim 10 or 11, wherein the terminator is selected from the group consisting of GAL10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); fructose-bisphosphate aldolase terminator (FBAlt); TEF1; i so- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator.

13. A method for producing D-mannitol, the method comprising:contacting a substrate with the engineered cell of any proceeding claim, wherein the engineered cell produces at least 20 g / L, at least 25 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, or at least 70 g / L mannitol after 120 hours.

14. The method of claim 13, wherein the substrate comprises starch, glucose, sucrose, cellulosic biomass, or combinations thereof.

15. Use of the engineered cell of any one of claims 1-12 to produce D-mannitol.

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