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Title:
HETEROLOGOUS PROTEASE EXPRESSION FOR IMPROVING ALCOHOLIC FERMENTATION
Document Type and Number:
WIPO Patent Application WO/2018/141872
Kind Code:
A1
Abstract:
The present disclosure relates to proteases for improving alcoholic fermentation. The proteases are expressed from a recombinant host cell. The present disclosure also provides a population of recombinant host cells expressing an heterologous protease that can be used in combination with recombinant host cells expressing an heterologous glucoamylase and/or an heterologous glycerol reduction system.

Inventors:
BARRETT, Trisha (2885 South Road, Bradford, Vermont, 05033, US)
RICE, Charles F (135 Dolly Road, Hopkinton, New Hampshire, 03229, US)
ARGYROS, Aaron (2 King Road, Etna, New Hampshire, 03750, US)
Application Number:
EP2018/052572
Publication Date:
August 09, 2018
Filing Date:
February 01, 2018
Export Citation:
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Assignee:
LALLEMAND HUNGARY LIQUIDITY MANAGEMENT LLC (Népfürdõ utca 22. Building B, 13th Floor, 1138 Budapest, 1138, HU)
International Classes:
C12N15/81; C12R1/865
Domestic Patent References:
WO2014151318A22014-09-25
WO2012138942A12012-10-11
Foreign References:
EP3091070A12016-11-09
US20120032443W2012-04-05
US20110039192W2011-06-03
US8956851B22015-02-17
US20140051355W2014-08-15
Other References:
DATABASE Geneseq [online] 17 February 2011 (2011-02-17), "P. pastoris GS115 aspartic-type endopeptidase homolog protein #5.", XP002779294, retrieved from EBI accession no. GSP:AYN05412 Database accession no. AYN05412
CHI Z ET AL: "Saccharomycopsis fibuligera and its applications in biotechnology", BIOTECHNOLOGY ADVANCES, ELSEVIER PUBLISHING, BARKING, GB, vol. 27, no. 4, 1 July 2009 (2009-07-01), pages 423 - 431, XP026127892, ISSN: 0734-9750, [retrieved on 20090327], DOI: 10.1016/J.BIOTECHADV.2009.03.003
DATABASE UniProt [online] 16 August 2004 (2004-08-16), "SubName: Full=YALI0F27071p {ECO:0000313|EMBL:CAG78744.1};", XP002779295, retrieved from EBI accession no. UNIPROT:Q6C080 Database accession no. Q6C080
DATABASE Geneseq [online] 3 February 2011 (2011-02-03), "Candida albicans SC5314 protein SEQ ID:32873.", XP002779296, retrieved from EBI accession no. GSP:ATZ46549 Database accession no. ATZ46549
"Computational Molecular Biology", 1988, OXFORD UNIVERSITY PRESS
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computer Analysis of Sequence Data", 1994, HUMANA PRESS
"Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
"Sequence Analysis Primer", 1991, STOCKTON PRESS
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151 - 153
"GenBank", Database accession no. CAC83969.1
"GenBank", Database accession no. XP_003677629.1
GUO ZP; QIU CY; ZHANG L; DING ZY; WANG ZX; SHI GY: "Expression of aspartic protease from Neurospora crassa in industrial ethanol-producing yeast and its application in ethanol production", ENZYME MICROB TECHNOL., vol. 48, no. 2, 8 February 2011 (2011-02-08), pages 148 - 154, XP027585728
JOHNSTON DB; MCALOON AJ: "Protease increases fermentation rate and ethanol yield in dry-grind ethanol production", BIORESOUR TECHNOL., vol. 154, February 2014 (2014-02-01), pages 18 - 25, XP028817433, DOI: doi:10.1016/j.biortech.2013.11.043
Attorney, Agent or Firm:
NICHOLLS, James Ronald (14 South Square, Gray's Inn, London Greater London WC1R 5JJ, WC1R 5JJ, GB)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1 . A first recombinant yeast host cell comprising a first genetic modification allowing the expression of an heterologous protease, wherein the heterologous protease is:

a) a polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92;

b) a variant having at least 70% identity to the polypeptide of a) and exhibiting proteolytic activity; or

c) a fragment having at least 70% identity to the polypeptide of a) or the variant of b) and exhibiting proteolytic activity.

2. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease is the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52.

3. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease is the variant of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52.

4. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease is the fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52.

5. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease has the amino acid sequence of SEQ ID NO: 2, is the variant of the polypeptide of SEQ ID NO: 2 or is the fragment of the polypeptide of SEQ ID NO: 2.

6. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease has the amino acid sequence of SEQ ID NO: 14, is the variant of the polypeptide of SEQ ID NO: 14 or is the fragment of the polypeptide of SEQ ID NO: 14.

7. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease has the amino acid sequence of SEQ ID NO: 40, is the variant of the polypeptide of SEQ ID NO: 40 or is the fragment of the polypeptide of SEQ ID NO: 40.

8. The first recombinant yeast host cell of claim 1 , wherein the heterologous protease has the amino acid sequence of SEQ ID NO: 52, is the variant of the polypeptide of SEQ ID NO: 52 or is the fragment of the polypeptide of SEQ ID NO: 52.

9. The first recombinant yeast host cell of any one of claims 1 to 8, having a second genetic modification allowing the expression of an heterologous glucoamylase.

10. The first recombinant yeast host cell of claim 9, wherein the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 91 , is a variant of the amino acid sequence of SEQ ID NO: 91 , is a fragment of the amino acid sequence of SEQ ID NO: 91 or of the variant.

1 1 . The first recombinant yeast host cell of any one of claims 1 to 10 having a third genetic modification for reducing the production of one or more native enzymes that function to produce glycerol or regulate glycerol synthesis.

12. The first recombinant yeast host cell of claim 1 1 , wherein the third genetic modification is for reducing the production of one or more native enzymes that function to produce glycerol.

13. The first recombinant yeast host cell of claim 12, wherein the third genetic modification is for reducing or inhibiting in the expression of the gene encoding the GPD2 polypeptide.

14. The first recombinant yeast host cell of any one of claims 1 to 13 having a fourth genetic modification for reducing the production of one or more native enzymes that function to catabolize formate.

15. The first recombinant yeast host cell of claim 14, wherein the fourth genetic modification is for reducing or inhibiting the expression of the genes encoding the FDH 1 polypeptide and the FDH2 polypeptide.

16. The first recombinant yeast host cell of any one of claims 1 to 15 being from the genus Saccharomyces.

17. The first recombinant yeast host cell of claim 16 being from the species Saccharomyces cerevisiae.

18. A cellular population comprising:

a first recombinant yeast host cell comprising the first genetic modification defined in any one of claims 1 to 8; and

a second recombinant yeast host cell comprising the second, the third and/or the fourth genetic modification defined in any one of claims 9 to 15.

19. The cellular population of claim 18, wherein the first recombinant yeast host cell lacks the second, the third or the fourth genetic modification defined in any one of claims 9 to 15.

20. The cellular population of claim 18, wherein the first recombinant yeast host cell lacks the second, the third and the fourth genetic modification defined in any one of claims 9 to 15.

21 . The cellular population of any one of claims 18 to 20, wherein the second recombinant yeast host cell comprises the second, the third or the fourth genetic modifications as defined in any one of claims 9 to 15.

22. The cellular population of any one of claims 18 to 20, wherein the second recombinant yeast host cell comprises the second, the third and the fourth genetic modifications as defined in any one of claims 9 to 15.

23. The cellular population of any one of claims 18 to 22, wherein the first recombinant yeast host cell is from the genus Saccharomyces.

24. The combination of claim 22, wherein the first recombinant yeast host cell is from the species Saccharomyces cerevisiae.

25. The cellular population of any one of claims 18 to 24, wherein the second recombinant yeast host cell is from the genus Saccharomyces.

26. The combination of claim 25, wherein the second recombinant yeast host cell is from the species Saccharomyces cerevisiae.

27. A process for promoting ethanolic fermentation, the process comprising fermenting a medium with the first recombinant yeast host cell defined in any one of claims 1 to 17 or with the cellular population defined in any one of claims 18 to 26.

28. The process of claim 27, wherein the medium comprises raw starch.

29. The process of claim 27 or 28, wherein the medium is derived from corn.

30. The process of claim 27 or 28, wherein the medium is derived from barley.

31 . The process of claim 30, wherein the barley is malted barley.

32. A method of producing an heterologous protease in a first recombinant yeast host cell, the method comprising culturing a first recombinant yeast host cell as defined in any one of claims 1 to 17 under conditions allowing the expression of the heterologous protease.

33. The method of claim 32, further comprising, prior to the culturing step, introducing a first genetic modification as defined in any one of claims 1 to 8 in a yeast cell to provide the first recombinant yeast host cell.

34. The method of claim 33, further comprising, prior to the culturing step, introducing a second, third and/or fourth genetic modification as defined in any one of claims 9 to 15 in the yeast cell to provide the first recombinant yeast host cell.

35. The method of any one of claims 32 to 34, further comprising substantially isolating the heterologous protease from the first recombinant yeast host cell.

36. A recombinant heterologous protease obtainable by the method of claim 35.

37. A composition comprising an heterologous protease as defined in any one of claims 1 to 8 or 35.

38. The composition of claim 37 being obtainable from a first recombinant yeast host cell defined in any one of claims 1 to 17.

39. The composition of claim 37 or 38, further comprising a glucoamylase as defined claim 10.

40. The composition of any one of claims 37 to 39 further comprising a medium.

41 . The composition of claim 40, wherein the medium comprises raw starch.

42. The composition of claim 40 or 41 , wherein the medium is derived from corn.

43. The composition of claim 40 or 41 , wherein the medium is derived from barley.

44. The composition of claim 43, wherein the barley is malted barley.

Description:
HETEROLOGOUS PROTEASE EXPRESSION FOR IMPROVING ALCOHOLIC

FERMENTATION

TECHNOLOGICAL FIELD

The present disclosure relates to the heterologous polypeptides, especially heterologous proteases, for improving alcoholic fermentation.

BACKGROUND

Saccharomyces cerevisiae is used in the commercial production of distilled spirits and fuel ethanol. This organism is proficient in fermenting glucose to ethanol, often to concentrations greater than 20% w/v. However, S. cerevisiae's ability to generate a nitrogen source is limited which either slows down fermentation (for distilled spirits production) or requires the exogenous addition of nitrogen sources such as urea (for bioethanol production).

Corn is a feedstock for both distilled spirits and fuel ethanol. In the mashing process, corn is both thermally and enzymatically liquefied using a-or beta amylase prior to fermentation in order to break down long chain starch polymers into smaller dextrins. This can come either through addition of an external enzyme preparation, or as in with distilled spirits, through the addition of malted barley The mash is then cooled and inoculated with S. cerevisiae along with the exogenous addition of purified glucoamylase, an exo-acting enzyme, which will further break down the dextrin into utilizable glucose molecules.

It has been shown that the addition of commercial proteases such as FERMGEN® increases the rate of fermentation by supplying free amino acids via hydrolysis of protein found in the corn along with a decrease in the supply of additional nitrogen, resulting in a cost savings up to 4 cents per gallon (Johnston and McAloon, 2014). Adequate nitrogen content and other yeast nutrients contribute to the overall efficiency of the corn fermentation. Along with being a source of free amino nitrogen, protein is also a major component within the binding matrix of corn. Currently, commercial proteases are added to these industrial fermentations, which can be costly to corn ethanol plants. Addition of protease to the fermentation can also increase the ethanol yield (Johnston and McAloon, 2014), so even small increases such as 1 % can translate into an extra billion gallons of ethanol per year.

There is thus a need to provide alternative fermenting materials and processes to improve alcoholic fermentation by increase the available nitrogen to the fermenting organisms.

BRIEF SUMMARY

The present disclosure relates to the use of heterologous proteases expressed from a recombinant yeast host cell for improving alcoholic fermentation. In some embodiments, the heterologous proteases increases the fermentation rate, increases ethanol yields and/or decreases the production of glycerol by the fermenting recombinant host cells. According to a first aspect, the present disclosure provides a first recombinant yeast host cell comprising a first genetic modification allowing the expression of an heterologous protease. The heterologous protease can be a polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92. The heterologous protease can be a variant having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92 and exhibiting proteolytic activity. The heterologous protease can be a fragment having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92 or the variant described herein and exhibiting proteolytic activity. In an embodiment, the heterologous protease is the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In still another embodiment, the heterologous protease is the variant of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In still another embodiment, the heterologous protease is the fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In a further embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 2, is the variant of the polypeptide of SEQ ID NO: 2 or is the fragment of the polypeptide of SEQ ID NO: 2. In still another embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 14, is the variant of the polypeptide of SEQ ID NO: 14 or is the fragment of the polypeptide of SEQ ID NO: 14. In yet another embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 40, is the variant of the polypeptide of SEQ ID NO: 40 or is the fragment of the polypeptide of SEQ ID NO: 40. In a further embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 52, is the variant of the polypeptide of SEQ ID NO: 52 or is the fragment of the polypeptide of SEQ ID NO: 52. In yet another embodiment, the first recombinant yeast host cell has a second genetic modification allowing the expression of an heterologous glucoamylase, such as, for example, the heterologous glucoamylase has the amino acid sequence of SEQ ID NO: 91 , is a variant of the amino acid sequence of SEQ ID NO: 91 or is a fragment of the amino acid sequence of SEQ ID NO: 91 or of the variant described herein. In still a further embodiment, the first recombinant yeast host cell has a third genetic modification for reducing the production of one or more native enzymes that function to produce glycerol or regulate glycerol synthesis. In some embodiments, the third genetic modification is for reducing the production of one or more native enzymes that function to produce glycerol, such as, for example, wherein the third genetic modification is for reducing or inhibiting in the expression of the gene encoding the GPD2 polypeptide. In yet another embodiment, the first recombinant yeast host cell has a fourth genetic modification for reducing the production of one or more native enzymes that function to catabolize formate, such as, for example, wherein the fourth genetic modification is for reducing or inhibiting the expression of the genes encoding the FDH1 polypeptide and the FDH2 polypeptide. In an embodiment, the first recombinant yeast host cell is from the genus Saccharomyces, such as, for example from the species Saccharomyces cerevisiae. According to a second aspect, the present disclosure provides a cellular population comprising a first recombinant yeast host cell comprising the first genetic modification defined herein and a second recombinant yeast host cell comprising the second, the third and/or the fourth genetic modification herein. In an embodiment, the first recombinant yeast host cell lacks the second, the third or the fourth genetic modification defined herein. In another embodiment, the first recombinant yeast host cell lacks the second, the third and the fourth genetic modification defined herein. In yet another embodiment, the second recombinant yeast host cell comprises the second, the third or the fourth genetic modifications as defined herein. In yet another embodiment, the second recombinant yeast host cell comprises the second, the third and the fourth genetic modifications as defined herein. In an embodiment, the first recombinant yeast host cell and/or the second recombinant yeast host cell is from the genus Saccharomyces, such as, for example, from the species Saccharomyces cerevisiae.

According to a third aspect, the present disclosure provides a process for promoting ethanolic fermentation, the method comprising fermenting a medium with the first recombinant yeast host cell defined herein or with the cellular population defined herein. In an embodiment, the medium comprises raw starch. In another embodiment, the medium comprises lignocellulose. In another embodiment, the medium is derived from corn. In still another embodiment, the medium is derived from barley, such as, for example, malted barley.

According to a fourth aspect, the present disclosure provides a method of producing an heterologous protease in a first recombinant yeast host cell, the method comprising culturing a first recombinant yeast host cell as defined herein under conditions allowing the expression of the heterologous protease. In an embodiment, the method further comprises introducing a first, second, third and/or fourth genetic modification as defined herein to obtain the first recombinant yeast host cell. Alternatively or in combination, the method can further comprise substantially isolating the heterologous protease from the first recombinant yeast host cell.

According to a fifth aspect, the present disclosure provides a recombinant heterologous protease obtainable by the method described herein. The heterologous protease can be a polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92. The heterologous protease can be a variant having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92 and exhibiting proteolytic activity. The heterologous protease can be a fragment having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92 or the variant described herein and exhibiting proteolytic activity. In an embodiment, the heterologous protease is the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In still another embodiment, the heterologous protease is the variant of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In still another embodiment, the heterologous protease is the fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In a further embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 2, is the variant of the polypeptide of SEQ ID NO: 2 or is the fragment of the polypeptide of SEQ ID NO: 2. In still another embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 14, is the variant of the polypeptide of SEQ ID NO: 14 or is the fragment of the polypeptide of SEQ ID NO: 14. In yet another embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 40, is the variant of the polypeptide of SEQ ID NO: 40 or is the fragment of the polypeptide of SEQ ID NO: 40. In a further embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 52, is the variant of the polypeptide of SEQ ID NO: 52 or is the fragment of the polypeptide of SEQ ID NO: 52.

According to a sixth aspect, the present disclosure provides a composition comprising an heterologous protease as defined herein. The heterologous protease can be a polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92. The heterologous protease can be a variant having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92 and exhibiting proteolytic activity. The heterologous protease can be a fragment having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42, 52 or 92 or the variant described herein and exhibiting proteolytic activity. In an embodiment, the heterologous protease is the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In still another embodiment, the heterologous protease is the variant of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In still another embodiment, the heterologous protease is the fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In a further embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 2, is the variant of the polypeptide of SEQ ID NO: 2 or is the fragment of the polypeptide of SEQ ID NO: 2. In still another embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 14, is the variant of the polypeptide of SEQ ID NO: 14 or is the fragment of the polypeptide of SEQ ID NO: 14. In yet another embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 40, is the variant of the polypeptide of SEQ ID NO: 40 or is the fragment of the polypeptide of SEQ ID NO: 40. In a further embodiment, the heterologous protease has the amino acid sequence of SEQ ID NO: 52, is the variant of the polypeptide of SEQ ID NO: 52 or is the fragment of the polypeptide of SEQ ID NO: 52. In an embodiment, the heterologous protease is obtainable/obtained from a first recombinant yeast host cell as defined herein. Alternatively or in combination, the composition can further comprise a glucoamylase as defined herein, further comprising a medium which can, for example comprise raw starch. In an embodiment, the medium is derived from corn or from barley (and, in some instances, can be derived from malted barley).

BRIEF DESCRIPTION OF THE DRAWINGS 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:

Fig. 1 compares the absolute protease activity (using azoalbumin as a substrate) when expressed in an heterologous fashion in Saccharomyces cerevisiae. Results are provided as normalized protease activity in function of the heterologous protease expressed (refer to Table 1 for a description of the proteases used).

Fig. 2 compares the ethanol and glycerol yield of M2390, M10874, M10885, M1 1589 and M12184 strains during corn fermentation. Results are provided as g of ethanol (first four bars for each strain tested) or glycerol /L (last bar for each strain tested).

Fig. 3 compares the ethanol and glycerol yield of M2390, M10874, M10885, M12982 and M10890 strains during corn fermentation. Results are provided as g of ethanol (first four bars for each strains tested) or glycerol /L (last bar for each strain tested).

Fig. 4 compares the amino acid sequences of proteases MP818 (SEQ ID NO: 14), MP812 (SEQ ID NO: 2), MP914 (SEQ ID NO: 52) and MP831 (SEQ ID NO: 40). Consensus sequence is provided as SEQ ID NO: 92.

DETAILED DESCRIPTION

The present disclosure provides recombinant yeast host cell expressing an heterologous proteases for increasing the fermentation rate as well as overall ethanol yield. In some embodiments, the recombinant yeast host cell expressing the heterologous proteases can also decrease glycerol production during fermentation and can even decrease the cost of adding purified enzymes to the fermentation medium.

Proteases are a class of enzymes capable of hydrolyzing polypeptide chains by breaking the peptide bonds linking amino acids. Proteases can release amino acids from the terminal end of a protein (e.g., exopeptidase) or internally (e.g., endopeptidase). There are six categories of proteases which are defined by their mode of action. These include aspartic, glutamic and metallo proteases which activate a water molecule to break the peptide bond as well as serine, threonine and cysteine proteases which create an intermediate product by covalently linking the enzyme to the peptide bond, and then a water molecule is activated to break the bond. Proteases can further be broken down into families, subfamilies and clans. Proteases can also be classified by their optimal pH: neutral, acid, or alkaline. The MEROPS database is dedicated to the classification of known proteases and their function (http://merops.sanger.ac.uk/). Recombinant yeast host cells

The present disclosure provides a recombinant yeast host cell expressing (and in some embodiments secreting) an heterologous protease. As used in the context of the present disclosure, the "recombinant yeast host cell" includes at least one genetic modification. In the context of the present disclosure, when recombinant yeast host cell is qualified has "having a genetic modification "or as being "genetically engineered", it is understood to mean that it has been manipulated to either add at least one or more heterologous or exogenous nucleic acid residue and/or remove at least one endogenous (or native) nucleic acid residue. The genetic manipulations did not occur in nature and is the results of in vitro manipulations of the recombinant host cell. When the genetic modification is the addition of an heterologous nucleic acid molecule, such addition can be made once or multiple times at the same or different integration sites. Also, the genetic modification can include introducing one or more nucleic acid molecule which may have been endogenous to the recombinant yeast host cell, provided that this modification be added at a different locus than the endogenous locus. When the genetic modification is the modification of an endogenous nucleic acid molecule, it can be made in one or both copies of the targeted gene.

When expressed in a recombinant yeast host cells, the polypeptides described herein are encoded on one or more heterologous nucleic acid molecule. The term "heterologous" when used in reference to a nucleic acid molecule (such as a promoter or a coding sequence) 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 natural location in the organism's genome. The heterologous nucleic acid molecule is purposively introduced into the recombinant host cell. The term "heterologous" as used herein also refers to an element (nucleic acid or protein) that is derived from a source other than the endogenous source. Thus, for example, a heterologous element 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). The term "heterologous" is also used synonymously herein with the term "exogenous".

The present disclosure also provides a method of producing the recombinant yeast host cell by introducing one or more genetic modifications (usually by introducing one or more heterologous nucleic acid molecules) in a yeast cell to provide a recombinant yeast host cell. In an embodiment, an heterologous nucleic acid encoding an heterologous protease is introduced into yeast cell to provide the recombinant yeast host cell. In some embodiments, the method comprises placing the recombinant yeast host cell under conditions so as to favor the expression of the heterologous protease (encoded by an heterologous nucleic acid molecule) by the recombinant yeast host cell.

When an heterologous nucleic acid molecule is present in the recombinant host cell, it 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 host cell. For example, genetic elements can be placed into the chromosomes of the host cell as opposed to in a vector such as a plasmid carried by the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The heterologous nucleic acid molecule can be present in one or more copies in the yeast host cell's genome. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the yeast's genome. In such embodiment, the nucleic acid molecule can be stable and self-replicating.

In the context of the present disclosure, the recombinant host cell can be a recombinant yeast host cell. Suitable recombinant yeast host cells can be, for example, from the genus Saccharomyces, Kluyveromyces, Arxula, Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia. Suitable yeast species can include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis. In some embodiments, the recombinant yeast host cell is from the following species: Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe or Schwanniomyces occidentalis. In some embodiment, the recombinant host cell can be an oleaginous yeast cell. For example, the recombinant oleaginous yeast host cell can be from the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiments, the recombinant host cell can be an oleaginous microalgae host cell (e.g., for example, from the genera Thraustochytrium or Schizochytrium). In an embodiment, the recombinant yeast host cell is from the genus Saccharomyces and, in some embodiments, from the species Saccharomyces cerevisiae. In one particular embodiment, the recombinant yeast host cell is Saccharomyces cerevisiae.

In some embodiments, heterologous nucleic acid molecules which can be introduced into the recombinant host cells 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.

The heterologous nucleic acid molecules of the present disclosure comprise a coding region for the heterologous polypeptide. A DNA or RNA "coding region" 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 under the 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 site, effector binding site and stem- loop structure. 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.

The nucleic acid molecules described herein can comprise 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.

The heterologous nucleic acid molecule 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, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. In the heterologous nucleic acid molecule described herein, the promoter and the nucleic acid molecule coding for the heterologous polypeptide are 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 is physically associated to the nucleotide acid molecule coding for the heterologous polypeptide in a manner that allows, under certain conditions, for expression of the heterologous protein from the nucleic acid molecule. In an embodiment, the promoter can be located upstream (5') of the nucleic acid sequence coding for the heterologous protein. In still another embodiment, the promoter can be located downstream (3') of the nucleic acid sequence coding for the heterologous protein. In the context of the present disclosure, one or more than one promoter can be included in the heterologous nucleic acid molecule. When more than one promoter is included in the heterologous nucleic acid molecule, each of the promoters is operatively linked to the nucleic acid sequence coding for the heterologous protein. The promoters can be located, in view of the nucleic acid molecule coding for the heterologous protein, upstream, downstream as well as both upstream and downstream. In the context of the present disclosure, it is possible to use a constitutive or an inducible promoter for expressing the heterologous proteins.

"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 heterologous polypeptide. The promoter can be heterologous or derived from a strain being from the same genus or species as the recombinant host cell. In an embodiment, the promoter is derived from the same genus or species of the yeast host cell and the heterologous polypeptide is derived from different genus that the host cell.

First genetic modification allowing the expression of an heterologous protease

As indicated in the Example below, the expression of an heterologous protease in a recombinant yeast host cell increases the fermentation rate, increases ethanol yield and/or decrease glycerol production. The Example below also shows that supplementing the fermentation medium with purified proteases does not further increase the fermentation rate, the ethanol yield or decrease glycerol production. As such, the recombinant yeast host cell of the present disclosure include a genetic modification allowing the expression of one or more heterologous protease. As used in the present disclosure, the term "heterologous protease" refers to a polypeptide which was not natively found in the recombinant yeast host cell or which is expressed at a different locus than the native locus in the recombinant yeast host cell.

The disclosure provides a recombinant yeast host cell comprising a first genetic modification allowing the expressing any heterologous protease, except the one disclosed in Guo et al., 201 1 . The recombinant yeast host cell of the present disclosure can express one or more heterologous proteases. In an embodiment, the heterologous protease is an aspartic protease or a protease susceptible of having aspartic-like activity. The heterologous protease can be derived from a known protease expressed in a prokaryotic (such as a bacteria) or a eukaryotic cell (such as a yeast, a mold, a plant or an animal).

Embodiments of aspartic proteases which can be used according to the present disclosure are shown in Figure 4. In some embodiments, the protease (its variant or fragment) has any one of the amino acid sequences shown in Figure 4, including the consensus sequence (SEQ ID NO: 92).

Table 1 . Characteristics of the proteases presented in Figure 4.

MP # Gene MEROPS Sequence Peptidase Active Site

Organism

(SEQ ID #) Name ID Length Unit Residues

MP812 D82, Y134,

Candida albicans SAP1 A01.014 391 43-380

(2) D267

Aspergillus MP818 D102, Y144,

PEP1 A01.026 395 74-392

fumigatus (14) D284

Candida MP914 D82, Y134,

SAP1 A01.014 391 43-380

dubliniensis (52) D267

Saccharomycopsi MP831 D93, Y132,

PEP1 unassigned 390 55-389

s fibuligera (40) D282 In an embodiment, the proteases (their variants or fragments) have the consecutive amino acids of the peptidase subunit defined in Table 1 . For example, the protease can have residues 43 to 380 of SEQ ID NO: 2, residues 74 to 392 of SEQ ID NO: 14, residues 43 to 380 of SEQ ID NO: 52 or residues 55 to 389 of SEQ ID NO: 40. In still another embodiment, the proteases (their variants or fragments) have the active sites residues of the proteases defined in Table 1 . For example, the proteases can have residues corresponding to D82, Y134 and D267 of SEQ ID NO: 2, residues corresponding to D102, Y144 and D284 of SEQ ID NO: 14, residues corresponding to D82, Y134 and D267 of SEQ ID NO: 52 or residues corresponding to D93, Y132 and D282 of SEQ ID NO: 40.

In an embodiment, the heterologous protease can be derived from a fungal organism. For example, the heterologous protease can be derived from the genus Candida, Clavispora, Saccharomyces, Yarrowia, Meyerozyma, Aspergillus or Saccharomycopsis. When the heterologous protease is derived from the genus Candida, it can be derived from the species Candida albicans, Candida dubliniensis or Candida tropicalos. When the heterologous protease is derived from Candida albicans, it can have the amino acid of SEQ ID NO: 2. When the heterologous protease is derived from Candida dubliensis, it can have the amino acid sequence of SEQ ID NO: 52. When the heterologous protease is derived from Candida tropicalis, it can have the amino acid sequence of SEQ ID NO: 38. When the heterologous protease is derived from the genus Clavispora, it can be derived from the species Clavispora lusitaniae. When the heterologous protease is derived from the species Clavispora lusitaniae, it can have the amino acid sequence of SEQ ID NO: 6 or 30. When the heterologous protease is derived from the genus Saccharomyces, it can be derived from the species Saccharomyces cerevisiae. When the heterologous protease is derived from the species Saccharomyces cerevisiae, it can have the amino acid sequence of SEQ ID NO: 8. When the heterologous protease is derived from the genus Yarrowia, it can be derived from the species Yarrowia lipolytica. When the heterologous protease is derived from the species Yarrowia lipolytica, it can have the amino acid sequence of SEQ ID NO: 10. When the heterologous protease is derived from the genus Meyerozyma, it can be derived from the species Meyerozyma guilliermondii. When the heterologous protease is derived from the species Meyerozyma guilliermondii, it can have the amino acid sequence of SEQ ID NO: 12. When the heterologous protease is derived from the genus Aspergillus, it can be derived from the species Aspergillus fumigatus. When the heterologous protease is derived from the species Aspergillus fumigatus, it can have the amino acid sequence of SEQ ID NO: 14. When the heterologous protease is derived from the species Saccharomycopsis, it can be derived from the species Saccharomycopsis fibuligera. When the heterologous protease is derived from the species Saccharomycopsis fibuligera, it can have the amino acid sequence of SEQ ID NO: 40. In an embodiment, the heterologous protease can be derived from a bacterial organism. For example, the heterologous protease can be derived from the genus Bacillus. When the heterologous protease is derived from the genus Bacillus, it can be derived from the species Bacillus subtilis, it can have the amino acid sequence of SEQ ID NO: 36.

In an embodiment, the heterologous protease can be derived from a plant. For example, the heterologous protease can be derived from the genus Ananas. When the heterologous protease is derived from the genus Ananas, it can be derived from the species Ananas comosus, it can have the amino acid sequence of SEQ ID NO: 42.

In an embodiment, the heterologous protease is a polypeptide having an amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. In still another embodiment, the heterologous protease is a polypeptide having an amino acid sequence of SEQ ID NO: 2, 14, 40 or 52. In yet a further embodiment, the heterologous protease is a polypeptide having the amino acid sequence of SEQ ID NO: 2. In yet a further embodiment, the heterologous protease is a polypeptide having the amino acid sequence of SEQ ID NO: 14. In yet a further embodiment, the heterologous protease is a polypeptide having the amino acid sequence of SEQ ID NO: 40. In yet a further embodiment, the heterologous protease is a polypeptide having the amino acid sequence of SEQ ID NO: 52.

The present disclosure also provides using variants of the polypeptides described herein as the heterologous protease. A "variant" comprises at least one amino acid difference (substitution or addition) when compared to the amino acid sequence of the polypeptides having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. The variants do exhibit protease activity, such as aspartic protease activity. Protease activity can be measured by various techniques known in the art, including methods using azoalbumin as a substrate. In an embodiment, the variant exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% proteolytic activity when compared to the proteolytic activity of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. In an embodiment, the variant exhibits at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. The term "percent identity", as known in the art, is a relationship between two or more polypeptide 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.

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. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non- conservative substitutions, such as replacing a basic amino acid with a hydrophobic one, are also well-known in the art.

A variant can be also 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 protease (e.g., hydrolysis of proteins). 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 protease (e.g., the hydrolysis of proteins). For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protease.

In an embodiment, the heterologous protease is a fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the protease or variant of the protease. The fragment of the protease exhibits proteolytic activity. In an embodiment, the fragment of the protease exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the protease activity of the full-length amino acid of of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. The protease fragments can also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of of SEQ ID NO: 2, 6, 8, 10, 12, 14, 30, 36, 38, 40, 42 or 52. The fragment can be, for example, a truncation of one or more amino acid residues at the amino-terminus, the carboxy terminus or both terminus of the protease polypeptide or variant. Alternatively or in combination, the fragment can be generated from removing one or more internal amino acid residues. In an embodiment, the alpha-amylase fragment has at least 100, 150, 200, 250, 300, 350 or 400 or more consecutive amino acids of the protease or the variant.

Second genetic modification allowing the expression of an heterologous glucoamylase

The recombinant yeast host cell having the first genetic modification allowing the expression of the heterologous protease can include one or mode additional genetic modifications.

For example, the recombinant yeast host cell can include a second genetic modification allowing the expression of an heterologous glucoamylase. Alternatively, the recombinant yeast host cell comprising the first genetic modification can be used in combination with another recombinant yeast host cell comprising the second genetic modification allowing the expression of an heterologous glucoamylase. Polypeptides having glucoamylase activity (also referred to as glucoamylases) are exo-acting enzymes capable of terminally hydrolyzing starch to glucose. Glucoamylase activity can be determined by various ways by the person skilled in the art. For example, the glucoamylase activity of a polypeptide can be determined directly by measuring the amount of reducing sugars generated by the polypeptide in an assay in which raw or gelatinized (corn) starch is used as the starting material.

In the context of the present disclosure, the heterologous glucoamylase can be derived from a yeast, for example, from the genus Saccharomycopsis and, in some instances, from the species S. fibuligera. The heterologous glucoamylase can be encoded by the glu0111 gene from S. fibuligera or a glu0111 gene ortholog. An embodiment of glucoamylase polypeptide of the present disclosure is the GLU01 1 1 polypeptide (GenBank Accession Number: CAC83969.1 ). The GLU01 1 1 polypeptide includes the following amino acids (or correspond to the following amino acids) which are associated with glucoamylase activity and include, but are not limited to amino acids located at positions 41 , 237, 470, 473, 479, 485, 487 of SEQ ID NO: 91. The heterologous glucoamylase can be a variant glucoamyase having the amino acids located at positions 41 , 237, 470, 473, 479, 485, 487 of SEQ ID NO: 91 . The heterologous glucoamylase can be a fragment of SEQ ID NO: 91 having to amino acids located at positions 41 , 237, 470, 473, 479, 485, 487 of SEQ ID NO: 91 . It is possible to use a polypeptide which does not comprise its endogenous signal sequence. Embodiments of heterologous glucoamylase have been also been described in PCT/US2012/032443 and PCT/US201 1/039192. In the context of the present disclosure, a "glu01 1 1 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 glu01 1 1 ortholog retains the same function, e.g. it can act as a glucoamylase. Glu01 1 1 gene orthologs includes but are not limited to, the nucleic acid sequence of GenBank Accession Number XP_003677629.1 (Naumovozyma castellii) XP_003685231 .1 {Tetrapisispora phaffii), XP_455264.1 {Kluyveromyces lactis), XP_446481 .1 {Candida glabrata), EER33360.1 {Candida tropicalis), EEQ36251.1 {Clavispora lusitaniae), ABN68429.2 {Scheffersomyces stipitis), AAS51695.2 {Eremothecium gossypii), EDK43905.1 {Lodderomyces elongisporus), XP_002555474.1 {Lachancea thermotolerans), EDK37808.2 {Pichia guilliermondii), CAA86282 {Saccharomyces cerevisiae), XP_003680486.1 {Torulaspora delbrueckii), XP_503574.1 {Yarrowia lipolytica), XP_002496552.1 {Zygosaccharomyces rouxii), CAX42655.1 {Candida dubliniensis), XP_002494017.1 {Komagataella pastoris) and AET38805.1 {Eremothecium cymbalariae).

Still in the context of the present disclosure, a variant of the heterologous glucoamylase can be used. A variant comprises at least one amino acid difference (substitution or addition) when compared to the amino acid sequence of the glucoamylase polypeptide of SEQ ID NO: 91 . The glucoamylase variants do exhibit glucoamylase activity. In an embodiment, the variant glucoamylase exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the glucoamylase activity of the amino acid of SEQ ID NO: 91 . The glucoamylase variants also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 91. The term "percent identity", as known in the art, is a relationship between two or more polypeptide 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. The variant glucoamylases 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. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as replacing a basic amino acid with a hydrophobic one, are also well-known in the art.

A variant glucoamylase can also 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 glucoamylase. 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 glucoamylase (e.g., the hydrolysis of starch into glucose). For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the glucoamylase.

The present disclosure also provides expressing fragments of the glucoamylases polypeptides and glucoamylase variants described herein. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the glucoamylase polypeptide or variant and still possess the enzymatic activity of the full-length glucoamylase. In an embodiment, the glucoamylase fragment exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the full-length glucoamylase of the amino acid of SEQ ID NO: 91. The glucoamylase fragments can also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 91 . The fragment can be, for example, a truncation of one or more amino acid residues at the amino-terminus, the carboxy terminus or both termini of the glucoamylase polypeptide or variant. Alternatively or in combination, the fragment can be generated from removing one or more internal amino acid residues. In an embodiment, the glucoamylase fragment has at least 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutive amino acids of the glucoamylase polypeptide or the variant.

Third genetic modification for reducing glycerol levels The recombinant host cell comprising the first genetic modification (and optionally the second genetic modification) can also include a third genetic modification for reducing the production of one or more native enzymes that function to produce glycerol or regulate glycerol synthesis. Alternatively, the recombinant yeast host cell comprising the first genetic modification (and optionally the second and/or third genetic modification) can be used in combination with another recombinant yeast host cell comprising the third genetic modification for reducing the production of one or more native enzymes that function to produce glycerol or regulate glycerol synthesis (and optionally the second genetic modification).

As used in the context of the present disclosure, the expression "reducing the production of one or more native enzymes that function to produce glycerol or regulate glycerol synthesis" refers to a genetic modification which limits or impedes the expression of genes associated with one or more native polypeptides (in some embodiments enzymes) that function to produce glycerol or regulate glycerol synthesis, when compared to a corresponding strain which does not bear the third genetic modification. In some instances, the third genetic modification reduces but still allows the production of one or more native polypeptides that function to produce glycerol or regulate glycerol synthesis. In other instances, the third genetic modification inhibits the production of one or more native enzymes that function to produce glycerol or regulate glycerol synthesis. In some embodiments, the recombinant host cells bear a plurality of third genetic modifications, wherein at least one reduces the production of one or more native polypeptides and at least another inhibits the production of one or more native polypeptides.

As used in the context of the present disclosure, the expression "native polypeptides that function to produce glycerol or regulate glycerol synthesis" refers to polypeptides which are endogenously found in the recombinant yeast host cell. Native enzymes that function to produce glycerol include, but are not limited to, the GPD1 and the GPD2 polypeptide (also referred to as GPD1 and GPD2 respectively) as well as the GPP1 and the GPP2 polypeptides (also referred to as GPP1 and GPP2 respectively). Native enzymes that function to regulating glycerol synthesis include, but are not limited to, the FPS1 polypeptide as well as the STL1 polypeptide. The FPS1 polypeptide is a glycerol exporter and the STL1 polypeptide functions to import glycerol in the recombinant yeast host cell. By either reducing or inhibiting the expression of the FPS1 polypeptide and/or increasing the expression of the STL1 polypeptide, it is possible to control, to some extent, glycerol synthesis. In an embodiment, the recombinant yeast host cell bears a genetic modification in at least one of the gpdl gene (encoding the GPD1 polypeptide), the gpd2 gene (encoding the GPD2 polypeptide), the gppl gene (encoding the GPP1 polypeptide), the gpp2 gene (encoding the GPP2 polypeptide), the fpsl gene (encoding the FPS1 polypeptide) or orthologs thereof. In another embodiment, the recombinant yeast host cell bears a genetic modification in at least two of the gpdl gene (encoding the GPD1 polypeptide), the gpd2 gene (encoding the GPD2 polypeptide), the gppl gene (encoding the GPP1 polypeptide), the gpp2 gene (encoding the GPP2 polypeptide), the fpsl gene (encoding the FPS1 polypeptide) or orthologs thereof. In still another embodiment, the recombinant yeast host cell bears a genetic modification in each of the gpdl gene (encoding the GPD1 polypeptide), the gpd2 gene (encoding the GPD2 polypeptide) and the fpsl gene (encoding the FPS1 polypeptide) or orthologs thereof. Examples of recombinant yeast host cells bearing such genetic modification(s) leading to the reduction in the production of one or more native enzymes that function to produce glycerol or regulating glycerol synthesis are described in WO 2012/138942. Preferably, the recombinant yeast host cell has a genetic modification (such as a genetic deletion or insertion) only in one enzyme that functions to produce glycerol, in the gpd2 gene, which would cause the host cell to have a knocked-out gpd2 gene. In some embodiments, the recombinant yeast host cell can have a genetic modification in the gpdl gene, the gpd2 gene and the fpsl gene resulting is a recombinant yeast host cell being knockout for the gpdl gene, the gpd2 gene and the fpsl gene. In still another embodiment (in combination or alternative to the "first" genetic modification described above), the recombinant yeast host cell can have a genetic modification in the stl 1 gene (e.g., a duplication for example) for increasing the expression of the STL1 polypeptide. In an embodiment, the recombinant yeast host cell can have a genetic modification in the gpd2 genes.

Fourth genetic modification for maintaining or increasing formate levels

The recombinant host cell comprising the first genetic modification (and optionally the second and/or third genetic modification) can also include a fourth genetic modification for reducing the production of one or more native enzymes that function to catabolize formate. Alternatively, the recombinant yeast host cell comprising the first genetic modification (and optionally the second, third and/or fourth genetic modification) can be used in combination with another recombinant yeast host cell comprising the fourth genetic modification for reducing the production of one or more native enzymes that function to catabolize formate (and optionally the second and/or third genetic modification).

As used in the context of the present disclosure, the expression "for reducing the production of one or more native enzymes that function to catabolize formate". As used in the context of the present disclosure, the expression "native polypeptides that function to catabolize formate" refers to polypeptides which are endogenously found in the recombinant host cell. Native enzymes that function to catabolize formate include, but are not limited to, the FDH1 and the FDH2 polypeptides (also referred to as FDH1 and FDH2 respectively). In an embodiment, the recombinant yeast host cell bears a genetic modification in at least one of the fdhl gene (encoding the FDH1 polypeptide), the fdh2 gene (encoding the FDH2 polypeptide) or orthologs thereof. In another embodiment, the recombinant yeast host cell bears genetic modifications in both the fdhl gene (encoding the FDH1 polypeptide) and the fdh2 gene (encoding the FDH2 polypeptide) or orthologs thereof. Examples of recombinant yeast host cells bearing such genetic modification(s) leading to the reduction in the production of one or more native enzymes that function to catabolize formate are described in WO 2012/138942. Preferably, the recombinant yeast host cell has genetic modifications (such as a genetic deletion or insertion) in the fdhl gene and in the fdh2 gene which would cause the host cell to have knocked-out fdhl and fdh2 genes.

In some embodiments, the recombinant yeast host cell can include a further genetic modification for increasing the production of an heterologous enzyme that function to anabolize (form) formate. As used in the context of the present disclosure, "an heterologous enzyme that function to anabolize formate" refers to polypeptides which may or may not be endogeneously found in the recombinant yeast host cell and that are purposefully introduced into the recombinant yeast host cells. In some embodiments, the heterologous enzyme that function to anabolize formate is an heterologous pyruvate formate lyase (PFL), an heterologous acetaldehyde dehydrogenases, an heterologous alcohol dehydrogenases, and/or and heterologous bifunctional acetylaldehyde/alcohol dehydrogenases (AADH) such as those described in US Patent Serial Number 8,956,851 and PCT/US2014/051355. More specifically, PFL and AADH enzymes for use in the recombinant yeast host cells can come from a bacterial or eukaryotic source. Heterologous PFL of the present disclosure include, but are not limited to, the PFLA polypeptide, a polypeptide encoded by a pfla gene ortholog, the PFLB polyeptide or a polypeptide encoded by a pflb gene ortholog. Heterologous AADHs of the present disclosure include, but are not limited to, the ADHE polypeptides or a polypeptide encoded by an adhe gene ortholog. In an embodiment, the recombinant yeast host cell of the present disclosure comprises at least one of the following heterologous enzymes that function to anabolize formate: the PFLA polypeptide, the PFLB polypeptide and/or the ADHE polypeptide. In an embodiment, the recombinant yeast host cell of the present disclosure comprises at least two of the following heterologous enzymes that function to anabolize formate: the PFLA polypeptide, the PFLB polypeptideand/ or the ADHE polypeptide. In another embodiment, the recombinant yeast host cell of the present disclosure comprises the following heterologous enzymes that function to anabolize formate : the PFLA polypeptide, the PFLB polypeptide and the ADHE polypeptide.

Additional genetic modifications

The recombinant host cell can be further genetically modified to allow for the production of additional heterologous polypeptides. In an embodiment, the recombinant yeast host cell can be used for the production of an enzyme, and especially an enzyme involved in the cleavage or hydrolysis of its substrate (e.g., a lytic enzyme and, in some embodiments, a saccharolytic enzyme). In still another embodiment, the enzyme can be a glycoside hydrolase. In the context of the present disclosure, the term "glycoside hydrolase" refers to an enzyme involved in carbohydrate digestion, metabolism and/or hydrolysis, including amylases (other than those described above), cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, trehalases, pectinases, and pentose sugar utilizing enzymes.

The additional heterologous polypeptide can be an "amylolytic enzyme", an enzyme involved in amylase digestion, metabolism and/or hydrolysis. The term "amylase" refers to an enzyme that breaks starch down into sugar. All amylases are glycoside hydrolases and act on a-1 ,4- glycosidic bonds. Some amylases, such as γ-amylase (glucoamylase), also act on a-1 ,6- glycosidic bonds. Amylase enzymes include oamylase (EC 3.2.1 .1 ), β-amylase (EC 3.2.1 .2), and γ-amylase (EC 3.2.1 .3). The oamylases are calcium metalloenzymes, unable to function in the absence of calcium. By acting at random locations along the starch chain, oamylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, oamylase tends to be faster-acting than β-amylase. Another form of amylase, β- amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second a-1 ,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. Another amylolytic enzyme is oglucosidase that acts on maltose and other short malto-oligosaccharides produced by o, β-, and γ-amylases, converting them to glucose. Another amylolytic enzyme is pullulanase. Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan is regarded as a chain of maltotriose units linked by alpha- 1 ,6-glycosidic bonds. Pullulanase (EC 3.2.1.41 ) is also known as pullulan-6-glucanohydrolase (debranching enzyme). Another amylolytic enzyme, isopullulanase, hydrolyses pullulan to isopanose (6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1 .57) is also known as pullulan 4-glucanohydrolase. An "amylase" can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis, including oamylase, β -amylase, glucoamylase, pullulanase, isopullulanase, and alpha-glucosidase.

The additional heterologous polypeptide can be a "cellulolytic enzyme", an enzyme involved in cellulose digestion, metabolism and/or hydrolysis. The term "cellulase" refers to a class of enzymes that catalyze cellulolysis (i.e. the hydrolysis) of cellulose. Several different kinds of cellulases are known, which differ structurally and mechanistically. There are general types of cellulases based on the type of reaction catalyzed: endocellulase breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains; exocellulase cleaves 2-4 units from the ends of the exposed chains produced by endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose. There are two main types of exocellulases (or cellobiohydrolases, abbreviate CBH) - one type working processively from the reducing end, and one type working processively from the non- reducing end of cellulose; cellobiase or beta-glucosidase hydrolyses the exocellulase product into individual monosaccharides; oxidative cellulases that depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase (acceptor); cellulose phosphorylases that depolymerize cellulose using phosphates instead of water. In the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to beta-glucose. A "cellulase" can be any enzyme involved in cellulose digestion, metabolism and/or hydrolysis, including an endoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase, acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin, pectinase, and feruoyl esterase protein.

The additional heterologous polypeptide can have "hemicellulolytic activity", an enzyme involved in hemicellulose digestion, metabolism and/or hydrolysis. The term "hemicellulase" refers to a class of enzymes that catalyze the hydrolysis of cellulose. Several different kinds of enzymes are known to have hemicellulolytic activity including, but not limited to, xylanases and mannanases.

The additional heterologous polypeptide can have "xylanolytic activity", an enzyme having the is ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses. The term "xylanase" is the name given to a class of enzymes which degrade the linear polysaccharide beta-1 ,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls. Xylanases include those enzymes that correspond to Enzyme Commission Number 3.2.1.8. The heterologous protein can also be a "xylose metabolizing enzyme", an enzyme involved in xylose digestion, metabolism and/or hydrolysis, including a xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, and a xylose transaldolase protein. A "pentose sugar utilizing enzyme" can be any enzyme involved in pentose sugar digestion, metabolism and/or hydrolysis, including xylanase, arabinase, arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase, arabinosidase, and arabinofuranosidase, arabinose isomerase, ribulose-5-phosphate 4- epimerase, xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, and/or xylose transaldolase.

The additional heterologous polypeptide can have "mannanic activity", an enzyme having the is ability to hydrolyze the terminal, non-reducing β-D-mannose residues in β-D-mannosides. Mannanases are capable of breaking down hemicellulose, one of the major components of plant cell walls. Xylanases include those enzymes that correspond to Enzyme Commission Number 3.2.25.

The additional heterologous polypeptide can be a "pectinase", an enzyme, such as pectolyase, pectozyme and polygalacturonase, commonly referred to in brewing as pectic enzymes. These enzymes break down pectin, a polysaccharide substrate that is found in the cell walls of plants.

The additional heterologous polypeptide can have "phytolytic activity", an enzyme catalyzing the conversion of phytic acid into inorganic phosphorus. Phytases (EC 3.2.3) can be belong to the histidine acid phosphatases, β-propeller phytases, purple acid phosphastases or protein tyrosine phosphatase-like phytases family.

Cellular populations

The present disclosure also provides cellular population comprising the recombinant yeast host cell comprising the first genetic modification. In an embodiment, the cellular population comprises or consists essentially of one or more of the recombinant yeast host cell comprising the first genetic modification (and in an embodiment, lacking the second, the third, the fourth and/or a further genetic modification). In some embodiments, the cellular population can also include non-genetically modified fermenting yeasts.

In yet another embodiment, the cellular population comprises a first recombinant yeast host cell (comprising at least the first genetic modification) and a second recombinant yeast host cell (comprising at least the second, third and/or fourth genetic modification) and optionally non- genetically-modified fermenting yeasts. In still another embodiment, the cellular population comprises a first recombinant yeast host cell (comprising the first genetic modification) and a second recombinant yeast host cell (comprising at least the second, third or fourth genetic modification) and optionally non-genetically-modified fermenting yeasts. In yet a further embodiment, the cellular population comprises a first recombinant yeast host cell (comprising the first genetic modification) and a second recombinant yeast host cell (comprising at least two of the second, third or fourth genetic modification) and optionally non-genetically-modified fermenting yeasts. In still another embodiment, the cellular population comprises a first recombinant yeast host cell (comprising the first genetic modification) and a second recombinant yeast host cell (comprising the second and third genetic modifications) and optionally non-genetically-modified fermenting yeasts. In another embodiment, the cellular population comprises a first recombinant yeast host cell (comprising the first genetic modification) and a second recombinant yeast host cell (comprising the second, third and fourth genetic modifications) and optionally non-genetically-modified fermenting yeasts.

The cellular population can be provided in a liquid or solid form (e.g., in some embodiments in a freeze-dried form or as a cream yeast). The cellular population can be provided as a single unit comprising both the first recombinant yeast host cell and the second recombinant yeast host cell. Alternatively, the cellular population can be provided in two units each comprising the first recombinant yeast host cell and the second recombinant yeast host cell.

The recombinant yeast host cells of the cellular population can be from the same or from different genus. In an embodiment, the recombinant yeast host cells of the cellular population can be from the same or different species. In still another embodiment, the recombinant yeast host cells of the cellular population are from the genus Saccharomyces and, in further embodiment, from the species Saccharomyces cerevisiae. Process for using the recombination yeast host cells and the cellular populations and associated compositions

As indicated herein, the use of a recombinant yeast host cell comprising the first genetic modification during allows to increase the fermentation rate and the ethanol yield when compared to a corresponding fermentation made by yeast cells lacking the first genetic modification.

Embodiments in which the cellular population does not include a recombinant yeast host cell comprising the second, third and/or fourth genetic modifications as described herein are especially useful for the production of distilled spirits. In such embodiments, the first recombinant yeast host cell (comprising the first genetic modification) or a cellular population comprising same can be used to ferment a medium to make ethanol. The distilled spirits fermentation medium can comprise, for example, a grain (barley, rye, corn, sorghum, wheat, rice, millet, buckwheat), a fruit (grape, apple, pear, plum, apricots, quinces, pineapple, juniper berry, bananas, plantain, gougi, coconut, ginger, pomace, cashew) and/or a vegetable (cassava, potato, sugar cane, molasses, agave). The distilled spirit can be, but is not limited to scotch whisky, rye whisky, vodka, brandy, cognac, vermouth, armagnac, calvados, cider, rhum. After fermentation, the fermentation medium can be distilled into the distilled spirit.

Embodiments in which the cellular population comprises recombinant yeast host cells comprising the first, second, third and/or fourth genetic modifications as well as cellular populations comprising same can be useful for the production of ethanol for biofuel applications. In some embodiment, a cellular population comprising the first recombinant yeast host cell comprising the first genetic modification and the second recombinant yeast host cell comprising the second, third and fourth genetic modifications can be used for the production of ethanol for biofuel applications. Broadly, the process comprises combining a substrate to be hydrolyzed (optionally included in a fermentation medium) with the recombinant host cells of the cellular populations. In an embodiment, the substrate to be hydrolyzed is a lignocellulosic biomass and, in some embodiments, it comprises starch (in a gelatinized or raw form). In some embodiments, the use of recombinant host cells avoids the need of adding additional external source of purified enzymes during fermentation to allow the breakdown of starch.

The production of ethanol 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, when a thermotolerant yeast cell is used in the process, 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. In some embodiments, the process can be used to produce 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, or at least about 500 mg per hour per liter.

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 enzyme based assays.

Heterologous protease

The present disclosure also provides the heterologous proteases disclosed herein expressed in a recombinant form. The heterologous proteases can be obtained by recombinant production in the first recombinant yeast host cell. In some embodiments, the method comprises culturing the recombinant yeast host cell of the present disclosure under conditions so as to allow the expression of the heterologous protease. The culturing step can be a continuous culture, a batch culture or a fed-batch culture. For example, the culture medium can comprise a carbon source (such as, for example, molasses, sucrose, glucose, dextrose syrup, ethanol and/or corn steep liquor), a nitrogen source (such as, for example, ammonia) and a phosphorous source (such as, for example, phosphoric acid). The method can further comprises, for example, a step of introducing the first, second, third and/or fourth genetic modification as described herein prior to the culturing step. The method can also comprises, in some instances, removing at least one component for the medium or substantially isolating the heterologous protease from the medium. The medium component that can be removed include, without limitation, water, amino acids, peptides and proteins, nucleic acid residues and nucleic acid molecules, cellular debris, fermentation products, etc. In an embodiment, the method can also comprise substantially isolating the cultured yeast recombinant host cells (e.g., the biomass) from the components of the culture medium. As used in the context of the present disclosure, the expression "substantially isolating" refers to the removal of the majority of the components of the culture medium from the cultured recombinant yeast host cells. In order to do so, the cultured recombinant yeast host cells can be centrifuged (and the resulting cellular pellet comprising the propagated recombinant yeast host cells can optionally be washed), filtered and/or dried (optionally using a vacuum-drying technique). The heterologous proteases can be provided in an isolated form or can be provided as a composition. The composition can optionally include a component from a medium (which can comprise raw starch, for example, derived from corn and/or barley) and/or a glucoamylase as described herein.

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.

EXAMPLE

Table 2. Description of the enzymes used in the Example.

Designation Description

1 ) Organism

2) Merops ID

3) EC#

4) Accession #

5) Alternative name

6) Type

7) SEQ ID NO

MP812 1 ) Candida albicans

2) A01.014

3) 3.4.23.24

4) C4YSF6

5) SAP1 , candidapepsin-1

6) Aspartic

7) SEQ ID NO: 2

MP813 1 ) Aspergillus fumigatus

2) A01.018

3) Unknown

4) 042630

5) pep2

6) Unknown

7) SEQ ID NO: 4

MP814 1 ) Clavispora lusitaniae

2) A01.018

3) Unknown

4) C4Y7E6

5) Saccharopepsin

6) Aspartic

7) SEQ ID NO: 6 Designation Description

MP815 1 ) Saccharomyces cerevisiae

2) A01.018

3) 3.4.23.25

4) P07267

5) saccharopepsin, PEP4

6) Aspartic

7) SEQ ID NO: 8

MP816 1 ) Yarrowia lipolytica

2) A01.018

3) Q6C080

4) Saccharopepsin

5) None

6) Aspartic

7) SEQ ID NO: 10

MP817 1 ) Meyerozyma guilliermondii

2) A01.018

3)

4) A5DLJ4

5) PGUG_04145

6) Putative aspartic

7) SEQ ID NO: 12

MP818 1 ) Aspergillus fumigatus

2) A01.026

3) 3.4.23.18

4) P41748

5) pepl

6) Aspartic

7) SEQ ID NO: 14

MP819 1 ) Saccharomyces cerevisiae

2) A01.030

3) 3.4.23.41

4) P32329

5) YPS1

6) Aspartic

7) SEQ ID NO: 16 Designation Description

MP820 1 ) Yarrowia lipolytica

2) A01.030

3) Unknown

4) Q6CAN1

5) YALI0D01331p

6) Aspartic

7) SEQIDNO: 18

MP821 1 ) Meyerozyma guilliermondii

2) A01.030

3) Unknown

4) A5DF74

5) PGUG_01925

6) Putative aspartic

7) SEQIDNO:20

MP822 1 ) Saccharomyces cerevisiae

2) A01.035

3) Unknown

4) Q12303

5) YPS3

6) Unknown

7) SEQIDNO:22

MP823 1 ) Candida tropicalis

2) A01.037

3) 3.4.23.24

4) Q00663

5) SAPT1

6) Aspartic

7) SEQIDNO:24

MP824 1 ) Clavispora lusitaniae

2) A01.038

3) Unknown

4) C4Y9C0

5) Candiparapsin

6) Unknown

7) SEQIDNO:26 Designation Description

MP825 1 ) Meyerozyma guilliermondii

2) A01.038

3)

4) A5DHF0

5) PGUG_02701

6) Putative aspartic

7) SEQIDNO:28

MP826 1 ) Clavispora lusitaniae

2) A01.067

3) Unknown

4) C4Y3R6

5) candiapepsin SAP9

6) Unknown

7) SEQIDNO:30

MP827 1 ) Candida albicans

2) A01.067

3) 3.4.23.24

4) 042779

5) SAP9

6) Aspartic

7) SEQIDNO:32

MP828 1 ) Meyerozyma guilliermondii

2) A01.067

3) Unknown

4) A5D9Q1

5) PGUG_00002

6) Putative aspartic

7) SEQIDNO:34

MP829 1 ) Bacillus subtilis

2) M04.014

3) 3.4.24.28

4) A0A0A0TWG6

5) nprE

6) Metalloprotease

7) SEQIDNO:36 Designation Description

MP830 1 ) Candida tropicalis

2) Unassigned

3) Unknown

4) Q9Y776

5) SAPT4

6) Aspartic

7) SEQIDNO:38

MP831 1 ) Saccharomycopsis fibuligera

2) Unassigned

3) 3.4.23.-

4) P22929

5) PEP1

6) Aspartic

7) SEQIDNO:40

MP832 1 ) Ananas comosus

2) C01.028

3) 3.4.22.33

4) 023791

5) Unknown

6) Unknown

7) SEQIDNO:42

MP833 1 ) Ananas comosus

2) C01.005

3) 3.4.22.32

4) P14518

5) Unknown

6) Unknown

7) SEQIDNO:44

MP860 1 ) zea mays Vignain like

2) C1A

3)

4) B6TYM9

5) vignain like

6) Unknown

7) SEQIDNO:46 Designation Description

MP861 1 ) zea mays cysteine protease

2) 1C1A

3)

4) B4FS90

5) cysteine protease 1

6) Unknown

7) SEQIDNO:48

MP862 1 ) zea mays cysteine protease 1 (2)

2)

3)

4) B6T669

5) cysteine protease 1

6) Unknown

7) SEQIDNO:50

MP914 1 ) Candida dubliniensis

2) A01.014

3) 3.4.23.24

4) B9WJ11

5) SAP1

6) Aspartic

7) SEQIDNO:52

MP915 1 ) Candida orthopsilosis

2) A01.014

3) 3.4.23.24

4) H8X9C8

5) CORT_0F03710

6) Aspartic

7) SEQIDNO:54

MP916 1 ) Meyerozyma guilliermondii

2) Unassigned

3) 3.4.23.24

4) A5DL07

5) PGUG_03958

6) Aspartic

7) SEQIDNO:56 Designation Description

MP917 1 ) Scheffersomyces stipites

2) Unassigned

3) 3.4.23.24

4) A3LZH2

5) PICST_63754

6) Aspartic

7) SEQIDNO:58

MP918 1 ) Lodderomyces elongisporus

2) A01.038

3) 3.4.23.24

4) A5DXL7

5) candidapepsin-1

6) Aspartic

7) SEQIDNO:60

MP919 1 ) Candida albicans

2) A01.060

3) 3.4.23.24

4) P0DJ06

5) SAP2

6) Aspartic

7) SEQIDNO:62

MP920 1 ) Candida albicans SC5314

2) A01.061

3) 3.4.23.24

4) P0CY29

5) SAP3

6) Aspartic

7) SEQIDNO:64

MP921 1 ) Candida dubliniensis CD36

2) A01.061

3) 3.4.23.24

4) B9WEB2

5) SAP3

6) Aspartic

7) SEQIDNO:66 Designation Description

MP922 1 ) Neurospora tetrasperma

2) A01.UPA

3) Unknown

4) F8MN20

5) NEUTE1DRAFT_100918

6) pepsin-like proteinases

7) SEQIDNO:68

MP923 1 ) Podospora anserine

2) Unknown

3) A01.UPA

4) B2AWU0

5) PODANS_7_8310

6) aspartic acid protease

7) SEQIDNO:70

MP924 1 ) Grossmannia clavigera

2) Unknown

3) A01.UPA

4) F0XHL4

5) CMQ_2598

6) aspartic acid protease

7) SEQIDNO:72

MP925 1 ) Chaetomium thermophilum

2) Unknown

3) A01.UPA

4) G0S4R8

5) CTHT_0023290

6) aspartic acid protease

7) SEQIDNO:74

MP926 1 ) Myceliophthora thermophila ATCC 42464]

2) Unknown

3) A01.UPA

4) G2QBW3

5) MYCTH_2305028

6) pepsin like protease

7) SEQIDNO:76 Designation Description

MP927 1 ) Magnaporthe oryzae 70-15

2) Unknown

3) A01.UPA

4) G4N837

5) candidapepsin-3

6) pepsin-like proteinases

7) SEQIDNO:78

MP928 1 ) Kluveromyces lactis

2) Unknown

3) A01.030

4) Q6CPL3

5) KLLA0_E04049g

6) pepsin-like proteinases

7) SEQIDNO:80

MP929 1 ) Ashbya gossypii ATCC 10895

2) Unknown

3) A01.035

4) Q750Y1

5) AGOS_AGL192W

6) pepsin-like proteinases

7) SEQIDNO:82

MP930 1 ) Thielavia terrestris NRRL 8126

2) Unknown

3) A01.UPA

4) G2RAU9

5) THITE_2155501

6) pepsin like protease

7) SEQIDNO:84

MP931 1 ) Neurospora crassa

2) Unknown

3) A01.015

4) Q7RZM6

5) NCU00338

6) Unknown

7) SEQIDNO:86 Designation Description

MP932 1 ) Aspergillus niger

2) Unknown

3) A01.UPA

4) E2PT33

5) An18g01320

6) Unknown

7) SEQ ID NO: 88

MP933 1 ) Bacillus amyloliquefaciens

2) Unknown

3) M04.014

4) E1 UT71

5) Unknown

6) nprE

7) SEQ ID NO: 90

Table 3. Description of the S. cerevisiae strains presented in the Example.

Designation Protease expressed Other transgenes Genes expressed inactivated

M2390 (wild-type, None None None control)

M 10874 Gene encoding Candida None Afcyl

albicans SAP1

(UniProtKB Accession

C4YSF6) (MP812)

M 10877 Gene encoding None Afcyl

Clavispora lusitaniae

Saccharopepsin

(UniProtKB Accession

C4Y7E6) (MP814)

M 10885 Gene encoding None Afcyl

Aspergillus fumigatus

PEP1 (UniProtKB

Accession P41748)

(MP818)

M 10890 Gene encoding None Afcyl

Saccharomycopsis

fibuligera PEP1

(UniProtKB Accession

P22929) (MP831 )

M 12982 Gene encoding Candida None Afcyl

dubliniensis SAP1

(UniProtKB Accession

B9WJ1 1 ) (MP914) Designation Protease expressed Other transgenes Genes expressed inactivated

M1 1259 Gene encoding Candida None None

albicans SAP1

(UniProtKB Accession

C4YSF6) expressed on

plasmid (MP812)

M1 1260 Gene encoding None None

Aspergillus fumigatus

PEP1 (UniProtKB

Accession P41748)

expressed on plasmid

(MP818)

M1 1262 Gene encoding None None

Clavispora lusitaniae

Saccharopepsin

(UniProtKB Accession

C4Y7E6) expressed on

plasmid (MP814)

M12184 Gene encoding Candida Saccharomycopsis Agpd2

albicans SAP1 fibuligera glu01 1 1

Afdhl (UniProtKB Accession (GeneBank Accession

C4YSF6) (MP812) CAC83969.1 ) Afdh2

Gene encoding the PFLA Afcyl polypeptide (UniProtKB

Accession A1A239)

Gene encoding the PFLB

polypeptide (UniProtKB

Accession A1A240)

Gene encoding the

ADHE polypeptide

(UniProtKB Accession

A1A067)

Gene encoding

Saccharomyces

cerevisiae STL1

(GeneBank Accession

NP_010825

Designation Protease expressed Other transgenes Genes expressed inactivated

M12106 Gene encoding Gene encoding Agpd2

Aspergillus fumigatus Saccharomycopsis

Afdhl PEP1 (UniProtKB fibuligera glu01 1 1

Accession P41748) (GeneBank Accession Afdh2 (MP818) CAC83969.1 )

Afcyl

Gene encoding the PFLA

polypeptide (UniProtKB

Accession A1A239)

Gene encoding the PFLB

polypeptide (UniProtKB

Accession A1A240)

Gene encoding the

ADHE polypeptide

(UniProtKB Accession

A1A067)

Gene encoding

Saccharomyces

cerevisiae STL1

(GeneBank Accession

NP_010825

M1 1589 None Gene encoding Agpd2

Saccharomycopsis

Afdhl fibuligera glu01 1 1

(GeneBank Accession Afdh2 CAC83969.1 )

Afcyl

Gene encoding the PFLA

polypeptide (UniProtKB

Accession A1A239)

Gene encoding the PFLB

polypeptide (UniProtKB

Accession A1A240)

Gene encoding the

ADHE polypeptide

(UniProtKB Accession

A1A067)

Gene encoding

Saccharomyces

cerevisiae STL1

(GeneBank Accession

NP_010825) Designation Protease expressed Other transgenes Genes

expressed inactivated

M8841 None Gene encoding Agpd2

Saccharomycopsis

Afdhl

fibuligera glu01 1 1

(GeneBank Accession Afdh2

CAC83969.1 )

Afcyl

Gene encoding the PFLA

polypeptide (UniProtKB

Accession A1A239)

Gene encoding the PFLB

polypeptide (UniProtKB

Accession A1A240)

Gene encoding the

ADHE polypeptide

(UniProtKB Accession

A1A067)

M 12962 (wild-type None None None

distilling strain)

M 14028 Afcyl Gene encoding Afcyl

Saccharomycopsis

fibuligera PEP1

(UniProtKB Accession

P22929) (MP818)

Heterologous protease candidates (summarized in Table 2 above), including three native S. cerevisiae proteases (PEP4, YPS1 , YPS3), were expressed in an industrial yeast background. The nucleic acid encoding each of these proteins were codon optimized and then integrated onto the chromosome under control of the yeast constitutive promoter, tef2p (e.g., promoter of the gene encoding the TEF2 polypeptide). These enzymes utilize native signal sequences if from fungal origin or the S. cerevisiae invertase if from bacterial origin. Each of the recombinant yeast host cell was assayed for secreted protease activity using azoalbumin as a substrate. Briefly, cells were grown at 35°C for 72 hours (h), centrifuged and cell supernatant was added in a 1 :1 ratio with a 1 % azoalbumin solution and incubated at 35°C for 4 h. Undigested protein was precipitated with TCA and incubated on ice for 30 minutes (min). The mixture was then filtered and absorbance of filtrate read at 410 nm. The results of the normalized protease activity are presented in Figure 1. MP812 (C. albicans SAP1 ), MP814 (CI. lusitaniae saccharopepsin), MP818 (A. fumigatus PEP1 ), MP831 (S. fibuligera PEP1 ), and MP914 (C. dubliniensis SAP1 ) were found to increased activity. A few other proteases had a moderate of activity (MP813, MP815. MP816, MP817, MP826). Several had little to no activity compared to the wild-type strain.

Next, a subset of these yeast-made proteases were tested in conventional corn mash fermentation in combination with glucoamylase and urea. Strains were inoculated into 20% total solid (TS) corn supplemented with 100% or 50% of a purified glucoamylase enzyme (100% = 0.48 amyloglucosidase unit (AGU)/gram of total solids (gTs); 50% = 0.24 AGU/gTs) and either 650 ppm or 325 ppm urea. Ethanol and glycerol productions were measured at different points in time with HPLC. Table 4 below compares ethanol and glycerol production over time in MP2390 (wild-type), M1 1589, M10874 (expressing MP812 in MP2390 background), M12184 (expressing MP812 in M1 1589 background), M10885 (expressing MP818 in M2390 background) or M12106 (MP818 in M1 1589 background) strains. As shown in Table 4, strains expressing protease demonstrate improved kinetics, reduced glycerol production and/or urea displacement over parental control.

Table 4. Ethanol and glycerol yield of corn fermentation with M2390, M10874, M10885, M1 1589, M12184 and M12106 strain in the presence of 100% or 50% GA and 650 or 325 ppm of urea. Results are provided as g of ethanol or glycerol /L.

Strains M2390 (wild-type), M10874 (MP814 expressed in a M2390 background), M10885 (MP818 expressed in a M2390 background), M1 1589, M12184 (MP812 expressed in a M1 1589 background), M12982 (MP914 expressed in a M2390 background) and M10890 (MP831 expressed in a M2390 background) strains were inoculated into a 23% Ts corn mash fermentation (in the absence of urea supplementation) and in the presence or absence a commercial protease (AYF 1 17™, in purified form). Protease-expressing strains in a M2390 background were dosed at 100% glucoamylase (0.48 AGU/gTs) whereas protease-expressing strains in a M1 1589 background were dosed at 50% glucoamylase (0.24 AGU/gTs). Ethanol and glycerol productions were measured at different points in time with HPLC. Results of this fermentation are shown in Figures 2 and 3 indicate that, when an heterologous protease is expressed, there is no advantage of supplementing the fermentation medium with a purified protease to increase ethanol yield or reduce glycerol production.

Strain M12962 and M12028 were submitted to a 1 .072 OG malted barley fermentation. Briefly, dry malted barley was mashed to create wort with a specific gravity of 1.072. The recombinant strains were tested in shake flasks in this substrate and metabolites were measured by HPLC. As shown in Table 5 below, the M14028 strain has improved kinetics, reduced glycerol (e.g., 14% reduction) and increase in ethanol content (e.g., increase of 1 .5%) after 52 h of fermentation.

Table 5. Metabolic profile of wild-type distilling strain (M12962) and M12028 strain (MP818 expressed in M12962 background) during malted barley fermentation.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. REFERENCES

Guo ZP, Qiu CY, Zhang L, Ding ZY, Wang ZX, Shi GY. Expression of aspartic protease from Neurospora crassa in industrial ethanol-producing yeast and its application in ethanol production. Enzyme Microb Technol. 201 1 Feb 8;48(2):148-54.

Johnston DB, McAloon AJ. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production. Bioresour Technol. 2014 Feb;154:18-25.

PCT/US2012/032443

PCT/US201 1/039192

WO 2012/138942