BR112017013172B1 - METHOD FOR SACCHARIFICATION OF A STARCH SUBSTRATE - Google Patents

Abstract

Methods of saccharifying a starch substrate are provided, comprising contacting the starch substrate with a glucoamylase consisting of or comprising the amino acid sequence of Fusarium verticillioides glucoamylase (Fv-GA) and further contacting the starch substrate with at least one additional glucoamylase. Additional methods of saccharifying and fermenting a starch substrate to prepare a final product, a biochemical final product, and a fermentable beverage using a combination of Fv-GA and at least one additional glucoamylase are provided.

Description

SEQUENCE LISTING

[001] Also attached is a listing of sequences comprising SEQ ID NOs: 1-12, which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present disclosure relates generically to glucoamylase (GA) enzymes and methods of using GAs are also described, particularly in mixtures, in processes relevant to the production of sugars, syrups and biochemical products, including ethanol.

BACKGROUND OF THE INVENTION

[003] Industrial fermentations predominantly employ glucose as a raw material for the production of a plurality of proteins, enzymes, alcohols and other chemical end products. Typically, glucose is the product of starch processing, which is conventionally a two-step enzymatic process that catalyzes the degradation of starch, involving liquefaction and saccharification.

[004] During liquefaction, insoluble granular starch is transformed into a paste in water, gelatinized with heat and hydrolyzed by a thermostable alpha-amylase. During saccharification, the soluble dextrins produced in liquefaction are further hydrolyzed by glucoamylases, producing a high-glucose syrup containing more than 95% glucose.

[005] Glucoamylases are exo-acting carbohydrases, capable of hydrolyzing both linear and branched glucosidic bonds in starch (e.g., amylose and amylopectin) to produce fermentable sugars from starch (e.g., a starch substrate liquefied by enzymes ). Fermentable sugars, e.g. low molecular weight sugars such as glucose, can then be converted to fructose by other enzymes (e.g. glucose isomerases); crystallized; or used in fermentations to produce numerous end products (e.g., alcohols, monosodium glutamate, succinic acid, vitamins, amino acids, 1,3-propanediol, and lactic acid).

[006] Considering the central role that glucoamylases play in the generation of glucose from starch, it will be an advantage in the art to provide new mixtures of glucoamylase (GA) enzymes with improved properties for this conversion. Examples of improved properties of GA mixtures include, but are not limited to: production of sugars, syrups and biochemicals, including but not limited to ethanol.

SUMMARY OF THE INVENTION

[007] Methods of saccharifying a starch substrate are provided, comprising contacting the starch substrate with a glucoamylase consisting of or comprising the amino acid sequence of Fusarium verticillioides glucoamylase (Fv-GA) and further contacting the starch substrate with hair minus one additional glucoamylase. Additional methods of saccharifying and fermenting a starch substrate to prepare a final product, a biochemical final product, and a fermentable beverage using a combination of Fv-GA and at least one additional glucoamylase are provided. Embodiments of the invention include: 1. A method of saccharifying a starch substrate, comprising contacting the starch substrate with a glucoamylase consisting of the Fv-GA sequence (SEQ ID NO: 1), having at least 85% identity with the sequence of Fv-GA (SEQ ID NO: 1), having at least 85% identity with a catalytic domain of Fv-GA (SEQ ID NO: 1), having at least 85% identity with a linker and a catalytic domain of Fv-GA (SEQ ID NO: 1) or having at least 85% identity with an active fragment of Fv-GA (SEQ ID NO: 1) having glucoamylase activity and contacting the starch substrate with at least one additional glucoamylase .

[008] In some embodiments of method 1, the first listed glucoamylase has 90%, 95%, 98% or 99% identity with the Fv-GA sequence (SEQ ID NO: 1). 2. The method of 1, wherein the additional glucoamylase comprises Afu-GA (SEQ ID NO: 3) or An-GA (SEQ ID NO: 7). 3. The method of 1 or 2, wherein the additional glucoamylase is Afu-GA (SEQ ID NO: 3) or a glucoamylase having at least 85% identity with SEQ ID NO: 3. 4. The method of 1 or 2 2, wherein the additional glucoamylase is An-GA (SEQ ID NO: 7) or a glucoamylase having at least 85% identity with SEQ ID NO: 7. 5. The method of any one of 1 - 4, wherein saccharification of the starch substrate yields a syrup rich in glucose. 6. The method of any one of 1-5, wherein the high glucose syrup comprises an amount of glucose selected from the list consisting of at least 95.5% glucose, at least 95.6% glucose, at least 95 .7% glucose, at least 95.8% glucose, at least 95.9% glucose, at least 96% glucose, at least 96.1% glucose, at least 96.2% glucose, at least at least 96.3% glucose, at least 96.4% glucose, at least 96.5% glucose and at least 97% glucose. 7. The method of any one of 1-6, further comprising fermenting the glucose-rich syrup into a final product. 8. The method of 7, in which saccharification and fermentation are carried out as a simultaneous saccharification and fermentation process (SSF). 9. Method 7 or 8, in which the final product is alcohol. 10. Method 9, in which the final product is ethanol. 11. The anyone from 1 - 10 method, in which the starch substrate is about 5% to 99%, 15% to 50%, or 40-99% dry solids (DS). 12. The method of any one of 1-11, wherein each glucoamylase is dosed at a range of about 0.01 to about 10 glucoamylase units (GAU) per gram of dry starch solids (dss). 13. The method of any one of 1 - 12, in which the starch substrate is selected from wheat, barley, corn, rye, rice, sorghum, bran, cassava, grain sorghum Milo, millet, potato, sweet potato, tapioca and any combination of these. 14. The method of any one of 1 - 13, wherein the starch substrate comprises liquefied starch, gelatinized starch or granular starch. 15. The method of any one of 1 - 14, further comprising adding to the starch substrate a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a pullulanase, a β-amylase, an α- amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a hydrolase, an alpha-glucosidase, a beta-glucosidase or a combination of these. 16. The method of 15, in which a pullulanase is also added. 17. A method of saccharifying and fermenting a starch substrate to prepare a final product, comprising contacting the starch substrate with a glucoamylase having at least 85% identity with Fv-GA (SEQ ID NO: 1), and a glucoamylase having at least 85% identity with Tr-GA variant CS4 (SEQ ID NO: 9).

[009] In some embodiments of method 17, the first listed glucoamylase has 90%, 95%, 98% or 99% identity with the Fv-GA sequence (SEQ ID NO: 1). In some embodiments of method 17, the second listed glucoamylase has 90%, 95%, 98% or 99% identity with the CS4 variant Tr-GA sequence (SEQ ID NO: 9). 18. The method of 17, in which saccharification and fermentation are carried out as a simultaneous saccharification and fermentation process (SSF). 19. The method of 17 or 18, in which the final product is alcohol. 20. The method of any one of 17 - 19, in which the final product is ethanol. 21. The method of 20, in which the saccharified and fermented starch substrate results in a reduced level of DP4+ and an increased final ethanol concentration. 22. The method of 21, in which the reduced level of DP4+ is lower than would be achieved under the same saccharification and fermentation conditions, using the same dose of Fv-GA total glucoamylase or Tr-GA variant CS4 individually. 23. The method of 21, in which the increased final ethanol concentration is greater than would be achieved under the same saccharification and fermentation conditions using the same dose of Fv-GA total glucoamylase or Tr-GA variant CS4 individually. 24. A method of saccharifying and fermenting a starch substrate to prepare a biochemical end product, comprising contacting the starch substrate with a glucoamylase having at least 85% identity with Fv-GA (SEQ ID NO: 1), and with a glucoamylase having at least 85% identity with Hg-GA (SEQ ID NO: 5), under conditions suitable for biochemical fermentation.

[0010] In some embodiments of method 24, the first listed glucoamylase has 90%, 95%, 98% or 99% identity with the Fv-GA sequence (SEQ ID NO: 1). In some embodiments of method 24, the second listed glucoamylase has 90%, 95%, 98% or 99% identity with the Hg-GA sequence (SEQ ID NO: 5). 25. The method of claim 24, wherein the final product is a biochemical product selected from the group consisting of an amino acid, an organic acid, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, sodium gluconate. calcium, potassium gluconate, glucono-delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaconic acid, 1,3-propanediol, biodiesel and isoprene. 26. A method of producing a fermented beverage, wherein the method comprises the step of contacting a mash and/or a wort with a glucoamylase having at least 85% identity with Fv-GA (SEQ ID NO: 1), and contacting the starch substrate with at least one additional glucoamylase.

[0011] In some embodiments of method 26, the first listed glucoamylase has 90%, 95%, 98% or 99% identity with the Fv-GA sequence (SEQ ID NO: 1).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the % glucose at the end of 48 hr for Afu-GA (80 μg/g ds), Fv-GA (80 μg/g ds) and the Afu-GA + Fv-GA mixture (40 μg/g ds + 40 μg/g ds).

[0013] Figure 2 is a graph based on data from Table 6, showing the effect of the ratio of Afu-GA and Fv-GA glucoamylases on the final % glucose after 48 hr of saccharification.

[0014] Figure 3 shows the % glucose at 48 hr of incubation in two different pH conditions.

[0015] Figure 4, which is based on data from Table 7, shows the % glucose at 72 hr for OPTIMAX™ 4060 VHP and additional mixing with Afu-GA or Fv-GA glucoamylases.

[0016] Figure 5 shows the % w/v of DP4+ at 54 hr for the mixture CS4 + Fv-GA, GA CS4 alone and Fv-GA alone.

[0017] Figure 6 shows the % w/v of ethanol at 54 hr for the mixture CS4 + Fv-GA, GA CS4 and Fv-GA.

DETAILED DESCRIPTION

[0018] The invention will now be described in detail by way of reference using only the following definitions and examples. All patents and publications, including all sequences disclosed in such patents and publications, referred to herein are expressly incorporated by reference.

[0019] Unless otherwise defined 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”, 3rd EDITION, John Wiley and Sons, Ltd., New York (2007), and Hale & Marham, “THE HARPER COLLINS DICTIONARY OF BIOLOGY”, Harper Perennial, NY (1991) provide the skilled artisan with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. Numeric ranges include the numbers that define the range. Unless otherwise noted, nucleic acids are written from left to right in a 5' to 3' orientation; Amino acid sequences are written from left to right in amino to carboxy orientation. Practitioners are particularly directed to Green and Sambrook Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press 2012, and Ausubel FM et al., 1993, for definitions and terms of the technique. It should be understood that this invention is not limited to the particular methodology, protocols and reagents described, as these may vary.

[0020] The titles provided here are not limitations of the various aspects or embodiments of the invention that may be taken 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.

[0021] All publications cited here are expressly incorporated here by reference for the purpose of describing and disclosing compositions and methodologies that may be used in connection with the invention.

1. DEFINITIONS

[0022] The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein", and "peptide" and are used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as an "enzyme". Conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal (i.e., N→C) orientation.

[0023] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded, and can have chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. As such, the present invention contemplates every possible variant nucleotide sequence encoding GA or an amino acid variant thereof, all of which are possible given the degeneracy of the genetic code. Unless otherwise noted, nucleic acid sequences are presented in a 5'-to-3' orientation.

[0024] Nucleic acid and amino acid sequences that are referred to here as being "non-naturally occurring" are those that are not found in nature, that is, they are the product of human manipulation and/or synthesis.

[0025] "Glycoamylase" or "GA" or "GA enzyme" or "GA polypeptide" as used here is defined as the class of enzymes amyloglucosidases (EC 3.2.1.3, glucoamylase, α-1,4-D-glucan glucohydrolase ). These are exo-acting enzymes that catalyze the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides. Enzymes are also capable of hydrolyzing α-1,6 and α-1,3 bonds, although generally at much lower rates than the hydrolysis of α-1,4 bonds.

[0026] A "variant" of an enzyme, protein, polypeptide, nucleic acid, or polynucleotide as used herein means that the variant is derived from a parent polypeptide or parent nucleic acid (e.g., native parent polypeptide or nucleic acid, wild-type or other defined) that includes at least one modification or change compared to that parent. Thus, a variant may have a few mutations compared to a parent, where by "a few" is intended to mean 1 to 10 mutations. For example, a variant having 1 to 10 amino acid substitutions compared to SEQ ID NO:1 can be referred to as a Fv-GA variant having some substitutions. Changes/modifications may include a replacement of an amino acid/nucleic acid residue in the parent with a different amino acid/nucleic acid residue at one or more sites, deletion of an amino acid/nucleic acid residue (or a series of amino acid residues /nucleic acid) in the parent at one or more sites, insertion of an amino acid/nucleic acid residue (or a series of amino acid/nucleic acid residues) in the parent at one or more sites, truncation of amino acid sequences /or carboxy-terminal or 5' and or 3' nucleic acid sequences, and any combination thereof. A variant Fv-GA enzyme according to aspects of the invention retains starch hydrolysis activity, but may have an altered property in some specific aspect, for example, an improved property. For example, a variant Fv-GA enzyme may have an altered pH optimum, improved thermostability, improved hydrolysis of one or more substrates (e.g., DP1 (e.g., glucose), DP3 (e.g., maltotriose, panose), DP4 ( e.g. maltotetraose), and/or pullulan) or a combination thereof. In certain embodiments, the variant Fv-GA enzyme contains an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, or an enzymatically active fragment thereof. Likewise, An-GA variant, Afu-GA variant, Hg-GA variant, Tr-GA variant CS4 variants and Tr-GA variants are contemplated being at least 60%, 70%, 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective SEQ ID NOs, or an enzymatically active fragment thereof.

[0027] "Combinatorial variants" are variants comprising two or more mutations, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more substitutions, deletions and/or insertions.

[0028] A "progenitor" polynucleotide, polypeptide, or enzyme sequence (e.g., a "progenitor Fv-GA enzyme"), or equivalents thereof, as used herein relates to a polynucleotide, polypeptide, or enzyme sequence that was used as starting point or template for designing a variant polynucleotide, polypeptide, or enzyme. It is further noted that the words "progenitor" and "paternal" are used interchangeably in this context. A "parent Fv-GA enzyme" as used herein means a polypeptide that in its mature form comprises an amino acid sequence that has at least 60% identity with SEQ ID NO:4, including amino acid sequences having at least 60%, 70%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 (which provides the amino acid sequence of the mature form of wild-type GA from Fusarium verticillioides) or an allelic variant thereof or a fragment that has starch hydrolysis activity.

[0029] The term "wild-type" relates to a naturally occurring polypeptide or nucleic acid sequence, that is, one that does not include a man-made variation.

[0030] The term "heterologous" when used with respect to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally in the same relationship to each other in nature. For example, the nucleic acid is typically produced recombinantly, having two or more sequences, e.g., from unrelated genes, arranged to produce a new functional nucleic acid, e.g., a promoter from one source and a coding region from another. source. Similarly, a heterologous polypeptide will often refer to two or more subsequences that are not in the same relationship to each other in nature (for example, a fusion polypeptide).

[0031] The term "recombinant" when used with reference to, for example, a cell, or nucleic acid, polypeptide, or vector, indicates that the cell, nucleic acid, polypeptide, or vector, has been modified by the introduction of a nucleic acid or heterologous polypeptide or by altering a native nucleic acid or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. A "recombinant" composition requires manipulation by a human and consequently excludes products from nature.

[0032] The terms "isolated" or "purified" as used herein relate to a nucleic acid or polynucleotide that is removed from the environment where it is naturally produced. In general, in an isolated or purified nucleic acid or polypeptide sample, the nucleic acid(s) or polypeptide(s) of interest is(are) present at an absolute or relative concentration increased by compared to the environment where they are naturally produced. In some cases, an isolated or purified nucleic acid or polynucleotide is generated synthetically.

[0033] The term "enriched" when describing a component or material in a composition (e.g., a polypeptide or polynucleotide) means that the component or material is present at a relatively increased concentration in that composition compared to the initial composition from the which the enriched composition was generated. For example, an enriched GA composition (or sample) is such that the relative or absolute GA concentration is increased compared to the initial fermentation product of the host organism.

[0034] As used here, the term "promoter" relates to a nucleic acid sequence whose function is to direct the transcription of a downstream gene. The promoter will generally be appropriate for the host cell where the target gene is being expressed. The promoter, in conjunction with other nucleic acid sequences regulating transcription and translation (also called "control sequences"), is necessary to express a given gene. In general, transcription and translation regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. An example of an inducible promoter useful in the present invention is the T. reesei (H. jecorina) cbh1 promoter which is deposited in GenBank under Accession Number D86235. In another aspect the promoter is a cbh II or H. jecorina xylanase promoter. Examples of suitable promoters include the promoter of the glucoamylase genes of A. awamori or A. niger (Nunberg, J. H. et al. (1984) Mol. Cell. Biol. 4, 2306-2315; Boel, E. et al. (1984 ) EMBO J. 3, 1581-1585), the carboxyl protease gene from Mucor miehei, the glucoamylase I gene from Hypocrea jecorina (Shoemaker, S. P. et al. (1984) European Patent Application No. EPO0137280A1), the trpC gene from A. nidulans (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45 ) the alcA gene of A. nidulans (Lockington, R. A. et al. (1986) Gene 33, 137-149), the tpiA gene of A. nidulans (McKnight, G. L. et al. (1986) Cell 46, 143-147) , the amdS gene of A. nidulans (Hynes, M. J. et al. (1983) Mol. Cell Biol. 3, 1430-1439), the xln1 gene of H. jecorina, the cbh2 gene of H. jecorina, the eg1 gene of H. jecorina, the H. jecorina eg2 gene, the H. jecorina eg3 gene, and higher eukaryotic promoters such as the SV40 early promoter (Barclay, S. L. and E. Meller (1983) Molecular and Cellular Biology 3, 2117-2130 ).

[0035] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader, that is, a signal peptide, is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, are contiguous and in reading frame. However, enhancers do not have to be contiguous. Binding is achieved by binding at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. Thus, the term "operably linked" relates to a functional link between a nucleic acid expression control sequence (such as a promoter, or series of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0036] The term "signal sequence", "signal peptide", "secretory sequence", "secretory peptide", "secretory signal sequence", "secretory signal peptide" and the like designates a peptide sequence that, as a component of a larger polypeptide , directs the larger polypeptide through a secretory pathway of a cell where it is synthesized, as well as nucleic acids encoding such peptides. In general, the larger polypeptide (or protein) is usually cleaved to remove the secretory/signal peptide during transit through the secretory pathway, where the cleaved form of the polypeptide (i.e., the form without the signal/secretory peptide) is many sometimes referred to herein as the "mature form" of the polypeptide. For example, SEQ ID NO:2 provides the amino acid sequence of F. verticilloides GA (Fv-GA) with the signal peptide (i.e., full-length Fv-GA).

[0037] As used here, the term "vector" relates to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of appropriate expression vectors is within the knowledge of those skilled in the art.

[0038] Accordingly, an "expression cassette" or "expression vector" is a recombinantly or synthetically generated nucleic acid construct, with a series of specified nucleic acid elements that allow transcription of a particular nucleic acid into a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0039] As used herein, the term "plasmid" relates to a circular double-stranded (ds) DNA construct that forms an extrachromosomal self-replicating genetic element when present in many bacteria and some eukaryotes. Plasmids can be employed for any of different purposes, for example, as cloning vectors, propagation vectors, expression vectors, etc.

[0040] As used herein, the term "selectable marker" relates to a nucleotide sequence or polypeptide encoded by it that is capable of expression in cells and in which expression of the selectable marker in cells confers the ability to be differentiated from cells that do not express the selectable marker. In certain embodiments, a selectable marker allows a cell expressing it to grow in the presence of a corresponding selective agent, or under corresponding selective growth conditions. In other embodiments, a selectable marker allows a cell that expresses it to be identified and/or isolated from cells that do not express it by virtue of a physical characteristic, for example, by differences in fluorescence, immunoreactivity, etc.

[0041] In general, nucleic acid molecules encoding Fv-GA or a variant Fv-GA will hybridize, under conditions of moderate to high stringency, with the wild-type sequence provided here as SEQ ID NO:2 (gene of native Fv-GA). However, in some cases a nucleotide sequence encoding Fv-GA is employed that has substantially different codon usage, whereas the enzyme encoded by the nucleotide sequence encoding Fv-GA has the same or substantially the same amino acid sequence as the native enzyme. For example, the coding sequence can be modified to facilitate faster expression of Fv-GA in a particular prokaryotic or eukaryotic expression system, according to the frequency with which a particular codon is used by the host (commonly referred to as "optimization of codons"). Te'o, et al. (2000), for example, describes the optimization of genes for expression in filamentous fungi. Such nucleic acid sequences are sometimes referred to as "degenerate" or "degenerate sequences".

[0042] A nucleic acid sequence is considered “selectively hybridizable” with a reference nucleic acid sequence if the two sequences specifically hybridize to each other under moderate to high stringency hybridization and washing conditions. Hybridization conditions are based on the melting temperature (Tf) of the complex or probe that binds to the nucleic acid. For example, “maximum stringency” typically occurs at about Tf-5°C (5° below probe Tf); “high stringency” at about 5-10° below Tf; “moderate” or “intermediate stringency” at about 10-20° below probe Tf; and “low stringency” at about 20-25° below Tf. Functionally, maximum stringency conditions can be used to identify sequences having strict identity or near-strict identity to the hybridization probe, whereas high stringency conditions can be used to identify sequences having about 80% or more sequence identity to the hybridization probe. probe.

[0043] Moderate and high stringency hybridization conditions are well known in the art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, and in Ausubel, F.M., et al., 1993, expressly incorporated by title reference here). An example of high stringency conditions includes hybridization at about 42°C in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg/mL denatured carrier DNA followed by washing twice in 2X SSC. and 0.5% SDS at room temperature and twice more in 0.1X SSC and 0.5% SDS at 42°C.

[0044] As used herein, the terms "transformed", "stably transformed" or "transgenic" with reference to a cell mean that the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome or as a plasmid episomal that is maintained over multiple generations.

[0045] As used here, the term “expression” relates to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process generally includes both transcription and translation.

[0046] The term "introduced" in the context of insertion of 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 cell or prokaryotic in which the nucleic acid sequence can be incorporated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or expressed transiently (e.g., transfected mRNA).

[0047] By the term "host cell" is intended to mean a cell that contains a vector and supports the replication, and/or transcription and/or transcription and translation (expression) of the expression construct. Host cells for use in the present invention can be prokaryotic cells, such as E. coli, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells. In certain embodiments, host cells are filamentous fungi.

[0048] When an amino acid position (or residue) in a first polypeptide is designated as being "equivalent" to an amino acid position in a related second polypeptide, it means that the amino acid position of the first polypeptide corresponds to the position noted in the second polypeptide related by one or more of (i) alignment of the primary sequences (see description of sequence alignment and sequence identity below); (ii) structural homology of sequences; or (iii) analogous functional property. Thus, an amino acid position in a first GA enzyme (or a variant thereof) can be identified as "equivalent" (or "homologous") to an amino acid position in a second GA enzyme (or even multiple different GA enzymes).

[0049] Primary sequence alignment: Equivalent amino acid positions can be determined using primary amino acid sequence alignment methodologies, many of which are known in the art. For example, by aligning the primary amino acid sequences of two or more different GA enzymes, it is possible to designate an amino acid position number of one GA enzyme as being equivalent to the position number of another of the aligned GA enzymes. In this way, the numbering system originating from the amino acid sequence of a GA enzyme (e.g., the Fv-GA enzyme denoted in SEQ ID NO: 1) can be used to identify equivalent (or homologous) amino acid residues in other GA enzymes. .

[0050] Structural sequence homology: In addition to determining "equivalent" amino acid positions using primary sequence alignment methodologies, "equivalent" amino acid positions can also be defined by determining homology at the secondary and/or tertiary structure level . For example, for a glucoamylase whose tertiary structure has been determined by x-ray crystallography, equivalent residues may be defined as those for which the atomic coordinates of two or more of the backbone atoms of a particular amino acid residue of the glucoamylase are within 0.13 nm and preferably 0.1 nm after alignment with Fv-GA (N in N, CA in CA, C in C, and O in O). Alignment is achieved after the best model has been oriented and positioned to give maximum overlap of atomic coordinates of non-hydrogen protein atoms of the glucoamylase in question with Fv-GA. The best model is the crystallographic model that gives the highest resolution available. When two or more different models have equal resolution, the model with the lowest R factor is used for experimental diffraction data, using the equation below.

[0051] Analogous functional property: Equivalent amino acid residues in a first polypeptide that are functionally analogous to a specific residue of a related second polypeptide (for example, a first glucoamylase and Fv-GA) are defined as those amino acids in the first polypeptide that adopt a conformation such that they alter, modify, or contribute to the polypeptide structure, binding to substrates, or catalysis in a defined manner and assigned to a specific residue of the second related polypeptide. When a tertiary structure has been obtained by x-ray crystallography for the first polypeptide, amino acid residues of the first polypeptide that are functionally analogous to the second polypeptide occupy an analogous position in that, although the backbone atoms of the given residue may not satisfy the equivalence criteria based on the occupancy of a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue are within 0.13 nm of the corresponding side chain atoms of the second polypeptide.

[0052] The term "improved property" or "improved performance" and the like with respect to a variant enzyme (e.g., a GA variant) is defined herein as a characteristic or activity associated with a variant enzyme that is improved compared to the their respective parent enzyme. Improved properties include, but are not limited to, improved thermostability or altered temperature-dependent activity profile, improved activity or stability at a desired pH or pH range, improved specificity for substrates, improved specificity for products, and improved stability in the presence of a chemical compound or other component in a step of the starch conversion process, etc. Improved performance can be determined using a particular assay(s) including, but not limited to: (a) expression, (b) hydrolytic activity on substrate DP2, (c) hydrolytic activity on substrate DP7, (d) hydrolytic activity on panose substrate, (e) hydrolytic activity on pullulan substrate, (f) hydrolytic activity on granular corn starch (CS), (g) thermostability, (h) glucose inhibition, (i) reversal activity, and (j) hydrolytic activity on an amylopectin substrate.

[0053] The term "enhanced thermostability" with respect to a variant protein (e.g., a GA variant) is defined herein as a variant enzyme exhibiting retention of a greater fraction of enzymatic activity after a period of incubation at an elevated temperature relative to the progenitor enzyme. Such a variant may or may not exhibit an altered thermal activity profile relative to the parent. For example, a variant may have an improved ability to undergo refolding after incubation at elevated temperature relative to the parent.

[0054] By "improved product specificity" is intended to mean a variant enzyme exhibiting an altered product profile compared to the parent enzyme, wherein the altered product profile of the variant is improved in a given application compared to the parent. A "product profile" is defined here as the chemical composition of the reaction products produced by the enzyme of interest.

[0055] By "enhanced substrate specificity" is intended to mean a variant enzyme that targets a specific substrate (or class of substrates) for hydrolysis in a manner different from the parent enzyme, such that the variant has improved performance in a given application in comparison with the parent.

[0056] By "enhanced chemical stability" it is intended to mean that a variant enzyme exhibits retention of enzymatic activity after a period of incubation in the presence of a chemical or chemicals that reduce the enzymatic activity of the parent enzyme under the same conditions. Variants with improved chemical stability are more capable of catalyzing a reaction in the presence of such chemicals compared to the parent enzyme.

[0057] A "pH range", in relation to an enzyme, relates to the range of pH values at which the enzyme exhibits catalytic activity.

[0058] The terms "pH stable" and "pH stability", with respect to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values over a predetermined period of time (e.g. example, 15 min., 30 min., 1 hour).

[0059] As used herein, "starch" refers to any material comprised of complex plant polysaccharide carbohydrates comprised of amylose and amylopectin with the formula (C6H10O5)x, where "X" can be any number. In particular, the term relates to any plant-based material including but not limited to grains, grasses, tubers and roots and more specifically wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, potato, sweet potato and tapioca. "Granular starch" relates to uncooked (raw) starch that has not been subjected to gelatinization, where "starch gelatinization" means solubilization of a starch molecule to form a viscous suspension.

[0060] "Degree of polymerization (DP)" relates to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DP1 are monosaccharides, such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP7 designates polymers with seven anhydroglucopyranose units.

[0061] As used here, "starch hydrolysis" and the like relates to the cleavage of glucosidic bonds with the addition of water molecules. Thus, enzymes having "starch hydrolysis activity" catalyze the cleavage of glucosidic bonds with the addition of water molecules.

[0062] As used herein, "fermentable sugars" relates to saccharides that can be metabolized under fermentation conditions. These sugars typically refer to glucose, maltose and maltotriose (DP1, DP2 and DP3).

[0063] As used here, "total sugar content" relates to the total sugar content present in a starch composition.

[0064] "Percent sequence identity" or grammatical equivalents means that a particular sequence has at least a certain percentage of amino acid residues identical to those present in a reference sequence specified using an alignment algorithm. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). “Software” for performing BLAST analyzes is publicly available by the National Center for Biotechnology Information (<www(dot)ncbi(dot)nlm(dot)nih(dot)gov>). This algorithm involves first identifying pairs of high-scoring sequences (HSPs) by identifying short words of length W in the search string, which match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a string. of the database. These initial word hits in the neighborhood act as starting points for finding longer HSPs containing the same words. The word hits are expanded in both directions along each of the two sequences being compared as much as the cumulative alignment score can be increased. The span of word hits is terminated when: the cumulative alignment score decreases by the amount X from a maximum value reached; the cumulative score goes to zero or lower; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectancy (E) of 10, M'5, N'-4 and a comparison of both tapes.

[0065] The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). A measure of similarity provided by the BLAST algorithm is the probability of the smallest sum (P(N)), which provides an indication of the probability of a match between two nucleotide or amino acid sequences occurring by chance. For example, an amino acid sequence is considered similar to a protease if the probability of the smallest sum in a comparison of the test amino acid sequence with a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0066] Alignment algorithms can also be employed to identify an amino acid residue (or residues) in a first amino acid sequence (e.g., Fv-GA) that corresponds to an amino acid residue (or residues) in a second amino acid sequence (e.g., homologous GA enzymes from other species, e.g., GA from T. reesei).

[0067] When questions of percentage sequence identity or corresponding amino acid residues between two sequences arise, alignment using the CLUSTAL W algorithm with predefined parameters will govern. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Predefined parameters for the CLUSTAL W algorithm are: Gap opening penalty: 10.0 Gap extension penalty: 0.05 Protein weight matrix: BLOSUM series DNA weight matrix: IUB % Delay of divergent sequences: 40 Gap Separation Distance: 8 DNA Transitions Weight: 0.50 Hydrophilic Residue List: GPSNDQEKR Negative Matrix Usage: OFF Residue Specific Penalty Toggle: ON Hydrophilic Penalty Toggle: ON Gap Separation Penalty Toggle OFF end.

II. MOLECULAR BIOLOGY

[0068] Embodiments of the present invention provide for the expression of a desired glucoamylase enzyme (or combination of glucoamylase enzymes) from nucleic acids encoding glucoamylase under the control of a functional promoter in a host cell of interest, for example, a filamentous fungus . Consequently, this invention is based on some routine techniques in the field of recombinant genetics. Basic texts revealing examples of suitable recombinant genetic methods are indicated above.

[0069] Any method known in the art that can introduce mutations into a progenitor nucleic acid/polypeptide is contemplated by the present invention.

[0070] The present invention relates to the expression, purification and/or isolation and use of variant GA enzymes. These enzymes can be prepared by recombinant methods using any of some glucoamylase genes known in the art (for example, the F. verticillioides glucoamylase gene comprising SEQ ID NO:2). Any convenient method for introducing mutations may be employed, including site-directed mutagenesis. As indicated above, mutations (or variations) include substitutions, additions, deletions or truncations that will correspond to one or more amino acid changes in the expressed GA variant. Again, site-directed mutagenesis and other methods of incorporating amino acid changes into proteins expressed at the DNA level can be found in numerous references, e.g., Green and Sambrook, et al. 2012 and Ausubel, et al.

[0071] DNA encoding an amino acid sequence variant of a parent GA is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared DNA encoding the progenitor GA enzyme.

[0072] Site-directed mutagenesis is a method that can be used in the preparation of replacement variants. This technique is well known in the art (see, for example, Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad.Sci. USA 82:488 (1987) ). In summary, when performing site-directed mutagenesis of DNA, the starting DNA is altered first by hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize a full-length second strand, using the hybridized oligonucleotide as a primer, and using the single-strand starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA.

[0073] PCR mutagenesis is also suitable for producing amino acid sequence variants of the progenitor GA. See Higuchi, in “PCR Protocols”, pages 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acid Res 17:723-733 (1989). In summary, when small amounts of template DNA are used as starting material in a PCR, primers whose sequence differs slightly from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence. only in the positions where the primers differ from the template.

[0074] Another method of preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34:315323 (1985). The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If such restriction sites do not exist, they can be generated using the oligonucleotide-mediated mutagenesis method described above to introduce them into appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to effect its linearization. A double-stranded oligonucleotide encoding the DNA sequence between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, in which the two strands of the oligonucleotide are synthesized separately and then hybridized together. using common techniques. This double-stranded oligonucleotide is referred to as a cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid so that it can be directly ligated into the plasmid. This plasmid now contains the mutated DNA sequence.

[0075] Alternatively, or additionally, the desired amino acid sequence encoding a desired glucoamylase can be determined, and a nucleic acid sequence encoding such an amino acid sequence variant can be generated synthetically.

[0076] The desired glucoamylase(s) prepared in this way may be subject to additional modifications, often depending on the intended use of the glucoamylase. Such modifications may involve additional alteration of the amino acid sequence, fusion to heterologous polypeptide(s) and/or covalent modifications.

III. GA AND VARIANT GA POLYPEPTIDES AND NUCLEIC ACIDS THAT ENCODE THEM

[0077] Glycoamylases (GAs) are produced by numerous strains of bacteria, fungi, yeast and plants. Many fungal glucoamylases are secreted from the cell, for example from Aspergillus strains (Svensson et al., Carlsberg Res. Commun. 48:529-544 (1983); Boel et al., EMBO J. 3:1097-1102 ( 1984); Hayashida et al., Agric. Biol. Chem. 53: 923-929 (1989); U.S. Patent No. 5,024,941; U.S. Patent No. 4,794,175 and WO 88/09795); Talaromyces (U.S. Patent No. 4,247,637; U.S. Patent No. 6,255,084; and U.S. Patent No. 6,620,924); Rhizopus (Ashikari et al., Agric. Biol. Chem. 50: 957-964 (1986); Ashikari et al., App. Microbio. Biotech. 32: 129-133 (1989) and U.S. Patent No. 4,863,864); Humicola (WO 05/052148 and U.S. Patent No. 4,618,579); and Mucor (Houghton-Larsen et al., Appl. Microbiol. Biotechnol. 62: 210-217 (2003)). Many of the genes encoding these enzymes have been cloned and expressed in yeast, fungal and/or bacterial cells.

[0078] Commercially, glucoamylases are very important enzymes and have been used in a wide variety of applications that require the hydrolysis of starch (for example, for the production of glucose and other monosaccharides from starch). Glycoamylases are used to produce high-fructose corn sweeteners, which comprise more than 50% of the sweetener market in the United States. In general, glucoamylases can be, and usually are, used with alpha-amylases in starch hydrolysis processes to hydrolyze starch to dextrins and then glucose. Glucose can be used directly; be converted to fructose by other enzymes (e.g. glucose isomerases); crystallized; or used in fermentations to produce numerous end products (e.g., ethanol, citric acid, succinic acid, ascorbic acid intermediates, glutamic acid, glycerol, 1,3-propanediol, and lactic acid).

[0079] Glycoamylases can consist of as many as three distinct structural domains, a catalytic domain of approximately 450 residues that is structurally conserved in all glycoamylases, generally followed by a linker region consisting of 30 to 80 residues that are connected to a starch-binding domain (SBD) of approximately 100 residues (also referred to as the carbohydrate-binding domain, or CBD). The structure of Trichoderma reesei glucoamylase (TrGA) with all three regions intact was determined at a resolution of 1.8 Angstrom. See WO 2009/048488 and WO 2009/048487, incorporated herein by reference. Using the determined coordinates, the structure was aligned with the coordinates of the catalytic domain of the glucoamylase of Aspergillus awamori strain X100 that was previously determined (Aleshin, A.E., Hoffman, C., Firsov, L.M., and Honzatko, R.B. glucoamylase from Aspergillus awamori var. The structures of the catalytic domains of these two glucoamylases overlap very closely, and it is possible to identify equivalent residues based on this structural overlap. It is also believed that all glucoamylases share the basic structure.

[0080] Given the well-known relationship between structure and function of glucoamylases, variants of glucoamylases having altered properties have been successfully created and characterized. Certain variants exhibit improved properties compared to the parental glucoamylases. Examples of improved properties include increased thermostability and increased specific activity. Methods of preparation and characterization of T. reesei GA variants with altered properties have been described in WO 2009/067218 (incorporated herein by reference).

[0081] Protein (amino acid) and nucleotide sequences of GA enzymes are described here including:

[0082] Fusarium verticillioides GA Protein Sequence (Fv-GA) (SEQ ID NO: 1)

[0083] Fusarium verticillioides GA DNA Nucleotide Sequence (Fv-GA) (SEQ ID NO: 2)

[0084] GA Protein Sequence from Aspergillus fumigatus (Afu-GA) (SEQ ID NO: 3)

[0085] Nucleotide Sequence of GA from Aspergillus fumigatus (Afu-GA) (SEQ ID NO: 4)

[0086] Humicola grisea GA Protein Sequence (Hg-GA) (SEQ ID NO: 5)

[0087] Humicola grisea GA Nucleotide Sequence (Hg-GA) (SEQ ID NO: 6)

[0088] Aspergillus niger GA Protein Sequence (An-GA) (SEQ ID NO: 7)

[0089] Nucleotide Sequence of GA from Aspergillus niger (An-GA) (SEQ ID NO: 8)

[0090] Trichoderma reesei GA Protein Sequence (Tr-GA) Mature variant CS4 (SEQ ID NO: 9)

[0091] GA Protein Sequence from Trichoderma reesei (Tr-GA) Mature (SEQ ID NO: 10)

[0092] Trichoderma reesei GA Protein Sequence (Tr-GA) Progenitor (SEQ ID NO: 11)

[0093] Trichoderma reesei GA Nucleotide Sequence (Tr-GA) (SEQ ID NO: 12)

[0094] Variant GA enzymes are also described here. Variant GA enzymes have one or more mutations, as shown herein, relative to a parent GA enzyme, wherein the parent GA enzyme has at least 60% (i.e., 60% or more) amino acid sequence identity to SEQ ID NO:4, including at least 61%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to and including 100% amino acid sequence identity with SEQ ID NO:4. Variant GA enzymes (i.e., having one or more mutations) may have at least 60% (i.e., 60% or more) amino acid sequence identity with SEQ ID NO:4, including at least 61%, 65% , 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:4. In certain embodiments, the parent GA is selected from: a strain of Trichoderma (e.g., T. reesei, T. longibrachiatum, T. strictipilis, T. asperellum, T. konilangbra, T. citrinoviride, T. pseudokoningii and T. hazianum ), a strain of Aspergillus (e.g. A. aculeatus, A. niger, A. nidulans, A. kawachi, A. awamori, A. clavatus, A. terreus, A. fumigates, and A. orzyae ), a strain of Talaromyces (e.g. T. emersonii, T. thermophilus, and T. duponti), a strain of Trametes (e.g. Trametes cingulata), a strain of Hypocrea (e.g. H. gelatinosa, H. orientalis, H. vinosa, H. .jecorina, H. schweinitzii, and H. citrina), a strain of Fusarium (e.g., F. oxysporum, and F. roseum), a strain of Humicola (e.g., H. grisea, H. insolens, and H. lanuginosis ), a strain of Saccharomycopsis (e.g., S. fibuligera), Scytalidium thermophilum, Podospora anderina, or their respective corresponding anamorph, teleomorph, or holomorph forms.

[0095] In some cases, the progenitor GA is GA from Fusarium verticillioides (Fv-GA). Additionally, the variant GA enzyme has starch hydrolysis activity (or is a fragment of variant GA having starch hydrolysis activity) wherein, in certain embodiments, the variant GA has an improved property compared to the parent GA (as detailed here).

[0096] Aspects of the present invention provide variants of a parent GA enzyme, wherein the variant has starch hydrolysis activity, has at least 60% (e.g., at least 80%) sequence identity with SEQ ID NO: 1, and has at least one improved property relative to the selected parent GA enzyme of: (a) expression, (b) hydrolytic activity on DP2 substrate, (c) hydrolytic activity on DP7 substrate, (d) hydrolytic activity on panose substrate, (e) hydrolytic activity on pullulan substrate, (f) hydrolytic activity on granular corn starch (CS), (g) thermostability, (h) glucose inhibition, (i) reversal activity, and (j) hydrolytic activity on an amylopectin substrate.

[0097] In certain embodiments, a GA variant has at least two, at least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, or more improved properties (selected from the list above) relative to the parent GA enzyme. In certain embodiments, a variant GA enzyme comprises an amino acid mutation in one or more amino acid positions in Fv-GA (as denoted in SEQ ID NO:1). Since certain GA progenitor enzymes according to aspects of the invention may not have the same amino acid as wild-type Fv-GA, amino acid positions corresponding to the residues noted above may also be designated either by position number alone.

[0098] Alignment of amino acid sequences to determine homology can be determined using a "sequence comparison algorithm". Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by visual inspection or MOE by Chemical Computing Group, Montreal Canada. See also the description of "percent sequence identity" provided in the Definitions section above.

IV. RECOMBINANT GA EXPRESSION

[0099] Aspects of the present invention include methods and compositions relating to the generation of nucleic acids encoding GA enzymes and GA enzyme variants, host cells containing such nucleic acids, the production of GAs or GA variants by such host cells, and the isolation, purification and/or use of GA variants.

[00100] As such, embodiments of the invention provide host cells that have been transduced, transformed or transfected with an expression vector comprising a nucleic acid sequence encoding a desired GA or GA variant. For example, a filamentous fungal cell or yeast cell is transfected with an expression vector having a biologically active promoter or promoter fragment or one or more (e.g., a series of) enhancers that act on the host cell lineage, operably linked to a DNA segment encoding a desired GA or GA variant, such that the desired GA or GA variant is expressed in the cell line.

A. Nucleic Acid Constructs/Expression Vectors.

[00101] Natural or synthetic polynucleotide fragments encoding a desired GA or GA variant can be incorporated into heterologous nucleic acid constructs or vectors, capable of introduction into, and replication in, a host cell of interest (e.g., a fungal cell filamentous or yeast). The vectors and methods disclosed herein are suitable for use in host cells for expressing a desired GA or GA variant. Any vector can be used as long as it meets the desired replication/expression characteristics in the host cell(s) where it is introduced (such characteristics being generally defined by the user). Large numbers of suitable vectors and promoters are known to those skilled in the art, some of which are commercially available. Cloning and expression vectors are also described in Sambrook et al., 1989, Ausubel FM et al., 1989, and Strathern et al., 1981, each of which is expressly incorporated herein by reference. Appropriate expression vectors for fungi are described in van den Hondel, C.A.M.J.J. et al. (1991) In: Bennett, J.W. and Lasure, L.L. (editors) “More Gene Manipulations in Fungi”. Academic Press, pages 396-428. The appropriate DNA sequence can be inserted into a plasmid or vector (collectively referred to herein as "vectors") by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by common procedures. Such procedures and related subcloning procedures are considered to be within the scope of knowledge of those skilled in the art.

[00102] Recombinant host cells comprising the coding sequence for a desired GA or GA variant can be produced by introducing a heterologous nucleic acid construct comprising the desired GA coding sequence or GA variant into the desired host cells (e.g., as described in more detail below). For example, a desired GA coding sequence or GA variant can be inserted into a suitable vector in accordance with well-known recombinant techniques and used to transform a filamentous fungus capable of expressing GA. As noted above, due to the inherent degeneracy of the genetic code, other nucleic acid sequences encoding substantially the same amino acid sequence or a functionally equivalent amino acid sequence can be used to clone and express a desired GA or GA variant. It is therefore appreciated that such substitutions in the coding region are encompassed in the sequence variants encompassed by the present invention.

[00103] The present invention also includes recombinant nucleic acid constructs comprising one or more of the nucleic acid sequences encoding desired GAs or GA variants as described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.

[00104] Heterologous nucleic acid constructs may include the coding sequence for a desired GA or GA variant: (i) isolated; (ii) in combination with additional coding sequences; as fusion polypeptide or signal peptide coding sequences, wherein the desired GA coding sequence or GA variant is the dominant coding sequence; (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment in which the desired GA coding sequence or GA variant is a heterologous gene.

[00105] In one aspect of the present invention, a heterologous nucleic acid construct is employed to transfer a nucleic acid sequence encoding a desired GA or GA variant to a host cell in vitro, e.g., to established strains of filamentous fungi and yeasts. Long-term production of a desired GA or GA variant can be achieved by generating a host cell that exhibits stable expression of the GA or GA variant. Thus, it follows that any effective method for generating stable transformants can be used in the practice of the invention. Suitable vectors are typically equipped with a nucleic acid sequence encoding a selectable marker, insertion sites, and suitable control elements, such as promoter and termination sequences. The vector may comprise regulatory sequences, including, for example, non-coding sequences, such as introns and control elements, i.e., promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in cells. host cells (and/or in a vector or host cell environment where a modified soluble protein antigen coding sequence is not normally expressed), operably linked to the coding sequence. Many suitable vectors and promoters are known to those skilled in the art, many of which are commercially available and/or are described in Sambrook, et al., (supra).

[00106] Examples of suitable promoters include constitutive promoters and inducible promoters, examples of which include a CMV promoter, an SV40 early promoter, an RSV promoter, an EF-1α promoter, a tet-responsive element (TRE)-containing promoter ) in the Tet-On or Tet-Off system as described (ClonTech and BASF), the beta-actin promoter and the metallothionin promoter which can be upregulated by addition of certain metal salts. A promoter sequence is a DNA sequence that is recognized by the particular host cell for expression purposes. It is operably linked to a DNA sequence encoding a GA or variant GA polypeptide. Such linkage comprises positioning the promoter relative to the start codon of the DNA sequence encoding the GA or variant GA polypeptide in the expression vector so that the promoter can drive transcription/translation of the sequence encoding the GA or variant GA. The promoter sequence contains transcription and translation control sequences that mediate the expression of GA or variant GA polypeptide. Examples include the promoters of genes encoding glucoamylase, alpha-amylase, or alpha-glucosidase from Aspergillus niger, Awamori, or A. oryzae; the gpdA or trpC genes of A. nidulans; the cbh1 or trp1 genes from Neurospora crassa; the genes encoding aspartic proteinase from A. niger or Rhizomucor miehei; the genes cbh1, cbh2, egl1, egl2 from H. jecorina, or encoding other cellulases.

[00107] The choice of the appropriate selectable marker will depend on the host cell and appropriate markers for different hosts are well known in the art. Typical selectable marker genes include argB from A. nidulans or H. jecorina, amdS from A. nidulans, pyr4 from Neurospora crassa or H. jecorina, pyrG from Aspergillus niger or A. nidulans. Additional examples of suitable selectable markers include, but are not limited to, trpc, trp1, oliC31, niaD or leu2, which are included in heterologous nucleic acid constructs used to transform a mutant strain such as trp-, pyr-, leu- and the like.

[00108] Such selectable markers confer on transformants the ability to use a metabolite that is not usually metabolized by filamentous fungi. For example, the H. jecorina amdS gene, which encodes the enzyme acetamidase, allows transforming cells to grow on acetamide as a nitrogen source. The selectable marker (e.g. pyrG) can restore the ability of an auxotrophic mutant strain to grow in a selective minimal medium or the selectable marker (e.g. olic31) can confer on transformants the ability to grow in the presence of an inhibitory drug or antibiotic.

[00109] The coding sequence of the selectable marker is cloned into any suitable plasmid using methods generally employed in the art. Examples of suitable plasmids include pUC18, pBR322, pRAX and pUC100. The pRAX plasmid contains AMA1 sequences from A. nidulans, which enable replication in A. niger.

[00110] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which belong to the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al., 1989; Freshney, 1987; Ausubel, et al., 1993; and Coligan et al., 1991.

B. Host Cells and Culture Conditions for the Production of GA Enzymes

[00111] After DNA sequences encoding GA or GA variants have been cloned into DNA constructs, the DNA is used to transform microorganisms. The microorganism to be transformed for the purpose of expressing a GA or GA variant according to the present invention can be chosen from a wide variety of host cells. The sections below are provided as examples of host cells/microorganisms and are not intended to limit the scope of host cells that may be employed in practicing aspects of the present invention.

(i) Filamentous Fungi

[00112] Aspects of the present invention include filamentous fungi that have been modified, selected and cultivated in an effective manner to produce production or expression of GA or desired GA variant relative to the corresponding untransformed parental filamentous fungi.

[00113] Examples of parent filamentous fungal species that can be treated and/or modified for desired glucoamylase expression include, but are not limited to, Trichoderma, Penicillium sp., Humicola sp., including Humicola insolens; Aspergillus sp., including Aspergillus niger, Chrysosporium sp., Myceliophthora sp., Fusarium sp., Hypocrea sp., and Emericella sp.

[00114] Cells expressing a desired GA or GA variant are cultured under conditions typically employed to cultivate the parental fungal lineage. Generally, cells are grown in a standard medium containing physiological salts and nutrients, as described in Pourquie, J. et al., “Biochemistry and Genetics of Cellulose Degradation”, publishers Aubert, J. P. et al., Academic Press, pages 7186, 1988 and Ilmen, M. et al., Appl. Environ. Microbiol. 63:1298-1306, 1997. Standard culture conditions are known in the art, for example, cultures are incubated at 28 ° C in agitated cultures or fermentors until desired levels of expression of the desired GA variant are achieved.

[00115] Culture conditions for a particular filamentous fungus can be found, for example, in scientific literature and/or from the source of fungi such as the American Type Culture Collection (ATCC). After fungal growth has been established, the cells are exposed to conditions effective to cause or allow expression of a desired GA or GA variant.

[00116] In cases where a desired GA coding sequence or GA variant is under the control of an inducible promoter, the inducing agent, for example, a sugar, metal salt or antibiotic, is added to the medium at a concentration effective to inducing expression of the desired GA or GA variant.

[00117] In one embodiment, the strain is a strain of Aspergillus niger, which is a useful strain for obtaining overexpressed protein. For example, A. niger var awamori dgr246 is known to secrete high amounts of secreted cellulases (Goedegebuur et al, Curr. Genet (2002) 41: 89-98). Other strains of Aspergillus niger var awamori are known as GCDAP3, GCDAP4 and GAP3-4 (Ward et al, 1993, Appl. Microbiol. Biotechnol. 39:738-743).

[00118] In another embodiment, the strain is a strain of Trichoderma reesei, which is a useful strain for obtaining overexpressed protein. For example, it is known that RL-P37, described by Sheir-Neiss, et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984) secretes high amounts of cellulase enzymes. Functional equivalents of RL-P37 include strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921) of Trichoderma reesei. It is contemplated that these strains will also be useful in overexpressing GA or variant GA.

[00119] When it is desired to obtain a GA or GA variant in the absence of potentially harmful native glucoamylase activity, it is useful to obtain a host cell strain that has had one or more glucoamylase genes deleted prior to the introduction of a construct or plasmid. DNA containing the DNA fragment encoding the desired GA or GA variant. Such strains may be prepared in any convenient manner, for example by the method disclosed in U.S. Patent No. 5,246,853 and WO 92/06209, the disclosures of which are incorporated herein by reference. By expressing a desired GA or GA variant in a host microorganism devoid of one or more glucoamylase genes (e.g., the endogenous glucoamylase gene of a host cell), identification and subsequent purification procedures, when desired, are simplified.

[00120] Gene deletion can be achieved by inserting a form of the desired gene to be deleted or destroyed into a plasmid by methods known in the art. The deletion plasmid is then cut at appropriate restriction enzyme site(s) internal to the coding region of the desired gene, and the coding sequence of the gene or part thereof is replaced with a selectable marker. Flanking DNA sequences from the site of the gene to be deleted or destroyed, for example from about 0.5 to about 2.0 kb, may remain on either side of the selectable marker gene. A suitable deletion plasmid will generally have unique restriction enzyme sites present therein to allow the fragment containing the deleted gene, including flanking DNA sequences, and the selectable marker gene to be removed as a single linear portion.

[00121] In certain embodiments, more than one copy of DNA encoding a desired GA or GA variant may be present in a host strain to facilitate overexpression of the GA or GA variant. For example, a host cell may have multiple copies of a desired GA or GA variant integrated into the genome or, alternatively, include a plasmid vector that is capable of autonomous replication in the host organism.

(ii) Yeast

[00122] The present invention also contemplates the use of yeast as a host cell for the production of one or more desired GA. Several different genes encoding hydrolytic enzymes have been expressed in various strains of the yeast S. cerevisiae. These include sequences encoding two endoglucanases (Penttila et al., 1987), two cellobiohydrolases (Penttila et al., 1988), and a beta-glucosidase from Trichoderma reesei (Cummings and Fowler, 1996), a xylanase from Aureobasidlium pullulans (Li and Ljungdahl , 1996), a wheat alpha-amylase (Rothstein et al., 1987), etc.

[00123] (iii) Others

[00124] It is further contemplated that, in some embodiments, expression systems may be employed in host cells other than filamentous fungal cells or yeast cells, including insect cell or bacterial cell expression systems. Some of the bacterial host cells may, for example, be such that they are also an ethanologen, such as an engineered Zymomonas mobilis, which is not only capable of expressing the enzyme(s)/variant(s) of interest but is also capable to metabolize certain monomeric and other fermentable sugars, transforming them into ethanol. The selection of a host cell may be determined by the wishes of the user of the GA or GA variants described herein, and thus no limitation in this regard is intended.

C. Introduction of a Nucleic Acid Sequence Encoding a Desired GA into Host Cells.

[00125] The invention further provides cells and cellular compositions that have been genetically modified to comprise a nucleic acid sequence encoding a desired exogenously provided GA or GA variant. A progenitor cell or cell line can be genetically modified (e.g., transduced, transformed, or transfected) with a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc., as further described above.

[00126] The transformation methods of the present invention can result in the stable integration of all or part of the transformation vector into the host cell genome. However, transformation resulting in the maintenance of a self-replicating extrachromosomal transformation vector is also contemplated.

[00127] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells can 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 of introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material in a host cell (see, e.g., Sambrook et al., supra). Essentially, the particular genetic manipulation procedure used must be capable of successfully introducing a polynucleotide (e.g., an expression vector) into the host cell that is capable of expressing the desired GA or GA variant.

[00128] Many standard transfection methods can be used to produce Trichoderma reesei cell lines that express large amounts of the heterologous polypeptide. Some of the published methods for introducing DNA constructs into Trichoderma strains include: Lorito, Hayes, DiPietro, and Harman, 1993, Curr. Genet. 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174; Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-164; for Aspergillus: Yelton, Hamer, and Timberlake, 1984, Proc. Natl. Academic. Sci. USA 81: 1470-1474; for Fusarium: Bajar, Podila and Kolattukudy, 1991, Proc. Natl. Academic. Sci. USA 88: 8202-8212; for Streptomyces: Hopwood et al., 1985, “The John Innes Foundation”, Norwich, UK and for Bacillus: Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, FEMS Microbiol. Lett. 55: 135-138. An example of a suitable transformation process for Aspergillus sp. can be found in Campbell et al. “Improved transformation efficiency of A.niger using homologous niaD gene for nitrate reductase”. Curr. Genet. 16:53-56; 1989.

[00129] Additionally, heterologous nucleic acid constructs comprising a nucleic acid sequence encoding a desired glucoamylase can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection.

D. Analysis of Nucleic Acid Coding Sequences and/or GA Protein Expression.

[00130] To evaluate the expression of a desired GA variant by a cell line that has been transformed with a nucleic acid construct encoding the desired GA variant, assays can be conducted at the protein level, the RNA level, or using functional bioassays. particulars regarding the activity and/or production of glucoamylase.

[00131] In general, assays employed to analyze the expression of a desired GA variant include, but are not limited to, Northern blot, spot blot (DNA or RNA analysis), RT-PCR (polymerase chain reaction with reverse transcriptase), or in situ hybridization using an appropriately labeled probe (based on the nucleic acid coding sequence) and conventional Southern blotting and autoradiography.

[00132] Additionally, the production and/or expression of a desired GA variant can be measured in a sample directly, for example, by assays for activity, expression and/or production of glucoamylase. Such assays are described, for example, in Becker et al., Biochem J. (2001) 356:19-30 and Mitsuishi et al., FEBS (1990) 275:135138, each of which is expressly incorporated herein by way of reference. The ability of a GA to hydrolyze isolated soluble and insoluble substrates can be measured using assays described in Srisodsuk et al., J. Biotech. (1997) 57:49-57 and Nidetzky and Claeyssens, Biotech. Bioeng. (1994) 44:961-966. Useful substrates for evaluating glucoamylase include soluble starch, amylopectin, DP7, paranitrophenyl glucoside. Additionally, protein expression can be evaluated by immunological methods, such as ELISA, competitive immunoassays, radioimmunoassays, Western blotting, indirect immunofluorescent assays and similar. Some of these assays can be performed using commercially available reagents and/or kits designed to detect GA enzymes. Such immunoassays can be used to qualitatively and/or quantitatively evaluate the expression of a desired GA or GA variant. The details of such methods are known to those skilled in the art and many reagents for implementing such methods are commercially available. In certain embodiments, an immunological reagent may be employed that is specific for a desired GA enzyme or GA variant but not for its parent GA, e.g., an antibody that is specific for a substitution in GA or a GA fusion partner or variant of GA (e.g., an N- or C-terminal tail sequence, e.g., a hexa-Histidine tail or a FLAG tail). Thus, aspects of the present invention include using a purified form of a desired GA or GA variant to produce either monoclonal or polyclonal antibodies specific to the expressed polypeptide for use in various immunoassays. (See, for example, Hu et al., 1991).

V. METHODS FOR ENRICHMENT, ISOLATION AND/OR PURIFICATION OF GA OR GA VARIANT POLYPEPTIDE

[00133] In general, a desired GA or GA variant polypeptide produced in a host cell culture is secreted into the medium (producing a culture supernatant containing the GA or GA variant) and can be enriched, purified or isolated, by example, by removing unwanted components from the cell culture medium. However, in some cases, a desired GA or GA variant polypeptide may be produced in a cellular form (e.g., cytoplasmic, periplasmic, or otherwise cell-associated) requiring recovery from a cellular lysate/homogenate. The desired GA or GA variant polypeptide is collected from the cells or cell supernatants where it was produced using techniques routinely employed by those skilled in the art. Examples include, but are not limited to, filtration (e.g., ultra- or microfiltration), centrifugation, density gradient fractionation (e.g., density gradient ultracentrifugation), affinity chromatography (Tilbeurgh et al., 1984), ion exchange chromatographic methods (Goyal et al., 1991; Fliess et al., 1983; Bhikhabhai et al., 1984; Ellouz et al., 1987), including ion exchange using materials with high resolving power (Medve et al. , 1998), hydrophobic interaction chromatography (Tomaz and Queiroz, 1999), and two-phase partitioning (Brumbauer, et al., 1999).

[00134] Although enriched, isolated or purified GA or GA variant polypeptide is sometimes desired, in some embodiments, a host cell expressing a GA or GA variant polypeptide is employed directly in a process that requires glucoamylase activity. Thus, enrichment, isolation or purification of the desired GA or GA variant polypeptide is not always required to obtain a GA or GA variant polypeptide composition for application in a desired assay or process that requires, or will benefit from, glucoamylase activity. . In one such example, yeast cells expressing GA or a GA variant can be added directly to a fermentation process such that the yeast cell expressing the GA or GA variant directly into the fermentation broth where its glucoamylase activity converts it to a substrate. non-fermentable sugars into fermentable sugars so that the yeast cell converts them directly into a desired product, for example, into ethanol (see, for example, Ilmén et al., “High level secretion of cellobiohydrolases by Saccharomyces cerevisiae” Biotechnology for Biofuels 2011 , 4:30).

SAW. COMPOSITIONS

[00135] Compositions are contemplated that include one or more GA or variant GA as disclosed herein. The GAs and variant GAs described herein can be used in enzymatic compositions including but not limited to starch hydrolysis and saccharification compositions, cleaning compositions and detergents (e.g., laundry detergents, dishwashing detergents, and for cleaning hard surfaces), alcohol and/or biochemical fermentation compositions, and in animal feed compositions. Furthermore, variant glucoamylases can be used in bakery applications such as bread and cake production, brewing industry, healthcare, textiles, food, environmental waste conversion processes, biopaste processing, and biomass conversion applications. .

[00136] In some embodiments, an enzyme composition including one or more GAs or variant GAs covered by the disclosure (e.g., obtained in culture medium or recovered and purified from culture medium) will be used in combination with any one or a combination of following enzymes: alpha-amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases, transferases, phytases, laccases, oxidases, redox enzymes, esterases, cutinases, granular starch hydrolysis enzyme and other glycoamylases.

[00137] In some representative compositions, one or more variant GA or GAs as described herein will be combined with an alpha-amylase, such as fungal alpha-amylases (e.g., derived from a Trichoderma sp.) or bacterial alpha-amylases (e.g. , derived from a Bacillus sp.), including its variants, chimeras, and hybrids. In certain embodiments, the alpha-amylase is an acid-stable alpha-amylase. In certain embodiments, alpha-amylase is a granular starch hydrolysis enzyme (GSHE). Commercially available alpha-amylases contemplated for use in the GA or variant GA compositions as described herein are available (e.g., from Danisco US Inc. or Novozymes).

[00138] In other embodiments, one or more GAs or variant GAs as described here can be combined with other GAs, whether variant or native. In some embodiments, the GAs of the disclosure will be combined with one or more GAs derived from Aspergillus strains or variants thereof, such as A. oryzae, A. niger, A. kawachi, and A. awamori; glucoamylases derived from Humicola strains or their variants; glycoamylases derived from Talaromyces strains or variants thereof, particularly T. emersonii, T. stipitatus; glucoamylases derived from Athelia strains or variants thereof, particularly A. rolfsii; glucoamylases derived from Penicillium strains or their variants, particularly P. chrysogenum; P. oxalicum; Trametes, Thermomyces, Neurospora, Phanerochaete, Ganoderma, Neosartoryia, Schizophyllum, Acremonium, Artomyces, Athelia, Aureobasidium, Byssocorticium, Chrysosporium, Coniochaeta, Disporotrichum, Fusarium, Gibberella, Gloeophillum, Leucopaxillus, Meripilus, Mucor, Myceliophthora, Sporotrichum, Neurospora , Nigrofomes, Pachykytospora, Penicillium, Piromyces, Rhizomucor, Rhizopus, Schizophyllum, Steccherinum, Subulispora, Syncephalastrum, Talaromyces, Thermoascus, Thermomyces, Thielavia, Trametes, Valsaria; and glucoamylases derived from Trichoderma strains or variants thereof, particularly T. reesei.

VII. GA UTILITY AND GA VARIANTS

[00139] As detailed above, GAs are very important commercial enzymes used in a wide variety of applications that require the hydrolysis of starch substrates into fermentable sugars (e.g. glucose, maltose, maltotriose, etc.). GAs are used in processes to produce high-fructose corn sweeteners, which comprise more than 50% of the sweetener market in the United States, as well as in processes for the direct production of glucose. In general, glucoamylases can be, and usually are, used with alpha-amylases in starch hydrolysis processes to hydrolyze starch to dextrins and then to glucose. Glucose can be used directly; be converted to fructose by other enzymes (e.g. glucose isomerases); crystallized; or used in fermentations to produce numerous end products (e.g., ethanol, citric acid, succinic acid, ascorbic acid intermediates, glutamic acid, glycerol, 1,3-propanediol, and lactic acid).

[00140] One or more GA or GA variant or combinations thereof may also be effective in the direct hydrolysis of starch for the production of syrup and/or biochemical products (e.g., alcohols, organic acids, amino acids, other biochemicals and biomaterials) where the reaction temperature is lower than the gelatinization temperature of the substrate.

[00141] Additionally, one or more GA or GA variant or combinations thereof may be useful in a one-step enzymatic conversion process, simultaneous liquefaction and saccharification (SLS), to produce glucose-rich syrup from gelatinized starch using a liquefaction alpha-amylase and a saccharification glucoamylase above the starch gelatinization temperature. Starch concurrently undergoes gelatinization, liquefaction, and saccharification during simultaneous liquefaction and saccharification in the disclosed methods. In contrast, granular starch still maintains the crystalline structure during incubation in a "no-cook" process or a direct starch-in-glucose (DSTG) process. Consequently, due to the immediate gelatinization of starch at its operating temperature, SLS significantly reduces the time required for starch solubilization compared to DSTG.

[00142] Given the commercial importance of GAs, it can be appreciated that the nucleic acids encoding the desired GA or GA variant, the GA polypeptides or the desired GA variant and compositions comprising the same are useful in a wide variety of applications. The improved property or properties of the GAs or GA variants described herein can be exploited in many ways. For example, GAs or GA variants with improved performance under heat stress conditions can be used to increase starch hydrolysis activity in processes conducted at elevated temperatures (e.g., temperatures at which the parent GA would perform poorly), allowing for a user to reduce the total amount of GA employed (compared to the use of the parent GA). Other improved properties of GA or GA variant polypeptides may be explored, including GAs or GA variants having altered pH optima, increased stability or activity at a specific pH, increased specific activity for a substrate, and/or high level expression in a host cell of interest.

[00143] A starch hydrolysis composition containing a desired GA or GA variant as described here has application in the production of ethanol. The ethanol obtained from this process can be additionally used as an octane booster or directly as a fuel instead of gasoline, which is advantageous as ethanol as a fuel source is more environmentally friendly than petroleum products. It is known that the use of ethanol will improve air quality and possibly reduce local ozone and smoke levels. Furthermore, the use of ethanol instead of gasoline may have strategic importance in reducing the impact exerted by sudden changes in supplies of non-renewable energy and petrochemical products.

[00144] Separate saccharification and fermentation is a process by which starch present in a raw material, for example, corn, is converted into glucose and subsequently an ethanologen (for example, a strain of yeast) converts the glucose into ethanol. Simultaneous saccharification and fermentation (SSF) is a process by which starch present in a raw material is converted into glucose and, at the same time and in the same reactor, an ethanologen converts glucose into ethanol. Thus, the GA or GA variants of the invention have application in these two processes for the degradation of starch-containing raw material for the purpose of generating ethanol.

[00145] In some embodiments, a GA or variant of GA as described here is expressed in an ethanologen so that the enzymatic activity of the variant GA expressed by the ethanologen generates glucose that can be converted to ethanol by the ethanologen.

[00146] GA or variants of GA as described here have application in the generation of host cells for the preparation of biochemicals of interest. As such, aspects of the present disclosure include methods of preparing a biochemical by obtaining a host cell expressing a GA or GA variant as described herein and culturing the host cell under conditions to prepare the biochemical of interest. It is anticipated that such conditions may include, for example, pH conditions of pH 3-8 and, for example, temperature conditions of 25-95°C. The host cell may include additional modifications to promote the preparation of the desired biochemical product, for example, to express homologous or heterologous genes, additional variant genes, and/or to delete or otherwise inactivate the expression of one or more endogenous genes. Biochemicals of interest include, but are not limited to, alcohols (ethanol, methanol, butanol, etc.) and other organic compounds, including volatile organic molecules (e.g., isoprene).

[00147] It should be noted that GAs or GA variants with decreased thermostability have application, for example, in areas where it is required that enzymatic activity be neutralized at lower temperatures, so as not to affect other enzymes that may be present.

[00148] One aspect of the invention relates to the use of the GA or GA variant polypeptide according to the invention in the production of a fermented beverage, such as beer. As such, aspects of the present disclosure include a method of providing/producing a fermented beverage comprising the step of contacting a mash and/or a wort with a GA or GA variant polypeptide as described herein. A further aspect relates to a method of providing a fermented beverage comprising the following steps: (a) preparing a mash, (b) filtering the mash to obtain a wort, and (c) fermenting the wort to obtain a fermented beverage, as a beer, in which a GA or GA variant polypeptide is added: (i) to the mash of step (a) and/or (ii) to the wort of step (b) and/or (iii) to the wort of step ( w).

[00149] According to yet another aspect, a fermented beverage, such as a beer, is produced or provided by a method comprising the step(s) of (1) contacting a mash and/or a wort with a GA or GA variant polypeptide as described herein; and/or (2) (a) preparing a mash, (b) filtering the mash to obtain a wort, and (c) fermenting the wort to obtain a fermented beverage, such as beer, in which a GA or GA variant polypeptide is added: (i) to the mash from step (a) and/or (ii) to the must from step (b) and/or (iii) to the must from step (c).

[00150] Aspects of the invention also include a fermented beverage, such as a beer, produced using a GA or GA variant as described above.

[00151] The term "beer" is intended to include any fermented wort produced by the fermentation/brewing process of a starch-containing plant material. Beer is often brewed from malt or adjunct, or any combination of malt and adjunct such as starch-containing plant material.

[00152] As used herein, the term "malt" is understood to mean any malted cereal grain, such as malted barley or wheat.

[00153] As used herein, the term "adjunct" relates to any plant material containing starch and/or sugar that is not malt, such as barley or wheat malt. As examples of adjuncts, materials such as common granulated corn, refined granulated corn, ground brewer's yeast, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, roasted cereal, flakes can be mentioned. of cereals, rye, oats, corn (maize), potatoes, tapioca, cassava and syrups, such as corn syrup, sugar cane syrup, invert sugar syrup, barley and/or wheat syrups, and the like can be used as a source of starch.

[00154] As used herein, the term "mash" relates to an aqueous paste of any plant material containing starch and/or sugar as grain ready for milling, for example comprising crushed barley malt, crushed barley, and/or other adjunct or a combination of these, mixed with water later to be separated into wort and mash pomace.

[00155] As used here, the term "wort" relates to the unfermented liquid flow following extraction of ground malt during mashing.

[00156] In another aspect the invention relates to a method of preparing/producing a fermented beverage such as beer comprising mixing the polypeptide of the invention with malt or adjunct.

[00157] Examples of beers produced in accordance with the above uses and methods (i.e., in which a variant of GA as described herein is used) include, but are not limited to, the following: fully malted beer, beer brewed under from "Reinheitsgebot", “ale”, “India Pale Ale” (IPA), “lager”, “bitter”, “Happoshu” (second category beer), third category beer, “dry” beer, “near” beer , light beer, low-alcohol beer, low-calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like, but also alternative cereal and malt beverages such as fruit flavored malt beverages, e.g. citrus flavored, such as lemon, orange, lime, or berry flavored malt beverages, flavored malt liquor beverages, e.g. “malt liquor” flavored with vodka, rum or tequila, or coffee-flavored malt drinks, such as “malt liquor” flavored with coffee, and the like.

[00158] As noted from the above, GA or GA variant polypeptides (and the nucleic acids that encode them) with improved properties compared to their progenitor GA enzymes have application in improving any of some assays and processes that employ cellobiohydrolases.

EXAMPLES

[00159] The present invention is described in more detail in the following examples which are not intended, in any way, to limit the scope of the invention as claimed. It is intended that the attached Figures be considered integral parts of the specification and description of the invention. All references cited are specifically incorporated herein by way of reference for everything described therein.

Example 1 I. Effect of enzyme concentration on the comparison of GA from A. niger (An-GA) and GA from Fusarium verticillioides (Fv-GA) during saccharification

[00160] A series of experiments were performed to determine whether the new glucoamylase mixture (An-GA + Fv-GA) produces a higher glucose yield compared to the final glucose yield with individual glucoamylases (An-GA, Fv -GA) under a typical saccharification condition. Experiments were performed using liquefied corn starch with 33.5% ds and the pH of the liquefied starch was adjusted to pH 4.4 using dilute acid. The liquefied starch was placed in a water bath maintained at 60 °C and enzyme(s) were added. All Fv-GA only treatments had 0.32 SSUs of AkAA acid-stable alpha-amylase to match the acid-stable alpha-amylase present in the commercial An-GA product. The liquefied starch was kept at 60 °C for 48 hrs and samples were collected throughout the incubation to analyze the saccharide profile. Glucoamylase dosages are presented in GAUs/g of corn starch DS and/or μg/g of ds and saccharide profiles at various ranges are presented in Tables 1, 2 and 3.

[00161] Table 1 shows the saccharide profile for OPTIMAX™ L-400 (name of DuPont Industrial Bioscience's An-GA glucoamylase commercial product) at various dosages. It can be seen that, at various dosages, An-GA is unable to reach >95.5% glucose at the end of 48 hr.

[00162] Table 2 shows the saccharide profile for Fv-GA at various dosages. It can be seen that, at various dosages, Fv-GA is also unable to reach >95.5% glucose at the end of 48 hr.

[00163] Table 3 shows the saccharide profile for the mixtures of An-GA (OPTIMAX™ L-400) + Fv-GA at various mixing ratios, an unexpected increase in the final glucose yield greater than 95 was observed, 5%.

[00164] Table 1. Distribution of saccharides for various dosages of An-GA (OPTIMAXTM L-400) Table 2. Distribution of saccharides for various dosages of Fv-GA (Fusarium GA)

[00165] Table 3. Distribution of saccharides for various dosage mixing ratios of An-GA (OPTIMAX™ L-400) and Fv-GA (GA from Fusarium verticilloides)

[00166] The data shown in Tables 1-3 corroborates a surprising finding that the An-GA + Fv-GA mixture performed better and produced >95.5% glucose while An-GA and Fv-GA individually used did not produce >95.5% glucose. The discovered enzyme mixture is beneficial for glucose syrup producers as the mixture of An-GA + Fv-GA gave a higher glucose yield.

[00167] EXAMPLE 2

[00168] I. Effect of different glycoamylases (purified glycoamylases from Trichoderma reesei (Tr-GA), Humicola grisea (H-GA), Aspergillus fumigatus (Afu-GA), and Fusarium verticilloides (Fv-GA)) with An-GA in glucose production during standard saccharification

[00169] Experiments were conducted to determine the effect of mixing different glucoamylases on glucose production when mixed with An-GA. The experiments were carried out using liquefied starch at 33.5% ds and the pH of the liquefied starch was adjusted to pH 4.4. The liquefied starch was placed in a water bath maintained at 60 °C and enzyme(s) were added. The liquefied starch was maintained at 60 °C for 48 hrs and samples were collected throughout the incubation to analyze the saccharide profile (shown in Table 4).

[00170] Table 4 shows the saccharide profile for treatments with different glucoamylases added at a dosage of 10 μg/g ds (purified glucoamylase protein) with An-GA (OPTIMAX™ L-400). Among all glucoamylases tested, only Fv-GA was effective when mixed with An-GA yielding higher glucose up to more than 95.5% at 48 hr.

[00171] Table 4. Distribution of saccharides for mixtures of An-GA (OPTIMAXTM L-400) with various glucoamylases

EXAMPLE 3 I. Effect of different glycoamylases (purified glycoamylases from Trichoderma reesei (Tr-GA), Humicola grisea (H-GA), Aspergillus niger (An-GA), and Fusarium verticilloides (Fv-GA)) with GA from A. fumigatus ( Afu-GA) in glucose production during standard saccharification

[00172] A set of experiments were carried out to determine whether different glycoamylases exhibit an effect with Afu-GA similar to what they exhibited when mixed with An-GA (discussed in Example 2). The experiments were carried out using liquefied starch at 33.5% ds and the pH of the liquefied starch was adjusted to pH 4.4. The liquefied starch was placed in a water bath maintained at 60 °C and enzyme(s) were added. Only purified enzyme proteins were used for these experiments so that all enzyme treatments had 0.32 SSUs of AkAA acid-stable alpha-amylase to match the commercial An-GA product. The liquefied starch was maintained at 60 °C for 48 hrs and samples were collected throughout the incubation to analyze the saccharide profile (shown in Table 5).

[00173] Table 5 shows the saccharide profile for the various glucoamylases added with Afu-GA at a dosage of 10 μg/g ds. Similar to the observation made in Example 2 with An-GA, among all the glucoamylases tested only Fv-GA was effective when mixed with Afu-GA to increase glucose to greater than 95.5% at 48 hr. This observation shows that Fv-GA increases glucose production when mixed with An-GA or Afu-GA, whereas other glucoamylases tested have no significant effect when mixed with An-GA and Afu-GA.

[00174] Table 5. Distribution of saccharides for mixtures of Afu-GA (GA from A. fumigatus) with various glucoamylases.

EXAMPLE 4 I. Effect of the mixture of Afu-GA and Fv-GA on DP1 (% glucose)

[00175] Experiments were conducted with Afu-GA, Fv-GA and a mixture of Afu-GA + Fv-GA to determine the effect on the % glucose produced under a typical saccharification condition. The experiments were carried out using liquefied starch at 33.5% ds and the pH of the liquefied starch was adjusted to pH 4.4. The liquefied starch was placed in a water bath maintained at 60 °C and enzyme(s) were added. All enzyme treatments had 0.32 SSUs of AkAA acid-stable alpha-amylase. The liquefied starch was maintained at 60 °C for 48 hrs and samples were collected throughout the incubation to analyze the saccharide profile (shown in Figure 1).

[00176] Figure 1 shows the % glucose at the end of 48 hr for Afu-GA (80 μg/g ds), Fv-GA (80 μg/g ds) and the Afu-GA + Fv-GA mixture (40 μg/g ds + 40 μg/g ds). The data in the graph shows that the mixture of Afu-GA + Fv-GA is successful in achieving the highest % glucose at 48 hr compared to the case where Afu-GA and Fv-GA are used alone at an equal dosage. of total glucoamylase protein.

EXAMPLE 5 I. Effect of different mixing ratios of Afu-GA and Fv-GA

[00177] Experiments were conducted with different mixing ratios of Afu-GA and Fv-GA to determine the optimal ratio of glucoamylases (proteins) to be used for maximum glucose production under a typical saccharification condition. The experiments were carried out using liquefied starch at 33.5% ds and the pH of the liquefied starch was adjusted to pH 4.4. The liquefied starch was placed in a water bath maintained at 60 °C and enzyme(s) were added. Only purified enzyme proteins were used in this set of experiments so that all enzyme treatments had 0.32 SSUs of AkAA acid-stable alpha-amylase to match the acid-stable alpha-amylase present in the commercial An-GA product. The liquefied starch was maintained at 60 °C for 48 hrs and samples were collected throughout the incubation to analyze the saccharide profile (shown in Table 6).

[00178] Table 6 shows the saccharide profile for the new mixture with different protein ratios of Afu-GA and Fv-GA. When Afu-GA is used alone at 80 μg/g ds, it does not reach 95.5% glucose at 48 hrs. But when Afu-GA is mixed with Fv-GA at various ratios (keeping total protein constant at 80 μg/g ds), all mixtures achieve >95.5% glucose at 48 hrs. Thus the mixture of Afu-GA + Fv-GA with equal total glucoamylase protein resulted in the highest level of glucose production compared to Afu-GA alone.

[00179] Table 6. Distribution of saccharides for mixtures of Afu-GA and Fv-GA at various protein ratios

[00180] Figure 2 is a graph based on data from Table 6, showing the effect of the ratio of Afu-GA and Fv-GA glucoamylases on the final % glucose after 48 hr of saccharification. It was found that the ideal mixture for optimizing glucose yield was the intermediate level protein ratio shown as 40 + 40. Although all mixtures achieved >95.5% glucose at 48 hrs, it was discovered that, surprisingly , the ratio of intermediate level proteins shown as 40 + 40 for Afu-GA and FV-GA reached >96% glucose at 48 hrs.

EXAMPLE 6 I. Effect of different mixing ratios of Afu-GA and Fv-GA at various pH conditions

[00181] A set of experiments was carried out with a mixture of Afu-GA + Fv-GA and compared with results for a commercial product, OPTIMAX 4060 VHP. The experiments were carried out using liquefied starch at 33.5% ds and the pH of the liquefied starch was adjusted to pH 4.4 and/or 5.5. The liquefied starch was placed in a water bath maintained at 60 °C and enzyme(s) were added. All enzyme treatments with purified non-commercial enzymes had 0.32 SSUs of AkAA acid-stable alpha-amylase to match the commercial product. The liquefied starch was maintained at 60°C for 48 hrs and samples were collected throughout the incubation to analyze the saccharide profile (shown in Figure 3).

[00182] Figure 3 shows the % glucose at 48 hr of incubation at two different pH conditions. The commercial product, OPTIMAXTM 4060 VHP, has reasonable performance at pH 4.5 and produces >95.5% glucose but does not perform well at pH 5.5 and only reaches 90.93% glucose at 48 hr. OPTIMAXTM 4060 VHP contains an A. niger glucoamylase, acid-stable alpha-amylase and pullulanase that helps achieve 95.5% glucose at pH 4.5 but suffers at high pH values, e.g. > 5, 0, since pullulanase is deactivated at higher pH values. This deactivation leads to a drop in the performance of the OPTIMAX 4060 VHP product at high pH. The Afu-GA + Fv-GA blend maintains performance over a wide pH range, pH 4.5 to 5.5 and consistently achieves >95.5% glucose.

EXAMPLE 7 I. Effect of a debranching enzyme, Pullulanase (PU), on a range of mixing ratios of Afu-GA and Fv-GA.

[00183] A set of experiments were carried out with the commercial product OPTIMAX™ 4060 VHP as it already contains a pullulanase debranching enzyme that helps it reach 95.5% glucose. Two different glucoamylases (Afu-GA and Fv-GA) were mixed in addition to the standard dosage of 0.16 GAUs/g ds of OPTIMAX™ 4060 VHP. Additional glucoamylases Afu-GA and Fv-GA were added at 10 μg/g ds.

[00184] Table 7 shows the saccharide profile for OPTIMAX™ 4060 VHP and for treatments in which additional glucoamylases Afu-GA or Fv-GA are added in addition to the standard dosage of OPTIMAX™ 4060 VHP. The standard dosage of OPTIMAX™ 4060 VHP achieves 95.52% glucose in 72 hr. The addition of Afu-GA glucoamylase does not increase the % glucose at 72 hr saccharification time and achieves 95.6% glucose which is similar to the standard dosage of OPTIMAX™ 4060 VHP.

[00185] Table 7. Distribution of saccharides for OPTIMAX™ 4060 VHP and additional mixing with Afu-GA or Fv-GA glucoamylases.

[00186] Figure 4 which is based on data from Table 7, shows the % glucose at 72 hr for OPTIMAX™ 4060 VHP and additional mixing with Afu-GA or Fv-GA glucoamylases.

[00187] Although each test condition achieved >95% glucose after 72 hrs, the combination of OPTIMAX™ 4060 VHP + Fv-GA has an unexpected improved effect of % glucose increased to 96.35%, which is significantly higher than that observed for the standard dosage of OPTIMAX™ 4060 VHP alone or treatment with OPTIMAX™ 4060 VHP + Afu-GA.

EXAMPLE 8 I. The effect of Fv-GA and additional GA mixture on simultaneous saccharification and fermentation (SSF) in a corn-to-ethanol conversion process.

[00188] A liquefied corn product was obtained from a commercial fuel alcohol producer. The specifications of the corn liquefied product were 32.84% ds and pH 3.5. The pH of the liquefied corn was adjusted to 4.5 with 4 N sodium hydroxide. The experimental SSF process was conducted in repetitions simultaneously in 250 mL Erlenmeyer flasks using 100 g of the liquefied product per flask. Appropriate amounts of enzymes were added to the liquefied product along with dry yeast and 600 ppm urea. The dry yeast is allowed to hydrate for about 10 minutes before adding to the flasks. The enzyme doses tested were 1) Tr-GA variant CS4 ("CS4") at 25 μg g ds and Fv-GA at 25 μg g ds; 2) GA CS4 at 50 μg g of ds and 3) Fv-GA at 50 μg g of ds. The flasks were covered with rubber plugs and placed in a forced air incubator at 32 °C with shaking at 200 rpm for 54 hrs. Samples were taken at regular intervals for ethanol measurements.

[00189] The ethanol data provided in Table 8 shows that for the mixture of Fv-GA plus GA CS4 the ethanol rates were higher and the final ethanol concentrations were also the highest among the compared treatments. Mixing GAs at an equal total protein addition was better compared to adding individual GA at an equal total protein loading. This reveals a synergistic effect of Fv-GA with the other GA.

[00190] A similar effect was observed for the reduction of DP4+ as the mixture of Fv-GA plus CS4 achieved the lowest level of DP4+ compared to individual GA additions to an equal addition of total protein.

[00191] A lower level of DP4+ and a higher level of ethanol is important for the commercial ethanol producer as this advantage translates into higher conversion of the substrate into the desired final product, for example, ethanol.

[00192] Table 8 The distribution of % w/v of ethanol and % w/v of DP4+ for the mixture CS4 + Fv-GA, GA CS4 and Fv-GA showing lower % w/v of DP4+ and higher % w/v of ethanol to the GA mixture.

[00220] Figure 5 shows the % w/v of DP4+ at 54 hr for the mixture CS4 + Fv-GA, GA CS4 alone and Fv-GA alone. It is clear that lower % w/v DP4+ was advantageously obtained for the GA mixture. As indicated in Figure 5, about 0.3% w/v lower DP4+ was obtained for the GA mixture compared to GA CS4 or Fv-GA alone. GA CS4 alone reduced DP4+ to 1.03% w/v whereas the new GA mixture reduced DP4+ to 0.74% w/v which is a decrease of 25.2% DP4+ under similar condition. Use of the mixture can lead to a 1.03x reduction in DP4+ under similar conditions.

[00221] Figure 6 shows the % w/v of ethanol at 54 hr for the mixture CS4 + Fv-GA, GA CS4 and Fv-GA, in which a higher % w/v of ethanol was generated for the GA mixture . As indicated, the % w/v ethanol was about 0.2% higher for the GA mixture compared to either GA CS4 or Fv-GA alone. GA CS4 alone achieved an ethanol content of 14.98% v/v while the new GA blend achieved an ethanol content of 15.26% v/v which is an overall increase of 1.86% in production of ethanol relative to GA CS4 alone under similar conditions. The use of the mixture can lead to a 1.018x increase in ethanol production under similar conditions.

EXAMPLE 9 I. The effect of Fv-GA and an additional GA mixture on saccharification under biochemical fermentation conditions of pH 7.0 and at 37°C.

[00222] Experiments were conducted with Afu-GA, HGA, TrGA, Fv-GA and a mixture of HGA + Fv-GA to determine the effect of individual GA enzymes and GA mixtures on final % glucose. The experiments were carried out using liquefied starch at 33.5% ds and the pH of the liquefied starch was adjusted to pH 7.0. The liquefied starch was placed in a water bath maintained at 37 °C and enzyme(s) were added. The liquefied starch was maintained at 37 °C for 72 hrs and samples were collected throughout the incubation to analyze the saccharide profile.

[00223] The data in Table 9 shows that Fv-GA at 80 μg/g ds was much faster and achieved the highest final glucose % compared to other GAs (also added at 80 μg/g ds). Furthermore, Fv-GA added at 80 μg/g ds was slightly better compared to HGA at 160 μg/g ds.

[00224] The data also shows that the mixture of Fv-GA at 40 μg/g ds and HGA at 160 μg/g ds is better compared to HGA at 320 μg/g ds (Table 10). This shows an important and unexpected synergy between Fv-GA and HGA. The mixture of Fv-GA and HGA (total protein addition of 200 μg/g ds, with Fv-GA at 40 μg/g ds and HGA at 160 μg/g ds) achieves a lower amount of DP4+ and the higher amount of % DP1 compared to HGA even at 320 μg/g ds.

[00225] Table 9. Saccharide distribution for Afu-GA, Fv-GA, HGA, and TrGA at 80 μg/g protein dosage ds during saccharification under biochemical fermentation conditions of 37 °C and pH 7.0

[00226] Table 10. The distribution of saccharides for the mixture Fv-GA + HGA and HGA at higher dosages showing that the mixture of Fv-GA plus HGA has better performance compared to HGA at the highest dosage during saccharification under conditions biochemical fermentation at 37 °C and pH 7.0

EXAMPLE 10 I. The effect of Fv-GA and an additional GA mixture on the direct starch to glucose (DSTG) conversion process.

[00227] Experiments were conducted with Afu-GA, Fv-GA and a mixture of Afu-GA + Fv-GA to determine the effect of the enzyme and enzyme mixtures on the final % solubility and % glucose. The experiments were carried out using 32% ds crude tapioca starch and the pH of the tapioca starch paste was left as is at pH 5.2. Enzymes were added as such 1) Afu-GA at 80 μg/g ds 2) Afu-GA at 40 μg/g ds + Fv-GA at 40 μg/g ds and 3) Fv-GA at 80 μg /g of ds. All treatments also included 10 AAUs of the alpha-amylase product SPEZYME Xtra® (DuPont). The slurry was mixed at room temperature to mix the added enzymes. An aliquot of 35 g with 2 replicates for each treatment was weighed in LABOMAT steel beakers. The metal beakers were again adjusted in the LABOMAT for incubation at 60 °C for 72 hrs with constant mixing in the forward and counterclockwise direction at 60 rpm. Samples were collected throughout the incubation to analyze the saccharide profile.

[00228] The data in Table 11 shows that the mixture of Fv-GA plus Afu-GA reaches a % solubility of 96.51 in 72 hrs and the individually added Afu-G or Fv-GA reach 93.05 and 94. 89% solubility, respectively. Furthermore, the values of % DP1 and % DP4+ for the mixture of Fv-GA and Afu-GA were 96.51% and 0.71%, respectively. This shows that the mixture of Fv-GA plus Afu-GA unexpectedly achieved the lowest amount of DP4+ and the highest amount of % DP1 compared to individually added Afu-GA or Fv-GA.

[00229] Table 11. Distribution of saccharides and % solubility for AfuGA, mixture Fv-GA + Afu-GA and Fv-GA showing that the mixture of Fv-GA plus AfuGA has better performance compared to Afu-GA and Fv- GA individually added during DSTG of tapioca starch at 60 °C and pH 5.2

SEQUENCE LISTING SEQ ID NO: 1 Fv-GA Protein Sequence:

[00231] DLAYTVKFWNRTGYDLWEEVNGSSFFTLSASHRALVEGAAL AKKLGKSCSDCATNAPRVLCFMQSFWTGSYIDSNINVNDGRKGLDA NSILSSIHTFDPSSKCTDSTFQPCSSRALANHKEVVDSFRSIYGVNKN RGKGKAAAVGRYSEDVYYDGNPWYLATLAAAEQLYAAVYQWNKIGS ITVDSVSLPFFSDLVPKVSKGTYRK NSKTYKAIIKAVTSYADGFVAVVQ TYTPKDGSLAEQFDKSTGTPKSAVHLTWSYASFVGAAERRTGVVPP AWGESNANKVPAVCEAAPACDTTITFNVKNVDVTSDQKVYIVGGITQ LSNWAPADGIALEESTSTKGLWTVKVKIPSDTSFEYKYIKKTSDGTVT WESDPNNSAATGSKCGSSSTINDEWR

SEQ ID NO: 2 Fv-GA DNA Nucleotide Sequence:

[00231] ATGTTTACTCAGATCTTGTATGGCCTCACGGCCTTATCT GCCCTCCAAGGTCAGGTCACTGCATCACCAGGCGGTTCCAGCCT TGATCGCTTCATTTCCAAAGAGGCCGACATCTCCATCAAGGGCGT GCTTGCCAATATTGGCGCCGATGGCAAGCGAGCCCAAGGCGCTG CACCTGGCGCCGTTGTAGCAAGTCCATCGAGAACAGATCCAGACT GTAAGTCAATATTCTTGATA GTATCGCAAATGGATGACTGAGACGT TTTCTAGACTGGTACACATGGACCCGAGACTCCGCCTTGACATAC AAAGTCCTTGTTGAGCGCTTCATTCACGGAGACAAGTCTCTCCAG CGCAAGATCGACGAATATGTCTCTGCCCAAGCCAAGCTCCAGGG CGTCACCAACCCATCTGGCGGCCCCGAGTCAGGCGGCCTTGGGG AACCCAAGTTTCACGTCAACCTCACAGCTTTCACAGGATCCTGGG GTCGTCCTCAACGAGATGGCCCTCCTCTGAGAGCTACTGCATTGA CGCTGTACGCCAACTGGCTTGTTTCTCACGGCGACGCTCCAAG GCCGTCAACAAGGTTTGGCCTGTCATTGAGAAGGATCTTGCATAC ACCGTCAAGTTCTGGAACAGAACCGGTTACGATCTTTGGGAGGAG GTTAACGGATCTTCGTTCTTCACCCTCTCTGCTTCACATCGTGCTC TGGTTGAGGGAGCTGCTCTTGCTA AGAAGCTTGGCAAGTCTTGCT CCGACTGCGCAACCAACGCCCCCCGTGTTCTCTGCTTCATGCAAA GCTTCTGGACCGGCAGCTACATCGACTCGAACATCAATGTCAACG ATGGCCGCAAGGGTCTTGATGCCAACTCCATTCTGTCTTCTATTCA CACCTTTGATCCTTCTTCGAAGTGCACAGACTCTACCTTCCAGCCT TGTTCTTCAAGGGCTCTTGCGAACCACAAGGAAGTAGTGGACTCT TTCC GCTCCATCTATGGTGTCAACAAAAACAGAGGTAAAGGTAAA GCTGCTGCTGTCGGTCGATACAGTGAGGATGTGTACTACGACGGT AACCCTTGGTACTTGGCTACTCTTGCTGCTGCCGAGCAACTGTAC GCTGCTGTCTATCAATGGAACAAGATCGGTTCCATCACAGTTGATA GTGTGTCGCTCCCCTTTTTCAGTGACCTTGTACCAAAGGTTTCCAA GGGAACCTATCGCAAGAACAGCAAGACATACAA GGCTATTATCAA GGCTGTCACTTCATACGCTGATGGCTTTGTCGCCGTTGTGCAGAC CTATACTCCCAAAGATGGCTCCCTCGCAGAGCAGTTCGATAAGTC TACTGGAACTCCCAAGTCAGCTGTTCACCTAACCTGGTCCTACGC CTCCTTTGTCGGTGCTGCCGAGCGTCGTACTGGCGTCGTTCCTCC AGCTTGGGGCGAGAGCAACGCCAACAAGGTGCCTGCTGTTTGCG AAGCAGCTCCAGCCTGC GACACCACCATCACGTTCAATGTGAAGA ACGTTGATGTCACGTCGGACCAGAAGGTTTACATTGTTGGCGGGA TCACTCAACTTTCCAACTGGGCCCCTGCTGACGGCATTGCGCTTG AGGAATCCACGAGCACCAAGGGCTTGTGGACTGTGAAGGTCAAG ATTCCATCTGATACCAGCTTTGAGTATAAGTATATAAAGAAGACGA GTGATGGAACTGTTACATGGGAGAGTGACCCCAATAACAGTGCGG C GACGGGCAGCAAGTGCGGAAGCAGCAGTACCATCAACGATGAG TGGAGGTAA

SEQ ID NO: 3 Afu-GA Protein Sequence:

[00232] MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETT VALNGADGAYAKPGIIIASPSTSEPDYYTWTRDAALVTK VALVDLFRNLGLQKVITEYVNSQTVSNPSGglaepky Ratalidfgggglidngyssyavnni wpivrndlsyvsywsqywsqsgfdlweevnsfftvavvqhralvegst fakrvgascddsqilcymqsfwtgsyinantgggggggkda ntvlasihtfdpeagdttfcdpcsfqpcspralanhkvytdsfrsvytdsfsvytdsfrsvytdsfrsvytdsfrsvytdsfrsvytdsfrsvytdSfrsvytdsfrsvytdsfrsvytdsfrsvytdsfrs G IpqgaVsagrypedvyyngnpwflttlaaaaeqlydaiyqwkgsis itstslaffkdiyssaavgtyasstftdineadgyvsivqaha mnngslseqfdkssglslslslslrssltarsysyyyyyyyyya gasansvps TswpstttttttttSgtaetaCatPTAVTFNETTTYGENVGSISELGNGSSKYTSNLWYVSVTLPAGTTEYKYIRKESDGSIVWE SDPNSYTVSYTVSYTVSYTVSYTVSYTDTWQ

SEQ ID NO: 4 Afu-GA Nucleotide Sequence:

[00233] atgcctcgcctttcctacgcgctctgtgcgctgtctctcgggcatgctgctattgcagc tcctcagttatccgctcgtgctaccggcagcttggactcctggttgggtactgagaccaccgttgcgc tcaatggtattctggccaacatcggtgccgacggtgcttatgcgaagagc gctaagcctggcata atcattgccagtccgagcaccagcgaaccagactgtgagaaccttcctgaactggccctgtccgg cagtcattgacctcggtagactactatacctggacgagagatgctgctctcgtcacgaaagtcctg gtcgacctcttccgcaacggcaacctgggtctgcagaaagtcattaccgaatacgtcaactctca ggcgtacttgcagaccgtgtctaatccgtcgggtggtcttgcgagcggaggtctcgcggagcctaa gtacaacgtcgacatgacggcctttaccggagcctggggtcgtcctcagcgtgatggtccggctct gcgggccaccgccctcatcgactttggcaactggctgattgtatgttctccatacgagcccca gga agcgttgctgacgtctacaggacaacggctactccagctatgctgtcaacaacatctggcccattg tgcgcaacgacttgtcctacgtttctcagtactggagccagagtggctttggtgagtcccgactctct ggaagtttacaacgtgcatcgattactgacaattgagattctacgtgacagatctctgggaagaag tcaactcca tgtccttcttcaccgtcgctgtccagcaccgtgccctcgtggagggaagcacgttcgc taaacgggtgggagcgtcgtgctcgtggtgtgactcgcaggccccccagatcctctgctacatgc agagtttctggactggctcgtatatcaacgccaacaccggtggtggccggtccggcaaggatg cc aacaccgtcctcgccagcatccataccttcgaccccgaagccggctgcgacgatactactttcca gccctgctctcctcgggcccttgccaaccacaaggtgtacaccgattcgttccgctcggtctacgcg atcaactccggcatcccacagggcgctgccgtttccgctggccgctaccccgaggacgtctacta caacggcaac ccttggttcctcaccaccctcgccgctgccgagcagctctacgacgctatctacc agtggaagaagatcggttccatcagcatcaccagcacctccctcgccttcttcaaggacatctaca gctccgccgcggtcggcacctacgcctctagcacctccaccttcacggacatcatcaacgcggtc aagacctacgcagacggct acgtgagcatcgtccaggcacacgccatgaacaacggctcccttt cggagcaattcgacaagtcctctgggctgtccctctccgcccgcgatctgacctggtcctacgccg ctttcctcaccgccaacatgcgtcgtaacggcgtggtgcctgccccctggggcgccgcctccgcc aactccgtcccctcgtct tgctccatgggctcggccacgggcacctacagcaccgcgacagcca cctcctggcccagcacgctgaccagcggctcgccaggcagcaccaccaccgtgggcaccacg accagtaccacctctggcaccgccgccgagaccgcctgtgcgacccctaccgccgtggccgtc acctttaacgagatcgccaccaccacctacggcgagaat gtttacattgttgggtccatctccgag ctcgggaactgggataccagcaaagcagtggccctgagtgcgtccaagtatacctccagcaata acctctggtacgtgtccgtcaccctgccggctggcacgacattcgagtacaagtatatccgcaag gaaagcgatggctcgatcgtgtgggagagtgaccccaaccgctcg tatacggtgccggcagctt gtggagtgtctactgcgaccgagaatgatacttggcagtga

SEQ ID NO: 5 Hg-GA Protein Sequence:

[00234] MHTFSKLLVLGSAVQSALGRPHGSSRLQERAAVDTFINTE KPIAWNKLLANIGPNGKAAPGAAAGVVIASPSRTDPPYFFTWTPDAAL VLTGIIESLGHNYNTTLQTVIQNYVASQAKLQQVSNPSGTFADGSGLG EAKFNVDLTAFTGEWGRPQRDGPPLRAIALIQYAKWLIANGYKSTAK SVVWPV VKNDLAYTAQYWNETGFDLWEEVPGSSFFTIASSHRALTE GAYLAAQLDTECPPCTTVAPQVLCFQQAFWNSKGNYVVSTSTAGEY RSGKDANSILASIHNFDPEAGCDNLTFQPCSERALANHKAYVDSFRN LYAINKGIAQGKAVAVGRYSEDVYYNGNPWYLANFAAAEQLYDAIYV WNKQGSITVTSVSLPFFRDLVSS VSTGTYSKSSSTFTNIVNAVKAYAD GFIEVAAKYTPSNGALAEQYDRNTGKPDSAADLTWSYSAFLSAIDRR AGLVPPSWRASVAKSQLPSTCSRIEVAGTYVAATSTSFPSKQTPNPS AAPSPSPYPTACADASEVYVTFNERVSTAWGETIKVVGNVPALGNW DTSKAVTLSASGYKSNDPLWSITVPIKATGSAVQYKYIKVGTNG KITW ESDPNRSITLQTASSAGKCAAQTVNDSWR

SEQ ID NO: 6 Hg-GA Nucleotide Sequence:

[00235] atgcataccttctccaagctcctcgtcctgggctctgccgtccagtctgccctcggg cggcctcacggctcttcgcgtctccaggaacgcgctgccgttgataccttcatcaacaccgagaa gcccatcgcatggaacaagctgctcgccaacatcggccctaacggcaaagccgctcccggtgc cgcc gccggcgttgtgattgccagcccttccaggacggaccctccttgtacgtggtggcatggaat ggacccaagagactgttttagatgaaagagagtttctgctaaccgccacacccagacttcttcacc tggaccccggatgccgccctggtcctcaccggcatcatcgagtcccttggccacaactacaacac caccctgcagaccgtcat ccagaactacgtcgcgtcgcaggccaagctgcagcaggtctcgaa cccctcgggaaccttcgccgacggctcgggtctcggtgaggccaagttcaatgtcgacctcactg ccttcactggcgaatggggtcgccctcagagggacggcccgcccctgcgcgccatcgctctcatc cagtacgccaagtggctgatc gccaacggctacaagagcacggccaagagcgtcgtctggcc cgtcgtcaagaacgatctcgcctacacggcccagtactggaacgagaccggcttcgatctctgg gaggaggtccccggcagctcgttctttaccatcgccagctctcacaggggtgagtcatttattgttca gtgttttctcattgaataattaccggaatgcc actgacgccaaacagctctgactgagggtgcttacc tcgccgctcagctcgacaccgagtgcccgccctgcacgaccgtcgcccctcaggttctgtgcttcc agcaggccttctggaactccaagggcaactatgtcgtctcaacagtaagatccctacaccaaca aaaaaatcgaaaaggaacgttagctgacccttctag tcaacggcgggcgagtatcgctccgg caaggacgccaactcgatcctggcgtccatccacaacttcgaccctgaggccggctgcgacaa cctgaccttccagccctgcagcgagcgcgccctggccaaccacaaggcctatgtcgactcgttcc gcaacctctacgccatcaacaagggcatcgcccagggcaaggccgttg ccgtcggccgctact cggaggatgtctactacaacggcaacccgtggtacctggccaactttgccgccgccgagcagct ctacgacgccatctacgtgtggaacaagcagggctccatcaccgtgacctcggtctccctgccctt cttccgcgaccttgtctcgtcggtcagcaccggcacctactccaagagcagctcga ccttcaccaa catcgtcaacgccgtcaaggcctacgccgacggcttcatcgaggtggcggccaagtacacccc gtccaacggcgcgctcgccgagcagtacgaccgcaacacgggcaagcccgactcggccgcc gacctgacgtggtcgtactcggccttcctctcggccatcgaccgccgcgcgggtctcgtcc ccccg agctggcgggccagcgtggccaagagccagctgccgtccacctgctcgcgcatcgaggtcgcc ggcacctacgtcgccgccacgagcacctcgttcccgtccaagcagaccccgaacccctccgcg gcgccctccccgtccccctacccgaccgcctgcgcggacgctagcgaggtgtacgtcacc ttca acgagcgcgtgtcgaccgcgtggggcgagaccatcaaggtggtgggcaacgtgccggcgctg gggaactgggacacgtccaaggcggtgaccctgtcggccagcgggtacaagtcgaatgatccc ctctggagcatcacggtgcccatcaaggcgacgggctcggccgtgcagtacaagtatatca agg tcggcaccaacgggaagattacttgggagtcggaccccaacaggagcattaccctgcagacgg cgtcgtctgcgggcaagtgcgccgcgcagacggtgaatgattcgtggcgttaa

SEQ ID NO: 7 An-GA Protein Sequence:

[00236] MSFRSLLALSGLVCTGLANVISKRATWDSWLSNEATVART AILNNIGADGAWVSGADSGIVVASPSTDNPDYFYTWTRDSGLVLKTL VDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNV DETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWP LVRNDLSYVAQYWNQTGYDLW EVNGSSFFTIAVQHRALVEGSAFAT AVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSAKDANTLLL GSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSD SEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEV TDVSLDFFKALYSDATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHA AS NGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNVVPSASW GETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSG SVTSTSKTTATASKTSTSTSSTSCTTPTAVAVTFDLTATTTYGENIYLV GSISQLGDWETSDGIALSADKYTSSDPLWYVTVTLPAGESFEYKFIRI ESDDSVEWESDPNREYTVPQACGTSTATVTDTWR

SEQ ID NO: 8 An-GA Nucleotide Sequence:

[00237] atgtcgttccgatctctactcgccctgagcggcctcgtctgcacagggttggcaaat gtgatttccaagcgcgcgacctgggattcatggttgagcaacgaagcgaccgtggctcgtactgc catcctgaataacatcggggcggacggtgcttgggtgtcgggcgcggactctggcattg tcgttgc tagtcccagcacggataacccggactacttctacacctggactcgcgactctggtctcgtcctcaa gaccctcgtcgatctcttccgaaatggagataccagtctcctctccaccattgagaactacatctcc gcccaggcaattgtccagggtatcagtaacccctctggtgatctgtccagcggcgctggtctcggt gaaccca agttcaatgtcgatgagactgcctacactggttcttggggacggccgcagcgagatg gtccggctctgagagcaactgctatgatcggcttcgggcaatggctgcttgacaatggctacacca gcaccgcaacggacattgtttggcccctcgttaggaacgacctgtcgtatgtggctcaatactgga accagacaggatatgat ctctgggaagtcaatggctcgtctttctttacgattgctgtgcaacaccgc gcccttgtcgaaggtagtgccttcgcgacggccgtcggctcgtcctgctcctggtgtgattctcaggc acccgaaattctctgctacctgcagtccttctggaccggcagcttcattctggccaacttcgatagca gccgttcc gccaaggacgcaaacaccctcctgctgggaagcatccacacctttgatcctgaggc cgcatgcgacgactccaccttccagccctgctccccgcgcgcgctcgccaaccacaaggaggtt gtagactctttccgctcaatctataccctcaacgatggtctcagtgacagcgaggctgttgcggtgg gtcggtaccctgaggacac gtactacaacggcaacccgtggttcctgtgcaccttggctgccgca gagcagttgtacgatgctctataccagtgggacaagcaggggtcgttggaggtcacagatgtgtc gctggacttcttcaaggcactgtacagcgatgctactggcacctactcttcgtccagttcgacttata gtagcattgtagatgccg tgaagactttcgccgatggcttcgtctctattgtggaaactcacgccgca agcaacggctccatgtccgagcaatacgacaagtctgatggcgagcagctttccgctcgcgacc tgacctggtcttatgctgctctgctgaccgccaacaaccgtcgtaacgtcgtgccttccgcttcttggg gcgagacc tctgccagcagcgtgcccggcacctgtgcggccacatctgccattggtacctacag cagtgtgactgtcacctcgtggccgagtatcgtggctactggcggcaccactacgacggctaccc ccactggatccggcagcgtgacctcgaccagcaagaccaccgcgactgctagcaagaccagc accagtacgtcatcaacctcctg taccactcccaccgccgtggctgtgactttcgatctgacagcta ccaccacctacggcgagaacatctacctggtcggatcgatctctcagctgggtgactgggaaac cagcgacggcatagctctgagtgctgacaagtacacttccagcgacccgctctggtatgtcactgt gactctgccggctggtgagtcgtttgag tacaagtttatccgcattgagagcgatgactccgtggag tgggagagtgatcccaaccgagaatacaccgttcctcaggcgtgcggaacgtcgaccgcgacg gtgactgacacctggcggtag

SEQ ID NO: 9 Mature variant CS4 Tr-GA Protein Sequence:

[00238] SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPS TIDPDYYYMWTRDSALVFKNLIDRFTETYDAGLQRRIEQYITAQVTLQ GLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQRDGPALRAIALI GYSKWLINNNYQSTVSNVIWPIVRNDLNYVAQYWNQTGFDLWEEVN GSSFFTVANQHRALV EGATLAATLGQSGSAYSSVAPQVLCFLQRFW VSSGGYVDSNINTNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCS DKALSNLKVVVDSFRSIYGVNKGIPAGAAVAIGRYAEDVYYNGNPWY LATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSS SSTFTNIINAVSTYADGFLSEAAKYVPADGSLA EQFDRNSGTPLSAVH LTWSYASFLTAAARRAGIVPPSWANSSASTIPSTCCSGASVVGSYSRP TATSFPPSQTPKPGVPSGTPYTPLPCATPTSVAVTFHELVSTQFGHT VKVAGNAAALGNWSTSAAVALDAVNYRDNHPLWIGTVNLEAGDVVE YKYIIVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQS

SEQ ID NO: 10 Mature Tr-GA Protein Sequence:

[00239] SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPS TIDPDYYYMWTRDSALVFKNLIDRFTETYDAGLQRRIEQYITAQVTLQ GLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQRDGPALRAIALI GYSKWLINNNYQSTVSNVIWPIVRNDLNYVAQYWNQTGFDLWEEVN GSSFFTVANQHRALV EGATLAATLGQSGSAYSSVAPQVLCFLQRFW VSSGGYVDSNINTNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCS DKALSNLKVVVDSFRSIYGVNKGIPAGAAVAIGRYAEDVYYNGNPWY LATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSS SSTFTNIINAVSTYADGFLSEAAKYVPADGSLA EQFDRNSGTPLSALH LTWSYASFLTATARRAGIVPPSWANSSASTIPSTCCSGASVVGSYSRP TATSFPPSQTPKPGVPSGTPYTPLPCATPTSVAVTFHELVSTQFGQT VKVAGNAAALGNWSTSAAVALDAVNYADNHPLWIGTVNLEAGDVVE YKYINVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQS SEQ ID NO: 11 Parent Tr-GA Protein Sequence (632 amino acids): 1 MHVLSTAVLL GSVAVQKVLG RPGSSGLSDV TKRSVDDFIS TETPIALNNL 51 LCNVGPDGCR AFGTSAGAVI ASPSTIDPDY YYMWTRDSAL VFKNLIDRFT 101 ETYDAGLQRR IEQYITAQVT LQGLSNPSGS LADGSGLGEP KFELTLKPFT 151 GNWGRPQRDG PALRAIALIG YSKWLINNNY QSTVSNVIWP IVRNDLNYVA 201 QYWNQTGFDL WEEVNGSSFF TVANQHRALV EGATLAATLG QSGSAYSSVA 251 PQVLCFLQRF WVSSGGYVDS NINTNEGRTG KDVNSVLTSI HTFDPNLGCD 301 AGTFQPCSDK ALSNLKVVVD SFRSIYGVNK GIPAGAAVAI GRYAEDVYYN 351 GNPWYLATFA AAEQLYDAIY VWKKTGSITV TATSLAFFQE LVPGVTAGTY 401 SSSSSTFTNI INAVSTYADG FLSEAAKYVP ADGSLAEQFD RNSGTPLSAL 451 HLTWSYASFL TATARRAGIV PPSWANSSAS TIPSTCSGAS VV GSYSRPTA 501 TSFPPSQTPK PGVPSGTPYT PLPCATPTSV AVTFHELVST QFGQTVKVAG 551 NAAALGNWST SAAVALDAVN YADNHPLWIG TVNLEAGDVV EYKYINVGQD 601 GSVTWESDPN HTYTVPAVAC VTQVVKEDTW QS SEQ ID NO: 12 Nucleotide Sequence of Tr-GA (1899 bp): 1 ATGCACGTCC TGTCGACTGC GGTGCTGCTC GGCTCCGTTG CCGTTCAAAA 51 GGTCCTGGGA AGACCAGGAT CAAGCGGTCT GTCCGACGTC ACCAAGAGGT 101 CTGTTGACGA CTTCATCAGC ACCGAGACGC CTATTGCACT GAACAATCTT 151 CTTTGCAATG TTGGTCCTGA TGGATGCCGT GCATTCGGCA CATCAGCTGG 201 TGCGGTGATT GCATCTCCCA GCACAATTGA CCCGGACTAC TATTACATGT 251 GGACGCGAGA TAGCGCTCTT GTCTTCAAGA ACCTCATCGA CCGCTTCACC 301 GAAACGTACG ATGCGGGCCT GCAGCGCCGC ATCGAGCAGT ACATTACTGC 351 CCAGGTCACT CTCCAGGGCC TCTCTAACCC CTCGGGCTCC CTCGCGGACG 401 GCTCTGGTCT CGGCGAGCCC AAGTTTGAGT TGACCCTGAA GCCTTTCACC 451 GGCAACTGGG GTCGACCGCA GCGGGATGGC CCAGCTCTGC GAGCCATTGC 501 CTTGATTGGA TACTCAAAGT GGCTCATCAA CAACAACTAT CAGTCGACTG 551 TGTCCAACGT CATCTGGCCT ATTGTGCGCA ACGACCTCAA CTATGTTGCC 60 1 CAGTACTGGA ACCAAACCGG CTTTGACCTC TGGGAAGAAG TCAATGGGAG 651 CTCATTCTTT ACTGTTGCCA ACCAGCACCG AGCACTTGTC GAGGGCGCCA 701 CTCTTGCTGC CACTCTTGGC CAGTCGGGAA GCGCTTATTC ATCTGTTGCT 751 CCCCAGGTTT TGTGCTTTCT CCAACGATTC TGGTGTCGT CTGGTGGATA 801 CGTCGACT DC GAGGATGT GTACTACAAC CGTCGCCATT GGCCGGTATG 1051 GGCAACCCTT GCTGCCGAGC AGCTGTACGA GGTATCTTGC TACATTTGCT 1101 TGCCATCTAC CATCACGGTG ACCGCCACCT GTCTGGAAGA AGACGGGCTC 1151 CCCTGGCCTT GCGTGACGGC CGGGACCTAC CTTCCAGGAG CTTGTTCCTG 1201 TCCAGCAGCT ATCAA CGCCG TCTCGACATA CTTCGACCTT TACCAACATC 1251 CGCCGATGGC GTACGTCCCC GCCGACGGTT TTCCTCAGCG AGGCTGCCAA 1301 CGCTGGCCGA GCACTCCGCT GTCTGCGCTT GCAGTTTGAC CGCAACAGCG 1351 CACCTGACGT ACAGCCACGG CCCGTCGGGC GGTCGTACGC CTCGTTCTTG 1401 TGGCATCGTG CAGCGCTAGC ACGATCCCCT CCCCCCTCGT GGGCCAACAG 1451 CGACGTGCTC CCTACTCGCG TCCCACCGCC CGGCGCGTCC GTGGTCGGAT 1501 ACGTCATTCC CTCCGTCGCA GACGCCCAAG CCTGGCGTGC CTTCCGGTAC 1551 TCCCTACACG CCCCTGCCCT GCGCGACCCC AACCTCCGTG GCCGTCACCT 1601 TCCACGAGCT CGTGTCGACA CAGTTTGGCC AGACGGTCAA GGTGGCGGGC 1651 AACGCCGCGG CCCTGGGCAA CTGGAGCACG AGCGCCGCCG TGGCTCTGGA 1701 CGCCGTCAAC TATGCCGATA ACCACCCCCT GTGGATTGGG ACGGTCAACC 1751 TCGAGGCTGG AGACGTCGTG GAGTACAAGT ACATCAATGT GGGCCAAGAT 1801 GGCTCCGTGA CCTGGGAGAG TGA TCCCAAC CACACTTACA CGGTTCCTGC 1851 GGTGGCTTGT GTGACGCAGG TTGTCAAGGA GGACACCTGG CAGTCGTAA

Claims (4)

1. Method for saccharifying a starch substrate comprising at least 33.5% dry solids (ds) to produce glucose, characterized by the fact that it comprises contacting the starch substrate with an Fv-GA glucoamylase encoded by SEQ ID NO: 1, and a CS4 variant Tr-GA glucoamylase encoded by SEQ ID NO: 9. 2. Method according to claim 1, characterized by the fact that it further comprises fermenting glucose to produce alcohol. 3. Method according to claim 1, characterized by the fact that it further comprises fermenting glucose to produce ethanol. 4. Method according to claim 3, characterized by the fact that saccharification and fermentation are carried out as a simultaneous saccharification and fermentation process (SSF).