# Patent

## Title
Acid fungal protease in fermentation of insoluble starch substrates

## Abstract

The invention is directed to methods of producing ethanol and decreasing residual starch production in a no cook fermentation comprising contacting granular starch containing substrates with a granular starch hydrolyzing enzyme, a protease, and a fermenting microorganism under suitable fermentation conditions at a temperature below the starch gelatinization temperature of the starch substrate to produce ethanol, wherein the ethanol production is increased and the amount of residual starch is decreased compared to a substantially similar method conducted without the protease.

## Background
RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Patent Application No. 60/640,399, entitled Acid Fungal Proteases, filed Dec. 30, 2004 and U.S. Provisional Patent Application No. 60/648,233, entitled Acid Fungal Proteases, filed Jan. 27, 2005, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to methods for producing alcohol, such as ethanol during fermentation of granular starch containing substrates. The invention also relates to the inclusion of an acid fungal protease in the fermentation, which results in an increase in the production of ethanol and in a decrease in the amount of residual starch produced during the fermentation compared to fermentation without the protease.
BACKGROUND OF THE INVENTION
Industrial fermentation processes predominately use glucose as a feedstock for the production of numerous end products, such as proteins, enzymes, amino acids, organic acids, alcohols, biochemicals, pharmaceuticals and the like. In many of these applications glucose is produced from the enzymatic conversion of substrates, which contain starch. Starch accumulates as microscopic granules in plant material and the partial crystalline nature of these granules imparts insolubility in cold water. As a result, many methods have been described for solubilizing starch granules. In particular, many methods have been described for converting starch containing substrates to alcohols, such as ethanol. These methods include direct and indirect heating prior to fermentations (STARCH CHEMISTRY AND TECHNOLOGY, Eds. R. L. Whistler et al., 2nd Ed., (1984) Academic Press Inc,; STARCH CONVERSION TECHNOLOGY Eds., G. M. A. Van Beynum et al., Food Science and Technology Series, Marcel Dekker Inc, NY; and THE ALCOHOL TEXTBOOK 3rd Ed., Eds. K. Jacques et al., (1999), Nottingham University Press). Due to temperature requirements direct and indirect heating methods are energy demanding. Therefore, more recent attention in the industrial fermentation arts and especially in alcohol fermentation has been directed to low energy processes for hydrolyzing granular starch. Low energy processes reduce the need for energy intensive liquefaction cooking steps (U.S. Pat. No. 4,514,496; Ueda et al., (1980) J. Ferment. Tech., 58:237-242; Han et al., (1987) Biotechnol. and Bioeng., 30:225-232; WO 03/066826; WO 03/068976; WO 04/106533; WO 04/081193; WO 05/052148; and US Patent Publication 2005/0266543). A number of novel enzyme compositions capable of hydrolyzing granular (uncooked) starch to produce fermentable glucose in alcohol fermentation processes have been introduced into the market place in the last few years, for example STARGEN (Genencor International Inc.). However, there still remains a need for additional and more effective starch conversion methods, which yield alcohols and high quality fermentation byproducts such as distillers' feeds.
The use of proteases in yeast fermentations has been shown to increase the yield and rate of ethanol production from liquefied starch substrates (U.S. Pat. No. 5,231,017). The NSP24 proteases of the present invention are not only novel proteases but also may be used to increase the ethanol production from insoluble starch substrates.
SUMMARY OF THE INVENTION
The present invention relates to NSP24 proteases having at least 85% sequence identity to SEQ ID NO: 2 and biologically active fragments thereof. In one embodiment, the invention pertains to an NSP24 protease having at least 98% sequence identity to SEQ ID NO: 2 and biologically active fragments thereof, such as SEQ ID NO: 3. In further embodiments, the invention relates to the use of NSP24 proteases in yeast fermentations to produce ethanol.
In one aspect, the invention relates to NSP24 protease compositions for use in hydrolysis of insoluble starch containing substrates, such as grains and/or fractionated components thereof.
In another aspect, the invention relates to NSP24 protease compositions for use in ethanol production from yeast fermentations.
In a further aspect, the invention relates to a method for producing ethanol comprising saccharifying a milled insoluble starch substrate with a granular starch hydrolyzing enzyme composition and an NSP24 protease at a temperature below the starch gelatinization temperature of the substrate and fermenting the saccharified starch substrate under suitable fermentation conditions to produce at least 8% v/v ethanol.
In another aspect, the invention relates to a method of increasing ethanol production in a no cook fermentation comprising contacting a milled insoluble starch substrate with a granular starch hydrolyzing enzyme, an NSP24 protease, and a fermenting microorganism under suitable fermentation conditions at a temperature below the starch gelatinization temperature of the substrate to produce ethanol, wherein the rate of ethanol production is increased compared to a substantially similar method conducted without the NSP24 protease.
In yet another aspect, the invention relates to a method of producing ethanol in a no cook simultaneous saccharification and fermentation including milled grain substrates comprising adding an NSP24 protease to the simultaneous saccharification and fermentation.
In still a further aspect, the invention relates to a method for decreasing the amount of residual starch produced from the fermentation of granular starch containing substrates comprising fermenting a granular starch containing substrate with a protease, a granular starch hydrolyzing enzyme and a fermenting microorganism, wherein the granular starch containing substrate is not cooked and obtaining a fermented broth wherein the % residual starch in the broth is decreased compared to the amount of residual starch obtained from a substantially similar method without the protease. In some embodiments, the residual starch will be less than 15%. In other embodiments, the protease will be an NSP24 protease.
In yet another aspect, the invention relates to a method for producing an alcohol (e.g. ethanol) comprising pretreating a slurry of a starch substrate at a temperature of about 68° C. to 35° C. for a period of about 30 minutes to 5 hours, fermenting the slurry with a composition comprising yeast, granular starch hydrolyzing enzymes and an NSP24 protease for a period of 12 to 96 hours at a temperature between 10° C. and 50° C. to produce alcohol. In some embodiments, the alcohol is ethanol and in other embodiments the ethanol is recovered from the fermentation. In further embodiments, the pretreatment includes additional enzymes, such as glucoamylase, alpha amylases, proteases or combinations thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the nucleotide sequence of nsp24 (SEQ ID NO: 1) (FIG. 1A) and the predicted amino acid sequence (NSP24, SEQ ID NO: 2) encoded by nsp24 (FIG. 1B), wherein the putative gene intron sequence is identified in bolded format in FIG. 1A and in FIG. 1B the leader sequence is in bold, the prepro sequence is underlined and the mature NSP24 protein sequence, which is represented by SEQ ID NO: 3 starts with KYGAPIS.
FIGS. 2A-D illustrate the nucleotide sequence (SEQ ID NO: 4) of the pTrex3g_NSP24 cDNA clone obtained from Trichoderma reesei. The NSP24 gene sequence is underlined and the putative gene intron sequence is identified in bolded format.
FIG. 3 illustrates the pTrex3g_NSP24 vector and locations of restriction enzyme cleavage sites along the nucleotide sequence of FIG. 2.
FIG. 4 illustrates the effect of different levels of NSP24 protease on % ethanol (v/v) production during no cook fermentation conditions with degermed ground corn.
FIG. 5 illustrates the effect of NSP24 on % alcohol (v/v) production at 72 hrs during no cook yeast fermentations with fractionated corn endosperm and varying levels (%) of backset and reference is made to example 5.
FIG. 6 illustrates the effect of NSP24 (0.7 SAPU/g ds) on % alcohol (v/v) production at 72 hrs during no cook yeast fermentations with different levels (% DS) of fractionated corn endosperm and reference is made to example 6.



DETAIL DESCRIPTION OF THE INVENTION
The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Also, information regarding methods of preparation, expression, isolation and use of proteases may be obtained by review of U.S. Pat. No. 6,768,001.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with general dictionaries of many of the terms used in this invention.
The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
Numeric ranges are inclusive of the numbers defining the range.
Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to a composition comprising “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
Definitions—
“Protease” means a protein or polypeptide domain of a protein or polypeptide derived from a microorganism, e.g. a fungus, bacterium, or from a plant or animal, that has the ability to catalyze cleavage of peptide bonds at one or more of various positions of a protein backbone (e.g. E.C. 3.4).
An “acid protease” refers to a protease having the ability to hydrolyze proteins under acid conditions.
As used herein, “an NSP24 protease” means an enzyme having protease activity and having at least 85% identity with the sequence of SEQ ID NO: 2 or biologically active fragments thereof. The term “NSP24” means the protease having SEQ ID NO: 2 or SEQ ID NO: 3.
A “biologically active fragment” (e.g., a biologically active fragment of NSP24) means a polypeptide fragment having protease activity and one or more amino acid residues deleted from the amino and/or carboxyl terminus of an NSP24 protease sequence.
The term “isolated” or “purified” refers to a protease that is altered from its natural state by virtue of separating the protease from one or more or all of the naturally occurring constituents with which it is associated in nature.
The terms “peptides”, “proteins”, and “polypeptides” are used interchangeably herein.
As used herein, “percent (%) sequence identity” with respect to the amino acid or nucleotides sequences identified is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in a sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
As used herein the term “granular starch hydrolyzing enzyme composition” or “granular starch hydrolyzing enzyme” means an enzyme composition capable of hydrolyzing starch in granular form. The compositions may consist of one enzyme or a combination of enzymes.
As used herein the term “alpha-amylase (e.g., E.C. class 3.2.1.1)” refers to enzymes that catalyze the hydrolysis of alpha-1,4-glucosidic linkages. These enzymes have also been described as those effecting the exo or endohydrolysis of 1,4-α-D-glucosidic linkages in polysaccharides containing 1,4-α-linked D-glucose units. Another term used to describe these enzymes is “glycogenase”. Exemplary enzymes include alpha-1,4-glucan 4-glucanohydrase glucanohydrolase.
As used herein the term “glucoamylase” refers to the amyloglucosidase class of enzymes (e.g., E.C. 3.2.1.3, glucoamylase, 1,4-alpha-D-glucan glucohydrolase). These are exo-acting enzymes, which release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules. The enzyme also hydrolyzes alpha-1,6 and alpha-1,3 linkages although at a much slower rate than alpha-1,4 linkages.
As used herein “GAU” refers to a glucoamylase unit, which is the amount of enzyme that will produce 1 g of reducing sugar calculated as glucose per hour from a soluble starch substrate at pH 4.2 and 60° C.
As used herein “SAPU” refers a spectrophotometric acid protease unit, wherein 1 SAPU is the amount of protease enzyme activity that liberates one micromole of tyrosine per minute from a casein substrate under conditions of the assay.
As used herein “SSU” refers to soluble starch unit, 1 SSU is equivalent to the reducing power of 1 mg glucose released per minute at specific incubation conditions.
As used herein “starch” refers to any material comprised of the complex polysaccharide carbohydrates of plants comprised of amylose and amylopectin with the formula (C6H10O5)X, wherein X can be any number.
The term “insoluble starch” and “granular starch” are used interchangeably herein and refer to uncooked (raw) starch (e.g. starch that has not been subject to gelatinization).
As used herein the term “gelatinization” means solubilization of a starch molecule by cooking to form a viscous suspension.
The term “residual starch” refers to the remaining starch (soluble or insoluble) left in a fermentation broth after fermentation of a starch containing substrate.
The term “gelatinization temperature” refers to the temperature at which gelatinization begins. The exact temperature of gelatinization depends on the substrate and may vary depending on factors such as plant species and environmental and growth factors.
The phrase “below the gelatinization temperature” refers to a temperature that is less than the temperature that starts gelatinization.
As used herein the term “liquefaction” refers to the stage in starch conversion in which gelatinized starch is hydrolyzed to give low molecular weight soluble dextrins.
As used herein the term “saccharification” refers to enzymatic conversion of starch to glucose.
As used herein the term “soluble starch” or “soluble starch hydrolyzate” refers to soluble products resulting from starch hydrolysis, which may comprise mono-, di-, and oligosaccharides (e.g. glucose, maltose and higher sugars).
The term “milling” refers to the breakdown of substrates comprising starch (e.g. cereal grains) to smaller particles. In some embodiments, the term is used interchangeably with grinding.
The term “fractionated grain” refers to plant grains that include only a portion of the grain.
The term end-product refers to any carbon source derived molecular product which is enzymatically converted from a starch substrate.
The term “enzymatic conversion” refers to the modification of a substrate by enzyme action.
The term polysaccharide” means a compound having multiple monosaccharide units joined in a linear or branched chain. In some embodiments, the term refers to long chains with hundreds or thousands of monosaccharide units. Typical examples of polysaccharides are starch, cellulose and glycogen.
The term “monosaccharide” means a monomeric unit of a polymer such as starch wherein the degree of polymerization (DP) is 1 (e.g., glucose, mannose, fructose and galactose).
The term “disaccharide” means a compound that comprises two covalently linked monosaccharide units (DP2) (e.g., sucrose, lactose and maltose).
The term “DP3+” means polymers with a degree of polymerization greater than 3.
The term “oligosaccharide” means a compound having 2 to 10 monosaccharide units joined in glycosidic linkages.
The term “dry solids content (DS or ds)” refers to the total solids of a slurry in % on a dry weight basis.
The terms “distillers dried grain (DDG)” and “distillers dried grain with solubles (DDGS)” refer to useful co-products of grain fermentation processes.
The term “promoter” means a regulatory sequence involved in binding RNA polymerase to initiate transcription of a gene.
A “heterologous promoter”, as used herein is a promoter, which is not naturally associated with a gene or a purified nucleic acid.
A “purified preparation” or a “substantially pure preparation” of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs.
A “purified preparation of cells”, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A “substantially pure nucleic acid”, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (e.g., one at the 5′ end and one at the 3′ end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional NSP24 protease sequence.
“Homologous”, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
As used herein the term “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
As used herein, “expression vector” means a DNA construct including a DNA sequence which is operably linked to a suitable control sequence capable of affecting the expression of the DNA in a suitable host.
The term “expression” means the process by which a polypeptide is produced based on the nucleic acid sequence of a gene.
As used herein, “operably linked” means that a regulatory region, such as a promoter, terminator, secretion signal or enhancer region is attached to or linked to a structural gene and controls the expression of that gene.
As used herein, a substance (e.g. a polynucleotide or protein) “derived from” a microorganism means that the substance is native to the microorganism.
As used herein, “microorganism” refers to a bacterium, a fungus, a virus, a protozoan, and other microbes or microscopic organisms.
As used herein, “host strain” or “host cell” means a suitable host for an expression vector including DNA according to the present invention and includes progeny of said cells.
The term “filamentous fungi” refers to all filamentous forms of the subdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORY MYCOLOGY, Wiley, New York and AINSWORTH AND BISBY DICTIONARY OF THE FUNGI, 9th Ed. (2001) Kirk et al., Eds., CAB International University Press, Cambridge UK). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic.
As used herein, the term “Trichoderma” or “Trichoderma sp.” refer to any fungal genus previously or currently classified as Trichoderma. 
The phrase “simultaneous saccharification and fermentation (SSF)” refers to a process in the production of alcohols in which a microbial organism, such as an ethanol producing microorganism and at least one enzyme such as a granular starch hydrolyzing enzyme are in the same process step.
As used herein “ethanol producing microorganism” refers to a microorganism or cell with the ability to convert a sugar or oligosaccharide to ethanol. Examples of ethanol producing microorganisms include fungal microorganisms such as yeast. A preferred yeast includes strains of Saccharomyces, particularly, S. cerevisiae. 
As used herein the term “culturing” refers to growing a population of microbial cells under suitable conditions in a liquid or solid medium. In one embodiment, culturing refers to fermentative bioconversion of a starch substrate, such as a substrate comprising granular starch, to an end-product (typically in a vessel or reactor). Fermentation is the enzymatic and anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. While fermentation occurs under anaerobic conditions it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen.
As used herein the term “contacting” refers to the placing of the respective enzyme(s) in sufficiently close proximity to the respective substrate to enable the enzyme(s) to convert the substrate to the end-product. Those skilled in the art will recognize that mixing solutions of the enzyme with the respective substrates can effect contacting.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
As used herein, the terms “transformed”, “stably transformed” and “transgenic” used in reference to a cell means the cell has a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.
As used herein the term “heterologous” with reference to a polypeptide or polynucleotide means a polypeptide or polynucleotide that does not naturally occur in a host cell.
The term “overexpression” means the process of expressing a polypeptide in a host cell wherein a polynucleotide has been introduced into the host cell.
NSP24 Proteases and Polynucleotides Encoding the Same—
The invention relates to NSP24 proteases which in some embodiments are acid proteases, such as acid fungal proteases. The NSP24 protease have at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and biologically active fragments thereof.
In some embodiments, the NSP24 protease is a protease having at least 95% sequence identity to SEQ ID NO: 2 and biologically active fragments thereof. In some embodiments, the NSP24 protease is designated NSP24 and has the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, which is a biologically active fragment of SEQ ID NO: 2.
In some embodiments, the NSP24 protease is a biologically active fragment of a protease having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, biologically active fragments include proteases having at least 250 amino acid residues, at least 300 amino acid residues, at least 350 amino acid residues, at least 375 amino acid residues, and also at least 400 amino acid residues.
In other embodiments, biologically active fragments include at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% of a polypeptide sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% and at least 99% sequence identity with the protein sequence of SEQ ID NO: 2. In some embodiments, the NSP24 protease will comprise at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% and at least 98% of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 2.
In some embodiments, biologically active fragments are fragments that exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells, e.g., as a result of post-translational processing, e.g., as the result of the removal of an amino-terminal signal sequence, as well as those made in expression systems, e.g., in CHO cells. Biologically active fragments may be generated by proteolytic cleavage or alternative splicing events.
In some embodiments, a biologically active fragment will comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, and at least 100% of the protease activity of the NSP24 protease having SEQ ID NO: 2. In some preferred embodiments, a fragment possesses at least 40% or at least 90% of the protease activity of the NSP24 protease of SEQ ID NO: 2 in any in vivo or in vitro protease assay.
NSP24 fragments can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of a protease can be assessed by methods known to those skilled in the art as described herein.
The invention also includes protease analogs. The analogs are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., b or amino acids; and cyclic analogs. Analogs can differ from naturally occurring proteases, such as NSP24 having SEQ ID NO: 2 or SEQ ID NO: 3, in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include in vivo or in vitro chemical derivatization of the proteases encompassed by the invention. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. The NSP24 proteases of the invention may include conservative amino acid substitutions using L-amino acids, wherein one amino acid is replaced be another biologically similar amino acid. Nonlimiting examples of conservative replacements include Gly/Ala; Val/Ile/Leu; Lys/Arg; Asn/Gln; Glu/Asp; Ser/Cys/Thr; and Phe/Trp/Tyr.
The invention also relates to polynucleotide sequences encoding the NSP24 proteases encompassed by the invention. Some of these polynucleotides include: a) polynucleotides encoding an NSP24 protease having at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, and at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3; b) polynucleotides encoding the sequence of SEQ ID NO: 2; c) a polynucleotide having the sequence of SEQ ID NO: 1; d) polynucleotides encoding a biologically active fragment of an NSP24 protease; e) polynucleotides which have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% and at least 99% sequence identity to the sequence of SEQ ID NO:1; and f) polynucleotides which hybridize to a nucleic acid probe corresponding to the DNA sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, said fragment having at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 150 consecutive nucleotides.
Because of the degeneracy of the genetic code, more than one codon may be used to code for a particular amino acid. Therefore different DNA sequences may encode a polypeptide having the same amino acid sequence as the polypeptide of, for example SEQ ID NO: 2. The present invention encompasses polynucleotides which encode the same polypeptide.
A nucleic acid is hybridizable to another nucleic acid sequence when a single stranded form of the nucleic acid can anneal to the other nucleic acid under appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known in the art for hydridization under low, medium, high and very high stringency conditions (See, e.g., Sambrook (1989) supra, particularly chapters 9 and 11).
Homology or percent identity of sequences may be determined using computer programs. Methods for performing sequence alignments and determining sequence identity are known to the skilled artisan and may be performed without undue experimentation. See, for examples Ausubel et al., (1995) Current Protocols in Molecular Biology, Chap. 19 (Greene Publishing and Wiley-Interscience, NY) and the ALIGN program (Dayhoff (1978) in Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington D.C.). A number of algorithms are available for aligning sequences and determining identity (See, Needleman et al., (1970) J. Mol. Biol. 48:443; Smith et all., (1981) Adv. Appl. Math 2:482; Pearson et al., 91988) Proc. Natl. Acad. Sci 85:2444 and BLASTP, BLASTN and BLASTX algorithms). Various computer programs are known to those in the art including but not limited to ALIGN or Megalign (DNASTAR); WU-BLAST-2; GAP; BLAST; BESTFIT (Altschul et al., (1996) Meth. Enzyme 266:460-480); FASTA and TFASTA (GCG Version B, Madison, Wis.) and CLUSTRAL (Intelligenetics).
Expression of NSP24 Proteases in Host Cells—
In some embodiments, polynucleotides encoding the NSP24 proteases of the invention will be introduced into host cells. In some embodiments, the polynucleotide encodes a heterologous protease, and in other embodiments, the polynucleotide codes for an endogenous protease which is overexpressed in the host cell. Some preferred host cells include filamentous fungal cells. Non-limiting examples of filamentous fungal host cells include Trichoderma spp. (e.g. T. viride and T. reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum), Penicillium spp., Humicola spp. (e.g. H. insolens and H. grisea), Aspergillus spp. (e.g., A. niger, A. nidulans, A. orzyae, and A. awamori), Fusarium spp. (F. graminum), Neurospora spp., Hypocrea spp. and Mucor spp. Further, host cells may include Bacillus spp (e.g. B. subtilis, B. licheniformis, B. lentus, B. stearothremophilus and B. brevis) and Streptomyces spp. (e.g., S. coelicolor and S. lividans (TK23 and TK21)).
Vectors and DNA constructs comprising polynucleotides encoding an NSP24 protease encompassed by the invention comprise gene promoter sequences for example of filamentous fungi which are typically cloned into intermediate vectors before transformation into host cells, such as Trichoderma reesei cells for replication and/or expression. These intermediate vectors are typically prokaryotic vectors, e.g., plasmids, or shuttle vectors.
To obtain high-level expression of a cloned gene, the heterologous gene is preferably positioned about the same distance from the promoter as is in the naturally occurring gene. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. Those skilled in the art are aware that a natural promoter can be modified by replacement, substitution, addition or elimination of one or more nucleotides without changing its function. The practice of the invention encompasses and is not constrained by such alterations to the promoter.
The expression vector/construct typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the heterologous sequence. A typical expression cassette thus contains a promoter operably linked to the heterologous nucleic acid sequence and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
The practice of the invention is not constrained by the choice of promoter in the genetic construct. However, exemplary promoters are the Trichoderma reesei cbh1, cbh2, eg1, eg2, eg3, eg5, xln1 and xln2 promoters. Also promoters from A. awamori and A. niger glucoamylase genes (glaA) (Nunberg et al., (1984) Mol. Cell Biol. 4:2306-2315) and the promoter from A. nidulans acetamidase find use in the vectors. A preferred promoter for vectors used in Bacillus subtilis is the AprE promoter; a preferred promoter used in E. coli is the Lac promoter, a preferred promoter used in Saccharomyces cerevisiae is PGK1, a preferred promoter used in Aspergillus niger is glaA, and a preferred promoter for Trichoderma reesei is cbhI.
In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
Although any fungal terminator is likely to be functional in the present invention, some preferred terminators include: the terminator from Aspergillus nidulans trpC gene (Yelton, M. et al. (1984) PNAS USA 81:1470-1474, Mullaney, E. J. et al. (1985) MGG 199:37-45), a terminator from Aspergillus awamori or Aspergillus niger glucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell Biol. 4:2306, Boel, E. et al. (1984) EMBO J. 3:1581-1585), the terminator from the Aspergillus oryzae TAKA amylase gene, and the terminator from the Mucor miehei carboxylprotease gene (EPO Publication No. 0 215 594).
The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include bacteriophages λ and M13, as well as plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. Examples of suitable expression and/or integration vectors are provided in Sambrook et al., (1989) supra, Bennett and Lasure (Eds.) More Gene Manipulations in Fungi, (1991) Academic Press pp. 70-76 and pp. 396-428 and articles cited therein; U.S. Pat. No. 5,874,276 and Fungal Genetic Stock Center Catalogue of Strains, (FGSC, www.fgsc.net.).
Useful vectors may be obtained from Promega and Invitrogen. Some specific useful vectors include pBR322, pUC18, pUC100, pDONTM201, pENTRTM, pGEN®3Z and pGEN®4Z. However, the invention is intended to include other forms of expression vectors which serve equivalent functions and which are, or become, known in the art. Thus, a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1, pBR322, pMb9, pUC 19 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs e.g., the numerous derivatives of phage .lambda., e.g., NM989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids such as the 2.mu plasmid or derivatives thereof.
In some embodiments, an expression vector includes a selectable marker. Examples of selectable markers include ones which confer antimicrobial resistance. Nutritional markers also find use in the present invention including those markers known in the art as amdS, argB and pyr4. Markers useful for the transformation of Trichoderma are known in the art (see, e.g., Finkelstein, chapter 6, in Biotechnology of Filamentous Fungi, Finkelstein et al., EDS Butterworth-Heinemann, Boston Mass. (1992) and Kinghorn et al., (1992) Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London). In some embodiments, the expression vectors will also include a replicon, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of heterologous sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication or integration of the DNA in Trichoderma reesei. 
The methods of transformation of the present invention may result in the stable integration of all or part of the transformation vector into the genome of a host cell, such as a filamentous fungal host cell. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated.
Many standard transfection methods can be used to produce bacterial and filamentous fungal (e.g. Aspergillus or Trichoderma) cell lines that express large quantities of the protease. Some of the published methods for the introduction of DNA constructs into cellulase-producing strains of Trichoderma include Lorito, Hayes, DiPietro and Harman, (1993) Curr. Genet. 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, (1990) Curr. Genet. 17:169-174; and Penttila, Nevalainen, Ratto, Salminen and Knowles, (1987) Gene 6: 155-164, also see U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328 and Nevalainen et al., “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes” in Molecular Industrial Mycology, Eds, Leong and Berka, Marcel Dekker Inc., NY (1992) pp 129-148; for Aspergillus include Yelton, Hamer and Timberlake, (1984) Proc. Natl. Acad. Sci. USA 81: 1470-1474, for Fusarium include Bajar, Podila and Kolattukudy, (1991) Proc. Natl. Acad. Sci. USA 88: 8202-8212, for Streptomyces include Hopwood et al., 1985, Genetic Manipulation of Streptomyces: Laboratory Manual, The John Innes Foundation, Norwich, UK and Fernandez-Abalos et al., Microbiol 149:1623-1632 (2003) and for Bacillus include Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, (1990) FEMS Microbiol. Lett. 55: 135-138).
However, any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). Also of use is the Agrobacterium-mediated transfection method described in U.S. Pat. No. 6,255,115. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the gene. In one embodiment, the invention concerns a method for producing a protease encompassed by the invention (e.g. an NSP24 protease) which comprises introducing into a host cell a polynucleotide comprising a promoter operably linked to a nucleic acid encoding an NSP24 protease, culturing the host cell under suitable culture conditions for the expression and production of the protease, and producing said protease. In some preferred embodiments, the protease is an NSP24 protease having at least 95% sequence identity to SEQ ID NO: 2 or biologically active fragments thereof.
After the expression vector is introduced into the cells, the transfected or transformed cells are cultured under conditions favoring expression of genes under control of protease gene promoter sequences. Large batches of transformed cells can be cultured as described herein. Finally, product is recovered from the culture using standard techniques. Optimal conditions for the production of the proteins will vary with the choice of the host cell, and with the choice of protease protein to be expressed. Such conditions will be easily ascertained by one skilled in the art through routine experimentation or optimization. Assays to determine protease activity that find use in the present invention include, but are not limited to those described in WO 9934011 and U.S. Pat. No. 6,605,458.
The protease protein of interest may be isolated or recovered and may further be purified after expression in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Conventional procedures for recovering a protease from culture medium include separation by centrifugation or filtration, or if necessary, disrupting the cells and removing the supernatant from the cellular fraction and debris. In some cases, after clarification, the proteinaecous components of the supernatant or filtrate are precipitated by means of a salt, e.g., ammonium sulphate. The precipitated proteins are then solublized and may be purified by a variety of chromatographic procedures. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the protein of interest may be purified using a standard anti-protein of interest antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, Protein Purification (1982).
Cell Cultures—
Host cells and transformed cells can be cultured in conventional nutrient media. The specific culture conditions, such as temperature, pH and the like, may be those that are used for the host cell selected for expression, and will be apparent to those skilled in the art. In addition, preferred culture conditions may be found in the scientific literature such as Sambrook, (1982) supra; Kieser, T, M J. Bibb, M J. Buttner, K F Chater, and D. A. Hopwood (2000) PRACTICAL STREPTOMYCES GENETICS. John Innes Foundation, Norwich UK; Harwood, et al., (1990) MOLECULAR BIOLOGICAL METHODS FOR BACILLUS, John Wiley and/or from the American Type Culture Collection (ATCC; www.atcc.org). Stable transformants of fungal host cells, such as Trichoderma cells can generally be distinguished from unstable transformants by their faster growth rate or the formation of circular colonies with a smooth rather than ragged outline on solid culture medium. See, for example Davis et al., (1970) Methods Enzymol. 17:79-143).
In some embodiments of the present invention, fungal cells expressing a NSP24 protease according to the invention are grown under batch or continuous fermentation conditions. A classical batch fermentation is a closed system, wherein the composition of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism(s). In this method, fermentation is permitted to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase eventually die. In general, cells in log phase are responsible for the bulk of production of end product.
A variation on the standard batch system is the “fed-batch fermentation” system, which also finds use with the present invention. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolism repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and fed-batch fermentations are common and well known in the art.
Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth and/or end product concentration. For example, in one embodiment, a limiting nutrient such as the carbon source or nitrogen source is maintained at a fixed rate an all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.
Methods for the Production of Ethanol and By-products—
In one embodiment, the invention comprises methods for producing ethanol comprising saccharifying a milled insoluble starch substrate with a granular starch hydrolyzing enzyme composition and a protease at a temperature below the gelatinization temperature of the starch in the substrate. While in general acid proteases, such as proteases derived from fungal strains (e.g. Aspergillus, Mucor, Rhizopus, Trichoderma, Penicillium, and Endothia) may be useful in the methods of the invention, preferred proteases include NSP24 proteases as described above.
An insoluble starch containing substrate may be obtained from any plant material including any plant part containing starch, such as seeds, kernels, grains, leaves, tubers, roots, stems and the like. Particularly preferred plant sources include corn; wheat; rye; sorghum; rice; millet; barley; rice, milo, cassaya; legumes, such as beans and peas; sugarcane; potatoes; sweet potatoes; bananas; tapioca and mixtures thereof. Some preferred sources of insoluble starch substrates include corn, wheat, barely, rye and sorghum.
Various references have reported on the amount of starch found in cereal grains and reference is made to The Alcohol Textbook, 3rd Ed. K. Jacques et al., Eds. 1999, Nottingham University Press. For example corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch; wheat contains about 60-65% starch; and polished rice contains 70-72% starch.
The plant material may be by-products, such as corn fiber, corncobs, stover and other cellulose or hemicellulose containing plant residues and materials. In addition, insoluble starch substrates include fractionated plant material. Typical examples include fractionated corn, sorghum, barley and wheat. Some preferred embodiments include fractionated corn, such as endosperm, germ or fiber (bran) but particualrly endosperm for corn and gluten starch for wheat. Those of general skill in the art are well aware of methods available to prepare fractionated grains (See, U.S. Pat. No. 6,254,914, U.S. Pat. No. 6,899,910; U.S. Pat. No. 6,936,294; U.S. Pat. No. 6,936,110 and U.S. Pat. No. 6,408,105; Singh and Johnston (2004) Adv. Food and Nutr. Res. 48:151-171 and Singh et al., (1999) Cereal Chem 76:868-872).
Milling of the insoluble starch substrate may be by either wet milling or dry milling. In some preferred embodiments, the insoluble substrate is dry-milled. In general if the substrate (e.g. grain) is not fractionated the non-fermentable components of the grain (e.g. the fiber, protein and germ) are carried through the fermentation process and are recovered as the by-product, distillers dried grain and distillers dried grain with solubles.
In either wet or dry milling fractionation processes, mechanical grinding followed by density separation is used for the different fractions or corn germ, fiber and endosperm (Ponnampalan et al., (2004) Appl. Biochem. Biotech., 115:837-842) and Simms L., (1985) “The Technology of Corn Wet Milling” in Starch Conversion Technology, Ed. G. M. A. van Beynnum and J. A. Roels Marcel Dekker Inc., NY).
Dry milling of whole cereal grains is known in the art and includes the use of hammer mills and roller mills. Also dry mill fractionation processes are known (Brekk O. L. (1970) “Corn Dry Milling Industry” pages 262-291 in Corn Culture Products G. E. Inglett Ed., AVI Publishing, Westport Conn.). An example of a typical composition of dry milled whole ground corn (WC) as compared to fractionated corn (FC) is as follows: moisture—13% (WC) and 12% (FC); protein—7.8% (WC) and 7.0% (FC); fat—3.5% (WC) and 0.7% (FC); crude fiber—1.5% (WC) and 0.5% (FC); ash—1.3% (WC) and 0.4% (FC) and carbohydrate—72.9% (WC) and 79.4% (FC) (Duensing et al., Corn Dry Milling: Processes, Products and Applications in Corn: Chemistry and technology ED. White et al. However, with modernization and advancements in the dry fractionation of ground corn, the endosperm fraction may contain greater than 80% starch.
In some embodiments, a milled substrate will be prepared by e.g. grinding wherein at least 50%, at least 55%, at least 60%, at least 65% and at least 70% of the ground particles will pass through a sieve having an aperture size of about 600 micrometers (between 585 and 610) designated herein as a 30 mesh screen. In some embodiments, less than 85%, less than 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, or less than about 40% of the ground particles will pass through a 30 mesh screen. In some embodiments, at least 25%, at least 30%, at least 40%, at least 30% and also at least 35% of the milled substrate will be retained on a 30 mesh screen. Also in some embodiments, between 30%-80% of the milled particles will pass through a 30 mesh screen. In other embodiments, between 30%-75%, also between 30%-70%, also between 35%-65%, and also between 40%-60% of the milled particles will pass through a 30 mesh screen.
The insoluble starch substrate in some embodiments is mixed with mashing liquor to form a slurry. The mashing liquor may be water and process water, such as stillage (backset) or a mixture of both. Stillage is the distillation residue of the fermentation and may include sugars, nitrogen and other nutrients and enzymes. Stillage can be used in proportion of 1% to 95%, also in the range of 15% to 75% and about 30% to 60% instead of water.
The % dry solids (DS) of a slurry may be in the range of about 10% to about 55%, in the range of about 15% to about 50%, in the range of about 20% to about 50%, in the range of about 25% to about 50%, in the range of about 30% to about 45%.
Saccharification takes place by contacting a substrate with a granular starch hydrolyzing enzyme composition and an NSP24 protease of the invention as previously described in detail above.
The amount of an NSP24 protease used in the methods of the invention will vary between the range of 0.001 to 10 SAPU/g DS. In some embodiments, the amount of an NSP24 protease will be between the range of 0.01 and 10.0 SAPU/g DS. In other embodiments, the amount of NSP24 protease will be between the range of about 0.05 and 5.0 SAPU/g DS. In other embodiments, the amount of NSP24 protease will range from 0.05 to 5 kg/metric ton (MT). Other useful amounts include the range of from 0.1 to 1 kg/MT NSP24 protease.
Granular starch hydrolyzing enzymes and enzyme compositions are known in the art and include compositions supplying glucoamylases and alpha amylases as separate enzymes or in combinations. The enzymes may be wild type or genetically modified enzymes including variants and hybrids.
Non-limiting examples of glucoamylases having granular starch hydrolyzing activity are glucoamylases derived from Humicola (H. grisea) (WO 2005/052148; U.S. Pat. No. 4,618,579; Tosi et al., 91993) Can. J. Microbiol. 39:846-852; Campos et al., (1995), App and Environ. Microbiol. 61:2436-2438; EP 171218; and Allison et al., (1992) Cur. Genet 21:225-229); Aspergillus (A. niger A. awamori, and A. kawachi) (Ueda et al., (1981) Biotechnology. Bioeng 23: 291, Hayashida (1973) Agr. Biol. Chem. 39:2093-2099 and Hayashida et al., (1980) Agric. Biol. Chem. 53:923-929); Trichoderma (T. reesei); and Rhizopus (R. niveus and R. oryzae) (U.S. Pat. No. 4,514,496, U.S. Pat. No. 4,092,434, U.S. Pat. No. 4,863,864; Takahashi et al., J. Biochem. (1985) 98: 663-671 and Ashikari et al., (1986) Agric. Biol. Chem. 50:957 964). Non-limiting examples of alpha amylases having granular starch hydrolyzing activity are derived from Aspergillus (A. kawachi) (See, US Appln. 2005/0266543 and Ueda et al., (1980) J. Fermentation Technol. 58:237-242). Recently a number of patent applications have been published which are directed to various enzyme compositions, which include granular starch hydrolyzing activities and the use of these compositions in alcohol fermentations. These publications include for example WO 03/066826; WO 04/113551; WO 04/081193; WO 04/106533; WO 05/052148 and WO 05/087938. Some of the enzymes disclosed as having granular starch hydrolyzing activity include wild type enzyme and variants and hybrids thereof.
Commercially available granular starch hydrolyzing compositions are available from Genencor International Inc. (Danisco A/S), STARGEN001; Shin Nihon Chemical Co. Japan, CU CONC; Bicon, India LTD., Bangalore, India M1 Amano and Novozymes A/S.
While the saccharifying step of the invention includes a granular starch hydrolyzing enzyme and an NSP24 protease, additional enzymes may be added to the mixture, such enzymes include for example other glucoamylases and amylases.
The amount of glucoamylase employed in the present process can vary according to enzyme activity. In some embodiments, suitable amounts include 0.01 to 15 GAU/g DS and also 0.01 to 10.0 GAU/g ds. Also a useful amount of glucoamylase in a granular starch hydrolyzing composition is about 0.1 to 5 GAU/g DS, also about 0.2 to 1.5 GAU/g DS.
The temperature of the saccharification will be conducted at a temperature below the temperature of gelatinization of the substrates. The exact temperature used in accordance with the methods of the invention depends upon the specific starch substrate used. General starch gelatinization temperature ranges are disclosed in Swinkels pages 32-38 in STARCH CONVERSION TECHNOLOGY, eds Van Beynum et al., (1985) Marcel Dekker Inc., NY and THE ALCOHOL TEXTBOOK, A REFERENCE FOR THE BEVERAGE, FUEL AND INDUSTRIAL ALCOHOL INDUSTRIES, 3rd Ed., eds Jacques et al., 1999, Nottingham University Press, UK. In some embodiments, the methods according to the invention will be conducted at a temperature of at least about 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., and 65° C. In other embodiments, the temperature will be between about 10-65° C., between about 20-60° C., between about 30-65° C., between about 35-65° C., between about 40-65° C., and between about 45-65° C. In other embodiments, the temperature will be between about 10-45° C., between about 20-45° C., between about 25-40° C., between about 30-40° C. and between about 30-35° C. In some preferred embodiments, the starch substrate is never subjected to the thermal conditions used for liquefactions and generally will be below 68° C. The temperature may vary during the saccharification process but the temperature will always be maintained at a temperature below the temperature of gelatinization for the substrate(s) used in the process.
In some embodiments, the methods according to the invention will be conducted at a pH of about pH 3.0 to 6.5; about pH 3.0 to 6.0; about pH 3.5 to 5.5, about pH 3.5 to 5.0; and about pH 3.5 to 4.5. In addition the pH during this step may vary. For example the pH may be increased during the saccharification. For example the pH may be increased from a pH of about 3 to 5.0 to a pH of about 4 to 6.
In some embodiments, the residence time of the method is from about 2 to 300 hrs, but more typically from 2 to 120 hours. In some embodiments, the process is conducted from about 5 to 100 hours. In other embodiments, the process is conducted from about 5 to 80 hours. In still other embodiments, the process is conducted for at least 5 hours but less than 100 hours.
In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98% and 99% of the dry solids of the insoluble starch is hydrolyzed. In some embodiments, the insoluble starch substrate is completely hydrolyzed. In some embodiments, at least 90% of the granular starch is hydrolyzed in 100 hours. In certain embodiments, at least 90% of the granular starch substrate is hydrolyzed in a time period of 24 hours. In other embodiments, at least 95% of the granular starch substrate is hydrolyzed in a time period of 24 hours.
In some embodiments, the saccharification step is followed by fermentation. The saccharified mash is contacted with a fermenting organism under suitable fermentation conditions. In some preferred embodiments, the fermenting organism is a commercially available yeast, such as a strain of Saccharomyces cerevisiae (U.S. Pat. No. 4,316,956). Commercial sources of yeast include FALI; RED STAR (Red Star); RED STAR RED (Red Star); FERMIOL (DSM Specialties) and SUPERSTART (Alltech). The amount of starter yeast employed in the methods is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time, (e.g. to produce at least 15% ethanol from an insoluble corn substrate having between 25-40% DS in less than 72 hours). The use of yeast in fermentation is well known and reference is made to THE ALCOHOL TEXTBOOK, K. JACQUES ET AL., EDS. 1999, NOTTINGHAM UNIVERSITY PRESS, UK.
The fermentation may be conducted under the same conditions of temperature, pH and the like as described above for saccharification.
In some preferred embodiments, the method comprises a simultaneous saccharification and fermentation (SSF), wherein the saccharification step and fermentation step are carried out contemporaneously. The culture medium, which includes the insoluble starch substrate contacted with a granular starch hydrolyzing composition and NSP24 protease either supplied in a cell free filtrate or expressed from a fungal host cell in the culture, will also include fermenting organism such as yeast.
In some embodiments, the residence time for the simultaneous saccharification and fermentation will be from about 2 to 300 hours, but preferably from 2 to 120 hours. In some embodiments, the time is from about 5 to 100 hours. In other embodiments, the time is from about 5 to 80 hours. In still other embodiments, the time is for at least 5 hours but less than 100 hours. In other embodiments, the process time is for at least about 10 hours but less 100 hours at a temperature between 20-50° C. and preferably between 25-40° C. and a pH of 3.5 to 6.0.
In some embodiments at least 5 vol %, at least 8 vol %, at least 10 vol %, at least 12 vol %, at least 15 vol %, at least 18 vol % and at least 20 vol % of ethanol will be produced in the fermentation media. In other embodiments, the amount of ethanol produced in the fermentation media (broth) will be between 5-30% (v/v), also between 5-25% (v/v), and between 5-20% (v/v). In some embodiments, the amount of ethanol produced will be at least 16% in 72 hours.
In some embodiments the amount of ethanol produced is enhanced compared to the amount of ethanol produced from a substantially similar method, which does not include an NSP24 protease. The ethanol production from the fermentations encompassed by the methods of the invention may be increased by at least 0.5%, at least 0.75%, at least 1.0%, at least 1.25%. at least 1.5%, at least 1.75% and at least 2.0% over a substantially similar method. A substantially similar method means a method conducted under the same conditions of temperature, pH, residence time, fermenting organism, granular starch hydrolyzing enzymes and the like. The only essential difference being the inclusion of the protease and specifically NSP24 protease in the saccharifying/fermenting media.
Following fermentation, alcohol (e.g., ethanol) may be recovered from the fermentation media by means well known in the art. In some embodiments, the alcohol in the fermented medium may be distilled, for example by heating or vacuum distillation. Reference is made to THE ALCOHOL TEXTBOOK, A REFERENCE FOR THE BEVERAGE, FUEL AND INDUSTRIAL ALCOHOL INDUSTRIES 3RD ED., K. JACQUES ET AL., EDS. 1999, NOTTINGHAM UNIVERSITY PRESS, UK.
The ethanol may then be used, for example, as fuel ethanol or potable ethanol. The remaining residue, known as stillage may also be recovered and components of the stillage recycled for the next fermentation or the stillage may be separated into a soluble fraction or insoluble fraction.
When the stillage is separated for example by centrifugation or screening into a soluble fraction and an insoluble fraction, these fractions can be used to make distillers' solubles or distillers' dried solubles or mixed together to make distillers' dried grain plus solubles (DDGS). One skilled in the art is familiar with processes for forming DDGS and distillers' grains in general. In some embodiments of the invention, when % ethanol increases in the fermentation process, the amount of DDGS decreases. In some embodiments the % residual starch at the end of the fermentation may decrease even without significant increase in ethanol production. In one embodiment, an advantage of the method encompassed by the invention may be an increase in the % total protein in the DDGS. The DDGS may then be used for example, in an animal feed formulation.
In another embodiment, an advantage of including a protease in the methods encompassed by the invention is a decrease in the residual starch in a yeast fermentation of granular starch containing substrates such as a milled whole grain or fractionated grain. High levels of residual starch in a fermentation media (broth) from yeast fermentation using granular starch containing substrates may potentially result in problems in the distillation step, which could cause poor separation of insoluble solids from solubles during centrifugation. In addition, high levels of residual starch could potentially cause higher viscosity. In some embodiments of the invention, the addition of a protease, preferably an NSP24 protease (e.g. NSP24), during yeast fermentation of a granular starch containing substrate (e.g. a grain such as fractionated or whole corn) results in a reduction of the residual starch in the fermentation broth. The fermentation broth may then be used advantageously for further processing.
In some embodiments, the fermentation process (e.g. SSF) encompassed by the invention will result in byproducts (e.g. DDGS) containing less than 30%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 2% and less than 1% residual starch. In some embodiments, the amount of residual starch produced by the methods of the invention after 72 hours will be less than 15%, less than 12%, less than 10%, less than 8% and less than 5%. In some embodiments of the invention, the amount of residual starch produced by the methods of the invention will be at least about 50%, at least about 40%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10%, at least about 5.0%, at least about 3.0%, and at least about 2.0% less than a substantially similar method conducted without a protease and particularly an NSP24 protease.
In another embodiment, the insoluble (granular) starch substrate may be pretreated prior to saccharification. Pretreatment may include exposing the insoluble starch substrate preferably in slurry form to an elevated temperature, wherein the temperature is below the starch gelatinization temperature of the starch substrate.
In some embodiments, the heat pretreatment may be at a temperature of below 68° C. but above 40° C. In some embodiments, the pretreatment will be between 40-65° C., also between 40-60° C., also between 45-55° C. and between 42-52° C., at a pH range of 3.0 to 6.0, also between pH 3.0 and 5.5, also between a pH of 3.5 to 5.5, also between pH of 3.5 to 5.0 and also between pH 3.8 to 4.5. In some embodiments, when corn is the substrate the pretreatment will be conducted at about 55 to 65° C. and at a pH of about 3.5 to 4.2. In other embodiments, when rye is the substrate the pretreatment will be conducted at about 40 to 50° C.
In some embodiments, the pretreatment will be conducted for 15 minutes to 24 hours, also from 30 minutes to 18 hours, also from 30 minutes to 12 hours, also from 30 minutes to 8 hours and also from 1 hour to less than 4 hours.
In addition to the heat pretreatment, different enzymes may be included in the pretreatment. In some embodiments, the pretreatment will include the use of an alpha amylase, such as bacterial alpha amylase (e.g., SPEZYME and GZYME 997 (Genencor International, Inc.)) and optionally a cellulase (e.g. Cellulase 2000). In other pretreatment embodiments, the enzyme may include granular starch hydrolyzing enzymes (e.g. alpha amylase derived from Aspergillus kawachi or granular hydrolyzing glucoamylase derived from Humicola grisea)).
In some embodiments, the pretreatment may include an acid protease, such as an NSP24 protease encompassed by the present invention. However, other acid proteases may be used including GC106 (Genencor International). In some embodiments, the amount of an acid protease used in a pretreatment will be in the range of 0.05 to 15 SAPU/g DS, also the dosage of 0.1 to 10 SAPU/g DS; 0.1 to 5 SAPU/g DS; and 0.25 to 5 SAPU/g DS find use in the invention. In some preferred embodiments, 0.5 to 2.0 SAPU/g DS and even 0.5 to 1.0 SAPU/g DS of an NSP24 protease will find use in the pretreatments of the invention. In some embodiments, the pretreatment may increase the amount of ethanol that is produced by the fermentation of the insoluble substrates as compared to the amount of ethanol produced by the fermentation of the insoluble substrate without a pretreatment but under substantially similar conditions. In some embodiments, the amount of ethanol will be increase by at least 0.3%, at least 0.5%, at least 0.75% or even at least 1.0%. In some embodiments, the amount of residual starch will be decreased by at least 1%, at least 2% at least 3%, a least 4%, at last 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% compared to the % of residual starch in a substantially similar fermentation without the pretreatment.
Additional enzymes may be added to the fermentations described above. These enzymes may be wild type enzymes, hybrids or variants of wild type enzymes including glucoamylases; alpha amylases; pullulanases; cellulases, such as endoglucanases; hemicellulases, such as mannases; lipases (e.g. E.C. 3.1.1.3); glucose oxidases; pectinases; xylanases; cutinases (e.g. E.C. 3.1.1.74); transglucosidases; transferases and alpha 1,6, glucosidases (e.g., E.C. 3.2.1.20). Some preferred glucoamylases useful in the methods of the invention are produced by several strains of filamentous fungi and yeast. In particular, glucoamylases secreted from strains of Aspergillus and Trichoderma are commercially important. Sources of these glucoamylases include: Aspergillus niger G1 and G2 glucoamylase and variants thereof (Boel et al., (1984) EMBO J. 3:1097-1102; WO 92/00381; WO 00/04136 and U.S. Pat. No. 6,352,851); Aspergillus awamori glucoamylases (WO 84/02921); Aspergillus oryzae glucoamylases and variants thereof (Hata et al., (1991) Agric. Biol. Chem. 55:941-949) and Aspergillus shirousami (See Chen et al., (1996) Prot. Eng. 9:499-505; Chen et al. (1995) Prot. Eng. 8:575-582; and Chen et al., (1994) Biochem J. 302:275-281). Glucoamylases are also obtained from strains of Talaromyces such as those derived from T. emersonii, T. leycettanus, T. duponti and T. thermophilus (WO 99/28488; U.S. Pat. No. No. RE: 32,153; and U.S. Pat. No. 4,587,215); strains of Rhizopus, such as R. niveus and R. oryzae; strains of Mucor; strains of Trichoderma, such as T. reesei and T. viride, strains of Humicola, such as H. grisea (See, Boel et al., (1984) EMBO J. 3:1097-1102; WO 92/00381; WO 00/04136; Chen et al., (1996) Prot. Eng. 9:499-505; Taylor et al., (1978) Carbohydrate Res. 61:301-308; U.S. Pat. No. 4,514,496; U.S. Pat. No. 4,092,434; and Jensen et al., (1988) Can. J. Microbiol. 34:218-223), and glucoamylase obtained from Athelia roltsii and variants thereof (WO 04/111218).
Other additional enzymes, which find use in the present invention, include debranching enzymes such as pullulanases (E.C. 3.2.1.41) and isoamylases (E.C. 3.2.1.68). Such enzymes hydrolyze alpha-1,6-glucosidic bonds. Thus, during the hydrolysis of the starch, debranching enzymes remove successive glucose units from the non-reducing ends of the starch. Another enzyme that may be used in the methods of the invention are beta-amylases (E.C. 3.2.1.2). These are exo-acting maltogenic amylases, which catalyze the hydrolysis of 1,4-alpha-glucosidic linkages in amylose, amylopectin and related glucose polymers. Some of these enzymes are characterized as having an optimum pH range from 4.5 to 7.0 and optimum temperature range from 40° C. to 65° C. Commercial beta-amylases are available for example SPEZYME BBA and OPTIMALT from Genencor International Inc.
Additional enzymes may include alpha amylases, which may or may not be characterized by having granular starch hydrolyzing activity. Examples of alpha amylases include both bacterial and fungal alpha amylases and variants thereof, such as alpha amylases from Bacillus amyloliquefaciens, Bacillus stearothermophilus, B. licheniformis and variants or hybrids thereof (U.S. Pat. No. 5,093,257; U.S. Pat. No. 6,093,562; U.S. Pat. No. 5,736,499; U.S. Pat. No. 5,958,739; U.S. Pat. No. 6,436,888; U.S. Pat. No. 6,867,031; WO 96/39528; WO 96/23874 and WO 05/001064). Commercially available alpha amylases are SPEZYME FRED and SPEZYME ETHYL (Genencor International Inc.). Cyclodextrin glucanotransferases (CGTases) (E.C. 2.4.1.19) and variants thereof may also find use in the invention (U.S. Pat. No. 5,278,059; U.S. Pat. No. 5,545,587 and WO 05/003337).
The effective amount of these enzymes to be included in the methods of the invention can be readily determined by one skilled in the art.
In some embodiments, an antimicrobial may be added to the compositions and fermentation medium of the invention. Antimicrobials are compounds that kill or inhibit the growth of microorganisms.
MATERIAL AND METHODS
In the experimental section that follows, the following abbreviations apply: NSP24 (NSP24 of the invention which is overexpressed in Trichoderma reesei; AnGA (DISTILLASE comprising an Aspergillus niger GA (Genencor International Inc.,)); GA (glucoamylase); TrGA (a GA obtained from Trichoderma reesei); STARGEN 001 (a granular hydrolyzing enzyme composition including glucoamylase and alpha amylase activity (Genencor International Inc.,)); AkAA (an Aspergillus kawachi alpha amylase) GAU (glucoamylase unit); SAPU (spectrophotometric acid protease unit) wt % (weight percent); ° C. (degrees Centigrade); rpm (revolutions per minute); H2O (water); dH2O (deionized water); dIH2O (deionized water, Milli-Q filtration); aa or AA (amino acid); bp (base pair); kb (kilobase pair); kD or kDa (kilodaltons); g or gm (grams); μg (micrograms); mg (milligrams); μL (microliters); ml and mL (milliliters); mm (millimeters); μm (micrometer); M (molar); mM (millimolar); μM (micromolar); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); DO (dissolved oxygen); kg (kilogram); MT (Metric ton) and EtOH (ethanol).
The following assays and methods are used in the examples provided below:
Ethanol and Carbohydrate Determinations
Ethanol and carbohydrate composition of the samples were determined using the HPLC method as described herein:

    a) a 1.5 mL Eppendorf centrifuge tube was filled with fermentor beer and cooled on ice for 10 min;
    b) the sample tube was centrifuged for 1 min in Eppendorf table top centrifuge;
    c) a 0.5 mL sample of the supernatant was transferred to a test tube containing 0.05 mL of Kill solution (1.11N H2SO4) and allowed to stand for 5 min;
    d) 5.0 mL of water is added to the test tube sample and then filtered into a HPLC vial through 0.45 μm Nylon Syringe Filter; and
    e) run on HPLC.

HPLC Conditions:



a) Ethanol System: Column: Phenomenex Rezex Organic Acid Column (RHM-Monosaccharide) #00H 0132-KO (Equivalent to Bio-Rad 87H); Column Temperature: 60° C.; Mobile Phase: 0.01 N H2SO4; Flow Rate: 0.6 mL/min; Detector: RI; and Injection Volume: 20 μL.
b) Carbohydrate System: Column: Phenomenex Rezex Carbohydrate (RCM-Monosaccharide) #00H-0130-KO (Equivalent to Bio-Rad 87H); Column Temperature: 70° C.; Mobile Phase: Nanopure DI H2O; Flow Rate: 0.8 mL/min; Detector: RI; Injection Volume: 10 μL (3% DS material)
The column separates based on the molecular weight of the saccharides, which are designated as DP-1 (monosaccharides); DP-2 (disaccharides); DP-3 (trisaccharides) and DP>3 (oligosaccharide sugars having a degree of polymerization greater than 3).
Residual starch iodine test: A sample of the beer (fermentation broth) was centrifuged in 2 ml plastic centrifuge tubes. The supernatant was decanted and the tube containing the pellet was placed in an ice bath. Several drops of 0.025N iodine solution (0.1N iodine from VWR Cat. No. VW3207-1 diluted 4×) was added to the pellet and mixed. A positive (+) starch shows a range of color from blue to purple and the intensity of color is directly proportional to the concentration of starch. A negative result (−) remains yellowish.

Determination of total starch content: The enzyme-enzyme starch liquefaction and saccharification process (dual enzyme method) was used to determine the total starch content. In a typical analysis, 2 g of the dry sample was taken in a 100 ml Kohlraucsh flask and 45 ml of MOPS buffer, pH 7.0 was added. The slurry was well stirred for 30 min. SPEZYMETM FRED (1:50 diluted in water), 1.0 ml was added and heated to boiling for 3-5 min. The flask was placed in an autoclave maintained at 121° C. for 15 min. After autoclaving the flask was placed in a water bath at 95° C. and 1 ml of 1:50 diluted SPEZYME FRED was added and incubated for 45 min. The pH was adjusted to pH 4.2 and the temperature was reduced to 60° C. This was followed by addition of 20 ml acetate buffer, pH 4.2. Saccharification was carried out by adding 1.0 ml of 1:100 diluted OPTIDEXTM L-400 (Glucoamylase, Genencor International Inc.) and the incubation was continued for 18 hr at 60° C. The enzyme reaction was terminated by heating at 95° C. for 10 min. The total sugar composition was determined by HPLC analysis using glucose as a standard. The soluble starch hydrolysate from water extraction of a sample at room temperature without enzymatic treatment was subtracted from the total sugar.

Total protein analysis: The total nitrogen (N) in the sample preparations was determined using the Kjeldhal method (American Assoc. Cereal Chemists (AACC), (1983), Methods 22B60 8th Ed. St Paul, Minn.). Protein content was calculated by 6.25× total N.

EXAMPLES
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspect of the present invention and are not to be construed as limiting the scope thereof.
Example 1
Cloning of Protease, NSP24 and Transformation into Trichoderma reesei 
Genomic DNA was extracted from T. reesei strain QM6a. PCR primers were designed, based on the putative protease sequence found in contig 1-5500 of the T. reesei genome (Joint Genome Institute (JGI) T. reesei genome v1.0). The forward primer contained a motif for directional cloning into the pENTR/D vector (Invitrogen).
The sequence of the afp6f primer was CACCATGCAGACCTTTGGAGCT (SEQ ID NO: 5), and the sequence of the afp7r primer was TTATTTCTGAGCCCAGCCCAG (SEQ ID NO: 6). The 1.3 kb PCR product was purified by gel extraction (Gel Purification kit, Qiagen) and cloned into pENTR/D, according to the Invitrogen Gateway system protocol.
The vector was then transformed into chemically competent Top10 E. coli (Invitrogen) with kanamycin selection. Plasmid DNA, from several independent clones, was digested with restriction enzymes to confirm the correct size insert. The protease gene insert was sequenced (Sequetech, Mountain View, Calif.) from several clones. Plasmid DNA from one clone, pENTR/D—55.3, was added to the LR clonase reaction (Invitrogen Gateway system) with pTrex3g/amdS destination vector DNA. The pTrex3g vector is based on the E. coli pSL1180 (Pharmacia Inc., NJ), which is a pUC118 phagemid based vector and is described in WO 05/001036. Recombination, in the LR clonase reaction, replaced the CmR and ccdB genes of the destination vector with the T. reesei protease from pENTR/D—55.3. This recombination directionally inserted protease between the cbhI promoter and terminator of the destination vector. Recombination site sequences of 44 and 50 bp remained upstream and downstream, respectively, of the protease gene. An aliquot of the LR clonase reaction was transformed into chemically competent Top10 E. coli and grown overnight with carbenicillin selection. Plasmid DNA from several clones was digested with restriction enzymes to confirm the correct insert size. Plasmid DNA from clone, pTrex3g—55.3.1 was digested with XbaI to release the expression cassette including the cbhI promoter:NSP24 protease:terminator:amdS. This 5.8 kb cassette was purified by agarose gel extraction, using standard techniques, and transformed into a strain of T. reesei derived from the publicly available strain QM6a (See, WO 05/001036). Reference is made to FIGS. 1, 2 and 3.
A 2 cm2 agar plug from a plate of sporulated mycelia was inoculated into 50 ml of YEG broth in a 250 ml, 4-baffled shake flask and incubated at 37° C. for 16-20 hours at 200 rpm. The mycelia were recovered by transferring liquid volume into 50 ml conical tubes and spun at 2500 rpm for 10 minutes. The supernatant was aspirated off. The mycelial pellet was transferred into a 250 ml, 0.22 μm CA Corning filter bottle containing 40 ml of filter-sterilized β-D-glucanase (InterSpex Products, Inc.) solution and incubated at 30° C., 200 rpm for 2 hours. The mycelia were harvested through sterile Miracloth (CalBiochem, LaJolla, Calif.) into a 50 ml conical centrifuge tube, centrifuged at 2000 rpm for 5 minutes, aspirated. The pellet was washed once with 50 ml of 1.2M sorbitol, centrifuged again, aspirated, and washed with 25 ml of sorbitol/CaCl2. The protoplasts were counted using a hemocytometer, centrifuged, aspirated, and resuspended in a volume of sorbitol/CaCl2 sufficient to generate a protoplast concentration of 1.25×108/ml. Aliquots of 200 μl were used per transformation reaction. 20 μg of DNA (≧1 μg/ul) was placed into 15 ml conical tubes and the tubes were placed on ice. 200 μl of the protoplasts were added. 50 μl PEG mix was added and mixed gently and incubated on ice for 20 minutes. 2 ml of PEG mix was added to the tubes and incubated at room temperature for 5 minutes. 4 ml sorbitol/CaCl2 (for a total of 6.25 ml) was added to the tubes. This transformation mixture was divided into 3 aliquots of  ̃2 ml per each overlay. The 2 ml was added to a tube of melted acetamide sorbitol top agar and the overlay mixture was poured onto acetamide sorbitol plates for selection of transformants able to grow with acetamide as the sole nitrogen source. Plates were incubated at 28-30° C. until colonies appeared. Transformants were purified by repeat passage of single colonies on acetamide media (acetamide sorbitol recipe without the sorbitol).
Materials—
40 ml β-D-glucanase Solution: 600 mg β-D-glucanase; 400 mg MgSO4.7H2O and 40 ml 1.2 M sorbitol.
200 ml PEG Mix: 50 g PEG 4000 (BDH Laboratory Supplies Poole, England) and 1.47 g CaCl2 2H2O made up in Milli Q water
Sorbitol/CaCl2: 1.2M Sorbitol and 50 mM CaCl2 
For amdS selection, Acetamide Sorbitol plates and overlays were used. For spore purification, the same plates were used, but without sorbitol.
Acetamide Sorbitol Agar (Plates and Top Agar)
Acetamide (Aldrich 99% sublimed)—0.6 g/L; CsCl—1.68 g/L; Glucose—20 g/L; KH2PO4—20 g/L; MgSO4.7H2O—0.6 g/L; CaCl2.2H2O—0.6 g/L; 1000× salts (see below)—1 ml. pH adjusted to 5.5 and volume brought to 300 ml. Filter sterilized with 0.22 micron filter and warmed to 55° C. in an oven.
To 700 ml water Noble Agar (low-melt for top agar) 20 g and Sorbitol 218 g was added and then autoclaved. This mixture was cooled to 55° C., and filter sterilized, acetamide mix was added. Plates or tubes were poured.
1000× Salts—FeSO4.7H2O (0.5 g/100 ml); MnSO4.H2O (0.16 g/100 ml); ZnSO4.7H2O (0.14 g/100 ml); CoCl2.6H2O (0.1 g/100 ml) and filter sterilize with 0.22 micron filter.
Potato Dextrose Agar (PDA, Difco Dehydrated Culture Media)—Potatoes, infusion from 200 g/L; Dextrose, 20 g/L and Agar, 15 g/L were mixed well in 50-80% final volume of dH2O, and then brought to 100% final volume. This mixture was autoclaved, cooled to 55° C. and poured.

To make up 1% skim milk agar for a pH 3.5 media PDA was prepared as above and to 100 ml molten PDA, 1.8 ml 10% tartaric acid and 12.5 ml sterilized 8% skim milk was added and plates were poured. To pre-sterilize skim milk, 8% skim milk (Difco) was autoclaved for 10 minutes, 122-123° C., and chamber pressure during exposure of 32-35 psi. The mixture was removed, cooled and stored at room temperature.

Protease Expression was evaluated in transformants after 3 days growth in shake flasks. T. reesei culture media (Davis, et al., (1970) Methods Enzymol. 17:79-143) was inoculated with an agar plug. Cultures were incubated for 3 days at 30° C., with shaking. Culture broth was passed through a 0.22 micron filter, and the filtrate spotted onto 1% Skim milk agar. Clearing zones were observed following overnight incubation at room temperature.
Example 2
Effect of Protease During Hydrolysis of Granular Starch Substrate
Ground corn (#2 Yellow Dent), which is typically used in the ethanol industry was fractionated to obtain the endosperm fraction. Both the corn and endosperm/gluten fraction were ground to obtain a sample that would pass >95% through a 1.5 mm screen (Perten Laboratory Mill 3100, Sweden). The moisture content and the total starch content of whole ground and endosperm fractions were determined (each flask contained equal amounts of starch (32% DS for ground corn and 29.9% DS for the endosperm fraction). The pH was adjusted to 4.2 using 6NH2SO4. AnGA (DISTILLASE L-400, Genencor International, Inc.) or a TrGA added at 1.0 GAU/g ds and acid stable alpha amylase from Aspergillus kawachi (AkAA) obtained from expression in Trichoderma as described in USP 2005/0266543 was added at 3 SSU/g DS. NSP24 was added at 0.5 SAPU/g DS along with 400 ppm urea. 5.0 g Red Start Ethanol Red dry yeast (Lesaffre Yeast Corporation, Milwaukee, Wis.) in 45 ml of DI H2O was prepared and mixed in a 32 C water bath for 1 hour prior to inoculating the reaction flasks with 0.5 ml of the yeast slurry. The flasks were placed in a 32° C. water bath and the mash (slurry) mixed gently during the entire fermentation period. Samples were removed during the fermentation for HPLC analysis using HPLC column: Phenomenex Rezex Organic Acid Column (RHM-Monosaccaride) #00H 0132-KO (Equivalent to Bio-Rad 87H); Column temperature: 60 C; Mobile Phase: 0.01N H2SO4; Flow rate 0/6 mL/min; Detector: RI; and Injection Volume: 20 uL. The fermentation was terminated after 72 hours. The HPLC profiles for sugars, lactic acids glycerol and ethanol at various sample intervals is shown in Table 1. The mash was dried at 60° C. to obtain the DDGS and the starch content of the DDGS was determined by the dual enzyme method.







TABLE 1







% Ethanol Production and % Residual Starch in Fermentation with or without NSP24





















% Residual


GA-

% EtOH
% EtOH
% EtOH
% EtOH
% EtOH
Starch


source
NSP24
17 hr
25 hr
49 hr
65 hr
72 hr
72 hr

















AnGA









Whole
−−
9.18
10.51
16.16
17.77
17.67
3.38


ground
+
10.52
11.87
17.61
18.41
18.60
2.03


corn


Endosperm
−−
7.73
9.91
14.01
15.46
15.69
2.89



+
11.06
13.61
17.31
18.24
18.34
0.55


TrGA


Whole
−−
7.73
9.93
14.00
15.95
16.36
8.45


ground
+
8.11
10.51
15.04
17.05
17.40
6.49


corn


Endosperm
−−
7.31
9.47
13.31
14.91
15.33
4.50



+
10.51
12.97
17.11
17.95
18.14
1.15









The addition of NSP24 to the yeast fermentation medium containing granular starch substrate resulted in a higher level of alcohol and a lower level of residual starch content in the DDGS. The effect is even more pronounced with the fractionated corn endosperm compared to whole ground corn.

Example 3
Effect of protease on the alcohol yield and the residual starch content of the DDGS during the fermentation of granular starch containing substrates, whole ground corn and fractionated corn (degermed corn) with different particle sizes.
Yellow Dent corn # 2 was ground to obtain ground whole corn with varying particle sizes using a laboratory scale hammer mill (Perten Laboratory Mill 3100, Sweden) and degermed corn was used as an example for fractionated corn. The particle size distribution was measured using a standard sieve with different pore sizes. In Table 2 below 95% means about 95% of the particles passed through a 30 mesh screen (0.59 mm) and about 5% did not pass through the screen; they were reatined on the screen; 70% means about 70% of the particles passed through a 30 mesh screen (0.59 mm) and about 30% did not pass through the screen; they were retained on the screen; and 50% means about 50% of the particles passed through a 30 mesh screen (0.59 mm) and about 50% did not pass through the screen; they were reatined on the screen. Determinations were also made of the moisture content and the total starch content of whole ground corn. Fermentations were conducted in 125 ml flasks containing 100 g mash of ground whole corn (32% ds) or degermed fractionated corn (29.6% ds). Urea (400 ppm) was added to each of the reaction flasks and the pH was adjusted to 4.2 using 6 N H2SO4. STARGENTM 001, was added at 2.5 kg/MT corn DS. NSP24 was added at 0.5 SAPU/g ds. Five grams of Red Star Ethanol Red dry yeast (Lesaffre Yeast Corporation, Milwaukee, Wis.) in 45 mls of water was prepared and mixed in a 32° C. water bath for one hour prior to inoculating the flask; 0.5 ml of this yeast slurry was added to each flask. The flasks were placed in a 32° C. water bath and the mash mixed gently during the entire fermentation period. Samples were removed during fermentation for HPLC analysis using HPLC Column: Phenomenex Rezex Organic Acid Column (RHM-Monosaccharide) #OOH 0132-KO (Equivalent to Bio-Rad 87H); Column Temperature: 60° C.; Mobile Phase: 0.01 N H2SO4; Flow Rate: 0.6 mL/min; Detector: RI; and Injection Volume: 20 μL. The fermentations were terminated after 72 hours. The yeast fermentations produced a number of compounds including sugars, lactic acid, glycerol and ethanol at various sampling intervals and some of the levels as determined by HPLC are shown below. The mash was dried at 60° C. to obtain the DDGS, and the starch content of the DDGS at the 72 hour fermentation broth was determined by the dual enzyme method.







TABLE 2







Effect of NSP24 on the ethanol yield and the residual starch content


of the DDGS during the fermentation of granular starch containing whole


ground corn or fractionated corn containing different particle sizes

















% Residual




%


Starch




EtOH
% EtOH
% EtOH
DDGS



NSP24
24 hr
48 hr
71 hr
72 hr
















Whole ground







Corn


Particle size*


95%
−−
11.33
15.19
15.73
9.08



+
13.53
17.47
17.46
7.72






(1.73)


70%
−−
11.23
15.47
16.82
4.66



+
14.24
17.81
18.03
2.37






(1.21)


50%
−−
10.85
14.96
16.49
26.21



+
12.69
16.84
17.67
13.83






(1.18)


Corn Endosperm


Particle Size*


95%
−−
7.91
12.71
14.77
10.35



+
13.85
17.93
18.27
2.00






(3.50)


70%
−−
7.51
12.42
14.60
17.78



+
13.47
17.68
18.38
1.18






(3.78)


50%
−−
7.74
12.59
14.80
11.20



+
13.33
17.03
17.99
1.63






(3.19)





1. *is defined in example 3


2. + represents samples with NSP24 and −− represents samples without NSP24 addition.


3. ( ) represents the difference in the ethanol yield at 71 hrs between the +NSP24 samples and the −−NSP24 samples for the same granular starch substrate and the same particle size.






From the table it is observed that protease addition during the fermentation of the granular starch substrates (whole ground corn or fractionated corn) is a higher alcohol yield and lower residual starch in the DDGS. The effect is even more pronounced with coarser ground particles for ground whole or fractionated corn. These results suggest that the addition of protease allows one to use a coarser particle size of the grain substrates.

Example 4
Effect of Protease Concentration During Fermentation with a Medium Containing a Degermed Ground Corn Fraction (Endosperm)
100 g of a 29.5% DS mash of endosperm (degermed corn, 75.8% starch, particle size of 99.5%<30 mesh) was transferred to a 125 ml flask, 400 ppm of urea was added and the pH was adjusted to pH 4.5. NSP24 (1.0 SAPU/g ds) was added and followed by the addition of STARGEN 001 (Genencor International) at 2.8 Kgs/MT of starch. Flasks were inoculated with 0.5 ml of 20% yeast (Red Star Ethanol Red) and then placed in a water bath maintained at 32° C. The contents of the flasks were continuously stirred for uniform mixing during incubation. Samples were taken at different intervals of time for HPLC analysis. The residual starch and protein content of the DDGS at 72 hours was determined and some of the results are shown below.







TABLE 3







Effect of protease addition during the fermentation medium


containing degermed fractionated corn on the final alcohol


yield and residual starch in the DDGS.













NSP24
Urea

% w/v
% w/v
% v/v
% Starch


kg/MT
ppm
hrs
DP > 2
DP-1
EtOH
DDGS
















0
400
22
0.24
2.72
10.15





46
0.21
2.92
14.57




72
0.18
1.35
16.45
11.62


0.09
400
22
0.24
1.67
11.70




46
0.18
1.13
16.11




72
0.16
0.01
17.60
1.67


0.13
400
22
0.24
1.41
12.44




46
0.19
1.14
16.33




72
0.21
0.01
17.49
0.57


0.26
400
22
0.21
0.92
13.17




46
0.18
0.04
17.39




72
0.19
0.01
17.96
0.49


0.43
400
22
0.21
0.77
13.10




46
0.18
0.02
17.48




72
0.20
0.01
18.30
0.35


0.26
600
22
0.21
0.83
13.33




46
0.28
0.02
17.77




72
0.17
0.02
18.20
0.31


0.26
800
22
0.19
0.32
13.56




46
0.17
0.04
18.21




72
0.17
0.02
18.42
0.58


0.26
1000
22
0.21
0.60
13.62




46
0.18
0.01
18.15




72
0.18
0.01
18.57
0.34









As observed from the results illustrated above, the addition of NSP24 resulted in a reduction in the levels of residual starch content at the end of the fermentation. Addition of NSP24 to the fermentation medium containing non-cooked degermed fractionated corn also resulted in a faster fermentation rate and higher alcohol yield.

Example 5
Effect of Protease in a Yeast Fermentation Containing Granular Starch Substrate and Different Levels of Backset or Evaporator Concentrate
A 30% ds mash in water and water containing different percentages of backset, wherein the backset was obtained from a typical dry grind conventional fermentation was were prepared using a fractionated corn endosperm (containing 78.51% starch DS and moisture content _%). Fermentations were conducted in 125 ml flasks containing 100 g mash of endosperm. The pH was adjusted to 4.2 using 6 N H2SO4. GA from DISTILLASE L-400 (Genencor International Inc) was added at 1.0 GAU/gds and an acid stable alpha amylase from Aspergillus kawachi (US Pat. Pub. 2005/0266543) was added at 3 SSU/g ds. NSP24 was added at 0.5 SAPU/g ds. Five grams of Red Star Ethanol Red dry yeast (Lesaffre Yeast Corporation, Milwaukee, Wis.) in 45 mls of water was prepared and mixed in a 32° C. water bath for one hour prior to inoculating the flask; 0.5 ml of the yeast slurry was added to each flask. The flasks were placed in a 32° C. water bath and the mash mixed gently during entire fermentation period. During the fermentation, samples were removed for HPLC analysis using HPLC Column: Phenomenex Rezex Organic Acid Column (RHM-Monosaccharide) #00H 0132-KO (Equivalent to Bio-Rad 87H); Column Temperature: 60° C.; Mobile Phase: 0.01 N H2SO4; Flow Rate: 0.6 mL/min; Detector: RI; and Injection Volume: 20 μL. The fermentations were terminated after 72 hours. The HPLC profile was determined for compounds produced during the fermentation including sugars, lactic acid, glycerol and ethanol at various sampling intervals. The mash was dried at 60° C. to obtain the DDGS, and the starch content of the DDG S from 72 hr fermentation broth was determined by the dual enzyme method. Some of the data is presented below in Table 4 and FIG. 5.







TABLE 4







Effect of protease in a yeast fermentation containing fractionated


corn as the granular starch substrate with different levels


of backset or condensate water.



















% Residual




% v/v
% v/v
% v/v
% v/v
Starch




EtOH
EtOH
EtOH
EtOH
DDGS at


% Backset
NSP24
21.5 hr
28 hr
53 hr
72 hr
72 hr
















0
−−
6.16
7.99
12.69
14.17
3.92



+
11.47
14.45
18.25
18.55
0.62


20
−−
7.68
19.81
14.22
15.71
4.31



+
12.56
15.13
19.01
17.96
0.52


40
−−
8.65
10.7
14.89
15.8
4.06



+
12.42
14.68
18.72
18.6
0.88


60
−−
9.72
11.41
15.56
16.22
3.71



+
11.77
14.48
18.31
18.12
1.39









Example 6
Effect of Dry Solids (ds) of Fractionated Corn on the Final Alcohol Yield and the Residual Starch Under Yeast Fermentation Conditions with Added Protease
The ground whole corn % DS was run at 32%, 34%, 36%, 38% and 40% and the fractionated corn endosperm % DS was run at 30%, 32%, 34% and 36%. Fermentations were conducted in 125 ml flasks containing 100 g mash of endosperm. The pH was adjusted to 4.2 using 6 N H2SO4. 400 ppm of urea was added and STARGENTM 001 (Genencor International Inc.) was added at 1.39 GAU/g ds. NSP24 was added at 0.5 SAPU/g ds. Five grams of Red Star Ethanol Red dry yeast (Lesaffre Yeast Corporation, Milwaukee, Wis.) in 45 mls of water was prepared and mixed in a 32° C. water bath for one hour prior to inoculating the flask. 0.5 ml of the yeast slurry was added to each flask. The flasks were then placed in a 32° C. water bath for 36 hrs with continuous mixing and then the temperature of was decreased to 25° C. and maintained for 72 hours. During the fermentation, samples were removed for HPLC analysis under the conditions described in example 5. The fermentations were terminated after 72 hours. The HPLC profiles for the compounds produced during the fermentation, including sugars, lactic acid, glycerol and ethanol at various sampling intervals was determined. The mash was dried at 60° C. to obtain the DDGS, and the starch content of the DDG S from 72 hr fermentation broth was determined by the dual enzyme method. Some of the data is present in Table 5 and FIG. 6.







TABLE 5







Effect of NSP24 and fractionated corn DS on the production


ethanol and residual starch under yeast fermentation conditions


with granular starch hydrolyzing enzymes











% Residual



% Alcohol (v/v)
starch














% DS
NSP24
24 hr
48 hr
72 hr
72 hr


















30
−−
9.99
14.76
17.91
18.41




+
12.73
18.39
19.17
2.76



32
−−
10.23
15.55
18.01
12.47




+
13.41
18.71
19.96
2.73



34
−−
10.79
16.2
19.46
15.05




+
14.4
21.18
21.68
11.32



36
−−
12.32
17.78
20.69
15.73




+
15.21
20.96
22.33
16.21










Example 7
Residual Starch and Ethanol Production with and Without NSP24 Pretreatments at Different pH Levels
A 29% ds whole ground corn substrate (de-oil) was prepared using tap water. The moisture content of the milled corn was 15.77%. For the pretreatment, the mash was adjusted to a pH of 4.5 or 3.7 at 60° C. in a water bath maintained for 60 min. Pretreatment (PT) included addition of STARGEN 001 (Genencor International, 0.75 GAU/g AnGA) with or without NSP24 dosed at 0.2 kg/MT. After pretreatment water was added to adjust for evaporation. The pretreated (PT) samples and samples of a milled corn slurry that was not pretreated (No PT) were transferred to a flask for yeast fermentation. The yeast fermentation was carried out by adding 400 ppm of urea and 0.75 GAU/g of STARGEN 001 in the presence of 0.2 kg/MT NSP24 or in the absence of NSP24 at either pH 4.5 or 3.7 at 30° C. Samples were taken at different time intervals for HPLC analysis of ethanol as previously described. The residual starch (RS) in the fermentation broth at 68 hours was measured using the dual enzyme method, and the results are shown below in Table 6.












TABLE 6






% v/v
%






EtOH
v/v EtOH
% v/v EtOH
% v/v EtOH



Treatment
24 hrs
43 hrs
50 hrs
68 hrs
% RS




















PT pH 4.5
10.33
14.31
14.64
15.04
8.36


(−NSP24)


PT pH 4.5
10.48
13.64
14.34
14.84
9.12


(+NSP24)


PT, pH 3.7
10.52
13.61
14.32
14.90
8.80


(−NSP24)


PT pH 3.7
10.82
14.57
15.06
16.23
5.81


(+NSP24)


No PT pH 4.5
9.89
12.94
13.50
14.31
12.81


(−NSP24)


No PT, pH 4.5
9.85
13.19
14.37
14.65
11.90


(+NSP24)


No PT, pH 3.7
9.98
13.35
13.93
14.75
11.22


(−NSP24)


No PT, pH 3.7
10.41
14.03
14.74
15.66
10.45


(+NSP 24)

## Claims

1. A method for producing ethanol comprising:
a) saccharifying a milled insoluble starch substrate obtained from cereal grain at a temperature below the starch gelatinization temperature of the substrate with an isolated granular starch hydrolyzing enzyme composition and an isolated NSP 24 protease enzyme, wherein said NSP24 protease has at least 95% amino acid sequence identity to SEQ ID NO:3 and has protease activity; and then
b) fermenting the saccharified substrate under suitable fermentation conditions to produce at least 8% v/v ethanol.

2. The method according to claim 1, wherein the NSP24 protease comprises an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 3.

3. The method according to claim 2, wherein the NSP24 protease comprises an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 3.

4. The method according to claim 3, wherein the NSP24 protease comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 3.

5. The method according to claim 4, wherein the NSP24 protease comprises the amino acid sequence of SEQ ID NO: 3.

6. The method according to claim 1, wherein the granular starch hydrolyzing enzyme composition comprises a glucoamylase.

7. The method according to claim 1, wherein the granular starch hydrolyzing enzyme composition comprises an alpha amylase.

8. The method according to claim 7, wherein the alpha amylase is derived from an Aspergillus sp.

9. The method according to claim 1, wherein the milled insoluble starch substrate comprises fractionated grains.

10. The method according to claim 9, wherein the fractionated grain is corn.

11. The method according to claim 1, wherein at least 15% v/v ethanol is produced from the fermentation of a milled grain with 25% to 35% DS.

12. The method according to claim 1, wherein the saccharification and fermentation steps are carried out simultaneously.

13. The method according to claim 1, wherein the temperature below the starch gelatinization temperature is between 30° C. to 65° C.

14. The method according to claim 1 further comprising pretreating the milled insoluble starch substrate at a temperature of about 55° C. to 65° C. for about 30 minutes to 4 hours.

15. The method according to claim 14, wherein the pretreatment further comprises contacting the milled insoluble starch substrate with an enzyme composition.

16. The method according to claim 1 further comprising recovering the produced ethanol.

17. The method according to claim 1, wherein at least 12% v/v ethanol is produced.

18. The method according to claim 17, wherein at least 15% v/v ethanol is produced.

19. The method according to claim 1, wherein
the cereal grain is from corn,
the temperature below the starch gelatinization temperature of the substrate is a temperature less than 68° C.,
the granular starch hydrolyzing enzyme composition is obtained from an alpha amylase or a glucoamylase derived from an Aspergillus sp, a Humicola sp, a Rhizopus sp and/or a Trichoderma sp., and
the NSP24 protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3 and has protease activity.

20. The method according to claim 19 further comprising recovering the ethanol.