Recombinant yeast host cells comprising a heterologous ATP futile cycle

Abstract

The present disclosure concerns recombinant yeast host cells for the production of one or more fermentation products. The recombinant yeast host cells of the present disclosure comprise at least one first genetic modification for providing a heterologous ATP futile cycle. The recombinant yeast host cells of the present disclosure also comprise at least one second 5 genetic modification in an engineered metabolic pathway for decreasing the production of the fermentation by-product.

Description

[0001] RECOMBINANT YEAST HOST CELLS COMPRISING A HETEROLOGOUS ATP FUTILE CYCLE

[0002] CROSS-REFERENCE TO RELATED APPLICATION(S) AND DOCUMENT(S)

[0003] This application claims priority from U.S. provisional patent application 63 / 736,11 1 filed December 19, 2024 and herewith incorporated in its entirety. This application also includes a sequence listing in electronic format which is also incorporated in its entirety.

[0004] TECHNOLOGICAL FIELD

[0005] The present disclosure relates to recombinant yeast host cell that are being used to produce one or more fermentation products, such as, for example, an alcohol.

[0006] BACKGROUND

[0007] In the industry, increasing fermentation titers (such as ethanol titers) is of great importance to the commercial viability of the yeast fermentation process. Glycerol is a major by-product from Saccharomyces cerevisiae in industrial ethanol fermentation, whose production plays significant physiological roles in yeast metabolism including osmoregulation and maintaining redox balance under anaerobic conditions. Around 4% of the carbon source can be consumed to produce glycerol by yeast in typical industrial fermentations. Redirecting carbon flux from glycerol to ethanol would therefore contribute to maximizing the economic potential of the bioethanol industry. However, since glycerol serves as an essential electron sink for reoxidizing reducing equivalents (NADH) generated from glycolysis, conventional strategies to reduce glycerol production focused on engineering cellular redox metabolism to reduce formation of NADH. Reducing glycerol production can lead to a reduction in fermentation rate and / or kinetics.

[0008] There remains a need for a recombinant yeast which is capable of increasing its fermentation yield while reducing the accumulation of fermentation by-products.

[0009] SUMMARY

[0010] The present disclosure comprises a recombinant yeast host cell comprising a heterologous ATP futile cycle as well as an engineered metabolic pathway for decreasing the production of a fermentation by-product. The presence of the heterologous ATP futile cycle and the engineered metabolic pathway favor the production of one or more fermentation products. In some embodiments, the recombinant yeast host cell exhibits improved fermentation kinetics.

[0011] According to a first aspect, the present disclosure provides a recombinant yeast host cell capable of producing at least one fermentation product and at least one fermentation byproduct. The recombinant yeast host cell comprises at least one first genetic modification for providing a heterologous ATP futile cycle. The recombinant yeast host cell also comprises at least one second genetic modification in an engineered metabolic pathway for decreasing the production of the fermentation by-product. In an embodiment, the heterologous ATP futile cycle comprises at least one heterologous enzyme. In another embodiment, the at least one heterologous enzyme comprises a heterologous polypeptide having fructose-1 ,6- bisphosphatase activity. In a further embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity comprises a polypeptide which belongs to E.C. 3.1 .3.11 . In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is a fructose-1 ,6-bisphosphatase. In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is encoded by a fbp gene. In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is of eukaryotic origin. In another embodiment, the heterologous polypeptide having fructose-1 ,6- bisphosphatase activity is of prokaryotic origin. In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is from Yarrowia sp. In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is from Saccharomyces sp. In another embodiment, the heterologous polypeptide having fructose- 1 ,6-bisphosphatase activity is from Yarrowia lipolytica. In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is from Saccharomyces cerevisiae. In another embodiment, the heterologous polypeptide having fructose-1 ,6- bisphosphatase activity has the amino acid sequence of SEQ ID NO: 3 or 61 , or is a variant of the amino acid sequence of SEQ ID NO: 3 or 61 having fructose-1 ,6-bisphosphatase activity. In another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is encoded by the nucleic acid sequence of SEQ ID NO: 4 or 60, or is a variant of the nucleic acid sequence of SEQ ID NO: 4 or 60 encoding a polypeptide having fructose-1 ,6- bisphosphatase activity. In a further embodiment, the recombinant yeast host cell comprises a reduction in the expression or a deletion in at least one native gene encoding a polypeptide having 6-phosphofructo-2-kinase activity. In yet a further embodiment, the recombinant yeast host cell comprises the reduction in the expression or the deletion in the pfk26 gene, the pfk27 gene or both the pfk26 and the pfk27 genes. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity comprises a polypeptide which belongs to E.C. 2.7.1.90. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is a pyrophosphate-dependent phosphofructokinase (PPi-PFK). In still another embodiment, the heterologous polypeptide having fructose-1 ,6- bisphosphatase activity is encoded by a pfk gene. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is of eukaryotic origin. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is of prokaryotic origin. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is from Xanthomonas sp. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is from Porphyromas sp. In still another embodiment, the heterologous polypeptide having fructose- 1 ,6-bisphosphatase activity is from Porphyromas sp. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is from Xanthomonas campestris. In still another embodiment, the heterologous polypeptide having fructose-1 ,6- bisphosphatase activity is from Porphyromonas gingivalis. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity has the amino acid sequence of SEQ ID NO: 5, or 7, or is a variant of the amino acid sequence of SEQ ID NO: 5, or 7 having fructose-1 ,6-bisphosphatase activity. In still another embodiment, the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is encoded by the nucleic acid sequence of SEQ ID NO: 6, or 8 or is a variant of the nucleic acid sequence of SEQ ID NO: 6, or 8 encoding a polypeptide having fructose-1 ,6-bisphosphatase activity. In some embodiments, the at least one fermentation by-product comprises glycerol. In some embodiments, the recombinant yeast host cell comprises one or more copies of a heterologous nucleic molecule encoding the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity. In embodiments, the at least one heterologous enzyme comprises a heterologous polypeptide having glycerol kinase activity. In such embodiment, the heterologous polypeptide having glycerol kinase activity comprises a polypeptide: belongs to E.C. 2.7.1 .30; is a glycerol kinase; is encoded by a gut1 gene; is of eukaryotic or of prokaryotic origin; is from Saccharomyces sp.; is from Saccharomyces cerevisiae; has the amino acid sequence of SEQ ID NO: 55 or a variant of SEQ ID NO: 55 having glycerol kinase activity; and / or is encoded by the nucleic acid sequence of SEQ ID NO: 54 or is a variant of the nucleic acid sequence of SEQ ID NO: 54 encoding the polypeptide having glycerol kinase activity. In further embodiments, the engineered metabolic pathway for reducing glycerol comprises a heterologous polypeptide having STL1 activity; a heterologous glyceraldehyde-3-phosphate dehydrogenase polypeptide; a heterologous pyruvate decarboxylase, a reduction in the expression or a deletion in at least one native gene encoding a polypeptide having glycerol 3- phosphate dehydrogenase activity; and / or a reduction in the expression or a deletion in at least one native gene encoding a polypeptide glycerol-3-phosphate phosphatase activity. In yet another embodiment, the at least one native gene encoding a polypeptide having glycerol 3- phosphate dehydrogenase activity comprises GPD1 and / or GPD2. In yet another embodiment, the at least one native gene encoding a polypeptide having glycerol-3-phosphate phosphatase activity comprises GPP1 and / or GPP2. In some embodiments, the fermentation product comprises an alcohol. In yet another embodiment, the recombinant yeast host cell further comprises an engineered metabolic pathway for increasing the production of the at least one fermentation product. In a further embodiment, the recombinant yeast host cell is from the genus Saccharomyces sp. In still a further embodiment, the recombinant yeast host cell is from the species Saccharomyces cerevisiae. According to a second aspect, the present disclosure provides a blend of a first recombinant yeast host cell and a second yeast host cell, wherein the first recombinant yeast host cell is described herein and the second yeast host cell lacks a heterologous ATP futile cycle.

[0012] According to a third aspect, the present disclosure provides a process for making at least one fermentation product. The process comprises contacting the recombinant yeast host cell described herein or the blend described herein with a source of a carbohydrate under a condition allowing the conversion of at least a part of the carbohydrate into the at least one fermentation product. In an embodiment, the source of the carbohydrate comprises C6 carbohydrates. In another embodiment, the source of the carbohydrate comprises or is derived from corn, sugarcane and / or molasses.

[0013] DETAILED DESCRIPTION OF THE DRAWINGS

[0014] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

[0015] Figures 1A to 1 C provide embodiments of metabolic pathways for ethanol and glycerol production from glucose in Saccharomyces cerevisiae in which a heterologous ATP futile cycle has been introduced. (A) A heterologous ATP futile cycle can be introduced by the expression of a heterologous fructose-1 ,6-bisphophatase (FBPase). (B) A heterologous ATP futile cycle can be introduced by the expression of a heterologous pyruvate carboxylase (PYC) and a heterologous phosphoenolpyruvate carboxykinase (PCK). (C) A heterologous ATP futile cycle can be introduced by the expression of a heterologous glycerol kinase (GUT1).

[0016] Figure 2 compares the metabolites (net ethanol in g / L; net glycerol in g / L and glucose in g / L) measured at the end of fermentation of a control wildtype S. cerevisiae strain (Y1) or recombinant S. cerevisiae strains expressing a heterologous enzyme having fructose-1 ,6- bisphosphatase (FBPase) activity (Y3, Y4 and Y5). Results are provided as metabolite measured (in g / L) at the end of fermentation and the strain used.

[0017] Figure 3 compares the CO2production profile of a control wildtype S. cerevisiae strain (Y1) or recombinant S. cerevisiae strains expressing a heterologous enzyme having FBPase activity (Y3, Y4, and Y5). Results are provided as cumulative CO2measured / evolved (in mL at atmospheric pressure) in function of time (hours) and of the strain used.

[0018] Figure 4 compares the metabolites (net ethanol in g / L; net glycerol in g / L and glucose in g / L) measured at the end of fermentation of a control wildtype S. cerevisiae strain (Y1) or recombinant S. cerevisiae strains expressing a heterologous FBPase under the control of different promoters (Y3, Y6, Y7, Y8, Y9, Y10, Y1 1 , and Y12). Results are provided as metabolite measured (in g / L) at the end of fermentation and the strain used. Figures 5A and B compare the CO2production profile of a control wildtype S. cerevisiae strain (Y1) or recombinant S. cerevisiae strains expressing a heterologous FBPase activity under the control of different promoters (Y3, Y6, Y7, Y8, Y9, Y10, Y11 , and Y12). Results are provided as cumulative CO2measured / evolved (in mL at atmospheric pressure) in function of time (hours) and of the strain used (Figures 5A and 5B). Figure 5B is a magnified version of Figure 5A between 24 and 72 hours of fermentation.

[0019] Figure 6 compares the metabolites (net ethanol in g / L; net glycerol in g / L and glucose in g / L) measured at the end of fermentation of a control recombinant S. cerevisiae strain including a glycerol reduction technology and lacking a heterologous FBPase (Y13) or recombinant S. cerevisiae strains including a glycerol reduction technology and expressing a heterologous FBPase under the control of different promoters (Y14, Y15, and Y16). Results are provided as metabolite measured (in g / L) at the end of fermentation and the strain used.

[0020] Figure 7 compares the CO2production profile of a control recombinant S. cerevisiae strain including a glycerol reduction technology and lacking a heterologous FBPase (Y13) or recombinant S. cerevisiae strains including a glycerol reduction technology and expressing a heterologous FBPase under the control of different promoters (Y14, Y15, and Y16). Results are provided as cumulative CO2measured / evolved (in mL at atmospheric pressure) in function of time (hours) and of the strain used.

[0021] Figure 8 compares the metabolites (net ethanol in g / L; net glycerol in g / L and glucose in g / L) measured at the end of fermentation of a control wildtype S. cerevisiae strain (Y1), a recombinant S. cerevisiae strain including a glycerol reduction technology, a heterologous FBPase, and having its native pfk genes (Y15) and recombinant S. cerevisiae strains expressing a glycerol reducing technology, a heterologous FBPase as well as including a deletion in one or more of its native pfk genes (Y17, Y18, and Y19). Results are provided as metabolite measured (in g / L) at the end of fermentation and the strain used.

[0022] Figure 9 compares the CO2production profile of a control wildtype S. cerevisiae strain (Y1), a recombinant S. cerevisiae strain including a glycerol reduction technology, a heterologous FBPase, and having its native pfk genes (Y15) and recombinant S. cerevisiae strains expressing a glycerol reducing technology, a heterologous FBPase as well as including a deletion in one or more of native pfk genes (Y17, Y18, and Y19). Results are provided as cumulative CO2measured / evolved (in mL at atmospheric pressure) in function of time (hours) and of the strain used.

[0023] DETAILED DESCRIPTION

[0024] The present disclosure concerns recombinant yeast host cells for producing at least one fermentation product. As used in the context of the present disclosure, a “fermentation product” is the main substance produced when yeasts convert carbohydrates through the process of fermentation (which, for a yeast from Saccharomyces sp. is usually conducted in anaerobic conditions). The fermentation product can include, without limitation, an alcohol (like ethanol, isopropanol, 2-phenylethanol, 1 -propanol as well as a combination thereof), a ketone (like acetone), a terpene (like farnesene, carotenoid as well as a combination thereof), an organic acid (like 3-hydroxy-propionic acid as, p-coumaric acid well as combination thereof), a phenolic compound (like tyrosol, salidroside as well as a combination thereof), a polyester (like polyhydroxybutyrate), an ester (like ethyl acetate or a fatty acid ethyl ester), an aromatic, an amino acid, a peptide, a polypeptide (including but not limited to an enzyme) as well as combinations thereof. In some embodiments, the fermentation product comprises ethanol. In still other embodiments, the fermentation products comprise ethanol and acetone. In further embodiments, the fermentation products comprise ethanol, acetone and isopropanol.

[0025] ATP futile cycles (also sometimes simply referred to futile cycles) in yeasts are known in the art and have been described in Navas et al. , 1996; Grauslund et al. , 1999; Jardon et al. , 2008; Semkiv et al., 2014; Semkiv et al., 2016; Zahoor et al., 2020; and Yatabe et al., 2023. What has been shown in the Examples below and is disclosed herein is that combining a heterologous ATP futile cycle with another genetic modification for decreasing the production of a fermentation by-product is advantageous as it provide a further increase in the yield of the fermentation product and, in some embodiments, a further decrease in the yield in the fermentation by-product.

[0026] The recombinant yeast host cell of the present disclosure comprises at least one genetic modification for introd ucing / providing a heterologous ATP futile cycle. As used in the context of the present disclosure, a “heterologous ATP futile cycle” refers to a metabolic cycle (e.g., a combination of anabolic (ADP —> ATP) and catabolic (ATP —> ADP) biochemical reactions) that is introduced by the at least one first genetic modification. The presence of the “heterologous ATP futile cycle” results in the hydrolysis of one or more molecules of ATP in the recombinant yeast host cell when compared to a control yeast host cell. The control yeast host cell is identical to the recombinant yeast host cell but lacks the at least one first genetic modification for providing the heterologous ATP futile cycle. As such, the control yeast host cell includes the other genetic modifications (such as the at least one genetic modification in an engineered metabolic pathway for decreasing the production of the fermentation by-product) that may be present in the recombinant yeast host cell. The recombinant yeast host cell, when compared to a control yeast host cell, exhibits an increase in the yield of the at least one fermentation product, engineered metabolic pathway. In an embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% (and in some further embodiments with a total solids content between 32% and 36%)) when compared to a control yeast host cell. The increase in ethanol can be observed when using corn mash as a substrate (with a total solids content between 28% and 37% for example), or a sugarcane must as a substrate (with a total of reducing sugars between 100 and 300 g / L). In an embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.5 g / L or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% (and in some further embodiments with a total solids content between 32% and 36%)) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.3% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.4% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.5% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.6% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.7% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.8% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 0.9% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .0% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .1 % or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .2% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .3% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .4% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .5% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .6% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .7% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .8% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 1 .9% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 2.0% or more in ethanol yield at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 2.5% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 3.0% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 4.0% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 4.5% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 5.0% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 5.5% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation product is ethanol, the recombinant yeast host cell of the present disclosure exhibits an increase of at least 6.0% or more in ethanol yield at the end of the fermentation (when using a sugarcane must as a substrate with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell.

[0027] The heterologous ATP futile cycle can rely on native enzymes / proteins whose expression has been increased / derepressed by the presence of the at least one first genetic modification. In such embodiments, the at least one first genetic modification can include one or more genetic modifications in the regulatory sequence(s) (including the promoter(s) and / orthe terminator(s)) of the native enzymes / proteins so as to increase / derepress their expression to provide the heterologous ATP futile cycle. In another embodiment, the at least one first genetic medication can include the expression of a heterologous transcription factor whose expression will favor the expression of the native enzymes / proteins to provide the heterologous ATP futile cycle. Alternatively, or in combination, the at least one first genetic modification can include the deletion of a native transcription favor whose inactivation will favor the expression of the native enzymes / proteins to provide the heterologous ATP futile cycle.

[0028] The at least one first genetic modification can include the expression of one or more heterologous enzymes / proteins to provide the heterologous ATP futile cycle. In such embodiment, the at least one first genetic modification comprises the introduction of a heterologous nucleic acid molecule encoding the at least one heterologous enzyme / protein.

[0029] In some embodiments, the heterologous ATP futile cycle can rely on at least one native enzyme / protein expressed by the recombinant yeast host cell and on at least another heterologous enzyme / protein (or more than one heterologous enzymes / proteins) whose expression has been allowed by the at least one first genetic modification. More specifically, in such embodiments, the at least one first genetic modification comprises the introduction of a heterologous nucleic acid molecule encoding the at least one heterologous enzyme. Optionally still in such embodiment, the at least one first genetic modification can also comprise one or more genetic modifications in the regulatory sequence(s) (including the promoter(s) and the terminator(s)) and / or the transcription factor(s) regulating the expression of the native enzyme / protein so as to increase / derepress their expression.

[0030] In further embodiments, the heterologous ATP futile cycle can rely on at least two heterologous enzymes / proteins whose expression has been made possible by the presence of the at least one first genetic modification (e.g., by the introduction of one or more heterologous nucleic acid molecules encoding the at least two heterologous enzymes / proteins).

[0031] In an embodiment, in the recombinant yeast host cell of the present disclosure, the heterologous ATP futile cycle can be introduced in a step of the glycolytic pathway. As used in the present disclosure, the “glycolytic pathway” refers to the series of enzymatic reactions which convert glucose to pyruvate. As such, the heterologous ATP futile cycle can use a metabolite of the glycolytic pathway and generate a metabolite of the glycolytic pathway. The metabolite used from the glycolytic pathway can be the same as the metabolite generated in the glycolytic pathway. The metabolite used from the glycolytic pathway can be different from the metabolite generated in the glycolytic pathway.

[0032] In alternative embodiments, in the recombinant yeast host cell of the present disclosure, the heterologous ATP futile cycle can be included outside the glycolytic pathway and use metabolites which are not necessarily associated with the glycolytic pathway.

[0033] During the fermentation process, the recombinant yeast host cells of the present disclosure produce at least one fermentation by-product. As used in the context of the present disclosure, a “fermentation by-product” is another substance than a fermentation product produced when yeasts convert carbohydrates through the process of fermentation (which, for a yeast from Saccharomyces sp. is usually conducted in anaerobic conditions). The generation of the fermentation by-products can reduce the yield of the fermentation product, create stress (and in some embodiments cause oxidative stress) such as a redox imbalance in the recombinant yeast host cell, and / or can cause an increase in the amount of residual carbohydrates (such as, for example, residual glucose) at the end of the fermentation. Fermentation by-products include, without limitation, glycerol, a fusel alcohol (like isoamyl alcohol, 2-methyl-1 -butanol, isobutyl alcohol, 1 -butanol, 1 -pentanol, 1 -hexanol, 2-phenylethanol as well as combinations thereof), an organic acid (like lactic acid, acetic acid, formic acid and combinations thereof), an ester derived from an organic acid (like lactate, acetate, formate and combinations thereof), yeast biomass and combinations thereof. In a specific embodiment, the fermentation byproduct is or comprises glycerol.

[0034] Besides the heterologous ATP futile cycle, the recombinant yeast host cell of the present disclosure also includes at least one genetic modification in an engineered metabolic pathway for decreasing the production of the at least one fermentation by-product. As used in the present disclosure, an “engineered metabolic pathway for decreasing the production of the at least one fermentation by-product” refers to a metabolic pathway (e.g., one or more polypeptides or enzymes) involved (directly or indirectly) in the generation of the fermentation by-product which has been designed to reduce the production of the fermentation by-product. In some embodiments, the engineered metabolic pathway can include a reduction in the expression or the inactivation of one or more native polypeptides / enzymes involved in the production of the fermentation by-product. Alternatively, or in combination, the engineered metabolic pathway can include one or more heterologous polypeptides / enzymes whose activity will decrease the production of the fermentation by-product either directly by generating the fermentation product or indirectly by causing the inhibition in expression / activity of native polypeptide / enzyme involved in the production of the fermentation by-product. In some embodiments, the engineered metabolic pathway is designed to reduce the generation / accumulation of glycerol and can be referred to as a glycerol reduction pathway. What is surprisingly shown in the present application is that the presence of the heterologous ATP futile cycle further reduces the amount of the at least one by-product when combined with the engineered metabolic pathway. In an embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease in glycerol at the end of the fermentation. This decrease in glycerol can be observed when using corn mash as a substrate (with a total solids content between 28% and 37% (and in some further embodiments with a total solids content between 32% and 36%)) or a sugarcane must as a substrate (with a total of reducing sugars between 100 and 300 g / L). In an embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 1 g / L or more in glycerol at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% (and in some further embodiments with a total solids content between 32% and 36%)) when compared to a control yeast host cell. In another embodiment in which the fermentation byproduct is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 10% or more in glycerol at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 15% or more in glycerol at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 20% or more in glycerol at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 25% or more in glycerol at the end of the fermentation (when using corn mash as a substrate with a total solids content between 28% and 37% and in some embodiments between 32% and 36%) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 30% or more in glycerol at the end of the fermentation (when using sugarcane must with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 35% or more in glycerol at the end of the fermentation (when using sugarcane must with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation byproduct is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 40% or more in glycerol at the end of the fermentation (when using sugarcane must with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is glycerol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 45% or more in glycerol at the end of the fermentation (when using sugarcane must with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In an embodiment in which the fermentation by-product is a fusel alcohol (like n-propanol for example), the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 ppm or more in the fusel alcohol (when using corn mash as a substrate with a total solids content between 28% and 37% (and in some further embodiments with a total solids content between 32% and 36%) or sugarcane must with a total of reducing sugars between 100 and 300 g / L) when compared to a control yeast host cell. In another embodiment in which the fermentation by-product is a fusel alcohol, the recombinant yeast host cell of the present disclosure exhibits a decrease of at least 5%, 10%, 15%, 20%, 25% or more in the fusel alcohol at the end of the fermentation when compared to a control yeast host cell.

[0035] In some embodiments, the fermentation kinetic and / or rate of the recombinant yeast host cell comprising the heterologous ATP futile cycle(s) is similar or, in some embodiments, higher than the fermentation kinetic of the control yeast host cell. As used in the context of the present disclosure, the “fermentation kinetic” refers to the rate at which yeasts (recombinant yeast host cells or control yeast host cell for example) convert carbohydrates into the at least one fermentation product. In some embodiments, the fermentation kinetic specifically refers to the conversion of glucose into the at least one fermentation product and carbon dioxide by the recombinant yeast host cell during the fermentation process until the completion of the fermentation (also referred to as the fermentation drop). The skilled person would be able to determine when a fermentation would be considered as completed, for example when available carbon in the fermentation medium is exhausted and / orwhen no fermentation activity is observed from the yeasts in the fermentation medium. In some embodiments, the fermentation kinetic of the recombinant yeast host cell is at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increased when compared to the fermentation kinetic of the control yeast host cell. The fermentation kinetic can be measured using various methods known in the art, including, without limitation, monitoring the specific gravity of the fermentation medium throughout the fermentation process, measuring the amount and / or rate of production of the fermentation product(s), measuring the amount and / or rate of consumption of the carbohydrates (like glucose) in the fermentation medium, and / or measuring the amount and / or rate of production of carbon dioxide during fermentation. Embodiments to measure carbon dioxide during fermentation are well known in the art and include, without limitation, monitoring using an automated CO2 rate flow analyser. The flow rate analyser comprises a custom platform for the fermentation bottles with tubes connected to a needle in the septum of the fermentation bottles. The needle is connected to a mass flow meter via multichannel high-pressure liquid chromatography (HPLC) valves and solenoids to allow for multiplexing the measurement of multiple vessels. A computer program controls the valves and solenoids while recording data for each fermentation bottle individually and continually cycles between all active fermentation bottles. At the end of the fermentation, the collected data is outputted by CO2production rates (mL / min) and the integral of those rates to estimate the total CO2produced during the fermentation (mL).

[0036] Recombinant yeast host cell

[0037] The recombinant yeast host cells of the present disclosure include genetic modifications (at least one to provide a heterologous ATP futile cycle and at least another one in an engineered metabolic pathway to reduce the production of a fermentation by-product). In some embodiments, the recombinant yeast host cell of the present disclosure includes a genetic modification aimed at increasing the expression of a targeted native and / or heterologous gene. In such embodiment, the genetic modification can be the addition of one or more copies of the heterologous gene encoding a heterologous polypeptide / enzyme in the genome of the recombinant yeast host cell. Alternatively, or in combination, the genetic modification can be made in one or more regulatory sequences (e.g., promoter(s) and / or te rm inator(s)) of a native gene to modulate its expression. In such embodiment, the genetic modifications can be made in one, more than one or all copies of the targeted gene(s). Optionally, the recombinant yeast host cell of the present disclosure can include a genetic modification aimed at reducing the expression (an in some embodiments at inactivating) of a native gene (or a combination of native genes). The genetic modification can be made in one, more than one or all copies of the native gene(s). For example, the one or more genetic modifications can be made in one or more regulatory sequences of a native gene to modulate its expression (in one, more than one or all copies to the native gene(s)). In another example, the one or more genetic modifications can be made in the coding region of the native gene(s) by either deleting at least one nucleic acid residue (and in some embodiments, the entire coding region of the native gene(s)) and / or introducing at least one nucleic acid residues (and in some embodiments, introducing another coding region at the genomic position of the native gene(s)). In the context of the present disclosure, when a yeast host cell is qualified as being “recombinant” or “genetically engineered”, it is understood to mean that it has been manipulated to either add at least one or more heterologous nucleic acid residues, substitute and / or remove at least one native nucleic acid residue. In some embodiments, the one or more nucleic acid residues that are added can be derived from a heterologous cell or the recombinant host cell itself. In the latter scenario, the nucleic acid residue(s) is (are) added at a genomic location which is different than the native genomic location. The genetic manipulations did not occur in nature and are the results of in vitro manipulations of the parental yeast host cell.

[0038] The term “heterologous” when used in reference to a nucleic acid molecule (such as a regulatory sequence such as a promoter or a coding region) refers to a nucleic acid molecule that is not natively found in the recombinant host cell. “Heterologous” also includes a native coding region, or portion thereof, that is removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its native location in the organism's genome or as additional copies at its native location. The heterologous nucleic acid molecule is purposively introduced into the recombinant yeast host cell.

[0039] The term “heterologous” when used in reference to a polypeptide refers to a polypeptide that is not natively found in the recombinant yeast host cell or that is expressed from a genomic position that is not native in the recombinant yeast host cell. Thus, for example, a heterologous polypeptide could be derived from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications).

[0040] In some additional embodiments, the present disclosure also provides increasing or reducing the expression of a native gene ortholog of a native gene known to encode a native polypeptide. A “gene ortholog” is understood to be a gene in a different species that evolved from a common ancestral gene by speciation. In the context of the present disclosure, a gene ortholog encodes a polypeptide exhibiting the same biological function as the native polypeptide.

[0041] In some further embodiments, the present disclosure also provides increasing or reducing the expression of a native gene paralog of a native gene known to encode a native polypeptide. A “gene paralog” is understood to be a gene related by duplication within the genome. In the context of the present disclosure, a gene paralog encodes a polypeptide that could exhibit the same biological function than the native polypeptide.

[0042] The heterologous nucleic acid molecule present in the recombinant yeast host cell can be integrated in the host cell’s genome. The term “integrated” as used herein refers to genetic elements that are placed, through molecular biology techniques, into the genome of a recombinant yeast host cell. In some embodiments, the genetic elements can be placed into the chromosome(s) of the recombinant yeast host cell (e.g., be chromosomally integrated). The heterologous nucleic acid molecule(s) can be integrated at a neutral integration site. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination, a double strand break mechanism, Cre-LoxP mediated recombination, delito perfetto, meganuclease-mediated double strand break, MAD7, TALEN, and / or CRISPR / Cas9. Alternatively, or in combination, the genetic elements can be placed into the genome of the recombinant yeast host but outside the chromosome(s) and be independently replicating from the yeast host cell’s chromosome. The heterologous nucleic acid molecule(s) can be introduced in the host cell using a vector. A “vector,” e.g., a “plasmid”, “cosmid” or “artificial chromosome” (such as, for example, a yeast artificial chromosome) refers to an extra chromosomal element and is usually in the form of a circular double-stranded DNA molecule. Such vectors may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source. The heterologous nucleic acid molecule can be present in one or more copies in the recombinant yeast host cell’s chromosome and / or in the recombinant yeast host cell’s genome.

[0043] In some embodiments, heterologous nucleic acid molecules which can be introduced are codon-optimized with respect to the intended 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 the cells of a given organism by replacing at least one, or more than one, codons with one or more codons that are more frequently used in the genes of that organism. In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias 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 which occur most frequently in a reference set of highly expressed genes from an organism. The CAI of codon optimized heterologous nucleic acid molecule described herein corresponds to between about 0.8 and 1 .0, between about 0.8 and 0.9, or about 1 .0.

[0044] The heterologous nucleic acid molecules of the present disclosure can comprise a coding region for one or more heterologous polypeptides to be expressed by the recombinant yeast host cell. The DNA or RNA “coding region” of a heterologous nucleic acid molecule is a DNA or RNA molecule which is transcribed and / or translated into a polypeptide in a cell in vitro or in vivo when placed underthe control of appropriate regulatory sequences. “Suitable regulatory regions” refer 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, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing sites, effector binding sites, stem-loop structures and terminators. The boundaries of the coding region are determined by a start codon at the 5' (amino) terminus, and a translation stop codon at the 3' (carboxyl) terminus. A coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, 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 transcription termination sequence will usually be located 3' to the coding region. In an embodiment, the coding region can be referred to as an open reading frame. “Open reading frame” is abbreviated ORF and means a length of nucleic acid, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0045] The heterologous nucleic acid molecule(s) described herein can comprise a non-coding region, for example a transcriptional and / or translational control regions. “Transcriptional and translational control regions” are DNA regulatory regions, such as promoters, enhancers, 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.

[0046] In the heterologous nucleic acid molecules described herein, the promoter, the terminator and the nucleic acid molecule coding for the one or more polypeptides (such as the one or more enzymes) can be operatively linked to one another. In the context of the present disclosure, the expressions “operatively linked” or “operatively associated” refers to fact that the promoter and / or the terminator is physically associated to the nucleotide acid molecule coding for the one or more polypeptides in a manner that allows, under certain conditions, for expression of the one or more polypeptides from the heterologous nucleic acid molecule. In an embodiment, the promoter (or the combination of promoters) can be located upstream (5’) of the nucleic acid sequence coding for the one or more polypeptides. In still another embodiment, the terminator (or the combination of terminators) can be located downstream (3’) of the nucleic acid sequence coding for the one or more polypeptides. When more than one promoter or terminator is included in the heterologous nucleic acid molecule, each of the promoters or promoters is operatively linked to the nucleic acid sequence coding for the one or more heterologous polypeptides / enzymes.

[0047] “Promoter” refers to a DNA fragment 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 translation of mRNA into a polypeptide. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cells at most times at a substantial similar level are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. A promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of the polymerase. The promoter can be heterologous to the nucleic acid molecule encoding the one or more polypeptides / enzymes. The promoter can be heterologous or derived from a strain being from the same genus or species as the yeast host cell in which it is intended to be introduced. In an embodiment, the promoter is derived from the same genus or species of the recombinant yeast host cell, and the heterologous polypeptide is derived from different genus than the recombinant yeast host cell.

[0048] In some embodiments, the present disclosure concerns the expression of a heterologous polypeptide (including a heterologous enzyme), or a variant thereof (including a fragment thereof) in a recombinant yeast host cell. A variant comprises at least one amino acid difference when compared to the amino acid sequence of the wildtype polypeptide. The polypeptide “variants” have at least 50% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 55% or more identity to the corresponding wildtype heterologous polypeptides described herein.

[0049] The polypeptide “variants” have at least 60% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 65% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 70% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 75% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 80% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 85% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 90% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 95% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 96% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 97% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 98% or more identity to the corresponding wildtype heterologous polypeptides described herein. The polypeptide “variants” have at least 99% or more identity to the corresponding wildtype heterologous polypeptides 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 using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., 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 to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0050] The variants exhibit the biological activity associated with the wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 50% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 55% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 60% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 65% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 70% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 75% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 80% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 85% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 90% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 95% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 96% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 97% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 98% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 99% or more of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. The biological activity of the variants can be determined by methods and assays known in the art.

[0051] The variants described herein may be (i) one in which one or more of the amino acid residues are substituted 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 one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes 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 (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide.

[0052] A “variant” can be a conservative variant or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the polypeptide. 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 polypeptide. For example, the overall charge, structure, or hydrophobic- hydrophilic properties of the polypeptide can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the polypeptide more hydrophobic or hydrophilic, without adversely affecting the biological activity of the polypeptide. A “variant” can be a fragment of a heterologous wildtype polypeptide or fragment of a variant polypeptide. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the corresponding wildtype heterologous polypeptide or of the variant polypeptide. In some embodiments, polypeptide “fragments” have at least at least 50, 100, 200, 300, 400, 500 or more consecutive amino acids of the corresponding wildtype polypeptide or the variant. In some embodiments, the fragment corresponds to the wildtype polypeptide or variant polypeptide to which the signal sequence was removed. In some embodiments, the “fragments” have at least 50% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 55% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 60% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 65% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 70% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 75% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 80% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 85% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 90% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 95% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 96% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 97% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 98% or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, the “fragments” have at least 99%, or more identity to the corresponding wildtype polypeptides or variants. In some embodiments, fragments of the polypeptides can be employed for producing the corresponding full-length enzyme by peptide synthesis. Therefore, the fragments can be employed as intermediates for producing the full-length polypeptide.

[0053] In the context of the present disclosure, the fragments exhibit the biological activity of the heterologous wildtype polypeptide or of the variant polypeptide. In an embodiment, the fragment polypeptide exhibits at least 50% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 55% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 60% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 65% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 70% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 75% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 80% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 85% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 90% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 95% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 96% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 97% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 98% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. In an embodiment, the fragment polypeptide exhibits at least 99% or more of the biological activity of the corresponding heterologous wildtype polypeptide or of the variant. The biological activity of fragments can be determined by methods and assays known in the art.

[0054] The recombinant yeast host cell of the present disclosure has the ability (which can be intrinsic and / or provided or increased by the genetic modifications introduced) to convert a biomass into one or more fermentation products. In some embodiments, the recombinant yeast host cell can be used in the production of an alcohol or a combination of alcohols as the fermentation product(s). Suitable recombinant yeast host cells can be, for example, from the genus Blastobotrys (formely known as Arxula or Sympodiomyces), Candida, Debaryomyces, Hanseniaspora (formely known as Kloeckera), Kazachstania, Komagataella, Kluyveromyces, Ogataea, Pichia (formely known as Hansenula or Issatchenkia), Phaffia, Saccharomyces, Scheffersomyces, Schwanniomyces, or Yarrowia. Suitable yeast species can include, for example, Saccharomyces cerevisiae (including, but not limited to, var. diastaticus), Saccharomyces uvarum, Kazachstania bulderi, Kazachstania barnetti, Kazachstania exigua, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella phaffii, Candida albicans, Candida utilis, Scheffersomyces stipitis, Pichia kudriavzevii, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Blastobotrys adeninivorans, Debaryomyces hansenii, or Schwanniomyces polymorphus. In some embodiments, the host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiments, the host cell can be an oleaginous microalgae host cell (e.g., for example, from the genus Thraustochytrium or Schizochytrium). In an embodiment, the fermenting yeast or recombinant yeast host cell is from the genus Saccharomyces and, in some additional embodiments, from the species Saccharomyces cerevisiae.

[0055] In some embodiments, the recombinant yeast host cell can be used in the production of peptide or a polypeptide as the fermentation product(s). In some embodiments, the yeast is a budding yeast. In other embodiments, the yeast is methylotrophic (e.g., yeast able to utilize methanol as the sole carbon and energy source). Embodiments of methylotrophic yeasts include, but are not limited to Komagataella sp. and Ogataea sp. In some embodiments, the yeast is an oleaginous yeast (e.g., a yeast capable of accumulating more than 20% of its dry cell weight as lipids or triglycerides). In an embodiment, the recombinant yeast host cell is from Blastobotrys sp. In another embodiment, the recombinant yeast host cell is from Blastobotrys adeninivorans (basonym Trichosporon adeninivorans). In an embodiment, the recombinant microbial yeast host cell is from Candida sp. In another embodiment, the recombinant yeast host cell is from Candida albicans. In an embodiment, the recombinant microbial yeast host cell is from Cyberlindnera sp. In another embodiment, the recombinant yeast host cell is from Cyberlindnera jadinii (basonym Saccharomyces jadinii). In an embodiment, the recombinant yeast host cell is from the Debaryomyces sp. In another embodiment, the recombinant yeast host cell is from Debaryomyces hansenii. In another embodiment, the recombinant yeast host cell is from Debaryomyces hansenii. In an embodiment, the recombinant yeast host cell is from Hanseniaspora sp. (also known as Kloeckera sp.). In another embodiment, the recombinant yeast host cell is from Hanseniaspora guilliermondii. In another embodiment, the recombinant yeast host cell is from Hanseniaspora pseudoguilliermondii. In an embodiment, the recombinant yeast host cell is from the Kazachstania sp. In another embodiment, the recombinant yeast host cell is from Kazachstania bulderi (basonym Saccharomyces bulderi). In another embodiment, the recombinant yeast host cell is from Kazachstania barnettii (basonym Saccharomyces barnettii). In another embodiment, the recombinant yeast host cell is from Kazachstania exigua (basonym Saccharomyces exiguus). In an embodiment, the recombinant yeast host cell is from Kluyveromyces sp. In another embodiment, the recombinant yeast host cell is from Kluyveromyces lactis (basonym Torulaspora lactis). In another embodiment, the recombinant yeast host cell is from Kluyveromyces marxianus also known as Kluyveromyces fragilis (basonym Saccharomyces marxianus). In an embodiment, the recombinant microbial yeast host cell is from Komagataella sp. In another embodiment, the recombinant yeast host cell is from Komagataella phaffii. In an embodiment, the recombinant yeast host cell is from Limtongozyma sp. In another embodiment, the recombinant yeast host cell is from Limtongozyma cylindracea (basonym Candida cylindracea). In an embodiment, the recombinant yeast host cell is from Lipomyces sp. In an embodiment, the recombinant yeast host cell is from Metschnikowia sp. In another embodiment, the recombinant yeast host cell is from Metschnikowia sinensis. In another embodiment, the recombinant microbial host cell / microbial cell yeast host cell is from Metschnikowia fructicola. In another embodiment, the recombinant yeast host cell is from Metschnikowia pulcherrima. In another embodiment, the recombinant yeast host cell is from Metschnikowia zobellii. In another embodiment, the recombinant yeast host cell is from Metschnikowia shanxiensis. In an embodiment, the recombinant yeast host cell is from Ogataea sp. In another embodiment, the recombinant yeast host cell is from Ogataea polymorpha (basonym Hansenula polymorpha). In another embodiment, the recombinant yeast host cell is from Ogataea methanolica (basonym Pichia methanolica). In an embodiment, the recombinant yeast host cell is from Pichia sp. (also known as Hansenula sp.). In an embodiment, the recombinant yeast host cell is from Rasamsonia sp. In another embodiment, the recombinant yeast host cell is from Rasamsonia emersonii. In an embodiment, the recombinant yeast host cell is from Saccharomyces sp. In another embodiment, the recombinant yeast host cell is from Saccharomyces cerevisiae. In yet another embodiment, the recombinant yeast host cell is from Saccharomyces cerevisiae var. diastaticus. In another embodiment, the recombinant yeast host cell is from Saccharomyces uvarum. In another embodiment, the recombinant yeast host cell is from Saccharomyces boulardii. In an embodiment, the recombinant yeast host cell is from Scheffersomyces sp. In another embodiment, the recombinant yeast host cell is from Scheffersomyces stipitis (basonym Pichia stipitis). In an embodiment, the recombinant yeast host cell is from Schwanniomyces sp. In another embodiment, the recombinant yeast host cell is from Schwanniomyces polymorphus (basonym Pichia polymorpha). In another embodiment, the recombinant yeast host cell is from Schwanniomyces occidentalis. In an embodiment, the recombinant yeast host cell is from Wickerhamomyces sp. In another embodiment, the recombinant yeast host cell is from Wickerhamomyces anomalus. In an embodiment, the recombinant yeast host cell is from Yarrowia sp. In another embodiment, the recombinant yeast host cell is from Yarrowia lipolytica.

[0056] The present disclosure provides methods for making the recombinant yeast host cell as described herein. Broadly, the method comprises introducing at least one first genetic modification for providing a heterologous ATP futile cycle as well as at least one second genetic modification to provide an engineered metabolic pathway for decreasing the production of the fermentation by-product, in any order or at the same time, in a parental yeast cell to obtain the recombinant yeast cell of the present disclosure. In some embodiments, the method can include introducing one or more heterologous nucleic acid molecules in a parental yeast cell to obtain the recombinant yeast host cell.

[0057] The present disclosure provides methods for the propagation of recombinant yeast cells of the present disclosure and ultimately the formulation of propagated recombinant yeast cells. In the propagation process, the recombinant yeast cell is placed in a culture medium under suitable conditions for cell growth. The culture medium can comprise a carbon source (such as, for example, molasses, sucrose, glucose, dextrose syrup, ethanol, corn, glycerol, corn steep liquor and / or a lignocellulosic biomass), a nitrogen source (such as, for example, ammonia or another inorganic source of nitrogen) and a phosphorous source (such as, for example, phosphoric acid or another inorganic source of phosphorous). The culture medium can further comprise additional micronutrients such as vitamins and / or minerals to support the propagation of the recombinant yeast cell.

[0058] The propagation can be conducted under conditions to allow cell growth and the accumulation of yeast biomass as well as to limit fermentation product production (and in some embodiments to limit ethanol production during propagation). The propagation process can be conducted in aerobic conditions. The propagation process can be conducted at a specific pH and / or a specific temperature to favor propagation. In embodiments in which the recombinant yeast cell is from the genus Saccharomyces, the process can comprise controlling the pH of the propagation medium to between about 3.0 to about 6.0, about 3.5 to about 5.5 or about 4.0 to about 5.5. In a specific embodiment, the pH is controlled at about 4.5. In another example, in embodiments in which the recombinant yeast cell is from the genus Saccharomyces, the process can comprise controlling the temperature of the propagation medium between about 20°C to about 40°C, about 25°C to about 30°C or about 30°C to about 35°C. In a specific embodiment, the temperature is controlled at between about 30°C to about 35°C (32°C for example).

[0059] In embodiments in which the heterologous ATP futile cycle is used to limit yeast biomass as a by-product, it may be advantageous to design the recombinant yeast host cell so that the heterologous ATP futile cycle be inactive during the propagation step and only active during the fermentation step. This can be done, for example, by using one or more promoters that limit (or avoid) expression of the native and / or heterologous polypeptides / enzymes of the ATP futile cycle during the propagation step and allows (or favors) the expression during the fermentation step. The process for propagating the recombinant yeast host cell can include a formulation step, e.g., a step of removing some or the majority of the water used during the propagation process. For example, the formulation step can include a step of dehydrating, filtering (including ultrafiltrating) and / or centrifuging the propagated recombinant yeast cell. The formulation step can include providing the recombinant yeast cells in the form of a cream and, in some additional embodiments, in the form of a stabilized liquid yeast formulation. The formulation can optionally include drying the propagated recombinant yeast cell to provide it in a dried form (active dry yeast or instant dry yeast). The drying step, when present, can include, for example, rotatory drum drying, spray-drying and / or fluid-bed drying.

[0060] Heterologous ATP futile cycles

[0061] As indicated above, the recombinant yeast host cell of the present disclosure comprises a heterologous ATP futile cycle (which, in some embodiments, is introduced in the yeast’s glycolytic pathway) which is provided by the one or more first genetic modifications introduced. In some embodiments, the heterologous ATP futile cycle comprises a heterologous enzyme or a combination of heterologous enzymes. Prior to the introduction of the at least one first genetic modification, the recombinant yeast host cell does not include an heterologous ATP futile cycle.

[0062] In Figures 1A to 1 C, an embodiment of a native yeast glycolytic pathway is provided:

[0063] - An enzyme having hexokinase activity (HXK) catalyses the formation of glucose- 6-phosphate (G6P) from glucose. In this reaction ATP is converted into ADP.

[0064] - An enzyme having phosphoglucoisomerase activity (PGI1) catalyses the formation of fructose-6-phosphate (F6P) from glucose-6-phosphate (G6P).

[0065] - Enzymes having phosphofructokinase activity (PFK1 / 2) catalyse the formation of fructose-1 ,6-bisphohate (F16P) from fructose-6-phosphate (F6P). In this reaction ATP is converted into ADP.

[0066] - An enzyme having fructose-1 ,6-bisphosphate aldolase activity (FBA1) catalyses the formation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3- phosphate (GAP) from fructose-1 ,6-bisphohate (F16P).

[0067] - An enzyme having triose phosphate isomerase activity (TPI1) catalyses the formation of glyceraldehyde-3-phosphate (GAP) from dihydroxyacetone phosphate (DHAP).

[0068] - Enzymes having glycerol-3-phosphate dehydrogenase activity (GPD1 / 2) catalyse the formation of glycerol-3-phosphate from dihydroxyacetone phosphate (DHAP). In this reaction, NADH is converted to NAD+.

[0069] - Enzymes having glycerol-1 -phosphohydrolase activity (GPP1 / GPP2) catalyse the formation of glycerol from glycerol-3-phosphate. - Enzymes having triose-phosphate activity (TDH1 / 2 / 3) catalyse the formation of 1 ,3 bis-phosphoglycerate (1 ,3-BPG) from glyceraldehyde-3-phosphate (GAP). In this reaction, NAD+ is converted to NADH.

[0070] - An enzyme having 3-phosphoglycerate kinase activity (PGK1) catalyses the formation of 3-phosphoglycerate (3-PG) from 1 ,3 bis-phosphoglycerate (1 ,3-BPG). In this reaction ADP is converted into ATP.

[0071] - An enzyme having glycerate phosphomutase activity (GPM1) (also known as phosphoglycerate mutase) catalyses the formation of 2-phosphoglycerate (2-PG) from 3-phosphoglycerate (3-PG).

[0072] - Enzymes having enolase activity (ENO1 / 2) catalyse the formation of phosphoenolpyruvate (PEP) from 2-phosphoglycerate (2-PG).

[0073] - Enzymes having pyruvate kinase activity (PYK1 / 2) catalyse the formation of pyruvate from phosphoenolpyruvate (PEP). In this reaction ADP is converted into ATP.

[0074] An embodiment of a heterologous enzyme which can be used to provide a heterologous ATP futile cycle is shown in Figure 1A. In such embodiment, a heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is introduced. The heterologous polypeptide having fructose-1 ,6-bisphosphatase activity catalyses the formation of fructose-6-phosphate (F6P) from fructose-1 ,6-biphosphate (F16BP). The native or heterologous phosphofructokinase(s) e.g., pfk1 / 2 on Figure 1A) catalyse the formation fructose-1 ,6-biphosphate (F16BP) from fructose-6-phosphate (F6P) and by doing so require the conversion of ATP into ADP. The introduction of a heterologous polypeptide having fructose-1 ,6-bisphosphatase activity creates a heterologous ATP futile cycle since it favors the hydrolysis of ATP into ADP.

[0075] One embodiment of a heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is a fructose-1 ,6-bisphosphatase (also known as fructose-1 ,6-diphosphatase, FBPase or FDPase). Fructose-1 ,6-bisphosphatases are enzymes associated with gluconeogenesis and are classified under E.C. 3.1.3.11. In some embodiments, the heterologous FBPase can be encoded by a fbp gene (such as, for example, from a fbp1, fbp2, fbp3, fbp4 or fbp5 gene). In some further embodiments, the heterologous FBPase can be encoded by a fbp1 gene. In some embodiments, the FBPase can have specificity towards a single substrate (single specificity), e.g., fructose-1 ,6-bisphophate. In additional embodiments, the FBPase can have specificity towards at least two substrates (dual specificity), e.g., fructose-1 ,6-bisphophate and sedoheptulose-1 ,7-bisphosphate. The heterologous FBPase can be of eukaryotic origin. The heterologous FBPase can also be of prokaryotic origin. In some embodiments, the heterologous FBPase is derived from Yarrowia sp., and, in some additional embodiments, the heterologous FBPase is derived from Yarrowia lipolytica. In additional embodiments, the heterologous FBPase has the amino acid sequence of SEQ ID NO: 3 or is a variant of the amino acid sequence of SEQ ID NO: 3 having fructose-1 ,6-bisphosphatase activity. In some further embodiments, the heterologous FBPase is encoded by the nucleic acid sequence of SEQ ID NO: 4 or is a variant of the nucleic acid sequence of SEQ ID NO: 4 encoding a polypeptide having fructose-1 ,6-bisphosphatase activity (such as, for example, a degenerate nucleic acid molecule encoding the polypeptide having the amino acid sequence of SEQ ID NO: 3 or encoding a variant of the amino acid sequence of SEQ ID NO: 3 having fructose-1 ,6- bisphosphatase activity).

[0076] Another embodiment of a heterologous polypeptide having fructose-1 ,6-bisphosphatase activity is a pyrophosphate-dependent phosphofructokinase (also known as PPi-PFK). PPi- PFK are enzymes associated with gluconeogenesis and are classified under E.C. 2.7.1.90. In some embodiments, the heterologous PPi-PFK can be encoded by a pfk gene. In some embodiments, the PPi-PFK can have specificity towards a single substrate (single specificity), e.g., fructose-1 ,6-bisphophate. In additional embodiments, the PPi-PFK can have specificity towards at least two substrates (dual specificity), e.g., fructose-1 ,6-bisphophate and sedoheptulose-1 ,7-bisphosphate. The heterologous PPi-PFK can also be of eukaryotic origin. The heterologous PPi-PFK can also be of prokaryotic origin. In some embodiments, the heterologous PPi-PFK is derived from Xanthomonas sp., and, in some additional embodiments, the heterologous PPi-PFK is derived from Xanthomonas campestris. In additional embodiments, the heterologous PPi-PFK has the amino acid sequence of SEQ ID NO: 5 or is a variant of the amino acid sequence of SEQ ID NO: 5 having fructose-1 ,6- bisphosphatase activity. In some further embodiments, the heterologous PPi-PFK is encoded by the nucleic acid sequence of SEQ ID NO: 6 or is a variant of the nucleic acid sequence of SEQ ID NO: 6 encoding a polypeptide having fructose-1 ,6-bisphosphatase activity (such as, for example, a degenerate nucleic acid molecule encoding the polypeptide having the amino acid sequence of SEQ ID NO: 5 or encoding a variant of the amino acid sequence of SEQ ID NO: 5 having fructose-1 ,6-bisphosphatase activity). In some embodiments, the heterologous PPi-PFK is derived from Porphyromonas sp., and, in some additional embodiments, the heterologous PPi-PFK is derived from Porphyromonas gingivalis. In additional embodiments, the heterologous PPi-PFK has the amino acid sequence of SEQ ID NO: 7 or is a variant of the amino acid sequence of SEQ ID NO: 7 having fructose-1 ,6-bisphosphatase activity. In some further embodiments, the heterologous PPi-PFK is encoded by the nucleic acid sequence of SEQ ID NO: 8 or is a variant of the nucleic acid sequence of SEQ ID NO: 8 encoding a polypeptide having fructose-1 ,6-bisphosphatase activity (such as, for example, a degenerate nucleic acid molecule encoding the polypeptide having the amino acid sequence of SEQ ID NO: 7 or encoding a variant of the amino acid sequence of SEQ ID NO: 7 having fructose-1 ,6- bisphosphatase activity). Another embodiment of a combination of heterologous enzymes which can be used to create a heterologous ATP futile cycle is shown in Figure 1 B. In such embodiment, a polypeptide having pyruvate carboxylase activity (PYC, PYC2 for example) and a polypeptide having phosphoenolpyruvate carboxykinase activity (PCK, PCK1 for example) are present (and at least one of the polypeptides or both polypeptides are heterologous). The polypeptide having pyruvate carboxylase activity catalyses the formation of oxaloacetate (OAA) from pyruvate (PYR). In this reaction ATP is converted into ADP. The polypeptide having phosphoenolpyruvate carboxykinase activity (PCK) catalyses the formation of phosphoenolpyruvate (PEP) from oxaloacetate (OAA). In this reaction, ATP is converted into ADP. The combination of a polypeptide having pyruvate carboxylase activity (PYC) and a polypeptide having phosphoenolpyruvate carboxykinase activity (PCK) creates a heterologous ATP futile cycle since it favors the hydrolysis of ATP into ADP. Pyruvate carboxylases are associated with gluconeogenesis and belong to E.C. 6.4.1 .1 . The polypeptide having pyruvate carboxylase activity can be of eukaryotic origin. The polypeptide having pyruvate carboxylase activity can be of prokaryotic origin. Phosphoenolpyruvate carboxykinases are associated with gluconeogenesis and belong to E.C. 4.1.1.31. The polypeptide having phosphoenolpyruvate carboxykinase activity can be of eukaryotic origin. The polypeptide having phosphoenolpyruvate carboxykinase activity can be of prokaryotic origin. In some embodiments, recombinant yeast host cells comprising the combination of polypeptides having pyruvate carboxylase activity (PYC) and polypeptides having phosphoenolpyruvate carboxykinase activity (PCK) can also comprise a native / heterologous polypeptide having pyruvate kinase activity (PYK2 for example). Polypeptides having pyruvate kinase activity catalyse the formation of pyruvate from phosphoenolpyruvate. In this reaction, ADP is converted into ATP. Polypeptides having pyruvate kinase activity are associated with gluconeogenesis and belong to E.C. 2.7.1.40. The polypeptide having pyruvate kinase activity can be of eukaryotic origin. The polypeptide having pyruvate kinase activity can be of prokaryotic origin.

[0077] A further embodiment that can be used to create a heterologous ATP futile cycle is shown in Figure 1 C. In such embodiment, a heterologous polypeptide having glycerol kinase activity (GUT1 for example) is introduced in the recombinant yeast host cell. Alternatively, or in combination, the native / heterologous polypeptide having glycerol kinase activity is expressed under the control of a constitutive promoter. The polypeptide having glycerol kinase activity catalyses the formation of glycerol-3-phosphate from glycerol. In this reaction ATP is converted into ADP. The polypeptide having glycerol kinase activity creates a heterologous ATP futile cycle since it favors the hydrolysis of ATP into ADP. Glycerol kinases are associated with gluconeogenesis and belong to E.C. 2.7.1.30. The glycerol kinase can be GUT1 and be encoded by the gut1 gene. The glycerol kinase can be GLPK and be encoded by a glpk gene. The glycerol kinase can be GK and be encoded by the g / rgene. The glycerol kinase can be of prokaryotic origin. In some embodiments, the glycerol kinase can be from Escherichia sp. and, in further embodiments, from Escherichia coli (UniProt: P0A6F3 for example). In some embodiments, the glycerol kinase can be from Bacillus sp. and, in further embodiments, from Bacillus substilis (UniProt: P18157 for example). In some embodiments, the glycerol kinase can be from Klebsiella sp. and, in further embodiments, from Klebsielle pneumoniae. In some embodiments, the glycerol kinase can be from Corynebacterium sp. and, in further embodiments, from Corynebacterium glutamicum (UniProt: Q8NLP9 for example). In some embodiments, the glycerol kinase can be from Pseudomonas sp. and, in further embodiments, from Pseudomonas aeruginosa (GenBank Accession Number Q9HY41 for example). The glycerol kinase can be of eukaryotic origin. In some embodiments, the glycerol kinase can be from Saccharomyces sp., and in further embodiments, from Saccharomyces cerevisiae. In additional embodiments, the glycerol kinase has the amino acid sequence of SEQ ID NO: 55 or is a variant of the amino acid sequence of SEQ ID NO: 55 having glycerol kinase activity. In some further embodiments, the glycerol kinase is encoded by the nucleic acid sequence of SEQ ID NO: 54 or is a variant of the nucleic acid sequence of SEQ ID NO: 54 encoding a polypeptide having glycerol kinase activity (such as, for example, a degenerate nucleic acid molecule encoding the polypeptide having the amino acid sequence of SEQ ID NO: 55 or encoding a variant of the amino acid sequence of SEQ ID NO: 55 having glycerol kinase activity). In some embodiments, the glycerol kinase can be from Streptomyces sp., and in further embodiments, from Streptomyces coelicolor (UniProt: Q9RJM2 for example). In some embodiments, the glycerol kinase can be from Thermus sp., and in further embodiments, from Thermus thermophilus (UniProt: Q53W24 for example). In some embodiments, the glycerol kinase can be from Homo sp., and in further embodiments, from Homo sapiens (UniProt: P32189 for example). In some embodiments, the glycerol kinase can be from Kluyveromyces sp., and in further embodiments, from Kluyveromyces lactis (GenBank Accession Q6CQ16 for example). ). In some embodiments, the glycerol kinase can be from Candida sp., and in further embodiments, from Candida albicans (GenBank Accession Q59KQ1 for example).Additional embodiments of the glycerol kinase include, without limitation Ambrosiozyma monospora (GenBank Accession Number GMG28666.1), Arxiozyma heterogenica (GenBank Accession Number KAK5782502.1), Australozyma saopauloensis (GenBank Accession Number XP_062876821 .1), Babjeviella inositovora (GenBank Accession Number XP_018985937.1), Brettanomyces bruxellensis (GenBank Accession Number XP_041135150.1), Brettanomyces naardenensis (GenBank Accession Number VEU20496.1), Brettanomyces nanus (GenBank Accession Number XP_038780845.1), Candida albicans (GenBank Accession Number EEQ43613.1), Candida arabinofermentans (GenBank Accession Number ODV85139.1), Candida boidinii (GenBank Accession Number GME89097.1 or OWB74874.1), Candida jaroonii (GenBank Accession Number CAH6721655.1), Candida metapsilosis (GenBank Accession Number XP_067549709.1), Candida oxycetoniae (GenBank Accession Number XP_049181824.1), Candida railenensis (GenBank Accession Number CAH2355137.1), Candida subhashii (GenBank Accession Number XP_049266066.1), Candida tropicalis (GenBank Accession Number ACK58624.1), Candida verbasci (GenBank Accession Number CAI5755906.1), Candidozyma auris (GenBank Accession Number QEL62856.1), Candidozyma duobushaemuli (GenBank Accession NumberXP_025339358.1), Candidozyma haemuli (GenBank Accession Number XP_025341769.1), Clavispora lusitaniae (GenBank Accession Number KAF5211893.1), Cyberlindnera fabianii (GenBank Accession Number ONH69976.1), Cyberlindnera jadinii (GenBank Accession Number CEP23419.1), Debaryomyces fabryi (GenBank Accession Number CUM54781 .1), Diutina rugosa (GenBank Accession Number XP_034012387.1), Eremothecium cymbalariae (GenBank Accession Number XP_003648501 .1), Eremothecium gossypii (GenBank Accession Number NP_983335.2), Eremothecium sinecaudum (GenBank Accession Number XP_017988013.1), Hanseniaspora osmophila (GenBank Accession Number OEJ85283.1), Henningerozyma blattae (GenBank Accession Number XP_004180713.1), Huiozyma naganishii (GenBank Accession Number XP_022466616.1), Hyphopichia burtonii (GenBank Accession Number XP_020076459.1), Kazachstania africana (GenBank Accession Number XP_003959067.1), Kluyveromyces dobzhanskii (GenBank Accession Number CDO94124.1), Kluyveromyces lactis (GenBank Accession Number XP_453973.1), Kluyveromyces marxianus (GenBank Accession Number QGN17983.1), Komagataella phaffii (GenBank Accession Number XP_002494228.1), Kuraishia capsulata (GenBank Accession Number XP_022459053.1), Lachancea dasiensis (GenBank Accession Number SCU93386.1), Lachancea fermentati (GenBank Accession Number SCW01112.1), Lachancea lanzarotensis (GenBank Accession Number XP_022626813.1), Lachancea meyersii (GenBank Accession Number SCV04269.1), Lachancea mirantina (GenBank Accession Number SCV03132.1), Lachancea nothofagi (GenBank Accession Number SCV00958.1), Lachancea sp. 'fantastica' (GenBank Accession Number SCU84894.1), Lachancea thermotolerans (GenBank Accession Number XP_002553727.1), Lodderomyces elongisporus (GenBank Accession Number EDK45282.1), Maudiozyma barnettii (GenBank Accession Number XP_041405031 .1), Maudiozyma exigua (GenBank Accession Number KAG0657130.1), Maudiozyma humilis (GenBank Accession Number GMM58502.1), Maudiozyma saulgeensis (GenBank Accession Number SMN20460.1), Metschnikowia aff. pulcherrima (GenBank Accession Number QBM88691 .1), Metschnikowia bicuspidata (GenBank Accession Number RKP31626.1), Metschnikowia sp. (GenBank Accession Number GEQ68057.1), Meyerozyma sp. (GenBank Accession Number RLV86499.1), Millerozyma farinosa (GenBank Accession Number CCE79961.1), Monosporozyma servazzii (GenBank Accession Number CAL9735608.1), Monosporozyma unispora (GenBank Accession Number KAG0654880.1), Nakaseomyces bracarensis (GenBank Accession Number XP_070908693.1), Nakaseomyces glabratus (GenBank Accession Number XP_445857.1), Nakaseomyces glabratus (GenBank Accession Number KTB15035.1), Naumovozyma castellii (GenBank Accession Number XP_003675944.1), Naumovozyma dairenensis (GenBank Accession Number XP_003671439.1), Ogataea parapolymorpha (GenBank Accession Number KAG7885669.1), Ogataea philodendri (GenBank Accession Number XP_046062722.1), Ogataea polymorpha (GenBank Accession Number XP_018210634.1), Pachysolen tannophilus (GenBank Accession Number ODV95577.1), Priceomyces carsonii (XP_062812346.1), Saccharomyces cerevisiae (GenBank Accession Number GFP68323.1 , CAI6705653.1 , KAJ1046306.1 , KAJ1546157.1 , CAI7336891 .1 , EDZ71841.1 , NP_011831 .1), or EGA78648.1), Saccharomycodes ludwigii (GenBank Accession Number SSD61217.1), Saccharomycopsis crataegensis (GenBank Accession Number XP_064853249.1), Scheffersomyces coipomensis (GenBank Accession Number XP_064759409.1), Scheffersomyces spartinae (GenBank Accession Number XP_043050940.1), Scheffersomyces stipitis (GenBank Accession Number XP_001386679.2), Scheffersomyces xylosifermentans (GenBank Accession Number XP_064777089.1), Spathaspora passalidarum (GenBank Accession Number XP_007373787.1), Spathaspora sp. (GenBank Accession Number RLV95360.1), Suhomyces tanzawaensis (GenBank Accession Number XP_020065144.1), Sungouiella intermedia (GenBank Accession Number SGZ55650.1), Tetrapisispora phaffii (GenBank Accession Number XP_003687792.1), Torulaspora delbrueckii (GenBank Accession Number XP_003682760.1), Torulaspora globosa (GenBank Accession Number QLQ77929.1), Vanderwaltozyma polyspora (GenBank Accession Number XP_001643979.1), Wickerhamomyces anomalus (GenBank Accession Number XP_019039547.1), Wickerhamomyces ciferrii (GenBank Accession Number XP_011273316.1), Wickerhamomyces ciferrii (GenBank Accession Number

[0078] XP_011273652.1), Wickerhamomyces mucosus (GenBank Accession Number

[0079] KAH3676417.1), Wickerhamomyces pijperi (GenBank Accession Number KAH3688356.1), Yamadazyma tenuis (GenBank Accession Number XP_006690151 .1), or Zygosaccharomyces bailii (GenBank Accession Number SJM85937.1). Further embodiments of the glycerol kinase include, without limitation Saccharomyces cerevisiae (Uniprot P32190), Derxia gummosa (Uniprot A0A8B6X5W0), Diplodia seriata (Uniprot A0A0G2EA57), Cyberlindnera jadinii (Uniprot A0A0H5CFQ4), Metarhizium rileyi (Uniprot A0A162M6S4), Sugiyamaella lignohabitans (Uniprot A0A167EEH8), Pseudogymnoascus destructans (Uniprot A0A177A1 H5), Komagataella pastoris (Uniprot A0A1 B2JH13), Pseudogymnoascus verrucosus (Uniprot A0A1 B8GKW2), Phaffia rhodozyma (Uniprot A0A1C9UKZ1), Diplodia seriata (Uniprot A0A1S8B3K4), Rhizopus stolonifer (Uniprot A0A367IVA9), Rhizopus azygosporus (Uniprot A0A367IXJ7), Rhizopus azygosporus (Uniprot A0A367J0H1), Rhizopus stolonifer (Uniprot A0A367J4Q4), Rhizopus azygosporus (Uniprot A0A367JCC8), Rhizopus azygosporus (Uniprot A0A367JVU8), Rhizopus azygosporus (Uniprot A0A367K8F4), Rhizopus stolonifer (Uniprot A0A367KN77), Rhizopus stolonifer (Uniprot A0A367KV54), Rhizopus stolonifer (Uniprot A0A367KWD1), Candida viswanathii (Uniprot A0A367Y0F6), Candida viswanathii (Uniprot A0A367YA67), Coniochaeta pulveracea (Uniprot A0A420YMM0), Coniochaeta pulveracea (Uniprot A0A420YN84), Apiotrichum porosum (Uniprot A0A427Y5K1), Apiotrichum porosum (Uniprot A0A427Y8X3), Saitozyma podzolica (Uniprot A0A427YEB8), Zygosaccharomyces mellis (Uniprot A0A4C2EBZ5), Steccherinum ochraceum (Uniprot A0A4R0RCE7), Aspergillus tanner! (Uniprot A0A4S3J0P8), Synchytrium microbalum (Uniprot A0A507BR84), Synchytrium endobioticum (Uniprot A0A507D971), Powellomyces hirtus (Uniprot A0A507DVT6), Chytriomyces confervae (Uniprot A0A507F5Q9), Monascus purpureus (Uniprot A0A507QM95), Monascus purpureus (Uniprot A0A507QMC3), Puccinia graminis f sp. tritici (Uniprot A0A5B0P5M6, A0A5B0Q459, or A0A5B0RCN6), Metarhizium rileyi (Uniprot A0A5C6G3Q4), Aspergillus tanner! (Uniprot A0A5M9MVQ4), Petromyces alliaceus (Uniprot A0A5N7CQA5), Saccharomyces pastorianus (Uniprot A0A6C1 DUE3), Saccharomyces pastorianus (Uniprot A0A6C1 E7W0), Aspergillus chevalier! (Uniprot A0A7R7VEL3), Aspergillus puulaauensis (Uniprot A0A7R7XM78), Aspergillus kawachii (Uniprot A0A7R8A0H1), Aspergillus puulaauensis (Uniprot A0A7R8AP70), Derxia gummosa DSM 723 (Uniprot A0A8B6X3X2), Alectoria fallacina (Uniprot A0A8H3EX64), Heterodermia speciosa (Uniprot A0A8H3F0E3), Imshaugia aleurites (Uniprot A0A8H3FHB4), Petromyces alliaceus (Uniprot A0A8H6A678), Apophysomyces ossiformis (Uniprot A0A8H7BJ60), Apophysomyces ossiformis (Uniprot A0A8H7BPG8), Apophysomyces ossiformis (Uniprot A0A8H7BZ70), Apophysomyces ossiformis (Uniprot A0A8H7EVA3), Lyophyllum shimeji (Uniprot A0A9P3PSB8), Curvularia kusanoi (Uniprot A0A9P4TMJ5), Didymella heteroderae (Uniprot A0A9P4WS97), Linnemannia schmuckeri (Uniprot A0A9P5S606), Podila minutissima (Uniprot A0A9P5VIE0), Mortierella hygrophila (Uniprot A0A9P6FFI8), Lunasporangiospora selenospora (Uniprot A0A9P6FVG8), Mortierella alpina (Uniprot A0A9P6JAQ3), Entomortierella chlamydospora (Uniprot A0A9P6MS94), Actinomortierella ambigua (Uniprot A0A9P6PXD4), Mortierella polycephala (Uniprot A0A9P6Q2E8), Modicella reniformis (Uniprot A0A9P6STV9), Linnemannia gamsii (Uniprot A0A9P6ULG3), Dissophora globulifera (Uniprot A0A9P6UNF7), Maudiozyma exigua (Uniprot A0A9P6VWQ2), Rhodotorula mucilaginosa (Uniprot A0A9P6VYN0), Scheffersomyces spartinae (Uniprot A0A9P7VCP3), Linnemannia hyalina (Uniprot A0A9P7XM96), Purpureocillium takamizusanense (Uniprot A0A9Q8QKL6), Purpureocillium takamizusanense (Uniprot A0A9Q8VA66), Coemansia erecta (A0A9W7XUQ8), Tieghemiomyces parasiticus (Uniprot A0A9W7ZRE2), Mycoemilia scoparia (Uniprot A0A9W7ZVP8), Tieghemiomyces parasiticus (Uniprot A0A9W8A923), Dispira parvispora (Uniprot A0A9W8ARV8), Coemansia asiatica (Uniprot A0A9W8CHC6), Coemansia biformis (Uniprot A0A9W8CUS0), Coemansia thaxteri (Uniprot A0A9W8EKM8), Coemansia pectinata (Uniprot A0A9W8H1 K6), Coemansia javaensis (Uniprot A0A9W8H980), Coemansia guatemalensis (Uniprot A0A9W8HUE2), Coemansia brasiliensis (Uniprot A0A9W8I439), Coemansia spiralis (Uniprot A0A9W8KZE0), Coemansia aciculifera (Uniprot A0A9W8M5C4), Fusarium irregulare (Uniprot A0A9W8PFZ8), Fusarium falciforme (Uniprot A0A9W8R2P0), Fusarium falciforme (Uniprot A0A9W8RDQ1), Fusarium torreyae (Uniprot A0A9W8S4A7), Fusarium torreyae (Uniprot A0A9W8S5B6), Fusarium piperis (Uniprot A0A9W8TEV6), Fusarium irregulare (Uniprot A0A9W8U5M3), Fusarium piperis (Uniprot A0A9W8W4D4), Didymella glomerata (Uniprot A0A9W8WZ53), Gnomoniopsis smithogilvyi (Uniprot A0A9W8YXJ6), Didymella pomorum (Uniprot A0A9W8ZDS7), Didymosphaeria variabile (Uniprot A0A9W9CET9), Neocucurbitaria cava (Uniprot A0A9W9CN68), Cladophialophora chaetospira (Uniprot A0AA38X2N2), Knufia peltigerae (Uniprot A0AA38XXF6), Knufia peltigerae (Uniprot A0AA38XZ19), Cladophialophora chaetospira (Uniprot A0AA39CCC0), Knufia peltigerae (Uniprot A0AA39CSF9), Ramalina farinacea (Uniprot A0AA43U0Z4), Cutaneotrichosporon cavernicola (Uniprot A0AA48I524), Cutaneotrichosporon cavernicola (Uniprot A0AA48L4J0), Cutaneotrichosporon spelunceum (Uniprot A0AAD3Y7Z5), Cutaneotrichosporon spelunceum (Uniprot A0AAD3YDD9), Aspergillus nanangensis (Uniprot A0AAD4GSW2), Aspergillus nanangensis (Uniprot A0AAD4GU38), Recurvomyces mirabilis (Uniprot A0AAE0WQA4), Recurvomyces mirabilis (Uniprot A0AAE1C316), Emydomyces testavorans (Uniprot A0AAF0DM47), Emydomyces testavorans (Uniprot A0AAF0IM67), Conoideocrella luteorostrata (Uniprot A0AAJ0CJ90), Conoideocrella luteorostrata (Uniprot A0AAJ0FNX5), Extremus antarcticus (Uniprot A0AAJ0GJ29), Exophiala dermatitidis (Uniprot A0AAN6F460), Exophiala dermatitidis (Uniprot A0AAN6F5C7), Friedmanniomyces endolithicus (Uniprot A0AAN6FVR2), Exophiala dermatitidis (Uniprot A0AAN6ITL3), Tilletia horrida (Uniprot A0AAN6JN10), Tilletia horrida (Uniprot A0AAN6JZR2), Friedmanniomyces endolithicus (Uniprot A0AAN6K7N7), Friedmanniomyces endolithicus (Uniprot A0AAN6K804), Friedmanniomyces endolithicus (Uniprot A0AAN6KAU8), Mucor velutinosus (Uniprot A0AAN7DEG4), Lithohypha guttulata (Uniprot A0AAN7T607), Elasticomyces elasticus (Uniprot A0AAN7VT76), Knufia fluminis (Uniprot A0AAN8E902), Orbilia javanica (Uniprot A0AAN8N248), Arthrobotrys conoides (Uniprot A0AAN8NED4), Arthrobotrys conoides (Uniprot A0AAN8NG15), Cytospora paraplurivora (Uniprot A0AAN9U276), Diatrype stigma (Uniprot A0AAN9UVT2), Saxophila tyrrhenica (Uniprot A0AAV9PLR8), Vermiconidia calcicola (Uniprot A0AAV9Q5I9), Vermiconidia calcicola (Uniprot A0AAV9QBC9), Vermiconidia calcicola (Uniprot A0AAV9QBN9), Orbilia brochopaga (Uniprot A0AAV9UCE4), Orbilia blumenaviensis (Uniprot A0AAV9UGG7), Arthrobotrys musiformis (Uniprot A0AAV9VWC7), Orbilia ellipsospora (Uniprot A0AAV9XAK5), Orbilia ellipsospora (Uniprot A0AAV9XBR1), Orbilia ellipsospora (Uniprot A0AAV9XBU3), Paramarasmius palmivorus (Uniprot A0AAW0B8B6), Beauveria asiatica (Uniprot A0AAW0RUD2), Scheffersomyces stipitis (Uniprot A3M042), Saccharomyces cerevisiae (Uniprot A6ZSL8, G2WF33 or C7GIQ4), Komagataella phaffii (Uniprot F2QY70), Candida albicans (Uniprot Q59KQ1), Millerozyma farinosa (Q6TNG4), or Kluyveromyces marxianus (Uniprot W0TI27).

[0080] Another embodiment of a heterologous enzyme which can be used to provide a heterologous ATP futile cycle is a polypeptide having heterologous glucose-6-phosphatase activity. In such embodiment, a heterologous polypeptide having glucose-6-phosphatase activity is introduced in the recombinant yeast host cell. The heterologous polypeptide having glucose-6- phosphatase activity catalyses the formation of glucose from glucose-6-phosphate (G6P). The native or heterologous hexokinase catalyses the formation of glucose-6-phosphate (G6P) from glucose. In this reaction ATP is converted to ADP. The introduction of a heterologous enzyme having glucose-6-phosphatase activity (in combination with a hexokinase) creates a heterologous ATP futile cycle. Glucose-6-phosphatases are enzymes associated with gluconeogenesis and are classified under E.C. 3.1 .3.9. In some embodiments, the heterologous glucose-6-phosphatase can be of eukaryotic origin. The heterologous glucose- 6-phosphatase can be of prokaryotic origin.

[0081] Another embodiment of a combination of heterologous enzymes which can be used to provide a heterologous ATP futile cycle comprises enzymes capable of interconverting glucose and trehalose. Such combination can include, for example, a polypeptide having trehalase activity (such as an acid trehalase like NTH1 , NTH2 or ATH for example), a polypeptide having hexokinase activity (like HXK), a polypeptide having phosphoglucomutase activity (like PGM), a polypeptide having UDP-glucose pyrophosphorylase (UGP) activity, a polypeptide having trehalose-6-phosphate synthase (TPS1) activity, and a polypeptide having trehalose-6- phosphate phosphatase (TPS2) activity. In the combination, at least one of the polypeptides is heterologous. In some embodiments, all the polypeptides of this combination are heterologous. In such embodiment, the heterologous polypeptide(s) of the combination are introduced in the recombinant yeast host cell. The polypeptide having acid trehalase activity catalyses the formation of glucose from trehalose. Acid trehalases are classified under E.C. 3.2.1.28 and can be of eukaryotic or prokaryotic origin. The polypeptide having hexokinase activity catalyses the formation of glucose-6-phosphate from glucose. In this reaction, ATP is converted into ADP. Hexokinases are classified under E.C. 2.7.1.1 and can be of eukaryotic or prokaryotic origin. The polypeptide having phosphoglucomutase activity catalyses the formation of glucose-1 -phosphate from glucose-6-phosphate. Phosphoglucomutases are classified under E.C. 5.4.2.2 and can be of eukaryotic or prokaryotic origin. The polypeptide having UDP-glucose pyrophosphorylase activity catalyses the formation of UDP-glucose from glucose-1 -phosphate. UDP-glucose pyrophosphorylases (which are also known as UTP- glucose-1 -phosphate uridylyltransferases) are classified under E.C. 2.7.7.9 and can be of eukaryotic or prokaryotic origin. The polypeptide having trehalose-6-phosphate synthase activity catalyses the formation of trehalose-6-phosphate from UDP-glucose. Trehalose-6- phosphate synthases are classified under E.C. 2.4.1 .15 and can be of eukaryotic or prokaryotic origin. The polypeptide having trehalose-6-phosphate phosphatase activity catalyses the formation of trehalose from trehalose-6-phosphate. Trehalose-6-phosphate phosphatase are classified under E.C. 3.1 .3.12 and can be of eukaryotic or prokaryotic origin.

[0082] A further embodiment of a heterologous enzyme which can be used to provide a heterologous ATP futile cycle is a polypeptide having phosphatase activity which is capable of hydrolysing ATP into ADP. In such embodiment, the enzyme having phosphatase activity is introduced in the recombinant yeast host cell. The introduction of a heterologous enzyme having phosphatase activity creates a heterologous ATP futile cycle since it favors the hydrolysis of ATP into ADP. One embodiment of such phosphatase is PHO8 which is known to dephosphorylate many compounds, including ATP. The increase in the expression of native PHO8 and / or the heterologous expression of PHO8 (especially in the vacuoles) can be used to create a heterologous ATP futile cycle. One embodiment of such phosphatase is PHO5 which is known to dephosphorylate many compounds, including ATP. The increase in the expression of native PHO5 and / or the heterologous expression of can be used to create a heterologous ATP futile cycle. Another embodiment of such phosphatase is the N-terminal domain of the SSB1 ribosome associated chaperone which is known to have ATPase activity. The expression of the heterologous N-terminal domain of the SSB1 ribosome associated chaperone can be used to create a heterologous ATP futile cycle. Another embodiment of such phosphatase is the FO subunit of ATPases which is known to have ATPase activity. The expression of the heterologous FO subunit of an ATPase can be used to create a heterologous ATP futile cycle. A further embodiment of such phosphatase are apyrases (also referred to as ATP-diphosphohydrolase and classified under E.C. 3.6.1.5). Apyrases can catalyse the hydrolysis of ATP into ADP as well as ADP into AMP. The increase in the expression of a native apyrase and / or the heterologous expression of an apyrase can be used to create a heterologous ATP futile cycle. It will be recognized the rate of completion of the heterologous ATP futile cycle should be designed to achieve an increase in the production of the fermentation product. If the rate of completion of the heterologous ATP futile cycle is too high, this may cause stress to the yeast and ultimately reduce the yield of the fermentation product. Alternatively, if the rate of completion of the heterologous ATP futile cycle is too low, the benefits in the yield of the fermentation product may not be observed. To find the right balance in activity the rate of completion of the heterologous ATP futile cycle, there are many tools available to the skilled person. For example, it is possible to select a heterologous polypeptide / enzyme based on its intrinsic biological activity and choose, based on the circumstances, a heterologous polypeptide / enzyme which exhibits more or less biological activity. In another embodiment, it is possible to modify the amino acid sequence of the one or more heterologous polypeptides / enzymes to modulate its biological activity. In still another embodiment, it is possible to include one or more copies of the heterologous gene(s) encoding the heterologous polypeptide(s) / enzyme(s) in the recombinant yeast host cell. In yet another example, it is possible to select a promoter (or a combination of promoters) which either allow the expression of the one or more native / heterologous polypeptides / enzymes in a constitutive manner e.g., constitutive promoter or a combination of constitutive promoters) or in an inducible manner (e.g., inducible promoter or a combination of inducible promoters). In some embodiments, the promoter or the combination of promoters is / are derived from one or more promoters that is present in Saccharomyces cerevisiae.

[0083] In some embodiments, the promoter or the combination of promoters presents in the heterologous nucleic acid molecule encoding the heterologous enzyme comprises a constitutive promoter. Constitutive promoters include, but are not limited to the promoter of the tef2 gene (referred to as tef2p), the promoter of the cwp2 gene (referred to as cwp2p), the promoter of the ssa1 gene (referred to as ssal p), the promoter of the enol gene (referred to as enol p), the promoter of the hxk1 gene (referred to as hxkl p), the promoter of the pgk1 gene (referred to as pgk1 p), the promoter of the adh1 gene (referred to as adh1 p), the promoter of the rev1 gene (referred to as revl p), the promoter of the cyc1 gene (referred to as cyd p), and the promoter of the ste5 gene (referred to as ste5p), the promoter of the ccw12 gene (referred to as ccw12p), the promoter of the hxk2 gene (referred to as hxk2p), the promoter of the pyk1 gene (referred to as pykl p), the promoter of the tipi gene (referred to as tipl p) as well as functional variants or functional fragments thereof.

[0084] The promoter or combination of promoters that can be used to control the expression of the one or more heterologous enzymes can be inducible promoters. An inducible promoter is more active under certain circumstances and less active in other circumstances. In some embodiments, an inducible promoter is only active in the presence of an inducer (which can be, for example, a certain range of oxygen level, the presence or the absence of a chemical / biological entity, etc.). In some embodiments, the promoter or the combination of promoters present in the recombinant yeast host cell is / are capable of allowing the expression of the heterologous enzyme(s) during the growth phase (e.g., propagation) of the recombinant yeast host cell. The propagation promoters favor the expression (and in some embodiments only allows the expression) of the coding sequence they control during the propagation phase of the yeast. In other embodiments, the promoter or the combination of promoters is / are capable of allowing the expression of the heterologous enzyme during the stationary phase (e.g., fermentation) of the recombinant yeast host cell. The fermentation promoters favor the expression (and in some embodiments only allows the expression) of the coding sequence they control during the fermentation phase of the yeast. In embodiments in which the fermentation by-product is the yeast biomass, the use of a fermentation promoter to express the heterologous FBPase during fermentation but not during propagation may be advantageous.

[0085] Inducible promoters include, but are not limited to glucose-regulated promoters (e.g., the promoter of the hxt7 gene (referred to as hxt7p); the promoter of the ctt1 gene (referred to as cttl p); the promoter of the glo1 gene (referred to as glol p); the promoter of the ygp1 gene (referred to as ygpl p); the promoter of the gsy2 gene (referred to as gsy2p); the promoter of the gpm1 gene (referred to as gpml p); the promoter of the pgk1 gene (referred to as pgkl p), and / or the promoter of the tipi gene (referred to as tipl p)), molasses-regulated promoters (e.g., the promoter of the moll gene (referred to as mol1 p); heat shock-regulated promoters (e.g., the promoter of the glo1 gene (referred to as glol p); the promoter of the sti1 gene (referred to as stil p); the promoter of the ygp1 gene (referred to as ygpl p); the promoter of the ssa1 gene (referred to as ssal p), and / or the promoter of the gsy2 gene (referred to as gsy2p)), oxidative stress response promoters (e.g., the promoter of the cup1 gene (referred to as cupl p); the promoter of the ctt1 gene (referred to as cttl p); the promoter of the trx2 gene (referred to as trx2p); the promoter of the gpd1 gene (referred to as gpdl p); the promoter of the hsp12 gene (referred to as hsp12p); the promoter of the hsp150 gene (referred to as hsp150p); the promoter of the ssc1 gene (referred to as ssd p)); osmotic stress response promoters (e.g., the promoter of the ctt1 gene (referred to as cttl p); the promoter of the glo1 gene (referred to as glo1 p); the promoter of the gpd1 gene (referred to as gpd1 p); the promoter of the ygp1 gene (referred to as ygpl p); the promoter of the hor7 gene (referred to as hor7p); and / or the promoter of the stl1 gene (referred to as stl1 p)), nitrogen-regulated promoters (e.g., the promoter of the ygp1 gene (referred to as ygpl p)), anaerobic-regulated promoters (e.g., the promoter from the aox1 gene (referred to as aox1 p); the promoter of the tir1 gene (referred to as tirl p); the promoter of the pau5 gene (referred to as pau5p); the promoter of the dan1 gene (referred to as danl p), the promoter of the tdh1 gene (referred to as tdhl p); the promoter of the spi1 gene (referred to as spil p); the promoter of the hxk1 gene (referred to as hxkl p); the promoter of the anb1 gene (referred to as anbl p); the promoter of the hxt6 gene (referred to as hxt6p); the promoter of the trx1 gene (referred to as trxl p); the promoter of the aac3 gene (referred to as aac3p); the promoter of the hor7 gene (referred to as hor7p); the promoter of the adh1 gene (referred to as adh1 p); the promoter of the tdh2 gene (referred to as tdh2p); the promoter of the tdh3 gene (referred to as tdh3p); the promoter of the gdp1 gene (referred to as gpdl p); the promoter of the cdc19 gene (referred to as cdc19p); the promoter of the eno2 gene (referred to as eno2p); the promoter of the pdc1 gene (referred to as pdc1 p); the promoter of the hxt3 gene (referred to as hxt3p); and / orthe promoter of the tpi1 gene (referred to tpi1 p)), ethanol-regulated promoters (including ethanol responsive promoters), redox-regulated promoters (e.g., including, but not limited to the promoter of the gpd2 gene (referred as gpd2p)), sulfite-regulated promoters (e.g., including, but not limited to the promoter of the fzf1 gene (referred to as the fzfl p); the promoter of the ssu1 gene (referred to as ssul p); and / or the promoter of the ssu1-r gene (referred to as the ssu1-rp)); and stress-response promoters (e.g., including, but not limited to the promoter of the yap1 gene (referred to as yapl p)), the promoter of the ssa3 gene (referred to as ssa3p)) and / or the promoter of the hsp104 gene (referred to as hsp104p), a functional variant or a functional fragment thereof).

[0086] Promoters that can be included in the recombinant yeast host cell of the present disclosure include, without limitation, the promoter of the tdh1 gene (referred to as tdhl p), of the hor7 gene (referred to as hor7p), of the hsp150 gene (referred to as hsp150p), of the hxt7 gene (referred to as hxt7p), of the gpm1 gene (referred to as gpm1 p), of the pgk1 gene (referred to as pgk1 p), of the st!1 gene (referred to as stl 1 p), of the tef2 gene (referred to as tef2p), of the tdh3 gene (referred to as tdh3p), of the fba1 gene (referred to as fbal p), of the eno2 gene (referred to as eno2p), and / or of the hyp2 gene (referred to as hyp2p).

[0087] In some embodiments, the promoter from Saccharomyces cerevisiae that can be included in the recombinant yeast host cell to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the adh1 gene (referred to as adh1 p), the promoter of the cdc19 gene (referred to as cdc19p), the promoter of the cyc1 gene (referred to as cyc1 p), the promoter of the ccw12 gene (referred to as ccw12p), the promoter of the gpd1 gene (referred to as gpdl p), the promoter of the hor7 gene (referred to as hor7p), the promoter of the hxk2 gene (referred to as hxk2p), the promoter of the hxt3 gene (referred to as hxt3p), the promoter of the hxt7 gene (referred to as hxt7p), the promoter of the pyk1 gene (referred to as pykl p), the promoter of the ssa1 gene (referred to as ssa1 p), the promoter of the tdh1 gene (referred to as tdhl p), and / or the promoter of the tpi1 gene (referred to as tpil p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the adh1 gene (referred to as adhl p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the cdc19 gene (referred to as cdc19p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the cyc1 gene (referred to as cyd p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the ccw12 gene (referred to as ccw12p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the gpd1 gene (referred to as gpdl p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the hor7 gene (referred to as hor7p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the hxk2 gene (referred to as hxk2p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the hxt3 gene (referred to as hxt3p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the hxt7 gene (referred to as hxt7p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the pyk1 gene (referred to as pykl p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the ssa1 gene (referred to as ssal p).

[0088] In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the tdh1 gene (referred to as tdhl p). In additional embodiment, the promoter from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the promoter of the tpi1 gene (referred to as tpil p).

[0089] In some embodiments, the promoter can be obtained or derived from a native promoter present in Komagataella sp., such as, for example, Komagataella phaffii. Inducible promoters include, but are not limited to glucose-regulated promoters, fructose-regulated promoters, glycerol- regulated promoters, heat shock-regulated promoters, oxidative stress response promoters, osmotic stress response promoters, nitrogen-regulated promoters, and ethanol-regulated promoters. In an embodiment, ethanol-regulated promoters include, without limitation, the promoter from the adh2 gene, which is also known as the adh3 gene (referred to as adh2p). Constitutive promoters include, without limitation, the promoter from the spi1 gene (referred to as spil p). In an embodiment, the promoter is a promoter from the gap1 gene (referred to as gapl p). In an embodiment, the promoter is a promoter from the hgt1 gene (referred to as hgtl p). In an embodiment, the promoter is a promoter from the glc3 gene (referred to as glc3p).

[0090] In an embodiment, the promoter is a promoter from the acb2 gene (referred to as acb2p). In an embodiment, the promoter is a promoter from the pex8 gene (referred to as pex8p). In an embodiment, the promoter is a promoter from the urc1 gene (referred to as urd p). In an embodiment, the promoter is a promoter from the tpo3 gene (referred to as top3p). In an embodiment, the promoter is a promoter from the bio2 gene (referred to as bio2p). In an embodiment, the promoter is a promoter from the gut1 gene (referred to as gutl p). In an embodiment, the promoter is a promoter from the cat1 gene (referred to as catl p). In an embodiment, the promoter is a promoter from the ic!1 gene (referred to as icl1 p). In an embodiment, the promoter is a promoter from the gcw14 gene (referred to as gcw14p).

[0091] In some embodiments, the promoter can be obtained or derived from a native promoter present in Ogataea sp., such as, for example, Ogataea polymorpha. In an embodiment, the promoter is a promoter from the sori gene (referred to as sorl p), the O. polymorpha methanol oxidase mox1 gene (referred to as mox1 p), the O. polymorpha promoter from the gap1 gene (referred to as OpGAPI p), the O. polymorpha promoter from the gapdh gene (referred to as OpGAPDHp), the O. polymorpha promoter from the gcw14 gene (referred to as OpGCW14p), the O. Polymorpha promoter from the adh1 gene (referred to as OpADHI p), the O. polymorpha promoter from the Icl1 gene (referred to as OpICLI p), and / or the O. polymorpha promoter from the tef1 gene (referred to as OpTEFI p).

[0092] In specific embodiments, the heterologous nucleic acid molecule encoding the heterologous enzyme comprises at least one of the adh2 and / or spi1 variant promoters described in U.S. patent application published under 2024 / 0417741 and herewith incorporated by reference.

[0093] The terminator or combination of terminators that can be used to control the expression of the heterologous polypeptide(s) in the recombinant yeast host cell can be native or heterologous to the nucleic acid sequence encoding the heterologous polypeptide. In embodiments, the terminator or the combination of terminators can be obtained from Saccharomyces cerevisiae. In some embodiments, the terminator or the combination of terminators comprises the terminator from the adh1 gene (referred to as adhlt), the terminator from the adh3 gene (referred to as adh3t), the terminator from the cyc1 gene (referred to as cydt), the terminator from the dit1 gene (referred to as ditit), the terminator from the fbpal gene (referred to as fbpalt), the terminator from the gpm1 gene (referred to as gpmlt), the terminator from the hxt2 gene (referred to as hxt2t), the terminator from the Idp1 gene (referred to as idplt), the terminator from the Ira2 gene (referred to as ira2t), the terminator from the mfa2 gene (referred to as mfa2t), the terminator from the pdc1 gene (referred to as pddt), the terminator from the pma1 gene (referred to as pamlt), the terminator from the tdh3 gene (referred to as tdh3t), the terminator from the ts!1 gene (referred to as tsllt), and / or the terminator from the yhi9 gene (referred to as yhi9t). In additional embodiments, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprises the terminator from the adh1 gene (referred to as adhlt), the terminator from the adh3 gene (referred to as adh3t), the terminator from the cyd gene (referred to as cydt), the terminator from the dit1 gene (referred to as ditit), the terminator from the fbpal gene (referred to as fbpalt), the terminator from the gpm1 gene (referred to as gpmlt), the terminator from the hxt2 gene (referred to as hxt2t), the terminator from the Idp1 gene (referred to as idplt), the terminator from the Ira2 gene (referred to as ira2t), the terminator from the mfa2 gene (referred to as mfa2t), the terminator from the pdc1 gene (referred to as pddt), the terminator from the pma1 gene (referred to as pamlt), the terminator from the tdh3 gene (referred to as tdh3t), the terminator from the tsl1 gene (referred to as tsllt), and / or the terminator from the yhi9 gene (referred to as yhi9t). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprises the terminator from the adh1 gene (referred to as adhlt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the adh3 gene (referred to as adh3t). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the cyc1 gene (referred to as cydt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the dit1 gene (referred to as ditit). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the fbpal gene (referred to as fbpalt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the gpm1 gene (referred to as gpmlt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the hxt2 gene (referred to as hxt2t). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the idp1 gene (referred to as idplt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the ira2 gene (referred to as ira2t). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the mfa2 gene (referred to as mfa2t). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the pdc1 gene (referred to as pddt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the pma1 gene (referred to as pamlt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the tdh3 gene (referred to as tdh3t). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the tsl1 gene (referred to as tsilt). In additional embodiment, the terminator from Saccharomyces cerevisiae that can be used to express the polypeptide providing the heterologous ATP futile cycle comprise the terminator from the yhi9 gene (referred to as yhi9t).

[0094] Another example to adjust the rate of completion of the heterologous ATP futile cycle is to modulate (increase or decrease) the expression / activity of the native enzyme(s) that are involved. This can be done by introducing one or more modifications to the amino acid sequence(s) of the native enzyme(s) involved. This can also be done by modifying the regulatory sequences (such as the promoter(s)) of the native enzyme(s) involved. This can further be done by adding copies the native gene(s) encoding the native enzyme(s) associated with the heterologous ATP futile cycle. This can further be done by inactivating / deleting the native gene(s) encoding the native enzyme(s) associated with the heterologous ATP futile cycle. In this latest embodiment, care should be taken not to inactivate all copies of native gene(s) encoding the native enzyme(s) associated with the heterologous ATP futile cycle, because this would not allow the completion of the ATP futile cycle. In such embodiment, the recombinant yeast host cells of the present disclosure can further comprise a genetic modification in one or more native enzymes involved in the heterologous ATP futile cycle to reduce the expression or inactivate the gene(s) encoding the one or more native enzymes associated with the heterologous ATP cycle.

[0095] In a first example, when the one or more heterologous enzymes includes a polypeptide having fructose-1 ,6-bisphophatase activity, the expression of one or more native enzymes having phosphofructokinase activity can be reduced or abrogated (by genetic deletion for example). In a specific embodiment, when the one or more heterologous enzymes includes a polypeptide having fructose-1 ,6-bisphophatase activity, the expression of one or more native phosphofructose kinases can be reduced or abrogated. In yet another specific embodiment, when the one or more heterologous enzymes includes a polypeptide having fructose-1 ,6- bisphophatase activity, the expression of the pfk26 gene, the pfk27 gene or both the pfk26 and pfk27 genes can be inactivated, reduced or abrogated.

[0096] In a second example, when the one or more heterologous enzymes includes a polypeptide having pyruvate carboxylase activity and a polypeptide having phosphoenolpyruvate carboxykinase activity, the expression of one or more native enzymes having pyruvate kinase activity can be reduced or abrogated (by genetic deletion for example). In a specific embodiment, when the one or more heterologous enzymes includes a polypeptide having pyruvate carboxylase activity and a polypeptide having phosphoenolpyruvate carboxykinase activity, the expression of one or more native pyruvate kinase can be reduced or abrogated. In yet another specific embodiment, when the one or more heterologous enzymes includes a polypeptide having pyruvate carboxylase activity and a polypeptide having phosphoenolpyruvate carboxykinase activity, the expression of the pyk1 gene, the pyk2 gene or both the pyk1 and pyk2 genes can be inactivated, reduced or abrogated.

[0097] In a third example, when the one or more heterologous enzymes includes a polypeptide having glucose-6-phosphatase activity, the expression of one or more native enzymes having hexokinase activity can be reduced or abrogated (by genetic deletion for example). In a specific embodiment, when the one or more heterologous enzymes includes a polypeptide having glucose-6-phosphatase activity, the expression of one or more native hexokinases can be reduced or abrogated. In yet another specific embodiment, when the one or more heterologous enzymes includes a polypeptide having glucose-6-phosphatase activity, the expression of the hxk1 gene, the hxk2 gene or both the hxk1 and hxk2 genes can be inactivated, reduced or abrogated.

[0098] By-product reduction

[0099] The recombinant yeast host cells of the present disclosure also include at least one second genetic modification in an engineered metabolic pathway for decreasing the production of at least one fermentation by-product. The engineered metabolic pathway for decreasing the production of at least one fermentation by-product is used to increase the yield of the fermentation product(s), to reduce the stress on the recombinant yeast host cell during propagation and / or fermentation, to renderthe recombinant yeast host cell more robust during propagation and / or fermentation, to reduce the amount of residual carbohydrate at the end of the fermentation, to reduce biomass and / or to improve the fermentation kinetic. The combination of the heterologous ATP futile cycle with the engineered metabolic pathway for reducing the production of the at least one fermentation by-product leads to a further decrease in the yield of the fermentation by-product(s) The combination of the heterologous ATP futile cycle with the engineered metabolic pathway for reducing the production of the at least one fermentation by-product leads to a further increase in the yield of the fermentation product(s) of the recombinant yeast host cell (when compared to a control yeast host cell lacking the heterologous ATP futile cycle and comprised the engineered metabolic pathway). The recombinant yeast host cell of the present disclosure can comprise a single genetic modification in the engineered metabolic pathway for decreasing the production of the fermentation by-product(s). Alternatively, the recombinant yeast host cell of the present disclosure can comprise a plurality of genetic modifications in the engineered metabolic pathway for decreasing the production of the fermentation by-product(s).

[0100] In an embodiment, the fermentation by-product comprises glycerol. Glycerol is an important fermentation by-product known to limit production of a fermentation product like an alcohol e.g., ethanol). As shown on Figures 1A to 1 C, once dihydroxyacetone phosphate (DHAP) is formed, it can be converted into glycerol-3-phosphate (glycerol-3-P) by the native NAD- dependent glycerol-3-phosphate dehydrogenases (GPD1 and GPD2). This reaction also converts NADH into NAD+. Once glycerol-3-phosphate (glycerol-3-P) is formed, it can be converted to glycerol by the native glycerol-3-phosphate phosphatases (GPP1 or RHR2; GPP2 or HOR2).

[0101] As such, in embodiments in which the engineered metabolic pathway is for reducing the production of glycerol as a by-product, a genetic modification can be introduced in the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and / or encoding enzymes involved in the generation of glycerol (GPP1 / 2) so as to reduce or abrogate their expression. The genetic modification(s) can be made to the regulatory sequence(s) (and in some embodiment in the promoters)) of the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and / or encoding enzymes involved in the generation of glycerol (GPP1 / 2)) so as to reduce their expression. The genetic modification(s) can be made to the coding sequence(s) of the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and / or encoding enzymes involved in the generation of glycerol (GPP1 / 2) so as to reduce or abrogate their expression. Alternatively, or in combination, the coding sequences(s) can be modified to introduce one or more amino acid modifications to reduce the activity of the native enzymes involved in the generation which of glycerol-3-phosphate (GPD1 / 2) and / or the native enzymes involved in the generation of glycerol (GPP1 / 2). The coding sequence(s) can be modified to introduce a partial or total deletion the native genes encoding the glycerol-3- phosphate (GPD1 / 2) and / or in the native genes encoding enzymes involved in the generation of glycerol (GPP1 / 2). In one embodiment, the engineered metabolic pathway comprises a genetic modification to reduce or abrogate the expression of the native gpd1 gene. For example, in the recombinant yeast host cell of the present disclosure, one or all copies of the native gpd1 gene can be inactivated (either by removing at least a part or the entirety of the gpd1 coding sequence or by inserting at least one nucleic acid residue to disrupt the gpd1 coding sequence). In another embodiment, the engineered metabolic pathway comprises a genetic modification to reduce or abrogate the expression of the native gpd2 gene. For example, in the recombinant yeast host cell of the present disclosure, one or all copies of the native gpd2 gene can be inactivated (either by removing at least a part or the entirety of the gpd2 coding sequence or by inserting at least one nucleic acid residue to disrupt the gpd2 coding sequence). In still another embodiment, the engineered metabolic pathway comprises a genetic modification to reduce or abrogate the expression of the native gpp1 gene. For example, in the recombinant yeast host cell of the present disclosure, one or all copies of the native gpp1 gene can be inactivated (either by removing at least a part or the entirety of the gpp1 coding sequence or by inserting at least one nucleic acid residue to disrupt the gpp1 coding sequence). In yet another embodiment, the engineered metabolic pathway comprises a genetic modification to reduce or abrogate the expression of the native gpp2 gene. For example, in the recombinant yeast host cell of the present disclosure, one or all copies of the native gpp2 gene can be inactivated (either by removing at least a part or the entirety of the gpp2 coding sequence or by inserting at least one nucleic acid residue to disrupt the gpp2 coding sequence). Embodiments for reducing or abrogating the native genes involved glycerol synthesis (for the production of glycerol-3-phosphate or glycerol) are described in U.S. patents serial number 8956851 , 9605269, 10570421 , and U.S. patent application published under 2020 / 0224209 and are incorporated herewith in their entirety.

[0102] The expression of the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and encoding enzymes involved in the generation of glycerol (GPP1 / 2) is negatively regulated by the presence of intracellular glycerol levels. When the intracellular levels of glycerol are low, the expression of the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and encoding enzymes involved in the generation of glycerol (GPP1 / 2) is favored to increase intracellular glycerol levels. When the intracellular levels of glycerol are high, the expression of the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and encoding enzymes involved in the generation of glycerol (GPP1 / 2) is reduced to decrease intracellular glycerol levels. Since glycerol is a metabolite that can be exported outside the yeast cell and imported back inside the yeast cell, it is possible to reduce the production of glycerol by increasing the intracellular levels of this metabolite and to ultimately reduce the expression of the native gene(s) encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and encoding enzymes involved in the generation of glycerol (GPP1 / 2). The intracellular glycerol can be exported outside the cell by the native FPS1 plasma membrane channel. As such, the engineered metabolic pathway for reducing the production of glycerol can comprise a genetic modification for reducing or abrogating the expression of the native fps1 gene. For example, in the recombinant yeast host cell of the present disclosure, one or all copies of the native fps1 gene can be inactivated (either by removing at least a part or the entirety of the fps1 coding sequence or by inserting at least one nucleic acid residue to disrupt the fps1 coding sequence). This genetic modification can be made to the regulatory sequence, the coding sequence or both the regulatory sequence and the coding sequence of the native fps1 gene. Without wishing to be bound to theory, the decrease in the expression of the fps1 gene and / or in the activity of native FPS1 will limit the efflux of glycerol outside the yeast cell, maintain or increase glycerol levels inside the yeast cell and thus limit the expression of the native genes involved in glycerol-3-phosphate or glycerol synthesis. Embodiments for decreasing or abrogating expression of the native fps1 gene are disclosed in U.S. patents serial number 8956851 , 9988650, and 10570421 and are incorporated herein in their entirety. Extracellular glycerol can also be imported inside the cell by the STL1 glycerol proton symporter. STL1 can also be used to reduce the amount of biomass that is accumulated As such, the engineered metabolic pathway for reducing the production of glycerol and / or yeast biomass can comprise a genetic modification for increasing the expression of the native st!1 gene and / or the activity of the native STL1 polypeptide, and / or for providing expression of a heterologous STL1 polypeptide (by incorporating a heterologous st!1 gene in the recombinant yeast host cell). In one embodiment, the recombinant yeast host cell comprises a heterologous STL1 polypeptide encoding by a heterologous st!1 gene. In a specific embodiment, the heterologous STL1 is from Saccharomyces sp. and in yet another embodiment from Saccharomyces cerevisiae. In another embodiment, the heterologous STL1 polypeptide has the amino acid sequence of SEQ ID NO: 1 1 or is a variant thereof having STL1 activity. In still another embodiment, the heterologous st!1 gene comprises the nucleic acid sequence of SEQ ID NO: 12 or is a degenerate sequence encoding a heterologous STL1 polypeptide having the amino acid sequence of SEQ ID NO: 11 or a variant thereof having STL1 activity. In a specific embodiment, the heterologous STL1 is from Candida sp. and in yet another embodiment from Candida albicans. In another embodiment, the heterologous STL1 polypeptide has the amino acid sequence of SEQ ID NO: 21 or is a variant thereof having STL1 activity. In still another embodiment, the heterologous st!1 gene is a degenerate sequence encoding a heterologous STL1 polypeptide having the amino acid sequence of SEQ ID NO: 21 or a variant thereof having STL1 activity. In a specific embodiment, the heterologous STL1 is from Saccharomyces sp. and in yet another embodiment from Saccharomyces paradoxus. In another embodiment, the heterologous STL1 polypeptide has the amino acid sequence of SEQ ID NO: 22 or is a variant thereof having STL1 activity. In still another embodiment, the heterologous st!1 gene is a degenerate sequence encoding a heterologous STL1 polypeptide having the amino acid sequence of SEQ ID NO: 22 or a variant thereof having STL1 activity. In a specific embodiment, the heterologous STL1 is from Millerozyma sp. and in yet another embodiment from Millerozyma farinosa. In another embodiment, the heterologous STL1 polypeptide has the amino acid sequence of SEQ ID NO: 23 or is a variant thereof having STL1 activity. In still another embodiment, the heterologous st!1 gene is a degenerate sequence encoding a heterologous STL1 polypeptide having the amino acid sequence of SEQ ID NO: 23 or a variant thereof having STL1 activity. In a specific embodiment, the heterologous STL1 is from Zygosaccharomyces sp. and in yet another embodiment from Zygosaccharomyces rouxii. In another embodiment, the heterologous STL1 polypeptide has the amino acid sequence of SEQ ID NO: 62 or is a variant thereof having STL1 activity. In still another embodiment, the heterologous st!1 gene is encoded a degenerate sequence encoding a heterologous STL1 polypeptide having the amino acid sequence of SEQ ID NO: 62 or a variant thereof having STL1 activity. Additional embodiments of heterologous STL1 polypeptides include, without limitation, Alternaria alternata Gene ID: 29120952, Arthroderma benhamiae Gene ID: 9523991 , Ashbya gossypii Gene ID: 4620396, Aspergillus aculeatus Gene ID: 30979124 and 37149396, Aspergillus affinis Gene ID: 77740444, Aspergillus alliaceus Gene ID: 43625705, Aspergillus ambiguus Gene ID: 300566969, Aspergillus bombycis Gene ID: 34445379, Aspergillus brunneoviolaceus Gene ID: 37087045, Aspergillus caelatus Gene ID: 43652960, Aspergillus campestris Gene ID: 36548357, Aspergillus chevalieri Gene ID: 66978413, Aspergillus fijiensis Gene ID: 63861694, Aspergillus fischeri Gene ID: 4588532, Aspergillus flavus Gene ID: 7910112 and 64844654, Aspergillus foveolatus Gene ID: 300611243, Aspergillus fumigatus Gene ID: 3504696, Aspergillus glaucus Gene ID: 34462219, Aspergillus heteromorphus Gene ID: 37066129, Aspergillus homomorphus Gene ID: 37199866, Aspergillus japonicus Gene ID: 37174581 , Aspergillus karnatakaensis Gene ID: 300586267, Aspergillus lentulus Gene ID: 54321612, Aspergillus lucknowensis Gene ID: 98143732, Aspergillus melleus Gene ID: 70121650, Aspergillus multicolor Gene ID: 300600831 , Aspergillus mulundensis Gene ID: 38121015, Aspergillus nidulans Gene ID: 2868048, Aspergillus nomiae Gene ID: 26809275, Aspergillus novofumigatus Gene ID: 36535757, Aspergillus oryzae Gene ID: 5997623, Aspergillus pseudodeflectus Gene ID: 98155287, Aspergillus pseudonomiae Gene ID: 43662905, Aspergillus pseudotamarii Gene ID: 43636195, Aspergillus pseudoviridinutans Gene ID: 67004373, Aspergillus puulaauensis Gene ID: 64973440, Aspergillus saccharolyticus Gene ID: 37076903, Aspergillus stella-maris Gene ID: 300645384, Aspergillus steynii Gene ID: 36557451 , Aspergillus sydowii Gene ID: 63753730, Aspergillus tanner! Gene ID: 54327994, Aspergillus terreus Gene ID: 4322759, Aspergillus thermomutatus Gene ID: 38122002, Aspergillus udagawae Gene ID: 66991269, Aspergillus undulatus Gene ID: 300632358, Aspergillus uvarum Gene ID: 37141161 , Aspergillus versicolor Gene ID: 63729359, Aspergillus went!! Gene ID: 63753730, Aureobasidium namibiae Gene ID: 25414329, Australozyma saopauloensis Gene ID: 88174529, Brettanomyces nanus Gene ID: 62196552, Candida albicans Gene ID: 3640309, Candida dubliniensis Gene ID: 8048917, Candida oxycetoniae Gene ID: 73381756, Candida subhashii Gene ID: 73472945, Candida tropicalis Gene ID: 8299741 , Candidozyma auris Gene ID: 40026103, Candidozyma duobushaemuli Gene ID: 37002176, Candidozyma haemulid Gene ID: 37006475, Candidozyma pseudohaemuli Gene ID: 36565586, Clavispora lusitaniae Gene ID: 8500695, Colletotrichum gloeosporioides Gene ID: 18740172, Cordyceps militaris Gene ID: 18171218, Diplodia corticola Gene ID: 31017281 , Gaeumannomyces graminis var. tritici Gene ID: 20349750, Eremothecium cymbalariae Gene ID: 11472257, Eremothecium gossypii Gene ID: 4620396, Eremothecium sinecaudum Gene ID: 28724161 , Eutypa lata Gene ID: 19232829, Huiozyma naganishii Gene ID: 34526380, Hyphopichia burtonii Gene ID: 30996564, Isaria fumosorosea Gene ID: 30023973, Kluyveromyces lactis Gene ID: 2896463, Kluyveromyces marxianus Gene ID: 34717406, Lachancea lanzarotensis Gene ID: 34686274, Lachancea thermotolerans Gene ID: 8290820, Magnaporthe oryzae Gene ID: 2678012, Metarhizium majus Gene ID: 26274087, Metarhizium robertsii Gene ID: 19259252, Metschnikowia bicuspidate Gene ID: 30028727, Nannizzia gypsea Gene ID: 10032882, Neofusicoccum parvum Gene ID:19029314, Ogataea angusta Gene ID: 66126536, Ogataea haglerorum Gene ID: 66119151 , Ogataea philodendri Gene ID: 70238770, Ogataea polymorpha Gene ID: 28919645, Pachysolen tannophilus GenBank Accession Numbers JQ481633 and JQ481634, Paecilomyces variotii Gene ID: 39597871 , Paracoccidioides lutzii Gene ID: 9094964, Paraphaeosphaeria sporulosa Gene ID: 28767590, Penicillium alfredii Gene ID: 81390650, Penicillium angulare Gene ID: 81617699, Penicillium antarcticum Gene ID: 83195198, Penicillium argentinense Gene ID: 81360519, Penicillium arizonense Gene ID: 34579234, Penicillium atrosanguineum Gene ID: 81578497, Penicillium bovifimosum Gene ID: 81407739, Penicillium brevicompactum Gene ID: 81643672, Penicillium canariense Gene ID: 81426710, Penicillium canescens Gene ID: 83267935, Penicillium cataractarum Gene ID: 81443221 , Penicillium chermesinum Gene ID: 83206014, Penicillium cinerascens Gene ID: 83183487, Penicillium citrinum Gene ID: 81380679, Penicillium chrysogenum Gene ID: 8310605, 81453331 and 63706182, Penicillium concentricum Gene ID: 81457044, Penicillium coprophilum Gene ID: 81418015, Penicillium cosmopolitan urn Gene ID: 81377518, Penicillium crustosum Gene ID: 81568463, Penicillium daleae Gene ID: 81599896, Penicillium diatomitis Gene ID: 81622887, Penicillium digitatum Gene ID: 26229435 and 26236011 , Penicillium expansum Gene ID: 27676930, Penicillium hispanicum Gene ID: 81639179, Penicillium horde! Gene ID: 81590393, Penicillium lagena Gene ID: 81672425, Penicillium longicatenatum Gene ID: 81770981 , Penicillium maclennaniae Gene ID: 81662173, Penicillium macrosclerotiorum Gene ID: 81727558, Penicillium malachiteum Gene ID: 81748316, Penicillium manginii Gene ID: 81761776, Penicillium odoratum Gene ID: 81796491 , Penicillium oxalicum Gene ID: 74436605, Penicillium paradoxum Gene ID: 81829458, Penicillium psychrosexuale Gene ID: 81835253, Penicillium pulvis Gene ID: 81712875, Penicillium riverlandense Gene ID: 81853714, Penicillium robsamsonii Gene ID: 81879419, Penicillium roqueforti Gene ID: 62335017, Penicillium rubens Gene ID: 8315162, Penicillium solitum Gene ID: 63876134, Penicillium soppii Gene ID: 81892267, Penicillium subrubescens Gene ID: 81808551 , Penicillium taxi Gene ID: 81861867, Penicillium verrucosum Gene ID: 81874098, Penicillium vulpinum Gene ID: 81904237, Penicillium waksmanii Gene ID: 81924277, Phialophora attae Gene ID: 28742143, Pichia membranifaciens Gene ID: 30179780, Pochonia chlamydosporia Gene ID: 28856912, Priceomyces carsonii Gene ID: 87973686, Pyrenophora tritici-repentis Gene ID: 6350281 , Rasamsonia emersonii Gene ID: 25315795, Saccharomycopsis crataegensis Gene ID: 90071763, Saccharomyces eubayanus Gene ID: 28933551 , Saccharomyces kudriavzevii Gene ID: 80923449, Saccharomyces mikatae Gene ID: 80917592, Saccharomyces paradoxus Gene ID: 54629954, Saccharomycodes ludwigii Gene ID: 70108434, Scheffersomyces amazonensis Gene ID: 90006297, Scheffersomyces coipomensis Gene ID: 90034514, Scheffersomyces spartinae Gene ID: 66115672, Scheffersomyces stipitis Gene ID: 4838168, Scheffersomyces xylosifermentans Gene ID: 90045028, Scedosporium apiospermum Gene ID: 27721841 , Sphaerulina musiva Gene ID: 27905328, Suhomyces tanzawaensis Gene ID: 30984694, Talaromyces amestolkiae Gene ID: 63798394, Talaromyces atroroseus Gene ID: 31007540, Talaromyces rugulosus Gene ID: 55989578, Togninia minima Gene ID: 19329524, Torulaspora delbrueckii Gene ID: 11505245, Torulaspora globosa Gene ID: 59327441 , Trichomonascus vanleenenianus Gene ID: 303473671 , Trichophyton rubrum Gene ID: 10373998, Trichophyton verrucosum Gene ID: 9577427, Verticillium albo-atrum Gene ID: 9537052, Verticillium dahliae Gene ID: 20711921 , Wickerhamomyces anomalus Gene ID: 30202366, Yamadazyma tenuis Gene ID: 18247501 , and Zygotorulaspora mrakii Gene ID: 59234600. Embodiments of heterologous stl1 genes and heterologous STL1 polypeptides are disclosed in U.S. patent 10570421 , U.S. patent 11753656, and U.S. patent application published under U.S. 2020 / 0224209 and are incorporated herewith in their entirety.

[0103] Combinations in the modulation in the expression of native genes encoding native enzyme(s) involved in the generation of glycerol-3-phosphate (GPD1 / 2) and / or encoding enzymes involved in the generation of glycerol (GPP1 / 2) as well as the heterologous expression of the STL1 polypeptide are disclosed in U.S. patent 10570421 , and U.S. patent application published under 2020 / 0224209 and are incorporated herewith in their entirety.

[0104] In another embodiment in which the fermentation by-product comprises glycerol, it is possible to limit the production of glycerol by favoring the conversion of glyceraldehyde-3-phosphate (GAP) directly into 3-phosphoglycerate (3-PG) by incorporating a heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (e.g., an enzyme belonging to EC 1.2.1.9 (using NADP+ as a cofactor) or 1.2.1.90 (using either NAD+ or NADP+ as a cofactor)). Without wishing to be bound to theory, the activity of the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (i) limits the availability of glyceraldehyde-3-phosphate (GAP) to be converted to dihydroxyacetone phosphate (DHAP) (and ultimately to glycerol) and (ii) favors the production of ethanol as a fermentation product. In an embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from eukaryotic origin. In another embodiment, the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is of prokaryotic origin. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Streptococcus sp. and in yet another embodiment from Streptococcus mutans. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 9 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene comprises the nucleic acid sequence of SEQ ID NO: 10 or is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 9 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus thermophilus. In another embodiment, the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 25 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 25 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus macacae. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 26 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO:

[0105] 26 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus hyointestinalis. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 27 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO:

[0106] 27 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus urinalis. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 28 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 28 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus canis. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 29 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 29 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus thoraltensis. In another embodiment, the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 30 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 30 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus dysgalactiae. In another embodiment, the heterologous non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 31 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 31 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus pyogenes. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 32 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 32 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In some embodiments, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Streptococcus ictaluri. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 32 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 32 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Lactobacillus sp. and in yet another embodiment from Lactobacillus delbrueckii. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 24 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 24 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Clostridium sp. and in yet another embodiment from Clostridium perfringens. In another embodiment, the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 34 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 34 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Clostridium chromiireducens. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 35 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 35 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Clostridium botulinum. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 36 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 36 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Bacillus sp. and in yet another embodiment from Bacillus cereus. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 37 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 37 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is Bacillus anthracis. In another embodiment, the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 38 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 38 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Bacillus thuringiensis. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 39 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 39 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Pyrococcus sp. and in yet another embodiment from Pyrococcus furiosus. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 40 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 40 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Chlamydomonas sp. and in yet another embodiment from Chlamydomonas reinhardtii. In another embodiment, the heterologous nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 63 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 63 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In a specific embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is from Streptococcus sp. In another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase has the amino acid sequence of SEQ ID NO: 64 or is a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. In still another embodiment, the heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene is a degenerate sequence encoding a heterologous non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 64 or a variant thereof having non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity. Additional embodiments of glyceraldehyde-3-phosphate dehydrogenase can also be derived, without limitation, from the following (the number in brackets correspond to the Gene ID number): Triticum aestivum (543435); Streptococcus mutans (1028095); Streptococcus agalactiae (1013627); Streptococcus pyogenes (901445); Clostridioides difficile (4913365); Mycoplasma mycoides subsp. mycoides SC str. (2744894); Streptococcus pneumoniae (933338); Streptococcus sanguinis (4807521); Acinetobacter pittii (11638070); Clostridium botulinum A str. (5185508); Bacillus thuringiensis serovar konkukian str. (2857794); Bacillus anthracis str. Ames (1088724); Phaeodactylum tricornutum (7199937); Emiliania huxleyi (17251102); Zea mays (542583); Helianthus annuus (1 10928814); Streptomyces coelicolor (1101 118); Burkholderia pseudomallei (3097058, 3095849); and variants thereof. Further embodiments of glyceraldehyde-3-phosphate dehydrogenase can also be derived, without limitation, from the following (the number in brackets correspond to the Pubmed Accession number): Streptococcus macacae (WP_003081126.1), Streptococcus hyointestinalis (WP_115269374.1), Streptococcus urinalis (WP_006739074.1), Streptococcus canis ( WP_00304411 1 .1), Streptococcus pluranimalium (WP_104967491 .1), Streptococcus equi (WP_012678132.1), Streptococcus thoraltensis (WP_018380938.1), Streptococcus dysgalactiae (WP_138125971 .1), Streptococcus halotolerans (WP_062707672.1), Streptococcus pyogenes (WP_136058687.1), Streptococcus ictaluri (WP_008090774.1), Clostridium perfringens (WP_142691612.1), Clostridium chromiireducens (WP_079442081 .1), Clostridium botulinum (WP_012422907.1), Bacillus cereus (WP_000213623.1), Bacillus anthracis (WP_098340670.1), Bacillus thuringiensis (WP_087951472.1), Pyrococcus furiosus (WP_011013013.1) as well as variants thereof. Embodiments of heterologous genes encoding heterologous polypeptides having nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase activity are provided in U.S. patent application published under 2021 / 0380989 and PCT application published under W02024 / 040001 and are herewith incorporated in their entirety.

[0107] In another embodiment, the fermentation by-product comprises glycerol and / or one or more fusel alcohols. In such embodiment, it is possible to introduce a heterologous polypeptide having pyruvate decarboxylase (PDC) activity in the recombinant yeast host cell to reduce the production of glycerol and / or one or more fusel alcohols. Pyruvate decarboxylases (E.C. 4.1.1.1) are capable of converting 2-oxo carboxylate into aldehyde and CO2. In a specific embodiment, the polypeptide having heterologous pyruvate decarboxylase activity is from prokaryotic origin. In some additional embodiments, the heterologous pyruvate decarboxylase is from Gluconacetobacter sp. and in yet another embodiment from Gluconacetobacter diazotrophicus. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 46 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 46 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Zymobacter sp. and in yet another embodiment from Zymobacter palmae. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 41 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 41 or being a variant thereof having pyruvate decarboxylase activity. In another specific embodiment, the polypeptide having pyruvate decarboxylase activity is from eukaryotic origin. In some additional embodiments, the heterologous pyruvate decarboxylase is from Zymomonas sp. and in yet another embodiment from Zymomonas mobilis. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 13 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene comprises the nucleic acid sequence of SEQ ID NO: 14 or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 13 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Pisum sp. and in yet another embodiment from Pisum sativum. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 42 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 42 or being a variant thereof having pyruvate decarboxylase activity. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 43 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 43 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Saccharomyces sp. and in yet another embodiment from Saccharomyces cerevisiae. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 44 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 44 or being a variant thereof having pyruvate decarboxylase activity. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 45 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 45 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Kluyveromyces sp. and in yet another embodiment from Kluyveromyces lactis. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 47 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 47 or being a variant thereof having pyruvate decarboxylase activity. Additional embodiments of heterologous pyruvate decarboxylases include, for example, from Lactobacillus florum (Accession Number WP_009166425.1), Lactobacillus fructivorans (Accession Number WP_039145143.1), Lactobacillus lindneri (Accession Number WP_065866149.1), Lactococcus lactis (Accession Number WP_104141789.1), Carnobacterium gallinarum (Accession Number WP_034563038.1), Enterococcus plantarum (Accession Number WP_069654378.1), Clostridium acetobutylicum (Accession Number NP_149189.1), Bacillus megaterium (Accession Number WP_075420723.1), Kluyveromyces lactis (Accession Number CAA61155), Bacillus thuringiensis (Accession Number WP_052587756.1) as well as variants thereof. Embodiments of heterologous genes encoding heterologous polypeptides having pyruvate decarboxylase activity as well as heterologous polypeptides having pyruvate decarboxylase activity are provided in U.S. patent application published under 2024 / 0409964 and are herewith incorporated in their entirety.

[0108] In a further embodiment, the fermentation by-product comprises an organic acid. In some yet another embodiment, the fermentation by-product comprises acetate as an organic acid. In such embodiment, it is possible to introduce a heterologous polypeptide having acetylcoenzyme A synthetase (acetyl-coA synthetase or ACS) activity in the recombinant yeast host cell to reduce the production or accumulation of acetate. In a specific embodiment, the polypeptide having heterologous acetyl-coA synthetase activity is from prokaryotic origin. In some additional embodiment, the heterologous acetyl-coA synthetase is from Salmonella sp. and in yet another embodiment from Salmonella enterica. In another embodiment, the heterologous acetyl-coA synthetase has the amino acid sequence of SEQ ID NO: 51 or is a variant thereof having acetyl-coA synthetase activity. In still another embodiment, the heterologous acetyl-coA synthetase gene is a degenerate sequence encoding a heterologous acetyl-coA synthetase having the amino acid sequence of SEQ ID NO: 51 or being a variant thereof having acetyl-coA synthetase. In some additional embodiment, the heterologous acetyl-coA synthetase is from Acetobacter sp. and in yet another embodiment from Acetobacter aceti. In another embodiment, the heterologous acetyl-coA synthetase has the amino acid sequence of SEQ ID NO: 52 or is a variant thereof having acetyl-coA synthetase activity. In still another embodiment, the heterologous acetyl-coA synthetase gene is a degenerate sequence encoding a heterologous acetyl-coA synthetase having the amino acid sequence of SEQ ID NO: 52 or being a variant thereof having acetyl-coA synthetase. In some additional embodiment, the heterologous acetyl-coA synthetase is from Escherichia sp. and in yet another embodiment from Escherichia coll. In another embodiment, the heterologous acetyl-coA synthetase has the amino acid sequence of SEQ ID NO: 53 or is a variant thereof having acetyl-coA synthetase activity. In still another embodiment, the heterologous acetyl- coA synthetase gene is a degenerate sequence encoding a heterologous acetyl-coA synthetase having the amino acid sequence of SEQ ID NO: 53 or being a variant thereof having acetyl-coA synthetase. In another specific embodiments, the polypeptide having acetyl-coA synthetase activity is from eukaryotic origin. In some additional embodiment, the heterologous acetyl-coA synthetase is from Saccharomyces sp. and in yet another embodiment from Saccharomyces cerevisiae. In another embodiment, the heterologous acetyl-coA synthetase has the amino acid sequence of SEQ ID NO: 48 or is a variant thereof having acetyl-coA synthetase activity. In still another embodiment, the heterologous acetyl-coA synthetase gene is a degenerate sequence encoding a heterologous acetyl-coA synthetase having the amino acid sequence of SEQ ID NO: 48 or being a variant thereof having acetyl-coA synthetase. In another embodiment, the heterologous acetyl-coA synthetase has the amino acid sequence of SEQ ID NO: 49 or is a variant thereof having acetyl-coA synthetase activity. In still another embodiment, the heterologous acetyl-coA synthetase gene is a degenerate sequence encoding a heterologous acetyl-coA synthetase having the amino acid sequence of SEQ ID NO: 49 or being a variant thereof having acetyl-coA synthetase. In some additional embodiment, the heterologous acetyl-coA synthetase is from Zygosaccharomyces sp. and in yet another embodiment from Zygosaccharomyces bailii. In another embodiment, the heterologous acetyl-coA synthetase has the amino acid sequence of SEQ ID NO: 50 or is a variant thereof having acetyl-coA synthetase activity. In still another embodiment, the heterologous acetyl-coA synthetase gene is a degenerate sequence encoding a heterologous acetyl-coA synthetase having the amino acid sequence of SEQ ID NO: 50 or being a variant thereof having acetyl-coA synthetase. Embodiments of heterologous genes encoding heterologous polypeptides having acetyl-coA synthetase activity as well as heterologous polypeptides having acetyl-coA synthetase activity are provided in U.S. patent application 2022 / 0090045, PCT patent application published under WO2025 / 133860, PCT patent application PCT / IB2025 / 059790 filed on September 29, 2025, PCT patent application published under WQ2025 / 015396, and PCT patent application published under WQ2025 / 0941 16 and are herewith incorporated in their entirety.

[0109] Additional genetic modifications

[0110] In some embodiments, the recombinant yeast host cell of the present disclosure can include at least one genetic modification in an engineered metabolic pathway for increasing the production of the at least one fermentation product. As used in the context of the present disclosure, the “engineered metabolic pathway for increasing the production of the at least one fermentation product” refers to a metabolic pathway (e.g., a combination of polypeptides or enzymes) involved (directly or indirectly) in the generation of the fermentation product(s) which has been genetically engineered to increase the production of the fermentation product(s). In some embodiments, the engineered metabolic pathway can include an increase in the expression of one or more native polypeptides / enzymes involved in the production of the fermentation product(s). Alternatively, or in combination, the engineered metabolic pathway can include a heterologous polypeptide / enzyme whose activity will increase the production of the fermentation product(s).

[0111] Polypeptides having pyruvate decarboxylase (PDC) activity can be used to increase the yield in the fermentation product and / or the rate in the production of the fermentation product. Pyruvate decarboxylases (E.C. 4.1.1.1) are capable of converting 2-oxo carboxylate into aldehyde and CO2. In a specific embodiment, the polypeptide having heterologous pyruvate decarboxylase activity is from prokaryotic origin. In a specific embodiment, the polypeptide having heterologous pyruvate decarboxylase activity is from prokaryotic origin. In some additional embodiments, the heterologous pyruvate decarboxylase is from Gluconacetobacter sp. and in yet another embodiment from Gluconacetobacter diazotrophicus. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 46 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 46 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Zymobacter sp. and in yet another embodiment from Zymobacter palmae. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 41 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 41 or being a variant thereof having pyruvate decarboxylase activity. In another specific embodiment, the polypeptide having pyruvate decarboxylase activity is from eukaryotic origin. In some additional embodiments, the heterologous pyruvate decarboxylase is from Zymomonas sp. and in yet another embodiment from Zymomonas mobilis. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 13 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene comprises the nucleic acid sequence of SEQ ID NO: 14 or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 13 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Pisum sp. and in yet another embodiment from Pisum sativum. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 42 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 42 or being a variant thereof having pyruvate decarboxylase activity. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 43 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 43 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Saccharomyces sp. and in yet another embodiment from Saccharomyces cerevisiae. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 44 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 44 or being a variant thereof having pyruvate decarboxylase activity. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 45 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 45 or being a variant thereof having pyruvate decarboxylase activity. In some additional embodiments, the heterologous pyruvate decarboxylase is from Kluyveromyces sp. and in yet another embodiment from Kluyveromyces lactis. In another embodiment, the heterologous pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 47 or is a variant thereof having pyruvate decarboxylase activity. In still another embodiment, the heterologous pyruvate decarboxylase gene or is a degenerate sequence encoding a heterologous pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 47 or being a variant thereof having pyruvate decarboxylase activity. Additional embodiments of heterologous pyruvate decarboxylases include, for example, from Lactobacillus florum (Accession Number WP_009166425.1), Lactobacillus fructivorans (Accession Number WP_039145143.1), Lactobacillus lindneri (Accession Number WP_065866149.1), Lactococcus lactis (Accession Number WP_104141789.1), Carnobacterium gallinarum (Accession Number WP_034563038.1), Enterococcus plantarum (Accession Number WP_069654378.1), Clostridium acetobutylicum (Accession Number NP_149189.1), Bacillus megaterium (Accession Number WP_075420723.1), Kluyveromyces lactis (Accession Number CAA61155), Bacillus thuringiensis (Accession Number WP_052587756.1) as well as variants thereof. Embodiments of heterologous genes encoding heterologous polypeptides having pyruvate decarboxylase activity as well as heterologous polypeptides having pyruvate decarboxylase activity are provided in U.S. patent application published under 20240409964 and are herewith incorporated in their entirety. Polypeptides having pyruvate formate lyase activity (which includes pyruvate formate lyases also known as PFL) can be used to increase the production of a fermentation product like ethanol. Pyruvate formate lyase convert acetyl coenzyme-A into pyruvate, a precursor in the formation of ethanol. In one specific embodiment, the genetic modification in the engineered metabolic pathway for the production of the fermentation product can comprise the increase in the native expression of polypeptides having pyruvate formate lyase activity and / or the heterologous expression of polypeptides having pyruvate formate lyase activity. Embodiments of native and heterologous polypeptides having pyruvate formate lyase activity are described in U.S. patent 8956851 , and U.S. patent application published under 2022 / 0002661 and are herewith included in their entirety.

[0112] Polypeptides having alcohol dehydrogenase activity and / or acetaldehyde dehydrogenase activity can be used to increase the production of a fermentation product like ethanol. The polypeptides convert acetaldehyde into ethanol. An embodiment of a polypeptide having both alcohol dehydrogenase activity and acetaldehyde dehydrogenase activity is an acetaldehyde dehydrogenase (acetylating) (also referred to as ADHE) which is classified in EC number 1.2.1.10 and catalyze the conversion of an acetaldehyde, CoA and NAD(+) in acetyl-CoA and NADH. Another embodiment of polypeptides having alcohol dehydrogenase activity and / or acetaldehyde dehydrogenase activity is a combination of an alcohol dehydrogenase and an acetaldehyde dehydrogenase. In one specific embodiment, the genetic modification in the engineered metabolic pathway forthe production of the fermentation product can comprise the increase in the native expression of polypeptides having alcohol dehydrogenase activity and / or the heterologous expression of polypeptides having alcohol dehydrogenase activity. Embodiments of native and heterologous polypeptides having alcohol dehydrogenase activity are described in U.S. patent 8956851 , U.S. patent 9605269, U.S. patent 9988650, and U.S. patent 11753656 and are herewith included in their entirety.

[0113] Polypeptides having phosphoketolase activity (which includes phosphoketolases also known as PHK) can be used to increase the production of a fermentation product like ethanol, especially when a source of C5 carbohydrates is used during the fermentation. Polypeptides having phosphoketolase activity convert xylulose-5-phosphate intro glyceraldehyde-3- phosphate. In one specific embodiment, the genetic modification in the engineered metabolic pathway for the production of the fermentation product can comprise the increase in the native expression of polypeptides having phosphoketolase activity and / or the heterologous expression of polypeptides having phosphoketolase activity. As used herein, the terms "phosphoketolase" and "PHK" are intended to include the enzymes capable of converting D- xylulose 5-phosphate, D-fructose 6-phosphate, and / or D-sedoheptulose 7-phosphate to acetyl-phosphate. The phosphoketolase can have a single-specificity activity (e.g., singlespecificity phosphoketolase), and be only capable of converting D-xylulose 5-phosphate to D- glyceraldehyde 3-phosphate and acetyl-phosphate, converting D-fructose 6-phosphate to D- erythrose 4-phosphate and acetyl-phosphate, or converting D-sedoheptulose 7-phosphate into D-ribose 5-phosphate and acetyl-phosphate or can have a multiple-specificity / dual-specificity e.g., multiple-specificity or dual-specificity phosphoketolase). In one embodiment, the phosphoketolase is intended to have a single-specificity and is capable of converting D- fructose 6-phosphate into acetyl-phosphate. In another embodiment, the phosphoketolase is capable of at least converting D-fructose 6-phosphate into acetyl-phosphate. In some other embodiments, the phosphoketolase is intended to have dual-specificity and is capable of converting D-xylulose 5-phosphate and D-fructose 6-phosphate to acetyl-phosphate or is intended to be multiple-specificity and is capable of converting D-xylulose 5-phosphate, D- fructose 6-phosphate, and D-sedoheptulose 7-phosphate to acetyl-phosphate. Phosphoketolases include those enzymes that correspond to Enzyme Commission Number 4.1 .2.9 and 4.1 .2.22. In the context of the present disclosure, the enzyme having PHK activity is heterologous to the recombinant yeast host cell. In some embodiments, the enzyme having PHK activity is of prokaryotic, fungal or eukaryotic origin. In other embodiments, the enzyme having PHK activity can be encoded by a phk1 gene (e.g., PHK1) or a phk2 gene (e.g., PHK2). In an embodiment, the enzyme having PHK activity is of bacterial origin. In an embodiment, the enzyme having PHK activity is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 65 or be a variant of the amino acid sequence of SEQ ID NO: 65 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 65 (or a variant thereof). In an embodiment, the enzyme having PHK activity is derived from Bifidobacterium asteroides. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 66 or be a variant of the amino acid sequence of SEQ ID NO: 66 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66 (or a variant thereof). In some embodiments, the enzyme having PHK activity is derived from Bifidobacterium bifidum. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 67 or be a variant of the amino acid sequence of SEQ ID NO: 67 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 67 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Bifidobacterium gallicium. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 68 or be a variant of the amino acid sequence of SEQ ID NO: 68 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Bifidobacterium animalis. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 69 or be a variant of the amino acid sequence of SEQ ID NO: 69 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 69 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Bifidobacterium breve. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 70 or be a variant of the amino acid sequence of SEQ ID NO: 70 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 70 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Bifidobacterium longum. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 71 or be a variant of the amino acid sequence of SEQ ID NO: 71 having PHK activity. In addition, the enzyme having PHK activity can be encoded a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 71 (or a variant thereof). In an embodiment, the enzyme having PHK activity can be obtained from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 72 or be a variant of the amino acid sequence of SEQ ID NO: 72 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 72 (or a variant thereof). In an embodiment, the enzyme having PHK activity can be obtained from Lactiplantibacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 73 or be a variant of the amino acid sequence of SEQ ID NO: 73 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 73 (or a variant thereof). Alternatively, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 74 or be a variant of the amino acid sequence of SEQ ID NO: 74 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 74 (or a variant thereof). Alternatively, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 75 or be a variant ofthe amino acid sequence of SEQ ID NO: 75 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Lactiplantibacillus easel. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 76 or be a variant of the amino acid sequence of SEQ ID NO: 76 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Lactiplantibacillus acidophilus. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 77 or be a variant of the amino acid sequence of SEQ ID NO: 77 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 77 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Lactiplantibacillus pentosus. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 78 or be a variant of the amino acid sequence of SEQ ID NO: 78 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 78 (or a variant thereof). In an embodiment, the enzyme having PHK activity can be obtained from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 79 or be a variant of the amino acid sequence of SEQ ID NO: 79 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 79 (or a variant thereof). In an embodiment, the enzyme having PHK activity can be obtained from Oenococcus sp., and in further embodiments from Oenococcus oeni. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 80 or be a variant of the amino acid sequence of SEQ ID NO: 80 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 80 (or a variant thereof). In still another embodiment, the enzyme having PHK activity can be of fungal origin. In an example, the enzyme having PHK activity can be obtained from Aspergillus sp., and in further embodiments, the enzyme having PHK activity can be obtained from Aspergillus clavatus. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 81 or be a variant of the amino acid sequence of SEQ ID NO: 81 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 81 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Aspergillus nidulans. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 82 or be a variant of the amino acid sequence of SEQ ID NO: 82 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 82 (or a variant thereof). In some embodiments, the enzyme having PHK activity can be obtained from Aspergillus niger. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 83 or be a variant of the amino acid sequence of SEQ ID NO: 83 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 83 (or a variant thereof). In an embodiment, the enzyme having PHK activity can be obtained from Neurospora sp., and in further embodiments from Neurospora crassa. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 84 or be a variant of the amino acid sequence of SEQ ID NO: 84 having PHK activity. In addition, the enzyme having PHK activity can be encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 84 (or a variant thereof). In an embodiment, the enzyme having PHK activity can be obtained from Penicillium sp., and in further embodiments from Penicillium chrysogenum. In such embodiment, the enzyme having PHK activity can have the amino acid sequence of SEQ ID NO: 85 or be a variant of the amino acid sequence of SEQ ID NO: 85 having PHK activity. In addition, the enzyme having PHK activity can be encoded a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 85 (or a variant thereof). Embodiments of native and heterologous polypeptides having phosphoketolase activity are described in U.S. patent 9988650, PCT application published under WO2025 / 133860 and are herewith included in their entirety.

[0114] In some embodiments, the recombinant yeast host cell of the present disclosure can include at least one genetic modification for increasing the production and / or secretion of one or more saccharolytic enzyme. As used in the context of the present disclosure, a “saccharolytic enzyme” can be any enzyme involved in carbohydrate digestion, metabolism and / or hydrolysis, including amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes. In an embodiment, the saccharolytic enzyme is an amylolytic enzyme. The saccharolytic enzyme can be an amylolytic enzyme. As used herein, the expression “amylolytic enzyme” refers to a class of enzymes capable of hydrolyzing starch or hydrolyzed starch. Amylolytic enzymes include, but are not limited to alpha-amylases (EC 3.2.1 .1 , sometimes referred to fungal alpha-amylase, see below), maltogenic amylase (EC 3.2.1.133), glucoamylase (EC 3.2.1 .3), glucan 1 ,4-alpha- maltotetraohydrolase (EC 3.2.1.60), pullulanase (EC 3.2.1.41), iso-amylase (EC 3.2.1.68), glucanase (E.C. 3.2.1.11), and amylomaltase (EC 2.4.1.25). In a specific embodiment, the heterologous glucanase is from Nemania sp. and in additional embodiments, from Nemania serpens. In yet another embodiment, the heterologous glucanase has the amino acid sequence of SEQ ID NO: 91 or is a variant thereof having glucanase activity. In yet a further embodiment, the heterologous glucanase is encoded a degenerate sequence encoding the heterologous glucanase has the amino acid sequence of SEQ ID NO: 91 or is a variant thereof having glucanase activity.

[0115] In an embodiment, the recombinant yeast host cell is capable of expressing and secreting a heterologous alpha-amylase. In an embodiment, the heterologous alpha-amylase is of prokaryotic origin. In another embodiment, the heterologous alpha-amylase is of eukaryotic origin. In further embodiments, the heterologous alpha-amylase is of fungal origin. In a specific embodiment, the heterologous alpha-amylase is from Aspergillus sp. and in some additional embodiments, the heterologous alpha-amylase is from Aspergillus terreus. In yet another embodiment, the heterologous alpha-amylase has the amino acid sequence of SEQ ID NO: 1 or is a variant thereof having alpha-amylase activity. In yet a further embodiment, the heterologous alpha-amylase is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 2 or a degenerate sequence encoding the heterologous alphaamylase has the amino acid sequence of SEQ ID NO: 1 or is a variant thereof having alphaamylase activity. In a specific embodiment, the heterologous alpha-amylase is from Bacillus sp. and in some additional embodiments, the heterologous alpha-amylase is from Bacillus amyloliquefaciens. In a specific embodiment, the heterologous alpha-amylase is from Rhizomucor sp. and in some additional embodiments, the heterologous alpha-amylase is from Rhizomucor pusillus. In yet another embodiment, the heterologous alpha-amylase has the amino acid sequence of SEQ ID NO: 86 or 87 or is a variant thereof having alpha-amylase activity. In yet a further embodiment, the heterologous alpha-amylase is encoded a degenerate sequence encoding the heterologous alpha-amylase has the amino acid sequence of SEQ ID NO: 86 or 87 or is a variant thereof having alpha-amylase activity. In a specific embodiment, the heterologous alpha-amylase is from Thermomyces sp. and in some additional embodiments, the heterologous alpha-amylase is from Thermomyces lanuginosus. In yet another embodiment, the heterologous alpha-amylase has the amino acid sequence of SEQ ID NO: 88 or is a variant thereof having alpha-amylase activity. In yet a further embodiment, the heterologous alpha-amylase is encoded a degenerate sequence encoding the heterologous alpha-amylase has the amino acid sequence of SEQ ID NO: 88 or is a variant thereof having alpha-amylase activity. In some embodiments, the heterologous alpha-amylase is provided as a fusion polypeptide in which a carbohydrate binding module has been added to improve the raw starch activity of the enzyme. Embodiments of heterologous alphaamylases are disclosed in U.S. patent 1 1739312, U.S. patent 9206444, U.S. patent 1 1332728, U.S. patent 7659102, U.S. patent application published under 2023 / 0323327, U.S. patent application published under 2025 / 0019681 as well as U.S. provisional patent application 63 / 886,386 filed on September 23, 2025 and are herewith incorporated in their entirety.

[0116] In an embodiment, the recombinant yeast host cell is capable of expressing and secreting a heterologous glucoamylase. In an embodiment, the heterologous glucoamylase is of prokaryotic origin. In another embodiment, the heterologous glucoamylase is of eukaryotic origin. In a specific embodiment, the heterologous glucoamylase is from Rasamsonia sp. and in some additional embodiments, the heterologous glucoamylase is from Rasamsonia emersonii (also known as Talaromyces emersonii). In yet another embodiment, the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 15 or is a variant thereof having glucoamylase activity. In yet a further embodiment, the heterologous glucoamylase is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 16 or a degenerate sequence encoding the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 15 or is a variant thereof having glucoamylase activity. In a specific embodiment, the heterologous glucoamylase is from Penicillium sp. and in some additional embodiments, the heterologous glucoamylase is from Penicillium oxalicum. In yet another embodiment, the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 19 or is a variant thereof having glucoamylase activity. In yet a further embodiment, the heterologous glucoamylase is encoded by a degenerate sequence encoding the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 19 or is a variant thereof having glucoamylase activity. In a specific embodiment, the heterologous glucoamylase is from Saccharomycopsis sp. and in some additional embodiments, the heterologous glucoamylase is from Saccharomycopsis fibuligera. In yet another embodiment, the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 20 or is a variant thereof having glucoamylase activity. In yet a further embodiment, the heterologous glucoamylase is encoded by a degenerate sequence encoding the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 20 or is a variant thereof having glucoamylase activity. In a specific embodiment, the heterologous glucoamylase is from Trametes sp. and in some additional embodiments, the heterologous glucoamylase is from Trametes sanguinea. In yet another embodiment, the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 89 or is a variant thereof having glucoamylase activity. In yet a further embodiment, the heterologous glucoamylase is encoded by a degenerate sequence encoding the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 89 or is a variant thereof having glucoamylase activity. In a specific embodiment, the heterologous glucoamylase is from Neurospora sp. and in some additional embodiments, the heterologous glucoamylase is from Neurospora crassa. In yet another embodiment, the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 90 or is a variant thereof having glucoamylase activity. In yet a further embodiment, the heterologous glucoamylase is encoded by a degenerate sequence encoding the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 90 or is a variant thereof having glucoamylase activity. In some embodiments, the heterologous glucoamylase is provided as a fusion polypeptide in which a carbohydrate binding module has been added to improve the raw starch activity of the enzyme. Embodiments of heterologous glucoamylases are disclosed in U.S. patent 10570421 , U.S. patent 11198881 , U.S. patent 9206444, U.S. patent 11332728, U.S. patent 10100299, and U.S. provisional patent application 63 / 885,794 filed on September 22, 2025 and are herewith incorporated in their entirety.

[0117] In an embodiment, the recombinant yeast host cell is capable of expressing and secreting a heterologous trehalase. In an embodiment, the heterologous trehalase is of prokaryotic origin. In another embodiment, the heterologous trehalase is of eukaryotic origin. In a specific embodiment, the heterologous trehalase is from Neurospora sp. and in some additional embodiments, the heterologous trehalase is from Neurospora crassa. In yet another embodiment, the heterologous trehalase has the amino acid sequence of SEQ ID NO: 17 or is a variant thereof having trehalase activity. In yet a further embodiment, the heterologous trehalase is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 18 or a degenerate sequence encoding the heterologous trehalase has the amino acid sequence of SEQ ID NO: 17 or is a variant thereof having trehalase activity. Embodiments of heterologous trehalases are disclosed in U.S. patent 10570421 , U.S. patent application published under 2023 / 0193232, and U.S. patent application published under 2022 / 0220487 and are herewith incorporated in their entirety.

[0118] In an embodiment, the recombinant yeast host cell is capable of expressing and secreting a heterologous protease. In an embodiment, the heterologous protease is of prokaryotic origin. In another embodiment, the heterologous protease is of eukaryotic origin. Embodiments of heterologous proteases are disclosed in U.S. patent application published under 2020 / 0165592, U.S. continuation-in-part patent application published under 2025 / 0092379 and are herewith incorporated in their entirety.

[0119] In an embodiment, the recombinant yeast host cell of the present disclosure is engineered to produce ethanol as well as another fermentation co-product like acetone. As used in the context of the present disclosure, a fermentation co-product is a product made during the fermentation of ethanol which has economical value but is present in lower yield than the fermentation product. In an embodiment, the fermentation co-product can be separated (physically or chemically) from the fermentation product. In order to generate the fermentation product and the fermentation co-product, the recombinant yeast host cell includes additional genetic modifications. Genetic modifications and heterologous enzymes involved in the generation of fermentation product(s) and co-product(s) have been disclosed in U.S. patent application published under 2022 / 0090045, PCT application published under WQ2025 / 015396, PCT application published under WQ2025 / 094116, PCT application published under WQ2025 / 133860, PCT application PCT / IB2025 / 059790 filed on September 29, 2025, U.S. provisional application 63 / 889,764 filed on September 29, 2025, and U.S. provisional application 63 / 889,757 filed on September 29, 2025 and are herewith incorporated in their entirety.

[0120] In an embodiment, the recombinant yeast host cell of the present disclosure is engineered to express a peptide or a polypeptide (which includes an enzyme). In order to generate the peptide or the polypeptide, the recombinant yeast host cell includes additional genetic modifications (like the introduction of one or more heterologous nucleic acid molecules encoding the heterologous peptide or polypeptides). Embodiments associated with the expression of heterologous peptides or polypeptides have been described in U.S. patent application published under 2022 / 0127564, U.S. patent application published under 2025 / 0346873, U.S. patent application published under 2024 / 0084244, U.S. patent application published under 2025 / 0197830, PCT application published under W02025 / 057132, and U.S. provisional patent application 63 / 740,120 filed on December 30, 2024 and are herewith incorporated in their entirety.

[0121] Applications of the recombinant yeast host cells

[0122] The present disclosure provides a process for making one or more fermentation products using the recombinant yeast host cell. The production of the one or more fermentation products is favored in the recombinant yeast host cell (when compared to a corresponding control yeast host cell) because it comprises (i) a heterologous ATP futile cycle and (ii) an engineered metabolic pathway for limiting the production of one or more fermentation by-products.

[0123] Broadly, the process of the present disclosure comprises contacting the recombinant yeast host cell with a source of a carbohydrate under conditions to allow the conversion of at least in part of the carbohydrate into the at least one fermentation products (e.g., fermenting step). In some embodiments, the fermentation step can be conducted until its completion (e.g., fermentation drop), for example when available carbohydrates in the fermentation medium are exhausted and / or when no fermentation activity is observed from the recombinant yeast host cells in the fermentation medium. The source of the carbohydrate present in a fermentation medium (sometimes referred to as a biomass) can include, in some embodiments, a C6 carbohydrate (like glucose for example). The source of the carbohydrate present in a fermentation medium (sometimes referred to as a biomass) can include, in some additional embodiments, a C5 carbohydrate (like arabinose and / or xylose for example). The process can optionally include a step of isolating the at least one fermentation product from the fermented fermentation medium (using distillation and / or mechanical separation for example). When the process is used to make both a fermentation product and a fermentation co-product, the process can optionally include a step of isolating the fermentation co-product from the fermented fermentation medium and / or from the fermentation product. In some embodiments, the process can include, prior to the contacting / fermenting step, a step of propagating the recombinant yeast host cell. When the process is used to make a biofuel, the process can also optionally include a step of recuperating the distiller’s grain after fermentation for animal nutrition.

[0124] In some embodiments, the at least one fermentation product comprises ethanol. In some additional embodiments, the at least one fermentation product comprises ethanol makes up the majority of fermentation products being produced by the recombinant yeast host cell. In additional embodiments, the one or more fermentation products can comprise, without limitation, acetone, farnesene, 3-hydroxy-propionic acid, p-coumaric acid, 2-phenylethanol, tryosol, salidroside, polyhydroxybutyrate, carotenoid, a fatty acid ethyl ester, ethyl acetate, isopropanol, 1 -propanol, ethanol or combinations thereof. In some embodiments, the fermentation products can comprise at least two distinct fermentation products such as at least two of any one of the following: acetone, farnesene, 3-hydroxy-propionic acid, p-coumaric acid, 2-phenylethanol, tryosol, salidroside, polyhydroxybutyrate, carotenoid, a fatty acid ethyl ester, ethyl acetate, isopropanol, 1 -propanol, or ethanol. In some specific embodiments, the fermentation product can comprise acetone and ethanol. In some embodiments, the fermentation products can comprise at least three distinct fermentation products such as at least three of any one of the following: acetone, farnesene, 3-hydroxy-propionic acid, p- coumaric acid, 2-phenylethanol, tryosol, salidroside, polyhydroxybutyrate, carotenoid, a fatty acid ethyl ester, ethyl acetate, isopropanol, 1 -propanol, or ethanol. In some specific embodiments, the fermentation product can comprise acetone, isopropanol and ethanol. In some embodiments, the fermentation product comprises an amino acid or a combination of different amino acids. In another embodiment, the fermentation product comprises a peptide. In yet another embodiment, the fermentation product comprises a polypeptide.

[0125] In some embodiments, the process of the present disclosure comprises a single fermentation (batch, fed-batch or continuous). In other embodiments, the process of the present disclosure comprises a plurality of fermentations in which the recombinant yeast host cells are recycled between two rounds of consecutive fermentations. In some embodiments, the recombinant yeast host cells are only exogenously added in the initial fermentation cycle and are then recycled in one or more further fermentation cycles. In such embodiment, each fermentation cycle of the process includes contacting a fermentation medium (comprising a fermentable carbohydrate) with a fermenting population under conditions to allow the conversion of the fermentable carbohydrate in a fermentation product (e.g., fermentation). At the end of the fermentation, the fermenting population present in the fermented fermentation medium is substantially isolated from the fermented fermentation medium and use to initiate another fermentation cycle. It is understood that, in such embodiments, the initial fermenting population consists essentially in the recombinant yeast host cells of the present disclosure and that, during the plurality of the fermentation cycles, the recycled fermenting population can include some contaminating wild (non-genetically modified) yeasts. The plurality of fermentation cycles can include at least one continuous fermentation. The plurality of fermentation cycles can only include continuous fermentations. The plurality of fermentation cycles can include at least one batch fermentation. The plurality of fermentation cycles can only include batch fermentations. The processes of the present disclosure can include an initial fermentation cycle at least one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 39 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 49 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 59 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 69 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 79 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 89 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 99 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 109 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 119 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 129 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 139 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 149 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 159 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 169 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 179 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 189 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 199 further fermentation cycles. Processes comprising a plurality of fermentation cycles have been disclosed in U.S. patents serial number 12247208 and 12385052 and are herewith incorporated by reference in their entirety.

[0126] Each fermentation cycle (irrespective of its type) comprises a step of contacting the fermentation medium with a fermenting population of recombinant yeast host cells. The fermenting population will ferment (e.g., convert some of the biomass into a fermentation product) the fermentation medium to generate a fermented medium (which comprises the one or more fermentation products, and optionally a fermentation co-product and / or a fermentation by-product). The contacting step can include simultaneously adding the fermenting population and the fermentation medium to a fermenter. The contacting step can include adding the fermentation medium to a fermenter and subsequently adding the fermenting population to the fermentation medium. The contacting step can include adding fermentation medium to a fermenter already containing the fermentation population. After the fermentation, a fermented medium will be obtained, and it comprises a fermentation product and a fermented population. In a process comprising a batch fermentation cycle, the fermenting population is contacted e.g., pitched) with a fermentation medium in a fermenter. The fermenting population can be added to the fermenter prior to, at the same time and / or after the fermentation medium has been added. In a process comprising a continuous fermentation cycle, the fermenting population can be added to the series of fermenters prior to, at the same time and / or after the fermentation medium has been added.

[0127] The fermentation medium that can be fermented by the recombinant yeast host cells described herein includes any type of fermentable carbohydrate known in the art and described herein. In one embodiment, the carbohydrate source is derived from a biomass. For example, the biomass can include, but is not limited to, starch, sugar and lignocellulosic materials comprising lignocellulosic fibers. In specific embodiments, the biomass comprises C6 carbohydrates like glucose. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. The starch present in the biomass can be totally or in part in a raw form or in a gelatinized form. When the biomass comprises or is derived from corn, it can include a corn mash. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, molasses or cane. In other embodiments, the biomass comprises C5 carbohydrates (like arabinose and xylose). The terms “lignocellulosic material”, “lignocellulosic substrate” and “cellulosic biomass” mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and / or agricultural residues, forestry residues and / or forestry wastes, paper-production sludge and / or waste paper sludge, waste -water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants and sugar-processing residues. The terms “hemicellulosics”, “hemicellulosic portions” and “hemicellulosic fractions” mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (i.e., comprising mannan, glucomannan and galactoglucomannan), pectins e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan) and proteoglycans (e.g., arabinogalactan-protein). In some embodiments, the biomass can include and / or be supplemented with citric acid (especially when acetic acid or acetate is the first metabolic product).

[0128] In a non-limiting example, the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; sugar cane must; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; succulents, such as but not limited to, agave; and forestry wastes, such as but not limited to, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials. Other lignocellulosic materials are agricultural wastes, such as cereal straws, including wheat straw, barley straw, canola straw and oat straw; corn fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.

[0129] Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (Avicel), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.

[0130] It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and / or insoluble cellulose, where the insoluble cellulose may be in a crystalline or noncrystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, molasses, sugarcane, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.

[0131] Paper sludge is also a viable feedstock for lactate or acetate production. Paper sludge is solid residue arising from pulping and paper-making and is typically removed from process wastewater in a primary clarifier. The cost of disposing of wet sludge is a significant incentive to convert the material for other uses, such as conversion to ethanol.

[0132] In specific embodiments, the fermentation medium comprises sugarcane or a sugarcane derivative. After it has been harvested, the sugarcane is pressed or diffused to generate sugarcane juice and a solid fibrous residue, the cane bagasse. In the context of the present disclosure, sugarcane juice is considered to be a sugarcane derivative. The sugarcane juice can be clarified and concentrated by evaporation until sucrose crystallization is observed. The clarified sugarcane juice and the concentrated sugarcane juice are considered sugarcane derivatives. The sucrose crystals obtained after crystallization can be collected by centrifugation, generating a sucrose saturated viscous phase, called “cane molasses”. Cane molasses, which is also considered to be a sugarcane derivative, can include between 45 to 60 % sucrose and 5 to 20 % glucose plus fructose. In some embodiments, the fermentation medium comprises, as a sugarcane derivative, a sugarcane juice. In another embodiment, the fermentation medium comprises, as a sugarcane derivative, a cane molasses. In still another embodiment, the fermentation medium comprises, as a sugarcane derivative, both sugarcane juice and a cane molasses.

[0133] In specific embodiments, the fermentation medium includes a carbohydrates source as fermentable materials which contain C6 sugar as for example fructose, glucose, galactose, sucrose, maltose, maltotriose, starch or fructans, as well as their degradation products. As an example, the carbohydrates source can be or comprise a fruit (apple, grape, pears, plums, cherries, peaches), a plant (sugar cane, sugar beet, agave, ginger), a sugar material (honey, molasses), a starchy material (rice, rye, corn, sorghum, millet, barley, wheat, triticale, potatoes, cassava) or a derived product (grape must, apple mash, malted grain (or cereal), crushed fruit, fruit puree, fruit juice, fruit must, plant mash, plant juice, gelatinized and saccharified starch from different plant origins as rice, corn, sorghum, wheat, barley). In another embodiment, the fermentation medium or mixture can be or comprise a starchy material. In the context of the present disclosure, a “starchy material” refers to a material that contains starch that could be converted into alcohol by a yeast during alcoholic fermentation. Starchy material could be for example, gelatinized and saccharified starch from cereals, grains (wheat, barley, rice, buckwheat) or grain derived products (malted grain or a wort) or vegetable (potatoes, cassava). In yet another embodiment, the fermentation medium can be or comprise, but is not limited to, barley, wheat, rye, oats, corn, maize, buckwheat, millet, rice, sorghum, including variants of these cereals that have been subject to the malting, cooking (torrefaction) or micronization process, or a combination thereof. In one embodiment, the malted grain (or cereal) is malted barley, malted wheat, malted rye, malted oats, malted corn, malted buckwheat, malted millet, malted rice, and malted sorghum. In another embodiment, the torrefied grain (or cereal) are torrefied barley, torrefied wheat, torrefied rye, torrefied oats, torrefied corn, torrefied buckwheat, torrefied millet, torrefied rice and torrefied sorghum. In yet another embodiment, the micronized grain (or cereal) is micronized barley, micronized wheat, micronized rye, micronized oats, micronized corn, micronized buckwheat, micronized millet, micronized rice and micronized sorghum. In another embodiment, the carbohydrate source comprise barley. In some embodiments, the recombinant yeast host cell of the present disclosure can be submitted to a method for making a beverage such as an alcoholic beverage. In such embodiments, the alcoholic beverage has between 1 to 99 %ABV, between 1 to 80 %ABV, between 1 to 20 %ABV, between 5 to 99 %ABV, between 5 to 80 %ABV, between 5 to 60 %ABV, between 5 to 40 %ABV, between 20 to 80 %ABV, between 30 to 80 %ABV, or between 35 to 80 %ABV. In an embodiment, the beverage is beer and has between 0.5% to 1 %ABV. Examples of alcoholic beverage products include, but are not limited to beer, brandy, cachaga, Cognac, mezcal, whisky, whiskey (for example bourbon, rye whiskey, wheat whiskey), gin, tequila, rum (for example rhum agricole), wine, mead, sake, baiju, shochu, soju, cider, perry, arrack, jenever, vermouth, Armagnac, korn, raki, pulque, basi, vodka, poitin, akvavit, aquavit, absinthe, spirits, new-make spirit, white dog, or moonshine.

[0134] The fermentation of the process can be performed at temperatures of at least about 25°C, about 28°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 50°C. In some embodiments, the process can be conducted at temperatures above about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 50°C.

[0135] In some embodiments, prior to fermentation, a step of liquefying starch can be included in the process. In such embodiment, the liquefied starch is then submitted to a following fermentation step. The liquefaction of starch can be performed at a temperature of between about 70°C- 105°C to allow for proper gelatinization and hydrolysis of the starch. In an embodiment, the liquefaction occurs at a temperature of at least about 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or 105°C. Alternatively, or in combination, the liquefaction occurs at a temperate of no more than about 105°C, 100°C, 95°C, 90°C, 85°C, 80°C, 75°C or 70°C. In yet another embodiment, the liquefaction occurs at a temperature between about 80°C and 85°C (which can include a thermal treatment spike at 105°C).

[0136] During fermentation, the pH of the fermentation medium can be equal to or below 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7., 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 , 4.0 or lower. In an embodiment, the pH of the fermentation medium (during fermentation) is between 4.0 and 5.5.

[0137] In some embodiments, the processes of the present disclosure comprise producing ethanol at a particular rate. For example, in some embodiments, ethanol is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1 .0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, at least about 1 g per hour per liter, at least about 1 .5 g per hour per liter, at least about 2 g per hour per liter, at least about 2.5 g per hour per liter, at least about 3 g per hour per liter, at least about 3.5 g per hour per liter, at least about 4 g per hour per liter, at least about 4.5 g per hour per liter, at least about 5 g per hour per liter, at least about 5.5 g per hour per liter, at least about 6 g per hour per liter, at least about 6.5 g per hour per liter, at least about 7 g per hour per liter, at least about 7.5 g per hour per liter, at least about 8 g per hour per liter, at least about 8.5 g per hour per liter, at least about 9 g per hour per liter, at least about 9.5 g per hour per liter, at least about 10 g per hour per liter, at least about 10.5 g per hour per liter, at least about 11 g per hour per liter, at least about 11 .5 g per hour per liter, at least about 12 g per hour per liter, at least about 12.5 g per hour per liter, at least about 13 g per hour per liter, at least about 13.5 g per hour per liter, at least about 14 g per hour per liter, at least about 14.5 g per hour per liter or at least about 15 g per hour per liter.

[0138] In some embodiments, the processes of the present disclosure can produce ethanol at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1 .0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, at least about 1 g per hour per liter, at least about 1 .5 g per hour per liter, at least about 2 g per hour per liter, at least about 2.5 g per hour per liter, at least about 3 g per hour per liter, at least about 3.5 g per hour per liter, at least about 4 g per hour per liter, at least about 4.5 g per hour per liter, at least about 5 g per hour per liter, at least about 5.5 g per hour per liter, at least about 6 g per hour per liter, at least about 6.5 g per hour per liter, at least about 7 g per hour per liter, at least about 7.5 g per hour per liter, at least about 8 g per hour per liter, at least about 8.5 g per hour per liter, at least about 9 g per hour per liter, at least about 9.5 g per hour per liter, at least about 10 g per hour per liter, at least about 10.5 g per hour per liter, at least about 11 g per hour per liter, at least about 1 1 .5 g per hour per liter, at least about 12 g per hour per liter, at least about 12.5 g per hour per liter, at least about 13 g per hour per liter, at least about 13.5 g per hour per liter, at least about 14 g per hour per liter, at least about 14.5 g per hour per liter, at least about 15 g per hour per liter or more than a control recombinant yeast host cell and grown under the same conditions. Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzymebased assays.

[0139] In the process described herein, it is possible to add an exogenous source (e.g., to dose) of an enzyme to facilitate saccharification or improve fermentation yield. As such, the process can comprise including one or more doses of one or more exogenous enzymes during the liquefaction / saccharification and / or the fermentation step. The exogenous enzyme can be provided in a purified form or in combination with other enzymes (e.g., a cocktail). In the context of the present disclosure, the term “exogenous” refers to a characteristic of the enzyme, namely that it has not been produced during the saccharification or the fermentation step by the recombinant yeast host cell, but that it was produced prior to the saccharification or the fermentation step. The exogenous enzyme that can be used prior to, during and / or after the saccharification / fermentation process can include, without limitation, an alpha-amylase, a glucoamylase, a protease, a phytase, a pullulanase, a fiber-degrading enzyme (such as, for example a cellulase, and / or a xylanase), a trehalase, or any combination thereof.

[0140] In the process described herein, it is possible to add a nitrogen source (usually urea or ammonia) to facilitate liquefaction / saccharification or improve fermentation yield. As such, the process can comprise including one or more amount of the nitrogen source prior to or during the saccharification and / or the fermentation step.

[0141] The processes of the present disclosure include those associated with the production of peptides or polypeptides (including, without limitation, enzymes) as the fermentation product(s). In such processes, recombinant yeast host cells can be cultured or propagated to allow the expression and the accumulation of the peptides / polypeptides. The growth phase can be optionally followed by a stationary phase. The growth phase as well as the stationary phase can be conducted in a culture medium allowing the cell growth and division of the recombinant yeast host cells under conditions (agitation, temperature, oxygen concentration, etc.) to favor the expression and accumulation, and optionally the secretion, of the peptides / polypeptides. In some embodiments, during or after the growth phase, the recombinant yeast host cells can be placed in the presence of an inducer which can allow the expression of the heterologous peptides / polypeptides.

[0142] In the processes for making peptides and polypeptides, during the growth phase, the recombinant yeast host cell is placed in contact with a source of metabolizable carbon sources. When the recombinant yeast host cell is Komagataella phaffii or Saccharomyces cerevisiae, the source of metabolizable carbon is a C1-C6 carbon source. In some embodiments, the source of metabolizable carbon can include a C2 carbon the source, including, without limitation, ethanol. Alternatively, or in combination, the source of metabolizable carbon can include, without limitation glucose, sucrose, fructose, glycerol, or a combination thereof. In a specific embodiment, during the growth phase, the recombinant yeast host cell is contacted with glucose as the sole source of metabolizable carbohydrate. In another specific embodiment, during the growth phase, the recombinant yeast host cell is contacted with glycerol as the sole source of metabolizable carbohydrate. In still another embodiment, during the growth phase, the recombinant yeast host cell is contacted with fructose as the sole source of metabolizable carbohydrate. In still another embodiment, during the growth phase, the recombinant yeast host cell is contacted with sucrose as the sole source of metabolizable carbohydrate. In some embodiments, the growth phase is performed as a continuous fermentation. In alternative embodiments, the growth phase is performed as a batch fermentation. In yet further embodiments, the growth phase is performed as a fed batch fermentation. The expression of the heterologous peptides or polypeptides can be performed, at least in part, in aerobic conditions. The expression of the heterologous peptides or polypeptides can be performed, at least in part, in anaerobic conditions.

[0143] In some embodiments, the process can further include a step of purifying (at least in part) the heterologous peptides / polypeptides from the recombinant yeast host cell. The purifying step refers to a step of physically dissociating, at least in part, the expressed peptides / polypeptides from the components of the recombinant yeast host cell having expressed same. The expression “substantially purified form” refers to the fact that the expressed peptides / polypeptides have been physically dissociated from some (and in some embodiments from the majority) of the components of the recombinant yeast host cells having expressed the peptides / polypeptides. In an embodiment, a composition comprising the expressed peptides / polypeptides in substantially purified form is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% pure. In some embodiments, the composition comprising the expressed peptides / polypeptides lacks a detectable amount of deoxyribonucleic acids from the recombinant yeast host cell used to express it. The purification step can include, without limitation, a filtrating step, a centrifugating step, a dialysing step, a precipitation step, an affinity capture step, a chromatographic step, etc.

[0144] In embodiments in which the peptide / polypeptide is intended to be expressed intracellularly, the method can include a cell-permeabilizing and / or cell-lysing step (after the expression step). The person skilled in the art will recognize that are many ways of permeabilizing and / or lysing recombinant yeast host cells. For example, the yeast cells can be homogenized (for example using a bead-milling technique, a bead-beating, or a high-pressure homogenization technique) and, as such, the processes can include a homogenizing step. In another example, the cells can be enzymatically treated, and as such, the method can include an enzyme treatment step. In still another embodiment of the processes of the present disclosure, the recombinant yeast host cells can be treated in basic or acidic conditions, and as such, the method can include a pH treatment step. In yet other embodiments of the processes of the present disclosure, the recombinant yeast host cells can be submitted to osmotic pressure and, as such, the processes can include a salt treatment step. In still yet further embodiments of the processes of the present disclosure, the recombinant yeast host cells can be submitted to a heat pressure and, as such, the processes can include a cold treatment or a heat treatment step.

[0145] In some embodiments, the propagated recombinant yeast host cells can be lysed using autolysis (which can optionally be performed in the presence of additional exogenous enzymes). For example, the propagated recombinant yeast host cells may be subject to a combined heat and pH treatment for a specific amount of time (e.g., 6, 12, 18, 24, 36, 48 h or more) in order to cause the autolysis of the propagated recombinant yeast host cells to provide the lysed recombinant yeast host cells. For example, the propagated recombinant yeast host cells can be submitted to a temperature of between about 40°C to about 70°C or between about 50°C to about 60°C. The propagated recombinant yeast host cells can be submitted to a temperature of at least about 40°C, 41 °C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C. Alternatively or in combination the propagated recombinant yeast host cells can be submitted to a temperature of no more than about 70°C, 69°C, 68°C, 67°C, 66°C, 65°C, 64°C, 63°C, 62°C, 61 °C, 60°C, 59°C, 58°C, 57°C, 56°C, 55°C, 54°C, 53°C, 52°C, 51 °C, 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41 °C or 40°C. In another example, the propagated recombinant yeast host cells can be submitted to a pH between about 3.5 and 8.5, between about 5.0 and 7.5, or between about 5.0 and 6.0. The propagated recombinant yeast host cells can be submitted to a pH of at least about, 3.5, 3.6,

[0146] 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,

[0147] 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,

[0148] 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4 or 8.5. Alternatively or in combination, the propagated recombinant yeast host cells can be submitted to a pH of no more than 8.5, 8.4, 8.3, 8.2, 8.1 , 8.0, 7.9, 7.8,

[0149] 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1 , 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7,

[0150] 5.6, 5.5, 5.4, 5.3., 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 , 3.9, 3.8, 3.7, 3.6 or 3.5. If necessary, the lysed recombinant yeast host cell can be submitted to a centrifugation and / or a filtration step to purify, at least in part, the peptides / polypeptides.

[0151] The processes can also include a drying step (before, after, or both before and after the purifying step). The drying step can include, for example, roller-drying, electrospray-drying, freeze-drying, spray-drying, lyophilization, and / or fluid-bed drying. The processes can also include a washing step (before, after, or both before and after the purifying step). In embodiments in which the drying step includes spray-drying, a carrier which is inert to the activity of the peptide / polypeptide can be used. Such carriers include, without limitations, salts such as starch, a salt (like NaCI or KCI) or maltodextrin.

[0152] The processes of the present disclosure can be used to provide the peptides / polypeptides in a yeast composition comprising living yeasts. In such embodiment, afterthe growth phase (and optionally the stationary phase), the recombinant yeast host cells can be substantially separated from the medium, optionally washed and / or dried, so as to be formulated in a yeast composition (in which part or all of the medium used has been removed). Embodiments of microbial compositions made from a recombinant yeast host cells (referred to as a yeast composition), include but are not limited to, a yeast cream, a stabilized liquid yeast, an active dry yeast or an instant dry yeast.

[0153] The processes can also be used to make a yeast product (e.g., a composition derived from a recombinant yeast host cell having expressed the peptides / polypeptides). In such embodiments, after growth, the yeasts can be substantially separated from the medium, optionally washed, lysed, submitted to a soluble / insoluble separation and / or dried, so as to be formulated in a yeast product. Embodiments of yeast products made from a recombinant yeast host cells, include but are not limited to, a yeast autolysate, yeast cell walls, or a yeast extract. The yeast products of the present disclosure can include, besides the peptide / polypeptide, at least one component of a recombinant yeast host cell. The “at least one component of a recombinant yeast host cell” can be an intracellular component and / or a component associated with the microbial host cell’s wall or membrane. The “at least one component of a recombinant yeast host cell” can include a protein, a peptide or an amino acid, a carbohydrate and / or a lipid. The “at least one component of a recombinant yeast host cell” can include a recombinant yeast host cell’s organelle.

[0154] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

[0155] EXAMPLE I

[0156] The Saccharomyces cerevisiae strains characterized in Example I are described in Table 1 . Table 1 . Description of the various Saccharomyces cerevisiae strains characterized in Example

[0157] Laboratory scale fermentations. The strains were added to a 30-gram commercial corn mash medium (having total solids of 31 .98%) at an inoculum size of 0.12 ODeoo / kg in 60-ml serum bottles attached to a CO2pressure monitoring system. The fermentations were performed for 72 hours with a temperature staging of 33°C for 24 hours and 31°C for 48 hours. Each fermentation was conducted twice. At the end of the fermentations, samples were subjected to HPLC analysis to determine glucose, glycerol, ethanol, isopropanol and / or acetone levels. Various heterologous enzymes (including those having fructose-1 ,6-bisphosphatase activity) were chromosomally integrated at a neutral integration site in different S. cerevisiae strains. Among the heterologous enzymes tested, those derived from Yarrowia lipolytica (expressed in strain Y3), Xanthomonas campestris (expressed in Y4), and Porphyromonas gingivalis (expressed in strain Y5) significantly reduced glycerol production (Figure 2) when compared to the wildtype yeast (Y1). In addition, under the conditions tested, the expression of the Yarrowia lipolytica FBPase (expressed in strain Y3) did not impair fermentation kinetics (Figure 3).

[0158] The effect of different fermentation promoters to express the Yarrowia lipolytica fructose-1 ,6- bisphosphatase (FBPase) was then determined during fermentation. The recombinant strains expressing a heterologous FBPase under the control of various promoters exhibited an increase in ethanol production, and a decrease glycerol production (Figure 4) while maintaining fermentation kinetics (Figures 5A and 5B) when compared to the wildtype strain (Y1).

[0159] The effect of the heterologous expression of the Y. lipolytica in an ethanol-producing yeast strain engineered to reduce the production of glycerol was determined. The strains expressing a heterologous FBPase exhibited an increase in ethanol production, a decrease in glycerol production when compared to the parental strain Y13 (Figure 6). In addition, strain Y15 also exhibited improved fermentation kinetics (Figure 7).

[0160] The effect of the heterologous expression of the Y. lipolytica in an acetone / ethanol-producing yeast strain which was further engineered to reduce the production of glycerol was determined. The acetone / ethanol-producing strain expressing a heterologous FBPase exhibited an increase in ethanol production, and a decrease in glycerol production when compared to the parental strain (data not shown). In addition, the acetone / ethanol-producing strain expressing a heterologous FBPase exhibited improved fermentation kinetics (data not shown).

[0161] The activity of some FBPases, like the Y. lipolytica FBPase, is known to be allosterically inhibited by fructose 2,6-bisphosphate (F26BP). In Saccharomyces cerevisiae, fructose 2,6- bisphosphate is synthesized from fructose-6-phosphate by a 6-phosphofructo-2-kinase (known as PFK26 or PFK27). It was thus determined if the deletion of PFK26 / 27 could further increase the effects of the heterologous FBPase from Y. lipolytica on fermentation performances. As indicated in Table 1 , PFK26 and PFK27 were deleted individually or in combination in strains Y17, Y18, and Y19. When compared to the wildtype (Y1) or the parental strain (Y15), single knockout of PFK26 (in strain Y17) and PFK27 (in strain Y18) or dual knockout of PFK26 / PFK27 (in strain Y19) further reduced glycerol and increased ethanol while maintaining the fermentation kinetics (Figures 8 and 9).

[0162] EXAMPLE II

[0163] Table 2. Description of the various Saccharomyces cerevisiae strains characterized in Example II. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0164] The strains were added to a 4-gram commercial corn mash medium (having total solids of 32.4%) at an inoculum size of 0.06 g of dry cell weight (DCW) / liter (L) in mini vials. The fermentations were performed for 51 hours in permissive conditions with a temperature of 33.3°C. At the end of the fermentations (drop), samples were subjected to HPLC analysis to determine glycerol and ethanol levels.

[0165] As shown in Table 3, the deletion of gpd1 decreased glycerol levels (compare Y22 / Y23 to Y21). As also shown in Table 3, the combination of the deletion of gpd1 and the heterologous expression of a FBPase further decreased glycerol levels (compare Y24 / Y25 to Y22 / Y23). Table 3. Metabolites measured at the end of the fermentations (in g / L).

[0166] The strains were added to a 25-gram commercial corn mash medium with high solids (having total solids of 35.5%) at an inoculum size of 0.06 g DCW / L in serum vessels sealed with rubber stoppers and aluminum crimps. The bottles were attached to a gas manifold system and the headspace flushed with a sterile gas mixture of CO2-N2 (purged) to remove residual oxygen ensuring anaerobic conditions at the start of fermentation. The fermentations were performed for 73 hours under permissive-purged conditions with a temperature staging of 33.1 °C to 30.6°C. At 24h (for permissive only) and at the end of the fermentations (drop), samples were subjected to HPLC analysis to determine glycerol levels.

[0167] As shown in Table 4, the presence of a heterologous expression of a FBPase or GUTI helped reducing the accumulation of glycerol in both permissive and purged conditions.

[0168] Table 4. Glycerol measured during and at the end of the permissive fermentations (in g / L) in both permissive and purged conditions.

[0169] EXAMPLE III

[0170] Table 5. Description of the various Saccharomyces cerevisiae strains characterized in Example III. Both the Y27 and Y28 strains both expressed a Streptococcus mutans NADP+- dependent glyceraldehyde-3-phosphate dehydrogenase (having the nucleic acid sequence of SEQ ID NO: 10 and encoding the amino acid sequence of SEQ ID NO: 9), a Saccharomyces cerevisiae STL1 polypeptide (having the nucleic acid sequence of SEQ ID NO: 12 and encoding the amino acid sequence of SEQ ID NO: 11), a Zymomonas mobilis pyruvate decarboxylase (having the nucleic acid sequence of SEQ ID NO: 14 and encoding the amino acid sequence of SEQ ID NO: 13), a Neurospora crassa trehalase (having the nucleic acid sequence of SEQ ID NO: 18 and encoding the amino acid sequence of SEQ ID NO: 17), and a Aspergillus terre us alpha-amylase (having the nucleic acid sequence of SEQ ID NO: 1 and encoding the amino acid sequence of SEQ ID NO: 2). Strains Y27 and Y28 also included deletions in the zwf1 and st!1 native genes. The additional genetic modifications included in strains Y27, and Y28 (if any) are provided in Table 5 (N.A. = no additional native gene deleted). The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0171] The strains (either in as pure cultures or blends) were added to a 25-gram commercial corn mash medium (having total solids of 33.5%) at an inoculum size of 4.47% v / v (inoculum from an 150 gram mash propagation incubated 9 h at 34°C (having total solids of 19%) at an inoculum size of 0.181 g DCW / L) in serum vessels. Prior to fermentation, the medium was supplemented with a 20%, 30% or a 62% dose of an exogenous glucoamylase where 100% enzyme inclusion is dosed at 2.111 E-04 v / v. The fermentations were performed for 57 hours in purged conditions (in which the headspace has been flushed with CO2 / N2) with a temperature staging of 32°C to 31 .5°C. At the end of the fermentations, samples were subjected to HPLC analysis to determine iso-amyl alcohol, iso-butanol, active amyl alcohol, and N-propanol levels.

[0172] As shown in Table 6, the use of a strain expressing a heterologous FBPase alone or in a blend with another strain which does not express the heterologous FBPase decreased the accumulation of fusel alcohols during fermentation (compare Y28 and Y27 / Y28 blend to Y27). Table 6. Amount of fusel alcohols (in ppm) obtained at the end of fermentation (drop). The blend comprises 10% of Y27 and 90% of Y28 (at inoculation). Results are shown as averages of all glucoamylase doses in mg / L.

[0173] EXAMPLE IV

[0174] Table 7. Description of the various Saccharomyces cerevisiae strains characterized in Example IV. Strains Y28 and Y29 both expressed a Streptococcus mutans NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (having the nucleic acid sequence of SEQ ID NO: 10 and encoding the amino acid sequence of SEQ ID NO: 9), a Saccharomyces cerevisiae STL1 polypeptide (having the nucleic acid sequence of SEQ ID NO: 12 and encoding the amino acid sequence of SEQ ID NO: 11), a Zymomonas mobilis pyruvate decarboxylase (having the nucleic acid sequence of SEQ ID NO: 14 and encoding the amino acid sequence of SEQ ID NO: 13), a Neurospora crassa trehalase (having the nucleic acid sequence of SEQ ID NO: 18 and encoding the amino acid sequence of SEQ ID NO: 17), a Aspergillus terreus alpha-amylase (having the nucleic acid sequence of SEQ ID NO: 1 and encoding the amino acid sequence of SEQ ID NO: 2) as well as a Rasamsonia emersonii glucoamylase (having the nucleic acid sequence of SEQ ID NO: 16 and encoding the amino acid sequence of SEQ ID NO: 15). Strains Y28 and Y29 also included deletions in the zwf1 and stl1 native genes. The additional genetic modifications included in strains Y28 and Y29 (if any) are provided in Table 7 (N.A. = no additional heterologous gene expression or native gene deleted). The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0175] StrainY28 (100%) or a blend of the strains (Y28 / Y29 : 90 weight % of Y28 and 10 weight % of Y29) were added to a 25-gram commercial corn mash medium (having total solids of 36.8%) at an inoculum size of 0.06 g DCW / L in serum vessels. The fermentations were performed for 63 hours in permissive conditions at a temperature staging of 32°C to 31 ,5°C. At the end of the fermentations, samples were subjected to HPLC analysis to determine ethanol and glycerol levels.

[0176] As shown in Table 8, the Y28 / Y29 blend comprising a strain expressing a heterologous FBPase generated more ethanol and less glycerol than strain Y28 expressing a heterologous FBPase (alone).

[0177] Table 8. Ethanol and glycerol field (in g / L) at the end of fermentation (drop).

[0178] EXAMPLE V

[0179] Table 9. Description of the various Saccharomyces cerevisiae strains characterized in Example V. Strain Y1 is a wild-type non-genetically modified strain. Strains Y13 and Y28 both expressed a Streptococcus mutans NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (having the nucleic acid sequence of SEQ ID NO: 10 and encoding the amino acid sequence of SEQ ID NO: 9), a Saccharomyces cerevisiae STL1 polypeptide (having the nucleic acid sequence of SEQ ID NO: 12 and encoding the amino acid sequence of SEQ ID NO: 11), a Zymomonas mobilis pyruvate decarboxylase (having the nucleic acid sequence of SEQ ID NO: 14 and encoding the amino acid sequence of SEQ ID NO: 13), a Neurospora crassa trehalase (having the nucleic acid sequence of SEQ ID NO: 18 and encoding the amino acid sequence of SEQ ID NO: 17), a Aspergillus terreus alpha-amylase (having the nucleic acid sequence of SEQ ID NO: 1 and encoding the amino acid sequence of SEQ ID NO: 2) as well as a Rasamsonia emersonii glucoamylase (having the nucleic acid sequence of SEQ ID NO: 16 and encoding the amino acid sequence of SEQ ID NO: 15). Strains Y13 and Y28 also included deletions in the zwf1 and stl1 native genes. The additional genetic modifications included in strains Y13 and Y28 (if any) are provided in Table 9 (N.A. = no additional heterologous gene expression or native gene deleted). The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0180] Strains were added to a 25 mL container comprising in a Verduyn medium at an inoculum size of 0.06 g DCW / L. The fermentations were performed for 24 hours at a temperature of 32°C. At the end of the fermentations, samples were subjected to HPLC analysis to determine ethanol and glycerol levels. The dry cell weight (DCW) was also determined at the end of the fermentation.

[0181] As shown in Table 10, the expression of a heterologous FBPase increased the amount of ethanol produced, decreased the amount of glycerol accumulated as well as decreased the dry cell weight obtained at the end of the fermentation (compare Y13 with Y28).

[0182] Table 10. Ethanol, glycerol and dry cell weight (all in g / L) determined at the end of the fermentation.

[0183] EXAMPLE VI

[0184] Table 11. Description of the various Saccharomyces cerevisiae strains characterized in Example VI. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0185] The strains were added to a 30-gram commercial corn mash medium (having total solids of 32%) at an inoculum size of 0.12 ODeoo / kg in 60-ml serum bottles attached to a CO2pressure monitoring system. The fermentations were performed for 72 hours with a temperature staging of 33.1 °C for 24 hours and 31 ,0°C for 48 hours. At the end of the fermentations, samples were subjected to HPLC analysis to determine glycerol and ethanol levels.

[0186] As shown in Table 12, the expression of a heterologous FBPase, a heterologous STL1 and a heterologous GAPN in strain Y62 further reduced glycerol production when compared to strain Y1 1 which only expressed a heterologous FBPase. Table 12. Ethanol and glycerol yields (in g / L) obtained at the end of the fermentation.

[0187] EXAMPLE VII

[0188] Table 13. Description of the various Saccharomyces cerevisiae strains characterized in

[0189] Example VII. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0190] The strains were added to a 25-gram commercial corn mash medium (having total solids of 33.14%) at an inoculum size 0.06 g DCW / L in serum vessels. The fermentations were incubated for 51 hours in permissive conditions with a temperature of 33.3°C. At the end of the fermentations (drop), samples were subjected to HPLC analysis to determine glycerol and ethanol levels.

[0191] As shown in Table 14, the expression of a heterologous FBPase in strains having a single glycerol reduction technology maintained or increased ethanol production while lowering glycerol accumulation (compare strain Y33 with Y32; strain Y35 with Y34, strain Y37 with Y36, strain Y39 with Y38 and strain Y31 with strain Y30).

[0192] Table 14. Ethanol and glycerol yields (in g / L) obtained at the end of the fermentation.

[0193] EXAMPLE VIII

[0194] Table 15. Description of the various Saccharomyces cerevisiae strains characterized in

[0195] Example VIII. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0196] The strains were added to serum vessels containing 20 mL commercial sugarcane must (~100 g / L total fermentable sugars) with a 40 pL inoculum from a YPD4% overnight culture. The fermentations were performed for 23 hours with a temperature of 33°C. At the end of the fermentations (drop), samples were subjected to HPLC analysis to determine the levels of various metabolites.

[0197] As shown in Table 16, strains expressing a heterologous FBPase produced more ethanol, generated less glycerol and isoamyl alcohol when compared to strains lacking such FBPase (compare strain Y41 with Y40, strain Y43 with Y42, strain Y45 with Y44, strain Y47 with Y46, strain 49 with Y58, strain 51 with Y50). The strains expressing a heterologous FBPase also finished fermentation sooner than strains lacking such FBPase (data not shown).

[0198] Table 16. Ethanol and glycerol yields (in g / L) as well as isoamyl alcohol (in ppm) obtained at the end of the fermentation.

[0199] EXAMPLE IX

[0200] Table 17. Description of the various Saccharomyces cerevisiae strains characterized in

[0201] Example IX. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0202] The strains were added to serum vessels containing 20 mL commercial sugarcane must (~100 g / L total fermentable sugars) with a 40 pL inoculum from a YPD4% overnight culture. The fermentations were performed for ~70 hours in permissive conditions with a temperature of 33°C. At the end of the fermentations (drop), samples were subjected to HPLC analysis to determine the levels of various metabolites. The fermentation end time was determined as the time (hour) when the pressure measurement reached 344.7 Pa.

[0203] As shown in Table 18, the strain expressing additional S. cerevisiae FBPase produced more ethanol, generated less glycerol and fermented faster when compared to a corresponding control strain lacking such FBPase (compare strain Y52 with Y51).

[0204] Table 18. Change (in %) in ethanol and glycerol yields as well as fermentation time when compared to control strain Y40.

[0205] EXAMPLE X

[0206] Table 19. Description of the various Saccharomyces cerevisiae strains characterized in

[0207] Example X. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0208] The strains were added to serum vessels containing 20 mL commercial sugarcane must (~100 g / L total fermentable sugars) with a 40 pL inoculum from a YPD4% overnight culture. The fermentations were performed for ~70 hours in permissive conditions with a temperature of 33°C. At the end of the fermentations (drop), samples were subjected to HPLC analysis to determine the levels of various metabolites. The fermentation end time was determined as the time (hour) when the pressure measurement reached 344.7 Pa. As shown in Table 20, strains expressing a heterologous FBPase produced more ethanol, generated less glycerol and fermented faster when compared to strains lacking such FBPase (compare strains Y54 and Y55 with Y53). In addition, the combined expression of the heterologous FBPase and the heterologous PDC allowed strains to produce more ethanol, generate less glycerol and ferment faster when compared to strains expressing the heterologous PDC alone (compare strains Y55 with Y53). The addition of two copies of the gene encoding the heterologous FBPase further increased ethanol yield and further decreased glycerol production (compare strain Y57 with Y56).

[0209] Table 20. Change (in %) in ethanol and glycerol yields as well as fermentation time when compared to control strain Y40.

[0210] EXAMPLE XI

[0211] Table 21. Description of the various Saccharomyces cerevisiae strains characterized in Example XI. The heterologous genes were chromosomally integrated in their respect recombinant yeast host cells.

[0212]

[0213] Strains were inoculated into 150-gram of diluted commercial cane molasses (diluted to obtain ~140 g / L fermentable sugars) supplemented with 652 ppm urea and 5 ppm Bactenix® V60. Yeast was added at 266 ppm, and fermentations were conducted in non-baffled batch flasks for 48 h at 35 C under permissive conditions. At the end of the fermentations (drop), samples were subjected to HPLC analysis to determine the levels of various metabolites.

[0214] As shown in Table 22, the strains expressing a heterologous FBPase produced more ethanol, and generated less glycerol when compared to the strains lacking such FBPase (compare strain Y59 with strain Y58, and strains Y61 with Y60).

[0215] Table 22. Change (in %) in ethanol and glycerol yields when compared to control strain Y58.

[0216] REFERENCES

[0217] Navas M A, Gancedo J M. The regulatory characteristics of yeast fructose-1 , 6-bisphosphatase confer only a small selective advantage. Journal of bacteriology, 1996, 178(7): 1809-1812.

[0218] Grauslund, M., Lopes, J.M. and R0nnow, B., 1999. Expression of GUT1 , which encodes glycerol kinase in Saccharomyces cerevisiae, is controlled by the positive regulators Adrl p, Ino2p and Ino4p and the negative regulator Opil p in a carbon source-dependent fashion. Nucleic acids research, 27(22), pp.4391 -4398.

[0219] Jardon, R., Gancedo, C. and Flores, C.L., 2008. The gluconeogenic enzyme fructose-1 , 6- bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates. Eukaryotic cell, 7(10), pp.1742-1749. Semkiv M V, Dmytruk K V, Abbas C A, et al. Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase. BMC biotechnology, 2014, 14: 1-9

[0220] Semkiv M V, Dmytruk K V, Abbas C A, et al. Activation of futile cycles as an approach to increase ethanol yield during glucose fermentation in Saccharomyces cerevisiae.

[0221] Bioengineered, 2016, 7(2): 106-111

[0222] Yatabe F, Seike T, Okahashi N, et al. Improvement of ethanol and 2, 3-butanediol production in Saccharomyces cerevisiae by ATP wasting. Microbial Cell Factories, 2023, 22(1): 204.

[0223] Zahoor, A., Messerschmidt, K., Boecker, S. and Klamt, S., 2020. ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production. Biotechnology for Biofuels, 13(1), p.185.

Claims

WHAT IS CLAIMED IS:1 . A recombinant yeast host cell capable of producing at least one fermentation product and at least one fermentation by-product, the recombinant yeast host cell comprising:• at least one first genetic modification for providing a heterologous ATP futile cycle; and• at least one second genetic modification in an engineered metabolic pathway for decreasing the production of the fermentation by-product.

2. The recombinant yeast host cell of claim 1 , wherein the heterologous ATP futile cycle comprises at least one heterologous enzyme.

3. The recombinant yeast host cell of claim 2, wherein the at least one heterologous enzyme comprises a heterologous polypeptide having fructose-1 ,6-bisphosphatase activity.

4. The recombinant yeast host cell of claim 3, wherein the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity comprises a polypeptide which:• belongs to E.C. 3.1 .3.11 ;• is a fructose-1 ,6-bisphosphatase;• is encoded by a fbp gene;• is of eukaryotic origin or of prokaryotic origin;• is from Yarrowia sp. or Saccharomyces sp.;• is from Yarrowia lipolytica or Saccharomyces cerevisiae;• has the amino acid sequence of SEQ ID NO: 3 or61 , or is a variant of the amino acid sequence of SEQ ID NO: 3 or 61 having fructose-1 ,6-bisphosphatase activity; and / or• is encoded by the nucleic acid sequence of SEQ ID NO: 4 or 60, or is a variant of the nucleic acid sequence of SEQ ID NO: 4 or 60 encoding a polypeptide having fructose-1 ,6-bisphosphatase activity.

5. The recombinant yeast host cell of claim 3 or 4 comprising a reduction in the expression or a deletion in at least one native gene encoding a polypeptide having 6- phosphofructo-2-kinase activity.

6. The recombinant yeast host cell of claim 5 comprising the reduction in the expression or the deletion in the pfk26 gene, the pfk27 gene or both the pfk26 and the pfk27 genes.

7. The recombinant yeast host cell of any one of claims 3 to 6, wherein the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity comprises a polypeptide which:• belongs to E.C. 2.7.1 .90;• is a pyrophosphate-dependent phosphofructokinase (PPi-PFK);• is encoded by a pfk gene;• is of eukaryotic origin or of prokaryotic origin;• is from Xanthomonas sp. , or Porphyromas sp. ;• is from Xanthomonas campestris, or Porphyromonas gingivalis;• has the amino acid sequence of SEQ ID NO: 5 or 7, or is a variant of the amino acid sequence of SEQ ID NO: 5 or 7 having fructose-1 ,6-bisphosphatase activity; and / or• is encoded by the nucleic acid sequence of SEQ ID NO: 6 or 8, or is a variant of the nucleic acid sequence of SEQ ID NO: 6 or 8 encoding a polypeptide having fructose-1 ,6-bisphosphatase activity.

8. The recombinant yeast host cell of any one of claims 3 to 7 comprising one or more copies of a heterologous nucleic molecule encoding the heterologous polypeptide having fructose-1 ,6-bisphosphatase activity.

9. The recombinant yeast host cell of any one of claims 2 to 8, wherein the at least one heterologous enzyme comprises a heterologous polypeptide having glycerol kinase activity.

10. The recombinant yeast host cell of claim 9, wherein the heterologous polypeptide having glycerol kinase activity comprises a polypeptide:• belongs to E.C. 2.7.1 .30;• is a glycerol kinase;• is encoded by a gut1 gene;• is of eukaryotic or of prokaryotic origin;• is from Saccharomyces sp.;• is from Saccharomyces cerevisiae;• has the amino acid sequence of SEQ ID NO: 55 or a variant of SEQ ID NO: 55 having glycerol kinase activity; and / or• is encoded by the nucleic acid sequence of SEQ ID NO: 54 or is a variant of the nucleic acid sequence of SEQ ID NO: 54 encoding the polypeptide having glycerol kinase activity.11 . The recombinant yeast host cell of any one of claim 1 to 10, wherein the at least one fermentation product comprises an alcohol.

12. The recombinant yeast host cell of any one of claims 1 to 11 , wherein the at least one fermentation by-product comprises glycerol.

13. The recombinant yeast host cell of claim 12, wherein the engineered metabolic pathway for decreasing the production of the fermentation by-product comprises:• a heterologous polypeptide having STL1 activity;• a heterologous glyceraldehyde-3-phosphate dehydrogenase polypeptide;• a heterologous pyruvate decarboxylase polypeptide;• a reduction in the expression or a deletion in at least one native gene encoding a polypeptide having glycerol 3-phosphate dehydrogenase activity; and / or• a reduction in the expression or a deletion in at least one native gene encoding a polypeptide glycerol-3-phosphate phosphatase activity.

14. The recombinant yeast host cell of claim 13, wherein the at least one native gene encoding a polypeptide having glycerol 3-phosphate dehydrogenase activity comprises GPD1 and / or GPD2.

15. The recombinant yeast host cell of claim 13 or 14, wherein the at least one native gene encoding a polypeptide having glycerol-3-phosphate phosphatase activity comprises GPP1 and / or GPP2.

16. The recombinant yeast host cell of any one of claims 1 to 15 further comprising an engineered metabolic pathway for increasing the production of the at least one fermentation product.

17. The recombinant yeast host cell of any one of claims 1 to 16 being from the genus Saccharomyces sp.

18. The recombinant yeast host cell of claim 17 being from the species Saccharomyces cerevisiae.

19. A blend of a first recombinant yeast host cell and a second yeast host cell, wherein the first recombinant yeast host cell is defined in any one of claims 1 to 18 and the second yeast host cell lacks a heterologous ATP futile cycle.

20. A process for making at least one fermentation product, the process comprising contacting the recombinant yeast host cell of any one of claims 1 to 18 or the blend of claim 19 with a source of a carbohydrate under a condition allowing the conversion of at least a part of the carbohydrate into the at least one fermentation product.

21. The process of claim 20, wherein the source of the carbohydrate comprises C6 carbohydrates.

22. The process of claim 20 or 21 , wherein the source of the carbohydrate comprises or is derived from corn, sugarcane, and / or molasses.

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