Aerobic fermentation processes for the production of xylitol with reduced ethanol production

Abstract

Disclosed herein are fermentation methods for the production of xylitol. In general, the methods described herein include supplementation of nitrogen into the fermentation media to improve xylitol production under aerobic conditions using a genetically engineered yeast capable of producing xylitol. The genetically engineered yeast may be a genetically engineered Moniliella pollinis cell.

Description

PT-2218-WO-PCTAEROBIC FERMENTATION PROCESSES FOR THE PRODUCTION OF XYLITOL WITH REDUCED ETHANOL PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S Provisional application No. 63 / 778,519 filed March 27, 2025 and U.S Provisional application No. 63 / 846,289 filed July 18, 2025, each of 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-2218-WO-PCT. xml” which is 301,293 bytes in size created on March 11, 2026 and electronically submitted via Patent Center herewith the application is incorporated by reference in its entirety.BACKGROUND

[0003] Xylitol is a low-calorie sweetener used as a food additive and sugar substitute. Commonly used in drug, dietary supplement, confectionary, and toothpaste compositions, xylitol has also been associated with anticariogenic properties when used in chewing gums. Traditional methods of xylitol production, including chemically catalyzed hydrogenation of xylose hydrolyzed from biomass extracted xylan, are both monetarily and environmentally costly. These methods require high temperatures and pressures, large amounts of water, and metal catalysts that must be mined. 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 traditional xylitol processing methods.

[0004] In the development of microorganism-based fermentation strategies for the production of xylitol, production of metabolic pathway intermediates and alternative fermentation products are important considerations. For example, metabolic pathways active in the production of xylitol may have overlap with the metabolic pathways for the production of arabitol, erythritol, ribitol, and the like. Similarly, alternative fermentation products such as ethanol and glycerol may be produced. However, production of other metabolites and fermentation products pulls carbon away from the production of xylitol. While geneticPT-2218-WO-PCTmodifications can be used to alter the fermentation product profile to produce more xylitol and less ethanol, glycerol, etc., changing the fermentation conditions can also impact the byproduct profile. Accordingly, provided herein are fermentation methods for the improved production of xylitol while limiting and / or controlling the production of unwanted byproducts.SUMMARY

[0005] The present disclosure provides a fermentation method for the production of xylitol, the method comprising: contacting a fermentation medium comprising dextrose and nitrogen (N) with an engineered yeast cell, capable of producing xylitol, in aerobic conditions to produce xylitol; wherein nitrogen is added to the fermentation medium such that nitrogen is present in the fermentation medium for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% of total fermentation reaction time; and wherein xylitol production is increased relative to an equivalent fermentation method run without additional nitrogen such that nitrogen is present in the fermentation medium for less than 50% of the total fermentation reaction time.

[0006] The fermentation methods for the production of xylitol may include growing a genetically engineered Moniliella pollinis cell capable of producing xylitol and comprising an exogenous polynucleotide sequence encoding a xylitol -phosphate dehydrogenase (XPDH) enzyme comprising a 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 at least one of at least one of SEQ ID NOs:55-64 in a fermentation media comprising dextrose and an initial nitrogen concentration under aerobic conditions to produce xylitol; and feeding supplemental nitrogen into the fermentation media when the initial nitrogen concentration is depleted and / or when dissolved oxygen in the reaction is 0%; wherein titer, rate, and / or yield of xylitol is higher than an equivalent fermentation run without feeding supplemental nitrogen.

[0007] The initial nitrogen concentration in the fermentation medium may be 0.5 g / L to 5 g / L, 1 g / L to 4 g / L, 1.5 g / L to 3.5 g / L, or preferably about 2 g / L to 3.25 g / L. Nitrogen may be added to the fermentation medium such that the total nitrogen concentration over the course of the fermentation reaction is about 3 g / L to 7 g / L, 3.5 g / L to 6.5 g / L, 4 g / L to 6 g / L, or about 4.5 g / L to 5.5 g / L. The nitrogen may be added to the fermentation at a constant or exponential rate or may be added as one or more bolus doses during fermentation. The nitrogen may be added during the fermentation at a rate of about 25 mg N / h to 150 mg N / h, 50 mg N / h to 140PT-2218-WO-PCTmg N / h, 60 mg N / h to 135 mg N / h, or about 75 mg N / h to 130 mg N / h. Nitrogen may be added in one or more bolus doses of 0.1 g / L to 2.5 g / L, for example, 0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, 1.0 g / L, 1.25 g / L, 1.5 g / L, 1.75 g / L, 2.0 g / L, or about 2.5 g / L.

[0008] The fermentation method may include a growth phase and a production phase, and nitrogen is added during the production phase. The fermentation temperature may be at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C. The dissolved oxygen content may be at least 20%, at least 30%, at least 40%, or at least 50%. The volumetric oxygen uptake rate (OUR) may be at least 15 mmol / L / h, at least 20 mmol / L / h, at least 25 mmol / L / h, at least 30 mmol / L / h, at least 35 mmol / L / h, at least 40 mmol / L / h, at least 45 mmol / L / h, or at least 50 mmol / L / h.

[0009] Xylitol may be produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L-l h-1. Xylitol titer may be at least 50, at least 75, at least 100, at least 125, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 g / L when the fermentation is run at 35 °C for 96 hours. Xylitol production using the nitrogen supplemented fermentation methods described herein is at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more than an equivalent aerobic fermentation method run without additional nitrogen. Glycerol production in the fermentation reaction is reduced and / or wherein glycerol reconsumption rate increases in the fermentation reaction relative to an equivalent fermentation method run without additional nitrogen such that nitrogen is present in the fermentation medium for less than 50% of the total fermentation reaction time.

[0010] The initial concentration of dextrose in the fermentation medium may be at least 100 g / L. Additional dextrose may be fed to the fermentation such at the total dextrose concentration is at least 200 g / L, at least 300 g / L, or at least 400 g / L.

[0011] The engineered yeast cell used in the fermentation methods described here in comprises (i) an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a 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 at least one of at least one of SEQ ID NOs:55-64; and / or (ii) an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme comprising a 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 at least one of SEQ ID NOs:50 and 49 and an exogenous polynucleotide sequencePT-2218-WO-PCTencoding a xylitol dehydrogenase (XDH) enzyme comprising a 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 at least one of SEQ ID NOs:65, 66, and 67. The engineered yeast cell may comprise an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a 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 at least one of at least one of SEQ ID NOs:55-64; an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a sequence 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 at least one of SEQ ID NOs: 57, 58, 59, or 62; or an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57.

[0012] The engineered yeast cell used in the fermentation methods described herein may comprise at least one additional copy of a polynucleotide encoding a ribulose-5-phosphate epimerase (RPE) enzyme 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: 53 and 54. The engineered yeast cell may comprise an exogenous polynucleotide sequence encoding a xylitol-5-phosphate phosphatase (X5PP) enzyme comprising a 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 at least one of SEQ ID NOs: 33, 35, 36, 37, 38, 39, 43, and 45, preferably at least one of SEQ ID NOs: 33, 36, 37, 39, and 45; and / or at least one additional copy of a polynucleotide encoding an X5PP enzyme comprising 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:51, 52, 33, or 47, preferably SEQ ID NO:33.

[0013] The yeast cell may be an osmotol erant yeast cell. The yeast cell may be a cell of the subphylum Ustilaginomycotina. The yeast cell may be selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephaHs. Trychosporonoides nigre see ns, Pseudozyma isukubaensis. Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon. Yarrowia Upolylica. Saccharomyces cerevisiae. Penicillium, Torula, Pichia, Candida, Candida magnolias, and Aureobasidium. The yeast cell may be a species of yeast cell that natively includes an adenosine triphosphate (ATP) citrate lyase gene.

[0014] The yeast cell may comprise a deletion or disruption of a native gene encoding an erythrose reductase enzyme at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,PT-2218-WO-PCTat least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 11 and 15, preferably wherein the cell has a deletion of both alleles of the gene encoding the erythrose reductase enzyme. The yeast cell may comprise an exogenous polynucleotide sequence encoding a trehalase construct comprising a secretion signal operably linked to a trehalase enzyme at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs: 132 and 133, wherein the secretion signal is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs: 134-137; a genetic modification resulting in overexpression of a native enzyme with glycerol kinase activity, for example a glycerol kinase enzyme comprising a 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 at least one of SEQ ID NOs:92 and 103; a deletion or disruption of a native gene encoding a glycerol -3 -phosphate dehydrogenase 2a enzyme 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: 107; and / or a genetic modification that increases expression of a transketolase enzyme 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: 106.

[0015] One or more of the exogenous polynucleotide sequence in the genetically modified yeast may be operably linked to a heterologous or artificial promoter selected from the group consisting of pyruvate kinase 1 promoter (PYKlp; SEQ ID NO:68), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 69), glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:70), translational elongation factor 1 promoter (TEFp; SEQ ID NO:71), phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72), 3-phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73), enolase promoter (ENOlp ; SEQ ID NO:74), asparagine synthetase promoter (ASNSp; SEQ ID NO:75), 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76), RPL16B (SEQ ID NO:77), glycerol -3 -phosphate dehydrogenase 2a promoter (GPDIIap; SEQ ID NO: 114), GDN1 (SEQ ID NO: 116), EPDH1 (SEQ ID NO: 117), translation elongation factor 1A like gene 7 promoter (TEF7p; SEQ ID NO: 120); heat shock protein 90 promoter (HSP90p; SEQ ID NO: 121); translation elongation factor 1A like gene 4 promoter (TEF4p; SEQ ID NO: 122); translation elongation factor 2A like gene 6 promoter (TEF6p; SEQ ID NO: 123); transaldolase 1 promoter (TALlp; SEQ ID NO: 124); citrate synthase 1 promoter (CITlp; SEQ ID NO: 125); endoplasmic reticulum chaperone BiP (KAR2p; SEQ ID NO: 126); heat shock protein 88 promoter (HSP88p; SEQ IDPT-2218-WO-PCTNO: 127); heat shock protein 70 promoter (HPS70p; SEQ ID NO: 128); tubulin alpha-1 chain promoter (TUB Ip; SEQ ID NO: 129); glyceraldehyde-3 -phosphate dehydrogenase 2 promoter (TDH2p; SEQ ID NO: 130); translation elongation factor 1A like gene 5 promoter (TEF5p; SEQ ID NO: 131); translation elongation factor 3 promoter (TEF3p; SEQ ID NO: 132); thioredoxin peroxidase promoter (TPXp; SEQ ID NO: 133); alginate lyase promoter (ALPp; SEQ ID NO: 134); thiazole biosynthetic 4 promoter (THI4p; SEQ ID NO: 135); phosphoglucomutase / phospomannomutase promoter (PMMp; SEQ ID NO: 136); fructose-bisphosphate aldolase 1 promoter (FBAlp; SEQ ID NO: 137); alcohol dehydrogenase 1 promoter (ADHlp; SEQ ID NO: 138); and extracellular endoglucanase 1 promoter (GLXlp; SEQ ID NO: 139). One or more of the exogenous polynucleotide sequences in the genetically modified yeast cell may be operably linked to a heterologous or artificial promoter selected form the group consisting of6PGD terminator (6PGDt; SEQ ID NO:78); ASNS terminator (ASNSt; SEQ ID NO:79); ENO1 terminator (ENOlt; SEQ ID NO:80); hexokinase 1 terminator (HXKlt; SEQ ID NO:81); PGK1 terminator (PGKlt; SEQ ID NO:82); PGM1 terminator (PGMlt; SEQ ID NO:83); PYK1 terminator (PYKlt; SEQ ID NO:84); RPLA terminator (RPLAt: SEQ ID NO:85); transaldolase 1 terminator (TALlt; SEQ ID NO:86); TDH3 terminator (TDH3t; SEQ ID NO: 87); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:88); triosephosphate isomerase 1 terminator (TPIlt; SEQ ID NO:89); MpTEFl terminator (MpTEFlt; SEQ ID NO:90); TEF7 terminator (TEF7t; SEQ ID NO: 140); HSP90 terminator (HSP90t; SEQ ID NO: 141); TEF4 terminator (TEF4t; SEQ ID NO: 142); TEF6 terminator (TEF6t; SEQ ID NO: 143); CITI terminator (CITI It; SEQ ID NO: 144): KAR2 terminator (KAR2t; SEQ ID NO: 145); HPS88 terminator (HSP88t; SEQ ID NO: 146); HSP70 terminator (HSP70t; SEQ ID NO: 147); TUB1 terminator (TUBlt; SEQ ID NO: 148); TKL1 terminator (TALlt; SEQ ID NO: 149); TDH2 terminator (TDH2t; SEQ ID NO: 150); TEF5 terminator (TEF5t; SEQ ID NO: 151); TGL2 terminator (TGL2t; SEQ ID NO: 152); TEF3 terminator (TEF3t; SEQ ID NO: 153); TPX terminator (TPXt; SEQ ID NO: 154); ALP terminator (ALPt; SEQ ID NO: 155); plasma membrane ATPase terminator (PMAlt; SEQ ID NO: 156); THI4 terminator (THI4t; SEQ ID NO: 157); glucose-6-phosphate isomerase 1 terminator (PGIlt; SEQ ID NO: 158); PMM terminator (PMMt; SEQ ID NO: 159); FBA1 terminator (FBAlt; SEQ ID NO: 160); ADH1 terminator (ADHlt; SEQ ID NO: 161); sodium / potassium transporting ATPase alpha chain terminator (ATPlt; SEQ ID NO: 162); and GLX1 terminator (GLXlt; SEQ ID NO: 163). One or more of the exogenous polynucleotidePT-2218-WO-PCTsequences may be incorporated into the genome of the yeast cell at a locus selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdllA locus, the gpdllB locus, the RCSR26640 locus, and the RCSR18717 locus.

[0016] The initial nitrogen concentration in the fermentation medium may be 0.5 g / L to 5 g / L, 1 g / L to 4 g / L, 1.5 g / L to 3.5 g / L, or preferably about 2 g / L to 3.25 g / L. Nitrogen may be added to the fermentation medium such that the total nitrogen concentration over the course of the fermentation reaction is about 3 g / L to 7 g / L, 3.5 g / L to 6.5 g / L, 4 g / L to 6 g / L, or about 4.5 g / L to 5.5 g / L. The nitrogen may be added during the fermentation at a rate of about 25 mg N / h to 150 mg N / h, 50 mg N / h to 140 mg N / h, 60 mg N / h to 135 mg N / h, or about 75 mg N / h to 130 mg N / h. The fermentation method includes a growth phase and a production phase, and the nitrogen feed is added during the production phase. The fermentation temperature may be at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C.BRIEF DESCRIPTION OF THE FIGURES

[0017] 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.

[0018] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0019] FIG. 1 shows the native pentose phosphate pathway (dotted lines and arrows) and the native glycolysis pathways (solid lines and arrows) in Moniliella pollinis.

[0020] FIG. 2 shows a graph of the agitation rate of vessels 1, 2, and 7 as outlined in Example 2.

[0021] FIG. 3 shows a graph of xylitol production in the reactions outlined in Example 2.

[0022] FIG. 4 shows a graph of dextrose consumption in the reactions outlined in Example 2.

[0023] FIG. 5 shows a graph of glycerol production and consumption in the reactions outlined in Example 2.

[0024] FIG. 6 shows a graph of ethanol production in the reactions outlined in Example 2.

[0025] FIG. 7 shows a graph of xylitol production in the reactions outlined in Example 3.

[0026] FIG. 8 shows a graph of glycerol production and consumption in the reactions outlined in Example 3.PT-2218-WO-PCT

[0027] FIG. 9 shows a graph of ethanol production in the reactions outlined in Example 3.

[0028] FIG. 10 shows a graph of the optical density at 600 nm (OD600) in the reactions outlined in Example 3.

[0029] FIG. 11 shows a graph of the oxygen uptake rate (OUR) in the reactions outlined in Example 3.

[0030] FIG. 12 shows a graph of dextrose consumption in the reactions outlined in Example 3.

[0031] FIG. 13 shows a graph of xylitol production in the reactions outlined in Example 4.

[0032] FIG. 14 shows a graph of glycerol production and consumption in the reactions outlined in Example 4.

[0033] FIG. 15 shows a graph of ammonia consumption in the reactions outlined in Example 4.DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] 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 all the 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%PT-2218-WO-PCTto 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.

[0037] 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.

[0038] This disclosure relates to methods for fermenting a yeast cell under aerobic conditions with supplemental nitrogen to improve production of xylitol while controlling or reducing production of glycerol and ethanol. In general, the yeast cells used in the fermentation methods described herein are recombinant cells engineered to produce xylitol that include a biosynthetic pathway for the conversion of xylulose-5-phosphate to xylitol. When these recombinant cells are fermented under microaerobic conditions, production of xylitol is increased (e.g., increase in rate, titer, and / or yield) and production of glycerol is reduced. However, microaerobic conditions produce excessive amounts of ethanol that can hinder fermentation scale-up due to the volatility and environmental health and safety risks of ethanol. As described herein, growth of these recombinant cells under aerobic conditions with supplemental nitrogen results in improved xylitol production and reduced glycerol production as seen with fermentation under microaerobic conditions but without the high ethanol production associated with microaerobic fermentation conditions.

[0039] The fermentation methods include the step of fermenting a substrate using the genetically engineered yeast to produce xylitol. The fermentation method can include additional steps, as would be understood by a person skilled in the art. For example, additional process steps may include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, adjusting media components, isolating the xylitol from the fermentation broth, and the like.

[0040] 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 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C, preferably about 35 °C. However,PT-2218-WO-PCTa skilled artisan will recognize that the fermentation temperature is not limited to any specific range or temperature recited herein and may be modified as appropriate.

[0041] The fermentation process can be run at various cell concentrations. In some embodiments, the cell dry weight at the end of fermentation can be 5 to 40, 8 to 30, or 10 to 20 g cell dry weight / L. Further, the pitch density or pitching rate of the fermentation process can vary. In some embodiments, the pitch density can be 0.05 to 11, 0.1 to 10, or 0.25 to 8 g cell dry weight / L.

[0042] Yeast fermentation processes are typically run with a controlled amount of oxygen in the reaction, for example, under anaerobic, microaerobic, or aerobic conditions. As used herein, “aerobic” refers to fermentation reaction conditions in which oxygen is readily available at high levels. For example, when the yeast is Moniliella pollinis, aerobic fermentation will typically have a dissolved oxygen content of at least 20%, at least 30%, at least 40%, or at least 50% during the majority of the production phase, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the fermentation time in the production phase has a dissolved oxygen content of at least 20%, at least 30%, at least 40%, or at least 50%.

[0043] As used herein, “microaerobic” refers to fermentation reaction conditions in which oxygen is available but at very low or limited levels compared to aerobic fermentation, generally where the rate of oxygen fed to the fermentation system is the same or similar to the rate at which oxygen is consumed by the yeast. For example, when the yeast is a Moniliella pollinis, microaerobic fermentation will typically have a dissolved oxygen content of less than 10 %, preferably less than 5% for a period of time during the fermentation, e.g., for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the total fermentation time has a dissolved oxygen content of less than 10 %, preferably less than 5%.

[0044] Herein, the dissolved oxygen content is measured relative to the maximum solubility of oxygen in the fermentation medium under air at atmospheric pressure. In other words, 100% dissolved oxygen (DO) is the maximum solubility of oxygen in the fermentation medium under air at atmospheric pressure.

[0045] The fermentation process can be run within certain oxygen uptake rate (OUR) ranges. The volumetric OUR of the fermentation process can be in the range of 15-80, 20-75, 25-70, 30-60, 30-50, or 40-50 mmol O2 / L / h. For example, the OUR may be at least 15 mmol / L / h, at least 20 mmol / L / h, at least 25 mmol / L / h, at least 30 mmol / L / h, at least 35 mmol / L / h, at least 40 mmol / L / h, at least 45 mmol / L / h, or at least 50 mmol / L / h.PT-2218-WO-PCT

[0046] The fermentation method is run using a suitable fermentation medium that includes a suitable fermentation substrate. The substrate of the fermentation method can include glucose, sucrose, galactose, mannose, molasses, xylose, fructose, hydrolysates of starch, lignocellulosic hydrolysates, or a combination thereof. One skilled in the art will recognize what fermentation substrate is suitable for a given fermentation organism and system.

[0047] In geographies with significant com, rice, or wheat refining, dextrose is a readily available substrate for microbial fermentation processes. The terms “glucose” and “dextrose” are used interchangeably herein and refer to D-glucose except where expressly indicated otherwise. The initial dextrose concentration of the fermentation may be at least 100, 200, 250, 300, 350, or at least 400 g / L dextrose when run as a batch fermentation. The initial dextrose concentration may be between 100 to 500, 150 to 450, 200 to 400, or 250 to 350 g / L when run as a batch fermentation. The fermentation process can be run as a dextrose-fed batch. When run in a fed batch fermentation process, the dextrose concentration may be at least 100, 200, 250, 300, 350, 400, or at least 450 g / L total over the course of the process. Further, the fermentation process can be a batch process, continuous process, or semi -continuous process, as would be understood by a person skilled in the art. One of skill in the art will recognize suitable initial and fed dextrose concentration for a microbial fermentation process.

[0048] In addition to the fermentation substrate, suitable fermentation medium may include micronutrients (e.g., amino acids, vitamins, and / or metals), salts, buffers, pH control agents, one or more sources of nitrogen, antifoaming agents, or combinations thereof.

[0049] The fermentation medium used in the fermentation process described includes nitrogen. The fermentation media used in the fermentation process described herein may include an initial nitrogen concentration of about 0.5 g / L to 5 g / L, 1 g / L to 4 g / L, 1.5 g / L to 3.5 g / L, or preferably about 2 g / L to 3.25 g / L. For example, the fermentation media may include an initial nitrogen concentration of about 2 g / L, about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, about 3.0 g / L, about 3.1 g / L, about 3.2 g / L, about 3.4 g / L, or about 3.5 g / L. Suitable fermentation medium components that are a source of nitrogen include, but are not limited to, inorganic nitrogen sources including urea, ammonia, ammonium sulfate, and nitrates, organic nitrogen sources including yeast extracts, plant meals and extracts, peptone, and the like, or combinations thereof.PT-2218-WO-PCT

[0050] Nitrogen included in the fermentation medium will typically be assimilable nitrogen. As used herein, “assimilable nitrogen” refer to nitrogen that is available to the fermentation organism, e.g., the yeast, to use during fermentation. Assimilable nitrogen is different than atmospheric nitrogen, N2, which is the most abundant gas in Earth’s atmosphere and cannot be used by fermentation organisms such as yeast as a nutrient source.

[0051] The fermentation processes described herein and run under aerobic conditions also includes supplemental nitrogen added and / or available over the course of the fermentation. Herein, “nitrogen supplementation” and “supplemental nitrogen” are used interchangeably and refer to the addition of nitrogen to a fermentation system. Suitable methods for nitrogen supplementation include, but are not limited to, increasing the initial amount of nitrogen in the fermentation media, feeding of nitrogen into the fermentation reaction, adding one or more boluses of nitrogen to the fermentation reaction, or combinations thereof.

[0052] In a standard fermentation reaction, the initial nitrogen present in the fermentation media will be consumed by yeast in the first X number of hours of the reaction, largely when the yeast is growing and replicating. In general, the fermentation processes described herein include supplemental nitrogen such that nitrogen is available in the fermentation for at least 1.5X, 2X, 2.5X, 3X the number of hours the nitrogen is available in the standard reaction without supplemental nitrogen. For example, in the fermentation methods described herein, nitrogen is available for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% of the total reaction time. The fermentation process may include a nitrogen concentration of at least 50 ppm for at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% of the total reaction time. The nitrogen availability in the fermentation media may be constant over a period of time or may be intermittent with two, three, four, five, or more periods of time during which nitrogen is available in the fermentation media. Without wishing to be bound by any particular theory or mode of action, the availability of nitrogen for longer periods of the total reaction time increases xylitol and reduces glycerol relative to an equivalent fermentation run under aerobic conditions but without supplemental nitrogen.

[0053] Nitrogen may be supplemented into the fermentation media such that the total concentration of nitrogen available in the fermentation system over the entire course of the reaction is about 3 g / L to 7 g / L, 3.5 g / L to 6.5 g / L, 4 g / L to 6 g / L, or about 4.5 g / L to 5.5 g / L. While the final concentration of nitrogen in the fermentation media at the end of fermentationPT-2218-WO-PCTmay be zero, the total concentration of nitrogen in the fermentation system will always be greater than zero, specifically it will be the initial concentration of nitrogen in the fermentation media, plus any nitrogen added to the fermentation media after the process begins.

[0054] When nitrogen is supplemented into fermentation system over a period of time, the rate of nitrogen supplementation will be at or below the rate at which the yeast in the fermentation system consumes nitrogen. When nitrogen is added at a rate higher than the rate at which the yeast in the fermentation system consumes nitrogen, the fermentation system will accumulate excess nitrogen in the fermentation media causing problems such as an increase in pH, higher than normal foaming, and the like, which may result in failure of the fermentation reaction before its completion. For example, when nitrogen is added to the fermentation media after the initial nitrogen is consumed, the nitrogen may be added at a rate of about 25 mg N / h to 150 mg N / h, 50 mg N / h to 140 mg N / h, 60 mg N / h to 135 mg N / h, or about 75 mg N / h to 130 mg N / h. The supplemental nitrogen may be added to the fermentation as a constant or exponential feed of nitrogen.

[0055] The supplemental nitrogen may be added as one or more boluses of nitrogen during fermentation. For example, one, two, three, four, five, or more boluses of nitrogen may be added during fermentation. The bolus dose may be between about 0.1 g / L to 2.5 g / L, for example, 0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, 1.0 g / L, 1.25 g / L, 1.5 g / L, 1.75 g / L, 2.0 g / L, or about 2.5 g / L.

[0056] The fermentation reactions described herein may include two phases, a first growth phase where the yeast is mostly growing and replicating and a second production phase in which the yeast is mostly metabolizing glucose to produce a fermentation product, such as xylitol. During the growth phase, the biomass increases exponentially, dissolved oxygen (DO) content decreases, carbon evolution rate (CER) increases, and the oxygen uptake rate (OUR) increases. After the CER peaks, the production phase begins, during which additional glucose and other components may be fed to the fermentation reaction, and the yeast begins to produce reaction products at a higher rate. The CER peak may be at about 50, 60, 70, 80, 90, 100, or about 110 mmol / L / h. During the production phase the OUR and DO remain relatively consistent until all the glucose is consumed. Nitrogen supplementation can happen during the growth phase, the production phase, or a combination thereof.

[0057] The fermentation process can be associated with various characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peakPT-2218-WO-PCTfermentation rate. These characteristics can be affected by the selection of the yeast and / or genetic modification of the yeast 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 yeast selection or modification and the selection of fermentation process conditions.

[0058] The xylitol production rate of the process may be at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h’1. The xylitol mass yield of the process may be at least 25, at least 30, at least 32, at least 34, at least 35, at least 38, or at least 40 percent. The final xylitol titer of the process may be at least 50, at least 75, at least 100, at least 125, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 g / L.

[0059] In general, the fermentation methods described herein are characterized by increased xylitol production relative to equivalent fermentation methods run under the same conditions but without the supplementation of nitrogen. Xylitol production may be increased by at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more xylitol than an equivalent aerobic fermentation method without the supplementation of nitrogen. For example, the fermentation methods described herein may produce at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more xylitol than an equivalent aerobic fermentation method run without nitrogen fed to the fermentation reaction in the growth and / or production phases.

[0060] The fermentation reactions described herein including supplementation of nitrogen may be characterized in a decrease in glycerol production and / or an increase in the rate of glycerol reconsumption relative to equivalent fermentation reactions run without supplemental nitrogen. For example, for fermentation reaction that include addition of a nitrogen feed during the production phase, less total glycerol may be produced and / or glycerol produced during the growth phase and early production phase may be consumed at a higher rate.

[0061] As described above, xylitol production under microaerobic conditions suffers from higher titers of ethanol production. In the fermentation methods described herein with the addition of supplemental nitrogen under aerobic conditions, there is no increase in ethanol production relative to an equivalent aerobic reaction run without supplemental nitrogen. For example, the final ethanol titer in the methods described herein may be less than 10 g / L, less than 8 g / L, less than 6 g / L, less than 5 g / L, less than 4 g / L, less than 3 g / L, or less than 2 g / L.PT-2218-WO-PCTPeak ethanol concentration in the fermentation methods described herein may be less than 10 g / L, less than 8 g / L, less than 6 g / L, less than 5 g / L, less than 4 g / L, less than 3 g / L, or less than 2 g / L.

[0062] In general, recombinant cells capable of producing xylitol 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). Yarrowia lipolytica, Issatchenkia orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Pichia pastoris, Candida (e.g., Candida magnolias, Candida ethanolica), Pichia deserticola, Pichia membranifadens, Pichia fermentans, Aspergillus, Trichoderma, Myceliphthora 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.

[0063] A suitable yeast cell may be a cell of the phylum Basidiomycota and the subphylum Ustilaginomycotina. Suitable yeast of the subphylum Ustilaginomycotina include, but are not limited to, Ustilago (e.g., U cynodontis, U maydis, U sphaerogena, U cordal, U scitaminea, U coicis, U syntherismae, U. esculenta, U neglecta, U crus-galli, Ustilago avenae), Sporisorium (e.g., Sporisorium exsertum), Moniliella (e.g., AL pollinis, M. tomentosa, M. acetoabutans, M. fonsecae, M. madida, M. megachiliensis, M. ocedocephalis, M. nigrescens), and Pseudozyma (e.g., Pseudozyma tsukubaensis), and Trichosporonoides (e.g., Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens). Yeast of the subphylum Ustilaginomycotina have been known and described inPT-2218-WO-PCTthe art as potential production organisms for valuable chemicals such as itaconate, malate, succinate, mannitol, and erythritol and other valuable biotechnological applications. See, for example, Geiser et al. (Prospecting the biodiversity of the fungal family Ustilaginacceae for the production of value-added 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 Biotechnol, 2010, 86:1017-1025).

[0064] The yeast cell may be an oleaginous yeast. Oleaginous yeast are those yeast that capable of producing significant amounts of lipids, for example, more than about 25 wt% of their cell dry weight as lipids. Oleaginous yeast may also be characterized as those including an adenosine triphosphate (ATP) citrate lyase (ACLY) gene encoding an enzyme capable of catalyzing the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA. Acetyl-CoA is a key building block for fatty acid synthesis, making its production important to a yeast ability to produce significant amounts of lipids. The genetically modified yeast described here in may be a yeast that natively includes an ATP citrate lyase gene.

[0065] A suitable yeast cell will have an active pentose phosphate pathway that produces ribulose-5-phosphate. As used herein “active pentose phosphate pathway” refers to expression of one or more functional enzymes which, together, convert glucose-6-phosphate, NADP+or NAD+, and water to NADPH or NADH, CO2, and ribulose-5-phosphate. Continuing in a non-oxidative phase, the pathway may also produce other pentose (i.e., 5-carbon) sugars. For example, the pentose phosphate pathway may produce ribulose-5-phosphate, ribose-5-phosphate, xylulose-5-phosphate, fructose 6-phosphate, combinations thereof, and the like, depending on the enzymatic activities present. The active pentose phosphate pathway may be native to the yeast cell, or it may be introduced into the yeast cell by genetic engineering.

[0066] 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.PT-2218-WO-PCT

[0067] The yeast cell may be an osmotol erant yeast cell. As used herein, “osmotol erant” refers to a yeast capable of growth and reproduction under conditions of high osmolarity, such as at least 10% (w / v), at least 20% (w / v), at least 30% (w / v), at least 40% (w / v), at least 50% (w / v), or at least 60% (w / v) glucose and / or at least 6% (w / v), at least 10% (w / v), at least 12% (w / v), at least 13% (w / v), at least 15% (w / v) sodium chloride. Species and strains of osmotolerant yeast are known and described in the art, including many species of yeast used in industrial fermentation processes. Likewise, methods for assaying yeast osmotolerance are known and described in the art. See, for example, Tiwari, S., et al., (“Nectar yeast community of tropical flowering plants and assessment of their osmotolerance and xylitol-producing potential,” Current Microbiology, 2022, 79:28).

[0068] The recombinant yeast cell may be a recombinant Moniliella cell, for example, a Moniliella pollinis cell. FIG. 1 shows the predicted native pentose phosphate and glycolysis pathways in Moniliella pollinis. Moniliella has previously been used 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).

[0069] Various plasmids and methods for transformation of Moniliella are also described in the Examples below. For example, Moniliella may be transformed using a bipartite polynucleotide sequence in which, following recombination, the exogenous polynucleotide of interest is integrated at the specified locus and the selection marker is expressible within the cell. Suitable selection markers are known and used in the art. The selectable marker may include, but is not limited to, amdS (for example broken into a 3’ portion, SEQ ID NO:1, and a 5’ portion, SEQ ID NO:2), G418 resistance gene (for example broken into a 3’ portion, SEQ ID NO:3, and a 5’ portion, SEQ ID NO:4), zeocin resistance gene (for example broken into a 3’ portion, SEQ ID NO:5, and a 5’ portion, SEQ ID NO:6), nourseothricin N-acetyl transferase (NAT) (for example broken into a 3’ portion, SEQ ID NO:7, and a 5’ portion, SEQ ID NO:8), and invertase gene (SUC2) (for example a 3’ portion of SEQ ID NOV and a 5’ portion of SEQ ID NO: 10).

[0070] The recombinant cells described herein include one or more exogenous polynucleotide sequences encoding one or more polypeptides that, when expressed, improve the fermentation of glucose to xylitol by the recombinant cells.PT-2218-WO-PCT

[0071] 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 ". lactis invertase gene is exogenous when introduced into S. cerevisiae.

[0072] 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. For the purposes of this application, the Moniliella pollinis cell “Moniliella tomentosa var pollinis TCV364” described in US 6,440,712, which is incorporated herein by reference in its entirety, and deposited under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) on March 28, 1997 under number MUCL40385, is considered the wild-type Moniliella pollinis cell.

[0073] 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 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 pathway is understood to include the co-factors, 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.PT-2218-WO-PCTTable 1 : Amino Acid three and one letter symbolsAmino Acid Three letter symbol One letter symbol Alanine Ala AArginine Arg RAsparagine Asn NAspartic acid Asp DCysteine Cys CGlutamic acid Glu EGlutamine Gin QGlycine Gly GHistidine His HIsoleucine He ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr TTryptophan Trp WTyrosine Tyr YValine Vai V

[0074] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed recombinant cells, compositions, and methods. Such sequences can be referred to as 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.PT-2218-WO-PCT

[0075] As used herein, the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well-known. 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) version 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.

[0076] 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.

[0077] 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 understoodPT-2218-WO-PCTby skilled persons and means the typical form and expression level 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.

[0078] 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. “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.

[0079] 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 (e.g., coding DNA) or a genomic DNA sequence (e.g., including both introns and exons).

[0080] 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.

[0081] 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 (e.g., polynucleotides encoding an erythrose reductase polypeptide) 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 polynucleotidePT-2218-WO-PCTsequences found in various species 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.

[0082] The fermentation methods described herein use a yeast cell capable of producing xylitol. Suitable yeast cells are described herein. In general, a yeast cell capable of producing xylitol as described herein include a biosynthetic pathway to produce xylitol from xylulose-5-phosphate. The biosynthetic pathway may be engineered into the organism by incorporating one or more exogenous polynucleotide sequence encoding heterologous enzymes. The biosynthetic pathway may also include one or more enzymes that are native to the host organism. The yeast cells may include a biosynthetic pathway that first converts xylulose-5-phosphate to xylitol -5 -phosphate using a xylitol phosphate dehydrogenase (XPDH) enzyme then converts xylitol-5-phosphate to xylitol using a phosphatase enzyme, for example a xylitol-5-phosphate phosphatase (X5PP) enzyme. The yeast cells may include a biosynthetic pathway that first converts xylulose-5-phosphate to xylulose using a xylulokinase (XKS) enzyme then concerts xylulose to xylitol using a xylitol dehydrogenase (XDH) enzyme. When the yeast cell is a Moniliella pollinis yeast cell, a cell capable of producing xylitol will include (i) an exogenous nucleic acid sequence encoding a xylitol phosphate dehydrogenase (XPDH) enzyme; or (ii) an exogenous nucleic acid sequence encoding a xylulokinase (XKS) enzyme and an exogenous nucleic acid sequence encoding a xylitol dehydrogenase (XDH) enzyme.

[0083] The recombinant cells described herein are capable of producing xylitol and may include an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme. The exogenous polynucleotide sequence may be an exogenous xylitol-phosphate dehydrogenase (XPDH) gene. A “xylitol-phosphate dehydrogenase gene” and an “XPDH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with xylitol-phosphate dehydrogenase activity. As used herein, “xylitol-phosphate dehydrogenase activity” refer to the ability to catalyze the conversion of xylulose-5-phosphate and NADPH or NADH to xylitol 5-phosphate and NADP+or NAD+. The XPDH gene may be derived from any suitable source. For example, the XPDH gene may be derivedPT-2218-WO-PCTfrom Clostridium difficile, Lactobacillus rhamnosus. Bacillus halodurans, Alkalihalobacillus ligniniphilus, Jeotgalibacillus soli, Heyndrickxia sporothermodurans, Clostridium fungisolvens, o Neobacillus cucumis. The XPDH gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:55-64. The XPDH gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:57, 58, 59, or 62. Additional description of recombinant cells capable of producing xylitol and including an exogenous polynucleotide encoding an enzyme with xylitol-phosphate dehydrogenase activity is provided in PCT Patent Publication No. WO / 2023 / 220545, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0084] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Clostridium difficile gene encoding the amino acid of SEQ ID NO: 55. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0085] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Clostridium difficile gene encoding the amino acid of SEQ ID NO: 56. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:56.

[0086] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, Lactobacillus rhamnosus gene encoding the amino acid of SEQ ID NO:57. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:57.

[0087] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Bacillus halodurans gene encoding the amino acid of SEQ ID NO: 58. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 58.PT-2218-WO-PCT

[0088] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Alkalihalobacillus ligniniphilus gene encoding the amino acid of SEQ ID NO:59. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:59.

[0089] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Jeotgalibacillus soli gene encoding the amino acid of SEQ ID NO:60. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:60.

[0090] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Heyndrickxia sporothermodurans gene encoding the amino acid of SEQ ID NO:61. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:61.

[0091] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Clostridium fungisolvens gene encoding the amino acid of SEQ ID NO:62. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:62.

[0092] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Neobacillus cucumis gene encoding the amino acid of SEQ ID NO:64. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:64.

[0093] The recombinant cells described herein are capable of producing xylitol and may include an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme. The exogenous polynucleotide sequence may be an exogenous xylulokinase (XKS) gene. In the art, some xylulokinase enzymes may also be referred to as xylulose sugar phosphatase enzymes. A “xylulokinase gene” and an “XKS gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with xylulokinase activity. As used herein, “xylulokinase activity” refer to the ability to catalyze the conversion of xylulose-5-phosphatePT-2218-WO-PCTand ADP to xylulose and ATP. The XKS gene may be derived from any suitable source. For example, the XKS gene may be derived from Saccharomyces cerevisiae. The XKS gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:50 and 49. Additional description of recombinant cells capable of producing xylitol and including a polypeptide with xylulokinase activity is provided in PCT Application Publication No. WO / 2023 / 220543, with a filing date of May 5, 2023, which is incorporated herein by reference in its entirety.

[0094] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Saccharomyces cerevisiae DOG1 sugar phosphatase gene encoding the amino acid of SEQ ID NO:50. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:50.

[0095] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Saccharomyces cerevisiae DOG2 sugar phosphatase gene encoding the amino acid of SEQ ID NO:49. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:49.

[0096] The recombinant cells described herein are capable of producing xylitol and may comprise an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme. The exogenous polynucleotide sequence may be an exogenous XDH gene. A “xylitol dehydrogenase gene” and an “XDH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with xylitol dehydrogenase activity. As used herein, “xylitol dehydrogenase activity” refer to the ability to catalyze the conversion of xylulose and NADH or NADPH to xylitol and NAD+or NADP+. The XDH gene may be derived from any suitable source. For example, the XDH gene may be derived from Pichia slipilis. Rhodobacteraceae bacterium, or Bemisia argentofolii . The XDH gene may encode an amino acid at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:65, 66, or 67. Additional description of recombinant cellsPT-2218-WO-PCTcapable of producing xylitol and including a polypeptide with xylulokinase activity and a polypeptide with xylitol dehydrogenase activity is provided in PCT Application Publication No. WO / 2023 / 220543, with a filing date of May 5, 2023, which is incorporated herein by reference in its entirety.

[0097] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a cofactor switched Pichia stipitis XDH gene encoding the amino acid of SEQ ID NO: 65. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0098] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Rhodobacteraceae bacterium SDR family oxidoreductase gene encoding the amino acid of SEQ ID NO:66. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ IDNO:66.

[0099] The recombinant cell may include an exogenous polynucleotide that is, or may be derived from, a Bemisia argentofolii (Silverleaf Whitefly) ketose reductase (sorbitol dehydrogenase) gene encoding the amino acid of SEQ ID NO:67. The exogenous polynucleotide may encode an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:67.

[0100] In addition to the xylitol biosynthetic pathway described above, the yeast cells described herein capable of producing xylitol may include genetic modifications that improve the production of xylitol. For example, the yeast cell capable of producing xylitol may also include an exogenous polynucleotide encoding a native or exogenous ribulose-5-phosphate epimerase (RPE) enzyme; have a genetic modification resulting in overexpression of a native RPE enzyme; include an exogenous polynucleotide sequence encoding an X5PP enzyme; overexpress a native X5PP enzyme; include an exogenous polynucleotide sequence encoding a transketolase (TKL) enzyme; and / or overexpress a TKL enzyme.

[0101] As used herein, “overexpression” refers to an expression level of a polypeptide that is higher than the expression level of the same polypeptide in the absence of a genetic modification or exogenous polynucleotide encoding said polypeptide in an equivalent cell.PT-2218-WO-PCT

[0102] Xylitol is a product of the pentose phosphate pathway, and increasing carbon flux into the pentose phosphate pathway should help in increasing xylitol yield and titer in fermentation. As demonstrated in FIG. 1 carbon can enter the pentose phosphate pathway from the “top” through the reaction of glucose-6-phosphate to gluconate-6-phosphate, or from the “bottom” through the transketolase (TKL) and transaldolase (TAL) enzyme catalyzed reactions of the glycolysis pathway intermediates fructose-6-phosphate and glyceraldehyde-3-phosphate. Without being bound by any particular theory, method, or mode of action, increasing expression of the transketolase enzyme (labeled TKL in FIG. 1) should increase carbon flux into the pentose phosphate pathway and subsequently increase xylitol rate, titer and / or yield.

[0103] The recombinant cells described herein are capable of producing xylitol and can be characterized by overexpression of a native enzyme with transketolase activity and / or include an exogenous polynucleotide sequence encoding a native or exogenous enzyme with transketolase activity. As used herein “transketolase enzyme” and “TKL enzyme” are used interchangeably and refer to an enzyme with transketolase activity. As used here in, “transketolase activity” refers to the ability to reversibly catalyze (i) the conversion of D-xylulose-5-phosphate and D-ribose-5-phosphate to sedoheptulose-7-phosphate and glyceraldehyde-3 -phosphate using a thiamine pyrophosphate (TPP) cofactor; and (ii) the conversion of D-xylulose-5-phosphate and erythrose-4-phosphate to fructose-6-phosphate and glyceraldehyde-3-phosphate. Polynucleotides encoding transketolase enzymes may be derived from any suitable source. For example, a polynucleotide encoding a transketolase enzyme may be derived from Moniliella pollinis. Additional description of recombinant cells capable of producing xylitol and including overexpression of a TKL enzyme and / or an exogenous polynucleotide sequence encoding a TKL enzyme is provided in US Provisional Patent Application No. 63 / 640,914, filed May 1, 2024, and US Provisional Patent Application No.63 / 640,918, filed May 1, 2024, each of which is incorporated herein by reference in its entirety.

[0104] The recombinant cell has an exogenous polynucleotide sequence that is or may be derived from ^Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO: 106. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO: 106.PT-2218-WO-PCT

[0105] The enzyme with transketolase activity may be native to the host cell. For example, when the host organism is M. pollinis, the transketolase enzyme may be an enzyme with a sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 106. The recombinant cell may comprise an exogenous polynucleotide encoding a transketolase enzyme with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 106. The recombinant cell may include a genetic modification that increases expression of a transketolase enzyme 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: 106. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native transketolase enzyme into the cell (e.g., integration of additional copies of the transketolase encoding polynucleotide into a non-native locus in the cell), insertion of a constitutive and / or strong promoter upstream of the coding region of the native transketolase enzyme encoding gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native transketolase enzyme encoding gene in the genome of the host cell. One of skill in the art will recognize that expression of a native transketolase enzyme encoding gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate.

[0106] The final step in the xylitol pathway, from xylitol 5 -phosphate to xylitol, requires a phosphatase enzyme. The Saccharomyces cerevisiae PYP1 (polyol phosphatase 1) gene encodes a sugar alcohol phosphatase that hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (D)-glycerol-3-phosphase (Xu et al., “Discovery and functional characterization of a yeast sugar alcohol phosphatase,” ACS Chem. Biol., 13, 2018, 3011-3020). PYP1 is a member of the haloacid dehalogenase (HAD)-like hydrolase superfamily (Kuznetsova, et a., “Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae '' J. Biol. Chem., 2015, 290, 18678-18698) and belongs to the enzyme class of sorbitol-6-phosphatases (Enzyme Commission (EC) 3.1.3.50). As xylitol 5-phosphate is a similar molecule to the known substrates of PYP1 it is demonstrated herein that one or more PYP-like enzymes or PYP orthologs have xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells described herein. E. coli HxpA (hexitol phosphatase A) is a HAD-like enzyme belonging to EC 3.1.3.50 with a similar substrate profile to PYP1 (Kuznetsova et al., “Genome wide analysis of substrate specificities of the EscherichiaPT-2218-WO-PCTcoli haloacid dehalogenase-like phosphate family,” 2006, J. Biol. Chem., 281, 36149-36161). Accordingly, it is also demonstrated herein that one or more HAD-like hydrolase enzymes or HAD-like hydrolase orthologs have xylitol-5-phosphate phosphatase activity and can be used to increase xylitol production in the recombinant cells describe here.

[0107] The recombinant cells described herein are capable of producing xylitol and may be characterized by overexpression of a native enzyme with xylitol-5-phosphate phosphatase (X5PP) activity and / or include an exogenous polynucleotide sequence encoding a native or exogenous enzyme with xylitol-5-phosphate phosphatase (X5PP) activity. In general, the recombinant cell(s) including overexpression of an X5PP enzyme or expressing an exogenous X5PP enzyme produce more xylitol than an equivalent cell lacking the exogenous X5PP enzyme or lacking overexpression of the X5PP enzyme. The enzyme may be any suitable enzyme with X5PP activity. As used herein, “X5PP enzyme” and “X5PP” are interchangeable and refer to an enzyme with X5PP activity. Herein, “xylitol-5-phosphate phosphatase activity” and “X5PP activity” are used interchangeably and refer to the ability to catalyze the conversion of xylitol-5-phosphate to xylitol and phosphate. Suitable X5PP enzymes may include a divalent metal cation, for example, Mg2+, Mn2+, or Co2+. Suitable enzymes with X5PP activity may include, but are not limited to, those classified under EC 3.1.3.50, for example, sugar alcohol phosphatases and HAD-like hydrolases. Polynucleotides encoding X5PP enzymes may be derived from any suitable source. For example, a polynucleotide encoding an X5PP enzyme may be derived from Moniliella pollinis, Saccharomyces cerevisiae. Lachancea dctsiensis. Tetrapisispora blattae, Saccharomyces pastorianus, Kazachstania Africana. Podospora comala. Geotrichum candidum. Ogattaea haglerorum. Debaryomyces fabryi, Monilinia fructicola, Nadsonia fulvescens var. elongata DSM 6958, Escherichia coli, Wicker hamomyces ciferrii, Bacillus amyloliquefaciens, and the like. The X5PP enzyme may be a polypeptide with an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NOs:33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. The X5PP enzyme may be a polypeptide with an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NOs:33, 35, 36, 37, 38, 39, 43, and 45. The X5PP enzyme may be a polypeptide with an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at leastPT-2218-WO-PCT90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of at least one of SEQ ID NOs:33, 36, 37, 39, and 45. Additional description of recombinant cells capable of producing xylitol and including a polypeptide with xylitol-5-phosphate phosphatase activity is provided in PCT Application Publication No. WO / 2024 / 229194, with a filing date of May 2, 2024, which is incorporated herein by reference in its entirety.

[0108] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO:33. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:33.

[0109] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO:34. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:34.

[0110] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from Lachancea dasiensis gene encoding the amino acid sequence of SEQ ID NO:35. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:35.

[0111] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Tetrapisispora blattae gene encoding the amino acid sequence of SEQ ID NO:36. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:36.

[0112] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces pastorianus gene encoding the amino acid sequence of SEQ ID NO:37. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:37.

[0113] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Kazachstania africana gene encoding the amino acid sequence ofPT-2218-WO-PCTSEQ ID NO:38. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:38.

[0114] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Podospora comata gene encoding the amino acid sequence of SEQ ID NO:39. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:39.

[0115] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Geotrichum candidum gene encoding the amino acid sequence of SEQ ID NO:40. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:40.

[0116] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Ogattaea haglerorum gene encoding the amino acid sequence of SEQ ID NO:41. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:41.

[0117] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from Debaryomyces fabryi gene encoding the amino acid sequence of SEQ ID NO:42. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:42.

[0118] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from Mondinia fructicola gene encoding the amino acid sequence of SEQ ID NO:43. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:43.

[0119] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Nadsonia fulvescens var. elongata DSM 6958 gene encoding the amino acid sequence of SEQ ID NO:44. The exogenous polynucleotide sequence may encodePT-2218-WO-PCTan amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:44.

[0120] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from an Escherichia coli gene encoding the amino acid sequence of SEQ ID NO:45. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:45.

[0121] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Wicker hamomyces ciferrii gene encoding the amino acid sequence of SEQ ID NO:46. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:46.

[0122] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Moniliella pollinis gene encoding the amino acid sequence of SEQ ID NO:47. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:47.

[0123] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Bacillus amyloliquefaciens gene encoding the amino acid sequence of SEQ ID NO:48. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:48.

[0124] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae DOG2 gene encoding the amino acid sequence of SEQ ID NO: 49. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:49.

[0125] The recombinant cell may comprise an exogenous polynucleotide sequence that is or may be derived from a Saccharomyces cerevisiae DOG1 gene encoding the amino acid sequence of SEQ ID NO: 50. The exogenous polynucleotide sequence may encode an amino acid sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, identical to SEQ ID NO:50.PT-2218-WO-PCT

[0126] The enzyme with X5PP activity may be native to the host cell. For example, when the host organism is M. pollinis, the X5PP enzyme may be an enzyme with a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to at least one of SEQ ID NOs:51, 52, 33, or 47. The recombinant cell may comprise an exogenous polynucleotide encoding an X5PP enzyme with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of SEQ ID NOs: 51, 52, 33, or 47. The recombinant cell may include a genetic modification that increases expression of an X5PP enzyme 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: 51, 52, 33, or 47. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native X5PP enzyme into the cell (e.g., integration of additional copies of the X5PP encoding polynucleotide into it non-native locus in the cell), insertion of a constitutive promoter upstream of the coding region of the native X5PP enzyme encoding gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native X5PP enzyme encoding gene in the genome of the host cell. One of skill in the art will recognize that expression of a native X5PP enzyme encoding gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate. Additional description of recombinant cells capable of producing xylitol and overexpressing a native enzyme with xylitol-5-phosphate phosphatase activity is provided in PCT Patent Application Publication No. WO / 2024 / 229194, filed May 2, 2024, which is incorporated herein by reference in its entirety.

[0127] The recombinant cells described herein are capable of producing xylitol and may include an exogenous polynucleotide encoding a native or exogenous ribulose-5-phosphate epimerase (RPE) enzyme or may have a genetic modification resulting in overexpression of a native RPE enzyme. In general, the recombinant cell(s) including overexpression of the RPE enzyme produce more xylitol than an equivalent cell lacking the RPE enzyme or lacking overexpression of the RPE enzyme. The RPE enzyme may be any suitable enzyme with ribulose 5-phosphate epimerase activity. As used herein, “ribulose 5-phosphate epimerase activity” and “RPE activity” are used interchangeably and refer to the ability to catalyze the conversion of ribulose-5-phosphate to xylulose-5-phosphate. The enzyme with RPE activity may be native to the host cell or the RPE enzyme may be an exogenous RPE enzyme. ForPT-2218-WO-PCTexample, when the host organism is M. pollinis, the RPE enzyme may be an enzyme with a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to at least one of SEQ ID NOs:53 and 54. The recombinant cell may comprise an exogenous polynucleotide encoding an RPE enzyme with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to at least one of SEQ ID NOs:53 and 54. The recombinant cell may include a genetic modification that increases expression of an RPE enzyme 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:53 and 54. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native RPE into the cell, insertion of a constitutive promoter upstream of the coding region of the native RPE gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native RPE gene in the genome of the host cell. One of skill in the art will recognize that expression of a native RPE gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate. Additional description of recombinant cells capable of producing xylitol and including an exogenous polynucleotide encoding a native or exogenous RPE enzyme or a genetic modification resulting in overexpression of a native RPE enzyme is provided in PCT Patent Publication No. WO / 2023 / 220547, filed May 5, 2023, which is incorporated herein by reference in its entirety.

[0128] 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 produces an 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 orPT-2218-WO-PCTdisrupted. 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.

[0129] Glycerol is a common byproduct of fermentation using yeast and is generally produced when yeast cells face osmotic stress to prevent dehydration of the cell by balancing the intracellular osmolarity with that of the fermentation broth. The production of glycerol is also important for redox balancing during fermentations and ensuring continued metabolism by recycling NADH. However, the synthetic pathway to glycerol diverts carbon away from the pentose phosphate pathway limiting the potential for xylitol production. The recombinant cells described herein have a deletion or disruption in one or more native gene encoding an enzyme involved in glycerol production. Deletion or disruption of one or more of these biosynthetic pathway enzymes decreases the ability of the recombinant cell to product glycerol and may, depending on the deletion or disruption, increase carbon flux into the fermentation pathway for the production of xylitol.

[0130] The recombinant cells described herein may include a deletion or disruption of a native glycerol-3 -phosphate dehydrogenase 2a (GPDIIa) gene. The native GPDIIa gene encodes an enzyme that has glycerol-3 -phosphate dehydrogenase 2a activity. As used herein “glycerol-3 -phosphate dehydrogenase 2a activity” and “GPDIIa activity” are used interchangeably and refer to enzymes that catalyze the reversible oxidation of glycerol-3 -phosphate and an NAD(P)+ cofactor to dihydroxyacetone phosphate and NAD(P)H. When the host contains multiple GPDIIa genes, it is preferred to delete or disrupt at least one of them. When the host cell contains multiple alleles of a given GPDIIa gene, it is preferred to delete or disrupt one allele or both alleles of a given GPDIIa gene.

[0131] When the recombinant cell is a Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of a GPDIIa 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: 107, also referred to as the RCSR26270 enzyme herein. When the recombinant cell is Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of a GPDIIa gene with a nucleotide sequence at least 70%, at least 75%,PT-2218-WO-PCTat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 108, also referred to as the RCSR26270 gene herein. The recombinant Moniliella pollinis cell may include a heterologous or native polynucleotide sequence incorporated at the GPDIIa locus defined by the flanking sequences of SEQ ID NO: 29 and SEQ ID NO:30 such that the native GPDIIa gene is deleted or disrupted.

[0132] The recombinant cells described herein may have a deletion or disruption in one or more native genes encoding an enzyme involved in erythritol fermentation or consumption. Deletion or disruption of one or more of these biosynthetic pathway enzymes decreases the ability of the recombinant cell to produce erythritol and may, depending on the deletion or disruption, increasing carbon flux into the fermentation pathway for the production of xylitol.

[0133] The recombinant cells described herein may include a deletion or disruption of a native erythrose reductase (ER) gene. The native ER gene encodes an enzyme that has erythrose reductase activity. As used herein “erythrose reductase activity” and “ER activity” are used interchangeably and refer to enzymes that catalyze the reversible conversion of erythrose or erythrose-4-phosphate to erythritol or erythritol-4-phosphate using a nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor. In the art, enzymes that catalyze the reversible conversion of erythrose-4-phosphate to erythritol-4-phosphate using a nicotinamide adenine dinucleotide (phosphate) (hydrogen) (NAD(P)(H)) cofactor may also be described as erythrose or erythritol phosphate dehydrogenases. When the host cell contains multiple ER genes, it is preferred to delete or disrupt at least one of them. When the host cell contains multiple alleles of a given ER gene, it is preferred to delete or disrupt one allele or both alleles of the given ER gene. Additional description of recombinant cells capable of producing xylitol and including a deletion or disruption of a native erythrose reductase gene is provided in PCT Patent Application Publication No. WO / 2024 / 229193, filed May 2, 2024, which is incorporated herein by reference in its entirety.

[0134] 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.

[0135] When the recombinant cell is a Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER gene encoding an amino acid sequence at least 70%,PT-2218-WO-PCTat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11, also referred to as the RCSR26640 enzyme herein. When the recombinant cell is Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER 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: 12, also referred to as the RCSR26640 gene herein. The recombinant Moniliella pollinis cell may include a heterologous or native polynucleotide sequence incorporated at the RCSR26640 locus defined by the flanking sequences of SEQ ID NO: 13 and SEQ ID NO: 14 such that the native RCSR26640 gene is deleted or disrupted.

[0136] When the recombinant cell is a Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER 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: 15, also referred to as the RCSR18717 enzyme herein. When the recombinant cell is Moniliella pollinis cell, the recombinant cell may comprise a deletion or disruption of an ER 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: 16, also referred to as the RCSR18717 gene herein. The recombinant Moniliella pollinis cell may include a heterologous or native polynucleotide sequence incorporated at the RCSR18717 locus defined by the flanking sequences of SEQ ID NO: 17 and SEQ ID NO: 18 such that the native RCSR18717 gene is deleted or disrupted.

[0137] In addition to reducing carbon flux into glycerol using the GPDIIa deletion described herein, glycerol may be further reduced by cellular consumption of glycerol. Accordingly, upregulation or overexpression of the enzymes in the glycerol catabolic pathway can reconsume the produced glycerol and convert it to dihydroxyacetone phosphate and ultimately glyceraldehyde-3 -phosphate which can then enter the pentose phosphate pathway. This glycerol recycle process can begin with the conversion of glycerol to glycerol-3 phosphate using a glycerol kinase enzyme. Without wishing to be bound by any particular theory, method, or mode of action, overexpressing a glycerol kinase enzyme will decrease the residual concentration of glycerol and increase the production of glycerol-3 phosphate that can then be converted to glyceraldehyde-3 -phopshate and recycle carbon back into the pentose phosphate pathway to increase xylitol production. Even in the absence of a xylitol synthetic pathway, over expression of a glycerol kinase enzyme should decrease the accumulation of glycerol.PT-2218-WO-PCT

[0138] The recombinant cells described herein may include an exogenous polynucleotide encoding a native glycerol kinase and / or may have a genetic modification resulting in overexpression of a native glycerol kinase. In general, cells overexpressing a glycerol kinase enzyme will accumulate less glycerol during fermentation than an equivalent cell lacking the glycerol kinase overexpression (e.g., wild-type expression of the glycerol kinase). The glycerol kinase enzyme may be any suitable enzyme with glycerol kinase activity. As used herein, “glycerol kinase enzyme” refers to an enzyme with glycerol kinase activity. As used herein, “glycerol kinase activity” refers to the ability of the polypeptide to catalyze the irreversible conversion of glycerol and adenosine triphosphate (ATP) to glycerol-3 phosphate and adenosine diphosphate (ADP). The glycerol kinase enzyme may be native to the host cell. For example, when the host cell is Moniliella pollinis. the glycerol kinase enzyme may be an enzyme with a 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 at least one of SEQ ID NOs:92 and 103. The enzyme of SEQ ID NO:103 is encoded by the M. pollinis gene RCSR17702 and the enzyme of SEQ ID NO:92 is encoded by the M. pollinis gene RCSR09543. The recombinant cell may comprise an exogenous polynucleotide encoding a glycerol kinase enzyme 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:92 and 103. The recombinant cell may include a genetic modification that increases expression of a glycerol kinase enzyme 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:92 and 103. The genetic modification may include, but is not limited to, insertion of additional copies of a nucleic acid encoding the native glycerol kinase enzyme into the cell, insertion of a constitutive promoter upstream of the coding region of the native glycerol kinase gene in the genome of the host cell, and / or modification of the existing promoter upstream of the coding region of the native glycerol kinase gene in the genome of the host cell. One of skill in the art will recognize that expression of a native glycerol kinase gene may be increased by a number of methods known in the art and will be able to select and apply such methods as appropriate.

[0139] 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,PT-2218-WO-PCTGPD1, CYB2A, CYB2B, g4240, YMR226, MDHB, AT02, Adh9091, Adhl202, ADE2, ADH2556, GAL6, MDH1, SCW11, ER1, ER3, pyrF, TRP3, gpdllA, and gpdllB loci. For example, in a M. pollinis host cells, suitable interaction loci may include, but are not limited to, the ER1 locus (defined as the locus flanked by SEQ ID NO: 19 and SEQ ID NO:20), the ER3 locus (defined as the locus flanked by SEQ ID NO:21 and SEQ ID NO:22), the PDC1 locus (defined as the locus flanked by SEQ ID NO:23 and SEQ ID NO:24), the pyrF locus (defined as the locus flanked by SEQ ID NO:25 and SEQ ID NO:26), the TRP3 locus (defined as the locus flanked by SEQ ID NO:27 and SEQ ID NO:28), the gpdllA locus (defined as the locus flanked by SEQ ID NO:29 and SEQ ID NO:30); the gpdllB locus (defined as the locus flanked by SEQ ID NO:31 and SEQ ID NO:32); the RCSR18717 locus (defined as the locus flanked by SEQ ID NO:17 and SEQ ID NO:18); and the RCSR26640 locus (defined as the locus flanked by SEQ ID NO: 13 and SEQ ID NO: 14). 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.

[0140] 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.

[0141] The exogenous polynucleotides 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 promoterPT-2218-WO-PCT(ADH1), hexokinase 2 promoter (HXK2), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp; SEQ ID NO: 68); 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:69); glyceraldehyde-3 -phosphate dehydrogenase 3 promoter (TDH3p; SEQ ID NO:70); translational elongation factor 1 promoter (TEFlp; SEQ ID NO:71); phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72); 3 -phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73); enolase promoter (ENOlp ; SEQ ID NO:74); asparagine synthetase promoter (ASNSp; SEQ ID NO:75); 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76); RPL16B (SEQ ID NO:77); glycerol-3 -phosphate dehydrogenase 2a promoter (GPDIIap; SEQ ID NO: 114); glycerol dehydrogenase NADP(H) 1 promoter (GDNlp; SEQ ID NO: 116); erythritol-P dehydrogenase 1 promoter (EPDH1; SEQ ID NO: 117), translation elongation factor 1A like gene 7 promoter (TEF7p; SEQ ID NO: 120); heat shock protein 90 promoter (HSP90p; SEQ ID NO: 121); translation elongation factor 1A like gene 4 promoter (TEF4p; SEQ ID NO: 122); translation elongation factor 2A like gene 6 promoter (TEF6p; SEQ ID NO: 123); transaldolase 1 promoter (TALlp; SEQ ID NO: 124); citrate synthase 1 promoter (CITlp; SEQ ID NO: 125); endoplasmic reticulum chaperone BiP (KAR2p; SEQ ID NO: 126); heat shock protein 88 promoter (HSP88p; SEQ ID NO: 127); heat shock protein 70 promoter (HPS70p; SEQ ID NO:128); tubulin alpha-1 chain promoter (TUBlp; SEQ ID NO:129); glyceraldehyde-3 -phosphate dehydrogenase 2 promoter (TDH2p; SEQ ID NO: 130); translation elongation factor 1A like gene 5 promoter (TEF5p; SEQ ID NO: 131); translation elongation factor 3 promoter (TEF3p; SEQ ID NO: 132); thioredoxin peroxidase promoter (TPXp; SEQ ID NO: 133); alginate lyase promoter (ALPp; SEQ ID NO: 134); thiazole biosynthetic 4 promoter (THI4p; SEQ ID NO: 135); phosphoglucomutase / phospomannomutase promoter (PMMp; SEQ ID NO: 136); fructose-bisphosphate aldolase 1 promoter (FBAlp; SEQ ID NO: 137); alcohol dehydrogenase 1 promoter (ADHlp; SEQ ID NO: 138); and extracellular endoglucanase 1 promoter (GLXlp; SEQ ID NO: 139).

[0142] 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; SEQ ID NO:78); ASNS terminator (ASNSt; SEQ ID NO:79); ENO1 terminator (ENOlt; SEQ ID NO:80); hexokinase 1 terminator (HXKlt; SEQ ID NO:81); PGK1 terminator (PGKlt; SEQ ID NO:82); PGM1 terminatorPT-2218-WO-PCT(PGMlt; SEQ ID NO:83); PYK1 terminator (PYKlt; SEQ ID NO:84); RPLA terminator (RPLAt: SEQ ID NO:85); transaldolase 1 terminator (TALlt; SEQ ID NO:86); TDH3 terminator (TDH3t; SEQ ID NO: 87); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:88); triosephosphate isomerase 1 terminator (TPIlt; SEQ ID NO:89); MpTEFl terminator (MpTEFlt; SEQ ID NO:90); TEF7 terminator (TEF7t; SEQ ID NO: 140); HSP90 terminator (HSP90t; SEQ ID NO: 141); TEF4 terminator (TEF4t; SEQ ID NO: 142); TEF6 terminator (TEF6t; SEQ ID NO: 143); CITI terminator (CITI It; SEQ ID NO: 144): KAR2 terminator (KAR2t; SEQ ID NO: 145); HPS88 terminator (HSP88t; SEQ ID NO: 146); HSP70 terminator (HSP70t; SEQ ID NO: 147); TUB1 terminator (TUBlt; SEQ ID NO: 148); TKL1 terminator (TALlt; SEQ ID NO: 149); TDH2 terminator (TDH2t; SEQ ID NO: 150); TEF5 terminator (TEF5t; SEQ ID NO: 151); TGL2 terminator (TGL2t; SEQ ID NO: 152); TEF3 terminator (TEF3t; SEQ ID NO: 153); TPX terminator (TPXt; SEQ ID NO: 154); ALP terminator (ALPt; SEQ ID NO: 155); plasma membrane ATPase terminator (PMAlt; SEQ ID NO: 156); THI4 terminator (THI4t; SEQ ID NO: 157); glucose-6-phosphate isomerase 1 terminator (PGIlt; SEQ ID NO: 158); PMM terminator (PMMt; SEQ ID NO: 159); FBA1 terminator (FBAlt; SEQ ID NO: 160); ADH1 terminator (ADHlt; SEQ ID NO: 161); sodium / potassium transporting ATPase alpha chain terminator (ATPlt; SEQ ID NO: 162); and GLX1 terminator (GLXlt; SEQ ID NO: 163).

[0143] 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.

[0144] The polypeptides 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 laboratory methods 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 a polypeptide as described herein. As usedPT-2218-WO-PCTherein, 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 double-stranded DNA loop into which additional DNA segments may be integrated.EXAMPLES

[0145] 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.

[0146] Throughout the Examples, strain numbering and sequence identification numbers are used consistently. For example, strain 1-16 in Example 1 is the same as strain 1-16 in Examples 2, 3, etc.Example 1 - Genetically Modified AfomZ / eZZa / wZZ / w.s Strains

[0147] Strain 1-1 is the Moniliella pollinis host strain “Moniliella tomentosa var pollinis TCV364” described in US 6,440,712, which is incorporated herein by reference in its entirety, and deposited under the Budapest Treaty at BCCM / MUCL (Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain by Eridania Beghin Say, Vilvoorde R&D Centre, Havenstraat 84, B-1800 Vilvoorde) on March 28, 1997 under number MUCL40385.Strain 1-2

[0148] SEQ ID NO:91 contained a 5’ flanking DNA for targeted chromosomal integration into the ER1 loci (SEQ ID NO: 19); an expression cassette for the XPDH homolog from Lactobacillus rhamnosus encoding the amino acid sequence of SEQ ID NO:57, under the control of the PYK1 promoter of SEQ ID NO:68 and the PGD terminator of SEQ ID NO:78; and a 5’ portion of the G418 selectable marker (SEQ ID NO:4). SEQ ID NO:93 contained (i) a 3’ portion of the G418 selectable marker (SEQ ID NO:3); ii) an expression cassette encoding the XPDH homolog from Lactobacillus rhamnosus of SEQ ID NO:57, under the control of thePT-2218-WO-PCTPYK1 promoter of SEQ ID NO:68 and the PGD terminator of SEQ ID NO:78; and (iii) a 3’ flanking DNA for targeted chromosomal integration into the ER1 locus (SEQ ID NO:20).

[0149] Strain 1-1 was transformed with SEQ ID NO: 91 and SEQ ID NO: 93 by first protoplasting the parent strain by adding an enzyme mixture containing 0.6M MgSO4, 7.5 g / L driselase, and 12.5 g / L Trichodermaharzianum lysing enzyme to a mycelial pellet of the parent strain. Protoplasts were then pelleted, washed with 0.6M MgSO4, and resuspended in STC medium (0.6M sucrose, 50 mM CaC12, 10 mM Tris-HCl, pH 7.5). Bipartite transformations were prepared by adding 100 pg single stranded salmon sperm DNA and 1.5 to 5 pg each of the 5’ and 3’ DNA transformation fragments (3-10 pg total; see Table 4 for list of fragments) to approximately 200 pL protoplast mixture (108cells / mL). 1 mL 50% PEG in STC medium was then added to the salmon sperm DNA, transformation DNA, and protoplast mixture and the resulting combination was incubated for 15 minutes at room temperature. Following incubation, recovery broth (0.4M 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, 100 rpm, for 16 to 24 hours. Following the incubation, protoplasts were pelleted by centrifugation and resuspended in 1 mL PBS.

[0150] The resuspended protoplasts were plated on PDA + 250 mg / L geneticin (G418) selection plates and incubated at 30-35 °C for at least 2-4 days until transformants grow. Resulting transformants were streaked for single colony isolation on PDA + geneticin (G418) plates and a single colony was selected. The selected colony was evaluated by colony PCR for integration of one copy of the L. rhamnosus XPDH encoding sequence and one additional copy (beyond the existing wild-type copies) of the RPE encoding sequences. A PCR verified isolate was designated strain 1-2.Strain 1-3

[0151] Strain 1-2 was transformed as outlined above with SEQ ID NO: 94 and SEQ ID NO: 95. The transformation fragment of SEQ ID NO: 94 contained, in order, a 5’ ER3 flanking sequence (SEQ ID NO:21), a MpPYKl promoter (SEQ ID NO:68), a gene encoding the M. pollinis RPE2 polypeptide of SEQ ID NO: 54, a MpPYK terminator (SEQ ID NO: 84), and a 5’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:6). The transformation fragment of SEQ ID NO:95 contained, in order, a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO: 5), and a 3’ ER3 flanking sequence (SEQ ID NO:22).PT-2218-WO-PCTTransformants were selected on PDA + zeocin selection plates and incubated at 35 °C for at least 2 days until transformants grow. Resulting transformants were streaked for single colony isolation on PDA + zeocin plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the gene encoding the M. pollinis RPE2 polypeptide. A PCR verified isolate was designated strain 1-3.Strain 1-4

[0152] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 1-3 was used to generate Moniliella pollinis strain with reduced foaming during shake flask fermentation. Strains with low-foaming phenotypes were selected based on visual evaluation of foaming in a shake flask fermentation compared to foaming of the parent 1-4 strain. The resulting low-foaming strain, containing two copies of an exogenous polynucleotide sequence encoding the XPDH of SEQ ID NO: 57 integrated at the ER1 locus and one copy of a polynucleotide sequence encoding the RPE of SEQ ID NO: 54 integrated at the ER3 locus, was designated 1-4.Strain 1-5

[0153] Strain 1-4 was transformed with the Cre recombinase plasmid of SEQ ID NO:96 using the transformation method outlined above. The resulting transformants were evaluated by colony PCR for removal of the zeocin and geneticin (G418) resistance selection markers. A PCR verified isolate was designated strain 1-5.Strain 1-6

[0154] Strain 1-5 was grown non- selectively on YPD plates to allow for the loss of the plasmid of SEQ ID NO:96. Biomass was struck for single colonies and evaluated by PCR to confirm loss of the plasmid. A PCR verified isolate was designated strain 1-6.Strain 1-7

[0155] Strain 1-6 was transformed with SEQ ID NO:97 and SEQ ID NO:94 as outlined above. SEQ ID NO: 97 contained a 3’ portion of a zeocin resistance gene expression cassette (SEQ ID NO:5), a MpPGKl promoter (SEQ ID NO:73), a gene encoding the X5PP polypeptide of SEQ ID NO:33, a Mp6PGD terminator (SEQ ID NO:78), and a 3’ ER3 flanking sequencePT-2218-WO-PCT(SEQ ID NO:22). Transformants were selected on PDA + zeocin selection plates and incubated at 35 °C for at least 2 days until transformants grow. Resulting transformants were streaked for single colony isolation on PDA + zeocin plates and single colonies were selected. Selected colonies were evaluated by colony PCR for integration of the M. pollinis X5PP gene at the ER3 locus. A PCR verified isolate was designated strain 1-7.Strain 1-8

[0156] Strain 1-7 was transformed with SEQ ID NO:98 using the transformation protocol outlined above and positive transformants were selected using nourseothricin selection plates. SEQ ID NO: 98 contains a deletion construct to remove one copy of the RSCR18717 gene (SEQ ID NO: 16) and a nourseothricin resistance gene expression cassette. A PCR verified isolate, in which one copy of the RCSR18717 gene was knocked out, was designated strains 1-8.Strain 1-9

[0157] Strain 1-8 was transformed with SEQ ID NO:99 using the transformation protocol outlined above. SEQ ID NO:99 contained a construct to loop out the zeocin and nourseothricin resistance selection markers. A PCR verified isolate, in which zeocin and nourseothricin resistance selection markers were removed, was designated strain 1-9.Strain 1-10

[0158] Strain 1-9 was transformed with SEQ ID NO:98 using the transformation protocol outlined above and positive transformants were selected using nourseothricin selection plates. A PCR verified isolate, in which both copies of the RCSR18717 gene were knocked out, was designated strains 1-10.Strain 1-11

[0159] Strain 1-10 was transformed with SEQ ID NO: 100 and SEQ ID NO: 101 using the transformation protocol outlined above and positive transformants were selected using geneticin (G418) selection plates. SEQ ID NO: 101 contained a 3’ portion of a G418 resistance gene expression cassette (SEQ ID NO:3), and a 3’ gpdllB flanking sequence (SEQ ID NO:32). SEQ ID NO: 100 contained a 5’ gpdllB flanking sequence (SEQ ID NO:31), an MpPYKl promoter (SEQ ID NO:68), a polynucleotide encoding the M. pollinis X5PP enzyme of SEQPT-2218-WO-PCTID NO:33, an MpENOl terminator (SEQ ID NO:80), an MpPGKl promoter (SEQ ID NO:73), a polynucleotide sequence encoding the M. pollinis X5PP enzyme of SEQ ID NO:33, an MpTDH3 terminator (SEQ ID NO:87), and a 5’ portion of a G418 resistance marker (SEQ ID NO:4). A PCR verified isolate, containing two copies of the gene encoding the X5PP of SEQ ID NO:33, was designated strain 1-11.Strain 1-12

[0160] Strain 1-11 was transformed with SEQ ID NO: 102 using the transformation protocol outlined above. SEQ ID NO: 102 contains a construct to loop out the nourseothricin and geneticin (G418) resistance selection markers. A PCR verified isolate, in which both the nourseothricin and geneticin resistance selection markers were removed, was designated strain 1-12.Strain 1-13

[0161] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 1-12 was used to generate Moniliella pollinis strain with improved xylitol production rate in shake flask fermentation. Strains with high xylitol rates were selected based on xylitol production (as measured by HPLC) in a shake flask fermentation compared to xylitol production in the parent 1-12 strain. A resulting strain with improved xylitol rate, which contained 2 copies of the gene encoding the XPDH of SEQ ID NO:57, 2 additional copies of the gene encoding the RPE of SEQ ID NO: 54 (for 3 copies total), 3 additional copies of the gene encoding the X5PP of SEQ ID NO:33 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, was designated strain 1-13.Strain 1-14

[0162] UV mutagenesis (using a Hoefer UV Crosslinker at an energy of 360 uJ / cm3) and selection of strain 1-13 was used to generate Moniliella pollinis strain with improved xylitol production rate in shake flask fermentation. Strains with high xylitol rates were selected based on xylitol production (as measured by HPLC) in a shake flask fermentation compared to xylitol production in the parent 1-13 strain. A resulting strain with improved xylitol rate, which contained 2 copies of the gene encoding the XPDH of SEQ ID NO:57, 2 additional copies of the gene encoding the RPE of SEQ ID NO: 54 (for 3 copies total), 3 additional copies of thePT-2218-WO-PCTgene encoding the X5PP of SEQ ID NO:33 (for 5 copies total), and knock out of both alleles of the RCSR18717 gene, was designated strain 1-14.Strain 1-15

[0163] Strain 1-14 was transformed with SEQ ID NO: 109 and SEQ ID NO: 110 using the transformation protocol outlined above and positive transformants were selected using geneticin (G418) selection plates. SEQ ID NO: 109 contained a 3’ portion of a G418 resistance marker gene (SEQ ID NO:3), the GPDIIa promoter (SEQ ID NO: 114), a polynucleotide sequence encoding the amino acid of SEQ ID NO: 54, the ENO1 terminator, and a 3’ RCSR26640 flanking sequence (SEQ ID NO: 14). SEQ ID NO: 110 contained a 5’ RCSR26640 flanking sequence (SEQ ID NO: 13), the EPDH1 promoter (SEQ ID NO: 117), a polynucleotide encoding the amino acid of SEQ ID NO:57, the ENO1 terminator, and a 5’ portion of a G418 resistance marker gene (SEQ ID NO:4). A PCR verified isolate, containing a gene encoding the amino acid of SEQ ID NO:57 under the control of the EPDH1 promoter and a gene encoding the amino acid of SEQ ID NO: 54 under the control of the GPDIIa promoter, was designated strain 1-15.Strains 1-16

[0164] Strain 1-15 was transformed with SEQ ID NO: 111 and SEQ ID NO: 112 using the transformation protocol outlined above and positive transformants were selected using nourseothricin selection plates. SEQ ID NO:111 contained a 5’ portion of a nourseothricin resistance gene expression cassette (SEQ ID NO:8), the EPDH1 promoter (SEQ ID NO: 117), a polynucleotide sequence encoding the amino acid of SEQ ID NO:33, the ENO1 terminator (SEQ ID NO:80), and a 5’ gpdllb flanking sequence (SEQ ID NO:31). SEQ ID NO: 112 contained a 3’ portion of a nourseothricin resistance gene expression cassette (SEQ ID NO:7), the EPDH1 promoter (SEQ ID NO: 117), a polynucleotide sequence encoding the amino acid of SEQ ID NO:33, the TDH3 terminator (SEQ ID NO:87), and a 3’ gpdllb flanking sequence (SEQ ID NO:32). A PCR verified isolate, containing two copies of the gene encoding the X5PP of SEQ ID NO:33 under the control of the EPDH1 promoter at the gpdllb locus, was designated strain 1-16.PT-2218-WO-PCTExample 2 - 2 L Nitrogen Feed and Bolus Fermentation Assay

[0165] Strain 1-16 was assayed for the production of xylitol under aerobic and microaerobic conditions with various treatments of nitrogen feeding.

[0166] A 500 mL baffed flask containing 80 mL of a first seed media (225 g / L dextrose, 12 g / L yeast extract, and 1.25 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg))) was inoculated with about 1 mL of a glycerol stock of strain 1-16 and incubated at 30 °C and 250 rpm for 26 hours. A 2L reaction vessel containing 1.5 L of a second seed media (240 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 0.4 mL / L antifoam, 10 g / L yeast extract, 4 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine), 13 g / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / L potassium (K), and 5.3 g / L sulfur (S)), and 7.4 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg))) was inoculated with 3.9% of the reactor volume coming from the shake flask seed culture and incubated at 30 °C and 1000-1500 rpm (about 40% dissolved oxygen) until the OUR reached 60 mmol / L / hr.

[0167] The fermentations were run according to the conditions outlined in Table 4. In general, a 2 L reaction vessel containing 1.2 L of production media (316 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 1.17 mL / L antifoam, 16.9 g / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / L potassium (K), and 5.3 g / L sulfur (S)), 1.69 g / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg)), and 2.7 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine) ) was inoculated with 5.16% of the reactor volume from the 2L seed culture and incubated at 35 °C and pH 3.8, controlled using sodium hydroxide. For aerobic conditions, dissolved oxygen was maintained around 40% by agitation between 1000-1500 RPM. For microaerobic conditions, an initial fixed rate 1000 RPM was used followed by a fixed rate of 925 RPM for reaction vessel 1 and 900 RPM for reaction vessel 2. FIG. 2 shows the agitation rates for vessels 1, 2, and 7. During the fermentation, additional dextrose was fed to the fermentation at a rate of 9-12 g / L / h beginning when the carbon evolution rate (CER) hit 50 mmol / L / hr. The total dextrose concentration in the fermentation (both initial and fed) was 407 g / L and the fermentation was run until the dextrose was depleted. For nitrogen feeding in vessels 3, 4, and 5, a nitrogenPT-2218-WO-PCTsolution of 200 g / L urea was used to supplement nitrogen to the fermentation reaction il R Vteaconesseaccording to the rates outlined in Table 2 beginning approximately 41 hours into fermentation and continuing for the remainder of the fermentation.Table 2.diid C Tttononeseiili ( / ) I NLttnarogengli ii T Ntttoarogenn reacon1 Microaerobic 2.54 1.85 ( / )Lt ssemgy 0.00 51 of 110 46.36 2 Microaerobic 2.54 1.85 i fd ( Nttrogenee raemg 0.00 51 of 110 46.36 Aerobic & Low / h) Nnitrogen feeding3 2.54 3.36 37.30 110 of 110 100.00 (0.399 mL / h of200 f fi Httors oermenaonug / L urea) iih i Nttt reacon presenn w Aerobic & High di mea 4 nitrogen feeding2.54 5.61 149.10 110 of 110 f fi Ptttercen oermenaon 100.00 (planned) (1.596 mL / h of200iih i N tttme presenn w g / L urea)di mea Aerobic & Highnitrogen feeding4(only 2.85 g added 2.54 3.87 149.10 58 of 58 (actual)before fermentationfoamed out)Aerobic & Mediumnitrogen feeding5 2.54 4.08 74.50 110 of 110 100.00 (0.798 mL / h of 200g / L urea)Aerobic & Nitrogen6 2.54 2.33 - 53 of 110 48.18 bolus (4 mL of 200PT-2218-WO-PCTg / L urea added 60hours intofermentation)7 Aerobic 2.54 1.85 - 48 of 110 43.64

[0168] Results are shown in FIGS. 3-6 and Table 3 below. Vessel 4, which was run to evaluate a higher nitrogen feeding rate, produced too much foam after the 60-hour time point, and no further data was collected for this vessel. Overall, when strain 1-16 was grown under microaerobic conditions (vessels 1 and 2) more xylitol was produced, and the rate of xylitol production was higher than the strain grown under aerobic conditions (vessel 7). As shown in FIG. 3 and Table 3, under microaerobic conditions (vessels 1 and 2), the final xylitol titer was over 200 g / L whereas the final titer under aerobic conditions was only 122 g / L. Additionally, glycerol production was lower and ethanol production higher under microaerobic conditions than under aerobic conditions. However, growth of strain 1-16 under microaerobic conditions resulted in production of a significant amount of ethanol, up to almost 20 g / L ethanol at its peak and above 5 g / L at the end of fermentation. Production of large amount of ethanol during fermentation poses a challenge for industrial scale fermentation due to the volatile nature of ethanol and the need for additional costly ethanol mitigation.

[0169] However, when nitrogen is fed to the fermentation under aerobic fermentation conditions, xylitol rate and titer are improved although not to the level of production under microaerobic conditions. As shown in FIG. 3 and Table 3, vessels 3 and 5, which were run under aerobic conditions and including a nitrogen feed, produced around 140 g / L xylitol. As shown in FIG. 5, vessels 3 and 5 had a peak glycerol concentration similar to the aerobic fermentation in vessel 7, however glycerol was re-consumed at a significantly higher rate resulting in a lower glycerol titer at the end of fermentation than the aerobic conditions without the nitrogen feed. The conditions of vessel 6, aerobic conditions with a bolus of nitrogen introduced, showed less pronounced but similar results to the steady nitrogen feed of vessels 3 and 5. The biggest difference in all of the nitrogen feed conditions tested (vessels 2, 5, and 6) was a drastic reduction in the amount of ethanol produced compared to the microaerobic conditions. This shows that the addition of nitrogen to aerobic fermentation improves xylitol production and improves glycerol re-consumption but does not introduce the significant ethanol production problems seen under microaerobic conditions.Reaction o o ovessel y (g)y (g)ate / ate / AnlL AnlL 810 980 Time (h) r oo 910 110Is) Is) Is)010 50p Dextrose 50 bo o9Z60Z 50 o.... 185 32504 339 593660 Is).... 192 402 32 Xylitol o 595 6226909 00..... 33274 423 32290 372 693....... 1275 33996 182 427 095 32365 359 bo Glycerol ...... 33002 192 446 157 32039 748o111 Ethanol L / i 50 ........ 33375 193 416 072 32339 376 650 103....... 33293 197 393 082 32419 367 6171 1— k00 Time (h) ..... 2326 358 27521 2799 91000 10 10p o o O ........ 26647 2925 2516 386 22817 5018 3046 1042l / l o 1 Dextrose 00 o Ln Lo ........ 25697 3417 2773 396 22369 5513 3436 391........ 3625 21418 3538 2782 532 16007 6334 5151 o Xylitol o....... 24612 3696 2907 391 5960 3639 343 o...... 3018 2543 22047 5060 3197 4621 o Glycerol 50 bo....... 2722 2357 397 27011 4369 3064 397 oo 1— k ...... 10476 13550 3469 1749 16947 1957 o p1 Ethanol......... 9767 14659 2954 1918 6875 2375 18077 2621 1836...... 9692 11224 5998 3017 12238 6923... 6075 15958 323860........ 6980 11882 6535 079 312 13294 7451 076881 861....... 13974 10073 4967 047 6875 10431 143080 060....... 12911 9625 5956 064 7772 9753 6645180 160...... 030 19764 1671 896 1127 517....... 025 20067 1548 1062 21047 1292 766....... 8325 13559 5931 9175 13895 4377 021...... 13767 4454 021 015 14189 2362..... 1836 11800 7539 12120 7332PT-2218-WO-PCT7 26.19 109.44 78.55 0.87 6.18 115.81 75.62 0.50 7 109.75 0.28 122.41 59.56 0.85 — — — — —Example 3 - 2L Nitrogen Feed Fermentation Assay

[0170] Strain 1-16 was assayed for the production of xylitol under aerobic and microaerobic conditions with various treatments of nitrogen feeding.

[0171] A 500 mL baffed flask containing 80 mL of a first seed media (225 g / L dextrose, 12 g / L yeast extract, and 1.25 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg))) was inoculated with about 1 mL of a glycerol stock of strain 1-16 and incubated at 30 °C and 250 rpm for 26 hours. A 2L reaction vessel containing 1.5 L of a second seed media (240 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 0.4 mL / L antifoam, 10 g / L yeast extract, 4 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine), 13 g / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / L potassium (K), and 5.3 g / L sulfur (S)), and 7.4 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg))) was inoculated with 3.9% of the reactor volume coming from the shake flask seed culture and incubated at 30 °C and 1000-1500 rpm (about 40% dissolved oxygen) until the OUR reached 60 mmol / L / hr.

[0172] The fermentations were run according to the conditions outlined in Table 4. In general, a 2 L reaction vessel containing 1.2 L of production media (316 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 0.17 mL / L antifoam, 16.9 mL / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / L potassium (K), and 5.3 g / L sulfur (S)), 1.69 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg), and 2.7 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine) ) or reduced nitrogen media (316 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 0.17 mL / L antifoam, 11.32 g / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / L potassium (K), and 5.3 g / L sulfur (S)), 0.27 g / L monopotassium phosphate, 0.33 g / L ammonium phosphate, 1.69 g / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg), and 2.7 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine)) as outlined in TablePT-2218-WO-PCT4, wil R Vteaconesseas inoculated with 2.58% of the reactor volume from the 2L seed culture and incubated at 35 °C and pH 3.8, controlled using sodium hydroxide. For aerobic conditions, dissolved oxygen was maintained around 40% by agitation between 1000-1500 RPM. For microaerobic conditions, a fixed agitation rate of 900 RPM was used. During the fermentation, additional diid C Tttononesedextrose was fed to the fermentation at a rate of 9-12 g / L / h beginning when the carbon evolution rate (CER) hit 50 mmol / L / hr. The total dextrose concentration in the fermentation (both initial and fed) was 407 g / L and the fermentation was run until the dextrose was depleted. For nitrogen feeding in vessels 10-13, a nitrogen solution of 200 g / L urea was used to di Measupplement nitrogen to the fermentation reaction according to the rates outlined in Table 4 beginning about 42 hours into the fermentation and continuing for the remainder of the reaction. iili ( / ) I NLttnarogengli ii T Ntttoarogenn reaconTable 4.( / )Lt ssemgyi fd ( / h) N Nttrogenee raemgf fi Httors oermenaonuiih i Nttt reacon presenn wdi meaf fii Pttt tercen oermenaonme Production8 Aerobic 2.54 1.27 - 44 of 134 33%ih idi Ntt presenn mea w MediaProduction9 Microaerobic 2.54 1.85 - 73 of 112 65% MediaAerobic & 75 mgProduction10 N / h nitrogen 2.54 4.69 75.00 102 of 112 91% MediafeedingAerobic & 100Production11 mg N / h nitrogen 2.54 5.11 100.00 112 of 112 100% MediafeedingPT-2218-WO-PCTAerobic & 125Production12 mg N / h nitrogen 2.54 5.47 125.00 112 of 112 100% MediafeedingAerobic,Reduced Initial Reduced13 Nitrogen & 75 Nitrogen 1.78 4.15 75.00 112 of 112 100% mg N / h nitrogen MediafeedAerobic & Reduced14 Reduced initial Nitrogen 1.78 0.89 - 61 of 134 46% nitrogen Media

[0173] Results are shown in FIGS. 7-11 and Table 5. In general, the results show that similar to Example 2, the addition of supplemental nitrogen (vessels 10-13) to the fermentation reaction improves xylitol production and reduces glycerol production relative to the control aerobic conditions (vessel 8) but produces significantly less xylitol than the control microaerobic conditions (vessel 9). As shown in FIGS. 10 and 11, vessel 10 had a slower growth rate (i.e., lower biomass as measured by OD600 and lower OUR) which resulted in lower-than-expected xylitol production and higher than expected glycerol production. However, if the growth rate was normal, the xylitol production would have been higher given that under this treatment the instantaneous xylitol production rate (1.7 g / L / h) after growth phase was higher than the aerobic conditions in vessel 8 (0.7 g / L / h).

[0174] FIG. 7 and Table 5 show that strain 1-16 produces the most xylitol, about 223 g / L, when fermented under microaerobic conditions (vessel 9). Fermentation under aerobic conditions produced about 140 g / L xylitol (vessel 8) and only about 90 g / L xylitol (vessel 14) when the initial nitrogen concentration is decreased and not supplemented during fermentation. Fermentation under aerobic conditions with fed nitrogen (vessels 11 and 12) produced about 160-165 g / L xylitol, which is significantly above the titer of xylitol produced under aerobic conditions without the nitrogen feed. Vessel 13, with fed nitrogen but a lower initial nitrogen concentration, produced about 116 g / L xylitol, which is above the xylitol produced by vessel 14 which lacked the fed nitrogen.PT-2218-WO-PCTil Rteaconesse v

[0175] Similar to the glycerol results shown in Example 2, fermentation of strain 1-16 under aerobic conditions with a supplemental nitrogen feed has the highest peak glycerol i (h) Tmeconcentration but re-consumed the produced glycerol at a rate higher than either the control aerobic conditions (vessel 8) or the microaerobic conditions (vessel 9). For the fermentation Dterosexreactions run with reduced initial nitrogen concentrations, glycerol production was slower but expected given the overall slower growth of strain 1-16 under these conditions.

[0176] Finally, FIG. 9 anlil Xtdoy Table 5 show that under all aerobic conditions (with and without fed nitrogen) ethanol production from strain 1-16 was significantly lower than ethanol production under microaerobic condill Gcerotyions. All aerobic conditions in a final ethanol titer at the end of fermentation of less than 2 g / L and a peak ethanol concentration of less than 6 g / L.hl EtanoHowever, when strain 1-16 is grown under microaerobic conditions peak ethanol concentration is over 16 g / L and the final ethanol titer is over 6 g / L. These results clearly indicate that i (h) Tmefermentation under aerobic conditions significantly reduces ethanol, and as a result mitigates the issues caused by excess ethanol production, and that addition of supplemental nitrogen Dteroseximproves xylitol production and glycerol re-consumption.lil XtoyTable 5.Analyte (g / L) Analyte (g / L)ll Gceroyhl Etano8 16.75 337.77 2.30 4.75 0.37 37.75 209.85 34.44 31.75 5.90 9 16.75 347.05 1.52 2.58 0.62 37.75 325.51 7.58 8.09 2.44 10 16.75 341.44 1.82 3.89 0.60 37.75 313.57 15.70 19.42 2.78 11 16.75 340.53 2.60 4.74 0.40 37.75 297.41 24.50 27.12 4.42 12 16.75 338.42 2.38 4.86 0.37 37.75 298.76 25.09 28.45 3.53 13 16.75 335.67 1.55 3.66 0.53 37.75 291.18 11.28 17.77 1.71 14 16.75 341.16 1.39 2.95 0.70 37.75 316.49 8.36 10.96 2.35 8 42.08 141.61 59.86 38.01 4.46 63.50 142.46 89.50 48.19 0.48 9 42.08 309.21 11.09 10.71 2.64 63.50 246.98 57.07 28.78 13.19 10 42.08 298.55 24.04 25.42 2.90 63.50 171.06 74.62 65.55 0.76PT-2218-WO-PCT11 42.08 277.69 36.14 33.50 2.66 63.50 59.34 123.02 72.79 2.34 12 42.08 274.81 36.19 34.46 2.36 63.50 71.38 114.46 77.24 1.79 13 42.08 291.38 16.60 22.88 2.14 63.50 207.95 58.44 57.99 1.29 14 42.08 296.04 11.93 14.24 2.80 63.50 139.95 61.63 44.33 5.26 8 72.92 123.39 94.26 56.41 0.38 87.00 74.94 106.03 64.00 0.21 9 72.92 189.46 93.42 31.99 15.82 87.00 76.74 159.20 32.37 16.39 10 72.92 99.15 88.04 88.71 0.72 87.00 3.95 114.29 109.49 0.30 11 72.92 2.17 151.42 67.83 0.27 87.00 0.89 164.72 35.18 0.14 12 72.92 2.08 146.27 77.14 0.81 87.00 0.83 155.86 52.14 0.31 13 72.92 130.31 78.96 76.20 0.79 87.00 63.82 102.36 88.27 1.08 14 72.92 81.33 89.43 54.11 0.83 87.00 213.39 70.55 49.72 0.35 8 93.75 55.85 112.05 62.03 0.36 109.75 19.49 128.91 48.36 0.15 9 93.75 32.52 190.51 30.17 15.44 109.75 1.33 223.18 13.90 6.29 10 93.75 1.92 121.62 90.17 0.09 — — — — — 11 93.75 0.82 165.66 29.75 0.41 — — — — — 12 93.75 1.33 160.51 47.93 0.15 — — — — — 13 93.75 37.21 116.78 90.65 0.62 — — — — — 14 93.75 197.40 72.82 55.82 0.22 109.75 160.27 83.81 67.80 0.24 8 115.50 11.03 133.37 41.02 0.23 133.92 4.90 139.58 35.16 0.25 14 115.50 144.98 84.82 68.44 2.24 133.92 127.76 89.85 70.41 1.34Example 4 - 2L Nitrogen Supplement Fermentation Assay

[0177] Strain 1-16 was assayed for the production of xylitol under aerobic conditions with various initial nitrogen concentrations.

[0178] A 500 mL baffed flask containing 80 mL of a first seed media (225 g / L dextrose, 12 g / L yeast extract, and 1.25 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg))) was inoculated with about 1 mL of a glycerol stock of strain 1-16 and incubated at 30 °C and 250 rpm for 26 hours. A 2L reaction vessel containing 1.5 L of a second seed media (240 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 0.4 mL / L antifoam, 10 g / L yeast extract, 4 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine), 13 g / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / LPT-2218-WO-PCTpotassium (K), and 5.3 g / L sulfur (S)), and 7.4 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg))) was inoculated with 3.9% of the reactor volume coming from the first seed culture and incubated at 30 °C and 1000-1500 rpm (about 40% dissolved oxygen) until the OUR reached 60 mmol / L / hr.

[0179] The fermentations were run according to the conditions outlined in Table 6. In general, a 2 L reaction vessel containing 1.2 L of production media (316 g / L dextrose, 0.054 g / L calcium chloride dihydrate, 0.12 g / L magnesium sulfate heptahydrate, 0.17 mL / L antifoam, 16.9 mL / L nutrient solution 2 (165.7 g / L nitrogen (N), 266 g / L phosphorus (P), 16.0 g / L potassium (K), and 5.3 g / L sulfur (S)), 1.69 mL / L nutrient solution 1 (6000 mg / L iron (Fe), 1195 mg / L zinc (Zn), 280 mg / L manganese (Mn), 150 mg / L copper (Cu), 28.0 g / L magnesium (Mg), and 2.7 mL / L vitamin mix (5 g / L thiamine-HCl and 50 g / L betaine) ), was inoculated with 2.58% of the initial volume coming from the 2L seed culture and incubated at 35 °C, 1000-1500 rpm (about 40% dissolved oxygen) and pH 3.8, controlled using sodium hydroxide. During the fermentation, additional dextrose was fed to the fermentation at a rate of 9-12 g / L / h beginning when the carbon evolution rate (CER) hit 50 mmol / L / hr. The total dextrose concentration in the fermentation (both initial and fed) was 407 g / L and the fermentation was run until the dextrose was depleted. For reaction vessels 17, 18, and 19, the initial production media contained 0.54 g / L, 1.09 g / L, or 1.63 g / L ammonium sulfate, respectively. In vessel 15, ammonium hydroxide was used to maintain the pH of the fermentation medium at or above 3.8. As a result, at least 0.22 g / L nitrogen was present in the fermentation medium in vessel 15 for the entire fermentation time (i.e., 86 or 86 hours, 100%).PT-2218-WO-PCTil R Vteaconesse Table 6Hours ofii (h) Ttmepon Percent of fermentationInitial fermentation Reaction reactionCondition Tested Nitrogen Total Nitrogen in time with N Vessel Dterosex System (g / L) withN(g / L) present in present inmedia lil Xtoy mediaNitrogen at leastAerobic ll Gceroy 0.22 g / L in15 (NH40H pH 2.54 fermentation 86 or 86 100% hl Etanocontrol) medium for entirereactionii (h) TtmeponAerobic control16 (NaOH pH 2.54 1.85 44 of 86 51% Dterosexcontrol)Aerobic +5%lil Xtoy17 nitrogen (NaOH 2.67 1.94 46 of 86 53% pH control)ll Gceroy Aerobic +10%18 Nitrogen (NaOH 2.80 2.03 47 of 86 55% hl Etano pH control)Aerobic +15%19 nitrogen (NaOH 2.93 2.12 48 of 86 56% pH control)Table 7Analyte (g / L) Analyte (g / L)15 14.75 328.48 2.17 4.09 1.86 21.25 312.37 4.48 6.56 1.27PT-2218-WO-PCT16 329.57 2.36 4.62 0.71 311.84 5.32 8.09 1.21 17 329.32 2.31 4.44 0.75 306.22 5.10 7.45 1.12 18 333.58 2.28 4.47 0.70 311.13 5.22 7.62 1.09 19 330.21 2.20 4.07 0.76 315.66 4.83 6.91 1.07 15 258.68 29.62 24.56 4.46 226.82 41.43 27.66 4.19 16 289.14 32.77 27.77 3.09 273.75 45.89 34.92 2.91 17 282.12 32.98 27.03 3.18 260.44 45.60 32.95 3.14 37.92 41.7518 273.26 34.80 26.89 4.23 243.31 49.22 32.58 4.40 19 185.09 44.38 29.16 4.62 181.87 67.47 34.33 5.19 15 258.68 29.62 24.56 4.46 226.82 41.43 27.66 4.19 15 217.95 63.92 33.08 5.13 182.85 84.18 33.06 5.50 16 251.12 60.61 42.86 2.49 201.57 73.05 49.34 1.24 17 46 234.83 59.78 39.39 3.07 50.5 196.74 77.49 47.31 1.84 18 223.12 70.28 40.62 4.65 181.98 87.53 45.81 3.14 19 161.84 92.40 38.31 5.61 133.12 103.61 36.55 3.89 15 107.24 129.19 25.94 6.63 22.44 181.54 23.40 8.83 16 130.40 93.92 68.60 0.65 71.36 105.70 86.51 0.68 17 61.77 117.58 97.98 62.24 0.71 73.5 66.32 119.81 83.15 0.51 18 96.77 119.23 65.42 0.67 28.41 129.73 81.51 0.67 19 111.32 141.47 53.27 0.80 31.79 142.65 66.70 0.72 15 0.15 215.46 9.58 4.43 0.07 208.24 0.41 0.06 16 25.45 113.34 95.08 0.37 0.19 126.39 69.39 0.55 17 85.78 17.11 120.39 84.87 0.45 110.5 0.23 129.65 58.76 0.53 18 0.22 139.62 87.64 0.50 0.26 141.94 52.07 0.47 19 0.17 153.55 74.17 0.58 0.17 146.53 41.25 0.43

[0180] As shown in FIGS. 13 and 15 and Table 7, reaction vessels 17, 18, and 19, which had a higher initial nitrogen concentration in the fermentation media, produced more xylitol than the control aerobic fermentation of vessel 16. These same vessels also showed lower overall glycerol production (FIG. 14 and Table 7) with similar rates of glycerol consumption as the control aerobic fermentation. Also, vessel 15, in which the pH was controlled with ammonium hydroxide instead of sodium hydroxide as in the aerobic control, showed higherPT-2218-WO-PCTxylitol production. The vessel with higher initial nitrogen concentrations has nitrogen present for a longer portion of the fermentation reaction than the control aerobic fermentation vessel and vessel 15 included nitrogen in the fermentation media for the entire reaction. In general, the results show that when nitrogen is present for a longer portion of the fermentation reaction time (FIG. 15 and Table 6), up to being present for the entire reaction, the fermentation produces more xylitol and less glycerol than the control aerobic reaction.CLAUSES

[0181] Clause 1. A fermentation method for the production of xylitol, the method comprising: contacting a fermentation medium comprising dextrose and nitrogen (N) with an engineered yeast cell, capable of producing xylitol, in aerobic conditions to produce xylitol; wherein nitrogen is added to the fermentation medium such that nitrogen is present in the fermentation medium for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% of total fermentation reaction time; and wherein xylitol production is increased relative to an equivalent fermentation method run without additional nitrogen such that nitrogen is present in the fermentation medium for less than 50% of the total fermentation reaction time.

[0182] Clause 2. The method of clause 1 , wherein the initial nitrogen concentration in the fermentation medium is 0.5 g / L to 5 g / L, 1 g / L to 4 g / L, 1.5 g / L to 3.5 g / L, or preferably about 2 g / L to 3.25 g / L.

[0183] Clause 3. The method of clause 1 or clause 2, wherein nitrogen is added to the fermentation medium such that the total nitrogen concentration over the course of the fermentation reaction is about 3 g / L to 7 g / L, 3.5 g / L to 6.5 g / L, 4 g / L to 6 g / L, or about 4.5 g / L to 5.5 g / L.

[0184] Clause 4. The method of any preceding clause, wherein the nitrogen is added to fermentation at a constant or exponential rate or is added as one or more bolus doses during fermentation.

[0185] Clause 5. The method of any preceding clause, wherein the nitrogen is added during the fermentation at a rate of about 25 mg N / h to 150 mg N / h, 50 mg N / h to 140 mg N / h, 60 mg N / h to 135 mg N / h, or about 75 mg N / h to 130 mg N / h.PT-2218-WO-PCT

[0186] Clause 6. The method of any preceding clause, wherein nitrogen is added in one or more bolus doses of 0.1 g / Lto2.5 g / L, for example, 0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, 1.0 g / L, 1.25 g / L, 1.5 g / L, 1.75 g / L, 2.0 g / L, or about 2.5 g / L.

[0187] Clause 7. The method of any preceding clause, wherein the fermentation method includes a growth phase and a production phase, and nitrogen is added during the production phase.

[0188] Clause 8. The method of any preceding clause, wherein the fermentation temperature is at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C.

[0189] Clause 9. The method of any preceding clause, wherein the volumetric oxygen uptake rate (OUR) is at least 15 mmol / L / h, at least 20 mmol / L / h, at least 25 mmol / L / h, at least 30 mmol / L / h, at least 35 mmol / L / h, at least 40 mmol / L / h, at least 45 mmol / L / h, or at least 50 mmol / L / h.

[0190] Clause 10. The method of any preceding clause, wherein the dissolved oxygen content is at least 20%, at least 30%, at least 40%, or at least 50%.

[0191] Clause 11. The method of any preceding clause, wherein the xylitol is produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h'1.

[0192] Clause 12. The method of any preceding clause, wherein the xylitol titer is at least 50, at least 75, at least 100, at least 125, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 g / L when the fermentation is run at 35 °C for 96 hours.

[0193] Clause 13. The method of any preceding clause, wherein xylitol production is at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more than an equivalent aerobic fermentation method run without additional nitrogen.

[0194] Clause 14. The method of any preceding clause, wherein glycerol production in the fermentation reaction is reduced and / or wherein glycerol reconsumption rate increases in the fermentation reaction relative to an equivalent fermentation method run without additional nitrogen such that nitrogen is present in the fermentation medium for less than 50% of the total fermentation reaction time.

[0195] Clause 15. The method of any preceding clause, wherein the initial concentration of dextrose is at least 100 g / L.PT-2218-WO-PCT

[0196] Clause 16. The method of any preceding clause, wherein additional dextrose is fed to the fermentation such at the total dextrose concentration is at least 200 g / L, at least 300 g / L, or at least 400 g / L.

[0197] Clause 17. The method of any preceding clause, wherein the engineered yeast cell comprises:(i) an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a 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 at least one of at least one of SEQ ID NOs:55-64; and / or(ii) an exogenous polynucleotide sequence encoding a xylulokinase (XKS) enzyme comprising a 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 at least one of SEQ ID NOs:50 and 49 and an exogenous polynucleotide sequence encoding a xylitol dehydrogenase (XDH) enzyme comprising a 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 at least one of SEQ ID NOs:65, 66, and 67.

[0198] Clause 18. The method of any preceding clause, wherein the engineered yeast cell comprises: an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a 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 at least one of at least one of SEQ ID NOs:55-64; an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a sequence 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 at least one of SEQ ID NOs: 57, 58, 59, or 62; or an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57.

[0199] Clause 19. The method of any preceding clause, wherein the engineered yeast cell comprises at least one additional copy of a polynucleotide encoding a ribulose-5-phosphate epimerase (RPE) enzyme 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:53 and 54.

[0200] Clause 20. The method of any preceding clause, wherein the engineered yeast cell comprises: an exogenous polynucleotide sequence encoding a xylitol-5-phosphate phosphatasePT-2218-WO-PCT(X5PP) enzyme comprising a 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 at least one of SEQ ID NOs: 33, 35, 36, 37, 38, 39, 43, and 45, preferably at least one of SEQ ID NOs: 33, 36, 37, 39, and 45; and / or at least one additional copy of a polynucleotide encoding an X5PP enzyme comprising 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:51, 52, 33, or 47, preferably SEQ ID NO:33.

[0201] Clause 21. The method of any preceding clause, wherein the yeast cell is an osmotol erant yeast cell.

[0202] Clause 22. The method of any preceding clause, wherein the yeast cell is a cell of the subphylum Ustilaginomycotina.

[0203] Clause 23. The method of any preceding clause, wherein the yeast cell is selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens. Pseudozyma Isukubaensis. Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon. Yarrowia lipolytica, Saccharomyces cerevisiae. Penicillium, Torula, Pichia, Candida, Candida magnolias, and Aureobasidiunr, and / or wherein the yeast cell is of a species comprising an adenosine triphosphate (APT) citrate lyase gene.

[0204] Clause 24. The method of any preceding clause, wherein the yeast cell comprises a deletion or disruption of a native gene encoding an erythrose reductase enzyme 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: 11 and 15, preferably wherein the cell has a deletion of both alleles of the gene encoding the erythrose reductase enzyme.

[0205] Clause 25. The method of any preceding clause, wherein the cell comprises: an exogenous polynucleotide sequence encoding a trehalase construct comprising a secretion signal operably linked to a trehalase enzyme at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs: 132 and 133, wherein the secretion signal is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs: 134-137; a genetic modification resulting in overexpression of a native enzyme with glycerol kinase activity, for example a glycerol kinase enzyme comprising a 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 at least one of SEQ IDPT-2218-WO-PCTNOs:92 and 103; a deletion or disruption of a native gene encoding a glycerol-3 -phosphate dehydrogenase 2a enzyme 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: 107; and / or a genetic modification that increases expression of a transketolase enzyme 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: 106.

[0206] Clause 26. The method of any one of clauses 17-25, wherein one or more of the exogenous polynucleotide sequence(s) is operably linked to a heterologous or artificial promoter selected from the group consisting of pyruvate kinase 1 promoter (PYKlp; SEQ ID NO: 68), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO: 69), glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:70), translational elongation factor 1 promoter (TEFp; SEQ ID NO:71), phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72), 3 -phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73), enolase promoter (ENO Ip ; SEQ ID NO: 74), asparagine synthetase promoter (ASNSp; SEQ ID NO:75), 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76), RPL16B (SEQ ID NO:77), glycerol-3 -phosphate dehydrogenase 2a promoter (GPDIIap; SEQ ID NO: 114), GDN1 (SEQ ID NO: 116), EPDH1 (SEQ ID NO: 117), translation elongation factor 1A like gene 7 promoter (TEF7p; SEQ ID NO: 120); heat shock protein 90 promoter (HSP90p; SEQ ID NO: 121); translation elongation factor 1 A like gene 4 promoter (TEF4p; SEQ ID NO: 122); translation elongation factor 2A like gene 6 promoter (TEF6p; SEQ ID NO: 123); transaldolase 1 promoter (TALlp; SEQ ID NO: 124); citrate synthase 1 promoter (CITlp; SEQ ID NO: 125); endoplasmic reticulum chaperone BiP (KAR2p; SEQ ID NO: 126); heat shock protein 88 promoter (HSP88p; SEQ ID NO: 127); heat shock protein 70 promoter (HPS70p; SEQ ID NO: 128); tubulin alpha-1 chain promoter (TUBlp; SEQ ID NO: 129); glyceraldehyde-3 -phosphate dehydrogenase 2 promoter (TDH2p; SEQ ID NO: 130); translation elongation factor 1A like gene 5 promoter (TEF5p; SEQ ID NO: 131); translation elongation factor 3 promoter (TEF3p; SEQ ID NO: 132); thioredoxin peroxidase promoter (TPXp; SEQ ID NO: 133); alginate lyase promoter (ALPp; SEQ ID NO: 134); thiazole biosynthetic 4 promoter (THI4p; SEQ ID NO: 135); phosphoglucomutase / phospomannomutase promoter (PMMp; SEQ ID NO: 136); fructose-bisphosphate aldolase 1 promoter (FBAlp; SEQ ID NO: 137); alcohol dehydrogenase 1 promoter (ADHlp; SEQ ID NO: 138); and extracellular endoglucanase 1 promoter (GLXlp; SEQ ID NO: 139).PT-2218-WO-PCT

[0207] Clause 27. The method of any one of clauses 17-26, wherein one or more of the exogenous polynucleotide sequences(s) is operably linked to a heterologous or artificial promoter selected form the group consisting of6PGD terminator (6PGDt; SEQ ID NO:78); ASNS terminator (ASNSt; SEQ ID NO:79); ENO1 terminator (ENOlt; SEQ ID NO:80); hexokinase 1 terminator (HXKlt; SEQ ID NO:81); PGK1 terminator (PGKlt; SEQ ID NO:82); PGM1 terminator (PGMlt; SEQ ID NO:83); PYK1 terminator (PYKlt; SEQ ID NO:84); RPLA terminator (RPLAt: SEQ ID NO:85); transaldolase 1 terminator (TALlt; SEQ ID NO: 86); TDH3 terminator (TDH3t; SEQ ID NO: 87); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:88); triosephosphate isomerase 1 terminator (TPIlt; SEQ ID NO:89); MpTEFl terminator (MpTEFlt; SEQ ID NO:90); TEF7 terminator (TEF7t; SEQ ID NO: 140); HSP90 terminator (HSP90t; SEQ ID NO: 141); TEF4 terminator (TEF4t; SEQ ID NO: 142); TEF6 terminator (TEF6t; SEQ ID NO: 143); CITI terminator (CITI It; SEQ ID NO: 144): KAR2 terminator (KAR2t; SEQ ID NO: 145); HPS88 terminator (HSP88t; SEQ ID NO: 146); HSP70 terminator (HSP70t; SEQ ID NO: 147); TUB1 terminator (TUBlt; SEQ ID NO: 148); TKL1 terminator (TALlt; SEQ ID NO: 149); TDH2 terminator (TDH2t; SEQ ID NO: 150); TEF5 terminator (TEF5t; SEQ ID NO: 151); TGL2 terminator (TGL2t; SEQ ID NO: 152); TEF3 terminator (TEF3t; SEQ ID NO: 153); TPX terminator (TPXt; SEQ ID NO: 154); ALP terminator (ALPt; SEQ ID NO: 155); plasma membrane ATPase terminator (PMAlt; SEQ ID NO: 156); THI4 terminator (THI4t; SEQ ID NO: 157); glucose-6-phosphate isomerase 1 terminator (PGIlt; SEQ ID NO: 158); PMM terminator (PMMt; SEQ ID NO: 159); FBA1 terminator (FBAlt; SEQ ID NO: 160); ADH1 terminator (ADHlt; SEQ ID NO: 161); sodium / potassium transporting ATPase alpha chain terminator (ATPlt; SEQ ID NO: 162); and GLX1 terminator (GLXlt; SEQ ID NO: 163).

[0208] Clause 28. The yeast cell of any preceding clause, wherein one or more of the exogenous polynucleotide sequence(s) is integrated into the genome of the yeast cell at a locus selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdllA locus, the gpdllB locus, the RCSR26640 locus, and the RCSR18717 locus.

[0209] Clause 29. A fermentation method for the production of xylitol, the method comprising: growing a genetically engineered Moniliella pollinis cell capable of producing xylitol and comprising an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a 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 toPT-2218-WO-PCTat least one of at least one of SEQ ID NOs:55-64 in a fermentation media comprising dextrose and an initial nitrogen concentration under aerobic conditions to produce xylitol; and feeding supplemental nitrogen into the fermentation media when the initial nitrogen concentration depleted and / or when dissolved oxygen in the reaction is 0%; wherein titer, rate, and / or yield of xylitol is higher than an equivalent fermentation run without feeding supplemental nitrogen.

[0210] Clause 30. The method of clause 29, wherein the initial nitrogen concentration in the fermentation medium is 0.5 g / L to 5 g / L, 1 g / L to 4 g / L, 1.5 g / L to 3.5 g / L, or preferably about 2 g / L to 3.25 g / L.

[0211] Clause 31. The method of clause 29 or clause 30, wherein nitrogen is added to the fermentation medium such that the total nitrogen concentration over the course of the fermentation reaction is about 3 g / L to 7 g / L, 3.5 g / L to 6.5 g / L, 4 g / L to 6 g / L, or about 4.5 g / L to 5.5 g / L.

[0212] Clause 32. The method of any one of clauses 29-31, wherein the nitrogen is added during the fermentation at a rate of about 25 mg N / h to 150 mg N / h, 50 mg N / h to 140 mg N / h, 60 mg N / h to 135 mg N / h, or about 75 mg N / h to 130 mg N / h.

[0213] Clause 33. The method of any anyone of clauses 29-32, wherein the fermentation method includes a growth phase and a production phase, and the nitrogen feed is added during the production phase.

[0214] Clause 34. The method of any one of clauses 29-33, wherein the fermentation temperature is at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C.

[0215] Clause 35. The method of any one of clauses 29-34, wherein the volumetric oxygen uptake rate (OUR) is at least 50, at least 60, or at least 70 mmol O2 / (L • h).

[0216] Clause 36. The method of any one of clauses 29-35, wherein the dissolved oxygen content is at least 20%, at least 30%, at least 40%, or at least 50% during the feeding of supplemental nitrogen.

[0217] Clause 37. The method of any one of clauses 29-36, wherein the xylitol is produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h’1.

[0218] Clause 38. The method of any one of clauses 29-37, wherein the xylitol titer is at least 50, at least 75, at least 100, at least 125, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 g / L when the fermentation is run at 35 °C for 96 hours.

[0219] Clause 39. The method of any one of clauses 29-38, wherein xylitol production is 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%,PT-2218-WO-PCTat least 50%, or at least 60% more than an equivalent aerobic fermentation method run without additional nitrogen.

[0220] Clause 40. The method of any one of clauses 29-39, wherein glycerol production in the fermentation reaction is reduced and / or wherein glycerol reconsumption rate increases in the fermentation reaction relative to an equivalent fermentation method run without feeding additional nitrogen.

[0221] Clause 41. The method of any one of clauses 29-40, wherein the initial concentration of dextrose is at least 100 g / L.

[0222] Clause 42. The method of any one of clauses 29-41, wherein additional dextrose is fed to the fermentation such at the total dextrose concentration is at least 200 g / L, at least 300 g / L, or at least 400 g / L.

Claims

PT-2218-WO-PCTCLAIMSWhat is claimed is:

1. A fermentation method for the production of xylitol, the method comprising:contacting a fermentation medium comprising dextrose and nitrogen (N) with an engineered yeast cell, capable of producing xylitol, in aerobic conditions to produce xylitol;wherein nitrogen is added to the fermentation medium such that nitrogen is present in the fermentation medium for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% of total fermentation reaction time; and wherein xylitol production is increased relative to an equivalent fermentation method run without additional nitrogen such that nitrogen is present in the fermentation medium for less than 50% of the total fermentation reaction time.

2. The method of claim 1, wherein the nitrogen is added to fermentation at a constant or exponential rate or is added as one or more bolus doses during fermentation, for example wherein nitrogen is added in one or more bolus doses of 0.1 g / L to 2.5 g / L, for example, 0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, 1.0 g / L, 1.25 g / L, 1.5 g / L, 1.75 g / L, 2.0 g / L, or about 2.5 g / L.

3. The method of any preceding claim, wherein the yeast cell is an osmotol erant yeast cell, for example, yeast cell is a cell of the subphylum Ustilaginomycotina.

4. The method of any preceding claim,wherein the yeast cell is selected from the group consisting of Trichosporonoides megachiliensis, Trychosporonoides oedocephalis, Trychosporonoides nigrescens. Pseudozyma Isukubaensis. Trigonopsis variabilis, Moniliella, Ustilaginomycetes, Trichosporon, Yarrowia lipolytica, Saccharomyces cerevisiae. Penicillium, Torula, Pichia, Candida, Candida magnolias, and Aureobasidiunr, and / orwherein the yeast cell is of a species comprising an adenosine triphosphate (APT) citrate lyase gene.

5. A fermentation method for the production of xylitol, the method comprising:PT-2218-WO-PCTgrowing a genetically engineered Moniliella sp. cell (e.g., Moniliella pollinis) capable of producing xylitol and comprising an exogenous polynucleotide sequence encoding a xylitol-phosphate dehydrogenase (XPDH) enzyme comprising a 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 at least one of at least one of SEQ ID NOs:55-64 in a fermentation media comprising dextrose and an initial nitrogen concentration under aerobic conditions to produce xylitol; andfeeding supplemental nitrogen into the fermentation media when the initial nitrogen concentration is depleted and / or when dissolved oxygen in the reaction is 0%;wherein titer, rate, and / or yield of xylitol is higher than an equivalent fermentation run without feeding supplemental nitrogen.

6. The method of any preceding claim, wherein the initial nitrogen concentration in the fermentation medium is 0.5 g / L to 5 g / L, 1 g / L to 4 g / L, 1.5 g / L to 3.5 g / L, or preferably about 2 g / L to 3.25 g / L.

7. The method of any preceding claim, wherein nitrogen is added to the fermentation medium such that the total nitrogen concentration over the course of the fermentation reaction is about 3 g / L to 7 g / L, 3.5 g / L to 6.5 g / L, 4 g / L to 6 g / L, or about 4.5 g / L to 5.5 g / L.

8. The method of any preceding claim, wherein the nitrogen is added during the fermentation at a rate of about 25 mg N / h to 150 mg N / h, 50 mg N / h to 140 mg N / h, 60 mg N / h to 135 mg N / h, or about 75 mg N / h to 130 mg N / h.

9. The method of any preceding claim, wherein the fermentation method includes a growth phase and a production phase, and nitrogen is added during the production phase.

10. The method of any preceding claim, wherein the fermentation temperature is at or between 25 °C to 45 °C, 30 °C to 40 °C, or 32 °C to 37 °C.

11. The method of any preceding claim, wherein the volumetric oxygen uptake rate (OUR) is at least 15 mmol O2 / (L • h), at least 20 mmol O2 / (L • h), at least 25 mmol O2 / (L • h), at leastPT-2218-WO-PCT30 mmol C>2 / (L • h), at least 35 mmol C>2 / (L • h), at least 40 mmol C>2 / (L • h), at least 45 mmol 02 / (L • h), or at least 50 mmol C>2 / (L • h); and / or wherein the dissolved oxygen content is at least 20%, at least 30%, at least 40%, or at least 50%.

12. The method of any preceding claim, wherein the xylitol is produced at a rate of at least 0.2, 0.3, 0.5, 0.75, or at least 1.0 g L'1h’1; and / or wherein the xylitol titer is at least 50, at least 75, at least 100, at least 125, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 g / L when the fermentation is run at 35 °C for 96 hours.

13. The method of any preceding claims, wherein xylitol production is at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more than an equivalent aerobic fermentation method run without additional nitrogen.

14. The method of any preceding claim, wherein glycerol production in the fermentation reaction is reduced and / or wherein glycerol reconsumption rate increases in the fermentation reaction relative to an equivalent fermentation method run without additional nitrogen such that nitrogen is present in the fermentation medium for less than 50% of the total fermentation reaction time.

15. The method of any preceding claim, wherein the initial concentration of dextrose is at least 100 g / L.

16. The method of any preceding claim, wherein additional dextrose is fed to the fermentation such at the total dextrose concentration is at least 200 g / L, at least 300 g / L, or at least 400 g / L.

17. The method of any preceding claim, wherein the engineered yeast cell comprises: an exogenous polynucleotide sequence encoding an XPDH enzyme comprising a sequence 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 at least one of SEQ ID NOs: 57, 58, 59, or 62; orPT-2218-WO-PCTan exogenous polynucleotide sequence encoding an XPDH enzyme comprising a sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57.

18. The method of any preceding claim, wherein the engineered yeast cell comprises at least one additional copy of a polynucleotide encoding a ribulose-5-phosphate epimerase (RPE) enzyme 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:53 and 54.

19. The method of any preceding claim, wherein the engineered yeast cell comprises: an exogenous polynucleotide sequence encoding a xylitol-5-phosphate phosphatase (X5PP) enzyme comprising a 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 at least one of SEQ ID NOs: 33, 35, 36, 37, 38, 39, 43, and 45, preferably at least one of SEQ ID NOs: 33, 36, 37, 39, and 45; and / orat least one additional copy of a polynucleotide encoding an X5PP enzyme comprising 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:51, 52, 33, or 47, preferably SEQ ID NO:33.

20. The method of any preceding claim, wherein the yeast cell comprises:a deletion or disruption of a native gene encoding an erythrose reductase enzyme 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: 11 and 15, preferably wherein the cell has a deletion of both alleles of the gene encoding the erythrose reductase enzyme.an exogenous polynucleotide sequence encoding a trehalase construct comprising a secretion signal operably linked to a trehalase enzyme at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs: 132 and 133, wherein the secretion signal is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to at least one of SEQ ID NOs: 134-137;a genetic modification resulting in overexpression of a native enzyme with glycerol kinase activity, for example a glycerol kinase enzyme comprising a sequence at least 70%, atPT-2218-WO-PCTleast 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:92 and 103;a deletion or disruption of a native gene encoding a glycerol -3 -phosphate dehydrogenase 2a enzyme 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: 107; and / ora genetic modification that increases expression of a transketolase enzyme 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: 106.

21. The method of any one of claims 5-20, whereinone or more of the exogenous polynucleotide sequence(s) is operably linked to a heterologous or artificial promoter selected from the group consisting of pyruvate kinase 1 promoter (PYKlp; SEQ ID NO: 68), 6-phosphogluconate dehydrogenase promoter (6PGDp; SEQ ID NO:69), glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p; SEQ ID NO:70), translational elongation factor 1 promoter (TEFp; SEQ ID NO:71), phosphoglucomutase 1 promoter (PGMlp; SEQ ID NO:72), 3 -phosphoglycerate kinase promoter (PGKlp; SEQ ID NO:73), enolase promoter (ENOlp ; SEQ ID NO:74), asparagine synthetase promoter (ASNSp; SEQ ID NO:75), 50S ribosomal protein LI promoter (RPLAp; SEQ ID NO:76), RPL16B (SEQ ID NO:77), glycerol -3 -phosphate dehydrogenase 2a promoter (GPDIIap; SEQ ID NO: 114), GDN1 (SEQ ID NO: 116), EPDH1 (SEQ ID NO: 117), translation elongation factor 1A like gene 7 promoter (TEF7p; SEQ ID NO: 120); heat shock protein 90 promoter (HSP90p; SEQ ID NO: 121); translation elongation factor 1A like gene 4 promoter (TEF4p; SEQ ID NO: 122); translation elongation factor 2A like gene 6 promoter (TEF6p; SEQ ID NO: 123); transaldolase 1 promoter (TALlp; SEQ ID NO: 124); citrate synthase 1 promoter (CITlp; SEQ ID NO: 125); endoplasmic reticulum chaperone BiP (KAR2p; SEQ ID NO: 126); heat shock protein 88 promoter (HSP88p; SEQ ID NO: 127); heat shock protein 70 promoter (HPS70p; SEQ ID NO:128); tubulin alpha-1 chain promoter (TUBlp; SEQ ID NO:129); glyceraldehyde-3 -phosphate dehydrogenase 2 promoter (TDH2p; SEQ ID NO: 130); translation elongation factor 1A like gene 5 promoter (TEF5p; SEQ ID NO: 131); translation elongation factor 3 promoter (TEF3p; SEQ ID NO: 132); thioredoxin peroxidase promoter (TPXp; SEQ ID NO: 133); alginate lyase promoter (ALPp; SEQ ID NO: 134); thiazole biosynthetic 4 promoter (THI4p; SEQ ID NO: 135); phosphoglucomutase / phospomannomutasePT-2218-WO-PCTpromoter (PMMp; SEQ ID NO: 136); fructose-bisphosphate aldolase 1 promoter (FBAlp; SEQ ID NO: 137); alcohol dehydrogenase 1 promoter (ADHlp; SEQ ID NO: 138); and extracellular endoglucanase 1 promoter (GLXlp; SEQ ID NO: 139); and / orone or more of the exogenous polynucleotide sequences(s) is operably linked to a heterologous or artificial promoter selected form the group consisting of6PGD terminator (6PGDt; SEQ ID NO:78); ASNS terminator (ASNSt; SEQ ID NO:79); EN01 terminator (ENOlt; SEQ ID NO:80); hexokinase 1 terminator (HXKlt; SEQ ID NO:81); PGK1 terminator (PGKlt; SEQ ID NO:82); PGM1 terminator (PGMlt; SEQ ID NO:83); PYK1 terminator (PYK It; SEQ ID NO: 84); RPL A terminator (RPLAt: SEQ ID NO: 85); transaldolase 1 terminator (TALlt; SEQ ID NO:86); TDH3 terminator (TDH3t; SEQ ID NO:87); translation elongation factor 2 terminator (TEF2t; SEQ ID NO:88); triosephosphate isomerase 1 terminator (TPIlt; SEQ ID NO:89); MpTEFl terminator (MpTEFlt; SEQ ID NO:90); TEF7 terminator (TEF7t; SEQ ID NO: 140); HSP90 terminator (HSP90t; SEQ ID NO: 141); TEF4 terminator (TEF4t; SEQ ID NO: 142); TEF6 terminator (TEF6t; SEQ ID NO: 143); CITI terminator (CITI It; SEQ ID NO: 144): KAR2 terminator (KAR2t; SEQ ID NO: 145); HPS88 terminator (HSP88t; SEQ ID NO: 146); HSP70 terminator (HSP70t; SEQ ID NO: 147); TUB1 terminator (TUB It; SEQ ID NO: 148); TKL1 terminator (TALlt; SEQ ID NO: 149); TDH2 terminator (TDH2t; SEQ ID NO: 150); TEF5 terminator (TEF5t; SEQ ID NO: 151); TGL2 terminator (TGL2t; SEQ ID NO: 152); TEF3 terminator (TEF3t; SEQ ID NO: 153); TPX terminator (TPXt; SEQ ID NO: 154); ALP terminator (ALPt; SEQ ID NO: 155); plasma membrane ATPase terminator (PMAlt; SEQ ID NO: 156); THI4 terminator (THI4t; SEQ ID NO: 157); glucose-6-phosphate isomerase 1 terminator (PGIlt; SEQ ID NO: 158); PMM terminator (PMMt; SEQ ID NO: 159); FBA1 terminator (FBAlt; SEQ ID NO: 160); ADH1 terminator (ADHlt; SEQ ID NO:161); sodium / potassium transporting ATPase alpha chain terminator (ATPlt; SEQ ID NO: 162); and GLX1 terminator (GLXlt; SEQ ID NO: 163); and / or one or more of the exogenous polynucleotide sequence(s) is integrated into the genome of the yeast cell at a locus selected from the ER1 locus, the ER3 locus, the PDC1 locus, the pyrF locus, the TRP3 locus, the gpdllA locus, the gpdllB locus, the RCSR26640 locus, and the RCSR18717 locus.

Images