Genetically modified yeast with reduced erythritol production

Abstract

This specification discloses genetically modified yeast cells characterized by the deletion or disruption of a native gene encoding an erythrose reductase enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of Sequence IDs 63 and 66.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 499,989, filed on 4 May 2023, which is incorporated herein by reference in its entirety.

[0002] References to sequence listings submitted via the Patent Center The contents of the sequence list XML file named "PT-1760-WO-PCT.xml", which was created on April 29, 2024, and submitted electronically through the Patent Center together with this application, and which is 177,337 bytes in size, are incorporated herein by reference in their entirety. [Background technology]

[0003] Erythritol is a natural tetracarbon sugar alcohol used as a food additive and sugar substitute. Erythritol is 60-70% sweeter than sucrose, but contains no calories, does not affect blood sugar, and does not cause tooth decay. Common uses of erythritol include beverages such as coffee, tea, nutritional supplements, juices, and soft drinks, as well as foods such as confectionery, biscuits, cookies, table sweeteners, and chewing gum. Erythritol is commercially produced through the fermentation of dextrose-containing substrates using yeast. While yeast has been used for many years in the production of erythritol by fermentation, improvements to the fermentation process are beneficial in increasing yield and production rate, resulting in a more sustainable and efficient fermentation process. Conversely, yeast used to produce erythritol may also be useful for the production of other polyols, and therefore, reducing erythritol production in yeast may also be beneficial. Accordingly, genetically modified yeast is provided herein, and in a fermentation process using this genetically modified yeast, erythritol production is regulated (i.e., increased or decreased). [Overview of the project]

[0004] This disclosure provides genetically modified yeast cells, which include deletions or disruptions of native genes encoding erythrose reductase enzymes that are 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 Sequence IDs 63 and 66. The yeast cells may be osmotically tolerant yeast cells. The yeast cells may also be cells of the subphylum Ustyraginomykothina. Yeast cells may be selected from the group consisting of Trichosporonoides megachiliensis, Trichosporonoides eoidocephalis, Trichosporonoides nigrecens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustyraginomisetes, Trichosporon, Yarowia liporitica, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium.

[0005] Yeast cells may be Moniliella polynis cells, in which the gene encoding the erythrose reductase enzyme 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 sequence numbers 61 and 64. The cells may have deletions of at least one allele of the gene encoding the erythrose reductase enzyme. The cells may have deletions of both alleles of the gene encoding the erythrose reductase enzyme.

[0006] When genetically modified cells are used in a fermentation process in the presence of dextrose, the titer and / or yield of erythritol production is reduced compared to the titer and / or yield of erythritol in an equivalent fermentation process using equivalent cells that do not have disruption or deletion of the native gene encoding erythritol reductase 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. 63 and 66.

[0007] In the genetically modified cells described herein, at least one copy of the gene encoding the erythrose reductase (ER) enzyme may be replaced with a native or exogenous polynucleotide sequence. The native or exogenous polynucleotide sequence may be operably linked to a heterologous or artificial promoter. Heterogeneous or artificial promoters 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).

[0008] Also provided herein are genetically engineered Moniliella polinis cells comprising the deletion or disruption of a native gene encoding an erythrose reductase enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 63 and 66. The native gene may be 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: 61 and 64.

[0009] In Moniliella polynis cells, the native gene may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61, and the ER enzyme may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 63. The native gene may be at least 80% identical to SEQ ID NO: 61, and the ER enzyme may be at least 80% identical to SEQ ID NO: 63. The native gene may be at least 90% identical to SEQ ID NO: 61, and the ER enzyme may be at least 85% identical to SEQ ID NO: 63. The native gene may be at least 95% identical to SEQ ID NO: 61, and the ER enzyme may be at least 90% identical to SEQ ID NO: 63. Both alleles of the native gene may be deleted if they are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61.

[0010] In Moniliella polynis cells, native genes may be 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 sequence numbers 64, and ER enzymes may be 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 sequence numbers 66. Native genes may be at least 80% identical to sequence number 64, and ER enzymes may be at least 80% identical to sequence number 66. Native genes may be at least 90% identical to sequence number 64, and ER enzymes may be at least 85% identical to sequence number 66. The native gene may be at least 95% identical to SEQ ID NO: 64, and the ER enzyme may be at least 90% identical to SEQ ID NO: 66. Both alleles of the native gene may be deleted if they are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 64.

[0011] This disclosure also provides a fermentation method comprising contacting a dextrose-containing substrate with genetically modified cells described herein, wherein fermentation of the substrate by the genetically modified cells produces less erythritol than that by equivalent cells without deletion or disruption. The fermentation temperature may be 25°C–45°C, 30°C–40°C, or 32°C–37°C (including the endpoints). The volume oxygen uptake rate (OUR) may be in the range of 5–80, 10–75, 15–70, 20–60, 30–50, or 40–50 mmol O2 / (L·h). The rate, titer, and / or yield of erythritol production may be reduced compared to equivalent fermentation performed using equivalent yeast cells in which the native gene encoding the erythritol reductase enzyme is not deleted or disrupted. The dextrose concentration may be at least 100 g / L.

[0012] When fermentation is carried out at 35°C for 96 hours, the erythritol titer can be less than 20, 18, 15, 12, 10, 5, 1, or 0.5 g / L. The erythritol production rate is 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, or 0.1 g / L. -1 h -1 It may be less than 1%. The yield of erythritol may be 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than 1%. The fermentation method may not produce erythritol, or the process may be substantially free of erythritol.

[0013] Furthermore, the use of genetically modified cells in fermentation processes as described herein is also provided herein. [Brief explanation of the drawing]

[0014] This patent or application includes at least one drawing made in color. A copy of the publication of this patent or patent application, including the color drawing, will be provided by the Patent Office upon request and payment of the necessary fees.

[0015] The drawings generally illustrate various embodiments considered herein, not as limitations, but as examples. [Figure 1] The natural pentose phosphate pathway (dotted line and arrow) and the natural glycolysis pathway (solid line and arrow) in Moniliella polynis are shown. [Figure 2] The enzyme activity of the in vitro assay described in Example 1 is shown. [Figure 3] The concentrations (g / L) of erythritol, xylitol, and glycerol metabolites after 96 hours of shaking flask fermentation of the strain outlined in Example 3 are shown. The data label reports the xylitol concentration. [Figure 4] The concentrations (g / L) of erythritol, xylitol, and glycerol metabolites after 96 hours of shaking flask fermentation of the strain outlined in Example 4 are shown. [Figure 5]Shows the concentrations (g / L) of erythritol, xylitol, and glycerol metabolites after 96 hours of shake flask fermentation of the strain outlined in Example 5. [Figure 6] Shows the concentrations (g / L) of erythritol, xylitol, and glycerol metabolites after 96 hours of shake flask fermentation of the strain outlined in Example 6. [Figure 7] Shows the concentrations (g / L) of erythritol, xylitol, and glycerol metabolites after 96 hours of shake flask fermentation of the strain outlined in Example 7. The data label reports the concentration of erythritol. [Figure 8] Shows the metabolite concentrations (g / L) for the fermentation outlined in Example 8. The data label reports the concentration of erythritol. **DETAILED DESCRIPTION OF THE INVENTION**

[0016] Here, specific aspects of the disclosed subject matter are referred to in detail, and experimental examples thereof are illustrated in part in the accompanying drawings. The disclosed subject matter is described with the appended claims, but it will be understood that the illustrated subject matter is not intended to limit the disclosed subject matter to the claimed scope.

[0017] In this document, the terms "a", "an", or "the" are used to include one or more than one unless the context clearly indicates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents referred to in this document are hereby incorporated by reference in their entirety as if each were individually incorporated by reference. If there is a lack of consistency in the use between this document and the documents incorporated by reference in this way, the use in the incorporated reference should be considered as supplementing the use in this document. In case of incompatible contradictions, the use in this document prevails.

[0018] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical limits explicitly listed as range limits, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly listed. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (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%). The notation "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise indicated. Similarly, the notation "approximately X, Y, or approximately Z)" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise indicated.

[0019] Unless otherwise stated, ppm (parts per million), percentages, and ratios are based on weight. Weight-based percentages are also referred to as weight % or %(weight) below.

[0020] This disclosure relates to various recombinant cells genetically engineered to reduce erythritol production. Generally, the recombinant cells described herein are capable of producing erythritol and are characterized by the deletion or disruption of a native gene encoding the erythritol reductase enzyme. This disclosure further provides a fermentation method using the genetically engineered yeast described herein.

[0021] Generally speaking, the recombinant cells described herein are yeast cells. As used herein, “yeast” refers to a eukaryotic unicellular microorganism classified as a member of the Kingdom Mycology. Yeasts are unicellular organisms that evolved from multicellular ancestors, and some species retain multicellular characteristics, such as forming strings of linked budding cells known as pseudohyphae or false hyphae. Yeast cells are also sometimes referred to as yeast-like cells in the Art, and as used herein, “yeast cells” encompass both yeast and yeast-like cells. Suitable yeasts and yeast-like host cells for modification include Saccharomyces cerevisiae, Chomagataera, Cluiveromyces (e.g., Cluiveromyces lactis, Cluiveromyces marsianus), Yarowia liporitica, Isatakenchia orientalis, Pichia galeiformis, Pichia YB-4149 (NRRL designated), Pichia pastris, Candida (e.g., Candida magnoliae, Candida etanorica), Pichia decericola, Pichia membranous membrane, and Pichia fur. Examples of yeast cells that may or may not be included are Mentans, Aspergillus, Trichoderma, Micerifsola thermophylla, Moniliella (e.g., Moniliella polinis), Pfaffia, Yamadajima, Hansenula, Pichia kudryavzevii, Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trichosporonoides eoidocephalis, Trichosporonoides nigrecens), Pseudozyma tsukubaensis, Trigonopsis variabilis, Penicillium and Torula. Those skilled in the art will understand the requirements for selecting suitable yeast cells, and the recombinant yeast cells of this disclosure are not limited to those expressly enumerated herein. Methods for genetically engineering yeast cells are known and described in the art, and those skilled in the art will understand the methods necessary to transform and genetically engineer suitable yeast cells.

[0022] Suitable yeast cells may be from the Basidiomycota and Ustyraginomikothina subphylum. Suitable yeasts from the Ustyraginomikothina subphylum include Ustyrago (e.g., U. synodontis, U. meidis, U. sphaerogena, U. cordal, U. sitaminea, U. coisis, U. syntelismae, U. esculenta, U. neglecta, U. crus galli, Ustyrago avenae), Sporisolium (e.g., Sporisolium excertum), and Moniliella (e.g., Moniliella polinis, M. to Examples include, but are not limited to, Mentha, M. acetabtans, M. foncsecae, M. madida, M. megachiliensis, M. osedocephalis, M. nigrecens, Pseudozyma (e.g., Pseudozyma tsukubaensis), and Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trichosporonoides eoidocephalis, Trichosporonoides nigrecens). Yeasts of the subphylum Ustyraginomikochina are known and described in the art as promising productive organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol, as well as other valuable biotechnology applications.For example, Geiser et al. (Prospecting the biodiversity of the fungal family Ustilaginacceae for the production of value-added chemicals ed chemicals,” Fungal Biol Biotechnol, 2014, 1:2), Feldbrugge et al., (“The biotechnological use and potential of plant pathogenic smut fungi”, Appl Microbiol Biotechnol,2013,97(8):3253-65), Guevarra et al.,("Accumulation of itaconic,2-hydroxyparaconic,itatartaric,and malic acids by strains of the genus Ustilago,Agric.Biol.Chem.,1990,54(9),2353-2358)", and Moon et al.,("Biotechnological production of erythritol and its applications,”Appl Microbiol See Biotechnol, 2010, 86:1017-1025.

[0023] Suitable yeast cells have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein, the "active pentose phosphate pathway" refers to the pathway that produces glucose-6-phosphate and NADP. + Or NAD+(NAD(P) +This refers to the expression of one or more functional enzymes that convert water to NADPH or NADH (NAD(P)H), CO2, and ribulose-5-phosphate. Following the non-oxidative step, this pathway may also produce other pentose (i.e., 5-carbon) sugars. For example, depending on the enzyme activity present, the pentose phosphate pathway may produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose-6-phosphate, combinations thereof, etc. The active pentose phosphate pathway may be naturally present in yeast cells or can be introduced into yeast cells by genetic engineering.

[0024] Yeast cells may be osmotically tolerant yeast cells. As used herein, “osmotically tolerant” means yeast capable of growing and regenerating under high osmotic conditions, e.g., at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v), or at least 60% (w / v) of glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), or at least 15% (w / v) of sodium chloride. Species and strains of osmotically tolerant yeast are known and described in the art and include many species of yeast used in industrial fermentation processes. Similarly, methods for assaying yeast osmotic tolerance are known and described in the art. For example, see Tiwari, S. et al., ("Nectar yeast community of tropical flowering plants and assessment of their osmotolerance and xylitol-producing potential", Current Microbiology, 2022, 79:28).

[0025] Recombinant yeast cells may be recombinant Moniliella cells, such as Moniliella polynis cells. Figure 1 shows the predicted natural pentose phosphate and glycolysis pathway in Moniliella polynis. Moniliella has been used for a long time in the fermentation production of erythritol, and methods for genetically modifying and fermenting Moniliella are known and described in the art. See, for example, Li et al. ("Methods for genetic transformation of filamentous fungi", 2017, Microb Cell Fact, 16:168).

[0026] Various plasmids and methods for the transformation of Moniliella are also described in the following examples. For example, Moniliella can be transformed using a binary polynucleotide sequence in which the desired exogenous polynucleotide is incorporated into a specific locus after recombination, and a selection marker can be expressed intracellularly. Suitable selection markers are known and described in the art. Selective markers may include, but are not limited to, amdS (e.g., degraded into the 3' portion (sequence number 43) and the 5' portion (sequence number 50)), the G418 resistance gene (e.g., degraded into the 3' portion (sequence number 48) and the 5' portion (sequence number 51)), the zeosin resistance gene (e.g., degraded into the 3' portion (sequence number 44) and the 5' portion (sequence number 45)), nulceotricin N-acetyltransferase (NAT) (e.g., degraded into the 3' portion (sequence number 47) and the 5' portion (sequence number 46)), and the invertase gene (SUC2) (e.g., the 3' portion (sequence number 49) and the 5' portion (sequence number 52)).

[0027] The recombinant cells described herein may include one or more exogenous polynucleotide sequences encoding one or more polypeptides that, when expressed, regulate the activity of the recombinant cells. For example, if genetically engineered cells can produce polyols (e.g., xylitol, ribitol, arabitol, etc.), the incorporation of one or more exogenous polynucleotide sequences encoding one or more polypeptides may increase the production of such polyols when genetically engineered yeast cells are used in a fermentation process to produce polyols from glucose.

[0028] The terms "glucose" and "dextrose" are used interchangeably herein and refer to D-glucose unless expressly indicated otherwise.

[0029] As used herein, “exogenous” refers to genetic material or its expression products that originate from outside the host organism. For example, exogenous genetic material or its expression products may be a modified form of genetic material native to the host organism, may originate from another organism, may be a modified form of a component derived from another organism, or may be a synthetically derived component. For example, the K. lactis invertase gene is exogenous when introduced into S. cerevisiae.

[0030] As used herein, “natural” means genetic material or its expression products found within the genome of wild-type host cells, apart from inter-individual variations that do not affect function or expression. For the purposes of this application, the Moniliella polynis cells “Moniliella tomentosa val polynis TCV364,” described in U.S. Patent No. 6,440,712 (which is incorporated herein by reference in its entirety) and deposited on March 28, 1997, under the Budapest Convention, with BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de l'Universite Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) under the number MUCL40385, are considered wild-type Moniliella polynis cells.

[0031] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequences and structures necessary to give the listed polymers their function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refers to a protein that catalyzes a chemical reaction. Any enumeration of a particular enzyme is understood to include cofactors, coenzymes, and metals necessary for the enzyme to function properly, either independently or as part of a biosynthetic pathway. Table 1 provides an overview of amino acids as understood in the art, along with their three-letter and one-letter symbols. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0032] [Table 1]

[0033] Mutants or sequences having substantial identity or homology to the polypeptides described herein may be used in the implementation of the recombinant cells, compositions, and methods disclosed herein. Such sequences may be referred to as mutant or modified sequences. That is, polypeptide sequences may be modified while still retaining the ability to exhibit the desired activity. Generally, mutant or modified sequences may have more than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild type, naturally occurring polypeptide sequences, or mutant polypeptides described herein.

[0034] As used herein, the terms “% sequence identity,” “% identity,” and “percent identity” are used interchangeably and refer to the percentage of residue matching between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods for amino acid and nucleic acid sequence alignment are well known. Sequence alignment and sequence identity generation include global and local alignments, which are performed using computational approaches. Alignment can be performed using default parameters with BLAST (Basic Local Alignment Search Tool) version 2.2.31 software from the National Center for Biological Information (NCBI). Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters. Maximum target sequences: 100, Short query: Automatically adjust parameters for short input sequences, Expected threshold: 10, Word size: 6, Maximum match in query range: 0, Matrix: BLOSUM62, Gap cost: (Presence: 11, Expansion: 1), Composition adjustment: Conditional composition score matrix adjustment, Filter: Not selected, Mask: Not selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequences: 100, Short query: Automatically adjust parameters for short input sequences, Expected threshold: 10, Word size: 28, Maximum match in query range: 0, Match / mismatch score: 1, -2, Gap cost: Linear, Filter: Low complexity region, Mask: Mask for lookup table only. Using the NCBI BLAST version 2.2.31 algorithm with default parameters, a sequence with an identity score of XX% (e.g., 80%) with a reference sequence is considered to have at least XX% identity with the reference sequence, or equivalently XX% sequence identity.

[0035] Polypeptide or polynucleotide sequence identity may be measured over the entire length of a defined polypeptide sequence, for example, as defined by a specific sequence number, or over a shorter length, for example, over the length of a fragment obtained from a larger defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are merely illustrative, and it is understood that any fragment length supported by the sequences shown herein, in tables, figures, or sequence listings may be used to describe the length over which the identity percentage can be measured.

[0036] Polypeptides disclosed herein may include “mutant” polypeptides, “mutants,” and “derivatives thereof.” As used herein, the term “wild-type” is a term understood by those skilled in the art and means a typical form of a polypeptide, such as that naturally occurring, which is distinguished from mutant or mutant forms. As used herein, “mutant,” “mutant,” or “derivative” means a polypeptide molecule having an amino acid sequence different from that of a reference protein or polypeptide molecule. A mutant or mutant may have one or more insertions, deletions, or substitutions of amino acid residues compared to the reference molecule.

[0037] The amino acid sequences of polypeptide variants, mutants, derivatives, or fragments intended herein may include conserved amino acid substitutions compared to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conserved amino acid substitutions compared to a reference molecule. A “conserved amino acid substitution” is a substitution from one amino acid to another that is predicted to interfere least as much with the properties of the reference polypeptide. In other words, a conserved amino acid substitution substantially preserves the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of substitution, e.g., a beta-sheet or alpha-helix structure, (b) the molecular charge and / or hydrophobicity at the site of substitution, and / or (c) the bulkiness of the side chain.

[0038] As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,” and “nucleic acid” are interchangeable 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 a sense strand or an antisense strand. DNA polynucleotides may be cDNA (e.g., coding DNA) or genomic DNA sequences (e.g., including both introns and exons).

[0039] A polynucleotide is said to encode a polypeptide if, in its natural state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce a polypeptide or fragment thereof. The antisense strand of such a polynucleotide is also said to encode a sequence.

[0040] Those skilled in the art will understand the degeneracy of the genetic code and that various polynucleotides can code for the same polypeptide. In some embodiments, a polynucleotide (e.g., a polynucleotide encoding an erythrose reductase enzyme polypeptide) can be codon-optimized for expression in specific cells, including but not limited to plant cells, bacterial cells, fungal cells, or animal cells. Polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, but any polynucleotide sequence encoding a desired form of the polypeptides described herein may be used. Thus, sequences not found in nature can be used. These may be desired, for example, to enhance the expression of polypeptides or proteins in heterologous expression systems. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, the genetic code, and human hands can also be used to generate degenerate coding sequences.

[0041] Recombinant cells described herein may include deletions or disruptions in one or more native genes. The phrase “deletion or disruption” refers to a state of a native gene in a recombinant cell having either a completely removed coding region (deletion) or an alteration (by deletion, insertion, or mutation, etc.) of the gene, its promoter, or its terminator, resulting in the gene no longer producing an active expression product, a significant reduction in the amount of expression product (e.g., at least 75% or at least 90%), or a significantly reduced activity (e.g., at least 75% or at least 90%) of the expression product. Deletions or disruptions can be achieved by genetic engineering, forced evolution, mutagenesis, RNA interference (RNAi), and / or selection and screening. Deletions or disruptions of native host cell genes may be closely related to the incorporation of one or more polynucleotide sequences (e.g., exogenous or native polynucleotide sequences) into the host cell at the locus of the host cell gene being deleted or disrupted. The inserted polynucleotide sequence may be designed to replace all or part of the host cell gene to be deleted or disrupted. The polynucleotide sequence may encode the gene product of interest, such as a polypeptide, enzyme, etc. Deletion or disruption may also be achieved using a deletion construct that does not contain the polynucleotide sequence to be incorporated. Other methods for gene disruption or deletion are known and described in the art.

[0042] The recombinant cells described herein may have deletions or disruptions in one or more native genes encoding enzymes involved in the fermentation or consumption of erythritol. The deletion or disruption of one or more of these biosynthetic pathway enzymes reduces the recombinant cell's ability to produce erythritol, thereby increasing the carbon flux to the fermentation pathway for the production of other polyols (e.g., xylitol, arabitol, ribitol, etc.) or other fermentation products.

[0043] The recombinant cells described herein may include deletion or disruption of the native erythritol reductase (ER) gene. The native ER gene encodes an enzyme that catalyzes the reversible conversion of erythritol or erythritol-4-phosphate using the nicotinamide adenine dinucleotide (phosphate)(hydrogen)(NAD(P)(H)) cofactor. If the host cell contains multiple ER genes, it is preferable to delete or disrupt at least one of them. If the host cell contains multiple alleles of a given ER gene, it is preferable to delete or disrupt one or both alleles of a given ER gene.

[0044] As used herein, "NAD(P)H" refers to nicotinamide adenine dinucleotide (phosphate) hydrogen, and includes both NADH and NADPH. As understood in the art, including phosphate (or the abbreviation P) in parentheses indicates that phosphate may or may not be present, and the name and abbreviation include both. Similarly, "NAD(H)" or "NADP(H)" refers to both the reduced and oxidized forms of the cofactor.

[0045] If the recombinant cells are Moniliella polynis cells, the recombinant cells may include deletions or disruptions of the ER gene encoding an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 66. If the recombinant cells are Moniliella polynis cells, the recombinant cells may include deletions or disruptions of the ER gene having a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 64.

[0046] If the recombinant cells are Moniliella polynis cells, the recombinant cells may include deletions or disruptions of the ER gene encoding an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 63. If the recombinant cells are Moniliella polynis cells, the recombinant cells may include deletions or disruptions of the ER gene having a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61.

[0047] Recombinant cells described herein may include one or more genetic modifications in which an exogenous nucleic acid is incorporated into the genome of a host cell. Those skilled in the art know how to select a suitable locus in the yeast genome for the incorporation of an exogenous nucleic acid. Suitable loci for incorporation include, but are not limited to, the PDC1, GPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, ATO2, Adh9091, Adh1202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdIIA, and gpdIIB loci. For example, in Moniliella polynis host cells, suitable interacting loci may include, but are not limited to, the ER1 locus (defined as the locus adjacent to SEQ ID NOs. 3 and SEQ ID NOs. 38), the ER3 locus (defined as the locus adjacent to SEQ ID NOs. 31 and SEQ ID NOs. 41), the PDC1 locus (defined as the locus adjacent to SEQ ID NOs. 28 and SEQ ID NOs. 40), the pyrF locus (defined as the locus adjacent to SEQ ID NOs. 29 and SEQ ID NOs. 39), the TRP3 locus (defined as the locus adjacent to SEQ ID NOs. 32 and SEQ ID NOs. 35), the gpdIIA locus (defined as the locus adjacent to SEQ ID NOs. 33 and SEQ ID NOs. 37), and the gpdIIB locus (defined as the locus adjacent to SEQ ID NOs. 34 and SEQ ID NOs. 42). Exogenous nucleic acids may also be incorporated into intergeneric regions or other locations in the host cell genome that are not specifically identified herein. Other suitable integration loci can be determined by those skilled in the art. Furthermore, those skilled in the art will be familiar with the use of sequences to design primers for verifying accurate gene integration at selected loci.

[0048] Recombinant cells may have one or more copies of a given exogenous nucleic acid sequence that is integrated into a host chromosome and replicated together with the chromosome into which it is integrated. For example, yeast cells may be transformed with a nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide described herein, and the polynucleotide sequence encoding the polypeptide may be integrated into the host chromosome in one or more copies. Recombinant cells may contain multiple copies (two or more) of a given polynucleotide sequence encoding a polypeptide described herein. Recombinant cells may have one, two, three, four, five, six, seven, eight, nine, ten, or more copies of the polynucleotide sequence encoding a polypeptide described herein that are integrated into the genome. The multiple copies of the polynucleotide sequence may all be integrated into a single locus, or they may be integrated into multiple loci.

[0049] Recombinant cells described herein are characterized by overexpression of an ER enzyme and / or may contain an exogenous polynucleotide sequence encoding the ER enzyme. The ER enzyme may be native to the host cell, or the ER enzyme may be an exogenous ER enzyme. For example, if the cell is Moniliella polynis, the ER enzyme may be an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with at least one of SEQ ID NOs: 63 and 66. Recombinant cells may contain an exogenous polynucleotide sequence encoding an ER enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with at least one of SEQ ID NOs: 63 and 66. Recombinant cells may include genetic modifications that increase the expression of the ER enzyme, which 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 sequence numbers 63 and 66. Genetic modifications may include, but are not limited to, the insertion of an additional copy of the nucleic acid encoding the native ER into the cell, the insertion of a constitutive promoter upstream of the coding region of the native ER gene in the host cell genome, and / or modification of a promoter located upstream of the coding region of the native ER gene in the host cell genome. Those skilled in the art will recognize that the expression of the native ER gene can be increased by many methods known in the art, and such methods can be selected and applied as needed. For example, the copy number of the native ER gene in the genetically modified cell can be increased by incorporating an additional copy of the native ER gene into the host cell genome.

[0050] As used herein, “overexpression” means an expression level of a polypeptide higher than the expression level of the same polypeptide in equivalent cells in the absence of the genetic modification or the exogenous polynucleotide encoding the polypeptide.

[0051] In addition to producing erythritol, the genetically modified yeast cells described herein can also produce other polyols (e.g., xylitol, arabitol, ribitol, etc.) or other fermentation products. Therefore, it may be desirable to increase or decrease erythritol production in the genetically modified cells based on other metabolites produced during the fermentation process and the desired yield of such metabolites.

[0052] For example, recombinant cells containing a deletion or disruption of the ER gene may also produce xylitol and may contain one or more exogenous polynucleotide sequences integrated into their genome. Examples of suitable recombinant yeast cells, polynucleotide sequences, and biosynthetic pathways for xylitol production are described in at least U.S. Provisional Applications 63 / 364,363, 63 / 364,370, 63 / 364,375, and 63 / 364,382, all filed on 9 May 2022, and are incorporated in their entirety by reference, respectively.

[0053] In another example, recombinant cells containing a deletion or disruption of the ER gene may also produce arabitol and may contain one or more exogenous polynucleotide sequences integrated into their genome. Examples of suitable recombinant yeast cells, polynucleotide sequences, and biosynthetic pathways for producing arabitol are described at least in U.S. Provisional Applications 63 / 364,359 and 63 / 364,380, both filed on 9 May 2022, which are incorporated herein by reference in their entirety.

[0054] Polynucleotides in recombinant cells described herein may be under the control of promoters. For example, native or exogenous polynucleotide sequences may be operably linked to heterologous or artificial promoters. Suitable promoters are well known and described in the art. Examples of promoters include pyruvate decarboxylase promoter (PDC), translation elongation factor 2 promoter (TEF2), SED1, alcohol dehydrogenase 1A promoter (ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYK1p, SEQ ID NO: 4), 6-phosphogluconate dehydrogenase promoter (6PGDp, SEQ ID NO: 6), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p, SEQ ID NO: 8), translation elongation factor 1 promoter (TEFp, SEQ ID NO: 9), Examples include, but are not limited to, modified TEFp (SEQ ID NO: 7), phosphoglucomutase 1 promoter (PGM1p, SEQ ID NO: 10), 3-phosphoglycerate kinase promoter (PGK1p, SEQ ID NO: 11), enolase promoter (ENO1p, SEQ ID NO: 12), asparagine synthetase promoter (ASNSp, SEQ ID NO: 13), 50S ribosomal protein L1 promoter (RPLAp, SEQ ID NO: 14), and RPL16B (SEQ ID NO: 15).

[0055] Polynucleotides in recombinant cells described herein may be under the control of terminators. For example, exogenous or native polynucleotides may be operably linked to heterologous or artificial terminators. Suitable terminators are well known and described in the art. Examples of terminators include the GAL10 terminator, PDC terminator, transaldolase terminator (TAL), 6PGD terminator (6PGDt, SEQ ID NO: 16), ASNS terminator (ASNSt, SEQ ID NO: 17), ENO1 terminator (ENO1t, SEQ ID NO: 18), hexokinase 1 terminator (HXK1t, SEQ ID NO: 19), PGK1 terminator (PGK1t, SEQ ID NO: 20), and PGM1 terminator (PGM1t, SEQ ID NO: 21). Examples of such terminators include, but are not limited to, PYK1 terminator (PYK1t, SEQ ID NO: 22), RPLA terminator (RPLAt, SEQ ID NO: 23), transaldolase 1 terminator (TAL1t, SEQ ID NO: 24), TDH3 terminator (TDH3t, SEQ ID NO: 25), translation elongation factor 2 terminator (TEF2t, SEQ ID NO: 26), triose phosphate isomerase 1 terminator (TPI1t, SEQ ID NO: 27), and MpTEF1 (SEQ ID NO: 60).

[0056] A promoter or terminator is "operably ligated" to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to the polynucleotide is such that the promoter or terminator may, in some cases, perform its transcriptional regulatory function.

[0057] Polypeptides described herein may be provided as part of a construct. As used herein, the term “construct” means 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. Recombinant polynucleotides are polynucleotides formed by laboratory methods, comprising polynucleotide sequences derived from at least two different natural sources, or they may be synthesized. Thus, constructs may include, for example, novel modifications to endogenous genes introduced by genome editing techniques. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. A construct may be a vector comprising a promoter operably ligated to a polynucleotide encoding a polypeptide described herein. As used herein, the term “vector” means a polynucleotide capable of transporting another polynucleotide to which it is ligated. A vector may also be a plasmid, which refers to a circular double-stranded DNA loop into which further DNA segments may be incorporated.

[0058] This disclosure also provides a fermentation method using recombinant cells as described herein. The fermentation method may be used to produce another polyol (e.g., xylitol, arabitol, ribitol, etc.) or another fermentation product. Therefore, it may be desirable to increase or decrease erythritol production in genetically modified cells based on other metabolites produced during the fermentation process and the desired yield of such metabolites.

[0059] The fermentation method includes the step of fermenting a substrate using the genetically modified yeast described herein. The fermentation method may include additional steps as will be understood by those skilled in the art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation liquid within a predetermined range, adjusting the pH during fermentation, and isolating the fermentation product from the fermentation liquid. The fermentation process may be a fully aerobic or partially aerobic process.

[0060] The fermentation method can be carried out using an appropriate fermentation substrate. Substrates for the fermentation method may include glucose, sucrose, galactose, mannose, molasses, xylose, fructose, starch hydrolysates, lignocellulose hydrolysates, or combinations thereof. Those skilled in the art will recognize which fermentation substrates are suitable for a given fermenting organism and fermentation system.

[0061] The fermentation process can be carried out under a variety of conditions. The fermentation temperature, i.e., the temperature of the fermentation liquid during processing, may be the ambient temperature. Alternatively, the fermentation temperature may be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C, preferably around 35°C. However, those skilled in the art will recognize that the fermentation temperature is not limited to the specific ranges or temperatures listed herein and may be changed as needed.

[0062] The fermentation process can be carried out within a specific oxygen uptake rate (OUR) range. The volume OUR of the fermentation process can be in the range of 5–80, 10–75, 15–70, 20–60, 30–50, or 40–50 mmol O2 / (L·h). In some embodiments, the specific OUR can be in the range of 0.05–10, 0.1–9, 0.5–8, 1.0–7, 1.5–6, 2–5, or 2.5–4 mmol O2 / (g cell dry weight·h). However, the volume OUR or specific OUR of the fermentation process is not limited to any specific rate or range listed herein.

[0063] The fermentation process can be carried out at various cell concentrations. In some embodiments, the dry cell weight at the end of fermentation can be 5-40, 8-30, or 10-20 g / L. Furthermore, the pitch density or pitting rate of the fermentation process can be varied. In some embodiments, the pitch density can be 0.05-11, 0.1-10, or 0.25-8 g / L.

[0064] When performed as a batch fermentation, the initial dextrose concentration of fermentation can be at least 100, 200, 250, 300, 350, or at least 400 g / L of dextrose. When performed as a batch fermentation, the initial dextrose concentration can be 100-500, 150-450, 400-200, or 250-350 g / L. When performed as a feed batch fermentation process, the dextrose concentration can be at least 100, 200, 250, 300, 350, 400, or at least 450 g / L.

[0065] The fermentation process can be associated with a variety of properties, including but not limited to fermentation rate, pathway fermentation yield, final potency, and peak fermentation rate. These properties can be influenced by the selection of yeast used in the fermentation process and / or genetic modification of the yeast. These properties can also be influenced by adjusting the fermentation process conditions. These properties can be adjusted through a combination of yeast selection or modification and selection of fermentation process conditions.

[0066] Using genetically modified cells that overexpress the ER gene, the erythritol production rate of the process is at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 gL. -1 h -1 The erythritol mass yield of this process may be at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 50 percent, at least 55 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, or at least 85 percent. The final erythritol titer of this process may be at least 5, 10, 20, 30, 50, 75, or 100 g / L.

[0067] Using genetically engineered cells in which at least one copy of the ER gene is deleted or disrupted, the erythritol production rate of the process is 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, or 0.1 gL.-1 h -1 It may be less than 1%. The mass yield of erythritol in this process may be 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than 1%. The final erythritol titer of the process may be less than 20, 18, 15, 12, 10, 5, 1, or 0.5 g / L.

[0068] The fermentation process can be carried out as a dextrose supply batch. Furthermore, the fermentation process can be a batch process, a continuous process, or a semi-continuous process, as can be understood by those skilled in the art. [Examples]

[0069] The present invention will be described in more detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Therefore, the present invention should not be construed as being limited in any way to the following examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0070] Throughout the examples, the strain numbering and sequence numbers are used consistently. For example, strains 1-7 in Example 2 are the same strains 1-7 in Examples 3, 4, 5, and 7.

[0071] Example 1: In vitro enzyme assay of erythrose reductase candidate Polynucleotides encoding suspected erythrose reductase (ER) enzymes (Table 2) were cloned into vectors containing T7 promoters and terminators for cell-free protein expression using purified recombinant T7 RNA polymerase for transcription and purified recombinant ribosomes for translation (New England Biolabs, PURExpress® In Vitro Protein Synthesis). Enzyme expression using an in vitro system was confirmed by gel electrophoresis (results not shown).

[0072] [Table 2]

[0073] The activity of cell-free synthesized proteins against four substrates (erythrose, erythrose-4-phosphate, xylulose, and xylulose-5-phosphate) was analyzed in conjunction with either NADPH cofactor or NADH cofactor. Enzyme assays were performed in 96-well plates using 180 μL of the reaction mixture outlined in Table 3 and 20 μL of the in vitro enzyme expression product. The reaction progress over 15 minutes was monitored by NAD(P)H concentration measured by absorbance at 340 nm. The results are shown in Table 4 and Figure 2.

[0074] [Table 3]

[0075] [Table 4]

[0076] Example 2 - Genetically modified Moniliella polinis strain The strains described in this example were developed to test in vivo regulation (increase or decrease) of erythritol production by knockout of a candidate ER gene, reduction of candidate ER enzyme expression, and / or overexpression of a candidate ER enzyme. A summary of the genetically modified strains is provided in Table 5.

[0077] Stock 1-1 Strain 1-1 is the Moniliella polnis host strain "Moniliella tomentosa var. polnis TCV364" described in U.S. Patent No. 6,440,712 (incorporated herein by reference in its entirety), and was deposited on March 28, 1997, under the Budapest Treaty, with the Belgian Coordinated Collections of Micro-organisms / Mycotheque de l’Universite Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde, under the accession number MUCL40385.

[0078] Strain 1-2 Strain 1-1 was transformed with SEQ ID NO: 2 and SEQ ID NO: 53 by first protoplasting the parental strain by adding an enzyme mixture containing 0.6 M MgSO4, 7.5 g / L driselase, and 12.5 g / L Trichoderma harzianum lysing enzyme to the mycelial pellets of the parental strain. The protoplasts were then pelleted, washed with 0.6 M MgSO4, and resuspended in STC medium (0.6 M sucrose, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). Binary transformation was carried out with 100 μg of single-stranded salmon sperm DNA and 1.5 - 5 μg (total 3 - 10 μg) of each of the 5' and 3' DNA transformation fragments in approximately 200 μL of protoplast mixture (10 8The mixture was prepared by adding it to cells / mL. Then, 1 mL of 50% PEG in STC medium was added to the salmon sperm DNA, transformed DNA, and protoplast mixture, and the resulting combination was incubated at room temperature for 15 minutes. After incubation, the recovered solution (0.4 M sucrose, 1 g / L yeast extract, 1 g / L malt extract, 10 g / L glucose, pH 4.5) was added to the mixture and incubated at 27°C and 100 rpm for 16-24 hours. After incubation, the protoplasts were pelletized by centrifugation and resuspended in 1 mL of PBS. The resuspended protoplasts were seeded on a PDA+250 mg / L Geneticin (G418) selective plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on the PDA+Geneticin (G418) plate for single colony isolation, and single colonies were selected. Selected colonies were evaluated by colony PCR for the incorporation of two copies of the L. rhamnosus XPDH sequence. PCR-validated isolates were designated as strains 1-2.

[0079] Sequence ID 53 contains (i) the 3' portion of the G418 selectable marker (SEQ ID NO: 48), ii) an expression cassette of the XPDH homolog from Lactobacillus rhamnosus of Sequence ID NO: 5, encoding the amino acid sequence of Sequence ID NO: 1, under the control of the PYK1 promoter of Sequence ID NO: 4 and the PGD terminator of Sequence ID NO: 16, and (iii) 3' flanking DNA for target chromosome integration into the ER1 locus (SEQ ID NO: 38). Sequence ID NO: 2 contains (i) 5' flanking DNA for target chromosome integration into the ER1 locus (SEQ ID NO: 3), (ii) an expression cassette of the XPDH homolog from Lactobacillus rhamnosus of Sequence ID NO: 5, encoding the amino acid sequence of Sequence ID NO: 1, under the control of the PYK1 promoter of Sequence ID NO: 4 and the PGD terminator of Sequence ID NO: 16, and (iii) the 5' portion of the G418 selectable marker (SEQ ID NO: 51). Xylitol-phosphate dehydrogenase (XPDH) enzyme converts xylitol-5-phosphate and NADPH or NADH into xylitol-5-phosphate and NADP. + or NAD +It catalyzes the conversion to [XPDH], and this activity is called "XPDH activity." The "XPDH gene" refers to the polynucleotide sequence that codes for the enzyme possessing XPDH activity.

[0080] Stocks 1-3 Strains 1-2 were transformed with SEQ ID NOs: 55 and 56, as outlined above. The transforming fragment for SEQ ID NOs: 55 contained, in order, a 5'ER3 flanking sequence (SEQ ID NOs: 31), an MpPYK1 promoter (SEQ ID NOs: 4), the gene encoding the Moniliella polynis RPE2 polypeptide (SEQ ID NOs: 54), an MpPYK terminator (SEQ ID NOs: 146), and the 5' portion of the zeosin resistance gene expression cassette (SEQ ID NOs: 45). The transforming fragment for SEQ ID NOs: 56 contained, in order, the 3' portion of the zeosin resistance gene expression cassette (SEQ ID NOs: 44), an MpTEF2 terminator (SEQ ID NOs: 26), and a 3'ER3 flanking sequence (SEQ ID NOs: 41). Transformants were selected on PDA+zeosin select plates and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on PDA+zeosin plates for single colony isolation, and single colonies were selected. The selected colonies were evaluated for the incorporation of the gene encoding the Moniliella polinis RPE2 polypeptide by colony PCR. The PCR-validated isolates were designated as strains 1-3. The ribulose 5-phosphate epimerase (RPE) enzyme catalyzes the conversion of ribulose-5-phosphate to xylulose-5-phosphate, and this activity is called "RPE" activity. An "RPE gene" refers to a polynucleotide sequence that encodes an enzyme with RPE activity, such as the RPE2 gene from Moniliella polinis that encodes the RPE enzyme RPE2.

[0081] Stock 1-4 UV mutagenesis (Hoefer UV Crosslinker at 360 μJ / cm²) 3Using the energy (used in the above) and selected strains 1-3, Moniliella polinis strains with reduced foaming during shaking flask fermentation were produced. Strains with a low-foaming phenotype were selected based on a visual evaluation of foaming during shaking flask fermentation compared to foaming in parent strains 1-3. The resulting low-foaming strains contained two copies of the exogenous polynucleotide sequence encoding XPDH of SEQ ID NO: 1, integrated into the ER1 locus, and one copy of the polynucleotide sequence encoding RPE of SEQ ID NO: 54, integrated into the ER3 locus, and were designated as 1-4.

[0082] Stocks 1-5 Strains 1-4 were transformed with the Cre recombinase plasmid of SEQ ID NO: 59 using the transformation method outlined above. The resulting transformants were evaluated by colony PCR for G418 removal and zeosin resistance selection markers. The PCR-validated isolates were designated as strains 1-5.

[0083] Stocks 1-6 Strains 1-5 were non-selectively grown on YPD plates to induce loss of plasmid SEQ ID NO: 59. Single colonies of biomass were attacked and evaluated by PCR to confirm plasmid loss. PCR-validated isolates were designated as strains 1-6.

[0084] Stock 1-7 Strains 1-6 were transformed with SEQ ID NO: 57 and SEQ ID NO: 55, as outlined above. SEQ ID NO: 57 contains the 3' portion of the zeosin resistance gene expression cassette (SEQ ID NO: 168), the MpPGK1 promoter (SEQ ID NO: 135), the polynucleotide sequence encoding the X5PP enzyme (SEQ ID NO: 200), the Mp6PGD terminator (SEQ ID NO: 140), and the 3'ER3 flanking sequence (SEQ ID NO: 165). Transformants were selected on a PDA+zeosin select plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA+zeosin plate for single colony isolation, and single colonies were selected. The selected colonies were evaluated for integration of the Moniliella polynis X5PP gene into the ER3 locus using colony PCR. PCR-validated isolates were designated as strains 1-7. The X5PP enzyme is a divalent metal cation, e.g., Mg 2+ Mn 2+ , or Cole 2+ In the presence of [X5PP], it catalyzes the conversion of xylitol-5-phosphate to xylitol and phosphate, and this is called "X5PP activity." The "X5PP gene" refers to a polynucleotide that encodes an enzyme possessing X5PP activity.

[0085] Stocks 1-8a~e Strains 1-7 were transformed with SEQ ID NOs: 75 and 58 using the transformation protocol outlined in Example 4. SEQ ID NOs: 75 contains a 5' gpdIIB flanking sequence (SEQ ID NOs: 34), an MpPGK1 promoter (SEQ ID NOs: 11), a copy of the RCSR18717 gene on SEQ ID NOs: 61, an MpTDH3 terminator (SEQ ID NOs: 25), and a 5' portion of the G418 resistance gene (SEQ ID NOs: 51). SEQ ID NOs: 58 contains a 3' fragment of the G418 resistance gene (SEQ ID NOs: 48), an MpTEF1 terminator (SEQ ID NOs: 60), and a 3' gpdIIB flanking sequence (SEQ ID NOs: 42). Transformants were selected on a PDA+G418 selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA+G418 plate for single colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the Moniliella polinis gene encoding the ER enzyme of sequence number 63. Sister isolates validated by PCR were designated strains 1-8a, 1-8b, 1-8c, 1-8d, and 1-8e.

[0086] Stock 1-9a~b Strains 1-7 were transformed with SEQ ID NO: 70 using the transformation protocol outlined above, and positive transformants were selected using a notheotricin selection plate. SEQ ID NO: 70 contains a deletion construct for removing one copy of the RSCR18717 gene. Two PCR-validated sister isolates with one copy of RCSR18717 knocked out were designated strains 1-9a and 1-9b.

[0087] Stock 1-10a~d Strain 1-9a was transformed with SEQ ID NO: 71 using the transformation protocol outlined in Example 4. SEQ ID NO: 71 contains constructs for looping out the zeosin and notheotricin resistance selection markers. Two PCR-validated sister isolates from which the zeosin and notheotricin resistance selection markers were removed were designated strains 1-10a and 1-10b.

[0088] Strain 1-9b was transformed with SEQ ID NO: 71 using the transformation protocol outlined in Example 4. SEQ ID NO: 71 contains constructs for looping out the zeosin and notheotricin resistance selection markers. Two PCR-validated sister isolates from which the zeosin and notheotricin resistance selection markers were removed were designated strains 1-10c and 1-10d.

[0089] Stock 1-11a~b Strain 1-10c was transformed with SEQ ID NO: 72 using the transformation protocol outlined in Example 4, and positive transformants were selected using a Geneticin (G418) selection plate. SEQ ID NO: 72 contains a deletion construct for removing the second copy (i.e., only the remaining copy) of the RCSR18717 gene. Two PCR-validated sister isolates in which both copies of the RCSR18717 gene were knocked out were designated strains 1-11a and 1-11b.

[0090] Stock 1-12a~b Strains 1-7 were transformed with SEQ ID NO: 73 using the transformation protocol outlined in Example 4, and positive transformants were selected using a notheotricin selection plate. SEQ ID NO: 73 contained a deletion construct for removing one copy of the RCSR26640 gene. Two PCR-validated sister isolates with one copy of the RCSR26640 gene knocked out were designated strains 1-12a and 1-12b.

[0091] Stock 1-13a~d Strain 1-12a was transformed with SEQ ID NO: 71 using the transformation protocol outlined in Example 4. SEQ ID NO: 71 contains constructs for looping out zeosin and notheotricin resistance selection markers. Two PCR-validated sister isolates from which the zeosin and notheotricin resistance selection markers were removed were designated strains 1-13a and 1-13b.

[0092] Strain 1-12b was transformed with SEQ ID NO: 71 using the transformation protocol outlined in Example 4. SEQ ID NO: 71 contains constructs for looping out the zeosin and notheotricin resistance selection markers. Two PCR-validated sister isolates from which the zeosin and notheotricin resistance selection markers were removed were designated strains 1-13c and 1-13d.

[0093] Stock 1-14a~c Strain 1-13c was transformed with SEQ ID NO: 73 using the transformation protocol outlined in Example 4, and positive transformants were selected on notheotricin select plates. Three PCR-validated sister isolates in which both copies of the RCSR26640 gene were knocked out were designated strains 1-14a, 1-14b, and 1-14c.

[0094] Stock 1-15a~b Strains 1-7 were transformed with SEQ ID NO: 74 using the transformation protocol outlined in Example 4, and positive transformants were selected using a notheotricin select plate. SEQ ID NO: 74 contained a deletion construct for removing a single copy of the RCSR11551 gene. Two PCR-validated sister isolates in which one copy of the RCSR11551 gene was knocked out were designated strains 1-15a and 1-15b.

[0095] Stock 1-16a~e Strain 1-1 was transformed with SEQ ID NOs. 75 and 58 using the transformation method outlined above. SEQ ID NOs. 75 contains a 5' gpdIIB flanking sequence (SEQ ID NOs. 34), an MpPGK1 promoter (SEQ ID NOs. 11), a copy of the RCSR18717 gene from SEQ ID NOs. 61, an MpTDH3 terminator (SEQ ID NOs. 25), and the 5' portion of the G418 resistance gene (SEQ ID NOs. 51). SEQ ID NOs. 58 contains a 3' fragment of the G418 resistance gene (SEQ ID NOs. 48), an MpTEF1 terminator (SEQ ID NOs. 60), and a 3' gpdIIB flanking sequence (SEQ ID NOs. 42). Transformants were selected on a PDA+G418 selection plate and incubated at 35°C for at least 2 days until the transformants grew. The resulting transformants were streaked on a PDA+G418 plate for single colony isolation, and single colonies were selected. The selected colonies were evaluated by colony PCR for integration of the Moniliella polinis gene encoding the ER enzyme of sequence number 63. Sister isolates validated by PCR were designated strains 1-16a, 1-16b, 1-16c, 1-16d, and 1-16e.

[0096] [Table 5]

[0097] Example 3: Shaking Flask Fermentation Assay Strains 1-7, 1-12a, 1-12b, 1-9a, 1-9b, 1-15a, and 1-15b were run in a shaking flask to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0098] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm path length cuvette. Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0099] A 250 mL baffle-free flask containing 20 mL of growth medium (Table 6) was inoculated with 0.4 mL of seed culture to form a growth culture. The growth culture was incubated at 35°C and 250 rpm. Samples were taken from the growth culture after incubation at 24, 48, 72, and 96 hours. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The results are shown in Tables 7 and 8, and the results at 72 hours are also shown in Figure 3.

[0100] Overall, strains 1-9a and 1-9b showed the greatest reduction in erythritol production (32%) compared to the parent (1-7).

[0101] [Table 6]

[0102] [Table 7]

[0103] [Table 8]

[0104] Example 4: Shaking Flask Fermentation Assay Strains 1-7, 1-9a, 1-9b, 1-10a, 1-10b, 1-10c, 1-10d, 1-12a, 1-12b, 1-13a, 1-13b, 1-13c, and 1-13d were run in a shaking flask to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0105] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm path length cuvette. Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0106] A 250 mL unbaffled flask containing 20 mL of growth medium (Table 6) was inoculated with 0.4 mL of seed culture to form a growth culture. The growth culture was incubated at 35°C and 250 rpm. Samples were taken from the growth culture after incubation at 24, 48, 72, and 96 hours. High-performance liquid chromatography with a refractive index detector was used to analyze the samples for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol. The results are shown in Table 9, and the results at 96 hours are also shown in Figure 4.

[0107] Overall, strains 1-10a to d showed reduced erythritol production compared to their grandparent strain 1-7. Erythritol production in strains 1-10a to d was similar to that of strains 1-9a to d, and all six of these strains have a knockout of one copy of the RCSR18717 gene. Strains 1-13a to d also showed a slight decrease in erythritol production compared to their grandparent strain 6-b, and this decrease is greater than the decrease seen in parent strains 1-12a to d.

[0108] [Table 9-1]

[0109] [Table 9-2]

[0110] Example 5: Shaking Flask Fermentation Assay Strains 1-7, 1-13c, 1-14a, 1-14b, and 1-14c were run in a shaking flask to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0111] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm path length cuvette. Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0112] A 250 mL unbaffled flask containing 20 mL of growth medium (Table 6) was inoculated with 0.4 mL of seed culture to form a growth culture. The growth culture was incubated at 35°C and 250 rpm. Samples were taken from the growth culture after incubation at 24, 48, 72, and 96 hours. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The results are shown in Table 10, and the results at 96 hours are also shown in Figure 5.

[0113] Overall, strains 1-13c, 1-14a, 1-14b, and 1-14c showed reduced erythritol production compared to strain 1-7. However, knockout of the second allele of RCSR26640 (i.e., strains 1-14a, 1-14b, and 1-14c) did not further reduce erythritol production beyond that of the single knockout strain (1-13c).

[0114] [Table 10]

[0115] Example 6: Shaking Flask Fermentation Assay Strains 1-7, 1-10a, 1-10c, and 1-11a-b were run in shaking flasks to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0116] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm path length cuvette. Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0117] A 250 mL unbaffled flask containing 20 mL of growth medium (Table 6) was inoculated with 0.4 mL of seed culture to form a growth culture. The growth culture was incubated at 35°C and 250 rpm. Samples were taken from the growth culture after incubation at 24, 48, 72, and 96 hours. High-performance liquid chromatography with a refractive index detector was used to analyze the samples for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol. The results are shown in Table 11. Figure 6 shows the results at 96 hours comparing strains 1-10a, 1-10c, 1-11a, and 1-11b.

[0118] Overall, strains 1-11a and 1-11b showed a significant decrease in erythritol production, with erythritol titers at 96 hours reduced to approximately 1.0 g / L. Two allele knockouts of the RCSR18717 gene in these strains showed a significant decrease in erythritol compared to both single knockout strains (1-10a and 1-10c) and grandparent strains (1-7) containing both wild-type alleles of RCSR18717.

[0119] [Table 11]

[0120] Example 7: Shaking Flask Fermentation Assay Strains 1-7 and 1-8a-e were run in a shaking flask to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0121] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm path length cuvette. Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0122] A 250 mL unbaffled flask containing 20 mL of production medium (Table 6) was inoculated with 0.4 mL of seed culture to form a production culture. The production culture was incubated at 35°C and 250 rpm. After 72 and 96 hours of incubation, samples were taken from the production culture. The samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The results are shown in Table 12. The results at 96 hours are shown in Figure 7. Overall, strains 1-8a to 8e, genetically engineered to overexpress the RCSR18717 gene, showed increased erythritol production compared to control strain 1-7, which had wild-type levels of RCSR18717 expression. Strains 1-8a to 8e showed approximately a 25% increase in erythritol titer compared to strain 1-7.

[0123] [Table 12]

[0124] Example 8: Shaking Flask Fermentation Assay Strains 1-7 and 1-16a-d were run in shaking flasks to evaluate glucose consumption and the production of ribitol, xylitol, erythritol, glycerol, and ethanol.

[0125] The strains were streaked onto YPD plates (20 g / L bacterial peptone, 10 g / L yeast extract, 20 g / L glucose, and 15 g / L agar) for biomass propagation and incubated at 30°C for 48–72 hours. Cells from the incubated YPD plates were scraped into 40 mL of nutrient-rich medium (170 g / L glucose, 10 g / L yeast extract) in a 250 mL baffle-less flask. Seed cultures were formed by incubating the cells at 30°C and 250 rpm until the optical density (OD600) reached 15–20. Optical density was measured at a wavelength of 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific) with a 1 cm path length cuvette. Seed cultures reached an OD600 of 15–20 in approximately 32–50 hours.

[0126] A 250 mL unbaffled flask containing 20 mL of growth medium (Table 6) was inoculated with 0.4 mL of seed culture to form a growth culture. The growth culture was incubated at 35°C and 250 rpm. Samples were taken from the growth culture after 24, 48, 72, and 100 hours of incubation. Samples were analyzed for glucose, ribitol, xylitol, erythritol, glycerol, and ethanol by high-performance liquid chromatography with a refractive index detector. The results are shown in Table 13. The results at 100 hours are shown in Figure 8. Overall, strains 1-16a to 1-16e, genetically engineered to overexpress the RCSR18717 gene, showed increased erythritol production compared to wild-type strain 1-1 with wild-type RCSR18717 expression. Erythritol yield was also higher in strains 1-16a to 1-16e than in strain 11-1 (Table 14).

[0127] Table 13

[0128] Table 14

Claims

1. Genetically modified yeast cells, Genetically modified yeast cells comprising deletion or disruption of a native gene encoding an erythrose reductase enzyme of 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% identity with at least one of Sequence IDs 63 and 66.

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

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

4. The yeast cell is selected from the group consisting of Trichosporonoides megachiliensis, Trichosporonoides eoidocephalis, Trichosporonoides nigrecens, Pseudozyma tsukubaensis, Trigonopsis variabilis, Moniliella, Ustyraginomisetes, Trichosporon, Yarowia liporitica, Saccharomyces cerevisiae, Penicillium, Torula, Pichia, Candida, Candida magnoliae, and Aureobasidium, according to any one of claims 1 to 3.

5. The yeast cell according to any one of claims 1 to 4, wherein the yeast cell is a Moniliella polynis cell, and the gene encoding the erythrose reductase enzyme 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 Sequence IDs 61 and 64.

6. The yeast cell according to claim 5, wherein the cell has a deletion of at least one allele of the gene encoding the erythrose reductase enzyme.

7. The yeast according to claim 5 or 6, wherein the cells have deletions of both alleles of the gene encoding the erythrose reductase enzyme.

8. Yeast cells according to any one of claims 1 to 7, wherein when the genetically modified cells are used in a fermentation process in the presence of dextrose, the titer and / or yield of erythritol production is reduced compared to the titer and / or yield of erythritol in an equivalent fermentation process using equivalent cells in which there is no disruption or deletion of a native gene encoding an erythritol reductase 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: 63 and 66.

9. The yeast cell according to any one of claims 1 to 8, wherein at least one copy of the gene encoding the erythrose reductase enzyme is substituted with a native or exogenous polynucleotide sequence.

10. The yeast cell according to claim 9, wherein the natural or exogenous polynucleotide sequence is operably linked to a heterogeneous or artificial promoter.

11. The yeast cell according to claim 9 or 10, wherein the promoter is a constitutive promoter.

12. The yeast cell according to any one of claims 9 to 11, wherein the heterologous or artificial promoter is selected from the group consisting of pyruvate kinase 1 promoter (PYK1p, SEQ ID NO: 4), 6-phosphogluconate dehydrogenase promoter (6PGDp, SEQ ID NO: 6), glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p, SEQ ID NO: 8), translation elongation factor 1 promoter (TEFp, SEQ ID NO: 9), modified TEFp (SEQ ID NO: 7), phosphoglucomutase 1 promoter (PGM1p, SEQ ID NO: 10), 3-phosphoglycerate kinase promoter (PGK1p, SEQ ID NO: 11), enolase promoter (ENO1p, SEQ ID NO: 12), asparagine synthase promoter (ASNSp, SEQ ID NO: 13), 50S ribosomal protein L1 promoter (RPLAp, SEQ ID NO: 14), and RPL16B (SEQ ID NO: 15).

13. Genetically engineered Moniliella polynis cells comprising deletion or disruption of a native gene encoding an erythrose reductase enzyme that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of sequence numbers 63 and 66.

14. The genetically engineered Moniliella polynis cell according to claim 13, wherein the native gene 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 sequence numbers 61 and 64.

15. Moniliella polynis cells according to claim 13 or 14, wherein the native gene is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61, and the ER enzyme is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

63.

16. Moniliella polynis cells according to claim 15, wherein the native gene is at least 80% identical to SEQ ID NO: 61, and the ER enzyme is at least 80% identical to SEQ ID NO:

63.

17. Moniliella polynis cells according to claim 15, wherein the native gene is at least 90% identical to SEQ ID NO: 61, and the ER enzyme is at least 85% identical to SEQ ID NO:

63.

18. Moniliella polynis cells according to claim 15, wherein the native gene is at least 95% identical to SEQ ID NO: 61, and the ER enzyme is at least 90% identical to SEQ ID NO:

63.

19. Moniliella polynis cells according to claim 13 or 14, wherein the native gene 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 Sequence ID No. 64, and the ER enzyme 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 Sequence ID No.

66.

20. Moniliella polynis cells according to claim 19, wherein the native gene is at least 80% identical to Sequence ID No. 64, and the ER enzyme is at least 80% identical to Sequence ID No.

66.

21. Moniliella polynis cells according to claim 19, wherein the native gene is at least 90% identical to SEQ ID NO: 64, and the ER enzyme is at least 85% identical to SEQ ID NO:

66.

22. Moniliella polynis cells according to claim 19, wherein the native gene is at least 95% identical to SEQ ID NO: 64, and the ER enzyme is at least 90% identical to SEQ ID NO:

66.

23. Moniliella polynis cells according to any one of claims 13 to 22, wherein both alleles of the aforementioned native gene are deleted.

24. Moniliella polynis cells according to any one of claims 13 to 18 and 23, wherein both alleles of the native gene that are 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 Sequence ID No. 61 are deleted.

25. Moniliella polynis cells according to any one of claims 13, 14, and 19-23, wherein both alleles of the native gene that are 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 Sequence ID No. 64 are deleted.

26. A fermentation method A method comprising contacting a dextrose-containing substrate with genetically modified yeast cells as described in any one of claims 1 to 25, wherein the fermentation of the substrate by the genetically modified cells produces less erythritol than fermentation using equivalent yeast cells that do not have a deletion or disruption of the gene encoding ER.

27. The fermentation temperature is 25°C to 45°C, 30°C to 40°C, or 32°C to 37°C (including the endpoints), and the volumetric oxygen uptake rate (OUR) is 5 to 80, 10 to 75, 15 to 70, 20 to 60, 30 to 50, or 40 to 50 mmol O ２ The method according to claim 26, wherein / (L・h).

28. The method according to claim 26 or 27, wherein the rate, titer, and / or yield of erythritol production are reduced compared to equivalent fermentation carried out using equivalent yeast cells in which the native gene encoding the erythritol reductase enzyme is not deleted or destroyed.

29. The method according to any one of claims 26 to 28, wherein the dextrose concentration is at least 100 g / L.

30. When the fermentation is carried out at 35°C for 96 hours, the erythritol titer is less than 20, 18, 15, 12, 10, 5, 1, and 0.5 g / L, and the erythritol production rate is 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, or 0.1 g / L. －１ h －１ The method according to any one of claims 26 to 29, wherein the yield of the erythritol is less than 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than 1%.

31. The method according to any one of claims 26 to 30, wherein the fermentation process does not produce erythritol.

32. Use of genetically modified yeast according to any one of claims 1 to 25 in a fermentation process.

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