Process for preparing a food product or animal feed

Description

1 / 96 PROCESS FOR PREPARING A FOOD PRODUCT OR ANIMAL FEED Divided from BR 11 2019 019106 2 STATEMENT REGARDING SEQUENCE LISTING

[0001] The sequence listing associated with this application is provided in text format in place of a paper copy and is incorporated herein by reference in the report. The name of the text file containing the sequence listing is “PCT - Sequence listing as filed”. The 224 KB text file was created on March 12, 2018 and is being submitted electronically. TECHNOLOGICAL FIELD

[0002] This report refers to a recombinant yeast host cell that expresses cell-associated enzymes and acts as a source of enzymatic activity in the production of food and / or feed, including those for the production of cooked products. BACKGROUND

[0003] Commercial yeast and commercial enzymes are commonly used to produce food and feed such as, for example, bread and other yeast-leavened baked goods. Commercial enzymes are also used without yeast for the preparation of food and feed, for example, in leavened and unleavened baked goods such as cakes and pita bread. Baker's yeasts are produced from strains of Saccharomyces cerevisiae by batch feed propagation and supplied in fresh or dried form. Enzymes for food and feed (such as enzymes for baking, brewing and feed) are produced from various plants, bacteria and fungi, often improved by genetic modification. Bacterial and fungal enzymes are usually expressed from organisms Petition 870260074630, dated 07 / 27 / 2026, page 5 / 103 2 / 96 producers during a sterile batch fermentation, separated, purified, concentrated, dried and supplied as granules.

[0004] Baking enzymes can be added to flour or dough used to prepare baked goods. They act on carbohydrates, proteins, and lipids during mixing, rising, and baking to facilitate processing and improve the appearance, texture, and quality of the finished product. For example, maltogenic alpha-amylase (MAA) is added to dough during the production of breads, cakes, and other baked goods. It acts on the amylopectin in wheat starch in the oven to inhibit starch retrogradation in the finished product and slow down the hardening rate.

[0005] Enzymes for food and feed in general, and cooking enzymes in particular, represent a significant part of the cost of producing cooked products. As such, there is an incentive to reduce the use or obsolete the use of purified exogenous enzymes for food and feed, such as cooking enzymes, in the process of preparing them. BRIEF SUMMARY

[0006] This report provides recombinant yeast host cells that have been genetically engineered to express one or more heterologous cell-associated enzymes for food and / or feed, as well as a process for their use or products derived from them for the preparation of food and / or feed products.

[0007] According to a first aspect, the present report provides a recombinant yeast host cell featuring a heterologous nucleic acid molecule encoding a heterologous enzyme associated with the cell for food and / or feed. The heterologous nucleic acid molecule is operationally associated with a heterologous promoter that allows the expression of the nucleic acid molecule. Petition 870260074630, dated 07 / 27 / 2026, p. 6 / 103 3 / 96 heterologous nucleic acid during propagation. In one embodiment, the heterologous nucleic acid molecule allows intracellular expression of the heterologous enzyme for food and / or feed. In another embodiment, the heterologous nucleic acid molecule allows expression of a membrane-associated heterologous enzyme for food and / or feed. For example, the heterologous nucleic acid molecule may allow expression of an immobilized heterologous enzyme for food and / or feed. In one embodiment, the immobilized heterologous enzyme for food and / or feed is a chimeric protein of formula (I): (NH2) FFE - L - TT (COOH) (I) where FFE is the enzyme for food and / or feed; L is present or absent and is an amino acid ligand; TT is an amino acid immobilization radical for the association of the enzyme for food and / or feed to a cell wall of the recombinant yeast host cell; and “-“ is an amide linkage. In the chimeric protein of formula (I), (NH2) indicates the location of the amino terminus of the chimeric protein and (COOH) indicates the location of the carboxyl terminus of the chimeric protein.

[0008] In another embodiment, the heterologous enzyme for food and / or feed is a chimeric protein of formula (II): (NH2) TT - L - FFE (COOH) (II) where FFE is the enzyme for food and / or feed; L is present or absent and is an amino acid ligand; TT is an amino acid immobilization radical for the association of the food and / or feed enzyme to a cell wall of the recombinant yeast host cell; and “-“ is a starch linkage. In the chimeric protein of formula (II), (NH2) indicates the location of the amino terminus of the chimeric protein and (COOH) indicates the location of the carboxyl terminus of the chimeric protein. In one embodiment, the heterologous nucleic acid molecule encodes the heterologous food enzyme, such as, for example, an alpha-acetolactate Petition 870260074630, dated 07 / 27 / 2026, p. 7 / 103 4 / 96 decarboxylase, an aminopeptidase, an amylase, a maltogenic amylase, an asparaginase, a bromelain, a carboxypeptidase, a catalase, a cellulase, a chymosin, a cyprosin, a ficin, a glucoamylase, a glucanase, a glucose oxidase, a glucose isomerase, a hemicellulase, a hexose oxidase, an inulinase, an invertase, a lactase, a lipase, a lipoxidase, a lysozyme, a mannanase, a milk coagulating enzyme, a pancreatin, a papain, a pectinase, a pentosanase, a pepsin, a phospholipase, a peroxidase, a protease, a pullulanase, a rennet, a transglutaminase, a trypsin, a urease and / or a xylanase. In yet another embodiment, the heterologous enzyme for food is a heterologous cooking enzyme, such as, for example, an amylolytic enzyme, a cellulase, a hemicellulase, an oxidase, an asparaginase, or a lipase.In yet another embodiment, the heterologous enzyme for food and / or feed is an amylolytic enzyme, such as, for example, a maltogenic alpha-amylase, a glucoamylase, an alpha-amylase, or a fungal amylase. In yet another embodiment, the heterologous enzyme for food and / or feed is an oxidase, such as, for example, a glucose oxidase. In another embodiment, the heterologous nucleic acid molecule encodes the heterologous enzyme for feed, such as, for example, a phytase, a beta-glucanase, a xylanase, an alpha-galactosidase, a protease, an amylase, a lipase, a mannanase, a cellulase, a hemicellulase, and / or a pectinase. In yet another embodiment, the heterologous enzyme for feed is phytase. In yet another embodiment of the chimeric protein, L is present and may comprise, for example, one or more G4S motifs (SEQ ID NO: 41) and / or one or more EA2K motifs (SEQ ID NO: 100) or EA3K motifs (SEQ ID NO: 101).In a further embodiment, the TT comprises a transmembrane domain, a variant thereof, or a fragment thereof. For example, the... Petition 870260074630, dated 07 / 27 / 2026, page 8 / 103 5 / 96 TT can be from a FLO1 protein. For example, TT can have the amino acid sequence of SEQ ID NO: 14, be a variant of the amino acid sequence of SEQ ID NO: 14, or be a fragment of the amino acid sequence SEQ ID NO: 14. In another embodiment, TT can be modified by a post-translational mechanism to present a glycosylphosphatidylinositol (GPI) anchor. For example, TT can be from an SED1 protein, a TIR1 protein, a CWP2 protein, a CCW12 protein, an SPI1 protein, a PST1 protein, or a combination of an AGA1 protein and an AGA2 protein. In a specific embodiment, TT is from the SPI1 protein and can have, for example, the amino acid sequence of SEQ ID NO: 74, be a variant of the amino acid sequence of SEQ ID NO: 74, or be a fragment of the amino acid sequence SEQ ID NO: 74.In a further embodiment, TT may be a fragment of the SPI protein and may have the amino acid sequence of SEQ ID NO: 76, 78, 80, or 82; be a variant of the amino acid sequence of SEQ ID NO: 76, 78, 80, or 82; or be a fragment of the amino acid sequence of SEQ ID NO: 76, 78, 80, or 82. In another specific embodiment, TT is from the CCW12 protein and may have, for example, the amino acid sequence of SEQ ID NO: 84; may be a variant of the amino acid sequence of SEQ ID NO: 84; or may be a fragment of the amino acid sequence of SEQ ID NO: 84. In yet another embodiment, TT may be a fragment of the CCW12 protein and may have the amino acid sequence of SEQ ID NO: 86, 88, 90, or 92. to be a variant of the amino acid sequence with SEQ ID NO: 86, 88, 90 or 92 or to be a fragment of the amino acid sequence with SEQ ID NO: 86, 88, 90 or 92.In another embodiment, TT is a combination of the AGA1 protein and the AGA2 protein and may present, for example, the amino acid sequence SEQ ID NO: 24, being a... Petition 870260074630, dated 07 / 27 / 2026, page 9 / 103 6 / 96 variant of the amino acid sequence of SEQ ID NO: 24, be a fragment of the amino acid sequence of SEQ ID NO: 24, present the amino acid sequence of SEQ ID NO: 26, be a variant of the amino acid sequence of SEQ ID NO: 26, or be a fragment of the amino acid sequence of SEQ ID NO: 26. In a further embodiment, the promoter is either a native promoter or a heterologous promoter. For example, the heterologous promoter may comprise the promoter of the tdhl gene, the hor7 gene, the hsp150 gene, the hxt7 gene, the gpml gene, the pgkl gene, and / or the stll gene. The heterologous promoter may comprise, for example, the promoter of the tdhl gene and / or the hor7 gene. In some embodiments, the heterologous nucleic acid molecule is operationally associated with a terminator which may be, for example, a native terminator or a heterologous terminator.In some embodiments, the heterologous terminator comprises a terminator of the ditl gene, the adh3 gene, the idpl gene, the gpml gene, the pmal gene, the tdh3 gene, the hxt2 gene, and / or the ira2 gene. The heterologous terminator may comprise, for example, the terminator of the ditl gene, the adh3 gene, and / or the idpl gene. In one embodiment, the membrane-associated heterologous polypeptide has a heterologous signaling peptide, such as, for example, the heterologous signaling peptide being from an invertase protein, an AGA2 protein, or a fungal amylase. In one embodiment, the heterologous signaling peptide is from the invertase protein and may have the amino acid sequence of SEQ ID NO: 68, be a variant of the amino acid sequence of SEQ ID NO: 68, or be a fragment of the amino acid sequence of SEQ ID NO: 68.In yet another embodiment, the heterologous signaling peptide is from the AGA2 protein and may have the amino acid sequence of SEQ ID NO: 69, be a variant of the amino acid sequence of SEQ ID NO: 69, or be a fragment of the amino acid sequence of SEQ ID NO: 69. Petition 870260074630, dated 07 / 27 / 2026, page 10 / 103 7 / 96 In yet another embodiment, the heterologous signaling peptide is from fungal amylase and may have the amino acid sequence of SEQ ID NO: 107, be a variant of the amino acid sequence of SEQ ID NO: 107, or be a fragment of the amino acid sequence of SEQ ID NO: 107. In some embodiments, the recombinant yeast host cell may be of the genus Saccharomyces sp. In some additional embodiments, the recombinant yeast host cell may be of the species Saccharomyces cerevisiae.

[0009] According to a second aspect, this report provides an additive comprising the enzyme for food and / or feed described herein. The additive may comprise or consist essentially of a yeast composition containing the recombinant yeast host cell as described herein. In one embodiment, the yeast composition may be provided in a live or deactivated form. The additive may comprise or consist essentially of a yeast product obtained from the recombinant yeast host cell described herein. In one embodiment, the yeast product may be a substantially purified enzyme for food and / or feed, a yeast extract, or a yeast fraction. The additive may be used as a food additive and / or a feed additive. In one embodiment, the food additive may be a dough conditioner.

[0010] According to a fourth aspect, this report provides a process for preparing a food product or feed. The process comprises incorporating the recombinant yeast host cell described herein or the additive described herein into the food product or feed. In one embodiment, the process further comprises fermenting the food product or feed in the presence of the recombinant yeast host cells and / or the additive. In another embodiment, the process comprises Petition 870260074630, dated 07 / 27 / 2026, page 11 / 103 8 / 96 additionally cooking the food product or feed in order to provide a cooked product. In such an embodiment, the process can be used to extend the shelf life of the cooked product and / or to improve the organoleptic properties of the cooked product. In yet another embodiment, the cooked product is bread. In one embodiment, the process can be used for the preparation of a food product. In yet another embodiment, the process can be used for the preparation of animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Having generically described the nature of the invention, reference will now be made to the accompanying drawings, which illustrate a preferred embodiment thereof, and in which:

[0012] Figures 1A and 1B provide the maltogenic amylase (MAA) enzyme activity measured in (Figure 1A) yeast cell pellets or (Figure 1B) wild-type yeast cream samples (M10474) or recombinant yeast host cells. In Figure 1A, the results are shown as maltogenic amylase activity (provided in MANU / mL) as a function of the yeast type tested (from left to right, M10474, T2986, T2987, T2988, T2989, T2990, T2991, T2944; strains are described in Table 1). In Figure 1B, the results are shown as maltogenic amylase activity (provided in MANU / mL) as a function of the type and batch of yeast tested (from left to right, M10474, M13819, M13822; the strains are described in Table 1).

[0013] Figures 2A to 2C provide crumb hardness (as measured in grams, left axis, shown in the dregs) and bread volume (as measured in cubic centimeters, right axis, as shown in the line labeled X) as a function of bread made with (from left to right) the wild type strain (M10474) without supplementation with Petition 870260074630, dated 07 / 27 / 2026, p. 12 / 103 9 / 96 enzyme, wild-type strain supplemented with 45 ppm of Novamyl®, wild-type strain supplemented with 90 ppm of Novamyl®, wild-type strain supplemented with 180 ppm of Novamyl®, strain M13822 (lot A), strain M13822 (lot B), M13819 (lot A) and M13819 (lot B) (the strains are described in Table 1). The results are shown at 5 (Figure 2A), 8 (Figure 2B) and 11 (Figure 2C) days after cooking.

[0014] Figures 3A to 3C provide the percent resilience as a function of bread made with (from left to right) the wild-type strain (M10474) without enzyme supplementation, wild-type strain supplemented with 45 ppm Novamyl®, wild-type strain supplemented with 90 ppm Novamyl®, wild-type strain supplemented with 180 ppm Novamyl®, strain M13822 (batch A), strain M13822 (batch B), M13819 (batch A) and M13819 (batch B). The results are shown for 5 (Figure 3A), 8 (Figure 3B) and 11 (Figure 3C) days after baking.

[0015] Figure 4 provides the glucoamylase enzyme activity measured in pellets (“bound”, light gray) and supernatant (“free”, dark gray) of cultured recombinant yeast host cells expressing a heterologous glucoamylase in the absence (strain M8498) and presence (strain M14244) of a Sed1 immobilizer. The results are shown as the percentage of glucoamylase activity as a function of the strain used.

[0016] Figure 5 provides the alpha-amylase enzymatic activity measured in pellets (“bound”, light gray) and supernatant (“free”, dark gray) of cultured recombinant yeast host cells expressing a heterologous alpha-amylase in the presence of a Sed1 immobilizer and a ligand (strain M14253), in the presence of a Sed1 immobilizer but without a ligand (M14254), and in the absence of a Sed1 immobilizer (strain Petition 870260074630, dated 07 / 27 / 2026, p. 13 / 103 10 / 96 M10074). The results are shown as the percentage of alpha-amylase activity as a function of the strain used.

[0017] Figure 6 provides the activity on wheat starch of several strains expressing a maltogenic amylase. Results are shown in MANU of wheat starch per mL (measured at OD 600 nm) for whole culture (left bars), supernatant (middle bars), and washed pellet (right bars) of strains M10474, M13822, M13819, M13979, and T3892 (strains are described in Table 1). Data for the “M” strains are the average of duplicate cultures. Data for T3892 include the average activity across all cultures of eight transforming isolates and the activity of the best-performing isolate (□ = best-performing isolate, whole culture; △ = best-performing isolate, supernatant; o = best-performing isolate, washed cell pellet). The graphs below indicate the predicted phenotypic location of the enzyme for each engineering strategy.

[0018] Figures 7A and 7B provide phytase activity in culture supernatant (gray bars) or associated with cells (bars with diagonal lines in Figure 7A or □ in Figure 7B) for strains expressing free or immobilized Citrobacter braakii phytase. The supernatant was incubated with 5 mM of a sodium phytate solution pH 5.5 for 30 minutes and the cells were incubated in the same solution for 2 hours. (Figure 7A) Absorbance at 700 nm was compared with a standard curve of known phosphate concentrations to express activity in FTUs. Absorbance was measured in the supernatant (gray bars) and in the cells (diagonal bars) in different strains (M12548, T2633, T2634, T2635, T2636, T2637 and T2638). (Figure 7B) the FTUs were compared between the different strains. The left vertical axis shows the supernatant activity and the FTU for each of the strains is provided as gray bars. The axis Petition 870260074630, dated 07 / 27 / 2026, page 14 / 103 Figure 11 / 96 on the right shows the cell-associated FTU activity and is provided as □ for each of the strains (M12548, T2633, T2634, T2635, T2636, T2637 and T2638). The values ​​for the parental strain and the cell-associated activity of the Pst1 immobilizer were obtained outside the range of the standard curve and, for this reason, below the detection limit.

[0019] Figure 8 provides phytase activity in culture supernatant (gray bars) or associated with cells (bars with diagonal lines) for strains expressing fused Escherichia coli phytase or with an N- or C-terminal immobilizer. The supernatant was incubated with 5 mM of a pH 5.5 sodium phytate solution for 30 minutes, and the cells were incubated in the same solution for 2 hours. The results are shown as optical density at 700 nm as a function of each strain (M11312, T2705, and T2706).

[0020] Figure 9 provides phytase activity in culture supernatant (gray bars) or associated with cells (bars with diagonal lines) for strains expressing fused E. coli phytase or N-terminal immobilizer phytase with or without AGA1 overexpression compared to E. coli phytase fused with a C-terminal Sed1 immobilizer. The supernatant was incubated with 5 mM of a pH 5.5 sodium phytate solution for 30 minutes and the cells were incubated in the same solution for 2 hours. The results are shown as the optical density at 700 nm as a function of each strain (M12550, M12795, M12983 and T2816).

[0021] Figure 10 provides the activity on wheat starch of strains expressing maltogenic amylase. The results are provided as the ratio of absorbance at 450 nm / optical density at 600 nm for the whole culture (left bars), the supernatant (middle bars) and washed cells (left bars) for the different strains (M10474, M13819, M13822, M14851, T4328, T4329, T4330, M12962, T4336, T4337 and Petition 870260074630, dated 07 / 27 / 2026, p. 15 / 103 12 / 96 (T4338). Data for the “M” strains are the average of duplicate cultures. Data for the “T” strains include the average activity of cultures from seven transformed isolates. Type 1 expression refers to the presence of an invertase peptide signal and a Spi1 immobilizer to generate an immobilized enzyme. Type 2 expression refers to the presence of an invertase peptide signal and the absence of an immobilizer to generate a secreted enzyme. Type 3 expression refers to the absence of a signal peptide and the absence of an immobilizer to generate an intracellular enzyme.

[0022] Figures 11A to 11C provide crumb hardness (as measured in grams, left axis, results shown in bars) of breads made with different dough conditioners on days 3 (Figure 11A), 7 (Figure 11B), and 11 (Figure 11C) after baking. Controls were made with or without the externally added Novamyl® maltogenic amylase (labeled “Control”) as indicated below the histogram. Control breads were compared to breads made with spray-dried M13979 homogenates (identified as “homo+spray” in the figures) or cream dosed for a specific Phadebas enzymatic activity as indicated below the histogram. All breads used wild-type yeast for leavening power.

[0023] Figures 12A to 12F provide the crumb hardness (Figure 12AC, as measured in grams) and resilience (Figure 12D-F, as measured in percentage) of loaves made with different dough conditioners on days 4 (Figure 12A and Figure 12D), 7 (Figure 12B and Figure 12E), and 10 (Figure 12C and Figure 12F) after baking. Controls were made with or without the externally added Novamyl® maltogenic amylase (labeled “Control”) as indicated below the histogram. Control loaves were compared with breads Petition 870260074630, dated 07 / 27 / 2026, page 16 / 103 13 / 96 made with M15532 yeast cream that was homogenized to release the intracellular enzyme and measured for a Phadebas assay as indicated below the histogram. All breads used wild yeast for the leavening power.

[0024] Figure 13 shows the alpha-amylase activity associated with yeast cell strains expressing various chimeric proteins comprising a thermotolerant alpha-amylase derived from Pyrococcus furiosus (SEQ ID NO: 71) in combination with different immobilizing radicals derived from the SPI1 protein or associated truncations (M15774, M15771, M15777, M15772 and M15222) compared to a control strain (M2390). The results are shown as absorbance at 540 nm as a function of the yeast strain used.

[0025] Figure 14 shows the alpha-amylase activity associated with yeast cell strains expressing various chimeric proteins comprising an alpha-amylase derived from Thermococcus hydrothermalis (SEQ ID NO: 72) in combination with different immobilizing radicals derived from the CCW12 protein or associated truncations (M15773, M15776, M16251 and M15215) compared to a control strain (M2390). The results are shown as absorbance at 540 nm as a function of the yeast strain used.

[0026] Figure 15 shows the alpha-amylase activity associated with yeast cell strains expressing various chimeric proteins comprising an alpha-amylase derived from T. hydrothermalis (SEQ ID NO: 72) in combination with a CCW12 protein-derived immobilizing radical and different ligands (M15785, M15786, M15782, M16252, M16221 and M16222) compared to a control strain (M2390). Results are shown as absorbance at 540 nm as a function of yeast strain. Petition 870260074630, dated 07 / 27 / 2026, p. 17 / 103 14 / 96

[0027] Figure 16 shows the alpha-amylase activity associated with yeast cell strains expressing various chimeric proteins comprising an alpha-amylase derived from P. furiosus (SEQ ID NO: 71), an immobilizing radical derived from the SPI1 protein, and different ligands (M15784, M15778, M15779, M15787, M15780, M15788, and M15783) compared to a control strain (M2390). The results are shown as absorbance at 540 nm as a function of the yeast strain.

[0028] Figure 17 shows the glucose oxidase (GO) activity associated with the whole culture (gray bars), washed cells (bars with diagonal lines), or the supernatant of broken washed cells (white bars) of yeast strains expressing an Aspergillus niger-derived glucose oxidase, expressed in a secreted (M16780) or intracellular (M16273) form, compared to a negative control strain (M10474) and a positive control amount of a commercially available purified glucose oxidase (positive control, Gluzyme Mono®). Results are shown as absorbance at 510 nm as a function of the yeast strain / control used.

[0029] Figure 18 shows the glucose oxidase (GO) activity associated with whole culture (gray bars), washed cells (bars with diagonal lines) of yeast strains expressing a glucose oxidase derived from Aspergillus niger, expressed in a secreted (M16780) or intracellular (M16273) form. The results are shown as absorbance at 510 nm (corrected to remove absorbance associated with the control strain M10474) as a function of the yeast strain used.

[0030] Figure 19 shows the fungal amylase (FA) activity associated with the whole culture (gray bars), washed cells (diagonal bars) or the supernatant of broken washed cells (bars Petition 870260074630, dated 07 / 27 / 2026, page 18 / 103 15 / 96 white yeast strains expressing a fungal amylase derived from Aspergillus oryzae expressed in a secreted form with different signaling peptides (S. cerevisiae invertase for M16772, native A. oryzae alpha-amylase signaling peptide for M16540) compared to a negative control strain (M10474) and a positive control amount of a commercially available purified fungal alpha-amylase (positive control, Fungamyl®). The results are shown as absorbance at 540 nm as a function of the yeast / control strain used.

[0031] Figure 20 shows the fungal amylase (FA) activity associated with whole culture (gray bars), washed cells (bars with diagonal lines), or the supernatant of broken washed cells (white bars) of yeast strains expressing a fungal amylase derived from Aspergillus oryzae expressed in a secreted form with different signaling peptides (S. cerevisiae invertase for M16772, native alpha-amylase signaling peptide from A. oryzae for M16540). The results are shown as absorbance at 540 nm (corrected to remove absorbance associated with the control strain M10474) as a function of the yeast strain used.

[0032] Figure 21 shows the evaluation of cell-associated glucose oxidase activity of a cell pellet of the M16780 strain using a baking test. The results are shown for control breads (prepared in the absence of an additive), for breads prepared with 10 ppm or 20 ppm of Gluzyme Mono®, or for breads prepared with a dosage of the cell pellet of the M16780 strain. DETAILED DESCRIPTION

[0033] This report provides recombinant yeast host cells that express a heterologous cell-associated enzyme for food and / or feed during their propagation phase. As such Petition 870260074630, dated 07 / 27 / 2026, page 19 / 103 In the context of this report, the term "propagation phase" refers to an expansion phase of a commercial process in which yeasts are propagated under aerobic conditions in order to maximize the conversion of a substrate into biomass. In some cases, the propagated biomass can be used in a subsequent fermentation step (usually under anaerobic conditions) in order to maximize the production of one or more desired metabolites and / or to prepare a fermented feed or feed product. The recombinant yeast host cells of this report are advantageous because they provide a lower-cost source of enzymatic activity than the purified products that are traditionally used.These recombinant yeast host cells can be advantageously used in various food and / or feed products, such as, for example, baked goods, even though the proof time and conditions do not provide an opportunity for the yeast to produce the enzymes in situ. These recombinant yeast host cells can also be used in other baked goods, fermented foods, non-fermented foods, and animal feed. Recombinant yeast host cells can be advantageously easily measured, dosed, and formulated. Recombinant yeast host cells

[0034] The recombinant yeast host cells in this report are intended for use in the preparation of products for human consumption (food) and / or animal feed. As used in the context of this report, the term "food and / or feed enzyme" refers to a protein exhibiting enzymatic activity and capable of being used in a process for the preparation of a food product or a feed product. In some embodiments, the "food and / or feed enzyme" refers to Petition 870260074630, dated 07 / 27 / 2026, p. 20 / 103 17 / 96 enzymes exhibiting applications in starch processing (e.g., starch-containing biomass). Food and feed enzymes include, without limitation, cooking enzymes, brewing enzymes, distilled beverage enzymes, winemaking enzymes, juice enzymes, starch processing enzymes, and feed enzymes. The recombinant yeast host cells of this report can optionally be used in a fermentation process. In one embodiment, the fermentation process can be relatively long, and the recombinant yeast host cells can be used, for example, in the preparation of distilled products, wine, and beer. In another embodiment, the fermentation process can be relatively short, and the recombinant yeast host cells can be used, for example, in the preparation of yeast-free baked goods.The recombinant yeast host cells of this report can also be used in a process that does not include a fermentation step. For example, the recombinant yeast host cell can be used for the preparation of food and beverages (e.g., unleavened baked goods (chemically leavened), dairy products, yeast extract, juices, fats and oils, as well as starch), or animal feed.

[0035] In one embodiment, the recombinant yeast host cells of this report express at least one enzyme for food and / or feed prior to the introduction of the heterologous nucleic acid molecules of this report and are genetically modified to express an additional cell-associated enzyme (a different one or the same one). In another embodiment, the recombinant yeast host cells of this report cannot Petition 870260074630, dated 07 / 27 / 2026, page 21 / 103 18 / 96 to be used in consolidated biological processing for the preparation of, for example, biofuels such as bioethanol.

[0036] The recombinant yeast host cells of this report may be provided in an active form (e.g., liquid (such as, for example, a yeast cream), compressed or fluid-bed dried yeast), in a semi-active form (e.g., liquid, compressed, or fluid-bed dried), in an inactive form (e.g., drum-dried or spray-dried), as well as a mixture thereof. For example, the recombinant yeast host cells may be a combination of active and semi-active form or inactive form in order to provide the proportion and dose of the enzyme required for the preparation of the feed or ration.

[0037] This report concerns recombinant yeast host cells that have been genetically engineered. The genetic modification(s) aim to increase the expression of a specific target gene (which is considered heterologous to the yeast host cell) and may be made at one or multiple genetic loci (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more). In the context of this report, when a recombinant yeast cell is qualified as being “genetically engineered,” it should be understood that this means it has been manipulated to have at least one or more exogenous nucleic acid residues added. In some embodiments, the one or more nucleic acid residues that are added may be derived from a heterologous cell or from the recombinant host cell itself. In this last scenario, the nucleic acid residue(s) is / are added to one or more genomic locations that are different from the native genomic location.Genetic manipulations do not occur in nature and are the result of in vitro manipulations of yeast. Petition 870260074630, dated 07 / 27 / 2026, page 22 / 103 19 / 96

[0038] When expressed in recombinant yeast host cells, the heterologous enzymes described herein are encoded on one or more heterologous nucleic acid molecules. The term “heterologous,” when used with reference to a nucleic acid molecule (such as a promoter, a terminator, or a coding s1q) or a protein (such as an enzyme), refers to a nucleic acid molecule or a protein that is not natively found in the recombinant host cell. “Heterologous” also includes a coding region / promoter / terminator, or a portion thereof, that has been removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, for example, not at its native location in the organism’s genome. The heterologous nucleic acid molecule is purposefully introduced into the recombinant host cell.For example, a heterologous element may be derived from a different host cell strain, or from an organism of a different taxonomic group (e.g., kingdom, phylum, class, order, family, genus, or species, or any subgroup within one of these classifications).

[0039] The heterologous nucleic acid molecule present in the recombinant host cell can be integrated into the host cell genome. The term “integrated” as used here refers to genetic elements that are placed, by means of molecular biology techniques, into the genome of a host cell. For example, genetic elements can be placed on the host cell chromosomes as opposed to in a vector such as a plasmid contained within the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The nucleic acid molecule Petition 870260074630, dated 07 / 27 / 2026, p. 23 / 103 The 20 / 96 heterologous nucleic acid molecule may be present in one or more copies (e.g., 2, 3, 4, 5, 6, 7, 8, or even more copies) in the yeast host cell genome. Alternatively, the heterologous nucleic acid molecule may be genome-independent of the yeast genome. In such an embodiment, the nucleic acid molecule may be both stable and self-replicating.

[0040] In the context of this report, the recombinant host cell is a yeast, and in some embodiments the yeast can be used in the production of food and / or feed. Suitable yeast host cells may be, for example, of the genera Saccharomyces, Kluyveromyces, Arxula, Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Torula, or Yarrowia. A suitable yeast species may include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, C. utilis, K. lactis, K. marxianus, or K. fragilis. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolitica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis. In one particular embodiment, the yeast is Saccharomyces cerevisiae. In some embodiments, the host cell may be an oleaginous yeast cell. For example, the oleaginous yeast host cell may be of the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon, or Yarrowia. In some alternative embodiments, the host cell may be an oleaginous microalgal host cell (e.g., of the genus Petition 870260074630, dated 07 / 27 / 2026, page 24 / 103 21 / 96 Thraustochytrium or Schizochytrium). In one embodiment, the recombinant yeast host cell is of the genus Saccharomyces, and in some embodiments, of the species Saccharomyces cerevisiae.

[0041] The recombinant yeast host cells of this report include a heterologous nucleic acid molecule intended to enable the expression of (e.g., encode) one or more heterologous enzymes for food and / or feed. In one embodiment, a heterologous enzyme is a food enzyme that may be, without limitation, alpha-acetolactate decarboxylase, aminopeptidase, amylase, maltogenic alpha-amylase, asparaginase, bromelain, carboxypeptidase, catalase, cellulase, chymosin (including chymosin A and B), cyprosin, ficin, glucoamylase (also known as amyloglucosidase or maltase), glucanase, glucose oxidase, glucose isomerase, hemicellulase, hexose oxidase, inulinase, invertase, lactase, lipase, lipoxidase, lysozyme, mannanase, milk coagulating enzyme, pancreatin, papain, pectinase, pentosanase, pepsin, phospholipase, peroxidase, protease, pullulanase, rennet (including bovine rennet), transglutaminase, trypsin, urease, and / or xylanase.In one embodiment, the heterologous enzyme for food and / or feed is a cooking enzyme. As used in the context of this report, the term "cooking enzyme" refers to a protein exhibiting enzymatic activity and capable of being used in a process for the preparation of a cooked product. In one embodiment, the heterologous nucleic acid molecule of the yeast host cells of this report encodes at least one heterologous cooking enzyme. Cooking enzymes include, without limitation, amylolytic enzymes (including, for example, maltogenic alpha-amylases, glucoamylases, alpha-amylases and fungal amylases), cellulases / hemicellulases, oxidases (including, for example, glucose oxidases), asparaginases and lipases. In another embodiment, the enzyme... Petition 870260074630, dated 07 / 27 / 2026, page 25 / 103 22 / 96 heterologous is a feed enzyme that may be, without limitation, a phytase, β-glucanase, xylanase, alpha-galactosidase, protease, amylase, lipase, mannanase, cellulase and / or hemicellulase-pectinases.

[0042] As used herein, the term “amylolytic enzyme” refers to a class of enzymes capable of hydrolyzing starch or hydrolyzed starch. In baking applications, amylolytic enzymes can participate in the release of fermentable sugars, increasing bread volume, reducing fermentation time, reducing staleness, and / or enhancing flavor. Amylolytic enzymes include, but are not limited to, alpha-amylases (EC 3.2.1.1, sometimes referred to as fungal alpha-amylases, as well as bacterial alpha-amylases, see below), maltogenic amylase (EC 3.2.1.133), glucoamylase (EC 3.2.1.3), glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60), pullulanase (EC 3.2.1.41), isoamylase (EC 3.2.1.68), and amylomaltase (EC 2.4.1.25). Fungal alpha-amylases can be used, for example, in the production of baked goods (e.g., yeast-leavened, chemically leavened, or unleavened products), juices, and fermented beverages (such as beers).Bacterial α-amylases can be used, for example, in the production of baked goods (e.g., yeast-leavened, chemically leavened, or unleavened products), fermented beverages (including beers, distilled beverages, and the like), as well as in starch processing. Maltogenic α-amylases can be used, for example, in the production of baked goods (e.g., yeast-leavened, chemically leavened, or unleavened products). In one embodiment, one or more amylolytic enzymes may be an α-amylase from Aspergillus oryzae (and exhibit, for example, the amino acid sequence SEQ ID NO: 2 or 105, a variant thereof, or a fragment thereof), a maltogenic α-amylase from Geobacillus stearothermophilus (and exhibit, for example, the sequence... Petition 870260074630, dated 07 / 27 / 2026, page 26 / 103 23 / 96 amino acid sequences of sequence IDs NO: 1, 51, 65 or 108, a variant thereof or a fragment thereof), a glucoamylase from Saccharomycopsis fibuligera (and display, for example, the amino acid sequence of sequence ID NO: 3, a variant thereof or a fragment thereof), a glucan 1,4-alpha-maltotetrahydrolase from Pseudomonas saccharophila (and display, for example, the amino acid sequence of sequence ID NO: 4, a variant thereof or a fragment thereof), a pullulanase from Bacillus naganoensis (and display, for example, the amino acid sequence of sequence ID NO: 5, a variant thereof or a fragment thereof), a pullulanase from Bacillus acidopullulyticus (and display, for example, the amino acid sequence of sequence ID NO: 6, a variant thereof or a fragment thereof), an isoamylase from Pseudomonas amyloderamosa (and present, for example, the amino acid sequence of SEQ ID NO: 7, a variant thereof or a fragment thereof), and / or an amylomaltase from Thermus thermophilus (and present, for example,the amino acid sequence of SEQ ID NO: 8, a variant thereof, or a fragment thereof.

[0043] As used herein, the term “cellulase / hemicellulase” refers to a class of enzymes capable of hydrolyzing cellulose, hemicellulose, or pentosans. In baking applications, cellulases and hemicellulases may participate in the establishment of a gluten network, providing a soluble dietary fiber, modulating dough viscosity, and / or modulating dough rheology. Cellulases / hemicellulases include, but are not limited to, a cellulase (EC 3.2.1.4) and an endoβ(1,4)D-xylanase (EC 3.2.1.8). In one embodiment, one or more cellulases / hemicellulases may be a Penicillium funiculosum cellulase (and present, for example, the amino acid sequence of SEQ ID NO: 42, a variant thereof, or a fragment thereof) and / or an endoB(1,4)D-xylanase from Rasamsonia emersonii (and present, for example, the amino acid sequence of SEQ ID NO: 42, a variant thereof, or a fragment thereof) and / or an endoB(1,4)D-xylanase from Rasamsonia emersonii (and present, for example, the amino acid sequence of SEQ ID NO: 42, a variant thereof, or a fragment thereof) Petition 870260074630, dated 07 / 27 / 2026, page 27 / 103 24 / 96 example, the amino acid sequence of SEQ ID NO: 43, a variant thereof or a fragment thereof).

[0044] As used herein, the term “oxidase” refers to a class of enzymes capable of catalyzing an oxidation-reduction reaction. The oxidase may be an oxidoreductase such as a hexose oxidase (including a glucose oxidase). Oxidases can be used in the production of baked goods (such as, for example, yeast-leavened products including bread). In some embodiments, oxidases (such as glucose oxidases) can improve the workability of dough. In baking applications, oxidases can participate in the control of Maillard reactions and / or the establishment of crumb structure. In one embodiment, one or more oxidases may be a glucose oxidase from Aspergillus niger (and have, for example, the amino acid sequence SEQ ID NO: 44 or 103, a variant thereof, or a fragment thereof).

[0045] As used herein, the term “asparaginase” refers to a class of enzymes capable of catalyzing the conversion of asparagine into aspartic acid and ammonia. Asparaginase can be used in the production of snacks, cereals (including breakfast cereals), as well as baked goods (e.g., leavened with yeast (including bread), chemically leavened or unleavened).

[0046] As used herein, the term “lipase” refers to a class of enzymes capable of hydrolyzing lipids. In baking applications, lipases can contribute to increasing bread volume, enhancing dough stability, providing anti-staleness, and / or facilitating the formation of emulsifiers. Lipases can be used, for example, in the production of baked goods (such as yeast-leavened products (including bread) and chemically leavened products). In one embodiment, one or more lipases may be a Petition 870260074630, dated 07 / 27 / 2026, page 28 / 103 25 / 96 triacylglycerol lipase from Thermomyces lanuginosis (and present, for example, the amino acid sequence of SEQ ID NO: 45, a variant thereof or a fragment thereof), a phospholipase A2 from Sus scrofa (and present, for example, the amino acid sequence of SEQ ID NO: 46, a variant thereof or a fragment thereof), a phospholipase A2 from Streptomyces vialaceoruber (and present, for example, the amino acid sequence of SEQ ID NO: 47, a variant thereof or a fragment thereof) and / or a phospholipase A2 from Aspergillus oryzea (and present, for example, the amino acid sequence of SEQ ID NO: 48, a variant thereof or a fragment thereof).

[0047] In one embodiment, the recombinant yeast host cell of the present report includes (and in one embodiment expresses) a nucleic acid molecule encoding for a maltogenic amylase. As used in the present report, the term “maltogenic amylase” refers to a polypeptide capable of hydrolyzing starch or starch hydrolyzed to maltose. Maltogenic amylases include, but are not limited to, fungal alpha-amylases (derived, for example, from Aspergillus sp. (e.g., A. niger, A. kawachi, and A. oryzae); Trichoderma sp. (e.g., T. reesie), Rhizopus sp., Mucor sp., and Penicillium sp.), acid-stable fungal amylase (derived, for example, from Aspergillus niger), beta-amylases (derived, for example, from plants (wheat, barley, rye, sorghum, soybean, sweet potato, rice) and microorganisms (Bacillus cereus, Bacillus polymixa, Bacillus megaterium, Arabidopsis thaliana), maltogenic amylases (EC3.2.1).133) (derived, for example, from microorganisms such as Bacillus subtilis, Geobacillus stearothermophilus, Bacillus thermoalkalophilus, Lactobacillus gasseri, Thermus sp.). In a specific embodiment, the recombinant yeast host cells of the present report include a heterologous nucleic acid molecule encoding maltogenic amylase. Petition 870260074630, dated 07 / 27 / 2026, p. 29 / 103 26 / 96 heterologous derivative of Geobacillus stearothermophilus and presenting, for example, the amino acid sequence of SEQ ID NO: 1, 51, 65 or 108, a variant thereof or a fragment thereof.

[0048] As used herein, the term “phosphatase” refers to a feed enzyme capable of catalyzing, in the presence of water, the cleavage of a phosphoric acid monoester into a phosphate ion and an alcohol. One embodiment of a phosphatase is a phytase, a protein exhibiting enzymatic activity and capable of hydrolyzing phytic acid (myo-inositol hexakisphosphate) into inorganic phosphorus. There are four distinct classes of phytase: histidine acid phosphatases (HAPS), beta-propeller phytases, purple acid phosphatases, and tyrosine phosphatase-like protein phytases (PTP-like phytases). Phytic acid has six phosphate groups that can be released by phytases in different proportions and in different orders. Phytases hydrolyze phosphates from phytic acid gradually, producing products that again become substrates for further hydrolysis.Phytases were grouped based on the first phytic acid phosphate position that is hydrolyzed: they are 3-phytase (EC 3.1.3.8), 4-phytase (EC 3.1.3.26), and 5-phytase (EC 3.1.3.72). In one embodiment, the phytase is derived from a bacterial species, such as, for example, Citrobacter sp. or Escherichia sp. In a specific embodiment, the heterologous phytase is derived from a Citrobacter sp., such as, for example, Citrobacter braakii, and may present, for example, the amino acid sequence of SEQ ID NO: 66, a variant thereof, or a fragment thereof. In another embodiment, the heterologous phytase is derived from an Escherichia sp., such as, for example, Escherichia coli, and may present, for example, the amino acid sequence of SEQ ID NO: 67, a variant thereof, or a fragment thereof. Petition 870260074630, dated 07 / 27 / 2026, p. 30 / 103 27 / 96

[0049] The heterologous enzyme for food and / or feed may be a variant of a known / native enzyme for food and / or feed. For example, in embodiments where the heterologous enzyme for food and / or feed is a heterologous cooking enzyme, the heterologous cooking enzyme may be a variant of a known / native cooking enzyme, for example, a variant of the heterologous cooking enzyme having the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 42, 43, 44, 45, 46, 47, 48, 51, 65, 66, 67, 103, 105 or 108. A variant comprises at least one different amino acid compared to the amino acid sequence of the native enzyme for food and / or feed. As used here, a variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the enzyme for food and / or feed.A substitution, insertion, or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the food and / or feed enzyme. For example, the overall charge, structure, or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Similarly, the amino acid sequence can be altered, for example, to make the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the food and / or feed enzyme. The food and / or feed enzyme variants exhibit at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the food and / or feed enzyme described herein. The term "percent identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.The level of identity can be determined conventionally. Petition 870260074630, dated 07 / 27 / 2026, page 31 / 103 28 / 96 using known computer programs. Identity can be easily calculated by known methods, including, but not limited to, those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods for identity determination are designed to provide the best match between the tested sequences. The methods for determining identity and similarity are codified in publicly available computer programs.Sequence alignments and percentile identity calculations can be performed using the Megalign program from the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The multiple alignments of the sequences described here were performed using the Clustal alignment method (Higgins and Sharp (1989) CABIOS. 5:151-153) with the standard parameters (GAP PENALTY=10, GAP LENGTH PENALTY ALT Y= 10). The standard parameters for pairwise alignments using the Clustal method were KTUPLB 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0050] The heterologous food and / or feed enzyme variant described herein (including the food and / or feed enzyme described herein) may be (i) one in which one or more of the amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be encoded by the genetic code, or (ii) one in which one or more of the residues Petition 870260074630, dated 07 / 27 / 2026, p. 32 / 103 29 / 96 amino acids include a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) one in which additional amino acids are fused to a mature polypeptide for polypeptide purification. A “variant” of the enzyme for food and / or feed may be a conservative variant or an allelic variant.

[0051] The heterologous enzyme for food and / or feed may be a fragment of a known / native enzyme for food and / or feed. In embodiments where the heterologous enzyme for food and / or feed is a heterologous cooking enzyme, the heterologous cooking enzyme may be a fragment of a known / native cooking enzyme or a fragment of a variant of a known / native cooking enzyme (such as, for example, a fragment of the cooking enzyme having the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 42, 43, 44, 45, 46, 47, 48, 51, 65, 66, 67, 103, 105 or 108 or a variant thereof). In one embodiment, a fragment corresponds to the known / native enzyme for food and / or feed from which the signal peptide sequence has been removed.The “fragments” of the enzyme for food and / or feed (including “fragments” of the cooking enzyme) contain at least 100, 200, 300, 400, 500, 600, 700 or more consecutive amino acids of the enzyme for food and / or feed. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the known / native enzyme for food and / or feed and still possess the enzymatic activity of the full-length enzyme for food and / or feed. In one embodiment, the fragment corresponds to the amino acid sequence of the enzyme that does not contain the signal peptide. In some embodiments, the fragments of the enzyme for food and / or feed... Petition 870260074630, dated 07 / 27 / 2026, p. 33 / 103 30 / 96 can be used for the production of the corresponding full-length enzyme for food and / or feed by peptide synthesis. In this way, the fragments can be used as intermediates for the production of full-length proteins.

[0052] In the recombinant yeast host cell of this report, the heterologous food and / or feed enzyme (including the cooking enzyme) is “cell-associated” to the recombinant yeast host cell insofar as it is designed to be expressed and remain physically associated with the recombinant yeast host cells. In one embodiment, the food and / or feed enzyme can be expressed inside the recombinant yeast host cell (intracellularly). In such an embodiment, the heterologous food and / or feed enzyme does not need to be associated with the walls of the recombinant yeast host cells. When the food and / or feed enzyme is intended to be expressed intracellularly, its signal peptide sequence, if present in the native sequence, can be deleted to allow intracellular expression.

[0053] In another embodiment, the heterologous enzyme for food / feed may be secreted; however, if so, it must remain physically associated with the recombinant yeast host cell. In one embodiment, at least a portion (usually at least one terminal) of the heterologous enzyme for food and / or feed is linked covalently, non-covalently, and / or electrostatically, for example, to the cell wall (and in some embodiments to the cytoplasmic membrane). For example, the heterologous enzyme for food and / or feed may be modified to contain one or more transmembrane domains, so as to exhibit one or more lipid modifications (myristoylation, palmitoylation, farnesylation, and / or prenylation), to Petition 870260074630, dated 07 / 27 / 2026, p. 34 / 103 31 / 96 interact with one or more membrane-associated proteins and / or for interactions with cellular lipid rafts. Although the heterologous enzyme for food and / or feed may not be directly bound to the membrane or cell wall (for example, such as when the binding occurs via an immobilizing radical), the protein is nevertheless considered a “cell-associated” heterologous enzyme for food and / or feed according to this report.

[0054] In some embodiments, the heterologous enzyme for food and / or feed may be expressed to be localized and associated with the cell wall of the recombinant yeast host cell. In some embodiments, the heterologous enzyme for food and / or feed is expressed to be localized on and associated with the outer surface of the host cell wall. Recombinant yeast host cells all have a cell wall (which includes a cytoplasmic membrane) defining the intracellular (e.g., facing inward toward the nucleus) and extracellular (e.g., facing outward) environments. The enzyme for food and / or feed may be localized (and, in some embodiments, physically associated with) the outer face of the recombinant host yeast cell wall and, in further embodiments, the outer face of the recombinant host yeast cytoplasmic membrane.In the context of this report, the expression "associated with the outer face of the cell wall / cytoplasmic membrane of the recombinant yeast host cell" refers to the ability of the heterologous enzyme for food and / or feed to physically integrate (covalently or non-covalently), at least in part, into the cell wall (and, in some embodiments, the cytoplasmic membrane) of the recombinant yeast host cell. This physical integration can be attributed to the presence, for example, of a transmembrane domain in the heterologous enzyme for food and / or feed. Petition 870260074630, dated 07 / 27 / 2026, page 35 / 103 32 / 96 ration, a domain capable of integrating with a cytoplasmic membrane protein in the heterologous enzyme for food and / or ration, a post-translational modification made in the heterologous enzyme for food and / or ration (e.g., lipidization), etc.

[0055] Some heterologous enzymes for food and / or feed (including cooking enzymes) exhibit an intrinsic ability to localize to and associate with the cell wall of a recombinant yeast host cell (i.e., being cell-associated). An example of a food and / or feed enzyme exhibiting the intrinsic ability to be cell-associated is shown in the radical of Figure 1A (e.g., column strain T2994 in Figure 1A). In this figure, results are presented for maltogenic alpha-amylase from Geobacillus stearothermophilus expressed in S. cerevisiae in the absence of an immobilizing radical and clearly show that this enzyme is intrinsically “cell-associated” and exhibits enzymatic activity (e.g., maltogenic alpha-amylase activity).

[0056] However, in some circumstances, it may be necessary to increase or provide cell association to some enzymes for food and / or feed, given that they exhibit insufficient intrinsic cell association or simply do not exhibit intrinsic cell association. In such an embodiment, it is possible to provide the heterologous enzyme for food and / or feed as a chimeric construct by combining it with an amino acid immobilizing radical which will provide or increase attachment to the cell wall of the recombinant yeast host cell. In such an embodiment, the chimeric enzyme for food and / or feed will be considered “immobilized”. It is preferred that the amino acid immobilizing radical of the chimeric protein be neutral with respect to the biological (enzymatic) activity of the heterologous enzyme for food and / or feed, for example, not interfering with the biological activity. Petition 870260074630, dated 07 / 27 / 2026, p. 36 / 103 33 / 96 (enzymatic) of the heterologous enzyme for food and / or feed. In some embodiments, the association of the amino acid immobilization radical with the heterologous enzyme for food and / or feed can increase the biological (enzymatic) activity of the heterologous enzyme for food and / or feed (when compared to the non-immobilized non-chimeric form).

[0057] In one embodiment, an immobilizing radical can be used to be expressed with the heterologous enzyme for food and / or feed in order to localize the enzyme to the wall of the recombinant yeast host cell. Several amino acid immobilizing radicals are known in the art and can be used in the chimeric proteins of this report.

[0058] The immobilizer radical can be a transmembrane domain found in another protein and allow the chimeric protein to present a transmembrane domain. In such an embodiment, the immobilizer radical can be derived from the FLO1 protein (presenting, for example, the amino acid sequence of SEQ ID NO: 10, a variant thereof or a fragment thereof, or be encoded by the nucleic acid sequence of SEQ ID NO: 9).

[0059] In yet another example, the amino acid immobilization radical can be modified after translation to include a glycosylphosphatidylinositol (GPI) anchor and allow the chimeric protein to present a GPI anchor. GPI anchors are glycolipids attached to the terminus of a protein (and, in some embodiments, to the carboxyl terminus of a protein) that allow the protein to anchor to the cytoplasmic membrane of a cell membrane. Amino acid immobilization radicals capable of providing a GPI anchor include, but are not limited to, those associated with / derived from an SED1 protein (presenting, for example, the sequence of Petition 870260074630, dated 07 / 27 / 2026, page 37 / 103 34 / 96 amino acids of SEQ ID NO: 12, a variant thereof or a fragment thereof, or encoded by the nucleic acid sequence of SEQ ID NO: 11), a TIR1 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 14, a variant thereof or a fragment thereof, or encoded by the nucleic acid sequence of SEQ ID NO: 13), a CWP2 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 16, a variant thereof or a fragment thereof, or encoded by the nucleic acid sequence of SEQ ID NO: 15), a CCW12 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 18 or 84, a variant thereof or a fragment thereof, or encoded by the nucleic acid sequence of SEQ ID NO: 17), an SPI1 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 20 or 74, a variant of these or a fragment of these or be encoded by the nucleic acid sequence of SEQ ID NO: 19),a PST1 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 22, a variant thereof, or a fragment thereof, or being encoded by the nucleic acid sequence of SEQ ID NO: 21) or a combination of an AGA1 protein and an AGA2 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 24, a variant thereof, or a fragment thereof, or being encoded by the nucleic acid sequence of SEQ ID NO: 23, or featuring, for example, the amino acid sequence of SEQ ID NO: 26, a variant thereof, or a fragment thereof, or being encoded by the nucleic acid sequence of SEQ ID NO: 25). In one embodiment, the immobilizing radical provides a GPI anchor and, in yet another embodiment, the immobilizing radical is derived from the SPI1 protein (featuring, for example, the amino acid sequence of SEQ ID NO: 20 or 74, a variant thereof or a fragment thereof, or being encoded by the nucleic acid sequence of SEQ ID NO: 19) or from, Petition 870260074630, dated 07 / 27 / 2026, p. 38 / 103 35 / 96 CCW12 protein (presenting, for example, the amino acid sequence of SEQ ID NO: 18 or 84, a variant thereof or a fragment thereof, or being encoded by the nucleic acid sequence of SEQ ID NO: 17).

[0060] In one embodiment, the immobilizer radical is a fragment of the SPI1 protein that has retained its ability to localize to the cell membrane. The SPI1 protein fragment comprises fewer than 129 consecutive amino acid residues from the amino acid sequence of SEQ ID NO: 74. For example, the SPI1 protein immobilizer radical fragment may comprise at least 10, 20, 21, 30, 40, 50, 51, 60, 70, 80, 81, 90, 100, 110, 111, or 120 consecutive amino acid residues from the amino acid sequence of SEQ ID NO: 74. In yet another embodiment, the SPI1 protein immobilizer radical fragment may comprise or consist essentially of the amino acid sequence presented in any of the SEQ ID NOs: 76, 78, 80, or 82.

[0061] In another embodiment, the immobilizing radical is a fragment of a CCW12 protein that has retained its ability to localize to the cell membrane. The CCW12 protein fragment comprises fewer than 112 consecutive amino acid residues from the amino acid sequence of SEQ ID NO: 84. For example, the CCW12 protein immobilizing radical fragment may comprise at least 10, 20, 24, 30, 40, 49, 50, 60, 70, 74, 80, 90, 99, 100, or 110 consecutive amino acid residues from the amino acid sequence of SEQ ID NO: 84. In yet another embodiment, the CCW12 protein immobilizing radical fragment may comprise or consist essentially of the amino acid sequence presented in any of the SEQ ID NOs: 86, 88, 90, or 92. Petition 870260074630, dated 07 / 27 / 2026, p. 39 / 103 36 / 96

[0062] The amino acid immobilizer radical may be a variant of a known / native amino acid immobilizer radical, for example, a variant of the amino acid immobilizer radical having the amino acid sequence SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 74, 76, 78, 80, 84, 82, 86, 88, 90 or 92. A variant comprises at least one different amino acid when compared with the amino acid sequence of the native amino acid immobilizer radical. As used herein, a variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the amino acid immobilizer radical (e.g., location on the outer face and anchoring of the heterologous enzyme to food and / or feed in the cytoplasmic membrane).A substitution, insertion, or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the amino acid immobilizer radical (e.g., location on the outer face and anchoring of the heterologous enzyme for food and / or feed in the cytoplasmic membrane). For example, the overall charge, structure, or hydrophilic-hydrophobic properties of the protein may be altered without adversely affecting a biological activity. Similarly, the amino acid sequence may be altered, for example, to make the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the amino acid immobilizer radical. The amino acid immobilizer radical variants exhibit at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid immobilizer radicals described herein.The term "percent identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Petition 870260074630, dated 07 / 27 / 2026, page 40 / 103 37 / 96 The level of identity can be conventionally determined using known computer programs. Identity can be easily calculated by known methods, including, but not limited to, those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods for identity determination are designed to provide the best match between the tested sequences. The methods for determining identity and similarity are codified in publicly available computer programs.Sequence alignments and percentile identity calculations can be performed using the Megalign program from the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The multiple alignments of the sequences described here were performed using the Clustal alignment method (Higgins and Sharp (1989) CABIOS. 5:151-153) with the standard parameters (GAP PENALTY=10, GAP LENGTH PENALTY ALT Y= 10). The standard parameters for pairwise alignments using the Clustal method were KTUPLB 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0063] The variants of amino acid immobilization radicals described herein may be (i) one in which one or more of the amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be encoded by the genetic code, or (ii) one in which one or more of the. Petition 870260074630, dated 07 / 27 / 2026, p. 41 / 103 38 / 96 amino acid residues include a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) one in which additional amino acids are fused to the mature polypeptide for polypeptide purification. An amino acid immobilizer “variant” can be a conservative variant or an allelic variant.

[0064] The amino acid immobilizer radical may be a fragment of a known / native amino acid immobilizer radical or a fragment of a variant of a known / native amino acid immobilizer radical (such as, for example, a fragment of the amino acid immobilizer radical having the amino acid sequence SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 74, 76, 78, 80, 82, 84, 86, 88, 90 or 92 or a variant thereof). The amino acid immobilizer radical “fragments” contain at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive amino acids from the amino acid immobilizer radical. A fragment comprises at least one fewer amino acid residue when compared to the amino acid sequence of the known / native amino acid immobilizer radical, and still possesses the biological activity of the full-length amino acid immobilizer radical (e.g., location in the cell wall).

[0065] In embodiments where an amino acid immobilization radical is desirable, the heterologous enzyme for food and / or feed may be provided as a chimeric protein expressed by the recombinant yeast host cell and have one of the following formulas (provided in the orientation from the amino terminal (NH2) to the carboxyl terminal (COOH)). FFE - L - TT (I) or TT - L - FFE (II) Petition 870260074630, dated 07 / 27 / 2026, p. 42 / 103 39 / 96

[0066] In both of these formulas, the residue “FFE” refers to the heterologous enzyme radical for food and / or feed, the residue “L” refers to the presence of an optional ligand, while the residue “TT” refers to an amino acid immobilization radical. In chimeric proteins of formula (I), the amino terminal of the amino acid immobilizer is located (directly or indirectly) at the carboxyl terminal (COOH or C) of the heterologous enzyme radical for food and / or feed. In chimeric proteins of formula (II), the carboxyl terminal of the amino acid immobilizer is located (directly or indirectly) at the amino terminal (NH2 or N) of the heterologous enzyme radical for food and / or feed.

[0067] In yet another embodiment, in the chimeric proteins of formula (I) and (II), the enzyme for food and / or feed may be a cooking enzyme. In such embodiments, the chimeric protein may have one of the following formulas (provided with the orientation of the amino terminal (NH2) to the carboxyl terminal (COOH)): BE - L - TT (Ia) or TT - L - BE (IIa)

[0068] In both of these formulas, the residue “BE” refers to the heterologous cooking enzyme radical, the residue “L” refers to the presence of an optional ligand, while the residue “TT” refers to an amino acid immobilization radical. In chimeric proteins of formula (Ia), the amino acid immobilizer is located (directly or indirectly) at the carboxyl terminal (COOH or C) of the heterologous cooking enzyme radical. In chimeric proteins of formula (IIa), the amino acid immobilizer is located (directly or indirectly) at the amino terminal (NH2 or N) of the heterologous enzyme radical for food and / or feed.

[0069] When the amino acid ligand (L) is absent, the amino acid immobilizing radical is directly associated with the heterologous enzyme for food and / or feed (or with the heterologous enzyme of Petition 870260074630, dated 07 / 27 / 2026, p. 43 / 103 40 / 96 cooking). In the chimeras of formulas (I) and (Ia), this means that the carboxyl terminal of the heterologous enzyme radical for food and / or feed (or the carboxyl terminal of the heterologous cooking enzyme radical) is directly associated (with an amide linkage) to the amino terminal of the amino acid immobilizer radical. In the chimeras of formulas (II) and (IIa), this means that the carboxyl terminal of the amino acid immobilizer radical is directly associated (with an amide linkage) to the amino terminal of the heterologous enzyme for food and / or feed (or the heterologous cooking enzyme).

[0070] In some embodiments, the presence of an amino acid linker (L) is desirable either to provide, for example, some flexibility between the heterologous enzyme radical for food and / or feed and the amino acid immobilizing radical or to facilitate the construction of the heterologous nucleic acid molecule. As used in this report, the “amino acid linker” or “L” refers to a stretching of one or more amino acids that separates the heterologous enzyme radical FFE or BE and the amino acid immobilizing radical TT (e.g., indirectly linking the heterologous enzyme for food and / or feed to the amino acid immobilizing radical TT). It is preferred that the amino acid linker be neutral, e.g., not interfering with the biological (enzymatic) activity of the heterologous enzyme for food and / or feed nor with the biological (cell-binding) activity of the amino acid immobilizing radical.In some embodiments, the amino acid ligand L can increase the biological activity of the heterologous enzyme radical for food and / or feed and / or the amino acid immobilization radical.

[0071] In cases where the ligand (L) is present in the chimeras of formulas (I) and (Ia), its amino terminus is associated (with an amide linkage) with the carboxyl terminus of the heterologous enzyme radical to Petition 870260074630, dated 07 / 27 / 2026, p. 44 / 103 41 / 96 food and / or feed and its carboxyl end is associated (with a starch linkage) to the amino end of the amino acid immobilization radical. In cases where the linker (L) is present in the chimeras of formulas (II) and (IIa), its amino end is associated (with a starch linkage) to the carboxyl end of the amino acid immobilization radical and its carboxyl end is associated (with a starch linkage) to the amino end of the heterologous enzyme radical for food and / or feed.

[0072] There are several amino acid linkers which include, without limitation, (G)n, (GS)n; (GGS)n; (GGGS)n; (GGGGS)n; (GGSG)n; (GSAT)n, where n = is an integer between 1 and 8 (or more). In one embodiment, the amino acid linker L is (GGGGS)n (also referred to as G4S) and, in still further embodiments, the amino acid linker L comprises more than one G4S motif (SEQ ID NO: 41). For example, the amino acid linker L may be (G4S)3 and have the amino acid sequence of SEQ ID NO: 93. In another example, the amino acid linker L may be (G)8 and have the amino acid sequence of SEQ ID NO: 94. In yet another example, the amino acid linker L may be (G4S)8 and have the amino acid sequence of SEQ ID NO: 95.

[0073] The amino acid linker may also be, in some embodiments, GSAGSAAGSGEF (SEQ ID NO: 96).

[0074] Additionally, there are amino acid linkers that include, without limitation, (EAAK)n and (EAAAK)n, where n = is an integer between 1 and 8 (or more). In some embodiments, one or more (EAAK)n / (EAAAK)n motifs may be separated by one or more additional amino acid residues. In one embodiment, the amino acid linker comprises one or more EA2K (SEQ ID NO: 100) or EA3K (SEQ ID NO: 101) motifs. In one embodiment, the amino acid linker may be (EAAK)3 and have the amino acid sequence of SEQ ID Petition 870260074630, dated 07 / 27 / 2026, p. 45 / 103 42 / 96 NO: 97. In another embodiment, the amino acid ligand can be (A(EAAAK)4ALEA(EAAAK)4A) and present the amino acid sequence of SEQ ID NO: 99.

[0075] Additional amino acid linkers include those featuring one or more (AP)n motifs where n = is an integer between 1 and 10 (or more). In one embodiment, the linker is (AP)io and features the amino acid sequence SEQ ID NO: 98.

[0076] In some embodiments, the linker also includes one or more HA tags (SEQ ID NO: 53). Tools for preparing recombinant yeast host cells

[0077] In order to prepare recombinant yeast host cells, heterologous nucleic acid molecules (also referred to as expression cassettes) are obtained in vitro and introduced into the yeast host cell in order to allow recombinant expression of the heterologous enzyme for food and / or feed.

[0078] The heterologous nucleic acid molecules of this report comprise a coding region for the heterologous polypeptide, for example, the heterologous enzyme for food and / or feed or a chimeric protein comprising it. A “coding region” of DNA or RNA is a DNA or RNA molecule (preferably a DNA molecule) that is transcribed and / or translated into a heterologous enzyme for food and / or feed in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. “Suitable regulatory regions” refers to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, processing or stability of RNA, or the translation of the region. Petition 870260074630, dated 07 / 27 / 2026, page 46 / 103 43 / 96 associated coding. Regulatory regions may include promoters, translation leader sequences, RNA processing site, effector binding site, and stem-loop structure. The boundaries of the coding region are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. A coding region may include, but is not limited to, prokaryotic regions, mRNA cDNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and a transcription termination sequence will usually be located 3' into the coding region. In one embodiment, the coding region may be referred to as an open reading frame.An "open reading frame" is abbreviated as ORF and stands for a length of nucleic acid, whether DNA, cDNA, or RNA, that comprises a translation initiation signal or start codon, such as an ATG or AUG, and a stop codon and can potentially be translated into a polypeptide sequence.

[0079] The heterologous nucleic acid molecules described herein may comprise transcription and / or translation control regions. “Transcription and / or translation control regions” are regulatory regions of DNA, such as promoters, amplifiers, terminators, and the like, that provide for the expression of a coding region in a host cell. In eukaryotic cells, polyadenylation signals are control regions.

[0080] In some embodiments, the heterologous nucleic acid molecules in this report include a promoter as well as a coding sequence for a heterologous enzyme for food and / or feed (including chimeric proteins comprising them). The heterologous nucleic acid sequence may also include a terminator. In Petition 870260074630, dated 07 / 27 / 2026, p. 47 / 103 44 / 96 heterologous nucleic acid molecules in this report, the promoter and terminator (when present) are operationally linked to the nucleic acid coding sequence of the heterologous enzyme for food and / or feed (including chimeric proteins comprising them), for example, controlling the expression and termination of the expression of the nucleic acid sequence of the heterologous enzyme for food and / or feed (including chimeric proteins comprising them). The heterologous nucleic acid molecules in this report may also include a nucleic acid encoding a signaling peptide, for example, a short peptide sequence to export the heterologous enzyme for food and / or feed out of the host cell.When present, the nucleic acid sequence encoding the signaling peptide is located directly upstream and is framed with the nucleic acid sequence encoding the heterologous enzyme for food and / or feed (including chimeric proteins comprising them).

[0081] In the heterologous nucleic acid molecule described herein, the promoter and the nucleic acid molecule encoding the heterologous enzyme for food and / or feed (including chimeric proteins comprising them) are operationally linked to each other. In the context of this report, the terms “operationally linked” or “operationally associated” refer to the fact that the promoter is physically associated with the nucleic acid molecule encoding the heterologous polypeptide in a manner that allows, under certain conditions, the expression of the heterologous protein from the nucleic acid molecule. In one embodiment, the promoter may be located upstream (5') of the nucleic acid sequence encoding the heterologous protein. In yet another embodiment, the promoter may be located downstream (3') of the nucleic acid sequence encoding Petition 870260074630, dated 07 / 27 / 2026, p. 48 / 103 45 / 96 for the heterologous protein. In the context of this report, one or more promoters may be included in the heterologous nucleic acid molecule. When more than one promoter is included in the heterologous nucleic acid molecule, each of the promoters is operationally linked to the nucleic acid sequence that codes for the heterologous protein. The promoters may be located, in view of the nucleic acid molecule that codes for the heterologous protein, upstream, downstream, as well as both upstream and downstream.

[0082] “Promoter” refers to a fragment of DNA capable of controlling the expression of a coding sequence or functional RNA. The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) from the heterologous nucleic acid molecule described herein. Expression may also refer to the translation of mRNA into a polypeptide. Promoters may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise segments of synthetic DNA. It should be understood by those skilled in the art that different promoters may direct expression at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cells most of the time at a substantially similar level are commonly called “constitutive promoters.”Promoters that cause a gene to be expressed during the propagation phase of a yeast cell are referred to here as "propagation promoters." Propagation promoters include both constitutive and inducible promoters, such as, for example, glucose-regulated promoters, molasses-regulated promoters, stress-response promoters (including osmotic stress response promoters), and so on. Petition 870260074630, dated 07 / 27 / 2026, page 49 / 103 46 / 96 aerobic regulated promoters. In the context of this report, it is important that the selected promoter allows the expression of the heterologous nucleic acid molecule during the propagation phase of the recombinant yeast host cell in order to allow a sufficient amount of the heterologous enzyme associated with the food and / or feed cell to be expressed. It is further recognized that since in most cases the exact boundaries of the regulatory sequences have not been completely defined, DNA fragments of different lengths may exhibit identical promoter activity. A promoter is generally linked at its 3' end by the transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above the base.Within the promoter, the transcription start site will be found (conveniently defined, for example, by mapping with S1 nuclease), as well as protein-binding domains (consensus sequences) responsible for polymerase binding.

[0083] The promoter can be native to or heterologous to the nucleic acid molecule encoding the heterologous polypeptide. The promoter can be heterologous or derived from a strain that is of the same genus or species as the recombinant host cell. In one embodiment, the promoter is derived from the same genus or species as the yeast host cell, and the heterologous polypeptide is derived from a different genus than the host cell. The promoter can be a single promoter or a combination of different promoters.

[0084] In this report, promoters that allow or favor the expression of heterologous proteins during the propagation phase of recombinant yeast host cells are preferred. Yeasts that are facultative anaerobes are able to Petition 870260074630, dated 07 / 27 / 2026, page 50 / 103 47 / 96 Respiratory reproduction under aerobic conditions and fermentative reproduction under anaerobic conditions. In many commercial applications, yeasts are propagated under aerobic conditions in order to maximize the conversion of a substrate into biomass. Optionally, the biomass can be used in a subsequent fermentation under anaerobic conditions to produce a desired metabolite. In the context of this report, it is important that the promoter or combination of promoters present in the heterologous nucleic acid be able to allow the expression of the heterologous enzyme for food and / or feed or its corresponding chimera during the propagation phase of the recombinant yeast host cell. This will allow the accumulation of the heterologous enzyme for food and / or feed associated with the recombinant yeast host cell before fermentation (if any).In some embodiments, the promoter allows expression of the heterologous enzyme for food and / or feed or its corresponding chimera during propagation, but not during fermentation (if any) of the recombinant yeast host cell.

[0085] Promoters can be native or heterologous to the heterologous gene encoding the heterologous protein. Promoters that can be included in the heterologous nucleic acid molecule can be constitutive or inducible promoters (such as those described in PerezTorrado et al., 2005). Inducible promoters include, but are not limited to, glucose-regulated promoters (e.g., the hxt7 gene promoter (referred to as hxt7p) and presenting the nucleic acid sequence SEQ ID NO: 30, a functional variant or a functional fragment thereof; the cttl gene promoter (referred to as ctt1p) and presenting the nucleic acid sequence SEQ ID NO: 60, a functional variant or a functional fragment thereof; the glol gene promoter (referred to as glo1p) and presenting the nucleic acid sequence Petition 870260074630, dated 07 / 27 / 2026, p. 51 / 103 48 / 96 nucleic acids of SEQ ID NO: 59, a functional variant or a functional fragment thereof; the promoter of the ygpl gene (referred to as ygp1p) and presenting the nucleic acid sequence of SEQ ID NO: 61, a functional variant or a functional fragment thereof; the promoter of the gsy2 gene (referred to as gsy2p) and presenting the nucleic acid sequence of SEQ ID NO: 53, a functional variant or a functional fragment thereof), molasses-regulated promoters (e.g., the promoter of the moll gene (referred to as mol1p) described in Praekelt et al., 1992 or presenting the nucleic acid sequence of SEQ ID NO: 64, a functional variant or a functional fragment thereof), heat shock-regulated promoters (e.g., the promoter of the glol gene (referred to as glo1p) and presenting the nucleic acid sequence of SEQ ID NO: 59, a functional variant or a functional fragment thereof;the promoter of the stil gene (referred to as sti1p) and presenting the nucleic acid sequence of SEQ ID NO: 56, a functional variant or a functional fragment thereof; the promoter of the ygpl gene (referred to as ygp1p) and presenting the nucleic acid sequence of SEQ ID NO: 61, a functional variant or a functional fragment thereof; the promoter of the gsy2 gene (referred to as gsy2p) and presenting the nucleic acid sequence SEQ ID NO: 53, a functional variant or a functional fragment thereof), oxidative stress response promoters (e.g., the promoter of the cupl gene (referred to as cup1p) and presenting the nucleic acid sequence SEQ ID NO: 58, a functional variant or a functional fragment thereof; the promoter of the cttl gene (referred to as ctt1p) and presenting the nucleic acid sequence of SEQ ID NO: 60, a functional variant or a functional fragment thereof;the promoter of the trx2 gene (referred to as trx2p) and presenting the nucleic acid sequence of SEQ ID NO: 55, a functional variant or a functional fragment thereof; the gene promoter; Petition 870260074630, dated 07 / 27 / 2026, p. 52 / 103 49 / 96 gpdl (referred to as gpd1p) and presenting the nucleic acid sequence of SEQ ID NO: 57, a functional variant or a functional fragment thereof; the hsp12 gene promoter (referred to as hsp12p) and presenting the nucleic acid sequence of SEQ ID NO: 63, a functional variant or a functional fragment thereof), osmotic stress response promoters (e.g., the cttl gene promoter (referred to as ctt1p) and presenting the nucleic acid sequence of SEQ ID NO: 60, a functional variant or a functional fragment thereof; the glol gene promoter (referred to as glo1p) and presenting the nucleic acid sequence of SEQ ID NO: 59, a functional variant or a functional fragment thereof; the gpd1 gene promoter (referred to as gpd1p) and presenting the nucleic acid sequence of SEQ ID NO: 57, a functional variant or a functional fragment thereof;the promoter of the ygpl gene (referred to as ygp1p) and presenting the nucleic acid sequence of SEQ ID NO: 61, a functional variant or a functional fragment thereof) and nitrogen-regulated promoters (e.g., the promoter of the ygp1 gene (referred to as ygp1p) and presenting the nucleic acid sequence of SEQ ID NO: 61, a functional variant or a functional fragment thereof).

[0086] Promoters that may be included in the heterologous nucleic acid molecule of this report include, without limitation, the promoter of the tdh1 gene (referred to as tdh1p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 27, a functional variant or a functional fragment thereof), of the hor7 gene (referred to as hor7p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 28, a functional variant or a functional fragment thereof), of the hsp150 gene (referred to as hsp150p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 29, a functional variant or a functional fragment thereof), of Petition 870260074630, dated 07 / 27 / 2026, p. 53 / 103 50 / 96 gene hxt7 (referred to as hxt7p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 30, a functional variant or a functional fragment thereof), of the gene gpml (referred to as gpm1p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 31, a functional variant or a functional fragment thereof), of the gene pgkl (referred to as pgk1p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 32, a functional variant or a functional fragment thereof) and / or of the gene stll (referred to as stl1p and featuring, for example, the nucleic acid sequence of SEQ ID NO: 33, a functional variant or a functional fragment thereof). In one embodiment, the promoter is or comprises the gene tdh1p and / or the gene hor7p. In yet another embodiment, the promoter comprises or consists essentially of the gene tdh1p and the gene hor7p. In an additional realization, the promoter is thd1p.

[0087] One or more promoters can be used to enable the expression of each heterologous polypeptide in the recombinant yeast host cell. In the context of this report, the term “functional fragment of a promoter” when used in combination with a promoter refers to a shorter nucleic acid sequence than that of the native promoter that retains the ability to control the expression of the nucleic acid sequence encoding the heterologous enzyme for food and / or feed or its chimera during the propagation phase of recombinant yeast host cells. Usually, functional fragments are either a 5' truncation and / or a 3' truncation of one or more nucleic acid residues of the native promoter nucleic acid sequence.

[0088] In some embodiments, nucleic acid molecules include one or a combination of terminator sequences in order to terminate heterologous enzyme translation into food and / or Petition 870260074630, dated 07 / 27 / 2026, p. 54 / 103 51 / 96 ration (or the chimeric protein comprising it). The terminator may be native to or heterologous to the nucleic acid sequence encoding the heterologous enzyme for food and / or ration or its corresponding chimera. In some embodiments, one or more terminators may be used. In some embodiments, the terminator comprises that of the ditl gene (referred to as dit1t and may present, for example, the nucleic acid sequence of SEQ ID NO: 34, a functional variant or a functional fragment thereof), the idpl gene (referred to as idp1t and may present, for example, the nucleic acid sequence of SEQ ID NO: 35, a functional variant or a functional fragment thereof), the gpml gene (referred to as gpm1t and may present, for example, the nucleic acid sequence of SEQ ID NO: 36, a functional variant or a functional fragment thereof), the pmal gene (referred to as pma1t and may present, for example, the nucleic acid sequence of SEQ ID NO: 37,a functional variant or a functional fragment thereof), of the tdh3 gene (referred to as tdh3t and may present, for example, the nucleic acid sequence of SEQ ID NO: 38, a functional variant or a functional fragment thereof), of the hxt2 gene (referred to as hxt2t and may present, for example, the nucleic acid sequence of SEQ ID NO: 39, a functional variant or a functional fragment thereof), of the adh3 gene (referred to as adh3t and may present, for example, the nucleic acid sequence of SEQ ID NO: 70, a functional variant or a functional fragment thereof) and / or of the ira2 gene (referred to as ira2t and may present, for example, the nucleic acid sequence of SEQ ID NO: 40, a functional variant or a functional fragment thereof). In one embodiment, the terminator is derived from the ditl gene (and may feature, for example, the nucleic acid sequence of SEQ ID NO: 34, a functional variant, or a functional fragment thereof). In another embodiment,the terminator comprises or is derived from the adh3 gene (and, Petition 870260074630, dated 07 / 27 / 2026, p. 55 / 103 52 / 96 may present, for example, the nucleic acid sequence of SEQ ID NO: 70, a functional variant, or a functional fragment thereof. In the context of this report, the expression "functional variant of a terminator" refers to a nucleic acid sequence that has been substituted at least one nucleic acid position when compared to the native terminator, which retains the ability to terminate the expression of the nucleic acid sequence encoding the heterologous protein or its corresponding chimera. In the context of this report, the expression "functional fragment of a terminator" refers to a nucleic acid sequence shorter than that of the native terminator, which retains the ability to terminate the expression of the nucleic acid sequence encoding the heterologous protein or its corresponding chimera.

[0089] In some embodiments, heterologous nucleic acid molecules include the coding sequence for one or a combination of signal peptide sequences that allow the export of the heterologous protein (or the chimeric protein comprising it) out of the yeast host cell wall. The signal peptide sequence may be simply added to the nucleic acid molecule (usually framed with the sequence encoding the heterologous food and / or feed enzyme) or replace the signal peptide sequence already present in the heterologous food and / or feed enzyme. The signal peptide sequence may be native to or heterologous to the nucleic acid sequence encoding the heterologous food and / or feed enzyme or its corresponding chimera. In some embodiments, one or more signal sequences may be merged.In some embodiments, the signaling sequence is from the gene that encodes the invertase protein (and may present, for example, the amino acid sequence of SEQ ID NO: 68, a variant thereof, or a... Petition 870260074630, dated 07 / 27 / 2026, p. 56 / 103 53 / 96 fragment thereof), the AGA2 protein (and may present, for example, the amino acid sequence of SEQ ID NO: 69, a variant thereof, or a fragment thereof) or the fungal amylase protein (and may present, for example, the amino acid sequence of SEQ ID NO: 107, a variant thereof, or a fragment thereof). In the context of this report, the expression “functional variant of a signal sequence” refers to a nucleic acid sequence that has been substituted at least one nucleic acid position when compared to the native signal sequence that retains the ability to direct the expression of the heterologous enzyme to food and / or feed or its corresponding chimera outside the cell.In the context of this report, the expression "functional fragment of a signaling sequence" refers to a shorter nucleic acid sequence than the native signaling sequence that retains the ability to direct the expression of the heterologous enzyme to food and / or feed or its corresponding chimera outside the cell.

[0090] In some embodiments where it is desirable to express the heterologous enzyme for food and / or feed within the recombinant yeast host cell (intracellularly), the heterologous nucleic acid molecule may exclude the coding portion for the signal peptide sequence found in the native gene encoding the enzyme for food and / or feed.

[0091] The heterologous nucleic acid molecule encoding the heterologous enzyme for food and / or feed, chimera, variant or fragment thereof, may be integrated into the genome of the yeast host cell. The term “integrated” as used herein refers to genetic elements that are placed, by means of molecular biology techniques, into the genome of a host cell. For example, genetic elements may be placed into the chromosomes of the host cell in Petition 870260074630, dated 07 / 27 / 2026, page 57 / 103 54 / 96 opposition to a vector such as a plasmid contained in the host cell. Methods for integrating genetic elements into the genome of a host cell are well known technically and include homologous recombination. The heterologous nucleic acid molecule may be present in one or more copies in the yeast host cell genome. Alternatively, the heterologous nucleic acid molecule may be genome-independent of the yeast genome. In such an embodiment, the nucleic acid molecule may be both stable and self-replicating.

[0092] This report also provides nucleic acid molecules for modifying the yeast host cell to allow the expression of heterologous feedstuff enzymes, chimeras, variants, or fragments thereof. The nucleic acid molecule may be DNA (such as complementary DNA, synthetic DNA, or genomic DNA) or RNA (including synthetic RNA) and may be provided in a single-stranded form (either sense or antisense strand) or a double-stranded form. The nucleic acid molecules contemplated may include alterations in coding regions, non-coding regions, or both. Examples are variant nucleic acid molecules containing alterations that produce silent substitutions, additions, or deletions, but which do not alter the properties or activities of the encoded feedstuff enzyme, chimeras, variants, or fragments.

[0093] In some embodiments, heterologous nucleic acid molecules that can be introduced into recombinant host cells are codon-optimized with respect to the recipient recombinant yeast host cell. As used herein, the term “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in Petition 870260074630, dated 07 / 27 / 2026, p. 58 / 103 55 / 96 cells of a given organism by replacing at least one, or more than one, codon with one or more codons that are most frequently used in the genes of that organism. In general, highly expressed genes in an organism are shifted to codons that are recognized by the most abundant tRNA species in that organism. A measure of this shift is the “codon adaptation index” or “CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those that occur most frequently in a reference set of highly expressed genes from an organism. The CAI of the codon-optimized heterologous nucleic acid molecule described here corresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, or about 1.0.

[0094] Heterologous nucleic acid molecules can be introduced into the yeast host cell using a vector. A “vector,” for example, a “plasmid,” “cosmid,” or “artificial chromosome” (such as, for example, a yeast artificial chromosome) refers to an extrachromosomal element and is usually in the form of a double-stranded DNA molecule. Such vectors can be autonomously replicating sequences, genome integration sequences, phages, or linear, circular, or supercoiled nucleotide sequences of single- or double-stranded DNA or RNA derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construct capable of introducing a promoter fragment and a DNA sequence of a selected gene along with the appropriate 3' untranslated sequence into a cell.

[0095] This report also provides nucleic acid molecules that are hybridizable to complementary nucleic acid molecules encoding heterologous polypeptides as well as variants or fragments. A nucleic acid molecule is “hybridizable” Petition 870260074630, dated 07 / 27 / 2026, page 59 / 103 56 / 96 to another nucleic acid molecule, such as cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to another nucleic acid molecule under appropriate temperature and ionic strength conditions of solution. The hybridization and washing conditions are well known and exemplified, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1. The temperature and ionic strength conditions determine the “rigor” of the hybridization. The rigor conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as duplicate genes, functional enzymes from closely related organisms.Post-hybridization washes determine the severity of the conditions. One set of conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min. For more stringent conditions, washes are performed at higher temperatures, identical to the above except for the temperature of the two final 30-min washes at 0.2X SSC, 0.5% SDS, which are increased to 60°C. Another set of highly stringent conditions uses two final washes at 0.1X SSC, 0.1% SDS at 65°C. An additional set of highly stringent conditions is defined by hybridization at 0.1X SSC, 0.1% SDS, 65°C and washing with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS. Petition 870260074630, dated 07 / 27 / 2026, pp. 60 / 103 57 / 96

[0096] Hybridization requires that two nucleic acid molecules contain complementary sequences, although depending on the rigor of the hybridization, base mismatches are possible. The appropriate rigor for nucleic acid hybridization depends on the length of the nucleic acids and the degree of complementation, variables well known in the technique. The greater the degree of similarity or homology between two nucleotide sequences, the higher the Tm value for nucleic acid hybrids presenting these sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations is reduced in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids longer than 100 nucleotides, equations have been derived for calculating Tm. For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of the mismatches becomes more important and the length of the oligonucleotide determines its specificity.In one embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably a length of at least 30 nucleotides. Furthermore, those skilled in the art will recognize that the temperature and salt concentration of the washing solution can be adjusted as needed according to factors such as the probe length. Processes for the propagation and formulation of recombinant yeast host cells.

[0097] This report allows the preparation of a yeast composition comprising the recombinant yeast host cell of this report. In some embodiments, the yeast composition can be used to reduce or eliminate the Petition 870260074630, dated 07 / 27 / 2026, pp. 61 / 103 58 / 96 requirement for supplementation in the food preparation process with exogenous (and purified / isolated) enzymes.

[0098] The process for preparing the yeast composition broadly comprises two steps: a first step of propagating the recombinant yeast host cell and a second step of formulating the yeast composition. As used in the context of this report, a “yeast composition” is a composition comprising the recombinant yeast host cell of this report which has been propagated. The yeast composition can be used, for example, in a subsequent fermentation (in order to provide the heterologous enzyme in situ during fermentation) or to prepare a food / feed product. In one embodiment, the recombinant yeast host cell is provided in an active or semi-active form in the yeast composition. For example, one embodiment of the yeast composition is a yeast cream made from the recombinant yeast host cell of this report.

[0099] The propagation step can be a continuous culture, a batch culture, or a fed-batch culture. In the propagation step, the recombinant yeast host cell is placed in a culture medium that can, in some embodiments, allow rapid growth. For example, the culture medium may comprise a carbon source (such as, for example, molasses, sucrose, glucose, dextrose syrup, ethanol, and / or corn steep liquor), a nitrogen source (such as, for example, ammonia), and a phosphorus source (such as, for example, phosphoric acid). The propagation step can be subdivided into two stages, an initial seeding stage and a subsequent large-scale propagation stage. During the propagation stage, it is possible to monitor and adjust the Petition 870260074630, dated 07 / 27 / 2026, pp. 62 / 103 59 / 96 temperature (usually around 32°C when the recombinant yeast host cell is of the species Saccharomyces cerevisiae), pH, and aeration conditions are adjusted to favor or optimize the division of the recombinant yeast host cell. For example, when batch feeding propagation is employed, high aeration and incremental carbohydrate addition can increase the biomass yield of the yeast composition.

[0100] In the formulation stage, the mixture obtained after propagation (comprising the recombinant yeast host cell(s)) can be modified. One of the advantages of the recombinant yeast host cells in this report is that the heterologous enzymatic activity for feed / feed is associated with the recombinant yeast host cell, which, for this reason, the concentration of biomass after propagation will also increase the amount / activity of the heterologous enzyme for feed / feed. In one embodiment for providing a yeast composition, at least one component of the mixture obtained after propagation is removed from the culture medium in order to provide the yeast composition. This component may be, without limitation, water, amino acids, peptides and proteins, nucleic acid residues and nucleic acid molecules, cell debris, fermentation products, etc.In one embodiment, the formulation step substantially involves isolating the propagated recombinant yeast host cells (e.g., biomass) from the culture medium components. As used in the context of this report, the expression "substantially in isolation" refers to the removal of most of the culture medium components from the propagated recombinant yeast host cells in order to provide the composition of... Petition 870260074630, dated 07 / 27 / 2026, pp. 63 / 103 60 / 96 yeast, the propagated recombinant yeast host cells can be centrifuged (and the resulting cell pellet comprising the propagated recombinant yeast host cells can optionally be washed), filtered and / or dried (optionally using a vacuum drying technique). The isolated recombinant yeast host cells can then be formulated into a yeast composition. The formulation step can, in some embodiments, preserve the viability (at least in part) of the recombinant yeast host cells. As such, the yeast composition can be provided in an active form or a semi-active form. The yeast composition can be provided in a liquid, semi-solid or dry form. In one embodiment, the yeast composition can be provided in the form of a yeast cream.

[0101] The yeast composition may additionally be modified into a yeast product. As used in the context of this report, a yeast product is a product obtained from the propagated recombinant yeast host cell comprising the heterologous enzyme for food and / or feed. The yeast product may be, for example, a yeast lysate (e.g., an autolysate), a yeast extract, a yeast fraction (e.g., yeast cell walls) and / or the heterologous enzyme for food and / or feed in a substantially isolated form.As used in the context of this report, the expression "substantial isolation / purification of heterologous enzyme for food and / or feed from lysed recombinant yeast host cells" refers to the removal of most components from the lysed recombinant yeast host cells of the heterologous enzyme for food and / or feed and providing it in an isolated / purified form. Petition 870260074630, dated 07 / 27 / 2026, pp. 64 / 103 61 / 96

[0102] Yeast compositions, as well as yeast products, can be provided as a food additive. As used in this report, the term "food additive" refers to a product used in human nutrition for the purpose of enhancing food quality or improving the production process. In such an embodiment, the yeast composition may also include, without limitation, a carrier (such as, for example, salt or wheat bran), a stabilizing agent and / or an oil. In a specific embodiment, the yeast composition may be provided as a live yeast composition (such as, for example, a yeast cream) suitable for downstream food preparation, as a deactivated yeast composition, as a yeast fraction and / or as a purified enzyme for food.In another specific embodiment, the yeast composition can be provided in a dry preparation (spray-dried, for example) suitable for downstream food preparation.

[0103] Yeast compositions can be provided as a feed additive. As used in this report, the term "feed additive" refers to a product used in animal nutrition for the purpose of increasing feed quality, the quality of animal-derived feed, and / or increasing animal performance and health (e.g., by providing greater digestibility of feed materials). In such an embodiment, the yeast composition may also include, without limitation, a carrier (such as, for example, salt or wheat bran), a stabilizing agent, and / or an oil. In a specific embodiment, the yeast composition may be provided as a live yeast composition (such as, for example, a yeast cream) suitable for downstream feed preparation, as a deactivated yeast composition, as a yeast fraction, and / or Petition 870260074630, dated 07 / 27 / 2026, pp. 65 / 103 62 / 96 a purified feed enzyme. In another specific embodiment, the yeast composition can be provided as a dry preparation (spray-dried, for example) suitable for downstream feed preparation. In one embodiment, the feed additive is added to the animal's diet to supplement it. Processes for the preparation of food and feed products.

[0104] The recombinant yeast host cell of this report was designed for use in the preparation of products for human consumption (food) or animal consumption (feed). This report, therefore, provides a process comprising the inclusion of the recombinant yeast host cell of this report in the food product or feed. In some embodiments, it may be advantageous to provide the recombinant yeast host cell of this report as a food additive or as a feed additive. In some embodiments, the process may also include fermentation of the product and / or cooking of the food product / feed. In cases where the process includes a fermentation step, the fermentation may be conducted (wholly or in part) in the presence of or by the recombinant yeast host cell described herein. The process of this report can be used to extend the shelf life of food products or feed.The enzymatic activity (associated with heterologous enzymes for food and / or feed, as well as chimeric proteins comprising them) of recombinant yeast cells can be measured before use and adjusted depending on the type of activity desired.

[0105] In one embodiment, food and feed products are cooked products. In such an embodiment, a recombinant yeast host cell expressing the enzyme associated with cooking is preferably used. Cooked products such as cooked products Petition 870260074630, dated 07 / 27 / 2026, pp. 66 / 103 63 / 96 leavened by yeast, can be fermented by the recombinant yeast host cell described herein. Yeast-leavened baked goods include, without limitation, bread, baked goods (including croissants), rolls, pita bread, tortillas, bagels, and pie or pizza crust and the like. When used during the preparation process of yeast-leavened products, recombinant yeast host cells may be the only fermenting organism added to the fermentable substrate. In other cases, recombinant yeast host cells may be mixed with non-recombinant yeasts (e.g., wild type) to provide the appropriate dose of heterologous baking enzyme activity.For example, a recombinant yeast host cell (which may be a recombinant Saccharomyces cerevisiae yeast host cell) can be combined in any proportion with a wild-type yeast host cell (which may be non-recombinant wild-type Saccharomyces cerevisiae). In one embodiment, the recombinant:wild-type ratio is between 1:100 and 100:1.

[0106] Amylolytic enzymes are of particular interest in the production of yeast-leavened baked goods because they promote the hydrolysis of starch (either in a crude or hydrolyzed form) and thus provide an energy source for fermenting yeasts to accelerate the fermentation process, increase CO2 production, increase ethanol production, and / or improve the organoleptic properties of the fermented product. Maltogenic amylases are, in particular, very useful in the bread-making process because they are known to extend shelf life while maintaining the softness and resilience of baked bread. Petition 870260074630, dated 07 / 27 / 2026, page 67 / 103 64 / 96

[0107] In another embodiment, the baked goods are not fermented by the recombinant yeast host cell described herein and are instead chemically leavened or unleavened. Chemically leavened and unleavened baked goods include, without limitation, cakes and pita bread.

[0108] In the process described herein, the recombinant yeast host cells of this report may be provided in an active form (e.g., liquid, compressed, or fluid-bed dried yeast), in a semi-active form (e.g., liquid, compressed, or fluid-bed dried), in an inactive form (e.g., drum-dried or spray-dried), as well as a mixture thereof. For example, the recombinant yeast host cells may be a combination of the active and semi-active or inactive form to provide the ratio and dose of the cooking enzyme required for the preparation of cooked products.

[0109] The present invention will now be more easily understood by reference to the following examples which are provided to illustrate the invention rather than to limit its scope. Petition 870260074630, dated 07 / 27 / 2026, pp. 68 / 103 65 / 96 EXAMPLE I - MATERIALS AND METHODS Table 1. Description of the yeast strains used in the examples. These strains were constructed with expression cassettes integrated into the FCY1 locus on each chromosome; the number of copies is provided in the table. The original base strain used for each strain is also provided in the table. Each integrated cassette included a copy of a heterologous enzyme, one or more promoters, and one or more terminators. In some cases, the signal peptide of the heterologous enzyme was replaced by another signal peptide as indicated in the table. When the heterologous enzyme is expressed in an immobilized form, the geometry of the immobilizer is provided (see definition of formulas I and II above), and the ligand as well as the immobilizer are provided. NA = not applicable. Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated by chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M2390 (Saccharomyces cerevisiae) None NANANANANANANANA 1Invertase = SEQ ID NO: 52, Aga2 = SEQ ID NO: 69, fungal amylase = SEQ ID NO: 1072HA = SEQ ID NO: 53; (G4S)2 = SEQ ID NO: 543Immobilizer Flo1 is a transmembrane domain located at the C-terminus = SEQ ID NO: 10; Immobilizer Sed1 is a GPI anchor located at the C-terminus = SEQ ID NO: 12; Immobilizer Tir1 is a fragment of the GPI mannoprotein located at the C-terminus = SEQ ID NO: 14; Immobilizer Cwp2 is a fragment of the GPI mannoprotein located at the C-terminus = SEQ ID NO: 16; Immobilizer Ccw12 is a fragment of the GPI mannoprotein located at the C-terminus = SEQ ID NO: 18; Spi1 immobilizer is a GPI anchor located at the C-terminus = SEQ ID NO: 20; Pst1 immobilizer is a GPI anchor = SEQ ID NO: 22; Aga1 / 2 immobilizer, Aga2 disulfide bond to Aga1; Aga1 has a GPI anchor, the enzyme is fused to Aga2 at the C-terminus = SEQ ID NO: 24; Aga1 / 2 immobilizer, Aga2 disulfide bond to Aga1; Aga1 has a GPI anchor, the enzyme is fused to Aga2 at the N-terminus = SEQ ID NO: 26. Petition 870260074630, dated 07 / 27 / 2026, pp. 69 / 103 66 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M 8498 Glucoamylase (SEQ ID NO: 29) M10474 1 TEF2p SED1t Secreted free Invertase None None M10074 Alpha-amylase (SEQ ID NO: 50) M10474 1 TEF2p SED1t Secreted free Invertase None None M10474 (Saccharomyces cerevisiae) None NANANANANANANANA M11312 Phytase (SEQ ID NO: 67) M2390 1 TEF2p ADH3t Secreted free Invertase NANA M12550 (Saccharomyces cerevisiae) None NANANANANANANANA M12548 (Saccharomyces boulardii) None NANANANANANANANA M12795 Phytase (SEQ ID NO: 67) M12550 1 TEF2p ADH3t Immobilized - Formula (II) Aga2 (G4S)2 Aga1 / 2 (Aga2 at the N-terminus of the enzyme) Petition 870260074630, dated 07 / 27 / 2026, pp. 70 / 103 67 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M12938 Phytase (SEQ ID NO: 67) M12550 1 TEF2p ADH3t Immobilized - Formula (II) Aga2 (G4S)2 Aga1 / 2 M12962 (Saccharomyces cerevisiae var diastaticus) None NANANANANANANANA M13819 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA / G4S Spi1 M13822 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Invertase None None M13979 Maltogenic alpha amylase (SEQ ID NO: 51) M10474 4 TDH1p / HOR7p DIT1t / IDP1t Immobilized - formula (I) Invertase (G4S)2 Spi1 Petition 870260074630, dated 07 / 27 / 2026, pp. 71 / 103 68 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M14244 Glucoamylase (SEQ ID NO: 29) M10474 1 TEF2p SED1t Immobilized - formula (I) Invertase HA / G4S Sed1 M14253 Alpha-amylase (SEQ ID NO: 50) M10474 1 TEF2p SED1t Immobilized - Formula (i) Invertase ha / g4s ligand Sed1 M14254 Alpha-amylase (SEQ ID NO: 50) M10474 1 TEF2p SED1t Immobilized - Formula (i) Invertase None Sed1 M14851 Maltogenic alpha-amylase (SEQ ID NO: 65) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Intracellular NANANA M15215 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 84 M15222 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 74 Petition 870260074630, dated 07 / 27 / 2026, pp. 72-103 69 / 96 Name | Heterologous enzyme expressed | Original base strain | Copies of the heterologous enzyme integrated per chromosome | Promoter | Terminator | Expression type | Signal peptide1 | Ligand2 | Immobilizer3 | M15532 | Maltogenic alpha-amylase (SEQ ID NO: 108) | M10474 | 2 TDH1p / HOR7p | DIT1t / IDP1t | Intracellular | NANANA | M15771 | Alpha-amylase (SEQ ID NO: 71) | M2390 | 1 TEF2p | ADH3t | Immobilized - Formula (i) | Invertase HA / (G4S)3 | SEQ ID NO: 78 | M15772 | Alpha-amylase (SEQ ID NO: 71) | M2390 | 1 TEF2p | ADH3t | Immobilized - Formula (i) | Invertase HA / (G4S)3 | SEQ ID NO: 82 | M15773 | Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 86 M15774 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 76 M15775 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 92 Petition 870260074630, dated 07 / 27 / 2026, pp. 73 / 103 70 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M15776 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 88 M15777 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 80 M15778 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 94 SEQ ID NO: 74 M15779 Alpha-amylase (SEQ M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 95 SEQ ID NO: 74 M15780 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 97 SEQ ID NO: 74 M15781 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 98 SEQ ID NO: 84 M15782 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ IDNO: 95 SEQ ID NO: 84 Petition 870260074630, dated 07 / 27 / 2026, pp. 74 / 103 71 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M15784 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 93 SEQ ID NO: 84 M15783 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 99 SEQ ID NO: 74 M15785 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 93 SEQ ID NO: 84 M15786 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 94 SEQ ID NO: 84 M15787 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 96 SEQ ID NO: 74 M15788 Alpha-amylase (SEQ ID NO: 71) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 98 SEQ ID NO: 74 M16221 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i)Invertase SEQ ID NO: 97 SEQ ID NO: 84 Petition 870260074630, dated 07 / 27 / 2026, pp. 75 / 103 72 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 M16222 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 99 SEQ ID NO: 84 M16251 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase HA / (G4S)3 SEQ ID NO: 90 M16252 Alpha-amylase (SEQ ID NO: 72) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase SEQ ID NO: 96 SEQ ID NO: 84 M16273 Glucose oxidase (SEQ ID NO: 103) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Intracellular NANANA M16540 Fungal amylase (SEQ ID NO: 105) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Fungal amylase NANA M16772 Fungal amylase (SEQ ID NO: 105) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Invertase NANA M16780 Glucose oxidase (SEQ ID NO: 103) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Invertase NANA Petition 870260074630, dated 07 / 27 / 2026, pp. 76 / 103 73 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 T2633 Phytase (SEQ ID NO: 66) M12548 1 TEF2p ADH3t Secreted free Invertase NANA T2634 Phytase (SEQ ID NO: 66) M12548 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Sed1 T2635 Phytase (SEQ ID NO: 66) M12548 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Tir1 T2636 Phytase (SEQ ID NO: 66) M12548 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Cwp2 T2637 Phytase (SEQ ID NO: 66) M12548 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Spi1 T2638 Phytase (SEQ ID NO: 66) M12548 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Pst1 T2705 Phytase (SEQ ID NO: 67) M2390 1 TEF2p ADH3t Immobilized - Formula (II) Aga2 (G4S)2 Aga 1 / 2 Petition 870260074630, dated 07 / 27 / 2026, pp. 77 / 103 74 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 T2706 Phytase (SEQ ID NO: 67) M2390 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Aga 1 / 2 T2816 Phytase (SEQ ID NO: 67) M12550 1 TEF2p ADH3t Immobilized - Formula (i) Invertase (G4S)2 Sed1 T2986 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA / (G4S)2 Flo1 T2987 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA / (G4S)2 Sed1 T2988 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA / (G4S)2 Tir1 Petition 870260074630, dated 07 / 27 / 2026, pp. 78 / 103 75 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated per chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 T2989 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA-(G4S)2 Cwp2 T2990 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA / (G4S)2 Ccw1 T2991 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase HA / (G4S)2 Spi1 T2994 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Invertase None None T3892 Maltogenic alpha-amylase (SEQ ID NO: 65) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Intracellular NANANA Petition 870260074630, dated 07 / 27 / 2026, pp. 79 / 103 76 / 96 Name | Heterologous enzyme expressed | Original base strain | Copies of the heterologous enzyme integrated per chromosome | Promoter | Terminator | Expression type | Signal peptide1 | Ligand2 | Immobilizer3 | T4328 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized Invertase (G4S)2 Spi1 | T4329 Maltogenic alpha-amylase (SEQ ID NO: 51) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Invertase NANA | T4330 Maltogenic alpha-amylase (SEQ ID NO: 65) M10474 2 TDH1p / HOR7p DIT1t / IDP1t Intracellular NANANA | T4336 Maltogenic alpha-amylase (SEQ ID NO: 51) M12962 2 TDH1p / HOR7p DIT1t / IDP1t Immobilized - Formula (i) Invertase (G4S)2 Spi1 T4337 Maltogenic alpha amylase (SEQ ID NO: 51) M12962 2 TDH1p / HOR7p DIT1t / IDP1t Secreted free Invertase NANA Petition 870260074630, dated 07 / 27 / 2026, pp. 80 / 103 77 / 96 Name Heterologous enzyme expressed Original base strain Copies of the heterologous enzyme integrated by chromosome Promoter Terminator Expression type Signal peptide1 Ligand2 Immobilizer3 T4338 Maltogenic alpha amylase (SEQ ID NO: 65) M12962 2 TDH1p / HOR7p DIT1t / IDP1t Intracellular NANANA Petition 870260074630, dated 07 / 27 / 2026, pp. 81 / 103 78 / 96

[0110] Cell growth. Cells were grown overnight in 5 mL of YPD (10 g / L yeast extract, 20 g / L bacteriological peptone, 40 g / L glucose). One (1) mL of the total culture was collected and the cells were pelleted by centrifugation. The cell-free supernatant was removed and stored for later analysis. The cell pellet was washed once and resuspended in deionized water.

[0111] Batch-fed fermentation seeding. A mixture of molasses was prepared (85% beet molasses, 15% cane molasses), diluted, and its pH was adjusted to pH 5.2 with sulfuric acid. The pure culture inoculum was diluted in sterile water and added to the molasses mixture with zinc sulfate, magnesium sulfate, biotin, thiamine, calcium pantothenate, and phosphoric acid. The yeasts were propagated at 32°C, pH 4.5, for 24 hours. The resulting propagated yeasts were centrifuged, and the propagation wort was washed using a laboratory Alfa Laval separator to obtain approximately 20% yeast solids. The yeast solids were treated with sulfuric acid, and the pH was subsequently adjusted with sodium hydroxide to provide the yeast cream.

[0112] Commercial batch fed fermentation. A mixture of molasses was prepared (85% beet molasses, 15% cane molasses), diluted, and its pH was adjusted to pH 5.2 with sulfuric acid. The yeast cream from the batch fed seeding was diluted in sterile water and added to the molasses mixture with zinc sulfate, magnesium sulfate, biotin, thiamine, and calcium pantothenate. The resulting propagated yeast was centrifuged, and the propagation wort was washed using a laboratory Alpha separator to obtain approximately 20% yeast solids.

[0113] Yeast cream and deactivated yeast cream. After fermentation, the collected fermentation wort was centrifuged and washed using a laboratory-scale GEA separator to prepare the Petition 870260074630, dated 07 / 27 / 2026, pp. 82 / 103 79 / 96 yeast cream with a final dry weight close to 20%. In order to prepare the deactivated yeast cream, approximately 600 g of yeast cream were heated in a temperature-controlled hot plate / shaker until reaching 75°C. The cream was kept at 75°C for 15 minutes and then removed from the heat source.

[0114] Spray drying. The spray-dried samples were prepared by drying at 150°C using a mini spray dryer (Buchi B-290). The feed rate was maintained to keep the outlet temperature around 80-85°C.

[0115] Ball milling / preparation of ball-milled homogenate. Yeast cream was broken up (with a typical cell disruption efficiency of >95%) by ball milling under the following ball mill conditions. Yeast cream (~20% solids) was ball milled using a 0.6 L chamber volume Dyno KDL at 4°C, using 0.5-0.75 mm glass beads filling the chamber to 80% with a 1.6 g / mL packing capacity and a 64 mm diameter stirrer with a peripheral speed of 10 m / s. The yeast cream flow rate was 6 kg / L / h.

[0116] Preparation of Instant Dry Yeast (IDY). After commercial fermentations aimed at producing IDY samples, the collected wort was centrifuged and washed using a laboratory-scale GEA separator to prepare a yeast cream with a final dry weight close to 20%. The cream was then filtered in a vacuum filtration system to prepare a yeast cake. In order to remove additional water, the yeast cake was further pressed to obtain a dry weight of approximately 35% before extrusion. The pressed cake was then extruded after being thoroughly mixed for a period of 5 minutes. The time addition rate was 1% based on the yeast dry matter. After extrusion, the yeast was dried in a laboratory-scale fluidized bed dryer (Aeromatic AG). The drying temperature was adjusted and controlled. Petition 870260074630, dated 07 / 27 / 2026, pp. 83 / 103 80 / 96 to 35-40°C. Drying lasted about 20-25 minutes to obtain a solids content above 94%. In terms of fermentation recipes, the significant difference from the IDY fermentation recipe is that it features a 2-hour maturation period at the end of fermentation, during which ammonia (N) feeding is stopped and the fermentation temperature is increased to 35°C.

[0117] Autolysis in the fermenter. At least 3 L (minimum working volume) of 20% solids cream was transferred to a 20 L fermenter (BiOENGiNEERiNG). Autolysis was performed at 55°C and pH 5.5 (automatic pH control with 2 N sulfuric acid) with gentle stirring at 70 rpm. The autolysate (~20% dry weight) was harvested after a 24-hour incubation and separated as described below.

[0118] Laboratory-scale autolysis. This autolysis is similar to the fermenter autolysis described above, but was performed on a smaller scale and with slightly different parameters. Yeast cream (20% solids) was subjected to autolysis and the pH was adjusted to pH 7. The mixture was incubated in 50 mL conical tubes in a water bath at 55°C for 48 hours.

[0119] Autolysate separation without washing. After autolysis in a fermenter, the total autolysate was separated at 11000 RCF for 10 minutes in 1 L bottles in a Sorvall Lynx 6000 centrifuge to obtain a soluble fraction (11-13% dry weight, yeast extract) and an insoluble fraction (yeast cell wall). Dry weight and enzymatic activity were measured for the total autolysate, yeast extract, and cell wall fractions for dry weight and MANU balances.

[0120] Autolysate separation with washing. Separations were performed by centrifuging the autolysate in a fermenter in 50 mL conical tubes for 10 minutes at 3000 RCF. Two additional washes were performed by adding water equal to the weight of the supernatant obtained in the centrifugation step. The yield of the YE (yeast extract) separation is calculated as the recovery of solids from Petition 870260074630, dated 07 / 27 / 2026, pp. 84 / 103 81 / 96 only from separation (WF = 0) and from separation plus one or two washes (WF = 1 or 2, respectively), in relation to the initial solids in the autolysate. The MANU of YE recovery is calculated (in Phadebas MANU) only from separation (WF = 0) and from separation plus one or two washes (WF = 1 or 2), in relation to the initial total Phadebas MANU in the autolysate.

[0121] Ultrafiltration. The autolysate in the fermenter was separated by centrifugation in 1 L bottles at 11000 RCF and the yeast extract fraction was further concentrated by ultrafiltration with a PES membrane with a molecular weight cutoff of 10 kDa (Millipore, Biomax-10). The retentate fraction is retained by the membrane and the permeate fraction passes through the membrane.

[0122] Maltogenic Amylase Assay. One New Maltogenic Amylase Unit, MANU, is the amount of enzyme that under standard conditions will cleave one micromole of maltotriose per minute. Prior to the assay for enzymatic activity, yeast cream samples were deactivated by incubation at 60°C for 10 minutes in MANU assay buffer (0.1 M citric acid, pH 5.0). The samples were then mixed with 20 mg / ml of maltotriose substrate and incubated at 37°C for 30 minutes. The reactions were stopped by the addition of an equal volume of 1 N sodium hydroxide stopping reagent. The glucose hydrolyzed by maltogenic amylase activity was measured after a 15-minute incubation at room temperature with glucose assay reagent (HK) (Sigma G3293). The absorbance was read at 340 nm in a spectrophotometer. Unknown samples were compared with a dose curve of Novamyl® with known enzymatic activity.This method was applied to generate only the results shown in Figure 1.

[0123] Assay of enzymatic activity in Phadebas MANU. Phadebas tablets contain a water-insoluble starch substrate and a blue dye, linked to the dye by crosslinks. The substrate is Petition 870260074630, dated 07 / 27 / 2026, pp. 85 / 103 82 / 96 hydrolyzed by maltogenic amylase, releasing the soluble blue dye. After the reaction and centrifugation, the absorbance of the solution was measured by spectrophotometry and is considered an approximation for enzymatic activity. For each sample, a Phadebas tablet was added to 4.9 mL of citrate-phosphate buffer (70 mM disodium hydrogen phosphate, 30 mM citric acid, pH 5.5), incubated in a water bath at 60°C for 5 minutes. Then, 0.1 mL of standard or sample, diluted in citrate-phosphate buffer, was added to the tablet and buffer solution and incubated for 15 minutes in a water bath at 60°C. The reaction was terminated by the addition of 1 mL of a 0.5 M sodium hydroxide solution and mixing. The tubes were centrifuged to remove solids and the absorbance of the substrate was measured at 620 nm with a spectrophotometer. The samples (dry or liquid) were compared with a dose curve of Novamyl® with known activity.This method was applied to generate all results in MANU, except for Figure 1.

[0124] Glucose oxidase assay. Cells were grown in batches in yeast extract peptone medium plus 2% glucose at 30°C for 24 hours. In order to obtain the washed, ruptured cell supernatant, the cells were killed by beating with glass beads for 2 x 1 min in assay buffer, with a one-minute rest between these. The supernatant was separated from the total lysate by centrifugation. Whole culture, supernatant, washed disrupted cell supernatant (reflecting cell-associated intracellular activity), washed cells, or a positive control of Gluzyme® (2.40 GODU / mL corresponding to 10000BG) were measured using the K-GLOX™ kit (Megazyme): samples in assay buffer (100 mM potassium phosphate, pH 7, containing 0.5 mg / mL BSA and 0.02% (w / v) sodium azide) were mixed with 90 mg / mL glucose and POD mixture and incubated at room temperature for 20 minutes. Absorbance was measured with a spectrophotometer at 510 nm. Petition 870260074630, dated 07 / 27 / 2026, pp. 86 / 103 83 / 96

[0125] Alpha-amylase assay (Figure 5). Alpha-amylase activity was measured by adding 25 μL of washed cells or cell-free supernatant to 25 μL of 5 mM p-nitrophenyl α-D-hexaoside in 50 mM sodium acetate, pH 5. The reaction was incubated at 35°C for 2 hours and stopped by the addition of 50 μL of 1M sodium bicarbonate. The cells were pelleted, 50 μL of the assay mixture were transferred to a microtiter plate, and the absorbance was measured at 405 nm. The activity of the cell fraction was represented as a percentage of the total activity (“bound” + “free”).

[0126] Alpha-amylase assay (Figures 13 to 16). Strains were initially grown in 600 µL of YPD40 at 35°C for 48 h in 96-well plates on a shaker at 900 rpm. Alpha-amylase activity was determined by adding 25 µL of washed cells or cell-free supernatant to 100 µL of 1% crude starch with 50 mM sodium acetate buffer (pH 5.2). The assay was treated for 30 min at 85°C using an Eppendorf Gradient Cycler. Reducing sugars were measured using a Dinitrosalicylic Acid (DNS) Reagent Solution method, using a 2:1 assay ratio of DNS:starch and boiled at 100°C for 5 min. Absorbance was measured at 540 nm.

[0127] Fungal amylase activity. Cells were grown in batches in yeast extract peptone medium plus 2% glucose at 30°C for 24 hours. The whole culture, supernatant, and / or supernatant of broken cells or washed cells were resuspended in assay buffer (70 mM disodium hydrogen phosphate, 30 mM citric acid, pH 5.5), mixed with 1% gelatinized wheat starch in assay buffer, and incubated at 30°C for 1 hour. 3,5-dinitrosalicylic acid (DNS) was added to react with the reducing ends and boiled at 99°C for 5 minutes. Absorbance was measured with a spectrophotometer at 540 nm. Petition 870260074630, dated 07 / 27 / 2026, page 87 / 103 84 / 96

[0128] Assay of wheat starch activity. Cells were grown in batch on yeast extract peptone medium plus 4% glucose at 35°C for 48 hours. The whole culture, supernatant, and washed cells resuspended in assay buffer (50 mM sodium acetate, pH 5) were mixed with 1% wheat starch in assay buffer and incubated at 60°C for 5 minutes. Then, 3,5-dinitrosalicylic acid was added to react with the reducing ends and boiled at 99°C for 5 minutes. Absorbance was measured with a spectrophotometer at 540 nm.

[0129] Phytase activity assay. A 2-fold serial dilution of 1 M monobasic potassium phosphate was prepared as a standard for calculating FTUs. 190 μL of a 5 mM sodium phytate solution, pH 5.5, was added to each well of a 96-well PCR plate. Standards or supernatants from overnight yeast cultures in yeast extract peptone medium with 4% glucose were combined with a 5 mM sodium phytate solution, pH 5.5, and incubated at 37°C for 30 min. Cell-associated samples were remeasured after 2 hours of incubation. Equal volumes of reaction and color-change solution (4 parts reagent A to 1 part reagent B, where reagent A is ammonium heptamolybdate-HCl in water and reagent B is 2.7% ferrous sulfate in water) were combined and incubated for 10 minutes at room temperature before pelleting at 3500 rpm for 3 minutes. The absorbance of each sample or standard was read at 700 nm in a spectrophotometer.

[0130] Baking test with Novamyl® as a control. Bread was made with the M10474 strain in the presence or absence of externally added doses of Novamyl® maltogenic amylase (as indicated in the figures) and was compared with bread made with strains expressing a cell-associated MAA (in the absence of Novamyl®). For bread made with yeast cream expressing MAA, yeast cream doses were normalized to 1000 MANU based on activity. Petition 870260074630, dated 07 / 27 / 2026, pages 88 / 103 The 85 / 96 enzymatic process shown in Figure 1B was supplemented with wild-type C yeast cream for sufficient leavening power. In summary, 1000 g of white flour, 600 g of water, 35 g of yeast cream (dosed at 30% solids), 70 g of dextrose, 30 g of canola oil, 20 g of salt, 0.06 g of ascorbic acid, 0.625 g of Novamyl® (when present), and 3.75 g of sodium stearoyl lactylate were combined in a bowl mixer, mixed for 1 minute at low speed and mixed for 10 minutes at high speed. Three 400 g pieces of dough were formed and risen for 7 minutes. The dough was then rolled to form loaves, placed in loaf pans, and left to rise at 44°C until it reached a height of 100 mm. The loaves were baked at 225°C for 17 minutes. The crumb hardness (an indicator of firmness), resilience, and bread volume were measured after baking.

[0131] Texture analysis. The texture analysis of the crumb was performed 5, 8, and 13 days after baking. The loaves were cut with an electric knife using a 2.5 cm gauge. Two slices in the middle of the bread were analyzed. The hardness and resilience of the crumb were evaluated using the TA-XT Plus Texture™ analyzer. The TA-3 probe was used to compress the crumb to a distance of 10 mm (40% compression). Five measurements per slice were taken on two slices for a total of 10 measurements. A macro was used to calculate the % resilience.

[0132] Baking test with Gluzyme® as a control. White bread was made with or without the addition of commercial glucose oxidase (Gluzyme Mono® 10000 BG, dosed at 100 or 200 GOU / kg of flour) or cell-associated yeast (dosed at 127 GOU / kg of flour). 1000 g of white flour, 600 g of water, 40 g of block yeast (~30% solids), 70 g of dextrose, 30 g of vegetable oil, 20 g of salt and 0.06 g of ascorbic acid were combined in a bowl mixer for 1 minute at low speed and for 9 minutes at high speed. Three 400 g pieces of dough were formed and placed in bread molds and Petition 870260074630, dated 07 / 27 / 2026, pp. 89 / 103 86 / 96 dough balls were allowed to rise at 44°C until they reached a height of 100 mm. The loaves were baked at 225°C for 17 minutes. The M16780 strain was grown in batches on a yeast extract peptone medium plus 4% glucose at 32°C for 24 hours. The cell pellet was obtained by centrifuging the whole YPD culture and removing the supernatant. The pellet was tested as described for the glucose oxidase method, and a volume equivalent to 127 GOU / kg of flour was dosed into the dough. Three pieces of dough were allowed to rise to a height of 100 mm and baked after measuring the oven height. The oven spring (oven height minus the height of the risen dough) was measured, and the crumb structure (higher score = finer crumb) was evaluated by visual inspection. EXAMPLE II - EXPRESSION OF MALTOGENIC ALPHA-AMYLASES ASSOCIATED WITH CELLS

[0133] The expression of heterologous MAA, especially in the presence of an immobilizer, provided the recombinant yeasts with maltogenic amylase activity both in the cell pellet (Figure 1A) and, on a larger scale, in the yeast cream (Figure 1B). In comparison, the corresponding wild-type strain failed to exhibit any maltogenic amylase activities (Figures 1A and 1B). The results shown in Figures 1A and 1B were obtained by strains expressing heterologous MAA from the tdhl and hor7 gene promoters. Similar results were obtained with a combination of only one promoter (from the hor7 gene; data not shown).

[0134] In order to determine the effect(s) of using yeasts expressing heterologous MAA in bread making, different types of bread were made with wild-type yeasts (supplemented or not with Novamyl®) or with recombinant yeasts expressing heterologous MAA. In bread making, quality is associated with softness in such a way that the ability to avoid the Petition 870260074630, dated 07 / 27 / 2026, pp. 90 / 103 87 / 96 Crude hardness is pursued. As shown in Figures 2A to 2C, the use of recombinant yeasts expressing heterologous MAA for bread preparation reduced crumb hardness when compared to bread made with only wild-type yeast not supplemented with Novamyl®. The use of recombinant yeasts expressing heterologous MAA provided breads exhibiting crumb hardness similar to those made with wild-type yeast supplemented with Novamyl®.

[0135] As also shown in Figures 2A to 2C, the use of yeasts expressing heterologous MAA maintained or even increased bread volume when compared with wild-type yeasts supplemented or not with Novamyl®.

[0136] Bread quality can also be assessed by measuring percent resilience, where an increase in percent resilience is desirable. As shown in Figures 3A to 3C, the use of yeasts expressing heterologous MAA even increased percent resilience when compared to wild-type yeasts that were not supplemented with Novamyl®.

[0137] A strain expressing intracellular G. stearothermophilus MAA (M14851) was propagated (batch aerobic feeding with molasses) and MANU activity was determined. As shown in Table 2, in the untreated whole must, between 25.6 and 39.3 of MANU activity was detected. After washing and concentrating the cream, MANU activity between 132 and 288 was detected. Table 2. Yeast biomass concentration concentrates cell-associated maltogenic amylase. Enzyme activity was determined in Phadebas assays compared to a Novamyl standard dose curve with known maltogenic amylase units (MANU). Petition 870260074630, dated 07 / 27 / 2026, pp. 91 / 103 88 / 96 MANU of Phadebas / ml Propagation of M14851 Total must (~6% solids) Washed and concentrated cream (19-20% solids) 1200617 25.6 112.2 1210617 35.6 232.0 1220617 39.3 287.6

[0138] Another strain expressing an immobilized G. stearothermophilus MAA (M13979) was propagated (batch aerobic feeding on molasses) and MANU activity was determined in several yeast preparations. The results are shown in Table 3. The data for yeast cream activity on day 1 after commercial propagation are the most representative measure of the cream in its original form. All other data were obtained 8 days after commercial propagation. Table 3. Phadebas MANU activity per gram of dry weight of various M13979 preparations. Enzyme activity was determined in Phadebas assays compared to a dose-resistance curve of Novamyl® enzyme standards with known maltogenic amylase units (MANUs). Phadebas MANU equivalent / gram of dry weight Sample M13979 1 day after propagation 8 days after propagation Cream 1087 3157 Homogenized ground by spheres 8698 Petition 870260074630, dated 07 / 27 / 2026, pp. 92 / 103 89 / 96 Phadebas MANU equivalent 1 gram dry weight Sample M13979 1 day after propagation 8 days after propagation Spray-dried cream 1121 Spray-dried deactivated cream 2039 Ball-ground homogenate, spray-dried 6721

[0139] Activities in MANU and wheat starch were determined in different preparations of a yeast strain that intracellularly expresses maltogenic alpha-amylase from G. stearothermophilus (M15532) and propagated (aerobic batch feeding in molasses). The results are provided in Tables 4 to 8 showing the effects of the different preparations on the observed enzymatic activity level. Table 4. Phadebas and wheat starch assays to measure maltogenic amylase activity in various M15532 preparations. Enzyme activity was determined in Phadebas assays by comparison with a dose curve of Novamyl® enzyme standards with known maltogenic amylase units (MANU). Propagation of M15532 Sample MANU form of Phadebas / g dry weight Wheat starch MANU / g dry weight High protein recipe 1060917 Untreated cream Liquid 287 574 Autolyzed cream by cooking in the laboratory (pH 7, 48 h, 55°C) (liquid) Liquid 17826 15328 Mix of 4 Untreated cream Liquid 96 Petition 870260074630, dated 07 / 27 / 2026, pages 93 / 103 90 / 96 M15532 Propagation Sample MANU Form of Phadebas / g dry weight Wheat starch MANU / g dry weight propagations (1280817, B300817, B310817, B300817) Autolyzed cream by cooking in the laboratory (pH 7, 48 h, 55°C) (liquid) Liquid 23614 12920 Homogenized ball-ground (liquid) Liquid 15916 12903 Homogenized ball-ground (spray-dried) Dry 10607 7764 Table 5. Results of enzyme activity in cream, autolyzed cream on a laboratory scale (incubated 48h at 5°C, pH 7) and samples of instant rehydrated dry yeast (IDY). Enzyme activity was determined in Phadebas assays in comparison with a dose curve of Novamyl® enzyme standards with known maltogenic amylase units (MANU). Sample of M15532 % of solids MANU of Phadebas / ml of sample MANU of Phadebas / g DCW Cream 17.9 58 325 Cream after 48 h, 55°C, pH 7 17.9 3572 19955 IDY rehydrated at 37°C 15.9 545 3438 IDY subjected to cold thermal shock 15.4 454 2958 Petition 870260074630, dated 07 / 27 / 2026, pp. 94 / 103 91 / 96 Table 6. Dry weight balances and enzymatic activity in preparations of autolysate, yeast extract, ultrafiltration retentate, and yeast cell wall before and after drying of different preparations of the yeast strain M15532. Enzymatic activity was determined in Phadebas assays in comparison with a dose curve of Novamyl® enzyme standards with known maltogenic amylase units (MANU). SAMPLE BEFORE DRYING dw MAA % MANU / gdw AUTOLYZED 18, 8983 YE 11.6 14422 10 kDa RETENTADO 15.1 83332 CW 37.0 883 Petition 870260074630, dated 07 / 27 / 2026, pages 95 / 103 AFTER DRYING dw NRC10NOV COZ. LAB NRC24NOV MANU / gdw MANU / gdw MANU / gdw % AV %RD AV %CV 91.3 13568 14812 6% 16864 17% 93.7 23067 27566 5% 24028 14% 93.7 25984 80817 5% 75552 15% 94.1 700 3757 4% 3421 4% MANUAL BALANCE PROCESS + DRYING dwt COOKING NRC BALANCING LAB. 24NOV 100 100 100 47 87 66 18 98 80 53 14 13 92 / 96 Table 7. Results of yeast extract separation from total autolysate and enzyme recovery with and without washing of yeast strain M15532. Yield of YE (yeast extract) separation is the recovery of solids from separation alone (WF = 0) and from separation plus one or two washes (WF = 1 or 2, respectively), relative to the initial solids in the autolysate. YE MANU recovery is the activity (in Phadebas MANU) from separation alone (WF = 0) and from separation plus one or two washes (WF = 1 or 2), relative to the initial total Phadebas MANU in the autolysate. Enzyme activity was determined in Phadebas assays compared to a dose curve of Novamyl® enzyme standards with known maltogenic amylase units (MANU). WASHING FACTOR (WF) YE SEPARATION YIELD (%) YE MANUAL RECOVERY (%) %DW IN YE 0 36 58 12.3 1 50 71 8.3 2 54 75 6.0 Table 8. Ultrafiltration results of yeast extract from M15532 with a molecular weight cutoff of 10 kDa. YE is the yeast extract, obtained by centrifuging the autolysate in a fermenter in 1-liter bottles for 10 minutes at 11000 RCF, to mimic industrial-scale separation. The retentate is the sample retained by ultrafiltration, and the permeate is the unretained sample. The MANU of Phadebas / ml was determined for each sample, and the MANU / g of DW (dry weight) was calculated based on the dry weight per sample. Enzyme activity was determined in Phadebas assays compared to a dose curve of Novamyl® enzyme standards with known maltogenic amylase units (MANU). Petition 870260074630, dated 07 / 27 / 2026, pages 96 / 103 93 / 96 Sample Concentration Factor %DW in sample MANU / mL MANU / g Balanced DW MANU (7°) Balanced DW (%) YE 1.0 11.6 1672 14422 100 100 10 kDa RETENTADO 3.5 15.5 12583 83332 222 38 10 kDa PERMEADO 10.9 22 203 1 67

[0140] Different preparations (e.g., cream and spray-dried) of yeast strains M13979 (expressing an immobilized MAA) and M15532 (expressing an intracellular MAA) were used to prepare types of bread. The use of yeast strains M13979 and M15532, when compared with control breads made in the absence of a dough conditioner, reduces the hardness of the bread crumb (Figures 11 and 12A) while maintaining its volume (Figure 11) and increasing its resilience (Figure 12B).

[0141] Wheat starch activity normalized for cell density was determined in whole culture, culture supernatant, and washed cells of several yeast strains expressing maltogenic alpha-amylase from G. stearothermophilus expressed in a secreted form, in an immobilized form, or expressed intracellularly as explained in the caption of Figure 10. The results are shown in Figure 8 and indicated that the highest activities are observed when MAA is expressed intracellularly. EXAMPLE III - EXPRESSION OF ALPHA-AMYLASES, GLUCOAMYLASES, PHYTASES, GLUCOSE OXIDASES AND HETEROLOGOUS FUNGAL AMYLASES

[0142] A heterologous glucoamylase (GA) was expressed in S. cerevisiae from the tef2 gene promoter. When GA was expressed as an immobilized enzyme, the activity associated with cell pelleting is increased (Figure 4). Petition 870260074630, dated 07 / 27 / 2026, pages 97 / 103 94 / 96

[0143] A heterologous alpha-amylase (AA) from the tef2 gene promoter. When AA was expressed as an immobilized enzyme, pellet-associated activity is increased, especially in the presence of a ligand (Figure 5).

[0144] Several yeast strain preparations expressing C. braakii phytase were obtained and their FTU activity was determined. Some strains expressed the phytase in a secreted form (T2633), while other strains expressed the phytase in an immobilized form (T2634, T2635, T2636, T2637, and T2638) using different immobilizers. The results are shown in Figures 7A and 7B for both the supernatant and the cells themselves.

[0145] Several yeast strain preparations expressing E. coli phytase were obtained and their FTU activity was determined. Some strains expressed phytase in a secreted form (M11312), other strains expressed phytase in an immobilized form (T2705, T2706, M12795, M12938, T2816) using different immobilizer configurations. The results are shown in Figures 8 and 9 for both the supernatant and the cells themselves.

[0146] Heterologous thermotolerant chimeric alpha-amylase-SPI1 constructs of P. furiosus and alpha-amylase-CCW12 constructs of T. hydrothermalis were obtained using various truncations of the immobilizing radicals. The alpha-amylase activity associated with washed cells of strains expressing chimeric polypeptides with truncated GPI anchoring portions was compared with non-truncated GPI anchoring portions, as shown in Figures 13 and 14.

[0147] As seen in Figure 13, the chimeric polypeptide with the full-length immobilizing radical (expressed from strain M15222) showed the same or greater alpha-amylase activity than the polypeptides with truncated immobilizing radicals (expressed from strains M15774 (length truncation 21 aa), M15771 Petition 870260074630, dated 07 / 27 / 2026, pp. 98 / 103 95 / 96 (51 aa length truncation), M1577 (81 aa length truncation) or M15772 (130 aa length truncation)).

[0148] As seen in Figure 14, chimeric polypeptides with a complete immobilizing radical (expressed from strain M15215) exhibited similar or higher alpha-amylase activity when compared to chimeric polypeptides with a truncated immobilizing radical (expressed from strains M15773 (24 aa truncation length), M15776 (49 aa truncation length), M16251 (74 aa truncation length) or M15775 (99 aa truncation length)).

[0149] Heterologous thermotolerant chimeric alpha-amylase-SPI1 constructs of P. furiosus and alpha-amylase-CCW12 constructs of T. hydrothermalis were obtained using various ligands and the same immobilizing radical. The alpha-amylase activity associated with washed cells of strains expressing chimeric polypeptides with different ligands is shown in Figures 15 and 16.

[0150] As seen in Figure 15, the alpha-amylase activity of all strains was higher than that of the control strain (M2390), regardless of the type of ligand used. Alpha-amylase activity was highest when ligand 7 (SEQ ID NO: 99) was used (strain M16222).

[0151] As seen in Figure 16, the alpha-amylase activity of all strains was higher than that of the control strain (M2390), regardless of the type of ligand used. Alpha-amylase activity was highest when ligand 5 (SEQ ID NO: 97) was used (strain M15780).

[0152] Heterologous chimeric glucose oxidase (GO) constructs were expressed intracellularly or in a secreted form. GO activity obtained from various cell fractions was compared with a control strain (M10474) or a positive control enzyme preparation of Gluzyme Mono® (Figure 17). GO activity associated with strains M16780 and M16273 was higher than that of the control GO activity associated with the parental strain M10474 (Figure 18). Petition 870260074630, dated 07 / 27 / 2026, pages 99 / 103 96 / 96

[0153] The M16780 strain was also used to supplement bread doughs which were compared with negative control breads (unsupplemented dough) and positive control breads (dough supplemented with Gluzyme Mono®). As shown in Figure 21, a greater oven spring and a finer crumb structure (which is observed for the M16780 cell pellet doughs) are indicators of glucose oxidase function in the supplemented dough.

[0154] Heterologous chimeric fungal amylase (FA) constructs were expressed in a secreted form. FA activity obtained from various fractions was compared with the control strain M10474 or a Fungamyl® enzyme preparation as a positive control (Figure 19). FA activity associated with strains M16772 and M16540 was higher than the activity of the control associated with the parental strain M10474 (Figure 20).

[0155] Although the invention has been described in connection with its specific embodiments, it should be understood that the scope of the claims should not be limited by the preferred embodiments presented in the examples, but should be given the broadest interpretation consistent with the description as a whole. REFERENCES Pérez-Torrado R, Bruno-Bárcena JM, Matallana E. Monitoring stressrelated genes during the process of biomass propagation of Saccharomyces cerevisiae strains used for wine making. Appl Environ Microbiol. 2005 Nov;71(11):6831-7. Praekelt UM, Meacock PA. MOL1, a Saccharomyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses. Yeast. 1992 Sep;8(9):699-710. Petição 870260074630, de 27 / 07 / 2026, pág. 100 / 103

Claims

1 / 2 CLAIMS 1. A process for the preparation of a food product or feed, said process CHARACTERIZED by comprising the inclusion of a recombinant yeast host cell, or an additive comprising the recombinant yeast host cell, in the food product or feed, wherein the recombinant yeast host cell has a heterologous nucleic acid molecule encoding a heterologous enzyme for food product or feed associated with the cell, wherein the heterologous enzyme for food product or feed associated with the cell is a glucose oxidase, wherein the heterologous nucleic acid molecule is operationally associated with a heterologous promoter that allows the expression of the heterologous nucleic acid molecule during the propagation of the recombinant yeast host cell.

2. Process according to claim 1, CHARACTERIZED in that it further comprises fermenting the food product or feed in the presence of the recombinant yeast host cell.

3. A process according to claim 1 or 2, CHARACTERIZED in that it further comprises cooking the food product or feed to provide a cooked product.

4. Process according to claim 3, CHARACTERIZED in that the baked product is bread.

5. Process, according to any one of claims 1 to 4, CHARACTERIZED in that the heterologous nucleic acid molecule allows: the intracellular expression of the heterologous enzyme for food and / or feed; the expression of a heterologous enzyme for food and / or feed associated with the membrane; or the expression of an immobilized heterologous enzyme for food and / or feed.

6. Process according to claim 5, CHARACTERIZED in that the immobilized heterologous enzyme for food and / or feed is a chimeric protein of formula (I) or (II): (NH2) FFE - L - TT (COOH) (I) (NH2) TT - L - FFE (COOH) (II) where: FFE is the enzyme for food and / or feed; L is present or absent and is an amino acid ligand; Petition 870260067563, dated 07 / 08 / 2026, p. 128 / 158 2 / 2 TT is an amino acid immobilization radical for the association of the enzyme for food and / or feed to the cell wall of the recombinant yeast host cell; (NH2) indicates the amino terminus of the chimeric protein; (COOH) indicates the carboxyl terminus of the chimeric protein; and “-” is an amide linkage.

7. Process, according to claim 6, CHARACTERIZED in that L is present and optionally comprises one or more G4S motifs (SEQ ID NO: 41) or one or more EA2K motifs (SEQ ID NO: 100) or EA3K motifs (SEQ ID NO: 101).

8. Process, according to claim 6 or 7, CHARACTERIZED in that TT comprises a transmembrane domain and, optionally, is of a FLO1 protein.

9. Process, according to any one of claims 6 to 8, CHARACTERIZED in that TT can be modified by a post-translational mechanism to present a glycosylphosphatidylinositol (GPI) anchor and, optionally, be of a SED1 protein, a TIR1 protein, a CWP2 protein, a CCW12 protein, an SPI1 protein, a PST1 protein or a combination of an AGA1 protein and an AGA2 protein.

10. Process, according to any one of claims 1 to 9, CHARACTERIZED in that the heterologous promoter comprises the promoter of the tdhl gene, the hor7 gene, the hsp150 gene, the hxt7 gene, the gpml gene, the pgkl gene and / or the stll gene.

11. Process, according to any one of claims 5 to 10, CHARACTERIZED in that the heterologous polypeptide for food and / or feed associated with the membrane or the heterologous enzyme for immobilized food and / or feed has a heterologous signaling peptide.

12. Process, according to any one of claims 1 to 11, CHARACTERIZED in that the genus is Saccharomyces.

13. Process, according to claim 12, CHARACTERIZED in that the species is Saccharomyces cerevisiae.

14. Process, according to any one of claims 1 to 13, CHARACTERIZED in that the glucose oxidase comprises the amino acid sequence of SEQ ID NO: 44 or 103, is a variant that exhibits at least 70% identity to the amino acid sequence of SEQ ID NO: 44 or 103, or is a fragment that exhibits at least 70% identity to the amino acid sequence of SEQ ID NO: 44 or 103. Petition 870260067563, dated 07 / 08 / 2026, pp. 129 / 158

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