Hybrid glucoamylase and methods of use thereof

Abstract

Described herein, inter alia, are hybrid glucoamylase polypeptides derived from glucoamylases from the Zygomycetes (e.g., Mucorales). Additionally, the present disclosure also relates to processes for producing fermentation products using the hybrid glucoamylase polypeptides disclosed herein, as well as methods for increasing starch digestibility in animals and methods for producing fermented beverages.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2021 / 141254, filed December 24, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (NB41866WOPCT2_sequencelisting.xml; size: 288 KB; creation date: December 19, 2022) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to compositions comprising hybrid glucoamylase polypeptides and methods for saccharifying starch substrates and using them to produce fermentation products. [Background technology]

[0004] Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by some filamentous fungi and yeasts.

[0005] The main application of glucoamylase is the saccharification of partially processed starch / dextrins to glucose, an essential substrate for many fermentation processes. Glucose can then be converted directly or indirectly into fermentation products using fermenting organisms. Examples of commercially available fermentation products include alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactate, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2), and more complex compounds.

[0006] The end product can also be a syrup. For example, the end product can be glucose, but can also be converted, for example by glucose isomerase, to fructose or to a mixture composed of approximately equal amounts of glucose and fructose. This mixture, or a mixture further enriched in fructose, is the most commonly used high fructose corn syrup (HFCS) commercially available worldwide.

[0007] Although a diverse group of microorganisms have been reported to produce glucoamylase, glucoamylase for commercial purposes has traditionally been produced using filamentous fungi, since they secrete large amounts of the enzyme extracellularly. However, commercially used fungal glucoamylases have certain limitations, such as slow catalytic activity or lack of stability, which leads to increased processing costs.

[0008] Thus, there is a continuing need for new glucoamylases and glucoamylase variants to improve the efficiency of saccharification and achieve high yields of fermentation products. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure relates to the identification and construction of hybrid (also known as "chimeric") glucoamylase polypeptides derived from glucoamylases of the Zygomycetes (e.g., Mucorales). Additionally, the present disclosure also relates to processes for producing fermentation products using the hybrid glucoamylase polypeptides disclosed herein, as well as methods for increasing starch digestibility in animals and methods for producing fermented beverages.

[0010] In some embodiments, provided herein is a hybrid polypeptide comprising: a) a first amino acid sequence comprising a catalytic module having glucoamylase activity or a functional fragment thereof; and b) a second amino acid sequence comprising a carbohydrate binding module (CBM) or a functional fragment thereof, wherein i) the first amino acid sequence comprising the catalytic module having glucoamylase activity or a functional fragment thereof is derived from Zygomycetes, and ii) the second amino acid sequence is located N-terminal and / or C-terminal to the first amino acid sequence. In some embodiments, the first amino acid sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:105, SEQ ID NO:110, SEQ ID NO:115, SEQ ID NO:120, or SEQ ID NO: 125. In some embodiments of any of the embodiments disclosed herein, the carbohydrate binding module belongs to a carbohydrate binding module family selected from the group consisting of CBM20, CBM21, CBM25, CBM26, CBM34, CBM41, and CBM45. In some embodiments, the polypeptide comprises a CBM at the N-terminus and C-terminus of the first amino acid sequence.In some embodiments of any of the embodiments disclosed herein, the second amino acid sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to one or more amino acid sequences selected from the group consisting of amino acids 25 to 130 of SEQ ID NO:1, amino acids 24 to 130 of SEQ ID NO:5, amino acids 22 to 130 of SEQ ID NO:7, amino acids 24 to 130 of SEQ ID NO:9, amino acids 24 to 130 of SEQ ID NO:11, amino acids 22 to 130 of SEQ ID NO:13, amino acids 25 to 130 of SEQ ID NO:15, amino acids 26 to 131 of SEQ ID NO:17, amino acids 25 to 130 of SEQ ID NO:19, amino acids 24 to 127 of SEQ ID NO:21, amino acids 23 to 145 of SEQ ID NO:23, amino acids 28 to 131 of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:39. In some embodiments of any of the embodiments disclosed herein, the polypeptide further comprises a linker region between the first amino acid sequence and the second amino acid sequence. In some embodiments of any of the embodiments disclosed herein, the polypeptide further comprises an oligopeptide at the C-terminus of the first amino acid sequence having at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:35.In some embodiments of any of the embodiments disclosed herein, the hybrid polypeptide is selected from the group consisting of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO: The polypeptide has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129. In some embodiments of any of the embodiments disclosed herein, the first amino acid sequence lacks a CBM. In some embodiments of any of the embodiments disclosed herein, the first amino acid sequence comprises SEQ ID NO:3. In some embodiments, the polypeptide further comprises an amino acid substitution at position 58 of SEQ ID NO:3. In some embodiments, the amino acid substitution is selected from the group consisting of V58P, V58G, V58A, V58L, V58I, V58F, V58Y, V58W, V58S, V66T, V58C, V58M, V58N, V58Q, V58D, V58E, V58K, V58R and V58H. A polynucleotide encoding any of the hybrid polypeptides disclosed herein.

[0011] In other aspects, provided herein is a vector comprising any of the polynucleotides disclosed herein.

[0012] In a further aspect, provided herein is a recombinant host cell comprising any of the polynucleotides disclosed herein or any of the vectors disclosed herein. In some embodiments, the host cell is an ethanol-producing microorganism. In some embodiments of any of the embodiments disclosed herein, the host cell is a yeast cell. In some embodiments of any of the embodiments disclosed herein, the host cell further expresses and secretes one or more additional enzymes selected from the group consisting of proteases, hemicellulases, cellulases, peroxidases, lipolytic enzymes, metallolipolytic enzymes, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, nucleases, oxidases, phenoloxidases, lipoxygenases, ligninases, alpha-amylases, pullulanases, phytases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, transferases, and combinations thereof.

[0013] In yet another aspect, provided herein is a method of producing a hybrid polypeptide in a host cell, comprising culturing any of the host cells disclosed herein under conditions suitable for expression and production of the hybrid polypeptide, in some embodiments, the method further comprises recovering the hybrid polypeptide from said culture.

[0014] In another aspect, provided herein is an enzyme composition comprising any of the hybrid polypeptides disclosed herein. In some embodiments, the enzyme composition is used in a starch conversion process.

[0015] In yet a further aspect, provided herein is a method of saccharifying a starch-containing material, comprising the steps of: i) contacting the starch-containing material with an alpha-amylase; and (ii) contacting the starch-containing material with any of the hybrid polypeptides disclosed herein, to produce at least 70% free glucose from the starch-containing material (substrate). In some embodiments, the method comprises performing steps (i) and (ii) sequentially or simultaneously. In some embodiments of any of the embodiments disclosed herein, the method further comprises pre-saccharification prior to the saccharification step ii). In some embodiments of any of the embodiments disclosed herein, step (i) is performed at or below the gelatinization temperature of the starch-containing material.

[0016] In another aspect, provided herein is a sugar produced by any of the methods disclosed herein.

[0017] In another aspect, provided herein is a method of producing a fermentation product from sugars produced by any of the methods disclosed herein, wherein the sugars are fermented by a fermenting organism. In some embodiments, the fermentation process occurs subsequent to or simultaneously with step (ii). In some embodiments of any of the embodiments disclosed herein, the fermentation product comprises ethanol. In some embodiments of any of the embodiments disclosed herein, the fermentation product comprises a non-ethanol metabolic product. In some embodiments, the metabolic product is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, organic acids, glucono delta-lactone, sodium erythorbate, omega-3 fatty acids, butanol, iso-butanol, amino acids, lysine, tyrosine, threonine, glycine, itaconic acid, 1,3-propanediol, vitamins, enzymes, hormones, isoprene, or other biochemicals or biological materials.

[0018] In still further aspects, provided herein are methods of applying any of the hybrid polypeptides disclosed herein to brewing.

[0019] In another aspect, provided herein is a method of applying any of the hybrid polypeptides disclosed herein to produce beer or a malt-based beverage.

[0020] In yet a further aspect, provided herein is a method for increasing starch digestibility in an animal comprising adding any of the hybrid polypeptides disclosed herein as a feed additive to the animal's feed.

[0021] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of that embodiment or aspect.

[0022] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosures of all patents, patent applications and other publications cited herein are incorporated by reference in their entirety for all purposes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Zygomycetes (e.g., Mucorales) are fungi in the order Eumycota that form elongated hyphae and a variety of asexual and sexual spore structures. Zygomycetes are fungi that grow in soil and dead plant material (Dijksterhuis & Samson, “Food Spoilage Microorganisms,” Woodhead Publishing Series in Food Science, Technology and Nutrition 2006, Pages 415-436, incorporated herein by reference). Many glucoamylase polypeptides found in Zygomycetes lack a carbohydrate-binding domain (CBD). As further described herein, the inventors of the present application have surprisingly discovered that the addition of one or more CBDs from enzymes found in other organisms to a glucoamylase polypeptide from a Zygomycete to form a chimeric polypeptide enhances the activity for starch hydrolysis compared to a Zygomycete glucoamylase polypeptide lacking a CBD.

[0024] I. Definition Prior to describing the present compositions and methods in detail, the following terms and abbreviations are defined.

[0025] Unless otherwise defined, all technical and scientific terms used have their usual meaning in the relevant scientific field. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, New York (1994) and Hale & Markham, Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide the usual meaning of many of the terms that describe this invention.

[0026] The term "glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) activity" is defined herein as an enzyme activity that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules.

[0027] "Catalytic domain" (CD) or "catalytic module" (CM), as used interchangeably herein, refer to the structural region of a polypeptide (such as a glucoamylase) that contains the active site for substrate (such as starch or related oligo- and polysaccharide molecules) hydrolysis.

[0028] "Chimeric polypeptide" or "hybrid polypeptide" or "hybrid glucoamylase polypeptide" or "chimeric glucoamylase polypeptide" are used interchangeably herein to refer to a polypeptide that comprises within it at least two polypeptides (or portions thereof, e.g., subsequences, peptides, or functional domains such as carbohydrate binding modules) from different sources. A chimeric or hybrid polypeptide may comprise two, three, four, five, six, seven, or more polypeptides or portions thereof from different sources, such as different genes, different cDNAs, or different animals or other species. A chimeric or hybrid polypeptide may comprise one or more linkers linking different polypeptides or portions thereof. Thus, the polypeptides or portions thereof may be directly linked, indirectly linked via linkers, or both, within a single chimeric polypeptide. A chimeric or hybrid polypeptide may comprise additional peptides, such as signal sequences, and sequences such as 6His and FLAG, that aid in purification or detection of the protein. Additionally, a chimeric or hybrid polypeptide may have amino acid or peptide additions to the N-terminus and / or C-terminus.

[0029] "Zygomycete fungi" refers to a former division or phylum of the fungal kingdom. Members currently belong to two phyla, Mucoromycota and Zoopagomycota, with approximately 1060 known species (Spatafora et al., 2016, Mycologia. 108(5):1028-1046, incorporated herein by reference). The "Mucorales" is the largest and best studied order of Zygomycetes. Members of this order are sometimes referred to as Mucorales.

[0030] The term "carbohydrate binding domain" (CBD) or "carbohydrate binding module" (CBM) or "starch binding module" (SBM) or "starch binding domain" (SBD), as used interchangeably herein, refers to a functional protein domain that is present in carbohydrate-active enzymes (e.g., endocellulases and exocellulases) and has carbohydrate-binding activity. In some instances, the carbohydrate binding domain may contribute to catalytic efficiency or thermostability, for example, by increasing enzyme-substrate complex formation. Although CBMs often naturally occur within larger enzymes (typically linked to one or more catalytic domains via a linker region), the term as used herein refers to an independent module. A CBM in its naturally occurring form may be located at the N-terminus, C-terminus, or internally of a polypeptide, but as used herein, may be a truncated form of its native form.

[0031] The term "linker" as used herein refers to an amino acid sequence that functions to link separate protein domains together.

[0032] As used herein, the term "pre-linker" refers to an amino acid sequence that functions to connect a catalytic domain (eg, the catalytic domain of a glucoamylase) and a linker.

[0033] The term "wild type" with reference to a polypeptide (such as a glucoamylase) refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion (such as the insertion of one or more CBMs) or deletion at one or more amino acid positions.

[0034] The terms "parent," "parental," or "reference" in reference to a polypeptide (such as a glucoamylase) may refer to a wild-type polypeptide or to a polypeptide into which one or more amino acid substitutions have been introduced, which is then used as a reference to compare performance characteristics of a polypeptide into which further amino acid substitutions or one or more functional protein domains (such as CBMs) have been introduced. In some embodiments, the parent polypeptide is a glucoamylase from the Zygomycetes or Mucorales (such as Saksenaea vasiformis). In some embodiments, the parent polypeptide is SEQ ID NO: 3 or 41.

[0035] The term "amino acid sequence" is synonymous with, and used interchangeably with, the terms "polypeptide," "protein," and "peptide." Such amino acid sequences, if they exhibit activity, may be referred to as "enzymes." Conventional one-letter or three-letter codes for amino acid residues are used, and amino acid sequences are represented in the standard amino-terminal to carboxy-terminal direction (i.e., N→C).

[0036] The term "mature polypeptide" is defined herein as a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

[0037] A "signal sequence" or "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside of a cell. The mature form of the extracellular protein lacks the signal sequence, which is cleaved during the secretion process. In some embodiments, SEQ ID NO: 45, 51, 57, 63, or 69 is a signal peptide.

[0038] The terms "nucleic acid" or "polynucleotide" are used interchangeably and include DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single-stranded or double-stranded and may be chemically modified. Because the genetic code is degenerate, more than one codon may be used to code for a particular amino acid, and the compositions and methods of the invention encompass nucleotide sequences that code for a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5'-to-3' orientation.

[0039] The term "coding sequence" refers to a polynucleotide sequence that directly specifies the amino acid sequence of a protein product of the polynucleotide sequence. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. A coding sequence can be DNA, cDNA, a synthetic, or recombinant nucleotide sequence.

[0040] The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of steps before appearing as a spliced ​​mature mRNA. These steps include the removal of intron sequences by a process called splicing. Thus, cDNA derived from mRNA lacks any intron sequences.

[0041] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., a hybrid glucoamylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from cells.

[0042] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence, which process includes both transcription and translation.

[0043] The term "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0044] "Expression vector" refers to a DNA construct that contains a DNA sequence encoding a polypeptide of interest, the coding sequence being operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0045] The term "control sequences" is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, and may be native or foreign to each other. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding the polypeptide.

[0046] The term "operably linked" means that the particular components are in a relationship, including but not limited to, juxtaposition, that permits them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0047] "Biologically active" refers to a sequence that has a particular biological activity, such as an enzymatic activity.

[0048] The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0049] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 (modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set at score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed, for example, with the XBLAST program parameters set at score-50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul, et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterative search that detects distant relationships (Id.) between molecules. When using BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Another computer program that can be used to generate multiple alignments of protein sequences is MUSCLE. Elements of the MUSCLE algorithm include fast distance estimation using k-mer counting, progressive alignment using a new profile function called logarithmic expectation score, and refinement using tree-dependent restriction partitioning. This program is described in MUSCLE: multiple sequence alignment with high accuracy and high throughput by Robert C. Edgar (2004) published in Nucleic Acids Res. 32: 1792-1797. Percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps allowed. In calculating percent identity, typically only exact matches are counted.

[0050] The term "homologous sequence" is defined herein as a predicted protein having an E-value (or expectation score) of less than 0.001 in a tfasty search (Pearson, WR, 1999, Bioinformatics Methods and Protocols, S. Misener and S. Krawetz, ed., pp. 185-219) with a glucoamylase, e.g., the glucoamylase of SEQ ID NO: 3 or 41.

[0051] As used herein, "equivalent position" or "corresponding position" means a position that is common to two amino acid sequences based on alignment of the amino acid sequence of a parent glucoamylase with a glucoamylase variant and alignment of the three-dimensional structure of the parent glucoamylase with that of the variant glucoamylase in three-dimensional space.

[0052] As used herein in reference to an amino acid residue position, "corresponding to" or "corresponding to" refers to the amino acid residue at the recited position in the protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to the recited residue in the protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related or reference protein.

[0053] As used herein, "performance index" or "PI" refers to the calculated activity per unit of enzyme compared to the parent molecule. In some aspects of any of the embodiments disclosed herein, the parent molecule used in calculating the performance index is a glucoamylase. In some embodiments, the parent molecule, by definition, has a performance index of 1. In other embodiments, a performance index greater than 1 (PI>1.0) indicates improved activity of the glucoamylase variant compared to the parent molecule.

[0054] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microorganism, e.g., an ethanol-producing microorganism, and at least one enzyme, e.g., an amylase, are present in the same process step. SSF includes the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) to sugars, e.g., glucose, and fermentation of the sugars to alcohol or other biochemicals or biological materials in the same reaction vessel.

[0055] A "slurry" is an aqueous mixture containing insoluble starch granules in water.

[0056] The term "total sugar content" refers to the total soluble sugar content present in a starch composition, including monosaccharides, oligosaccharides and polysaccharides.

[0057] The term "dry solids" (ds) refers to dry solids dissolved in water, dry solids dispersed in water, or a combination of both. Thus, dry solids include granular starch and its hydrolysis products, such as glucose.

[0058] The term "high DS" refers to an aqueous starch slurry having a dry solids content greater than 38% (wt / wt).

[0059] "Degree of polymerization (DP)" refers to the number of anhydroglucopyranose units (n) in a given sugar. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP4+ (>DP3) refers to polymers with a degree of polymerization greater than 3.

[0060] The term "contacting" refers to the placement of the referenced components (including but not limited to an enzyme, a substrate, and a fermenting organism) in sufficient proximity to affect a desired outcome, such as an enzyme acting on a substrate or a fermenting organism fermenting a substrate.

[0061] As used herein, the terms "yeast cell", "yeast strain", or simply "yeast" refer to organisms from the phyla Ascomycota and Basidiomycota. An exemplary yeast is Saccharomyces cerevisiae from the order Saccharomycetales. Specific examples of yeast include Saccharomyces spp., including but not limited to S. cerevisiae. Yeasts include organisms used in the production of fuel alcohol and organisms used in the production of potable alcohol, such as specialized proprietary yeast strains used to produce distinctive tasting beer, wine, and other fermented beverages.

[0062] "Ethanol-producing microorganism" refers to a microorganism with the ability to convert sugars or other carbohydrates into ethanol.

[0063] The term "biochemicals" refers to metabolic products of microorganisms such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega-3 fatty acids, butanol, iso-butanol, amino acids, lysine, itaconic acid, other organic acids, 1,3-propanediol, vitamins or isoprene, or other biomaterials.

[0064] The term "pullulanase", also called debranching enzyme (EC 3.2.1.41, pullulan 6-glucanohydrolase), is capable of hydrolyzing the alpha 1-6 glucosidic bonds in the amylopectin molecule.

[0065] In this specification, a certain range is indicated by a numerical value preceded by the term "about". The term "about" is used herein to literally support the exact number preceded by this term and a number close to or approximately the number preceded by this term. In determining whether a certain number is close to or approximately a specifically recited number, the close or approximate number not recited may be a number that provides a substantial equivalent to the specifically recited number in the context in which it is stated. For example, with respect to a certain numerical value, the term "about" refers to a range of -15% to +15% of this numerical value, unless the term is otherwise clearly defined in the context.

[0066] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise.

[0067] It is further noted that the claims may be drafted to exclude any optional element, in which case this draft is intended to serve as a predicate to the use of exclusive terminology, such as "sole," "only," or the like, in the context of reciting claim elements or using a "negative" limitation.

[0068] The term "comprising" and its cognates are used in their inclusive sense, i.e., equivalent to the term "including" and its corresponding cognates. Furthermore, it should be noted that the term "comprising" as used herein means including, but not limited to, the component listed before "comprising". The component before the term "comprising" is required or essential, but a composition including that component may further include other non-required or optional components.

[0069] It should also be noted that the term "consisting essentially of," as used herein, refers to a composition in which the component listed preceding the term is present in a total amount of less than 30% by weight of the total composition and in the presence of other known components that do not contribute to or interfere with the action or activity of the component in question.

[0070] It should also be noted that the term "consisting of," as used herein, means including and being limited to the components set forth before the term "consisting of." The component set forth before the term "consisting of" is thus necessary or essential, and no other components are included in the composition.

[0071] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitation were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0072] 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.

[0073] Other definitions of terms may be found throughout the specification.

[0074] II. Composition Provided herein are hybrid (also known as chimeric) polypeptides comprising a first amino acid sequence that contains a catalytic module (CM) or a functional fragment thereof having glucoamylase activity; and a second amino acid sequence that has a carbohydrate binding module (CBM) or a functional fragment thereof.

[0075] A. Catalyst Module In a first aspect, the present invention relates to a hybrid polypeptide comprising a catalytic module (CM) or a functional fragment thereof with glucoamylase activity (i.e. the ability to catalyze the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules) derived from the class Zygomycetes (e.g. Mucorales). Non-limiting examples of fungal species found within the Zygomycetes from which glucoamylase CM can be derived include those from Saksenaea vasiformis, Saksenaea oblongispora, Apophysomyces ossiformis, Apophysomyces elegans, Apophysomyces variabilis, and Apophysomyces trapeziformis.

[0076] Non-limiting examples of CMs with glucoamylase activity from Zygomycetes include those having an amino acid sequence that shares at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% and at least about 99%, or 100% amino acid sequence identity to the polypeptide of SEQ ID NO:3, 33, 41, 47, 49, 53, 55, 59, 61, 65, 67, 71, 73, 105, 110, 115, 120 or 125.

[0077] In some embodiments, the CM is separated from the CBM by a linker peptide sequence. Non-limiting examples of linker peptides include an oligopeptide having at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:29 (from Aspergillus niger), or an oligopeptide having at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:37 (from Trichoderma reesei). In some embodiments, the linker oligopeptide is located at the C-terminus of the CM. In further embodiments, the linker oligopeptide is located at the N-terminus of the CM.

[0078] In further embodiments, a pre-linker peptide sequence can separate the CD from the linker. Non-limiting examples of pre-linker peptides include an oligopeptide having at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 27 (from Aspergillus niger), or an oligopeptide having at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 35 (from Trichoderma reesei). In some embodiments, the pre-linker oligopeptide is located at the C-terminus of the CM. In further embodiments, the linker oligopeptide is located at the N-terminus of the CM.

[0079] In some embodiments, a CM having glucoamylase activity or a functional fragment thereof is a variant polypeptide that comprises an amino acid sequence that differs from the polypeptide of SEQ ID NO: 3, 33, 41, 47, 49, 53, 55, 59, 61, 65, 67, 71, 73, 105, 110, 115, 120 or 125 by 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or even 1 or fewer amino acids.

[0080] In some embodiments, a CM or functional fragment thereof with glucoamylase activity for use in the present invention further has one or more of pullulan and / or panose and / or maltodextrin hydrolysis activities. In further embodiments, a wild-type CM or functional fragment thereof with glucoamylase activity for use in the present invention lacks a CBM or an amino acid sequence encoding a functional CBM.

[0081] B. Carbohydrate-binding module (CBM) Carbohydrate-binding modules (CBMs), previously known as cellulose-binding domains, are protein domains found in carbohydrate-active enzymes (e.g., glycoside hydrolases). The majority of these domains have carbohydrate-binding activity. CBMs are classified into numerous families based on amino acid sequence similarity. Currently (December 2021), there are 88 CBM families in the CAZy database.

[0082] Examples of CBM families for use in any of the hybrid polypeptides disclosed herein include CBM families 20, 21, 25, 26, 34, 41, and 45. With reference to cazy.org / Carbohydrate-Binding-Modules, CBM family 20 includes modules that bind starch and cyclodextrin. Granular starch binding function has been demonstrated in several instances. CBM family 21 includes a module of about 100 residues and is found in many eukaryotic proteins involved in glycogen metabolism. Granular starch binding function has been demonstrated in one instance. CBM family 25 binds alpha-glucosaccharides, particularly those containing alpha-1,6 linkages, and granular starch. With reference to CBM family 26, starch binding function has been demonstrated in two instances. This module was previously known as the X22 module and is structurally related to the CBM25 module. For CBM family 34, a module of about 120 residues has been shown and granular starch-binding function has been demonstrated on the example of Thermoactinomyces vulgaris R-47 α-amylase 1 (TVAI). CBM family 41 contains a module of about 100 residues and is found mainly in bacterial pullulanases. An N-terminal module from Thermotoga maritima Pul13 has been shown to bind the α-glucans amylose, amylopectin, pullulan, and oligosaccharide fragments derived from these polysaccharides. CBM 45 has a module of about 100 residues and is found at the N-terminus of plastid α-amylases and α-glucan, water dikinases. Starch-binding activity has been demonstrated on the example of potato α-glucan, water dikinase.

[0083] In some embodiments, the CBM or functional fragment thereof has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 25-130 of SEQ ID NO: 1. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 24-130 of SEQ ID NO:5. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 22-130 of SEQ ID NO: 7. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 24-130 of SEQ ID NO:9. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 24-130 of SEQ ID NO: 11. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 22-130 of SEQ ID NO: 13.In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 25-130 of SEQ ID NO: 15. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 26-131 of SEQ ID NO: 17. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 25-130 of SEQ ID NO: 19. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 24-127 of SEQ ID NO:21. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 23-145 of SEQ ID NO: 23. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to amino acids 28-131 of SEQ ID NO: 25. In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO:31.In other embodiments, the CBM has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO:39.

[0084] In some embodiments, the CBM is a variant polypeptide comprising an amino acid sequence of amino acids 25 to 130 of SEQ ID NO: 1, amino acids 24 to 130 of SEQ ID NO: 5, amino acids 22 to 130 of SEQ ID NO: 7, amino acids 24 to 130 of SEQ ID NO: 9, amino acids 24 to 130 of SEQ ID NO: 11, amino acids 22 to 130 of SEQ ID NO: 13, or amino acids 25 to 130 of SEQ ID NO: 15. A variant polypeptide comprising an amino acid sequence that differs by 10 or less amino acids, 9 or less amino acids, 8 or less amino acids, 7 or less amino acids, 6 or less amino acids, 5 or less amino acids, 4 or less amino acids, 3 or less amino acids, 2 or less amino acids, or even 1 or less amino acid from amino acids 26 to 131 of SEQ ID NO: 17, amino acids 25 to 130 of SEQ ID NO: 19, amino acids 24 to 127 of SEQ ID NO: 21, amino acids 23 to 145 of SEQ ID NO: 23, amino acids 28 to 131 of SEQ ID NO: 25, SEQ ID NO: 31, or SEQ ID NO: 39.

[0085] The CBM, or functional fragment thereof, can also include a signal peptide in some non-limiting embodiments. Suitable examples of signal peptides include the amino acid sequences of SEQ ID NOs: 45, 51, 57, 63, or 69.

[0086] C. Chimeric Polypeptides Provided herein are hybrid polypeptides comprising any of the catalytic modules having glucoamylase activity or functional fragments thereof from the Zygomycetes (e.g., Mucorales) disclosed herein and one or more (e.g., any one, two, three or four) carbohydrate binding modules or functional fragments thereof (e.g., any of the CBMs disclosed herein).

[0087] Hybrid polypeptides include those disclosed herein, e.g., SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, 19, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129.

[0088] In some embodiments, the hybrid polypeptides disclosed herein (e.g., any of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129) have a CD improved starch hydrolysis activity (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more) compared to a parent glucoamylase polypeptide from a Zygomycetes (e.g., Mucorales) that has a CBD but lacks a CBD , 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 180%, 185%, 190%, 195%, 200% or more (including values ​​between these percentages). Starch hydrolysis activity can be determined by any method known in the art, including those shown in Examples 3 and 5 herein.

[0089] In other embodiments, the hybrid polypeptides disclosed herein (e.g., any of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129) are In some embodiments, the glucoamylase polypeptides exhibit improved thermostability (e.g., an improvement in thermostability of about any of the following percentages (including values ​​between these percentages)) compared to a parent glucoamylase polypeptide from the Zygomycetes (e.g., Mucorales) having a CD but lacking a CBD. The thermal stability of a polypeptide can be determined by any method known in the art.

[0090] In another aspect, the hybrid polypeptides disclosed herein may, in some embodiments, comprise conservative substitutions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129. Examples of conservative amino acid substitutions are listed below. Some conservative substitutions (i.e., mutations) can be made by genetic engineering, while others are made by introducing synthetic amino acids into the polypeptide by other means. TIFF2025501588000001.tif181161

[0091] Additionally, when a CD comprises the amino acid sequence of SEQ ID NO:3 (or any amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:3), the CD can further comprise an additional amino acid substitution at position 58. In some embodiments, the substitution comprises any of V58P, V58G, V58A, V58L, V58I, V58F, V58Y, V58W, V58S, V66T, V58C, V58M, V58N, V58Q, V58D, V58E, V58K, V58R or V58H.

[0092] D. Enzyme Compositions The present invention also relates to compositions comprising a hybrid polypeptide (such as any of the hybrid glucoamylase polypeptides disclosed herein) and / or a starch substrate. In some embodiments, hybrid polypeptides comprising an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequences of SEQ ID NOs: 75-89 and 106-129 can also be used in the enzyme compositions. Preferably, the compositions are formulated to provide desired properties such as low color, low odor, and acceptable storage stability at a temperature of about 4-40° C. and a pH of about 3-7.

[0093] The composition may include a hybrid glucoamylase polypeptide of the present invention as the primary enzyme component. In addition, the composition may include multiple enzyme activities such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, beta-amylase, isoamylase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, pullulanase, ribonuclease, transglutaminase, xylanase, or combinations thereof, which may be added in effective amounts as would be known to one of skill in the art.

[0094] The polypeptide compositions may be prepared according to methods known in the art and may be in the form of a liquid composition or in the form of a dry composition. For example, compositions containing the hybrid glucoamylase may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., and may further include any one or more of the additional enzymes listed herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, etc. Such compositions may work in combination with endogenous enzymes or other components already present in slurries, water baths, washers, food or beverage products, etc., such as endogenous plant (including algae) enzymes, residual enzymes from previous processing steps, etc. The polypeptides included in the compositions may be stabilized according to methods known in the art.

[0095] The composition may be a cell expressing a polypeptide, such as a cell capable of producing a product from fermentation. Such cells may be provided in liquid or dry form with suitable stabilizers. Such cells may further express additional polypeptides, such as those described above.

[0096] The dosage of the hybrid glucoamylase polypeptide compositions of the present invention, as well as other conditions used for the compositions, can be determined based on methods known in the art.

[0097] The composition is suitable for use in liquefaction, saccharification and / or fermentation processes, preferably starch conversion, especially for the production of syrups and fermentation products, such as ethanol. The composition is also suitable for use in animal nutrition (e.g., as an ingredient in animal feed) and fermented beverage products.

[0098] III. Method A. Generation of Hybrid Polypeptides The hybrid glucoamylase polypeptides disclosed herein can be produced in host cells, for example, by secretion or intracellular expression. Cultured cell material (e.g., whole cell broth) containing the hybrid glucoamylase polypeptide can be obtained after secretion of the hybrid glucoamylase polypeptide into the cell culture medium. Optionally, the hybrid glucoamylase polypeptide can be isolated from the host cells or from the cell broth, depending on the desired purity of the final glucoamylase. Genes encoding the hybrid glucoamylase polypeptides can be cloned and expressed according to methods well known in the art.

[0099] Suitable host cells include bacteria, fungi (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus species (such as, but not limited to, Aspergillus niger or Aspergillus oryzae), Trichoderma species (such as Trichoderma reesi), or Myceliopthora species (such as Myceliopthora thermophila). Other host cells include bacterial cells, such as Bacillus species (e.g., Bacillus subtilis or B. licheniformis), and Streptomyces species. Suitable yeast host organisms may be selected from Schizosaccharomyces species or Saccharomyces species, such as Saccharomyces cerevisiae, or species belonging to the Schizosaccharomyces genus, such as S. pombe. Strains of the methylotrophic yeast species Pichia pastoris may be used as host organisms.

[0100] In addition, the host may express one or more accessory enzymes, proteins, peptides, which may be beneficial for liquefaction, saccharification, fermentation, SSF, and downstream processes. Furthermore, the host cells may produce ethanol and other biochemicals or biomaterials in addition to the enzymes used to digest various feedstocks. Such host cells may be useful in fermentation processes or fermentation and saccharification processes to reduce or eliminate the need to add enzymes.

[0101] 1. Vector The DNA construct comprising the nucleic acid encoding the hybrid glucoamylase polypeptide disclosed herein can be constructed to be suitable for expression in host cells.Due to the known degeneracy of genetic code, various polynucleotides that code for the same amino acid sequence can be designed and produced by routine techniques.It is also known that depending on the desired host cell, codon optimization may be required before attempting expression.

[0102] A polynucleotide encoding a hybrid glucoamylase polypeptide of the present disclosure can be incorporated into a vector, which can be transferred to a host cell using known transformation techniques, such as those disclosed below.

[0103] A suitable vector may be one that can be transformed into and / or replicated within a host cell. For example, a vector containing a nucleic acid encoding a hybrid glucoamylase polypeptide disclosed herein may be transformed and / or replicated in a bacterial host cell as a means for propagation and amplification of the vector. The vector may also be suitably transformed into an expression host such that the coding polynucleotide is expressed as a functional glucoamylase enzyme.

[0104] Non-limiting representative useful vectors are pTrex3gM (see U.S. Patent Application Publication No. 2013 / 0323798) and pTTT (see U.S. Patent Application Publication No. 2011 / 0020899), which can be inserted into the genome of a host. Both vectors pTrex3gM and pTTT can be modified by routine techniques such that they contain and express a polynucleotide encoding a variant glucoamylase polypeptide of the invention.

[0105] An expression vector usually contains control nucleotide sequences, such as a promoter, an operator, a ribosome binding site, a translation initiation signal, and optionally a repressor gene or one or more activator genes. In addition, an expression vector may contain a sequence encoding an amino acid sequence capable of targeting the hybrid glucoamylase polypeptide to a host cell organelle, such as peroxisome, or a specific cell compartment. For expression under the direction of the control sequence, the nucleic acid sequence of the hybrid glucoamylase polypeptide is operably linked to the control sequence in a manner suitable for expression.

[0106] A polynucleotide encoding a hybrid glucoamylase polypeptide disclosed herein can be operably linked to a promoter to allow transcription in a host cell. The promoter can be any DNA sequence that shows transcriptional activity in the host cell of choice and can be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of promoters for directing transcription of a DNA sequence encoding a glucoamylase, inter alia, in a bacterial host include the promoter of the lac operon of E. coli, the promoter of the Streptomyces coelicolor agarase gene dagA or celA, the promoter of the Bacillus licheniformis amylase gene (amyL), the promoter of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoter of the Bacillus amyloliquefaciens amylase (amyQ), the promoter of the Bacillus subtilis xylA and xylB genes, and the like.

[0107] Examples of promoters useful for transcription in fungal hosts include those derived from genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and the like. When the gene encoding the hybrid glucoamylase polypeptide is expressed in a bacterial species, such as E. coli, suitable promoters can be selected from bacteriophage promoters, including, for example, the T7 promoter and the phage lambda promoter. Along these lines, examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal1 and Gal10 promoters of Saccharomyces cerevisiae, and the AOX1 or AOX2 promoters of Pichia pastoris. Expression in filamentous fungal host cells often includes the endogenous inducible promoter cbh1 from T. reesei. See Liu et al. (2008) Acta Biochim. Biophys. Sin (Shanghai) 40(2):158-65.

[0108] The coding sequence may be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the hybrid glucoamylase polypeptide gene to be expressed, or may be from a different genus or species from which the hybrid glucoamylase moiety is derived (i.e., the species from which the CM of the CBM is derived). The signal sequence and promoter sequence comprising the DNA construct or vector may be introduced into a fungal host cell and may be from the same source. For example, the signal sequence may be the Trichoderma reesei cbh1 signal sequence operably linked to the cbh1 promoter.

[0109] The expression vector may also contain a suitable transcription terminator and, in eukaryotes, a polyadenylation sequence operably linked to the DNA sequence encoding the glucoamylase. The termination and polyadenylation sequences may preferably be derived from the same source as the promoter.

[0110] Vectors may also contain selectable markers, such as genes whose products complement a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or genes that confer antibiotic resistance, such as ampicillin resistance, kanamycin resistance, chloramphenicol resistance, or tetracycline resistance. Additionally, vectors may contain Aspergillus selection markers (e.g., amdS, argB, niaD, and xxsC), markers that confer hygromycin resistance, or selection may be achieved by cotransformation as known in the art. See, e.g., PCT Application WO 91 / 17243.

[0111] 2. Transformation and Cultivation of Host Cells The isolated cell containing either the DNA construct or the expression vector is advantageously used as a host cell for recombinant production of the variant glucoamylase. The cell may be transformed with a DNA construct encoding the enzyme, conveniently by integrating the DNA construct into the host's chromosome (in one or more copies). This integration is generally considered to be advantageous, since the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct into the host's chromosome may be performed according to conventional methods, for example by homologous or non-homologous recombination. Alternatively, the cell may be transformed with an expression vector associated with various types of host cells.

[0112] Examples of suitable bacterial host organisms include gram-positive bacterial species such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis. thuringiensis, Streptomyces species such as Streptomyces murinus, Lactic acid bacteria species including Lactococcus species such as Lactococcus lactis, Lactobacillus species including Lactobacillus reuteri, Leuconostoc species, Pediococcus species, and Streptococcus species. Other strains of Gram-negative bacteria species belonging to the Enterobacteriaceae family including E. coli, or the Pseudomonadaceae family can be selected as host organisms.

[0113] Suitable yeast host organisms can be selected from biotechnologically relevant yeast species, including but not limited to yeast species such as Pichia species, Hansenula species, or Kluyveromyces species, Yarrowinia species, Schizosaccharomyces species, or Saccharomyces species, such as Saccharomyces cerevisiae, or species belonging to the Schizosaccharomyces genus, such as S. pombe. Strains of the methylotrophic yeast species Pichia pastoris can be used as host organisms. Alternatively, the host organism can be a Hansenula species.

[0114] Suitable host organisms among filamentous fungi include species of the genus Aspergillus, such as Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. In addition, strains of Fusarium species, such as Fusarium oxysporum, or strains of Rhizomucor species, such as Rhizomucor miehei, can be used as host organisms. Other suitable strains include species of the genera Thermomyces and Mucor. Additionally, Trichoderma species can be used as hosts. The hybrid glucoamylase polypeptide expressed by the fungal host cell can be glycosylated (i.e., will contain glycosyl moieties). The glycosylation pattern can be the same or different from that present in the wild-type glucoamylase. The type and / or degree of glycosylation can alter the enzymatic and / or biochemical properties.

[0115] It is advantageous to be able to delete a gene from an expression host and cure the gene defect by a transformed expression vector. Using known methods, a fungal host cell can be obtained with one or more inactivated genes. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned (e.g., the cbh1, cbh2, egl1 and egl2 genes) can be deleted. Gene deletion can be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0116] General transformation techniques are known in the art. See, for example, Sambrook et al. (2001), supra. Expression of heterologous proteins in Trichoderma is described, for example, in U.S. Pat. No. 6,022,725. See also, Cao et al. (2000) Science 9:991-1001, for transformation of Aspergillus strains. Genetically stable transformants can be constructed by a vector system in which the nucleic acid encoding glucoamylase is stably integrated into the host cell chromosome. Transformants are then selected and purified by known techniques.

[0117] 3. Expression and Fermentation Methods for producing the hybrid glucoamylase polypeptides disclosed herein can include culturing host cells under conditions conducive to production of the enzyme, and recovering the enzyme from the cells and / or culture medium.

[0118] The medium used to culture the cells can be any conventional medium suitable for growing the host cells and expressing the variant glucoamylase polypeptide. Suitable media and media components are available from commercial suppliers or can be prepared according to published recipes (e.g., as set forth in catalogues of the American Type Culture Collection).

[0119] Any of the fermentation methods well known in the art can be suitably used to ferment such transformed or derived fungal strains, hi some embodiments, the fungal cells are grown under batch or continuous fermentation conditions.

[0120] 4. Enrichment and Purification Methods Separation and concentration techniques are known in the art, and conventional methods can be used to prepare concentrated solutions or broths containing the hybrid glucoamylase polypeptides of the invention.

[0121] After fermentation, a fermentation broth is obtained and various suspended solids, such as microbial cells and residual raw fermentation materials, are removed to obtain a glucoamylase solution by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography, etc., are commonly used.

[0122] At times, it may be desirable to concentrate the solution or broth containing the hybrid glucoamylase polypeptide to optimize recovery. Use of a non-concentrated solution or broth will usually require increased incubation times to collect the enriched or purified enzyme precipitate.

[0123] B. Uses of Hybrid Glucoamylase Polypeptides The present invention is also directed to the use of the hybrid glucoamylase polypeptides or compositions of the present invention in liquefaction, saccharification and / or fermentation processes. The hybrid glucoamylase polypeptides or compositions may be used in a single process, such as a liquefaction process, a saccharification process, or a fermentation process. The variant glucoamylase polypeptides or compositions may also be used in combined processes, preferably in conjunction with starch conversion, such as in a liquefaction saccharification process, a liquefaction fermentation process, or a saccharification fermentation process.

[0124] 1. Saccharification Liquefied starch can be saccharified using alpha-amylase and hybrid glucoamylase polypeptides, optionally in the presence of other enzymes, to a syrup rich in sugars of lower DP (e.g., DP1+DP2). The exact composition of the saccharification product depends on the combination of enzymes used and the type of modified starch. Advantageously, the syrup obtained using the provided hybrid glucoamylase polypeptides has a weight percentage of DP1 of the total oligosaccharides in the saccharified starch of more than 90%, for example 90%-98% or 95%-97%. The weight percentage of DP2 in the saccharified starch can be as low as less than about 3%, for example 0-3% or 0-2.8%.

[0125] Liquefaction is generally carried out as a continuous process, whereas saccharification is often carried out as a batch process. Saccharification conditions depend on the nature of the liquefaction product and the type of enzyme available. In some cases, the saccharification process may include a temperature of about 60-65°C and a pH of about 4.0-4.5, e.g., pH 4.3. Saccharification may be carried out at a temperature of, e.g., about 40°C, about 50°C, or about 55°C to about 60°C, or about 65°C, and the liquefaction product may need to be cooled. If necessary, the pH may also be adjusted. Saccharification is usually carried out in a stirred tank that may take several hours to fill or empty. Enzymes are usually added in a percentage relative to the dry solids as the tank is filled, or as a single dose at the start of the filling stage. Saccharification reactions to produce syrups are typically carried out for about 24-72 hours, e.g., 24-48 hours. Pre-saccharification may be applied prior to saccharification in simultaneous saccharification and fermentation (SSF) at a temperature of 30-65° C., typically around 60° C., for typically 40-90 minutes.

[0126] 2. Hydrolysis of raw starch The invention provides for the use of the hybrid glucoamylase polypeptides of the invention to produce glucose and the like from raw or granular starch. In general, the hybrid glucoamylase polypeptides of the invention can be used in raw starch hydrolysis (RSH) or granular starch hydrolysis (GSH) processes, either alone or in the presence of alpha-amylase, to produce desired sugars and fermentation products. Granular starch is solubilized by enzymatic hydrolysis below gelatinization temperatures. Such "low temperature" systems (also known as "no cook" or "cold cook") have been reported to be capable of handling higher concentrations of dry solids (e.g., up to 45%) than conventional systems.

[0127] "Raw starch hydrolysis" processes (RSH) differ from conventional starch processing processes in that they involve sequential or simultaneous saccharification and fermentation of granular starch below the gelatinization temperature of the starch substrate, typically in the presence of at least a glucoamylase and / or an amylase.

[0128] The hybrid glucoamylase polypeptides of the invention may also be used in combination with enzymes that hydrolyze only alpha-(1,6)-glucosidic bonds in molecules that contain at least four glucosyl residues. Preferably, the hybrid glucoamylase polypeptides of the invention are used in combination with pullulanases or isoamylases. The use of isoamylases and pullulanases for debranching starch, the molecular properties of the enzymes, and the potential use of the enzymes with glucoamylases are described in GM A van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, 101-142.

[0129] 3. Fermentation Soluble starch hydrolysates, particularly glucose-rich syrups, can be fermented by contacting the starch hydrolysates with a fermentation organism, usually at a temperature of around 32°C, e.g., 30°C to 35°C. "Fermentation organism" refers to any organism, including bacterial and fungal organisms, suitable for use in a fermentation process and capable of producing a desired fermentation product. Particularly suitable fermentation organisms are capable of fermenting, i.e., converting, directly or indirectly, sugars, such as glucose or maltose, to the desired fermentation product. Examples of fermentation organisms include yeasts, e.g., Saccharomyces cerevisiae, and bacteria, e.g., Zymomonas mobilis, that express alcohol dehydrogenase and pyruvate decarboxylase. Ethanol-producing microorganisms can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. For example, improved strains of ethanol-producing microorganisms that can tolerate higher temperatures are known in the art and can be used. See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27:1049-56. Yeasts that can be used for alcohol production include, but are not limited to, Saccharomyces species, including S. cerevisiae, as well as Kluyveromyces species, Lachancea species, and Schizosaccharomyces species. Numerous yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high alcohol production, rapid growth rate, etc. The temperature and pH of the fermentation depend on the fermenting organism. Microorganisms that produce other metabolic products by fermentation, such as citric acid and lactic acid, are also known in the art. See, e.g., Papagianni (2007) Biotechnol. Adv. 25:244-63; John et al. (2009), Biotechnol. Adv. 27:145-52.

[0130] The saccharification and fermentation processes may be carried out as SSF processes. The SSF process may in some embodiments be carried out with fungal cells continuously expressing and secreting the variant glucoamylase throughout the SSF. Also, the fungal cells expressing the hybrid glucoamylase polypeptide may be fermenting microorganisms, e.g., ethanol producing microorganisms. Thus, ethanol production may be carried out using fungal cells expressing sufficient hybrid glucoamylase polypeptides such that relatively little or no exogenous enzyme addition is required. The fungal host cells may be selected from appropriately engineered fungal strains. Also, fungal host cells may be used that express and secrete other enzymes in addition to the hybrid glucoamylase polypeptide. Such cells may express amylases and / or pullulanases, phytases, alpha-glucosidases, isoamylases, beta-amylase cellulases, xylanases, other hemicellulases, proteases, beta-glucosidases, pectinases, esterases, oxidoreductases, transferases, or other enzymes. Fermentation may be followed by subsequent recovery of ethanol.

[0131] 4. Fermentation products The term "fermentation product" refers to a product produced by a process that involves a fermentation process using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol, ethylene glycol, propylene glycol, butanediol, glycerin, sorbitol, and xylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); ketones (e.g., acetic acid, acetic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); e.g., acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); alkenes (e.g., pentene, hexene, heptene, and octene); gases (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO)); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 , beta-carotene); as well as hormones.

[0132] In a preferred embodiment, the fermentation product is ethanol, such as fuel ethanol; potable ethanol, i.e. potable neutral spirits; or industrial ethanol, or products used in the potable alcohol industry (e.g. beer and wine), dairy industry (e.g. fermented dairy products), leather industry and tobacco industry. Preferred fermentation processes used include alcoholic fermentation processes well known in the art. Preferred fermentation processes are anaerobic fermentation processes well known in the art.

[0133] 5. Brewing The process of making beer is well known in the art (see, for example, Wolfgang Kunze (2004) "Technology Brewing and Malting," Research and Teaching Institute of Brewing, Berlin (VLB), 3rd edition). Briefly, the process includes (a) preparing a mash, (b) filtering the mash to prepare a wort, and (c) fermenting the wort to obtain a fermented beverage, such as beer.

[0134] A brewing composition comprising a hybrid glucoamylase polypeptide in combination with an amylase and optionally a pullulanase and / or an isoamylase may be added to the mash in step (a) above, i.e., during preparation of the mash. Alternatively or in addition, the brewing composition may be added to the mash in step (b) above, i.e., during filtration of the mash. Alternatively or in addition, the brewing composition may be added to the wort in step (c) above, i.e., during fermentation of the wort.

[0135] 6. Animal Nutrition The hybrid glucoamylase polypeptides and compositions described herein can be used as feed additives for animals to increase starch digestibility. Described herein are methods for increasing starch digestibility in animals by administering to the animal feed in combination with the hybrid glucoamylase polypeptides disclosed herein.

[0136] The term "animal" refers to any living organism belonging to the animal kingdom, including, but not limited to, mammals (except humans), non-human animals, farm animals, livestock, agricultural animals, zoo animals, breeding stock, etc. For example, all non-ruminant and ruminant animals can be mentioned. In one embodiment, the animal is a non-ruminant, i.e., monogastric animal. Examples of monogastric animals include, but are not limited to, pigs, swine, such as piglets, breeding pigs, sows; poultry, such as turkeys, ducks, chickens, broilers, laying hens, etc.; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In a further embodiment, the animal is a ruminant, including, but not limited to, cows, calves, goats, sheep, giraffes, bison, moose, elk, yak, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns, and nilgai.

[0137] The terms "animal feed", "feed", "feed ingredients" and "livestock feed" are used interchangeably and refer to a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and / or large grains (e.g., corn or sorghum); b) cereal by-products, such as corn gluten meal, distillers dried grains with solubles (DDGS) (particularly corn-based distillers dried grains with solubles (cDDGS), wheat bran, etc.); , wheat middlings, wheat shorts, rice bran, rice husk, oat husk, palm kernel, and citrus pulp; c) proteins obtained from sources such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, rapeseed, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) fats and oils obtained from vegetable and animal sources; and / or e) minerals and vitamins.

[0138] The digestibility of starch in feed is highly variable and depends on many factors, including the physical structure of both the starch and the feed matrix. It has been found that the digestibility of starch in animal feed can be improved by using at least one glucoamylase as a feed additive.

[0139] When used as a feed, such as a functional feed, or in the preparation of a feed, the enzyme or feed additive composition herein may be used in combination with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient, such as at least one ingredient selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben, and propylparaben.

[0140] Also, at least one hybrid glucoamylase polypeptide described herein (or an enzyme composition comprising at least one hybrid glucoamylase polypeptide described herein) can be homogenized to produce a powder. This powder can be mixed with other components known in the art. Optionally, the feedstuff can also contain additional minerals, such as calcium, and / or additional vitamins. In some embodiments, the feedstuff is a corn soybean meal mix.

[0141] In an alternative preferred embodiment, the enzyme composition comprising at least one hybrid glucoamylase polypeptide may be formulated into granules as described in WO 2007 / 044968 (referred to as TPT granules) or WO 1997 / 016076 or WO 1992 / 012645, which are incorporated herein by reference. "TPT" stands for Thermal Protection Technology. When the feed additive composition is formulated into a granule, the granule comprises a hydration barrier salt coated over the protein core. The benefits of such a salt coating are improved heat resistance, improved storage stability, and protection from other feed additives that would otherwise adversely affect the enzyme. Preferably, the salt used in the salt coating has a water activity of greater than 0.25 or a humidity of greater than 60% at 20°C. In some embodiments, the salt coating comprises Na2SO4.

[0142] Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumables contain one or more of the following: buffers, salts, sorbitol and / or glycerol.

[0143] Any of the hybrid glucoamylase polypeptides described herein for use as feed additives can be used alone or in combination with at least one direct-feeding microorganism. DFM categories include Bacillus spp., lactic acid bacteria, and yeasts. Additionally, any of the glucoamylases described herein for use as feed additives can be used alone or in combination with at least one essential oil, such as cinnamaldehyde and / or thymol. Still further, any of the glucoamylases described herein for use as feed additives can be used alone or in combination with at least one additional enzyme. Examples of such enzymes include, but are not limited to, phytase, xylanase, protease, amylase, glucanase, or other glucoamylase.

[0144] Also disclosed are methods of increasing the nutritional value of animal feed, wherein an effective amount of any of the hybrid glucoamylase polypeptides described herein can be added to the animal feed.

[0145] As used herein, the phrase "effective amount" relates to the amount of an active agent (e.g., any of the hybrid glucoamylase polypeptides disclosed herein), alone or in combination with one or more other active agents (e.g., but not limited to, one or more additional enzymes, one or more DFMs, one or more essential oils, etc.), required to impart improved performance to an animal in terms of one or more metrics.

[0146] The term "animal performance" as used herein may be determined by any metric such as, but not limited to, feed efficiency and / or weight gain of the animal and / or feed conversion ratio and / or digestibility of nutrients in the feed and / or digestible or metabolizable energy in the feed and / or the ability of the animal to avoid adverse effects of a disease or of the immune response of a subject.

[0147] Animal performance characteristics may include, but are not limited to, body weight; weight gain; bulk; body fat percentage; height; body fat distribution; growth; growth rate; egg size; egg weight; egg bulk; egg production rate; mineral absorption; mineral excretion, mineral retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake (ADFI); average daily gain (ADG); retention and / or secretion of any one or more of copper, sodium, phosphorus, nitrogen, and calcium; amino acid retention or absorption; mineralization, bone mineralization; carcass yield; and carcass quality.

[0148] "Improved animal performance in one or more metrics" means increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio and / or improved digestibility of nutrients or energy in the feed and / or improved nitrogen retention and / or improved ability to avoid the adverse effects of necrotic enteritis and / or improved immune response in a subject resulting from use of a feed comprising the feed additive composition described herein compared to a feed not containing said feed additive composition.

[0149] All references cited herein are incorporated by reference in their entirety for all purposes. To further illustrate the present compositions and methods, and their advantages, the following specific examples are provided by way of illustration and not by way of limitation. EXAMPLES

[0150] Example 1 Sequence of a hybrid variant of Saksenaea vasiformis B4078 glucoamylase The polypeptides encompassed by the present invention were generated by fusing a selection of carbohydrate-binding module (CBM)-containing polypeptides to the N-terminus and / or C-terminus of the catalytic domain of SvaGA1 (SEQ ID NO: 41) or the catalytic domain of variant SvaGA1v2 (SEQ ID NO: 3). In constructs with CBM+linker added to the N-terminus, the signal peptides of SvaGA1 and SvaGA1v2 (predicted by SignalP software version 4.0) (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786)) were replaced with the signal peptide naturally associated with the CBM sequence used.

[0151] The protein and DNA sequences of the various catalytic domains (CDs), pre-linkers (sequences between the CD and linker), linkers, and carbohydrate-binding modules (CBMs) evaluated in subsequent studies are shown in Table 1. The abbreviation "RSA" refers to the "Fungal Collection at the Rancho Santa Ana Botanic Garden." [Table 1-1] [Table 1-2] [Table 1-3] [Table 2]

[0152] The structures of glucoamylase protein variants based on the SvaGA1 catalytic domain constructed in the present invention are shown in Table 2. Many CBMs were incorporated into the N-terminus or C-terminus of the glucoamylase catalytic domain.

[0153] The sequence numbers of the hybrid variants are listed in Table 3 below. [Table 3]

[0154] Example 2 Expression of hybrid GA variants in Trichoderma reesei Polynucleotides encoding GA variant sequences (codon-modified sequences used as expression cassettes) were synthesized by Generay (Generay Biotech Co., Ltd, Shanghai, China) and inserted into the pGX256 expression vector, a derivative of pTTT (see US Patent Publication No. 2011 / 0020899). All plasmids were transformed into suitable Trichoderma reesei strains using protoplast transformation (Te'o et al., J. Microbiol. Methods 51:393-99, 2002). Transformants were selected and fermented by the method described in WO 2016 / 138315. Supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis.

[0155] Fungal cell cultures were grown in defined medium as described by Lv et al. (2012) in “Construction of two vectors for gene expression in Trichoderma reesei”. Plasmids 67:67-71. After 96 hours, the clarified culture broth was collected by centrifugation. The glucoamylase variants were purified by methods known in the art. Column chromatography fractions containing the target protein were pooled, concentrated, and equilibrated with 20 mM sodium acetate, pH 5.0, 150 mM sodium chloride using an Amicon Ultra-15 device with 10K MWCO. The purified samples were approximately 99% pure (by SDS-PAGE analysis) and were stored in 40% glycerol at -80°C until use.

[0156] Example 3 Maize starch hydrolysis activity of hybrid SvaGA1v2 variants In this assay, corn starch (Sigma, Cat. No. S4126) at 1% (w / v) in 50 mM sodium acetate buffer (pH 4.5) was used as substrate. The corn starch hydrolysis activity of the hybrid glucoamylase variants was tested in combination with fungal alpha-amylase from Aspergillus terreus. The reaction was started by adding 10 μL glucoamylase and 10 μL alpha-amylase to 90 μL substrate, with final doses of 10 ppm and 1.5 ppm for glucoamylase and alpha-amylase, respectively. Incubation was performed for 60 min in iEMS (32 °C, 900 rpm). Glucose release was then measured using the coupled glucose oxidase / peroxidase (GOX / HRP) and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (Anal. Biochem. 105 (1980), 389-397). The reaction mixture (10 μL) was transferred to 90 μL of ABTS / GOX / HRP solution (2.74 mg / mL ABTS, 0.1 U / mL HRP and 1 U / mL GOX). The absorbance at 405 nm was immediately measured at 11 s intervals for 5 min using a SoftMax Pro plate reader (Molecular Device). The output was the reaction velocity Vo, which was used to indicate the corn starch hydrolysis activity of the hybrid glucoamylase variants. As shown in Table 4, all hybrid SvaGA1v2 variants showed higher corn starch hydrolysis activity than SvaGA1v2. Furthermore, all of the hybrid variants also outperformed the commercially relevant Trichoderma reesei variant glucoamylase (previously described in WO 2009 / 067218) under the conditions tested. [Table 4]

[0157] Example 4 Sequences of hybrid variants of additional Mucorales-clade glucoamylases Polypeptides encompassed by the present invention were generated by fusing a selection of polypeptides containing a starch-binding domain (CBM) to the N-terminus and / or C-terminus of the catalytic domain of a Mucorales-clade glucoamylase or variant thereof. In constructs with a CBM+linker added to the N-terminus, the signal peptide of those glucoamylases (predicted by SignalP software version 4.0) (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786)) was replaced with the signal peptide naturally associated with the CBM sequence used.

[0158] The amino acid and DNA sequence numbers of the various catalytic domains (CDs), pre-linkers (sequences between the CD and linker), linkers, carbohydrate binding modules (CBMs) used in subsequent studies are listed in Table 5. [Table 5-1] [Table 5-2]

[0159] The structures of various hybrid glucoamylase variants constructed and evaluated in the present invention are shown in Table 6. Many CBMs were incorporated at the N-terminus or C-terminus of the glucoamylase catalytic domain. [Table 6-1] [Table 6-2]

[0160] The sequence numbers of the resulting hybrid variants are listed in Table 7. [Table 7-1] [Table 7-2]

[0161] Example 5 Maize starch hydrolysis activity of hybrid variants of additional Mucorales-clade glucoamylases In this assay, corn starch (Sigma, Cat. No. S4126) at 1% (w / v) in 50 mM sodium acetate buffer (pH 4.5) was used as substrate. The corn starch hydrolysis activity of the hybrid glucoamylase variants was tested in the presence of fungal alpha-amylase from Aspergillus terreus. The reaction was started by adding 10 μL glucoamylase and 10 μL alpha-amylase to 90 μL substrate, with final doses of 10 ppm and 1.5 ppm for glucoamylase and alpha-amylase, respectively. Incubation was performed for 60 min in iEMS (32 °C, 900 rpm). Glucose release was measured using the combined glucose oxidase / peroxidase (GOX / HRP) and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (Anal. Biochem. 105 (1980), 389-397). The reaction mixture (10 μL) was transferred to 90 μL of ABTS / GOX / HRP solution (2.74 mg / mL ABTS, 0.1 U / mL HRP and 1 U / mL GOX). The absorbance at 405 nm was immediately measured at 11 s intervals for 5 min using a SoftMax Pro plate reader (Molecular Device). The output was the reaction velocity Vo, which was used to measure the corn starch hydrolysis activity of the hybrid glucoamylase variants. As shown in Table 8, all hybrid glucoamylase variants showed higher corn starch hydrolysis activity when compared to the enzyme constructs lacking the CBM region at the N-terminus or C-terminus. Furthermore, all hybrid GAs were superior to the commercially relevant Trichoderma reesei variant glucoamylase (previously described in WO 2009 / 067218). [Table 8]

Claims

1. a) a first amino acid sequence comprising a catalytic module or a functional fragment thereof having glucoamylase activity; and b) a second amino acid sequence comprising a carbohydrate binding module (CBM) or a functional fragment thereof; Including, 1. A hybrid polypeptide, comprising: i) a first amino acid sequence comprising a catalytic module or a functional fragment thereof having glucoamylase activity, the first amino acid sequence being derived from Zygomycetes; and ii) the second amino acid sequence being located at the N-terminus and / or C-terminus of the first amino acid sequence.

2. 2. The hybrid polypeptide of claim 1, wherein the first amino acid sequence comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:105, SEQ ID NO:110, SEQ ID NO:115, SEQ ID NO:120, or SEQ ID NO:

125.

3. 2. The hybrid polypeptide of claim 1, wherein the CBM belongs to a carbohydrate binding module family selected from the group consisting of CBM20, CBM21, CBM25, CBM26, CBM34, CBM41 and CBM45.

4. The hybrid polypeptide of claim 3 , comprising a CBM at the N-terminus and C-terminus of the first amino acid sequence.

5. 4. The hybrid polypeptide of claim 3, wherein the second amino acid sequence comprises one or more amino acid sequences selected from the group consisting of amino acids 25 to 130 of SEQ ID NO:1, amino acids 24 to 130 of SEQ ID NO:5, amino acids 22 to 130 of SEQ ID NO:7, amino acids 24 to 130 of SEQ ID NO:9, amino acids 24 to 130 of SEQ ID NO:11, amino acids 22 to 130 of SEQ ID NO:13, amino acids 25 to 130 of SEQ ID NO:15, amino acids 26 to 131 of SEQ ID NO:17, amino acids 25 to 130 of SEQ ID NO:19, amino acids 24 to 127 of SEQ ID NO:21, amino acids 23 to 145 of SEQ ID NO:23, amino acids 28 to 131 of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:

39.

6. The hybrid polypeptide of claim 1 , further comprising a linker region between the first amino acid sequence and the second amino acid sequence.

7. The hybrid polypeptide of claim 1, further comprising an oligopeptide comprising the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 35 at the C-terminus of the first amino acid sequence.

8. 2. The hybrid polypeptide of claim 1, having an amino acid sequence comprising the amino acid sequence of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128 or SEQ ID NO:

129.

9. 2. The hybrid polypeptide of claim 1, wherein: (a) the first amino acid sequence lacks a CBM; and / or (b) the first amino acid sequence comprises SEQ ID NO: 3; and / or (c) the hybrid polypeptide further comprises an amino acid substitution at position 58 of SEQ ID NO: 3, wherein the amino acid substitution comprises V58P, V58G, V58A, V58L, V58I, V58F, V58Y, V58W, V58S, V66T, V58C, V58M, V58N, V58Q, V58D, V58E, V58K, V58R, or V58H.

10. A polynucleotide encoding the hybrid polypeptide of any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10.

12. 11. A recombinant host cell comprising the polynucleotide of claim 10 or a vector comprising said polynucleotide.

13. 13. The recombinant host cell of claim 12, which is an ethanol-producing microorganism.

14. 13. The recombinant host cell of claim 12, further expressing and secreting one or more additional enzymes selected from the group consisting of proteases, hemicellulases, cellulases, peroxidases, lipolytic enzymes, metallolipolytic enzymes, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, alpha-amylases, pullulanases, phytases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, transferases, and combinations thereof.

15. 13. A method for producing a hybrid polypeptide in a host cell, comprising culturing the host cell of claim 12 under conditions suitable for the expression and production of said hybrid polypeptide.

16. 10. A method for saccharifying a starch-containing material, comprising the steps of: (i) contacting the starch-containing material with an alpha-amylase; and (ii) contacting the starch-containing material with any one of the hybrid polypeptides of claims 1 to 9, wherein the method produces at least 70% free glucose from the starch-containing material (substrate).

17. 17. The method of claim 16, comprising carrying out steps (i) and (ii) sequentially or simultaneously.

18. A method for applying the hybrid polypeptide according to any one of claims 1 to 9 to brewing.

19. 10. The application of the hybrid polypeptide according to any one of claims 1 to 9 in the production of beer or malt-based beverages.

20. A method for increasing starch digestibility in an animal, comprising adding at least one hybrid polypeptide according to any one of claims 1 to 9 as a feed additive to the animal's feed.