COMBINED USE OF AT LEAST ONE ENDO-PROTEASE AND AT LEAST ONE EXO-PROTEASE IN AN SSF PROCESS FOR IMPROVING ETHANOL YIELD

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to processes for producing fermentation products from gelatinized and/or un-gelatinized starch-containing material, as well as to proteases for use in the methods of the invention.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, from starch-containing material is well-known in the art. Generally two different kinds of processes are used. The most commonly used process, often referred to as a “conventional process”, includes liquefying gelatinized starch at high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation carried out in the presence of a glucoamylase and a fermenting organism. Conventional starch-conversion processes, such as liquefaction and saccharification processes are described in, e.g., U.S. Pat. No. 3,912,590, EP252730 and EP063909.

Another well-known process, often referred to as a “raw starch hydrolysis”-process (RSH process) includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.

U.S. Pat. No. 5,231,017-A discloses the use of an acid fungal protease during ethanol fermentation in a process comprising liquefying gelatinized starch with an alpha-amylase.

WO 2003/066826 discloses a raw starch hydrolysis process (RSH process) carried out on non-cooked mash in the presence of fungal glucoamylase, alpha-amylase and fungal protease.

WO 2007/145912 discloses a process for producing ethanol comprising contacting a slurry comprising granular starch obtained from plant material with an alpha-amylase capable of solubilizing granular starch at a pH of 3.5 to 7.0 and at a temperature below the starch gelatinization temperature for a period of 5 minutes to 24 hours; obtaining a substrate comprising greater than 20% glucose, and fermenting the substrate in the presence of a fermenting organism and starch hydrolyzing enzymes at a temperature between 10° C. and 40° C. for a period of 10 hours to 250 hours. Additional enzymes added during the contacting step may include protease.

WO 2010/008841 discloses processes for producing fermentation products, such as ethanol, from gelatinized as well as un-gelatinized starch-containing material by saccharifying the starch material using at least a glucoamylase and a metalloprotease and fermenting using a yeast organism. Particularly the metallo protease is derived form a strain of Thermoascus aurantiacus.

WO 2014/037438 discloses serine proteases derived from Meripilus giganteus, Trametes versicolor, and Dichomitus squalens and their use in animal feed.

WO 2015/078372 discloses serine proteases derived from Meripilus giganteus, Trametes versicolor, and Dichomitus squalens for use in a starch wet milling process.

WO 2013/102674 discloses exo-proteases belonging to family S53.

S53 proteases are known in the art, e.g., a S53 peptide from Grifola frondosa with accession number MER078639. A S53 protease from Postia placenta (Uniprot: B8PMI5) was isolated by Martinez et al in “Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion”, 2009, Proc. Natl. Acad. Sci. USA 106:1954-1959.

Vanden Wymelenberg et al. have isolated a S53 protease (Uniprot: Q281W2) in “Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins”, 2006, Fungal Genet. Biol. 43:343-356. Another S53 polypeptide from Postia placenta (Uniprot:B8P431) has been identified by Martinez et al. in “Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion”, 2009, Proc. Natl. Acad. Sci. U.S.A. 106:1954-1959.

Floudas et al have published the sequence of a S53 protease in “The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes”, 2012, Science, 336:1715-1719. Fernandez-Fueyo et al have published the sequences of three serine proteases in “Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis”, 2012, Proc Natl Acad Sci USA. 109:5458-5463 (Uniprot:M2QQ01, Uniprot:M2QWH2, UniprotM2RD67).

It is an object of the present invention to identify protease mixtures that will result in an increased ethanol yield in a starch to ethanol process, when said proteases are added/are present during saccharification and/or fermentation.

SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that adding a mixture of endoprotease and exo-protease to the SSF process will result in an increased ethanol yield. The invention provides in a first aspect a process for producing a fermentation product from starch-containing material comprising:

a) saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material using a carbohydrate-source generating enzymes; and

b) fermenting using a fermenting organism; wherein

- steps a) and/or b) is performed in the presence of an endo-protease and an exo-protease mixture, and wherein the exo-protease makes up at least 5% (w/w) of the protease mixture on a total protease enzyme protein basis.

In a second aspect the invention provides a process for producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying starch-containing material at a temperature above the initial gelatinization temperature of said starch-containing material in the presence of an alpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

wherein steps b) and/or c) is performed in the presence of an endo-protease and an exo-protease mixture, and wherein the exo-protease makes up at least 5% (w/w) of the protease mixture on a total protease enzyme protein basis.

In a third aspect the invention relates to a composition suitable for use in the processes of the invention, more particularly a composition comprising a mixture of endo-protease and exo-protease, and wherein the exo-protease makes up at least 5% (w/w) of the protease mixture on a total protease enzyme protein basis, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, particularly at least 75%, more particularly the exo-protease makes up from between 5 to 95% (w/w) on a total protease enzyme protein basis, particularly 10 to 80% (w/w), particularly 15 to 70% (w/w), more particularly 20 to 60% (w/w), and even more particularly 25 to 50% (w/w) of the protease mixture in the composition on a total protease enzyme protein basis.

In a fourth aspect the present invention relates to a use of the composition according to the invention in saccharification of a starch containing material.

In a fifth aspect the present invention relates to a polypeptide having serine protease activity, and belonging to family S10, selected from the group consisting of: (a) a polypeptide having having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6; (b) a polypeptide encoded by a polynucleotide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 8; (c) a fragment of the polypeptide of (a), or (b) that has serine protease activity.

In a sixth aspect the present invention relates to a polypeptide having serine protease activity, and belonging to family S53, selected from the group consisting of:

(a) a polypeptide having having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 23; or

(b) a polypeptide encoded by a polynucleotide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 29; or

In a seventh aspect the present invention relates to A polypeptide having serine protease activity, and belonging to family S53, selected from the group consisting of:

(a) a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 25; or

(b) a polypeptide encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 30; or

The present invention also relates to polynucleotides encoding an serine protease of the invention; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the serine protease of the invention.

Definitions

Proteases: The term “protease” includes any enzyme belonging to the EC 3.4 enzyme group (including each of the eighteen subclasses thereof). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, Calif., including supplements 1-5 published in 1994, Eur. J. Biochem. 223: 1-5; 1995, Eur. J. Biochem. 232: 1-6; 1996, Eur. J. Biochem. 237: 1-5; 1997, Eur. J. Biochem. 250: 1-6; and 1999, Eur. J. Biochem. 264: 610-650 respectively. The nomenclature is regularly supplemented and updated; see e.g. the World Wide Web (WWW) at http://www.chem.qmw.ac.uk/iubmb/enzyme/index.html.

Proteases are classified on the basis of their catalytic mechanism into the following groups: Serine proteases (S), Cysteine proteases (C), Aspartic proteases (A), Metalloproteases (M), and Unknown, or as yet unclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J. Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), in particular the general introduction part.

Polypeptides having protease activity, or proteases, are sometimes also designated peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. Proteases may be of the exo-type (exo-peptidases) that hydrolyse peptides starting at either end thereof, or of the endo-type that act internally in polypeptide chains (endopeptidases).

S53 protease: The term “S53” means a protease activity selected from:

(a) proteases belonging to the EC 3.4.21 enzyme group; and/or

(b) proteases belonging to the EC 3.4.14 enzyme group; and/or

(c) Serine proteases of the peptidase family S53 that comprises two different types of peptidases: tripeptidyl aminopeptidases (exo-type) and endo-peptidases; as described in 1993, Biochem. J. 290:205-218 and in MEROPS protease database, release, 9.4 (31 Jan. 2011) (www.merops.ac.uk). The database is described in Rawlings, N. D., Barrett, A. J. and Bateman, A., 2010, “MEROPS: the peptidase database”, Nucl. Acids Res. 38: D227-D233.

For determining whether a given protease is a Serine protease, and a family S53 protease, reference is made to the above Handbook and the principles indicated therein. Such determination can be carried out for all types of proteases, be it naturally occurring or wild-type proteases; or genetically engineered or synthetic proteases.

The peptidases of the S53 family tend to be most active at acidic pH (unlike the homologous subtilisins), and this can be attributed to the functional importance of carboxylic residues, notably Asp in the oxyanion hole. The amino acid sequences are not closely similar to those in family S8 (i.e. serine endopeptidase subtilisins and homologues), and this, taken together with the quite different active site residues and the resulting lower pH for maximal activity, provides for a substantial difference to that family. Protein folding of the peptidase unit for members of this family resembles that of subtilisin, having the clan type SB.

S8 protease: Most members of this family are endopeptidases, and are active at neutral-mildly alkali pH. Many peptidases in the family are thermostable. Casein is often used as a protein substrate and a typical synthetic substrate is Suc-Ala-Ala-Pro-Phe-NHPhNO2. Most members of the family are nonspecific peptidases with a preference to cleave after hydrophobic residues. Link to S10 family definition for activity and specificities: http://merops.sanger.ac.uk/cgi-bin/famsum?family=S8.

S10 protease: The carboxypeptidases in family S10 show two main types of specificity. Some (e.g. carboxypeptidase C) show a preference for hydrophobic residues in positions P1 and P1″. Carboxypeptidases of the second set (e.g. carboxypeptidase D) display a preference for the basic amino acids either side of the scissile bond, but are also able to cleave peptides with hydrophobic residues in these positions. Link to S10 family definition for activity and specificities: http://merops.sanger.ac.uk/cgi-bin/famsum?family=S10.

Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of an enzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Fragment: The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has serine protease activity.

Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample; e.g. a host cell may be genetically modified to express the polypeptide of the invention. The fermentation broth from that host cell will comprise the isolated polypeptide.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having serine protease activity.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Protease activity: The term “protease activity” means proteolytic activity (EC 3.4). There are several protease activity types such as trypsin-like proteases cleaving at the carboxyterminal side of Arg and Lys residues and chymotrypsin-like proteases cleaving at the carboxyterminal side of hydrophobic amino acid residues.

Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperatures are 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95° C. Examples of general protease substrates are casein, bovine serum albumin and haemoglobin. In the classical Anson and Mirsky method, denatured haemoglobin is used as substrate and after the assay incubation with the protease in question, the amount of trichloroacetic acid soluble haemoglobin is determined as a measurement of protease activity (Anson, M. L. and Mirsky, A. E., 1932, J. Gen. Physiol. 16: 59 and Anson, M. L., 1938, J. Gen. Physiol. 22: 79).

For the purpose of the present invention, protease activity may be determined using assays which are described in “Materials and Methods”, such as the Kinetic Suc-AAPF-pNA assay, Protazyme AK assay, Kinetic Suc-AAPX-pNA assay and o-Phthaldialdehyde (OPA). For the Protazyme AK assay, insoluble Protazyme AK (Azurine-Crosslinked Casein) substrate liberates a blue colour when incubated with the protease and the colour is determined as a measurement of protease activity. For the Suc-AAPF-pNA assay, the colourless Suc-AAPF-pNA substrate liberates yellow paranitroaniline when incubated with the protease and the yellow colour is determined as a measurement of protease activity.

Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5′ and/or 3′ end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity.

Variant: The term “variant” means a polypeptide having protease activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved processes for producing ethanol from starch-containing materials by the combined use of at least one endo-protease and at least one exo-protease in an SSF process. More particularly the exo-protease should make up at least 5% (w/w) of the protease mixture on a total protease enzyme protein basis.

More specifically the present invention relates to a process for producing a fermentation product from starch-containing material comprising:

a) saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material using a carbohydrate-source generating enzymes; and

b) fermenting using a fermenting organism; wherein

steps a) and/or b) is performed in the presence of an endo-protease and an exo-protease mixture, and wherein the exo-protease makes up at least 5% (w/w) of the protease mixture on a total protease enzyme protein basis.

In a second aspect the invention provides a process for producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying starch-containing material at a temperature above the initial gelatinization temperature of said starch-containing material in the presence of an alpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

Processes for producing fermentation products, e.g., ethanol, from starch-containing materials are generally well known in the art. Generally two different kinds of processes are used. The most commonly used process, often referred to as a “conventional process”, includes liquefying gelatinized starch at high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation carried out in the presence of a glucoamylase and a fermenting organism. Another well-known process, often referred to as a “raw starch hydrolysis”-process (RSH process) includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.

Native starch consists of microscopic granules, which are insoluble in water at room temperature. When aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. At temperatures up to about 50° C. to 75° C. the swelling may be reversible. However, with higher temperatures an irreversible swelling called “gelatinization” begins. During this “gelatinization” process there is a dramatic increase in viscosity. Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch-containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers. The raw material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in the production of, e.g., syrups. Both dry and wet milling is well known in the art of starch processing and may be used in a process of the invention. Methods for reducing the particle size of the starch containing material are well known to those skilled in the art.

As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or “liquefied” so that it can be suitably processed. This reduction in viscosity is primarily attained by enzymatic degradation in current commercial practice.

Liquefaction is carried out in the presence of an alpha-amylase, preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase. In an embodiment, a phytase is also present during liquefaction. In an embodiment, viscosity reducing enzymes such as a xylanase and/or beta-glucanase is also present during liquefaction.

During liquefaction, the long-chained starch is degraded into branched and linear shorter units (maltodextrins) by an alpha-amylase. Liquefaction may be carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C. (e.g., 70-90° C., such as 77-86° C., 80-85° C., 83-85° C.) and an alpha-amylase is added to initiate liquefaction (thinning).

The slurry may in an embodiment be jet-cooked at between 95-140° C., e.g., 105-125° C., for about 1-15 minutes, e.g., about 3-10 minutes, especially around 5 minutes. The slurry is then cooled to 60-95° C. and more alpha-amylase is added to obtain final hydrolysis (secondary liquefaction). The jet-cooking process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6. The alpha-amylase may be added as a single dose, e.g., before jet cooking.

The liquefaction process is carried out at between 70-95° C., such as 80-90° C., such as around 85° C., for about 10 minutes to 5 hours, typically for 1-2 hours. The pH is between 4 and 7, such as between 4.5 and 5.5. In order to ensure optimal enzyme stability under these conditions, calcium may optionally be added (to provide 1-60 ppm free calcium ions, such as about 40 ppm free calcium ions). After such treatment, the liquefied starch will typically have a “dextrose equivalent” (DE) of 10-15.

Generally liquefaction and liquefaction conditions are well known in the art.

Alpha-amylases for use in liquefaction are preferably bacterial acid stable alphaamylases. Particularly the alpha-amylase is from an Exiguobacterium sp. or a Bacillus sp. such as e.g., Bacillus stearothermophilus or Bacillus licheniformis.

Saccharification may be carried out using conditions well-known in the art with a carbohydrate-source generating enzyme, in particular a glucoamylase, or a beta-amylase and optionally a debranching enzyme, such as an isoamylase or a pullulanase. For instance, a full saccharification step may last from about 24 to about 72 hours. However, it is common to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65° C., typically about 60° C., followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation (SSF) process. Saccharification is typically carried out at a temperature in the range of 20-75° C., e.g., 25-65° C. and 40-70° C., typically around 60° C., and at a pH between about 4 and 5, normally at about pH 4.5.

The saccharification and fermentation steps may be carried out either sequentially or simultaneously. In an embodiment, saccharification and fermentation are performed simultaneously (referred to as “SSF”). However, it is common to perform a pre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to 65° C., typically around 60° C. which is followed by a complete saccharification during fermentation referred to as simultaneous saccharification and fermentation (SSF). The pH is usually between 4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification and fermentation (SSF) process, there is no holding stage for saccharification, rather, the yeast and enzymes are added together and the process is then carried out at a temperature of 25-40° C., such as between 28° C. and 35° C., such as between 30° C. and 34° C., such as around 32° C. The SSF-process may be carried out at a pH from about 3 and 7, preferably from pH 4.0 to 6.5, or more preferably from pH 4.5 to 5.5.

In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Instead of the conventional process described above, the fermentation product, e.g., ethanol, may be produced from starch-containing material without gelatinization (i.e., without cooking) of the starch-containing material (often referred to as a “raw starch hydrolysis” process). The fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material and water. In one embodiment the process includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s) to produce sugars that can be fermented into the fermentation product by a suitable fermenting organism. In this embodiment the desired fermentation product, e.g., ethanol, is produced from un-gelatinized (i.e., uncooked), preferably milled, cereal grains, such as corn.

Accordingly, in this aspect the invention relates to processes for producing a fermentation product from starch-containing material comprising the steps of:

a) saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material using a carbohydrate-source generating enzymes; and

b) fermenting using a fermenting organism; wherein

steps a) and/or b) is performed in the presence of an endo-protease and an exo-protease mixture, and wherein the exo-protease makes up at least 5% (w/w) of the protease mixture.

In a particular embodiment steps a) and b) are performed simultaneously, wherein the saccharifying enzymes and fermenting organisms (e.g., yeast) are added together and then carried out at a temperature of 25-40° C. The SSF-process may be carried out at a pH from about 3 and 7, preferably from pH 4.0 to 6.5, or more preferably from pH 4.5 to 5.5. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

The term “initial gelatinization temperature” means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50° C. and 75° C.; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Stärke 44(12): 461-466. In one embodiment a temperature below the initial gelatinization temperature means that the temperature typically lies in the range between 30-75° C., preferably between 45-60° C. In a preferred embodiment the process is carried at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around 32° C.

As disclosed above in the background art section, the use of proteases during fermentation is known in the art, however, according to the present invention an increased ethanol yield may be obtained when saccharification and/or fermentation is performed in the presence of an endoprotease and exo-protease mixture. In particular the present inventors have found that, the exo-protease should make up at least 5% (w/w) of the protease mixture on a total protease enzyme protein basis.

In one embodiment the exo-protease makes up at least 10% (w/w) of the protease mixture on a total protease enzyme protein basis, such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, particularly at least 75%, more particularly the exo-protease makes up from between 5 to 95% (w/w) on a total protease enzyme protein basis, particularly 10 to 80% (w/w), particularly 15 to 70% (w/w), more particularly 20 to 60% (w/w), and even more particularly 25 to 50% (w/w) of the protease mixture in the composition on a total protease enzyme protein basis.

In another embodiment the endo-protease and exo-protease is present in a ratio of 5:2 micro grams enzyme protein (EP)/g dry solids (DS), particularly 5:3, more particularly 5:4.

The proteases used in a process of the invention are selected from endo-peptidases (endoproteases) and exo-peptidases (exo-proteases). Among endo-peptidases, serine proteases (EC 3.4.21) and metallo-proteases (EC 3.4.24) are especially relevant.

In a particular embodiment the endo-protease is selected from the group consisting of serine proteases belonging to family S53, S8, or from metallo proteases belonging to family M35.

In another particular embodiment the endo-protease is selected from A1 proteases.

The endo-protease is in one embodiment selected from a serine protease of family S53, such as from a strain of the genus Meripilus, more particularly Meripilus giganteus.

More particularly the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:

a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1, or the polypeptide of SEQ ID NO: 2.

The endo-protease is in a further embodiment selected from a serine protease of family S8, such as from a strain of the genus Pyrococcus or Thermococcus, particularly Pyrococcus furiosus, and Thermococcus litoralis.

More particularly the S8 protease is a polypeptide having serine protease activity, selected from the group consisting of:

In another particular embodiment the endo-protease is selected from metallo-proteases (see Handbook of Proteolytic Enzymes, A. J. Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998)); in particular, the proteases of the invention are selected from the group consisting of:

(a) proteases belonging to the EC 3.4.24 metalloendopeptidases;

(b) metalloproteases belonging to the M group of the above Handbook;

In one particular embodiment the endo-protease is selected from the M35 family, more particularly M35 protease derived from Thermoascus aurantiacus, the mature polypeptide of which comprises amino acids 1-177 of SEQ ID NO: 16 or a polypeptide having at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% identity to the polypeptide of SEQ ID NO: 16.

The exo-protease is preferably selected from a protease belonging to family S10, S53, M14, M28, particularly S10, more particularly S10 from Aspergillus or Penicillium, e.g., Aspergillus oryzae, Aspergillus niger, or Penicillium simplicissimum.

In one particular embodiment the S10 exo-protease is selected from a polypeptide having serine protease activity, selected from the group consisting of:

a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6, or the polypeptide of SEQ ID NO: 7.

In another particular embodiment the S10 exo-protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 31.

The exo-protease is in another embodiment selected from S53 exo-protease is derived from a strain of Aspergillus, Trichoderma, Thermoascus, or Thermomyces, particularly Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, Thermoascus thermophilus, or Thermomyces lanuginosus.

In one particular embodiment the S53 exo-protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or the polypeptide of SEQ ID NO: 20.

Before initiating the process a slurry of starch-containing material, such as granular starch, having 10-55 w/w % dry solids (DS), preferably 25-45 w/w % dry solids, more preferably 30-40 w/w % dry solids of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants.

In a particular embodiment, the process of the invention further comprises, prior to the conversion of a starch-containing material to sugars/dextrins the steps of:

(x) reducing the particle size of the starch-containing material; and

(y) forming a slurry comprising the starch-containing material and water.

In an embodiment, the starch-containing material is milled to reduce the particle size. In an embodiment the particle size is reduced to between 0.05-3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fits through a sieve with a 0.05-3.0 mm screen, preferably 0.1-0.5 mm screen.

After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted into a soluble starch hydrolyzate.

In an embodiment, the particle size is smaller than a #7 screen, e.g., a #6 screen. A #7 screen is usually used in conventional prior art processes.

Alpha-Amylase Present and/or Added in Liquefaction

In an embodiment the alpha-amylase is from the genus Bacillus, such as a strain of Bacillus stearothermophilus, in particular a variant of a Bacillus stearothermophilus alphaamylase, such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 15 herein.

In an embodiment the Bacillus stearothermophilus alpha-amylase has a double deletion of two amino acids in the region from position 179 to 182, more particularly a double deletion at positions I181+G182, R179+G180, G180+I181, R179+I181, or G180+G182, preferably I181+G182, and optionally a N193F substitution, (using SEQ ID NO: 15 for numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase has a substitution at position S242, preferably S242Q substitution.

In an embodiment the Bacillus stearothermophilus alpha-amylase has a substitution at position E188, preferably E188P substitution.

In an embodiment the alpha-amylase is selected from the group of Bacillus stearothermophilus alpha-amylase variants with the following mutations:

- I181*+G182*+N193F+E129V+K177L+R179E;
- I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;
- I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and
- I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 15 for numbering).

In an embodiment the alpha-amylase variant has at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO: 15.

It should be understood that when referring to Bacillus stearothermophilus alphaamylase and variants thereof they are normally produced in truncated form. In particular, the truncation may be so that the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 15 herein, or variants thereof, are truncated in the C-terminal preferably to have around 490 amino acids, such as from 482-493 amino acids. Preferably the Bacillus stearothermophilus variant alpha-amylase is truncated, preferably after position 484 of SEQ ID NO: 15, particularly after position 485, particularly after position 486, particularly after position 487, particularly after position 488, particularly after position 489, particularly after position 490, particularly after position 491, particularly after position 492, more particularly after position 493.

Glucoamylase Present and/or Added in Saccharification and/or Fermentation

The carbohydrate-source generating enzyme present during saccharification may in one embodiment be a glucoamylase. A glucoamylase is present and/or added in saccharification and/or fermentation, preferably simultaneous saccharification and fermentation (SSF), in a process of the invention (i.e., saccharification and fermentation of ungelatinized or gelatinized starch material).

In an embodiment the glucoamylase present and/or added in saccharification and/or fermentation is of fungal origin, preferably from a stain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii or a strain of Trametes, preferably T. cingulata, or a strain of Pycnoporus, preferably P. sanguineus, or a strain of Gloeophyllum, such as G. serpiarium, G. abietinum or G. trabeum, or a strain of the Nigrofomes.

In an embodiment the glucoamylase is derived from Talaromyces, such as a strain of Talaromyces emersonii, such as the one shown in SEQ ID NO: 11.

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 11;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 11.

In an embodiment the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ ID NO: 4 in WO 2011/066576, or SEQ ID NO: 12 herein.

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 12;

In an embodiment the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strain of Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferred embodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQ ID NO: 2 in WO 2011/068803.

In an embodiment the glucoamylase is derived from Gloeophyllum serpiarium, such as the one shown in SEQ ID NO: 13.

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 13;

In another embodiment the glucoamylase is derived from Gloeophyllum trabeum such as the one shown in SEQ ID NO: 14. In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 14;

In an embodiment the glucoamylase is derived from Trametes, such as a strain of Trametes cingulata, such as the one shown in SEQ ID NO: 10.

In one embodiemnt the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 10;

In an embodiment the glucoamylase is derived from a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO 2012/064351.

Glucoamylases may in an embodiment be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS, especially 0.1-0.5 AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from DuPont).

According to a preferred embodiment of the invention the glucoamylase is present and/or added in saccharification and/or fermentation in combination with an alpha-amylase. Examples of suitable alpha-amylase are described below.

Alpha-Amylase Present and/or Added in Saccharification and/or Fermentation

In an embodiment an alpha-amylase is present and/or added in saccharification and/or fermentation in the processes of the invention. In a preferred embodiment the alpha-amylase is of fungal or bacterial origin. In a preferred embodiment the alpha-amylase is a fungal acid stable alpha-amylase. A fungal acid stable alpha-amylase is an alpha-amylase that has activity in the pH range of 3.0 to 7.0 and preferably in the pH range from 3.5 to 6.5, including activity at a pH of about 4.0, 4.5, 5.0, 5.5, and 6.0.

In one embodiment the alpha-amylase is derived from the genus Aspergillus, especially a strain of A. terreus, A. niger, A. oryzae, A. awamori, or Aspergillus kawachii, or of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.

In a preferred embodiment the alpha-amylase present and/or added in saccharification and/or fermentation is derived from a strain of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, such as one shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucor pusillus alpha-amylase hybrid having an Aspergillus niger linker and starch-binding domain, such as the one shown in SEQ ID NO: 9 herein, or a variant thereof.

In an embodiment the alpha-amylase present and/or added in saccharification and/or fermentation is selected from the group consisting of.

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9;

(ii) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 9.

In a preferred embodiment the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID NO: 9 having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 9 for numbering).

In an embodiment the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably disclosed as SEQ ID NO: 9, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 9 for numbering), and wherein the alpha-amylase variant present and/or added in saccharification and/or fermentation has at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment the ratio between glucoamylase and alpha-amylase present and/or added during saccharification and/or fermentation may preferably be in the range from 500:1 to 1:1, such as from 250:1 to 1:1, such as from 100:1 to 1:1, such as from 100:2 to 100:50, such as from 100:3 to 100:70.

In one embodiment the alpha-amylase is present in an amount of 0.001 to 10 AFAU/g DS, preferably 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

In a further embodiment the alpha-amylase and glucoamylase is added in a ratio of between 0.1 and 100 AGU/FAU-F, preferably 2 and 50 AGU/FAU-F, especially between 10 and 40 AGU/FAU-F when saccharification and fermentation are carried out simultaneously.

Fermentation

The fermentation conditions are determined based on, e.g., the kind of plant material, the available fermentable sugars, the fermenting organism(s) and/or the desired fermentation product. One skilled in the art can easily determine suitable fermentation conditions. The fermentation may be carried out at conventionally used conditions. Preferred fermentation processes are anaerobic processes.

For example, fermentations may be carried out at temperatures as high as 75° C., e.g., between 40-70° C., such as between 50-60° C. However, bacteria with a significantly lower temperature optimum down to around room temperature (around 20° C.) are also known. Examples of suitable fermenting organisms can be found in the “Fermenting Organisms” section above.

For ethanol production using yeast, the fermentation may go on for 24 to 96 hours, in particular for 35 to 60 hours. In an embodiment the fermentation is carried out at a temperature between 20 to 40° C., preferably 26 to 34° C., in particular around 32° C.

The fermentation may include, in addition to a fermenting microorganisms (e.g., yeast), nutrients, and additional enzymes, including phytases. The use of yeast in fermentation is well known in the art.

Other fermentation products may be fermented at temperatures known to the skilled person in the art to be suitable for the fermenting organism in question.

Fermentation is typically carried out at a pH in the range between 3 and 7, preferably from pH 3.5 to 6, more preferably pH 4 to 5. Fermentations are typically ongoing for 6-96 hours.

The processes of the invention may be performed as a batch or as a continuous process. Fermentations may be conducted in an ultrafiltration system wherein the retentate is held under recirculation in the presence of solids, water, and the fermenting organism, and wherein the permeate is the desired fermentation product containing liquid. Equally contemplated are methods/processes conducted in continuous membrane reactors with ultrafiltration membranes and where the retentate is held under recirculation in presence of solids, water, and the fermenting organism(s) and where the permeate is the fermentation product containing liquid.

After fermentation the fermenting organism may be separated from the fermented slurry and recycled.

Starch-Containing Materials

Any suitable starch-containing starting material may be used in a process of the present invention. In one embodiment the starch-containing material is granular starch. In another embodiment the starch-containing material is derived from whole grain. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing starting materials, suitable for use in the processes of the present invention, include barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. The starch-containing material may also be a waxy or non-waxy type of corn and barley. In a preferred embodiment the starch-containing material is corn. In a preferred embodiment the starch-containing material is wheat.

Fermentation Products

The term “fermentation product” means a product produced by a method or process including fermenting using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂and CO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. In an preferred embodiment the fermentation product is ethanol.

Fermenting Organisms

The term “fermenting organism” refers to any organism, including bacterial and fungal organisms, such as yeast and filamentous fungi, suitable for producing a desired fermentation product. Suitable fermenting organisms are able to ferment, i.e., convert, fermentable sugars, such as arabinose, fructose, glucose, maltose, mannose, or xylose, directly or indirectly into the desired fermentation product.

Examples of fermenting organisms include fungal organisms such as yeast Preferred yeast include strains of Saccharomyces, in particular Saccharomyces cerevisiae or Saccharomyces uvarum; strains of Pichia, in particular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichia pastoris; strains of Candida, in particular Candida arabinofermentans, Candida boidinii, Candida diddensii, Candida shehatae, Candida sonorensis, Candida tropicalis, or Candida utilis. Other fermenting organisms include strains of Hansenula, in particular Hansenula anomala or Hansenula polymorpha; strains of Kluyveromyces, in particular Kluyveromyces fragilis or Kluyveromyces marxianus; and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.

Preferred bacterial fermenting organisms include strains of Escherichia, in particular Escherichia coli, strains of Zymomonas, in particular Zymomonas mobilis, strains of Zymobacter, in particular Zymobactor palmae, strains of Klebsiella in particular Klebsiella oxytoca, strains of Leuconostoc, in particular Leuconostoc mesenteroides, strains of Costridium, in particular Clostridium butyricum, strains of Enterobacter, in particular Enterobacter aerogenes, and strains of Thermoanaerobacter, in particular Thermoanaerobacter BG1L1 (Appl. Microbiol. Biotech. 77: 61-86), Thermoanarobacter ethanolicus, Thermoanaerobacter mathranii, or Thermoanaerobacter thermosaccharolyticum. Strains of Lactobacillus are also envisioned as are strains of Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, and Geobacillus thermoglucosidasius.

In an embodiment, the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.

In an embodiment, the fermenting organism is a C5 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.

The amount of starter yeast employed in fermentation is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time, (e.g., to produce at least 10% ethanol from a substrate having between 25-40% DS in less than 72 hours). Yeast cells are generally supplied in amounts of about 10⁴to about 10¹², and preferably from about 10⁷to about 10¹⁰, especially about 5×10⁷viable yeast count per mL of fermentation broth. After yeast is added to the mash, it is typically subjected to fermentation for about 24-96 hours, e.g., 35-60 hours. The temperature is between about 26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g., around pH 4-5.

Yeast is the preferred fermenting organism for ethanol fermentation. Preferred are strains of Saccharomyces, especially strains of the species Saccharomyces cerevisiae, preferably strains which are resistant towards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or 20 vol. % or more ethanol.

In an embodiment, the C5 utilizing yeast is a Saccharomyces cerevisea strain disclosed in WO 2004/085627.

In an embodiment, the fermenting organism is a C5 eukaryotic microbial cell concerned in WO 2010/074577 (Nedalco).

In an embodiment, the fermenting organism is a transformed C5 eukaryotic cell capable of directly isomerize xylose to xylulose disclosed in US 2008/0014620.

In an embodiment, the fermenting organism is a C5 sugar fermentating cell disclosed in WO 2009/109633.

Commercially available yeast include LNF SA-1, LNF BG-1, LNF PE-2, and LNF CAT-1 (available from LNF Brazil), RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC—North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

The fermenting organism capable of producing a desired fermentation product from fermentable sugars is preferably grown under precise conditions at a particular growth rate. When the fermenting organism is introduced into/added to the fermentation medium the inoculated fermenting organism pass through a number of stages. Initially growth does not occur. This period is referred to as the “lag phase” and may be considered a period of adaptation. During the next phase referred to as the “exponential phase” the growth rate gradually increases. After a period of maximum growth the rate ceases and the fermenting organism enters “stationary phase”. After a further period of time the fermenting organism enters the “death phase” where the number of viable cells declines.

Recovery

Subsequent to fermentation, the fermentation product may be separated from the fermentation medium. Thus in one embodiment the fermentation product is recovered after fermentation. The fermentation medium may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively, the fermentation product may be recovered by stripping. Methods for recovery are well known in the art.

Enzyme Compositions

The present invention also relates to a composition comprising a mixture of endo-protease and exo-protease, and wherein the exo-protease makes up at least 5% (w/w) of the protease in the mixture on a total protease enzyme protein basis, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, particularly at least 75%, more particularly the exo-protease makes up from between 5 to 95% (w/w) of the protease in the mixture on a total protease enzyme protein basis, particularly 10 to 80% (w/w), particularly 15 to 70% (w/w), more particularly 20 to 60% (w/w), and even more particularly 25 to 50% (w/w) of the protease mixture in the composition on a total protease enzyme protein basis.

In one embodiemnt the endo-protease is derived from proteases belonging to family S53, S8, M35, or A1 and the exo-protease is derived from proteases belonging to family S10, S53, M14, or M28.

In a particular embodiment the endo-protease is S53 from Meripilus giganteus and the exo-protease is S10 from Aspergillus oryzae, Aspergillus niger or Penicillium simplicissimum.

The endo-protease is preferable selected from a serine protease of family S53, such as e.g., S53 protease from Meripilus, particularly Meripilus giganteus, or a serine protease of family S8, such as e.g., S8 proteases from Pyrococcus, Thermococcus, particularly Pyrococcus furiosus, and Thermococcus litoralis, or a metallo-proteaase selected from the M35 family, more particularly M35 protease derived from Thermoascus aurantiacus.

In a particular embodiment the M35 metallo-protease is derived from Thermoascus aurantiacus, such as e.g., the mature polypeptide which comprises amino acids 1-177 of SEQ ID NO: 16 or a polypeptide having at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% identity to the polypeptide of SEQ ID NO: 16.

In anoter particular embodiment endo-protease may be a A1 protease.

In another specific embodiment the S53 endo-protease is selected from the group consisting of:

The exo-protease is preferably selected from a protease belonging to family S10, S53, M14, M28, particularly S10, or S53, more particularly S10 from Aspergillus or Penicillium, e.g., Aspergillus oryzae, Aspergillu niger, or Penicillium simplicissimum, or S53 exo-protease from Aspergillus, Trichoderma, Thermoascus, or Thermomyces, particularly Aspergillus oryzae, Trichoderma reesei, Thermoascus thermophilus, or Thermomyces lanuginosus.

In one specific embodiment the S10 exo-protease is selected from the group consisting of:

In another specific embodiment the S10 exo-protease is selected from the group consisting of a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6, or the polypeptide of SEQ ID NO: 7.

In another specific embodiment the S53 exo-protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or the polypeptide of SEQ ID NO: 20.

In one particular embodiment the endo-protease is a S53 protease from Meripilus giganteus, such as the one disclosed in SEQ ID NO: 2, and the exo-protease is a S10 protease from Aspergillus or Penicillium, particularly Aspergillus oryzae or Penicillium simplicissimum, such as the the S10 proteases disclosed in SEQ ID NO: 5 and SEQ ID NO: 7.

In another particular embodiment the endo-protease is a S53 protease from Meripilus giganteus, such as the one disclosed in SEQ ID NO: 2, and the exo-protease is a S53 protease from Aspergillus, Trichoderma, Thermoascus, or Thermomyces, particularly Aspergillus oryzae, Trichoderma reesei, Thermoascus thermophilus, or Thermomyces lanuginosus, selected from the group consisting of SEQ ID NO: 20, 22, 24, and 26.

The compositions may comprise the proteases as the major enzymatic components. Alternatively, the compositions may comprise multiple enzymatic activities, such as the endprotease/exo-protease and one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, alpha-amylase, beta-amylase, pullulanase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, protease, ribonuclease, transglutaminase, or xylanase. In one embodiment the composition further comprises a carbohydrate-source generating enzyme and optionally an alpha-amylase. In one particular embodiment the carbohydrate-source generating enzyme is selected from the group consisting of glucoamylase, alpha-glucosidase, maltogenic amylase, pullulanase and beta-amylase.

In particular, the carbohydrase-source generating enzyme is a glucoamylase and is present in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

In an embodiment the glucoamylase comprised in the composition is of fungal origin, preferably derived from a strain of Aspergillus, preferably Aspergillus niger, Aspergillus oryzae, or Aspergillus awamori, a strain of Trichoderma, especially T. reesei, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Gloeophyllum, e.g., a strain of Gloeophyllum sepiarum or Gloeophyllum trabeum; a strain of the genus Pycnoporus, e.g., a strain of Pycnoporus sanguineus; or a strain of the Nigrofomes, or a mixture thereof.

In an embodiment the glucoamylase is derived from Trametes, such as a strain of Trametes cingulata, such as the one shown in SEQ ID NO: 10.

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 10;

In an embodiment the glucoamylase is derived from Talaromyces, such as a strain of Talaromyces emersonii, such as the one shown in SEQ ID NO: 11,

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 11;

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 12;

In an embodiment the glucoamylase is derived from Gloeophyllum serpiarium, such as the one shown in SEQ ID NO: 13.

In an embodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 13;

In another embodiment the glucoamylase is derived from Gloeophyllum trabeum such as the one shown in SEQ ID NO: 14.

In an embodiment the glucoamylase is selected from the group consisting of.

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 14;

In an embodiment the glucoamylase is derived from a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO 2012/064351.

In addition to a glucoamylase the composition may further comprise an alpha-amylase. Particularly the alpha-amylase is an acid fungal alpha-amylase. A fungal acid stable alphaamylase is an alpha-amylase that has activity in the pH range of 3.0 to 7.0 and preferably in the pH range from 3.5 to 6.5, including activity at a pH of about 4.0, 4.5, 5.0, 5.5, and 6.0.

Preferably the acid fungal alpha-amylase is derived from the genus Aspergillus, especially a strain of A. terreus, A. niger, A. oryzae, A. awamori, or Aspergillus kawachii, or from the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from a strain of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, such as one shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucor pusillus alpha-amylase hybrid having an Aspergillus niger linker and starch-binding domain, such as the one shown in SEQ ID NO: 9 herein, or a variant thereof.

In an embodiment the alpha-amylase is selected from the group consisting of:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9;

In a preferred embodiment the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID NO: 9 having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N: Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 9 for numbering).

In an embodiment the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably disclosed as SEQ ID NO: 9, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 9 for numbering), and wherein the alpha-amylase variant has at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO: 9.

The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the composition may be in the form of granulate or microgranulate. The variant may be stabilized in accordance with methods known in the art.

The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.

The enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, as a source of the enzymes.

The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.

Uses of the Composition According to the Invention

The compositions according to the invention are contemplated for use in saccharification of starch. In one aspect the present invention thus relates to a use of the composition according to the present invention in saccharification of a starch containing material.

In one embodiment the use further comprises fermenting the saccharified starch containing material to produce a fermentation product. The starch material may be gelatinized or ungelatinized starch. Particularly the fermentation product is alcohol, more particularly ethanol.

In a particular embodiment saccharification and fermentation is performed simultaneously.

Polypeptides Having Serine Protease Activity

The present invention relates to polypeptides having serine exo-protease (peptidase) activity and which polypeptides further belong to the S10 carboxypeptidase family. In an embodiment, the present invention relates to a polypeptide having serine protease activity and belonging to family S10, selected from the group consisting of:

(a) a polypeptide having having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 8;

In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 70% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 75% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 80% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 85% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 95% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 100% of the serine protease activity of the mature polypeptide of SEQ ID NO: 6.

In an embodiment, the polypeptide has been isolated. A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof; or is a fragment thereof having serine protease activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 6. In another aspect, the polypeptide comprises or consists of amino acids 51 to 473 of SEQ ID NO: 6 disclosed herein as SEQ ID NO: 7.

In another embodiment, the present invention relates to an polypeptide having serine protease activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 8 or the cDNA sequence thereof of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated. In another embodiment the invention relates to polypeptides having serine exo-protease (peptidase) activity and which polypeptides further belong to the S53 family.

In particular the invention relates to polypeptide having serine protease activity, and belonging to family S53, selected from the group consisting of:

(a) a polypeptide having having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 23; or

In one embodiment the mature polypeptide is amino acids 208 to 614 of SEQ ID NO: 23, particularly amino acids 209 to 614 of SEQ ID NO: 23, more particularly amino acids 210 to 614 of SEQ ID NO: 23, more particularly amino acids 211 to 614 of SEQ ID NO: 23, more particularly amino acids 212 to 614 of SEQ ID NO: 23.

In particular the invention relates to polypeptide having serine protease activity, and belonging to family S53, selected from the group consisting of:

In one embodiment the mature polypeptide is amino acids 199 to 594 of SEQ ID NO: 25, particularly amino acids 200 to 594 of SEQ ID NO: 25, more particularly amino acids 201 to 594 of SEQ ID NO: 25, more particularly amino acids 202 to 594 of SEQ ID NO: 25, more particularly amino acids 203 to 594 of SEQ ID NO: 25.

In a particular embodiment the present invention relates to polypeptides having serine exo-protease (peptidase) activity and which polypeptides further belong to the S53 family, wherein the polypeptide comprises or consists of a polypeptide of SEQ ID NO: 23; or amino acids 208 to 614 of SEQ ID NO: 23, particularly amino acids 209 to 614 of SEQ ID NO: 23, more particularly amino acids 210 to 614 of SEQ ID NO: 23, more particularly amino acids 211 to 614 of SEQ ID NO: 23, more particularly amino acids 212 to 614 of SEQ ID NO: 23.

In a particularl embodiment the present invention relates to polypeptides having serine exo-protease (peptidase) activity and which polypeptides further belong to the S53 family, wherein the polypeptide comprises or consists of a polypeptide of SEQ ID NO: 25; or amino acids 199 to 594 of SEQ ID NO: 25, particularly amino acids 200 to 594 of SEQ ID NO: 25, more particularly amino acids 201 to 594 of SEQ ID NO: 25, more particularly amino acids 202 to 594 of SEQ ID NO: 25, more particularly amino acids 203 to 594 of SEQ ID NO: 25.

In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 6 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for [enzyme] activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Sources of Polypeptides Having Serine Protease Activity

A polypeptide having serine protease activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

In another aspect, the polypeptide is from Penicillium, Thermoascus, or Thermomyces, e.g., a polypeptide obtained from Penicillium simplicissimum, Therrnmoascus thermophilus, or Thermomyces lanuginosus.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to polynucleotides encoding a serine exo-protease polypeptide of family S10 or family S53. In an embodiment, the polynucleotide encoding the polypeptide has been isolated.

In one embodiment the polynucleotides encoding the exo-proteases of SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25 are disclosed herein as SEQ ID NO: 8, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 respectively.

The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NAS-BA) may be used. The polynucleotides may be cloned from a strain of [Genus], or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including variant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and variant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3′-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rmB).

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5′-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alphaamylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (omithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The host cell may be a eukaryote, such as a fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that are specific for the polypeptides.

The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the present invention expressing the polypeptide is used as a source of the polypeptide.

The invention is further disclosed in the below list of preferred embodiments.

Embodiment 1

A process for producing a fermentation product from starch-containing material comprising:

a) saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material using a carbohydrate-source generating enzymes; and

b) fermenting using a fermenting organism; wherein

Embodiment 2

A process for producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying starch-containing material at a temperature above the initial gelatinization temperature of said starch-containing material in the presence of an alpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

Embodiment 3

The process according to embodiments 1 or 2, wherein saccharification and fermentation is performed simultaneously.

Embodiment 4

The process according to any of the preceding embodiments, wherein the exo-protease makes up at least 10% (w/w) of the protease mixture on a total protease enzyme protein basis, such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, particularly at least 75%, more particularly the exo-protease makes up from between 5 to 95% (w/w) on a total protease enzyme protein basis, particularly 10 to 80% (w/w), particularly 15 to 70% (w/w), more particularly 20 to 60% (w/w), and even more particularly 25 to 50% (w/w) of the protease mixture in the composition on a total protease enzyme protein basis.

Embodiment 5

The process according to any of the preceding embodiments, wherein the endo-protease and exo-protease is present in a ratio of 5:2 micro grams enzyme protein (EP)/g dry solids (DS), particularly 5:3, more particularly 5:4.

Embodiment 6

The process according to any of embodiments 1-5, wherein the endoprotease is derived from proteases belonging to family S53, S8, M35, A1.

Embodiment 7

The process according to any of embodiments 1-5, wherein the exo-protease is derived from proteases belonging to family S10, S53, M14, M28.

Embodiment 8

The process of embodiment 6 wherein the S53 protease is derived from a strain of the genus Meripilus, more particularly Meripilus giganteus.

Embodiment 9

The process of any of embodiments 1-8, wherein the S53 protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1, or the polypeptide of SEQ ID NO: 2.

Embodiment 10

The process of embodiment 6, wherein the S8 protease is derived from a strain of the genus Pyrococcus, Thermococcus, particularly Pyrococcus furiosus, and Thermococcus litoralis.

Embodiment 11

The process of embodiment 10, wherein the S8 protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 3.

Embodiment 12

The process according to embodiments 7, wherein the S10 exo-protease is derived from a strain of Aspergillus or Penicillium, particularly Aspergillus oryzae, Aspergillus niger or Penicillium simplicissimum.

Embodiment 13

The process of embodiment 12, wherein the S10 exo-protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4, or the polypeptide of SEQ ID NO: 5.

Embodiment 14

Embodiment 15

Embodiment 16

The process according to embodiment 7, wherein the S53 exo-protease is derived from a strain of Aspergillus, Trichoderma, Thermoascus, or Thermomyces, particularly Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, Thermoascus thermophilus, or Thermomyces lanuginosus.

Embodiment 17

The process according to embodiment 16, wherein the S53 protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or the polypeptide of SEQ ID NO: 20.

Embodiment 18

Embodiment 19

Embodiment 20

Embodiment 21

The process according to embodiments 16, wherein the S53 protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 32.

Embodiment 22

The process of any of the preceding embodiments, wherein an alphaamylase is present or added during saccharification and/or fermentation.

Embodiment 23

The process according to embodiment 22, wherein the alpha-amylase is an acid alpha-amylase, preferably an acid fungal alpha-amylase.

Embodiment 24

The process according to embodiment 23, wherein the alpha-amylase is derived from the genus Aspergillus, especially a strain of A. terreus, A. niger, A. oryzae, A. awamori, or Aspergillus kawachii, or of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.

Embodiment 25

The process according to embodiment 24, wherein the alpha-amylase present in saccharification and/or fermentation is derived from a strain of the genus Rhizomucor, preferably a strain of Rhizomucor pusillus, such as a Rhizomucor pusillus alpha-amylase hybrid having a linker and starch-binding domain from an Aspergillus niger glucoamylase.

Embodiment 26

The process of embodiment 25, wherein the alpha-amylase present in saccharification and/or fermentation is selected from the group consisting of:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9;

Embodiment 27

The process of embodiment 26, wherein the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably disclosed as SEQ ID NO: 9, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

Embodiment 28

The process of any of embodiments 22-27, wherein the alpha-amylase is present in an amount of 0.001 to 10 AFAU/g DS, preferably 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

Embodiment 29

The process of any of embodiments 1-28, wherein the carbohydrate-source generating enzyme is selected from the group consisting of glucoamylase, alpha-glucosidase, maltogenic amylase, pullulanase, and beta-amylase.

Embodiment 30

The process of any of embodiments 1-29, wherein the carbohydrase-source generating enzyme is a glucoamylase and is present in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

Embodiment 31

The process of any of embodiments 28-30, wherein the alpha-amylase and glucoamylase is added in a ratio of between 0.1 and 100 AGU/FAU-F, preferably 2 and 50 AGU/FAU-F, especially between 10 and 40 AGU/FAU-F when saccharification and fermentation are carried out simultaneously.

Embodiment 32

The process of any of embodiments 29-31, wherein the glucoamylase is derived from a strain of Aspergillus, preferably Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Gloeophyllum, e.g., a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum; a strain of the genus Pycnoporus, e.g., a strain of Pycnoporus sanguineus; or a mixture thereof.

Embodiment 33

The process of embodiment 32, wherein the glucoamylase is derived from Trametes, such as a strain of Trametes cingulata, such as the one shown in SEQ ID NO: 10.

Embodiment 34

The process of embodiment 33, wherein the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 10;

Embodiment 35

The process of embodiment 32, wherein the glucoamylase is derived from Talaromyces, such as a strain of Talaromyces emersonii, such as the one shown in SEQ ID NO: 11.

Embodiment 36

The process of embodiment 35, wherein the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 11;

Embodiment 37

The process of embodiment 32, wherein the glucoamylase is derived from a strain of the genus Pycnoporus, such as a strain of Pycnoporus sanguineus such as the one shown in SEQ ID NO: 12.

Embodiment 38

The process of embodiment 37, wherein the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 12;

Embodiment 39

The process of embodiment 32, wherein the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum sepiarium shown in SEQ ID NO: 13.

Embodiment 40

The process of embodiment 39, wherein the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 13;

Embodiment 41

The process of embodiment 32, wherein the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum trabeum such as the one shown in SEQ ID NO: 14.

Embodiment 42

The process of embodiment 41, wherein the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 14;

Embodiment 43

The process of any of embodiments 1-42, wherein the fermentation product is recovered after fermentation.

Embodiment 44

The process of any of embodiments 1-43, wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol and/or industrial ethanol.

Embodiment 45

The process of any of embodiments 1-44, wherein the fermenting organism is yeast, preferably a strain of Saccharomyces, especially a strain of Saccharomyces cerevisiae.

Embodiment 46

The process of embodiment 1, wherein the starch-containing material is granular starch.

Embodiment 47

The process of embodiment 46, wherein the starch-containing material is derived from whole grain.

Embodiment 48

The process of any of embodiments 1-47, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice or potatoes.

Embodiment 49

The process of any of embodiments 1-48, wherein fermentation is carried out at a pH in the range between 3 and 7, preferably from 3.5 to 6, or more preferably from 4 to 5.

Embodiment 50

The process of any of embodiments 1-49, wherein the process is carried out for between 1 to 96 hours, preferably is from 6 to 72 hours.

Embodiment 51

The process of any of embodiments 1-50, wherein the dry solid content of the starch-containing material is in the range from 10-55 w/w-%, preferably 25-45 w/w-%, more preferably 30-40 w/w-%.

Embodiment 52

The process of any of embodiments 1-51, wherein the starch-containing material is prepared by reducing the particle size of starch-containing material to a particle size of 0.1-0.5 mm.

Embodiment 53

The process of embodiment 3, wherein the temperature during simultaneous saccharification and fermentation is between 25° C. and 40° C., such as between 28° C. and 35° C., such as between 30° C. and 34° C., such as around 32° C.

Embodiment 54

The process of embodiment 3, wherein the pH during simultaneous saccharification and fermentation is selected from the range 3-7, preferably 4.0-6.5, more particularly 4.5-5.5, such as pH 5.0.

Embodiment 55

The process of any of embodiments 2-54, wherein liquefaction is carried out at pH 4.0-6.5, preferably at a pH from 4.5 to 5.5, such as pH 5.0.

Embodiment 56

The process of any of embodiments 2-55, wherein the temperature in liquefaction is in the range from 70-95° C., preferably 80-90° C., such as around 85° C.

Embodiment 57

The process of embodiments 1 or 2, further comprising, prior to the step (a), the steps of:

x) reducing the particle size of starch-containing material;

y) forming a slurry comprising the starch-containing material and water.

Embodiment 58

The process of any of embodiments 2-57, wherein a pullulanase is present i) during fermentation, and/or ii) before, during, and/or after liquefaction.

Embodiment 59

A composition comprising a mixture of endo-protease and exo-protease, and wherein the exo-protease makes up at least 5% (w/w) of the protease in the mixture on a total protease enzyme protein basis, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, particularly at least 75%, more particularly the exo-protease makes up from between 5 to 95% (w/w) of the protease in the mixture on a total protease enzyme protein basis, particularly 10 to 80% (w/w), particularly 15 to 70% (w/w), more particularly 20 to 60% (w/w), and even more particularly 25 to 50% (w/w) of the protease mixture in the composition on a total protease enzyme protein basis.

Embodiment 60

The composition of embodiment 59, wherein the endo-protease is derived from proteases belonging to family S53, S8, M35, or A1 and the exo-protease is derived from proteases belonging to family S10, 553, M14, or M28.

Embodiment 61

The composition according to embodiment 60, wherein the endo-protease is S53 from Meripilus giganteus and the exo-protease is S10 from Aspergillus oryzae, Aspergillus niger or Penicillium simplicissimum.

Embodiment 62

The composition of embodiments 61, wherein the S53 protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1, or the polypeptide of SEQ ID NO: 2.

Embodiment 63

The composition of embodiment 61, wherein the S10 protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4, or the polypeptide of SEQ ID NO: 5.

Embodiment 64

The composition of embodiment 61, wherein the S10 exo-protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6, or the polypeptide of SEQ ID NO: 7.

Embodiment 65

Embodiment 66

The composition according to embodiment 59, wherein wherein the S53 exo-protease is derived from a strain of Aspergillus, Trichoderma, Thermoascus, or Thermomyces, particularly Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, Thermoascus thermophilus, or Thermomyces lanuginosus.

Embodiment 67

The composition according to embodiments 66, wherein the S53 exo-protease is a polypeptide having serine protease activity, selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or the polypeptide of SEQ ID NO: 20.

Embodiment 68

Embodiment 69

Embodiment 70

Embodiment 71

Embodiment 72

The composition of any of the embodiments 59-71, further comprising a carbohydrate-source generating enzyme selected from the group of glucoamylase, alpha-glucosidase, maltogenic amylase, and beta-amylase.

Embodiment 73

The composition of embodiment 72, wherein the carbohydrate-source generating enzyme is selected from the group of glucoamylases derived from a strain of Aspergillus, preferably Aspergillus niger or Aspergillus awamori, a strain of Trichoderma, especially T. reesei, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Gloeophyllum, e.g., a strain of Gloeophyllum sepiarum or Gloeophyllum trabeum; a strain of the genus Pycnoporus, e.g., a strain of Pycnoporus sanguineus; or a mixture thereof.

Embodiment 74

The composition of any of embodiments 59-73, further comprising an alpha-amylase selected from the group of fungal alpha-amylases, preferably derived from the genus Aspergillus, especially a strain of Aspergillus terreus, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, or Aspergillus kawachii, or of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.

Embodiment 75

A use of the composition according to any of embodiments 59-74 in saccharification of a starch containing material.

Embodiment 76

The use according to embodiment 75, further comprising fermenting the saccharified starch containing material to produce a fermentation product.

Embodiment 77

The use according to any of the embodiments 75-76, wherein the starch material is gelatinized or ungelatinized starch.

Embodiment 78

The use according to any of the embodiments 75-77, wherein the fermentation product is alcohol, particularly ethanol.

Embodiment 79

The use according to any of embodiments 75-78, wherein saccharification and fermentation is performed simultaneously.

Embodiment 80

A polypeptide having serine protease activity, and belonging to family S10, selected from the group consisting of:

(a) a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6.

Embodiment 81

The polypeptide of embodiment 80, comprising or consisting of SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 6.

Embodiment 82

The polypeptide of embodiments 80-81, wherein the mature polypeptide is amino acids 51 to 473 of SEQ ID NO: 6.

Embodiment 83

The polypeptide of any of embodiments 80-82, which is a variant of the mature polypeptide of SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion at one or several positions.

Embodiment 84

A polypeptide having serine protease activity, and belonging to family S53, selected from the group consisting of:

(a) a polypeptide having having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 23; or

Embodiment 85

A polypeptide having serine protease activity, and belonging to family S53, selected from the group consisting of:

Embodiment 86

The polypeptide of embodiment 84, wherein the mature polypeptide is SEQ ID NO: 24.

Embodiment 87

The polypeptide of embodiment 85, wherein the mature polypeptide is SEQ ID NO: 26.

Embodiment 88

A polynucleotide encoding a polypeptide of any of embodiments 80-87.

Embodiment 89

A nucleic acid construct or expression vector comprising the polynucleotide of embodiment 88 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

Embodiment 90

A recombinant host cell comprising the heterologous polynucleotide of embodiment 88 operably linked to one or more control sequences that direct the production of the polypeptide.

Embodiment 91

A method of producing a polypeptide of any of embodiments 80-87, comprising cultivating the host cell of embodiment 90 under conditions conducive for production of the polypeptide.

Embodiment 92

The method of embodiment 91, further comprising recovering the polypeptide.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

Enzyme Assays

Protease Assays

AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended in Borax/NaH₂PO₄buffer pH9 while stirring. The solution is distributed while stirring to microtiter plate (100 microL to each well), 30 microL enzyme sample is added and the plates are incubated in an Eppendorf Thermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzyme sample (100° C. boiling for 20 min) is used as a blank. After incubation the reaction is stopped by transferring the microtiter plate onto ice and the coloured solution is separated from the solid by centrifugation at 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant is transferred to a microtiter plate and the absorbance at 595 nm is measured using a BioRad Microplate Reader.

Kinetic Suc-AAPF-pNA Assay:

pNA substrate: Suc-AAPF-pNA (Bachem L-1400).
Temperature: Room temperature (25° C.)
Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to pH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 with HCl or NaOH.

20 μl protease sample (diluted in 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% Triton X-100). The increase in OD₄₀₅was monitored as a measure of the protease activity.

Endpoint Suc-AAPF-pNA Assay:

pNA substrate: Suc-AAPF-pNA (Bachem L-1400).
Temperature: controlled (assay temperature).
Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 4.0

200 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with the Assay buffer) were pipetted in an Eppendorf tube and placed on ice. 20 μl protease sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate (1400 rpm.). The incubation was stopped by transferring the tube back to the ice bath and adding 600 μl 500 mM H₃BO₃/NaOH, pH 9.7. The tube was mixed and 200 μl mixture was transferred to a microtiter plate, which was read at OD₄₀₅. A buffer blind was included in the assay (instead of enzyme). OD₄₀₅(Sample)−OD₄₀₅(Blind) was a measure of protease activity.

Protazyme AK Assay:

Substrate: Protazyme AK tablet (cross-linked and dyed casein; from Megazyme)
Temperature: controlled (assay temperature).
Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 6.5.

A Protazyme AK tablet was suspended in 2.0 ml 0.01% Triton X-100 by gentle stirring. 500 μl of this suspension and 500 μl assay buffer were dispensed in an Eppendorf tube and placed on ice. 20 μl protease sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate (1400 rpm.). The incubation was stopped by transferring the tube back to the ice bath. Then the tube was centrifuged in an ice cold centrifuge for a few minutes and 200 μl supernatant was transferred to a microtiter plate, which was read at OD₆₅₀. A buffer blind was included in the assay (instead of enzyme). OD₆₅₀(Sample)−OD₆₅₀(Blind) was a measure of protease activity.

Kinetic Suc-AAPX-pNA Assay:

pNA substrates: Suc-AAPA-pNA (Bachem L-1775)
- Suc-AAPR-pNA (Bachem L-1720)
- Suc-AAPD-pNA (Bachem L-1835)
- Suc-AAPI-pNA (Bachem L-1790)
- Suc-AAPM-pNA (Bachem L-1395)
- Suc-AAPV-pNA (Bachem L-1770)
- Suc-AAPL-pNA (Bachem L-1390)
- Suc-AAPE-pNA (Bachem L-1710)
- Suc-AAPK-pNA (Bachem L-1725)
- Suc-AAPF-pNA (Bachem L-1400)
Temperature: Room temperature (25° C.)
Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 4.0 or pH 9.0.

20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% Triton X-100). The increase in OD₄₀₅was monitored as a measure of the protease activity.

o-Phthaldialdehyde (OPA) Assay:

This assay detects primary amines and hence cleavage of peptide bonds by a protease can be measured as the difference in absorbance between a protease treated sample and a control sample. The assay is conducted essentially according to Nielsen et al. (Nielsen, P M, Petersen, D, Dampmann, C. Improved method for determining food protein degree of hydrolysis. J Food Sci, 2001, 66: 642-646).

500 μl of sample is filtered through a 100 kDa Microcon centrifugal filter (60 min, 11,000 rpm, 5° C.). The samples are diluted appropriately (e.g. 10, 50 or 100 times) in deionizer water and 25 μl of each sample is loaded into a 96 well microtiter plate (5 replicates). 200 μl OPA reagent (100 mM di-sodium tetraborate decahydrate, 3.5 mM sodium dodecyl sulphate (SDS), 5.7 mM di-thiothreitol (DDT), 6 mM o-phthaldialdehyde) is dispensed into all wells, the plate is shaken (10 sec, 750 rpm) and absorbance measured at 340 nm.

Assays for Glucoamylase Activity

Glucoamylase Units, AGU

The Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyses 1 micromole maltose per minute under the standard conditions (37° C., pH 4.3, substrate: maltose 100 mM, buffer acetate 0.1 M, reaction time 6 minutes as set out in the glucoamylase incubation below), thereby generating glucose.

glucoamylase incubation:

Substrate:
maltose 100 mM

Buffer:
acetate 0.1M

pH:
4.30 ± 0.05

Incubation temperature:
37° C. ± 1

Reaction time:
6 minutes

Enzyme working range:
0.5-4.0 AGU/mL

The analysis principle is described by 3 reaction steps:

Step 1 is an Enzyme Reaction:

Glucoamylase (AMG), EC 3.2.1.3 (exo-alpha-1,4-glucan-glucohydrolase), hydrolyzes maltose to form alpha-D-glucose. After incubation, the reaction is stopped with NaOH.

Steps 2 and 3 Result in an Endpoint Reaction:

Glucose is phosphorylated by ATP, in a reaction catalyzed by hexokinase. The glucose-6-phosphate formed is oxidized to 6-phosphogluconate by glucose-6-phosphate dehydrogenase. In this same reaction, an equimolar amount of NAD+ is reduced to NADH with a resulting increase in absorbance at 340 nm. An autoanalyzer system such as Konelab 30 Analyzer (Thermo Fisher Scientific) may be used.

Color reaction

Tris
approx. 35
mM

ATP
0.7
mM

NAD⁺
0.7
mM

Mg²⁺
1.8
mM

Hexokinase
>850
U/L

Glucose-6-P-DH
>850
U/L

pH
approx. 7.8

Temperature
37.0° C. ± 1.0° C.

Reaction time
420
sec

Wavelength
340
nm

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 AFAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.

Acid alpha-amylase, an endo-alpha-amylase (1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzes alpha-1,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.

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Standard Conditions/Reaction Conditions

- Substrate: Soluble starch, approx. 0.17 g/L
- Buffer Citrate, approx. 0.03 M
- Iodine (12): 0.03 g/L
- CaCl₂: 1.85 mM
- pH: 2.50±0.05
- Incubation 40° C.
- temperature:
- Reaction time: 23 seconds
- Wavelength: 590 nm
- Enzyme 0.025 AFAU/mL
- concentration:
- Enzyme working 0.01-0.04 AFAU/mL
- range:

A folder EB-SM-0259.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Determination of FAU-F

FAU-F fungal Alpha-Amylase Units (Eungamyl) is measured relative to an enzyme standard of a declared strength.

Reaction conditions

Temperature
37°
C.

pH
7.15

Wavelength
405
nm

Reaction time
5
min

Measuring time
2
min

A folder (EB-SM-0216.02) describing this standard method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003 M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Alpha-Amylase Activity (KNU-A)

Alpha amylase activity is measured in KNU(A) Kilo Novozymes Units (A), relative to an enzyme standard of a declared strength.

Alpha amylase in samples and α-glucosidase in the reagent kit hydrolyze the substrate (4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside (ethylidene-G₇PNP) to glucose and the yellow-colored p-nitrophenol.

The rate of formation of p-nitrophenol can be observed by Konelab 30. This is an expression of the reaction rate and thereby the enzyme activity.

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The enzyme is an alpha-amylase with the enzyme classification number EC 3.2.1.1.

Parameter
Reaction conditions

Temperature
37°
C.

pH
7.00 (at 37° C.)

Substrate conc.
Ethylidene-G₇PNP, R2: 1.86 mM

Enzyme conc. (conc. of high/low
1.35-4.07 KNU(A)/L

standard in reaction mixture)

Reaction time
2
min

Interval kinetic measuring time
7/18
sec.

Wave length
405
nm

Conc. of reagents/chemicals
α-glucosidase, R1: ≥3.39 kU/L

critical for the analysis

A folder EB-SM-5091.02-D on determining KNU-A actitvity is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Enzymes

Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylase with the mutations: I181′+G182*+N93F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to 491 amino acids (using SEQ ID NO: 15 for numbering).

Alpha-Amylase X: Bacillus stearothermophilus alpha-amylase with the mutations: I181*+G182*+N193F truncated to 491 amino acids (using SEQ ID NO: 15 for numbering).

Glucoamylase Po: Mature part of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 17 herein.

Protease Pfu: Protease derived from Pyrococcus furiosus shown in SEQ ID NO: 3 herein.

Glucoamylase Po 498 (GA498): Variant of Penicillium oxalicum glucoamylase having the following mutations: K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 17 for numbering).

Alpha-amylase blend A: Blend comprising Alpha-amylase AA369, glucoamylase GA498, and protease PfuS (dosing: 2.1 μg EP/g DS AA369, 4.5 μg EP/g DS GA498, 0.0385 μg EP/g DS PfuS, where EP is enzyme protein and DS is total dry solids)

Glucoamylase blend A: Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ ID NO: 34 in WO99/28448 and SEQ ID NO: 11 herein, Trametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 and SEQ ID NO: 10, and Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch binding domain (SBD) disclosed in SEQ ID NO: 9 herein having the following substitutions G128D+D143N using SEQ ID NO: 9 for numbering (activity ratio in AGU:AGU:FAU-F is about 29:8:1).

Example 1. Effect of Exo-Peptidase from A. oryzae Combination with Endo-Protease from M. giganteus for Increasing Ethanol Titer in Simultaneous Saccharification and Fermentation Process

Liquefaction was carried out in a metal canister using Labomat BFA-24 (Mathis, Concord, N.C.). In the canister was added 222 g of industrial produced ground corn to 377 g tap water and mixed well. The target dry solid was about 32% DS. pH was adjusted to pH 5.0 and dry solid was measured using moisture balance (Mettler-Toledo). Alpha-amylase blend A was dosed 0.03% (w/w) into the corn slurry and liquefaction took place in the Labomat chamber at 85° C. for 2 hr. After liquefaction, canister was cooled in ice-bath to room temperature and the liquefied mash was transferred to a container following by supplemented with 3 ppm of penicillin and 350 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via miniscale fermentations. Approximately 5 g of liquefied corn mash above was added to 15 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of glucoamylase blend A and appropriate amount of endo-protease from Meriphilus giganteus (SEQ ID NO: 2) with or without exo-peptidase namely carboxypeptidase from Aspergillus oryzae (SEQ ID NO: 5) as shown in table below followed by addition of 25 micro liters hydrated yeast per 5 g slurry. As control, only glucoamylase blend A was added and without addition of endo-protease or exo-peptidase. Actual glucoamylase and protease dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32° C. Three replicates were selected for 24 hours, 48 hour and 56 hour time point analysis. At each time point, fermentation was stopped by addition of 50 micro liters of 40% H₂SO₄, follow by centrifuging, and filtering through a 0.45 micrometer filter. Ethanol and oligosaccharides concentration were determined using HPLC.

Endo-protease
Exo-peptidase

from M.giganteus
from A. oryzae

Treatments
(μg/g DS)
μg/gDS

1. Control
—
—

2. Endo-protease only
5
—

3. Endo-protease only
7
—

4. Endo-protease only
9
—

5. Endo-protease + Exo-peptidase
5
2

6. Endo-protease + Exo-peptidase
5
4

As shown in result table below, combination of endo-protease with exo-peptidase increased ethanol yield with statistically significant compared to control or endo-protease alone.

Ethanol yield at 56 hour with different treatments of endo-protease without or with exo-peptidase.

Treatments
Ethanol (g/l)

1. Control
119.4

2. Endo-protease (5)
127.7

3. Endo-protease (7)
126.7

4. Endo-protease (9)
127.8

5. Endo-protease (5) + Exo-protease (2)
128.8

6. Endo-protease (5) + Exo-protease (4)
129.1

Example 2. Effect of Exo-Peptidase from P. simplicissimum Combination with Endo-Protease from M. giganteus for Increasing Ethanol Titer in Simultaneous Saccharification and Fermentation Process

An industrial prepared liquefied mash using alpha-amylase blend A was used for the experiment. The dry solid determined by moisture balance (Mettler-Toledo) was about 33% DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 350 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 5 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of glucoamylase blend A and appropriate amount of endo-protease from Meriphilus giganteus (SEQ ID NO: 2) with or without exo-peptidase namely carboxypeptidase from Penicillium simplicissimum (SEQ ID NO: 7) as shown in table below followed by addition of 25 micro liters hydrated yeast per 5 g slurry. As control, glucoamylase blend A and 350 ppm urea was added but no addition of endo-protease or exo-peptidase. Actual glucoamylase and protease dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32° C. Three replicates were selected for 24 hours, 48 hour and 54 hour time point analysis. At each time point, fermentation was stopped by addition of 50 micro liters of 40% H₂SO₄, follow by centrifuging, and filtering through a 0.45 micrometer filter. Ethanol and oligosaccharides concentration were determined using HPLC.

Endo-protease
Exo-peptidase

from
from P.

M. giganteus

simplicissimum

Treatments
(μg/g DS)
μg/gDS

1. Control
—
—

2. Endo-protease only
5
—

3. Endo-protease + Exo-peptidase
5
2

As shown in result tables below, combination of endo-protease with exo-peptidase increased ethanol yield with statistically significant compared to control or endo-protease alone. In particular, treatment with exo-peptidase from P. simplicissimum markedly enhanced yeast fermentation rate as showed at 24 hr the ethanol titer was much higher.

Ethanol yield at 24 hour of endo-protease without or with exo-peptidase.

Treatments
Ethanol (g/l)

1. Control
84.9

2. Endo-protease only
88.0

3. Endo-protease + Exo-peptidase
91.1

Ethanol yield at 48 hour of endo-protease without or with exo-peptidase.

Treatments
Ethanol (g/l)

1. Control
131.2

2. Endo-protease only
132.0

3. Endo-protease + Exo-peptidase
132.6

Fermentation completed reaching 48 hour and no further increase in ethanol titer upon 54 hour.

Example 3. Effect of Exo-Peptidase Tripeptidylaminopeptidase Combination with Endo-Protease for Increasing Ethanol Titer in Simultaneous Saccharification and Fermentation Process

Liquefaction was carried out in Labomat BFA-24 (Mathis, Switzerland). In the canister was added 150.2 g homemade ground corn to 250 g tap water and mixed well. The target dry solid was about 32.5% DS. pH was adjusted to pH 5.5 and dry solid was measured using moisture balance (Mettler-Toledo). Alpha-amylase X was dosed 0.045% (w/w) of the corn and liquefaction took place in the Labomat chamber at 85° C. for 2.5 hr.

After liquefaction, canister was cooled in ice-bath to room temperature and the liquefied mash was transferred to a container following by supplemented with 3 ppm of penicillin and 350 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 5 g of liquefied corn slurry above was added to 15 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of Glucoamylase blend A, and appropriate amount of endo-protease from Meriphilus giganteus (SEQ ID NO: 2) with or without exo-protease of tripeptidylaminopeptidase exo protease 1, 2, 3 and 4 the mature form of which are disclosed herein as SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26 respectively. The combinations are as shown below followed by addition of 20 micro liters hydrated yeast per 5 g slurry. As control, only glucoamylase was added and without addition of endo- or exo-protease. Actual glucoamylase and protease dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32 C. Three replicates were carried out for 52 hour time point analysis. At each time point, fermentation was stopped by addition of 50 micro liters of 40% H2SO4, centrifuging, and filtering through a 0.45 micrometer filter. Ethanol and oligosaccharides concentration were determined using HPLC.

Endo-
Exo-

protease
protease

dose
dose

Treatments
(μg/g DS)
μg/gDS

1. Control
—
—

2. Endo-protease (5)
5
—

3. Endo-protease (4.5) + Exo-protease 1 (0.5)
4.5
0.5

4. Endo-protease (3.75) + Exo-protease 1 (1.25)
3.75
1.25

5. Endo-protease (4.5) + Exo-protease 2 (0.5)
4.5
0.5

6. Endo-protease (3.75) + Exo-protease 2 (1.25)
3.75
1.25

7. Endo-protease (4.5) + Exo-protease 3 (0.5)
4.5
0.5

8. Endo-protease (3.75) + Exo-protease 3 (1.25)
3.75
1.25

9. Endo-protease (4.5) + Exo-protease 4 (0.5)
4.5
0.5

10. Endo-protease (3.75) + Exo-protease 4(1.25)
3.75
1.25

Exo-protease 1, 2, 3 or 4 which are SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26.

SEQ ID NO: 20

Aspergillus oryzae

SEQ ID NO: 22

Trichoderma reesei

SEQ ID NO: 24

Thermoascus thermophilus

SEQ ID NO: 26

Thermomyces lanuginosus

As shown in result table below, combination of endo-protease with exo-protease increased ethanol yield with statistically significant compared to endo-protease alone.

Ethanol yield at 52 hour with different treatments of endo-protease without or with exo-protease.

Treatments
Ethanol (g/l)

1. Control(200 ppm urea)
78.6

2. Endo-protease (5)
112.9

3. Endo-protease (4.5) + Exo-protease 1 (0.5)
114.9

4. Endo-protease (3.75) + Exo-protease 1 (1.25)
113.8

5. Endo-protease (4.5) + Exo-protease 2 (0.5)
113.8

6. Endo-protease (3.75) + Exo-protease 2 (1.25)
113.7

7. Endo-protease (4.5) + Exo-protease 3 (0.5)
114.3

8. Endo-protease (3.75) + Exo-protease 3 (1.25)
114.0

9. Endo-protease (4.5) + Exo-protease 4 (0.5)
113.6

10. Endo-protease (3.75) + Exo-protease 4 (1.25)
113.3

Example 4. Cloning and Expression of a $10 Peptidase from Penicillium simplicissimum

Gene

A fungal strain was isolated and based on both morphological and molecular characterization (ITS sequencing) classified as Penicillium simplicissimum. The Penicillium simplicissimum strain was annotated as Penicillium simplicissimum strain NN044175 and fully genome sequenced. The genomic DNA sequence of a S10 peptidase polypeptide encoding sequence was identified in the genome of Penicillium simplicissimum strain NN044175 and the genomic DNA sequence and deduced amino acid sequence are shown in SEQ ID NO: 18 and SEQ ID NO: 6, respectively. The genomic DNA sequence of 1618 nucleotides contains 4 introns of 53 bp (nucleotides 246 to 298), 44 bp (nucleotides 630 to 673), 51 bp (nucleotides 1188 to 1238), and 48 bp (nucleotides 1506 to 1553), respectively. The genomic DNA fragment encodes a polypeptide of 473 amino acids. The complementary DNA sequence is shown in SEQ ID NO: 8

Expression Vector

The Aspergillus expression vector pDau109 (WO 2005/042735) consists of an expression cassette based on the partly duplicated Aspergillus niger neutral amylase II (NA2) promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpl) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the vector is the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source and the amplicillin resistance gene (beta lactamase) allowing for facile selection for positive recombinant E. coli clones using commercially available and highly competent strains on commonly used LB ampicillin plates. pDau109 contains a multiple cloning site situated between the promoter region and terminator, allowing for insertion of the gene of interest in front of the promoter region.

Expression Cloning

The gene encoding the Penicillium simplicissimum S10 peptidase (SEQ ID NO: 18) was PCR amplified from genomic DNA isolated from Penicillium simplicissimum strain NN044175. The PCR product encoding the Penicillium simplicissimum S10 peptidase (SEQ ID NO: 18) was cloned into the pDau109 Aspergillus expression vector using the unique restriction sites BamHI and HindIII and transformed into E. coli (Top10, Invitrogen). Expression plasmids containing the insert were purified from the E. coli transformants, and sequenced with vector primers and gene specific primers in order to determine a representative plasmid expression clone that was free of PCR errors. The plasmid expression clone was transformed into A. oryzae and a recombinant A. oryzae clone containing the integrated expression construct were grown in liquid culture. Expression of the Penicillium simplicissimum S10 peptidase was verified by SDS-page. The enzyme containing supernatant was sterile filtered before purification.

Example 5. Characterization of the Penicillium simplicissimum S10 Peptidase (SEQ ID NO: 6)

Enzyme: Penicillium simplicissimum S10 mature peptidase disclosed in SEQ ID NO: 7.

Assays:

A Z-Ala-Lys-OH based end-point assay was used for obtaining the pH-profiles for the enzyme and the Temp-activity profile at pH 5. For the pH-stability profile the enzyme was diluted 10× in the assay buffers and incubated for 2 hours at 37° C. After incubation the enzyme samples were transferred to pH 5, before assay for residual activity.

End-Point Z-Ala-Xxx-OH Assay:

Z-Ala-Xxx-OH Substrates:

Z-Ala-Ala-OH (Bachem C-1045).

Z-Ala-Leu-OH (Bachem C-3155).

Z-Ala-Glu-OH (Bachem C-1075).

Z-Ala-Lys-OH (Bachem C-1140).

Z-Ala-Phe-OH (Bachem C-1155).

Z-Ala-His-OH (Bachem C-1120).

Z-Ala-Met-OH (Bachem C-1145)

Temperature: 37° C. except for Temp-activity profile.

Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to pH-values: 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 with HCl or NaOH.

100 μl Z-Ala-Xxx-OH substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 25× in 0.01% Triton X-100) was mixed with 150 μl Assay buffer in an Eppendorf tube and placed on ice. 50 μl peptidase sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate. The tube was then transferred back to the ice bath and when the tube had cooled, 500 μl Stop reagent (17.9 g TCA+29.9 g Na-acetate trihydrate+19.0 ml conc. CH₃COOH and deionised water ad 500 ml) was added and the tube was vortexed and left for 15 min at room temperature (to ensure complete precipitation). The tube was centrifuged (15000×g, 3 min, room temp), 30 μl supernatant was transferred to a microtiter plate and 225 μl freshly prepared OPA-reagent (3.81 g disodium tetraborate and 1.00 g SDS were dissolved in approx. 80 ml deionised water—just before use 80 mg ortho-phtaldialdehyde dissolved in 2 ml ethanol was added and then 1.0 ml 10% (w/v) DTE and finally the volume was adjusted ad 100 ml with deionised water) was added. After 2 minutes, A₃₄₀was read in a MTP reader. The A₃₄₀measurement relative to proper blinds (substrate blind and enzyme blind) was a measure of carboxypeptidase activity.

The protease disclosed as SEQ ID NO: 7 (Penicillium simplicissimum) was shown to have optimum activity at about pH 5, a pH stability profile with an optimum at pH 3-6, and a temperature optimum at around 55° C., pH 5.

The N-terminal was determined to start at position 46 in SEQ ID NO: 6 and thus the mature protease corresponds to SEQ ID NO: 7.

Example 6. Effect of Exo-Peptidase from A. niger in Combination with Endo-Protease from M. giganteus for Increasing Ethanol Titer in Simultaneous Saccharification and Fermentation Process

A liquefied mash using alpha-amylase X (pH=5.5, T=85° C.), was used for the experiment. The dry solid determined by moisture balance (Mettler-Toledo) was about 30% DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations at T=32° C. Approximately 5 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of glucoamylase blend A and appropriate amount of endo-protease from Meriphilus giganteus (SEQ ID NO: 2) with or without exo-peptidase namely carboxypeptidase from Aspergillus niger (SEQ ID NO: 31) as shown in table below followed by addition of 100 micro liters hydrated yeast per 5 g slurry. As control, glucoamylase blend A with no addition of endo-protease or exo-peptidase. Actual glucoamylase and protease dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32° C. Three replicates of each treatment were used during SSF. After 50 hours, fermentation was stopped by addition of 50 micro liters of 40% H₂SO₄, follow by centrifuging, and filtering through a 0.45 micrometer filter. Ethanol and oligosaccharides concentration were determined using HPLC.

Endo-protease
Exo-peptidase

from M. giganteus
from A. niger

Treatments
(μg/g DS)
μg/gDS

1. Control
—
—

2. Endo-protease only
2.5
—

3. Endo-protease + Exo-peptidase
2.5
2.5

As shown in result tables below, combination of endo-protease with exo-peptidase increased ethanol yield with statistically significant compared to control or endo-protease alone.

Ethanol yield at 50 hours of endo-protease without or with exo-peptidase.

Treatments
Ethanol (g/l)

1. Control
121.9

2. Endo-protease only
123.6

3. Endo-protease + Exo-peptidase
124.5

Example 7. Effect of Exo-Peptidase or Tripeptidylaminopeptidase (TPAP) from A. niger Combined with Endo-Protease from M. giganteus for Increasing Ethanol Titer in Simultaneous Saccharification and Fermentation Process

An industrial prepared liquefied mash using alpha-amylase X (pH=5.5, T=85° C.), was used for the experiment. The dry solid determined by moisture balance (Mettler-Toledo) was about 30% DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 5 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of glucoamylase blend A, and appropriate amount of endo-protease from Meriphilus giganteus (SEQ ID NO: 2) with or without exo-peptidase tripeptidylaminopeptidase from Aspergillus niger (SEQ ID NO: 32) as shown in the table below followed by addition of 100 micro liters hydrated yeast per 5 g slurry. As control, glucoamylase blend A with no addition of endo-protease or exo-peptidase. Actual glucoamylase and protease dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32° C. Three replicates of each treatment were used during SSF. After 50 hours, fermentation was stopped by addition of 50 micro liters of 40% H₂SO₄, follow by centrifuging, and filtering through a 0.45 micrometer filter. Ethanol and oligosaccharides concentration were determined using HPLC.

Endo-protease
Tripeptidylamino-

from
peptidase from

M. giganteus

A. niger

Treatments
(μg/g DS)
μg/gDS

1. Control
—
—

2. Endo-protease only
2.5
—

3. Endo-protease +
2.5
2.5

Tripeptidylaminopeptidase

As shown in the tables below, combination of endo-protease with tripeptidylaminopeptidase increased ethanol yield compared to control or endo-protease alone.

Ethanol yield at 50 hours of endo-protease without or with tripeptidylaminopeptidase.

Treatments
Ethanol (g/l)

1. Control
121.9

2. Endo-protease only
123.6

3. Endo-protease + Exo-peptidase
124.4

COMBINED USE OF AT LEAST ONE ENDO-PROTEASE AND AT LEAST ONE EXO-PROTEASE IN AN SSF PROCESS FOR IMPROVING ETHANOL YIELD

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