Glucoamylase variants and methods of use thereof

Abstract

Described herein are, inter alia, glucoamylase variants and methods of using same to saccharify starch substrates. Additionally, the disclosure also relates to processes for producing fermentation products and methods for increasing starch digestibility in animals, as well as methods for producing fermented beverages using said glucoamylase variants.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / CN2021 / 124148, filed October 15, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (NB42043-WO-PCT2_sequencelisting.xml; size: 62,000 bytes; creation date: October 11, 2022) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to compositions comprising glucoamylase variants and methods for saccharifying starch substrates and using same 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, as 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 increases processing costs.

[0008] Thus, there remains a 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 recombinant host cells, compositions comprising glucoamylases, and methods for saccharifying starch substrates using glucoamylases. Additionally, the disclosure also relates to processes for producing fermentation products and methods for increasing starch digestibility in animals, as well as methods for producing fermented beverages.

[0010] Thus, in some embodiments, provided herein are sequences comprising the amino acids at positions 20, 21, 23, 37, 49, 51, 52, 66, 67, 69, 73, 77, 79, 80, 81, 84, 92, 94, 102, 119, 121, 134, 140, 141, 143, 156, 157, 158, 164, 165, 166, 172, 192, 203, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 300, 301, 302, 303, 304, 30 243, 252, 253, 274, 278, 281, 290, 302, 310, 321, 338, 341, 350, 351, 352, 354, 370, 390, 403, 404, 405, 416, 418, 422, 430, 434, 440, 441, 444, 445, and / or 449, and / or glucoamylase variants or fragments thereof that contain one or more amino acid substitutions at residue positions corresponding to the equivalent positions in the parent glucoamylase. In some embodiments, the equivalent positions are determined by sequence identity and the parent glucoamylase has at least 80% sequence identity and less than 100% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37. In some embodiments, equivalent positions are determined by sequence identity and the parent glucoamylase comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37. In some embodiments of any of the embodiments disclosed herein, the parent glucoamylase is a Mucorales clade-group glucoamylase.In some embodiments of any of the embodiments disclosed herein, the one or more amino acid substitutions are selected from the group consisting of X020A / E / F / G / P;X021M / S / T / W;X023L / M;X037C / N;X049W;X051K / L / V / Y;X052F / N / P;X66A / C / F / M / P / T / W;X067A / C / M;X069A / C / K;X073N / P;X077M / P / S;X079C / R / T;X080H / K / N;X081N / S;X084L / V;X092C / I / M;X0 94A / G / Y;X102G;X119A / D / G;X121G / L / M / P / V;X134Q / S / W;X140C / I / Q;X141F / K;X143G; / W;X166A / F / G / H / R;X172G / L;X192F / R;X203C / M / Q / W / Y;X210A / F / G / I / L / M / N / W;X213H / R;X214A / C / E / G / L / T / Y;X215C / D / F / H / R / V / W; X218F / A / H / K / Q / V / Y;X221M / R / T;X222C / M / V;X233M / PT / Y;X235F / Y;X236A / Q / S;G238H / M / N / S / T / V; / V;X274A / D / K;X278A;X281D / P;F290M / V;X302F / H / K / M / P / Q / S / T / V / W;X310T / V / Y;X321D;X338I;X341M / T;X350T / C / E / I;X351E / V;X3 and / or X449L, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof.In order to identify the specifications of the keys, see Schedule 1 and the mounting brackets are S020A / E / F / G / P;K021M / S / 1. T / W;E023L / M;E037C;S049W;A051K / L / V / Y;G052F / N / P;X66A / C / F / M / P / T / W;S067A / C / M;V069A / C / K;K073N / P;T077M / P / S;A079C / R / T;G080H / K / N;D081N / S;I084L / V;V092C / I / M;F0 94A / G / Y;X102G;S119A / D / G;T121G / L / M / P / V;E134Q / S / W;M140C / I / Q;L141F / K;A143G;F156C / P;T157A / I;N158A / Y;I16 4L / T;Y165I / T / W;K166A / F / G / H / R;V172G / L;Y192F / R;D203C / M / Q / W / Y;R210A / F / G / I / L / M / N / W;D213H / R;N214A / C / E / G / L / Y;S215C / D / F / H / R / V / W;A218F / A / H / K / Q / V / Y;S221M / R / T;G222C / M / V;S233M / PT / Y;W235F / Y;D236A / Q / S;G238H / M / N / S / T / V;T243A / P / S;V252F / H;E253K;G274A / D / K;P278A;E281D / P;F290M / V;N302F / H / K / M / P / Q / S / T / V / W;N310T / V / Y;N32 1D;F338I;L341M / T;K350C / E / I;N351E / V;T352D / N / S;V354L / M;S370M / N / R;S390D / E / L;Q403G / K / R;Y404K / M;H405Q / S / Y;F416C / Y;R418E / W;Y422A / F / V;T430A / Q;A434S;A440G / H / L;Q441L / S / W;A444L / P / S;G445M / Y;In some embodiments of any of the embodiments disclosed herein, the variants are at positions 20, 21, 51, 79, 80, 92, 102, 121, 140, 143, 157, 158, 165, 166, 192, 203, 210, 213, 214, 215, 221, 222, 233, 235, 236, 238, 243, 252, 274, 278, 281, 290, 302, 310, 312, 314, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 338, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, The variant glucoamylase comprises one or more amino acid substitutions at residue positions corresponding to 321, 341, 350, 352, 354, 370, 390, 403, 404, 405, 418, 440, 441, and / or 444, and / or equivalent positions within the parent glucoamylase, wherein the variant glucoamylase exhibits improved hydrolysis of maltose compared to the parent glucoamylase or a fragment thereof lacking one or more of the substitutions. In some embodiments, the one or more amino acid substitutions are: X020A / P; X021S; X051L / V; X079C / T; X080K; X092M; X102G; X121G / P / M; X140I / C; X140C; X143G; X157I; X158A; X165W; X166G / R / F; X192F; X203W; X210L; X213R; X214A / E; X215D / C / F; X221R / T / M; X222M / C; X233M / Y / P; X235Y; X236A / Q; X238S / H; X243E / V; X252C / F; X274A; X278A; X281D; X290M / V; X302K / V / P / S / W; X310T / Y; X321D; X341M; X350I / T; X352D; X354L / M; X370M; X390E; X403G / R; X404K; X405Q / S / Y; X418W / E; X440G / H; X441S; and / or X444L, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof.In some embodiments, the one or more amino acid substitutions are S020A / P; K021S; A051L / V; A079C / T; G080K; V092M; X102G; T121G / P / M; M140I / C; M140C; A143G; T157I; N158A; Y165W; K166G / R / F; Y192F; D203W; R210L; D213R; N214A / E; S215D / C / F; S221R / T / M; G222M / C; S233M / Y / P;W235Y;D236A / Q;G238S / H;T243E / V;V252C / F;G274A;P278A;E281D;F290M / V;N302K / V / P / S / W;N310T / Y;N321D;L341M;K350I / T;T352D;V354L / M;S370M;S390E;Q403G / R;Y404K;H405Q / S / Y;R418W / E;A440G / H;Q441S; and / or A444L. In some embodiments of any of the embodiments disclosed herein, the variants are at positions 20, 21, 23, 37, 51, 52, 67, 69, 73, 77, 79, 80, 81, 84, 92, 94, 102, 119, 121, 140, 141, 143, 164, 165, 166, 172, 210, 213, 214, 215, 218, 221, 222, 233, 236, 238, 252, 253, 274, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 318, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 341, 342, , 290, 302, 321, 341, 350, 351, 352, 370, 390, 403, 404, 405, 418, 430, 440, 444, and / or 445, and / or equivalent positions in the parent glucoamylase, wherein the variant glucoamylase exhibits improved hydrolysis of panose compared to the parent glucoamylase or a fragment thereof lacking one or more of the substitutions.In some embodiments, the one or more amino acid substitutions are: X020P / A;X021T / W / M;X023M / L;X037C;X051L / V;X052N;X067C;X069C / A;X073N;X077S;X079C;X080H / K;X081N;X084V / L;X092C;X094Y;X102G;X119G;X121M / L / G / P;X140C / Q / I;X141F;X143G;X164L;X165W / T;X166R;X172G;X210L;X213H;X214A / E;X215D;X218Q / A / V / Y;X221R;X222 M / V / C; X233M / T / P; X236Q; X238H; X252F; X274D / K; X281D; X290M / V; X302S / K / W / Q / V; X321D; X341M / T; X350C / T; X351E; X352D / N; X370M; X390D / L / E; X403G; X403R / K; X404M / K; X405S / Q; X418E / W; X430A; X440G; X444P; and / or X445Y / M, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof. In some embodiments, the one or more amino acid substitutions are S020P / A;K021T / W / M;E023M / L;E037C;A051L / V;G052N;S067C;V069C / A;K073N;T077S;A079C;G080H / K;D081N;I084V / L;V092C;F094Y;X102G;S119G;T121M / L / G / P;M140C / Q / I;L141F;A143G;I164L;Y165W / T;K166R;V172G;R210L;D213H;N214A / E;S215D;S218Q / A / V / Y;S221R;G222M / V / C;S233M / T / P;D236Q;G238H;V252F;G274D / K;E281D;F290M / V;N302S / K / W / Q / V;N321D;L341M / T;K350C / T;N351E;T352D / N;S370M;S390D / L / E;Q403G;Q403R / K;Y404M / K;H405S / Q;R418E / W;T430A;A440G;A444P; and / or G445Y / M.In some embodiments of any of the embodiments disclosed herein, the variant is a sequence. Column number 4 position 20, 51, 69, 73, 77, 79, 80, 81, 84, 94, 119, 121, 134, 140, 141, 143, 156, 158, 165, 166, 203, 210, 214, 215, 218, 221, 222, 233, 235, 236, 252, 253, 274, 278, 281, 290, 302, 310, 321, 341, 350 , 352, 354, 370, 390, 416, 418, 434, 440, and / or 445, and / or equivalent positions within the parent glucoamylase, wherein the variant glucoamylase exhibits improved hydrolysis of pullulan compared to the parent glucoamylase or a fragment thereof lacking one or more of the substitutions. In some embodiments, the one or more amino acid substitutions are: X020F / A / G; X051K; X069C; X073P / N; X077M / P; X079C / T; X080K; X081N; X084V / L; X094G / A; X119G / D; X121L / M; X134S; X140I / C / Q; X141F; X143G; X156P / C; X158A; X165T; X166H / A / F; X203C / Q / W / Y; X210L / M / F / N / A / I; X214A / Y / L / C; X215V / C / W / D / H; X218V / K / A / Q; X221 R; X222M; X233M / P; X235Y; X236Q; X252F / H; X253K; X274K / D; X278A; X281D; X290M; X302P / S / V; X310T; X321D; X341M; X350I / T; X352D / S; X354L; X370M; X390D / L / E; X404K; X416Y; X418E / W; X434S; X440G; and / or X445M, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in the parent glucoamylase or fragment thereof.In some embodiments, the one or more amino acid substitutions are S020F / A / G; A051K; V069C; K073P / N; T077M / P; A079T; G080K; D081N; I084V / L; F094G / A; S119G / D; T121L / M; E134S; M140I / C / Q; L141F; A143G; F156P / C; N158A; Y165T; K166H / A / F; D203C / Q / W / Y; R210L / M / F / N / A / I; N214A / Y / L / C; S Includes one or more of: 215V / C / W / D / H; S218V / K / A / Q; S221R; G222M; S233M / P; W235Y; D236Q; V252F / H; E253K; G274K / D; P278A; E281D; F290M; N302P / S / V; N310T; N321D; L341M; K350I / T; T352D / S; V354L; S370M; S390D / L / E; Y404K; F416Y; R418E / W; A434S; A440G; and / or G445M. In some embodiments of any of the embodiments disclosed herein, the variants are at positions 21, 23, 37, 52, 66, 69, 73, 77, 79, 80, 81, 84, 92, 119, 121, 134, 140, 141, 157, 158, 164, 165, 210, 213, 214, 218, 221, 222, 233, 235, 236, 243, 252, 253, 274, 281, 290, 302, 341, 350, 362, 370, 372, 374, 376, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 4 The variant glucoamylases include one or more amino acid substitutions at residue positions corresponding to 351, 352, 370, 390, 403, 404, 405, 416, 418, 422, 440, 441, 444, and / or 445, and / or equivalent positions within a parent glucoamylase or fragment thereof, wherein the variant glucoamylase exhibits a higher performance index (PI) for saccharification of soluble starch compared to a parent glucoamylase lacking one or more of the substitutions.In some embodiments, the one or more amino acid substitutions are: X021T;X023M / L;X037C;X052N / F;X066A / F / T;X069C;X073N / P;X077P / M / S;X079R;X080N / H / K;X081N / S;X084L / V;X092I;X119A / G;X121M / L;X134S / Q;X140C / I / Q;X141F / K;X157A;X158A;X164L / T;X165W / T;X210W / G;X213H;X214G / Y;X218Q / F / A;X221R;X222M / V / C;X233M / P;X2 35F; X236Q; X243A; X252F; X253K; X274D / K / A; X281D; X290M / V; X302W / S / Q / K; X341M / T; X350C / E; X351E; X352D / N; X370M; X390D / L; X403G / R / K; X404I / M / K; X405S; X416C / Y; X418E / W; X422A; X440G; X441L; X444S / P; and / or X445M, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof. In some embodiments, the one or more amino acid substitutions are K021T; E023M / L; E037C; G052N / F; V066A / F / T; V069C; K073N / P; T077P / M / S; A079R; G080N / H / K; D081N / S; I084L / V; V092I; S119A / G; T121M / L; E134S / Q; M140C / I / Q; L141F / K; T157A; N158A; I164L / T; Y165W / T; R210W / G; D213H; N214G / Y; S218Q Includes one or more of: / F / A;S221R;G222M / V / C;S233M / P;W235F;D236Q;T243A;V252F;E253K;G274D / K / A;E281D;F290M / V;N302W / S / Q / K;L341M / T;K350C / E;N351E;T352D / N;S370M;S390D / L;Q403G / R / K;Y404M / K;H405S;F416C / Y;R418E / W;Y422A;A440G;Q441L;A444S / P; and / or G445M.In some embodiments of any of the embodiments disclosed herein, the variants are at positions 20, 21, 51, 67, 69, 73, 77, 79, 80, 81, 84, 102, 119, 121, 134, 140, 141, 143, 156, 157, 158, 165, 166, 172, 192, 203, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 252, 274, 281, 290, 302, 304, 306, 308, 309, 310, 311, 312, 313, 314, 315, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, , 310, 321, 341, 350, 351, 352, 354, 370, 390, 403, 404, 405, 418, 422, 434, 440, 444, and / or 445, and / or equivalent positions in the parent glucoamylase, wherein the variant glucoamylase exhibits improved hydrolysis of maltodextrin compared to the parent glucoamylase or a fragment thereof lacking one or more of the substitutions. In some embodiments, the one or more amino acid substitutions are: X020P / A / E; X021S / M; X051V / L / K / Y; X067A; X069C; X073P / N; X077P / M; X079C / T; X080K / H; X081N; X084L / V; X102G; X119G; X121M / G / V / P;X134S / Q;X140C / I;X141F;X143G;X156P / C;X157I;X158A;X165W / T;X166G;X172G ;X192F;X203W / C / M;X210L / M;X213R;X214A / E;X215D / C / F;X218V / Y / Q;X221R / T;X2 and / or X445M, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof.In some embodiments, the one or more amino acid substitutions are S020P / A / E; K021S / M; A051V / L / K / Y; S067A; V069C; K073P / N; T077P / M; A079C / T; G080K / H; D081N; I084L / V; P102G; S119G; T121M / G / V / P; E134S / Q; M140C / I; L141F; A143G; F156P / C; T157I; N158A; Y165W / T; K166G; V172G; Y192F; D203W / C / M; R210L / M; D213R; N214A / E; S21 5D / C / F;S218V / Y / Q;S221R / T;G222M / C;S233M / P / Y;W235Y;D236Q / A;V252F ;G274K / A / D;E281D;F290M / V;N302P / K / S / V / Q / W;N310T / Y;N321D;L341M;K 350I / T;N351V;T352D;V354L / M;S370M;S390E / L / D;Q403K / R / G;Y404K / M;H Contains one or more of 405Q / S / Y; R418W / E; Y422A; A434S; A440G / H; A444P; and / or G445M. In some embodiments of any of the embodiments disclosed herein, the variants comprise one or more amino acid substitutions at residue positions corresponding to positions 23, 51, 52, 66, 67, 77, 119, 121, 134, 140, 141, 156, 157, 165, 192, 213, 221, 222, 233, 236, 252, 281, 290, 302, 350, 352, 370, 390, 403, 404, 416, and / or 422 of SEQ ID NO:4, and / or the equivalent positions within a parent glucoamylase, and the variant glucoamylase exhibits improved thermostability compared to a parent glucoamylase or a fragment thereof lacking one or more of the substitutions.In some embodiments, the one or more amino acid substitutions are: X023M; X051Y; X052N; X066A / C / F / M / W; X067C; X077P; X119A; X121M; X134S; X140I / C; X141F; X156C; X157I; X165W / I; X192F; X213R; X221R; X222M; X233M; X236Q; X 252F; X281D; X290V; X302H / Q / W / S / K; X350E / C; X352D; X370M; X390L / D; X403R; X404K / M; X416Y; and / or X422A / V, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof. In some embodiments, the one or more amino acid substitutions include one or more of: E023M; A051Y; G052N; V066A / C / F / M / W; S067C; T077P; S119A; T121M; E134S; M140I / C; L141F; F156C; T157I; Y165W / I; Y192F; D213R; S221R; G222M; S233M; D236Q; V252F; E253F; E281D; F290V; N302H / Q / W / S / K; K350E / C; T352D; S370M; S390L / D; Q403R; Y404K / M; F416Y; and / or Y422A / V. In some embodiments of any of the embodiments disclosed herein, the variants are at positions 49, 51, 52, 66, 69, 77, 80, 94, 119, 134, 158, 164, 165, 172, 192, 213, 215, 218, 222, 233, 235, 236, 238, 243, 253, 274, 302, 338, 403, 405, 416, 418, 422, 430, 440, 441, 444, 445, and / or any of SEQ ID NO:4. In some embodiments, the variant glucoamylase comprises one or more amino acid substitutions at residue positions corresponding to 449 or 450, and / or equivalent positions in the parent glucoamylase, and the variant glucoamylase exhibits less conversion to sugars having a degree of polymerization (DP) of 2 or greater compared to the parent glucoamylase or a fragment thereof lacking one or more of the substitutions. In some embodiments, the one or more amino acid substitutions are X049W;X051Y;X052P;X66P;X069K / C;X077P / M;X080H;X094A / G;X119D;X134W / Q;X158Y;X164T / L;X165W / I;X172G;X192R / F;X213H;X215R;X218K;X222C;X233T / P;X235F;X236A;X238H / S / N;X243P / S;X243H / S / N ... and / or X449L, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof. In some embodiments, the one or more amino acid substitutions are S049W; A051Y; G052P; V66P; V069K / C; T077P / M; G080H; F094A / G; S119D; E134W / Q; N158Y; I164T / L; Y165W / I; V172G; Y192R / F; D213H; S215R; S218K; G222C; S233T / P; W Includes one or more of: 235F; D236A; G238H / S / N; T243P / S; E253K; G274K / A; N302H / F / P / M / Q / T; F338I; Q403G; H405Q / S; F416C / Y; F416Y; R418W / E; Y422A; T430A / Q; A440L / H; Q441W / L; A444L / P; G445Y; and / or F449L.In some embodiments of any of the embodiments disclosed herein, the variants comprise one or more amino acid substitutions at residue positions corresponding to positions 21, 23, 51, 52, 66, 67, 79, 81, 92, 119, 140, 158, 164, 172, 192, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 238, 243, 253, 281, 290, 302, 310, 351, 352, 354, 370, 403, 416, 422, 430, 441, and / or 445 of SEQ ID NO:4, and / or the equivalent positions within a parent glucoamylase, and the variant glucoamylase exhibits improved saccharification yields of glucose compared to a parent glucoamylase lacking one or more of the substitutions. In some embodiments, the one or more amino acid substitutions are X021S / W;X023L;X051Y / K;X052N;X066 / A / C / F / M / W;X067M / C / A;X079R;X081S;X092C / M;X119A;X140I;X158A / Y;X164L;X172L / G;X192F;X210W;X213H;X214Y;X215F / C / R;X218H / K / A;X221M / T;X222V;X233T; and / or X445Y, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase.In some embodiments, the one or more amino acid substitutions are K021S / W; E023L; A051Y / K; G052N; V066 / A / C / F / M / W; S067M / C / A; A079R; D081S; V092C / M; S119A; M140I; N158A / Y; I164L; V172L / G; Y192F; R210W; D213H; N214Y; S215F / C / R; S21 Includes one or more of: 8H / K / A; S221M / T; G222V; S233T; W235Y / F; D236S / A; G238T / V / M; T243A; E253K; E281P / D; F290V; N302K / Q / H / W; N310V / T; N351V; T352S; V354L; S370R; Q403K; F416Y; Y422F; T430A; Q441L; and / or G445Y. In some embodiments of any of the embodiments disclosed herein, the variants are at positions 20, 21, 23, 37, 51, 52, 66, 67, 69, 73, 77, 79, 80, 81, 84, 92, 94, 102, 119, 121, 134, 140, 141, 156, 157, 158, 164, 165, 166, 167, 169, 170, 172, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, , 166, 172, 192, 203, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 238, 243, 252, 253, 274, 278, 281, 290, 302, 310, 341, 350, 351, 352, 354, 370, 390, 403, 404, 405, 416, 418 , 422, 430, 440, 441, 444, and / or 445, and / or equivalent positions within a parent glucoamylase or fragment thereof, wherein the variant glucoamylase exhibits two or more of the following relative to a parent glucoamylase lacking one or more of the substitutions: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) improved hydrolysis of soluble starch; v) improved hydrolysis of maltodextrin; vi) improved thermostability; vii) less conversion to sugars with a DP of 2 or greater; and / or viii) improved saccharification yield of glucose.In some embodiments, the one or more amino acid substitutions are: X020A / P; X021M / S / T / W; X023L / M; X037C; X051K / L / V / Y; X052N; X066A / C / F / M / W; X067A / C; X069C; X073N / P; X077M / P / S; X079R / T; X080H / K; X081N / S; X084L / V; X092C / M; X094A / G; X10 2G;X119A / D / G;X121G / L / M / P;X134Q / S;X140C / I / Q;X141F;X156C / P;X157I;X158A / Y;X164L / T; ;X166G / F / R;X172G;X192F;X203C / W;X210L / M / W;X213H / R;X214A / E / Y;X215C / D / F / R;X218A / K / Q / V / Y;X22 1M / R / T;X222C / M / V;X233M / P / T / Y;X235F / Y;X236A / Q;X238H / S;X243A;X252F;X253K;X274A / D / K; 281D;X290M / V;X290V;X302H / K / P / Q / S / V / W;X310T / Y;X341M / T;X350C / E / I;X351E / V;X352D / N / S; ; X370M; X390D / E / L; X403G / K / R; X404K / M; X405Q / S / Y; X416C / Y; X418E / W; X422A; X430A; X440G / H; X441L; X444L / P; and / or X445M / Y, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof.If the housing of one of the keys is inserted in paragraph 1, one of the housing brackets is S020A / E / P;K02 1M / S / T / W;E023L / M;E037C;A051K / L / V / Y;G052N ;V066A / C / F / M / W;S067A / C;V069C;K073N / P;T077M / P / S;A079R / T;G080H / K;D081N / S;I084L / V;V0 92C / M;F094A / G;P102G;S119A / D / G;T121G / L / M / P;E134Q / S;M140C / I / Q;L141F;F156C / P;T157I;N 158A / Y;I164L / T;Y165I / T / W;K166G / F / R;V172G;Y192F;D203C / W;R210L / M / W;D213H / R;N214A / E / Y;S215C / D / F / R;S218A / K / Q / V / Y;S221M / R / T;G222C / M / V;S233M / P / T / Y;W235F / Y;D236A / Q;G238 H / S;T243A;V252F;E253K;G274A / D / K;P278A;E281D;F290M / V;F290V;N302H / K / P / Q / S / V / W;N310T / Y;L341M / T;K350C / E / I;N351E / V;T352D / N / S;V354L / M;S370M;S390D / E / L;Q403G / K / R;Y404K / M; H405Q / S / Y;F416C / Y;R418E / W;Y422A;T430A;A440G / H;Q441L;A444L / P;In some embodiments of any of the embodiments disclosed herein, the variants are: a) X236S and X281D; b) X215R and X441W; c) X321D and X434S; d) X143G and X434S; e) X079C and X143G; f) X350T and X434S; g) X351E and X403K; h) X052N and X084L; i) X243P and X290V; j) X290V and X350E. ;k) X302K and X441W;l) X156C and X404K;m) X067M and X404K;n) X052N and X141F;o) X052N and X351E;p) X233M and X445Y;q) X066C and X233M;r) X218H and X290V;s) X067M and X302H;t) X066C and X119A;u) X243P and X445Y;v) X192F and X243P;w) X156C and X2 43P;x) X023L and X066C;y) X023L-X119A;z) X020E and X192F;aa) X192F and X310V;bb) X023M and X302H;cc) X192F and X416Y;dd) X119A and X302H;ee) X235Y and X416Y;ff) X052N and X404K;gg) X023M and X449Y;hh) X158A and X172L;ii) X172L and X290V; or pp) X052N and X416Y, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof.In some embodiments, the two or more amino acid substitutions are: a) D236S and E281D; b) S215R and Q441W; c) N321D and A434S; d) A143G and A434S; e) A079C and A143G; f) K350T and A434S; g) N351E and Q403K; h) G052N and I084L; i) T243P and F290V; j) F290V and K350E; k) N302K and Q441W; l) F156C and Y404K; m) S067M and Y404K; n) G052N and L141F; o) G052N and N351E; p) S 233M and G445Y;q) A066C and S233M;r) S218H and F290V;s) S067M and N302H;t) A066C and S119A;u) T243P and G445Y;v) Y192F and T243P;w) F156C and T243P;x) E023L and A066C;y) E023L and S119A;z) S020E and Y192F;aa) Y192F and N310V;bb) E023M and N302H;cc) Y192F and F416Y;dd) S119A and N302H;ee) W235Y and F416Y;ff) G052N and Y40. 4K; gg) E023M and F449Y; hh) N158A and V172L; ii) V172L and F290V; jj) E023M and R210L; kk) R210L and F449Y; ll) T157I and E281D; mm) I164T and S215R; nn) M140C and Y422V; oo) S119A and N302K; or pp) G052N and F416Y. In some embodiments of any of the embodiments disclosed herein, the variants are: a) X141F, X281D, and X441W; b) X143G, X321D, and X434S; c) X321D, X350T, and X434S; d) X067M, X158A, and X281D; e) X156C, X192F, and X403K; f) X052N, X140C, and X422V; g) X066C, X119A, and X164T; h) X066C, X233M, and X445Y; i) X156C, X192F, and X243P; j) X023L, X066C, and X11 9A; k) X023M, X119A, and X404K; l) X310V, X416Y, and X445Y; m) X158A, X221R, and X290V; n) X023M, X052N, and X404K; o) X081S, X157I, and X236S; p) X243P, X302K, and X416Y; q) X140C, X302K, and X422V; or r) X052N, X416Y, and X445Y, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof.In some embodiments, the three or more amino acid substitutions are: a) L141F, E281D, and Q441W; b) A143G, N321D, and A434S; c) N321D, K350T, and A434S; d) S067M, N158A, and E281D; e) F156C, Y192F, and Q403K; f) G052N, M140C, and Y422V; g) A066C, S119A, and I164T; h) A066C, S233M, and G445Y; i) F156C, Y192F, and Q403K; 2F, and T243P; j) E023L, A066C, and S119A; k) E023M, S119A, and Y404K; l) N310V, F416Y, and G445Y; m) N158A, S221R, and F290V; n) E023M, G052N, and Y404K; o) D081S, T157I, and D236S; p) T243P, N302K, and F416Y; q) M140C, N302K, and Y422V; or r) G052N, F416Y, and G445Y. In some embodiments of any of the embodiments disclosed herein, the variant comprises four or more amino acid substitutions, including: a) X215R, X236S, X281D, and X441W; b) X052N, X084L, X140C, and X422V; c) X020E, X156C, X192F, and X243P; d) X023M, X221R, and X404K; e) X158A, X172L, X221R, and X290V; f) X140C, X165W, X302K, and X422V; or g) X119A, X253F, X310V, and X403K, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof. In some embodiments, the four or more amino acid substitutions include: a) S215R, D236S, E281D, and Q441W; b) G052N, I084L, M140C, and Y422V; c) S020E, F156C, Y192F, and T243P; d) E023M, S119A, S221R, and Y404K; e) N158A, V172L, S221R, and F290V; f) M140C, Y165W, N302K, and Y422V; or g) S119A, E253F, N310V, and Q403K.In some embodiments of any of the embodiments disclosed herein, the variant comprises five or more amino acid substitutions including X067M, X157I, X218H, X302H, and X416Y, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof. In some embodiments, the five or more amino acid substitutions comprise S067M, T157I, S218H, N302H, and F416Y. In some embodiments of any of the embodiments disclosed herein, the variant glucoamylase exhibits one or more of the following, relative to a parent glucoamylase lacking the substitution: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) higher PI of soluble starch saccharification (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrin; vi) improved thermostability; vii) less conversion to sugars with a DP of 2 or higher; and / or vii) improved saccharification yield of glucose. In some embodiments of any of the embodiments disclosed herein, the variant comprises substitutions at residue positions corresponding to positions a) 66, 67, and 69; b) 102, 119, and 121; and / or c) 143, 156, 164, 192, and 233 of SEQ ID NO:3, SEQ ID NO:4, and / or equivalent positions within the parent glucoamylase or fragments thereof. In some embodiments, the variant comprises amino acid substitutions including: a) X066A / C / F / M / P / T / W, X067A / C / M, and X069A / C / K; b) X102P / G, X119A / D / G, X121G / L / M / P / V; and / or c) X143G, X156C / P, X164L / T, X192F / R, and X233M / PT / Y. In some embodiments, the variant comprises amino acid substitutions including: a) V066A / C / F / M / P / T / W, S067A / C / M, and V069A / C / K; b) S102P / G, S119A / D / G, T121G / L / M / P / V; and / or c) A143G, F156C / P, I164L / T, Y192F / R, and S233M / PT / Y.In some embodiments of any of the embodiments disclosed herein, the glucoamylase variant further comprises an amino acid substitution at one or more residue positions corresponding to positions 66 and / or 102 of SEQ ID NO:4 and / or the equivalent positions in the parent glucoamylase. In some embodiments, the one or more amino acid substitutions comprise X066A / C / F / M / P / T / W and / or X102P / G, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in the parent glucoamylase. In some embodiments, the one or more amino acid substitutions comprise V066A / C / F / M / P / T / W and / or S102P / G. In some embodiments of any of the embodiments disclosed herein, the glucoamylase variant comprises an N-linked glycosylation at position N075 of SEQ ID NO:4 and / or the equivalent position in the parent glucoamylase. In some embodiments of any of the embodiments disclosed herein, the glucoamylase variant further comprises one or more amino acid substitutions at residue positions corresponding to positions 81, 83, 153, 370, or 372 of SEQ ID NO:4 and / or equivalent positions within the parent glucoamylase, wherein the variant glucoamylase exhibits increased glycosylation and increased thermostability compared to a parent glucoamylase lacking one or more of the substitutions. In some embodiments, the one or more substitutions comprise one or more substitutions at positions D81N, K83T, A153T, S370N, and / or A372S.

[0011] In other aspects, provided herein are polynucleotides encoding any of the glucoamylase variant polypeptides disclosed herein.

[0012] In another aspect, provided herein is a vector comprising any of the polynucleotides disclosed herein.

[0013] In a further aspect, provided herein is a host cell comprising any of the polynucleotides disclosed herein or any of the vectors disclosed herein. In some embodiments, the host cell is a bacterial host cell or a fungal host cell. In some embodiments of any of the embodiments disclosed herein, the host cell is an Aspergillus species, a Bacillus species, a Trichoderma species, a Pichia species, a Myceliophthora species, or a Saccharomyces species.

[0014] In another aspect, provided herein is an enzyme composition comprising any of the glucoamylase variants disclosed herein. In some embodiments of any of the embodiments disclosed herein, the composition is used in a starch conversion process. In some embodiments of any of the embodiments disclosed herein, the composition is used in an animal feed formulation. In some embodiments of any of the embodiments disclosed herein, the composition is used in an alcohol fermentation process. In some embodiments of any of the embodiments disclosed herein, the composition is used in a process for producing a fermented beverage.

[0015] In yet an additional aspect, provided herein is a method of producing a variant glucoamylase in a host cell, the method comprising: a) culturing a host cell transformed with any of the vectors disclosed herein in a culture medium under conditions suitable for producing the glucoamylase variant; and b) producing the variant. In some embodiments, the method further comprises recovering the glucoamylase variant from the culture medium. In some embodiments of any of the embodiments disclosed herein, the host cell is a bacterial host cell or a fungal host cell. In some embodiments of any of the embodiments disclosed herein, the host cell is an Aspergillus species, a Bacillus species, a Trichoderma species, a Pichia species, a Myceliophthora species, or a Saccharomyces species.

[0016] In another aspect, provided herein is a method of saccharifying a composition comprising starch to produce a composition comprising glucose, the method comprising: a) contacting the starch composition with any of the glucoamylase variant polypeptides disclosed herein; and b) saccharifying the starch composition to produce the glucose composition. In some embodiments, the composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch. In some embodiments of any of the embodiments disclosed herein, the method further comprises c) contacting the starch composition with an alpha-amylase. In some embodiments of any of the embodiments disclosed herein, the method further comprises d) contacting the starch composition with a pullulanase. In some embodiments of any of the embodiments disclosed herein, the method further comprises e) fermenting the glucose composition to produce a fermentation product. In some embodiments, the fermentation product is an alcohol. In some embodiments, the alcohol is ethanol or butanol. In some embodiments of any of the embodiments disclosed herein, the method further comprises f) adding one or more of additional glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, protease, pullulanase, β-amylase, additional α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, trehalase, isoamylase, oxidoreductase, esterase, transferase, pectinase, alpha-glucosidase, beta-glucosidase, lyase, hydrolase, or combinations thereof to the starch composition. In some embodiments of any of the embodiments disclosed herein, the fermentation is a simultaneous saccharification and fermentation (SSF) reaction.

[0017] 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.

[0018] 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. [Brief description of the drawings]

[0019] [Figure 1] 1 shows a multiple amino acid sequence alignment of Mucorales clade glucoamylases.

[0020] [Diagram 2] FIG. 1 shows a phylogenetic tree of predicted mature Mucorales clade glucoamylases and other fungal glucoamylases.

[0021] [Diagram 3] Electron density (2fo-fc) images shown at the Asn75 glycosylation site based on the X-ray diffraction structure determined for SvaGA1v2.

[0022] [Figure 4] 1 shows an image of the interaction of the glycan chain of residue Asn75 with the protein based on the X-ray diffraction structure determined for SvaGA1v2, with hydrogen bond interactions (<3.3 Å) indicated by dashed lines.

[0023] [Diagram 5] Highlighted in dark grey is an image of a helix containing the conserved Asp63 side chain and performance-improving mutations at positions 66, 67, and 69 based on the X-ray diffraction structure determined for SvaGA1v2.

[0024] [Figure 6] Highlighted in black are images of the loop from residues 100 to 129, predicted common acid residues, the conserved Asp side chain, and the amino acid positions of the performance-enhancing mutations based on the X-ray diffraction structure determined for SvaGA1v2.

[0025] [Figure 7] Based on the X-ray diffraction structure determined for SvaGA1v2, the four-helix bundle associated with the glycan chain at residue Asn75 is highlighted in dark grey, while the amino acid positions of some of the performance-enhancing mutations are shown as black spheres. The Asn75 glycan is shown as a stick model colored in black. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure relates to recombinant host cells, compositions comprising glucoamylases, and methods for saccharifying starch substrates using glucoamylases. Additionally, the disclosure also relates to processes for producing fermentation products and methods for increasing starch digestibility in animals, as well as methods for producing fermented beverages.

[0027] I. Definition Prior to describing the present compositions and methods in detail, the following terms and abbreviations are defined.

[0028] 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 meanings of many of the terms that describe this invention.

[0029] 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.

[0030] The term "glucoamylase variant" as used herein refers to a non-naturally occurring glucoamylase having at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) amino acid substitution(s) in a given parent glucoamylase amino acid sequence.

[0031] The term "wild-type" with respect to a polypeptide (such as a glucoamylase) refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. However, in another embodiment, non-limiting examples of wild-type glucoamylases include SEQ ID NOs: 4-37.

[0032] 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 that is then used as a reference to compare performance characteristics of polypeptides having further amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acid substitutions). In some embodiments, the parent polypeptide is SEQ ID NO: 2, 3, 5, or 6. In another embodiment, the parent polypeptide is SEQ ID NO:4-37.

[0033] 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 to carboxy terminal direction (i.e., N→C).

[0034] The term "mature polypeptide" is defined herein as a polypeptide in its final form after translation and any post-translational modifications (N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one embodiment, the predicted mature polypeptide is SEQ ID NO: 4 based on analysis of SignalP software version 4.0 (Nordahl Petersen et al. (2011) Nature Methods 8:785-786). In another embodiment, the mature polypeptide comprises amino acid positions 20-468 of SEQ ID NO: 2. In another embodiment, the mature polypeptide comprises amino acid positions 21-468 of SEQ ID NO: 2. In another embodiment, the mature polypeptide comprises amino acid positions 22-468 of SEQ ID NO: 2. In another embodiment, the mature polypeptide comprises amino acid positions 23-468 of SEQ ID NO: 2. In another embodiment, the mature polypeptide comprises amino acid positions 24-468 ​​of SEQ ID NO: 2. In another embodiment, the mature polypeptide comprises amino acid positions 25-468 of SEQ ID NO: 2.

[0035] 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 off during the secretion process. In some embodiments, SEQ ID NO:34 is the signal peptide. In other embodiments, the signal peptide comprises amino acid positions 1-20 of SEQ ID NO:2. In other embodiments, the signal peptide comprises amino acid positions 1-21 of SEQ ID NO:2. In other embodiments, the signal peptide comprises amino acid positions 1-22 of SEQ ID NO:2. In other embodiments, the signal peptide comprises amino acid positions 1-23 of SEQ ID NO:2. In other embodiments, the signal peptide comprises amino acid positions 1-24 of SEQ ID NO:2.

[0036] The terms "nucleic acid" or "polynucleotide" can be used interchangeably to encompass DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. Because the genetic code is degenerate, more than one codon can 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.

[0037] 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 a DNA, cDNA, synthetic, or recombinant nucleotide sequence.

[0038] 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.

[0039] A "synthetic" molecule is produced not by an organism but by in vitro chemical or enzymatic synthesis.

[0040] 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 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.

[0041] The term "glycosylation" as used herein refers to the attachment of a glycan to a molecule, such as a protein, such as a variant glucoamylase. Glycosylation can be an enzymatic reaction. The attachment formed can be via a covalent bond. The phrase "highly glycosylated" refers to a molecule, such as an enzyme, that is glycosylated at many sites, and at all or nearly all available glycosylation sites, such as N-linked glycosylation sites. Alternatively, or in addition, the phrase "highly glycosylated" can refer to extensive glycolytic branching (such as the size and number of glycolytic moieties associated with a particular N-linked glycosylation site) at all or substantially all N-linked glycosylation sites. In some embodiments, the engineered glucoamylase polypeptide is glycosylated at all or substantially all consensus N-linked glycosylation sites (i.e., NXS / T consensus N-linked glycosylation sites (X is any amino acid other than proline)). The glucoamylase can have various degrees of glycosylation. It is known that such glycosylation can improve stability during storage and use. In further embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consensus N-linked glycosylation sites (i.e., NXS / T consensus N-linked glycosylation sites, where X is any amino acid except proline) can be introduced into a glucoamylase (such as a variant glucoamylase) to improve one or more properties of the glucoamylase, such as, but not limited to, improving thermostability, activity, or saccharification yield.

[0042] The term "glycan" as used herein refers to the carbohydrate moiety of a complex carbohydrate such as a polysaccharide or oligosaccharide, or a glycoprotein. Glycans can be homopolymers or heteropolymers of monosaccharide residues. They can be linear or branched molecules.

[0043] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence, the process including both transcription and translation.

[0044] The term "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

[0045] "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 an appropriate ribosome binding site on the mRNA, an enhancer, and a sequence to control the termination of transcription and translation.

[0046] 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, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding the polypeptide.

[0047] 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.

[0048] The term "sequence motif" refers to a nucleotide or amino acid sequence pattern that is widespread and has proven or predicted biological significance. In the present invention, the sequence motif is an amino acid sequence motif identified within the Mucorales clade glucoamylases.

[0049] "Biologically active" refers to a sequence that has a particular biological activity, such as an enzymatic activity.

[0050] 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.

[0051] 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 log-expected score, and refinement using tree-dependent constraint partitioning. This program is described in MUSCLE: High-precision, high-throughput multiple sequence alignment 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.

[0052] 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, such as the glucoamylase of SEQ ID NO: 4.

[0053] As used herein, "equivalent positions" refers to positions that are common to the two amino acid sequences based on alignment of the amino acid sequence of a parent glucoamylase with a variant glucoamylase and alignment of the three-dimensional structure of the parent glucoamylase with that of the variant glucoamylase in three-dimensional space.

[0054] As used herein, "corresponding to" or "corresponding to" or "corresponding" in reference to an amino acid residue position 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.

[0055] 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.

[0056] As used herein, a "convert sugar" is a sugar that is formed when a monosaccharide (in a process defined herein as "conversion") is condensed with another monosaccharide (occasionally a disaccharide) in the presence of a catalyst (e.g., an acid) to form an oligosaccharide, e.g., (mostly) a disaccharide or (rarely) a trisaccharide. As a result, often times the converted sugar has bond linkages that are not present in the original starch composition. Examples of converted sugars include, for example, xylobiose (both α- and β-forms of (1,1), (1,2), (1,3), and (1,4)-linked xylobiose), O-α-D-xylopyranosyl-α-D-xylopyranoside, 3-O-α-D-xylopyranosyl-D-xylose, 2-O-α-D-xylopyranosyl-D-xylose, 4-O-α-D-xylopyranosyl-D-xylose, maltose, isomaltose, cellobiose, gentiobiose, 1,6-anhydro-β-D-glucofuranose, kojibiose, sophorose, nigerose, laminarabiose, and any combination thereof. Converted sugars are typically non-fermentable and therefore are considered waste products of the saccharification reaction.

[0057] 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, including glucose, and fermentation of the sugars to alcohol or other biochemicals or biological materials in the same reaction vessel.

[0058] A "slurry" is an aqueous mixture containing insoluble starch granules in water.

[0059] The term "total sugar content" refers to the total soluble sugar content present in a starch composition, including monosaccharides, oligosaccharides, and polysaccharides.

[0060] 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, including glucose.

[0061] The term "high DS" refers to an aqueous starch slurry having a dry solids content greater than 38% (wt / wt).

[0062] "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.

[0063] The term "contact" refers to the placing of 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.

[0064] 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 beverage alcohol, including specialized and proprietary yeast strains used to produce distinctive tasting beer, wine, and other fermented beverages.

[0065] "Ethanol-producing microorganism" refers to a microorganism with the ability to convert sugars or other carbohydrates into ethanol.

[0066] 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.

[0067] The term "pullulanase", also called debranching enzyme (EC 3.2.1.41, pullulan 6-glucanohydrolase), is capable of hydrolyzing the alpha 1-6 glucosidic bond in the amylopectin molecule.

[0068] In this specification, certain ranges are indicated by numerical values ​​preceded by the term "about". The term "about" is used in this specification to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the unrecited close or approximate number may be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented. For example, with respect to a numerical value, the term "about" refers to a range of -15% to +15% of the numerical value, unless the term is otherwise clearly defined in the context.

[0069] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0070] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as an antecedent basis for use of exclusive terminology, such as "solely," "only," and the like, in the context of reciting claim elements or using a "negative" limitation.

[0071] 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 preceding the term "comprising." The component preceding the term "comprising" is required or essential, but a composition including that component may further include other non-essential or optional components.

[0072] It should also be noted that the term "consisting essentially of" as used herein refers to a composition in which the component preceding said term is in the presence of other known components in a total amount of less than 30% by weight of the total composition and does not contribute to or interfere with the action or activity of said component.

[0073] It should also be noted that the term "consisting of" as used herein is meant to include and be limited to the element preceding the term "consisting of." Thus, the element preceding the term "consisting of" is required or essential, and no other element is present in the composition.

[0074] Every individual upper numerical limit given throughout this specification is intended to include every lower numerical limit, as if such lower numerical limit were expressly written herein. Every individual lower numerical limit given throughout this specification includes every higher numerical limit, as if such higher numerical limit were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0075] 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.

[0076] Throughout this specification, other definitions of terms may appear.

[0077] II. Variant Polypeptides with Glucoamylase Activity In a first aspect, the present invention relates to a variant polypeptide comprising an amino acid sequence having preferably at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and even at least 99% amino acid sequence identity to a polypeptide of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 having glucoamylase activity and having at least one amino acid substitution (e.g., any of the substitutions shown in Table 1). In another aspect, provided herein is a polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and even at least 99% amino acid sequence identity to a polypeptide comprising amino acid positions 20 to 468 of SEQ ID NO:2, amino acid positions 21 to 468 of SEQ ID NO:2, amino acid positions 22 to 468 of SEQ ID NO:2, amino acid positions 23 to 468 of SEQ ID NO:2, amino acid positions 24 to 468 of SEQ ID NO:2, or amino acid positions 25 to 468 of SEQ ID NO:2.

[0078] In some embodiments, the variant polypeptide comprises an amino acid sequence having at least 60% and less than 100% sequence identity to a polypeptide of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37. In other embodiments, the polypeptide comprises an amino acid sequence having at least 60% and less than 100% sequence identity to a polypeptide comprising amino acid positions 20-468 of SEQ ID NO:2, amino acid positions 21-468 of SEQ ID NO:2, amino acid positions 22-468 of SEQ ID NO:2, amino acid positions 23-468 of SEQ ID NO:2, amino acid positions 24-468 ​​of SEQ ID NO:2, or amino acid positions 25-468 of SEQ ID NO:2. In some embodiments, a polypeptide is non-naturally occurring (ie, does not occur in nature and is the product of human ingenuity).

[0079] In some embodiments, the glucoamylase variant polypeptide of the present invention is a homologous 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, 4 or less amino acids, 5 or less amino acids, 6 or less amino acids, 7 or less amino acids, 8 or less amino acids, 9 or less amino acids, 8 or less amino acids, 9 or less amino acids, 10 ...

[0080] In some embodiments, the variant polypeptide of the invention is a catalytic region comprising amino acids 18-449 of SEQ ID NO:4, as predicted by the ClustalX Hypertext Transfer Protocol Secure: / / world wide web.ncbi.nlm.nih.gov / pubmed / 17846036.

[0081] In some embodiments, the polypeptides of the invention have pullulan and / or panose and / or maltodextrin hydrolyzing activity.

[0082] In another aspect, the glucoamylase variant disclosed herein may, in some embodiments, comprise conservative substitutions of one or several amino acid residues compared to any one of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5-37, a polypeptide comprising amino acid positions 20-468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 21-468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 22-468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 23-468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 24-468 ​​of SEQ ID NO:2, or a polypeptide comprising amino acid positions 25-468 of SEQ ID NO:2. Exemplary conservative amino acid substitutions are listed below. Some conservative substitutions (i.e., mutations) can be made by genetic engineering, while others can be made by introducing synthetic amino acids into the polypeptide by other means. TIFF2024539622000002.tif199161

[0083] In some embodiments, the polypeptide of the present invention is a variant of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37, a polypeptide comprising amino acid positions 20 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 21 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 22 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 23 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 24 to 468 of SEQ ID NO:2, or a polypeptide comprising amino acid positions 25 to 468 of SEQ ID NO:2, or a fragment thereof, having glucoamylase activity. The variant glucoamylase can include a deletion, substitution (e.g., any of the amino acid substitutions shown in Table 1), insertion, or addition of one or more amino acid residues compared to the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37, or a homologous sequence thereof. In all cases, the phrase "one or more amino acid residues" refers to 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues.Any one of the polypeptides of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 to 37, a polypeptide comprising amino acid positions 20 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 21 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 22 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 23 to 468 of SEQ ID NO:2, a polypeptide comprising amino acid positions 24 to 468 of SEQ ID NO:2, or a polypeptide comprising amino acid positions 25 to 468 of SEQ ID NO:2. The amino acid substitutions (e.g., any of the amino acid substitutions shown in Table 1), deletions, and / or insertions of the tide can be at most 50, at most 40, at most 30, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, and even at most 1.

[0084] In some embodiments, the variant alteration comprises or consists of a substitution at a position corresponding to position 102 of the polypeptide of SEQ ID NO: 4, or at the corresponding position of any one of SEQ ID NOs: 5-37. In some embodiments, the amino acid at a position corresponding to position 102 of the polypeptide of SEQ ID NO: 4, or at the corresponding position of any one of SEQ ID NOs: 5-37, is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Ile, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val. In some embodiments, the variant alteration comprises or consists of a substitution S102P of the polypeptide of SEQ ID NO: 4, or at the corresponding position of any one of SEQ ID NOs: 5-37. In further embodiments, the variant comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0085] In some embodiments, the variant alteration comprises or consists of a substitution at a position corresponding to position 66 of the polypeptide of SEQ ID NO: 4, or at the corresponding position of any one of SEQ ID NOs: 2-37. In some embodiments, the amino acid at a position corresponding to position 66 of the polypeptide of SEQ ID NO: 4, or at the corresponding position of any one of SEQ ID NOs: 2-37, is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In some embodiments, the variant alteration comprises or consists of a substitution V66A at the polypeptide of SEQ ID NO: 4, or at the corresponding position of any one of SEQ ID NOs: 2-37.

[0086] In another embodiment, the variant alteration comprises or consists of substitutions at positions corresponding to position 66 and position 102 of the polypeptide of SEQ ID NO: 4, or at the corresponding positions of any one of SEQ ID NOs: 2-37. In some embodiments, the variant alteration comprises or consists of substitutions V66A and S102P of the polypeptide of SEQ ID NO: 4, or at the corresponding positions of any one of SEQ ID NOs: 2-37.

[0087] Alternatively, the amino acid changes may be of such a nature that they alter the physicochemical properties of the polypeptide, for example, they may improve the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0088] In one embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions exhibits a greater range (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, or greater (including any values ​​between these percentages to a greater extent). The variant glucoamylase may comprise a variant glucoamylase having a sequence corresponding to positions 20, 21, 51, 66, 79, 80, 92, 102, 121, 140, 143, 157, 158, 165, 166, 192, 203, 210, 213, 214, 215, 221, 222, 233, 235, 236, 238, 243, 252, 274, 278, 281, 290, 302, 310, 321, 341, 350, 352, 354, 370, 390, 403, 404, 405, 418, 440, 441, and / or 444 of SEQ ID NO:4, and / or The parent glucoamylase may have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acid substitutions) at the equivalent position in the parent glucoamylase (e.g., but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37).One or more substitutions at a particular position are: X020A / P;X021S;X051L / V;X066A / C / F / M / P / T / W;X079C / T;X080K;X092M;X102G;X121G / P / M;X140I / C;X140C;X143G;X157I;X158A;X165W;X166G / R / F;X192F;X203W;X210L;X213R;X214A / E;X215D / C / F;X221R / T / M;X222M / C;X233M / Y / P;X235Y;X236A / Q;X238S / H;X243E / V;X252C / F;X274A;X278A;X281D;X290M / V;X302K / V / P / S / W;X310T / Y;X321D;X341M;X350I / T;X352D;X354L / M;X370M;X390E;X403G / R;X404K;X405Q / S / Y;X418W / E;X440G / H;X441S; and / or X444L (X being the parent glucoamylase) or any amino acid corresponding to the equivalent position in the M / C;S233M / Y / P;W235Y;D236A / Q;G238S / H;T243E / V;V252C / F;G274A;P278A;E281D;F290M / V;N302K / V / P / S / W;N310T / Y;L341M;K350I;T352D;V354L / M;S370M;S390E;Q403G / R;Y404K;H405Q / S / Y;R418W / E;A440G / H;Q441S; and / or A444L. Any assay known in the art can be used to determine hydrolysis of a disaccharide (e.g., maltose), including the assays described in the Examples section.

[0089] In further embodiments, glucoamylase variants disclosed herein having one or more amino acid substitutions exhibit a greater degree of activity (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119 The enzymes can hydrolyze panose to a greater extent than any of the following percentages (including values ​​between these percentages): 1%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, or more. Panose is a trisaccharide consisting of a maltose molecule linked to a glucose molecule by an alpha-1,6 glycosidic bond that is commonly used as a substrate to help characterize the activity of starch-degrading enzymes.The variant glucoamylase may comprise a variant glucoamylase having a sequence corresponding to positions 20, 21, 23, 37, 51, 52, 67, 69, 73, 77, 79, 80, 81, 84, 92, 94, 102, 119, 121, 140, 141, 143, 164, 165, 166, 172, 210, 213, 214, 215, 218, 221, 222, 233, 236, 238, 252, 253, 274, 281, 290, 302, 321, 341, 350, 351, 352, 370, 390, 403, 404, 405, 418, 430, 440, 444, and / or 445 of SEQ ID NO:4, and / or can have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acid substitutions) at the equivalent positions in the parent glucoamylase (e.g., but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37).One or more substitutions at a particular position are: X020P / A;X021T / W / M;X023M / L;X037C;X051L / V;X052N;X067C;X069C / A;X073N;X077S;X079C;X080H / K;X081N;X084V / L;X092C;X094Y;X102G;X119G;X121M / L / G / P;X140C / Q / I;X141F;X143G;X164L;X165W / T;X166R;X172G;X210L;X213H;X214A / E;X215D;X218 Q / A / V / Y;X221R;X222M / V / C;X233M / T / P;X236Q;X238H;X252F;X274D / K;X281D;X290M / V;X302S / K / W / Q / V;X321D;X341M / T;X350C / T;X351E;X352D / N;X370M;X390D / L / E;X403G;X403R / K;X404M / K;X405S / Q;X418E / W;X430A;X440G;X444P; and / or X445Y / M (where X is the equivalent position in the parent glucoamylase). or any amino acid corresponding to S020P / A;K021T / W / M;E023M / L;E037C;A051L / V;G052N;S067C;V069C / A;K073N;T077S;A079C;G080H / K;D081N;I084V / L;V092C;F094Y;X102G;S119G;T121M / L / G / P;M140C / Q / I;L141F;A143G;I164L;Y165W / T;K166R;V172G;R210L;D213H;N214A / E;S 215D; S218Q / A / V / Y; S221R; G222M / V / C; S233M / T / P; D236Q; G238H; V252F; G274D / K; E281D; F290M / V; N302S / K / W / Q / V; N321D; L341M / T; K350C / T; N351E; T352D / N; S370M; S390D / L / E; Q403G; Q403R / K; Y404M / K; H405S / Q; R418E / W; T430A; A440G; A444P; and / or G445Y / M. Any assay known in the art can be used to determine the hydrolysis of panose, including the assays described in the Examples section.

[0090] In another embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions exhibits a greater range (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, Pullulan can be hydrolyzed to any of the following percentages (including values ​​between these percentages): 1%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, or greater. Pullulan is a polysaccharide polymer composed of maltotriose units (also known as α-1,4-; α-1,6-glucan). The three glucose units in maltotriose are linked by α-1,4 glycosidic bonds, while consecutive maltotriose units are linked to each other by α-1,6 glycosidic bonds. Pullulan is commonly used as a substrate to identify glucoamylases that can hydrolyze the alpha 1-6 bonds in starch.The variant glucoamylase may comprise a variant glucoamylase having a residue position corresponding to positions 20, 51, 69, 73, 77, 79, 80, 81, 84, 94, 119, 121, 134, 140, 141, 143, 156, 158, 165, 166, 203, 210, 214, 215, 218, 221, 222, 233, 235, 236, 252, 253, 274, 278, 281, 290, 302, 310, 321, 341, 350, 352, 354, 370, 390, 416, 418, 434, 440, and / or 445 of SEQ ID NO:4, and / or a variant glucoamylase having a residue position corresponding to positions 20, 51, 69, 73, 77, 79, 80, 81, 84, 94, 119, 121, 134, 140, 141, 143, 156, 158, 165, 166, 203, 210, 214, 215, 218, 221, 222, 233, 235, 236, 252, 253, 274, 278, 281, 290, 302, 310, 321, 341, 350, 352, 354, 370, 390, 41 The glucoamylase may have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 amino acid substitutions) at the equivalent position within the glucoamylase (e.g., but not limited to, any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOs:5-37).One or more substitutions at a particular position are: X020F / A / G;X051K;X069C;X073P / N;X077M / P;X079C / T;X080K;X081N;X084V / L;X094G / A;X119G / D;X121L / M;X134S;X140I / C / Q;X141F;X143G;X156P / C;X158A;X165T;X166H / A / F;X203C / Q / W / Y;X210L / M / F / N / A / I;X214A / Y / L / C;X215V / C / W / D / H;X218V / K / A / Q;X221R;X222M;X233M / P;X235Y;X236Q;X252F / H;X253K;X274K / D;X278A;X281D;X290M;X302P / S / V;X310T;X321D;X341M;X350I / T;X352D / S;X354L;X370M;X390D / L / E;X404K;X416Y;X418E / W;X434S;X440G; and / or X445M (where X is the equivalent position in the parent glucoamylase). , S020F / A / G;A051K;V069C;K073P / N;T077M / P;A079C / T;G080K;D081N;I084V / L;F094G / A;S119G / D;T121L / M;E134S;M140I / C / Q;L141F;A143G;F156P / C;N158A;Y165T;K166H / A / F;D203C / Q / W / Y;R210L / M / F / N / A / I;N214A / Y / L / C;S21 5V / C / W / D / H;S218V / K / A / Q;S221R;G222M;S233M / P;W235Y;D236Q;V252F / H;E253K;G274K / D;P278A;E281D;F290M;N302P / S / V;N310T;N321D;L341M;K350I / T;T352D / S;V354L;S370M;S390D / L / E;Y404K;F416Y;R418E / W;A434S;A440G; and / or G445M. Any assay known in the art can be used to determine the hydrolysis of pullulan, including the assays described in the Examples section.

[0091] In yet another embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions exhibits a higher performance index (PI) (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, %, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more (including any values ​​between these percentages). The variant glucoamylase may comprise a variant glucoamylase having a residue position corresponding to positions 21, 23, 37, 52, 66, 69, 73, 77, 79, 80, 81, 84, 92, 119, 121, 134, 140, 141, 157, 158, 164, 165, 210, 213, 214, 218, 221, 222, 233, 235, 236, 243, 252, 253, 274, 281, 290, 302, 341, 350, 351, 352, 370, 390, 403, 404, 405, 416, 418, 422, 440, 441, 444, and / or 445 of SEQ ID NO:4, and / or a variant glucoamylase having a residue position corresponding to positions 21, 23, 37 ... The cocoamylase may have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 amino acid substitutions) at equivalent positions within the cocoamylase (e.g., but not limited to, any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:5-37).One or more substitutions at a particular position are: X021T;X023M / L;X037C;X052N / F;X066A / F / T;X069C;X073N / P;X077P / M / S;X079R;X080N / H / K;X081N / S;X084L / V;X092I;X119A / G;X121M / L;X134S / Q;X140C / I / Q;X141F / K;X157A;X158A;X164L / T;X165W / T;X210W / G;X213H;X214G / Y;X218Q / F / A;X221R;X 222M / V / C;X233M / P;X235F;X236Q;X243A;X252F;X253K;X274D / K / A;X281D;X290M / V;X302W / S / Q / K;X341M / T;X350C / E;X351E;X352D / N;X370M;X390D / L;X403G / R / K;X404M / K;X405S;X416C / Y;X418E / W;X422A;X440G;X441L;X444S / P; and / or X445M (where X is the equivalent position in the parent glucoamylase). or any amino acid corresponding to any of the following: K021T;E023M / L;E037C;G052N / F;V066A / F / T;V069C;K073N / P;T077P / M / S;A079R;G080N / H / K;D081N / S;I084L / V;V092I;S119A / G;T121M / L;E134S / Q;M140C / I / Q;L141F / K;T157A;N158A;I164L / T;Y165W / T;R210W / G;D213H;N214G / Y;S218Q / F / A;S221R;G222M / V / C;S233M / P;W235F;D236Q;T243A;V252F;E253K;G274D / K / A;E281D;F290M / V;N302W / S / Q / K;L341M / T;K350C / E;N351E;T352D / N;S370M;S390D / L;Q403G / R / K;Y404M / K;H405S;F416C / Y;R418E / W;Y422A;A440G;Q441L;A444S / P; and / or G445M. PI can be determined based on the assays described in the Examples section.

[0092] In other embodiments, a glucoamylase variant disclosed herein having one or more amino acid substitutions exhibits improved hydrolysis of maltodextrins (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 1 The maltodextrins may exhibit an improvement in hydrolysis of any of the following percentages (including values ​​between these percentages): 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more. Maltodextrins are composed of D-glucose units linked in chains of variable length. The glucose units are primarily linked by α(1→4) glycosidic bonds, and maltodextrins are typically composed of a mixture of chains varying in length between 3 and 17 glucose units. Maltodextrins are produced using alpha amylase, thus mimicking the glucoamylase substrate.The variant glucoamylases include those having residue positions corresponding to positions 20, 21, 51, 67, 69, 73, 77, 79, 80, 81, 84, 102, 119, 121, 134, 140, 141, 143, 156, 157, 158, 165, 166, 172, 192, 203, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 252, 274, 281, 290, 302, 310, 321, 341, 350, 351, 352, 354, 370, 390, 403, 404, 405, 418, 422, 434, 440, 444, and / or 445 of SEQ ID NO:4, as well as or at an equivalent position within the parent glucoamylase (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57 amino acid substitutions).One or more substitutions at a particular position are: X020P / A / E;X021S / M;X051V / L / K / Y;X067A;X069C;X073P / N;X077P / M;X079C / T;X080K / H;X081N;X084L / V;X102G;X119G;X121M / G / V / P;X134S / Q;X140C / I;X141F;X143G;X156P / C;X157I;X158A;X165W / T;X166G;X172G;X192F;X203W / C / M;X210L / M;X213R;X214A / E;X215D / C / F;X218V / Y / Q;X221R / T;X222M / C;X233M / P / Y;X235Y;X236Q / A;X252F;X274K / A / D;X281D;X290M / V;X302P / K / S / V / Q / W;X310T / Y;X321D;X341M;X350I / T;X351V;X352D;X354L / M;X370M;X390E / L / D;X403K / R / G;X404K / M;X405Q / S / Y;X418W / E;X422A;X434S;X440G / H;X444P; and / or X445M; (where X is the equivalent position in the parent glucoamylase) or any amino acid corresponding to the amino acid position (S020P / A / E;K021S / M;A051V / L / K / Y;S067A;V069C;K073P / N;T077P / M;A079C / T;G080K / H;D081N;I084L / V;P102G;S119G;T121M / G / V / P;E134S / Q;M140C / I;L141F;A143G;F156P / C;T157I;N158A;Y165W / T;K166G;V172G;Y192F;D203W / C / M;R210L / M;D213R;N214A / E;S215D / C / F;S218V / Y / Q;S221R / T;G222M / C;S233M / P / Y;W235Y;D236Q / A;V252F;G274K / A / D;E281D;F290M / V;N302P / K / S / V / Q / W;N310T / Y;N321D;L341M;K350I / T;N351V;T352D;V354L / M;S370M;S390E / L / D;Q403K / R / G;Y404K / M;H405Q / S / Y;R418W / E;Y422A;A434S;A440G / H;A444P; and / or G445M.Any assay known in the art can be used to determine hydrolysis of maltodextrin, including those assays described in the Examples section.

[0093] In further embodiments, glucoamylase variants disclosed herein having one or more amino acid substitutions exhibit improved thermostability (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, In some embodiments, the improved thermal stability may be greater than or equal to 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more (including any values ​​between these percentages). The variant glucoamylase may have a residue position corresponding to positions 23, 51, 52, 66, 67, 77, 119, 121, 134, 140, 141, 156, 157, 165, 192, 213, 221, 222, 233, 236, 252, 281, 290, 302, 350, 352, 370, 390, 403, 404, 416, and / or 422 of SEQ ID NO:4, and / or ... The polypeptide may have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid substitutions) at equivalent positions within SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37, but are not limited to these.The one or more substitutions at a particular position may be X023M; X051Y; X052N; X066A / C / F / M / W; X067C; X077P; X119A; X121M; X134S; X140I / C; X141F; X156C; X157I; X165W / I; X192F; X213R; X221R; X222M; X233M; X236Q; X252F; X281D; X290V; X302H / Q / W / S / K; X350E / C; X352D; X370M; X390L / D; X403R; X404K / M; X416Y; and / or X422A / V (where X is the equivalent position in the parent glucoamylase). , or one or more of: E023M; A051Y; G052N; V066A / C / F / M / W; S067C; T077P; S119A; T121M; E134S; M140I / C; L141F; F156C; T157I; Y165W / I; Y192F; D213R; S221R; G222M; S233M; D236Q; V252F; E281D; F290V; N302H / Q / W / S / K; K350E / C; T352D; S370M; S390L / D; Q403R; Y404K / M; F416Y; and / or Y422A / V. Any assay known in the art can be used to determine the thermal stability of a polypeptide, including those assays described in the Examples section.

[0094] In another embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions exhibits reduced conversion (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, The improvement may be a conversion reduction of 6%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including any value between these percentages). The variant glucoamylase may have a residue position corresponding to positions 49, 51, 52, 66, 69, 77, 80, 94, 119, 134, 158, 164, 165, 172, 192, 213, 215, 218, 222, 233, 235, 236, 238, 243, 253, 274, 302, 338, 403, 405, 416, 418, 422, 430, 440, 441, 444, 445, and / or 449 of SEQ ID NO:4, and / or a ... For example, but not limited to, it may have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 amino acid substitutions) at the equivalent position within SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37.One or more substitutions at a particular position are: XS049W;X051Y;X052P;X066P;X069K / C;X077P / M;X080H;X094A / G;X119D;X134W / Q;X158Y;X164T / L;X165W / I;X172G;X192R / F;X213H;X215R;X218K;X222C;X233T / P;X235F;X236A; X238H / S / N; X243P / S; X253K; X274K / A; X302H / F / P / M / Q / T; X338I; X403G; X405Q / S; X416C / Y; X416Y; X418W / E; X422A; X430A / Q; X440L / H; X441W / L; X444L / P; X445Y; and / or X449L (where X is the equivalent position in the parent glucoamylase). or any amino acid corresponding to the amino acid sequence of S049W;A051Y;G052P;V066P;V069K / C;T077P / M;G080H;F094A / G;S119D;E134W / Q;N158Y;I164T / L;Y165W / I;V172G;Y192R / F;D213H;S215R;S218K;G222C;S233T / P;W23 and / or F449L. Any assay known in the art can be used to determine the formation of converted sugars during saccharification, including those described in the Examples section.

[0095] In further embodiments, glucoamylase variants disclosed herein having one or more amino acid substitutions exhibit improved saccharification yields (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, In some embodiments, the saccharification yield may be improved by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more (including any values ​​between these percentages). The variant glucoamylase may comprise a variant glucoamylase having a residue position corresponding to positions 21, 23, 51, 52, 66, 67, 79, 81, 92, 119, 140, 158, 164, 172, 192, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 238, 243, 253, 281, 290, 302, 310, 351, 352, 354, 370, 403, 416, 422, 430, 441, and / or 445 of SEQ ID NO:4, and / or ... The nucleic acid sequence may have one or more amino acid substitutions (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 amino acid substitutions) at the equivalent position within the nucleic acid sequence (e.g., but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any of SEQ ID NOs: 5-37).One or more substitutions at a particular position are: X021S / W;X023L;X051Y / K;X052N;X066A / C / F / M / W;X067M / C / A;X079R;X081S;X092C / M;X119A;X140I;X158A / Y;X164L;X172L / G;X192F;X210W;X213H;X214Y;X215F / C / R;X218H / K / A;X221M / T;X222V;X233T;X235Y / F;X236S / A;X238T / V / M;X243A;X253K;X281P / D;X290V;X302K / Q / H / W;X310V / T;X351V;X352S;X354L;X370R;X403K;X416Y;X422F;X430A;X441L; and / or X445Y (where X is the equivalent position in the parent glucoamylase). or any amino acid corresponding to K021S / W;E023L;A051Y / K;G052N;V066A / C / F / M / W;S067M / C / A;A079R;D081S;V092C / M;S119A;M140I;N158A / Y;I164L;V172L / G;Y192F;R210W;D213H;N214Y;S215F / C / R;S218H / K / A;S221M / T;G222V;S233T;W235Y / F;D236S / A;G238T / V / M;T243A;E253K;E281P / D;F290V;N302K / Q / H / W;N310V / T;N351V;T352S;V354L;S370R;Q403K;F416Y;Y422F;T430A;Q441L; and / or G445Y. Any assay known in the art can be used to determine saccharification yield, including those assays described in the Examples section.

[0096] Thus, in further embodiments, glucoamylase variants disclosed herein having one or more amino acid substitutions exhibit an improved (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more (including any values ​​between these percentages): i) improved hydrolysis of disaccharides (e.g., maltose); ii) greater hydrolysis of panose; iii) greater hydrolysis of pullulan; iv) higher PI of saccharification of soluble starch (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrins; vi) improved thermal stability; vii) less conversion to sugars with a DP of 2 or more;and / or viii) improved saccharification yield of glucose. The variant glucoamylase may exhibit two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) improvements in the saccharification yield of glucose. Residue positions corresponding to 221, 222, 233, 235, 236, 238, 243, 252, 253, 274, 278, 281, 290, 302, 310, 341, 350, 351, 352, 354, 370, 390, 403, 404, 405, 416, 418, 422, 430, 440, 441, 444, 445, and / or 449, and and / or one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 1 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71 amino acid substitutions. One or more substitutions at a particular position are:X020A / P;X021M / S / T / W;X023L / M;X037C;X051K / L / V / Y;X052N;X066A / C / F / M / W;X067A / C;X069C;X073N / P;X077M / P / S;X079R / T;X080H / K;X081N / S;X084L / V;X092C / M;X094A / G; X102G;X119A / D / G;X121G / L / M / P;X134Q / S;X140C / I / Q;X141F;X156C / P;X157I;X158A / Y;X164L / T ;X165I / T / W;X166G / F / R;X172G;X192F;X203C / W;X210L / M / W;X213H / R;X214A / E / Y;X215C / D / F / R;X218A / K / Q / V / Y;X221M / R / T;X222C / M / V;X233M / P / T / Y;X235F / Y;X236A / Q;X238H / S;X243A;X252F;X253K;X274A / D / K;X278A;X281D;X290M / V;X29 0V;X302H / K / P / Q / S / V / W;X310T / Y;X341M / T;X350C / E / I;X351E / V;X352D / N / S;X354L / M;X370M;X390D / E / L;X403G / K / R;X404I / K / M;X405Q / S / Y;X41 6C / Y;X418E / W;X422A;X430A;X440G / H;X441L;X444L / P; and / or X445M / Y (wherein X is any amino acid corresponding to the equivalent position in the parent glucoamylase), or one or more of S020A / P;K021M / S / T / W;E023L / M;E037C;A051K / L / V / Y;G052N;V066A / C / F / M / W;S067A / C;V069C;K073N / P;T077M / P / S;A079R / T;G080H / K;D081N / S;I084L / V;V092C / M ;F094A / G;P102G;S119A / D / G;T121G / L / M / P;E134Q / S;M140C / I / Q;L141F ;F156C / P;T157I;N158A / Y;I164L / T;Y165I / T / W;K166G / F / R;V172G;Y192 F;D203C / W;R210L / M / W;D213H / R;N214A / E / Y;S215C / D / F / R;S218A / K / Q / V / Y;S221M / R / T;G222C / M / V;S233M / P / T / Y;W235F / Y;D236A / Q;G238H / S;T2 43A;V252F;E253K;G274A / D / K;P278A;E281D;F290M / V;F290V;N302H / K / P / Q / S / V / W;N310T / Y;L341M / T;K350C / E / I;N351E / V;T352D / N / S;V354L / M;S370M;S390D / E / L;Q403G / K / R;Y404I / K / M;H405Q / S / Y;F416C / Y;R418E / W;Y422A;T430A;A440G / H;Q441L;A444L / P; and / or G445M / Y.

[0097] Multiple amino acid substitutions (e.g., any of the substitutions shown in Table 1), deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0098] Further provided herein are, in additional embodiments, a) X236S and X281D; b) X215R and X441W; c) X321D and X434S; d) X143G and X434S; e) X079C and X143G; f) X350T and X434S; g) X351E and X403K; h) X052N and X084L; i) X243P and X290V; j) X290V and X350E; k) X302K and X441W; l) X156C and and X404K;m) X067M and X404K;n) X052N and X141F;o) X052N and X351E;p) X233M and X445Y;q) X066C and X233M;r) X218H and X290V;s) X067M and X302H;t) X066C and X119A;u) X243P and X445Y;v) X192F and X243P;w) X156C and X243P;x) X023L and X066C;y) X023L-X119A;z) X020E and X192F;aa) X192F and X310V;bb) X023M and X302H;cc) X192F and X416Y;dd) X119A and X302H;ee) X235Y and X416Y;ff) X052N and X404K;gg) X023M and X449Y;hh) X158A and X172L;ii) X172L and X290V;jj) X023M and X210L;kk) X210L and X449Y;ll) X157I and X281 D; mm) X164T and X215R; nn) X140C and X422V; oo) X119A and X302K; or pp) X052N and X416Y, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase (for example, but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37).In other embodiments, the two or more amino acid substitutions are: a) D236S and E281D; b) S215R and Q441W; c) N321D and A434S; d) A143G and A434S; e) A079C and A143G; f) K350T and A434S; g) N351E and Q403K; h) G052N and I084L; i) T243P and F290V; j) F290V and K 350E;k) N302K and Q441W;l) F156C and Y404K;m) S067M and Y404K;n) G052N and L141F;o) G052N and N351E;p) S233M and G445Y;q) A066C and S233M;r) S218H and F290V;s) S067M and N302H;t) A066C and S119A;u) T243P and G445Y ;v) Y192F and T243P;w) F156C and T243P;x) E023L and A066C;y) E023L and S119A;z) S020E and Y192F;aa) Y192F and N310V;bb) E023M and N302H;cc) Y192F and F416Y;dd) S119A and N302H;ee) W235Y and F416Y;ff) G052N and Y40 4K; gg) E023M and F449Y; hh) N158A and V172L; ii) V172L and F290V; jj) E023M and R210L; kk) R210L and F449Y; ll) T157I and E281D; mm) I164T and S215R; nn) M140C and Y422V; oo) S119A and N302K; or pp) G052N and F416Y. The variant glucoamylase exhibits one or more of the following, relative to a parent glucoamylase lacking two or more of the substitutions: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) higher PI of soluble starch saccharification (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrins; vi) improved thermal stability; vii) less conversion to sugars with a DP of 2 or greater; and / or viii) improved saccharification yield of glucose.

[0099] Also provided herein are, in additional embodiments, a) X141F, X281D, and X441W; b) X143G, X321D, and X434S; c) X321D, X350T, and X434S; d) X067M, X158A, and X281D; e) X156C, X192F, and X403K; )X052N, X140C, and X422V;g)X066C, X119A, and X164T;h)X066C, X233M, and X445Y;i)X156C, X192F, and X243P;j)X023L, X066C, and X119A;k)X023M, X119A, and X404K;l)X310V, X4 or r) X052N, X416Y, and X445Y, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase (such as, but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37). In other embodiments, the three or more amino acid substitutions are: a) L141F, E281D, and Q441W; b) A143G, N321D, and A434S; c) N321D, K350T, and A434S; d) S067M, N158A, and E281D; e) F156C, Y192F, and Q403K; f) G052N, M140C, and Y422V; g) A066C, S119A, and I164T; h) A066C, S233M, and G445Y; i) F156C, Y192F , and T243P; j) E023L, A066C, and S119A; k) E023M, S119A, and Y404K; l) N310V, F416Y, and G445Y; m) N158A, S221R, and F290V; n) E023M, G052N, and Y404K; o) D081S, T157I, and D236S; p) T243P, N302K, and F416Y; q) M140C, N302K, and Y422V; or r) G052N, F416Y, and G445Y.The variant glucoamylase exhibits one or more of the following, relative to a parent glucoamylase lacking three or more of the substitutions: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) higher PI of soluble starch saccharification (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrins; vi) improved thermal stability; vii) less conversion to sugars with a DP of 2 or greater; and / or viii) improved saccharification yield of glucose.

[0100] Additionally, provided herein are, in additional embodiments, a) X215R, X236S, X281D, and X441W; b) X052N, X084L, X140C, and X422V; c) X020E, X156C, X192F, and X243P; d) X023M, X221R, and X404K; e) X158A, X172L, X221R, and X290V; f) X140C, X165W, X or g) glucoamylase variants having four or more substitutions at residue positions corresponding to X119A, X253F, X310V, and X403K, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase (e.g., but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37). In other embodiments, the four or more amino acid substitutions may be: a) S215R, D236S, E281D, and Q441W; b) G052N, I084L, M140C, and Y422V; c) S020E, F156C, Y192F, and T243P; d) E023M, S119A, S221R, and Y404K; e) N158A, V172L, S221R, and F290V; f) M140C, Y165W, N302K, and Y422V; or g) S119A, E253F, N310V, and Q403K. The variant glucoamylase exhibits one or more of the following, relative to a parent glucoamylase lacking four or more of the substitutions: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) higher PI of soluble starch saccharification (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrins; vi) improved thermal stability; vii) less conversion to sugars with a DP of 2 or greater; and / or viii) improved saccharification yield of glucose.

[0101] Further provided herein, in additional embodiments, are glucoamylase variants having five or more substitutions at residue positions corresponding to X067M, X157I, X218H, X302H, and X416Y, where X is any amino acid corresponding to the equivalent position in SEQ ID NO:4 and / or the equivalent position in a parent glucoamylase or fragment thereof (such as, but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37). In other embodiments, the four or more amino acid substitutions can be S067M, T157I, S218H, N302H, and F416Y. The variant glucoamylase exhibits one or more of the following, relative to a parent glucoamylase lacking five or more of the substitutions: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) higher PI of soluble starch saccharification (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrins; vi) improved thermal stability; vii) less conversion to sugars with a DP of 2 or greater; and / or viii) improved saccharification yield of glucose.

[0102] In further embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consensus N-linked glycosylation sites (i.e., NXS / T consensus N-linked glycosylation sites, where X can be any amino acid except proline and N(Asp) is glycosylated) can be introduced into any of the variant glucoamylases disclosed herein, or into the glucoamylase of SEQ ID NO: 4, to improve one or more properties of the glucoamylase. For example, but not limited to, i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) higher PI of soluble starch saccharification (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved hydrolysis of maltodextrin; vi) improved thermostability; vii) less conversion to sugars with a DP of 2 or more; and / or vii) improved saccharification yield of glucose, compared to a parent glucoamylase lacking one or more additional glycosylation sites. Amino acid substitutions introducing glycosylation sites into the variant glucoamylase polypeptide can be made at residue positions corresponding to positions 81, 83, 153, 370, or 372 of SEQ ID NO:4, and / or equivalent positions within the parent glucoamylase. In some embodiments, the one or more substitutions include one or more substitutions at positions D81N, K83T, A153T, S370N, and / or A372S.

[0103] As shown in Example 7, substitution of the Asn glycosylation site at position 75 was observed to cause a significant decrease in catalytic activity. Thus, in some embodiments, the variant glucoamylases disclosed herein have an N-linked glycosylation at position N075 of SEQ ID NO:4 and / or the equivalent position in the parent glucoamylase (such as, but not limited to, SEQ ID NO:2, SEQ ID NO:3, or any of SEQ ID NOs:5-37).

[0104] III. Production of glucoamylase The variant glucoamylases 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 glucoamylase can be obtained after secretion of the glucoamylase into the cell culture medium. Optionally, the glucoamylase can be isolated from the host cells or even from the cell broth, depending on the desired purity of the final glucoamylase. Genes encoding glucoamylases can be cloned and expressed according to methods well known in the art. 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 reesei), or Myceliophthora species (such as Myceliophthora thermophila). Other host cells include bacterial cells, such as Bacillus species (such as Bacillus subtilis or B. licheniformis), and Streptomyces species. Suitable yeast host organisms can be selected from the Schizosaccharomyces or Saccharomyces species, including Saccharomyces cerevisiae or Schizosaccharomyces species, such as S. pombe. Strains of the methylotrophic yeast species Pichia pastoris can be used as host organisms.

[0105] 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 co-saccharification and fermentation processes, to reduce or eliminate the need for added enzymes.

[0106] A. Vector The DNA constructs comprising the nucleic acids encoding the variant glucoamylase polypeptides disclosed herein can be constructed to be suitable for expression in host cells.Due to the known degeneracy of the genetic code, various polynucleotides encoding 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.

[0107] A polynucleotide encoding a variant 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.

[0108] 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 variant glucoamylase polypeptide disclosed herein may be transformed and / or replicated in a bacterial host cell as a means of propagating and amplifying 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.

[0109] Exemplary useful vectors include pTrex3gM (see U.S. Patent Publication No. 20130323798) and pTTT (see U.S. Patent Publication No. 20110020899), 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.

[0110] 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 glucoamylase to a host cell organelle, such as a peroxisome, or to a specific cell compartment. For expression under the direction of the control sequence, the nucleic acid sequence of the variant glucoamylase is operably linked to the control sequence in a manner suitable for expression.

[0111] A polynucleotide encoding a variant glucoamylase polypeptide disclosed herein can be operably linked to a promoter, allowing transcription in the 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 in a bacterial host include, inter alia, the promoter of the lac operon of E. coli, the promoter of the Streptomyces coelicolor agarose genes dagA or celA, the promoter of the amylase gene of Bacillus licheniformis (amyL), the promoter of the maltogenic amylase gene of Bacillus stearothermophilus (amyM), the promoter of the amylase gene of Bacillus amyloliquefaciens (amyQ), the promoter of the xylA and xylB genes of Bacillus subtilis, and the like.

[0112] 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 glucoamylase-encoding gene is expressed in a bacterial species, such as E. coli, a suitable promoter can be selected from bacteriophage promoters, including, for example, the T7 promoter and the phage lambda promoter. Along these lines, examples of promoters suitable 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.

[0113] 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 variant glucoamylase gene of interest to be expressed or may be from a different genus or species than the variant glucoamylase (i.e., the species from which the variant 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.

[0114] 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 suitably be derived from the same source as the promoter.

[0115] Vectors may also contain selectable markers, for example, 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, for example, WO 91 / 17243.

[0116] B. Transformation and Cultivation of Host Cells The isolated cells containing either the DNA construct or the expression vector are advantageously used as host cells for the recombinant production of the variant glucoamylase. The cells may be transformed with a DNA construct encoding the enzyme, conveniently by integrating the DNA construct into the host 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 chromosome may be carried out according to conventional methods, for example by homologous or non-homologous recombination. Alternatively, the cells may be transformed with expression vectors associated with various types of host cells.

[0117] 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.

[0118] Suitable yeast host organisms may be selected from biotechnologically relevant yeast species, such as, 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 may be used as host organisms. Alternatively, the host organism may be a Hansenula species.

[0119] 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. In addition, Trichoderma species may be used as hosts. The glucoamylase expressed by the fungal host cell may be glycosylated, i.e., contain glycosyl moieties. The glycosylation pattern may be the same as that present in the wild-type glucoamylase, or may be different. The type and / or degree of glycosylation may confer altered enzymatic and / or biochemical properties.

[0120] It is advantageous to delete genes from the expression host, where the gene defect can be cured by the transformed expression vector. Known methods can be used to obtain fungal host cells with one or more inactivated genes. In one non-limiting embodiment, any gene (e.g., cbh1, cbh2, egl1, and egl2 genes) can be deleted from a cloned Trichoderma spp. or other filamentous fungal host. 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.

[0121] 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 the glucoamylase is stably integrated into the host cell chromosome. Transformants are then selected and purified by known techniques. The deletion plasmid is then cut at an appropriate restriction enzyme site within the desired gene coding region, and the gene coding sequence or a portion thereof is replaced by a selectable marker. Flanking DNA sequences (preferably about 0.5-2.0 kb) from the locus of the gene to be deleted remain on either side of the marker gene. A suitable deletion plasmid will have unique restriction enzyme sites present in it to allow the fragment containing the gene to be deleted, including generally flanking DNA sequences and a selectable marker gene, to be removed as a single linear piece.

[0122] Depending on the host cell used, post-transcriptional and / or post-translational modifications may be made. A non-limiting example of a post-transcriptional and / or post-translational modification is "clipping" or "truncation" of the polypeptide. In another example, the clipping may be to take a mature glucoamylase polypeptide and further remove N- or C-terminal amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N- or C-terminal amino acids) to generate a truncated form of the glucoamylase that retains enzymatic activity.

[0123] Other examples of post-transcriptional or post-translational modifications include, but are not limited to, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine, or threonine phosphorylation. Those skilled in the art will understand that the type of post-transcriptional or post-translational modification that a protein may undergo may depend on the host organism in which the protein is expressed.

[0124] Further sequence modifications of the polypeptide after expression may occur. Sequence modifications include, but are not limited to, oxidation, deglycosylation, saccharification, etc. It is known that glycation can affect the activity of glucoamylases, especially when incubated with glucose or other reducing sugars at temperatures above 30° C. and neutral or alkaline pH. Protein engineering to eliminate lysine residues can be used to prevent such modifications. An example can be found in U.S. Pat. No. 8,507,240. For example, yeast expression can result in hyperglycosylated polypeptides that result in an apparent increase in molecular weight. Also, WO 2013 / 119470, published on August 15, 2013 (incorporated herein by reference), relates to phytases with increased stability believed to be due to increased glycosylation.

[0125] C. Expression and Fermentation Methods for producing the variant glucoamylases disclosed herein can include culturing host cells under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0126] The medium used to culture the cells may be any conventional medium suitable for growing the host cells and obtaining expression of the variant glucoamylase polypeptide. Suitable media and medium components are available from commercial suppliers or may be prepared according to published recipes (e.g., as set forth in catalogues of the American Type Culture Collection).

[0127] Any of the fermentation methods known in the art can be suitably used to ferment such transformed or derived fungal strains, in some embodiments, the fungal cells are grown under batch or continuous fermentation conditions.

[0128] D. Enrichment purification method Separation and concentration techniques are known in the art, and conventional methods can be used to prepare concentrated solutions or broths containing the variant glucoamylase polypeptides of the invention.

[0129] After fermentation, a fermentation broth is obtained and various suspended solids, such as microbial cells and residual raw fermentation material, 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, are commonly used.

[0130] At times, it may be desirable to concentrate the solution or broth containing the glucoamylase polypeptide to optimize recovery. Use of an unconcentrated solution or broth will typically require increased incubation times to collect the enriched or purified enzyme precipitate.

[0131] IV. Composition The present invention also relates to compositions comprising a polypeptide (such as a glucoamylase, e.g., any of the variant glucoamylases disclosed herein) and / or a starch substrate. In some embodiments, variant glucoamylases comprising an amino acid sequence that is 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 sequence of SEQ ID NO:4 (or any of SEQ ID NOs:2-30) can also be used in the enzyme composition. Preferably, the composition is formulated to achieve desired characteristics, 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.

[0132] The composition may include a variant glucoamylase polypeptide of the invention as the main 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 known to those skilled in the art.

[0133] 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 comprising the variant glucoamylase may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., and may further comprise 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 the slurry, water bath, washing machine, food or beverage product, etc., such as endogenous plant (including algae) enzymes, residual enzymes from a prior processing step, etc. The polypeptides included in the compositions may be stabilized according to methods known in the art.

[0134] 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 appropriate stabilizers. Such cells may further express additional polypeptides, such as those described above.

[0135] The dosage of the polypeptide composition of the present invention and other conditions under which the composition is used can be determined based on methods known in the art.

[0136] The compositions are 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 compositions are also suitable for use in animal nutrition (e.g., as a component of animal feed) and fermented beverage products.

[0137] V. Use and Methods The present invention also relates to the use of the variant glucoamylase polypeptides or compositions of the present invention in liquefaction processes, saccharification processes, and / or fermentation processes. The variant 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 a combination of processes, preferably in conjunction with starch conversion, such as a liquefaction saccharification process, a liquefaction fermentation process, or a saccharification fermentation process.

[0138] A. Glycation The liquefied starch can be saccharified using alpha-amylase and variant glucoamylase, 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 enzyme combination used and the type of modified starch. Advantageously, the syrup obtained using the provided variant glucoamylase can contain 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%.

[0139] 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 must 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 typically added in a constant ratio 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 added prior to saccharification in simultaneous saccharification and fermentation (SSF) at a temperature of 30-65° C., typically around 60° C., typically for 40-90 minutes.

[0140] B. Raw Starch Hydrolysis The invention provides for the use of variant glucoamylases of the invention to produce glucose and the like from raw or granular starch. In general, glucoamylases 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 processing higher concentrations of dry solids (e.g., up to 45%) than conventional systems.

[0141] The "raw starch hydrolysis" process (RSH) differs from conventional starch processing processes in that it involves 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.

[0142] The variant glucoamylases of the invention may also be used in combination with enzymes that hydrolyze only alpha-(1,6)-glucosidic linkages in molecules containing at least four glucosyl residues. Preferably, the variant glucoamylases 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.

[0143] C. Fermentation Soluble starch hydrolysates, particularly glucose-rich syrups, can be fermented by contacting the starch hydrolysates with a fermenting organism, typically at a temperature of around 32°C, e.g., 30°C to 35°C. "Fermenting 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 fermenting organisms are capable of fermenting, i.e., converting, directly or indirectly, sugars, such as glucose or maltose, to the desired fermentation product. Examples of fermenting 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 withstand high 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, Lachancea, and Schizosaccharomyces species. Several yeast strains are commercially available, many of which have been selected or engineered for desired characteristics, such as high alcohol production, rapid growth rate, etc. The temperature and pH of the fermentation will depend on the fermenting organism. Microorganisms that produce other metabolic products, such as citric acid and lactic acid, by fermentation 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.

[0144] 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. The fungal cells expressing the variant glucoamylase may also be fermenting microorganisms, such as ethanol producing microorganisms. Thus, ethanol production may be carried out using fungal cells expressing sufficient variant glucoamylase such that relatively little or no exogenous enzyme addition is required. The fungal host cells may be selected from appropriately engineered fungal strains. Fungal host cells may also be used that express and secrete other enzymes in addition to the variant glucoamylase. Such cells may express amylases and / or pullulanases, phytases, alpha-glucosidases, isoamylases, beta-amylases, cellulases, xylanases, other hemicellulases, proteases, beta-glucosidases, pectinases, esterases, oxidoreductases, transferases, or other enzymes. Fermentation may be followed by subsequent recovery of ethanol.

[0145] D. 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); for example, 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.

[0146] 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.

[0147] E. Brewing The process of producing 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.

[0148] The brewing composition comprising the variant glucoamylase 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.

[0149] F. Animal Nutrition The variant glucoamylases 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.

[0150] The term "animal" refers to any living organism belonging to the animal kingdom, including, but not limited to, mammals (except humans), non-human animals, domestic animals, livestock, farm 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, and laying hens; 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.

[0151] The terms "animal feed", "feed", "feed ingredient", 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) (especially corn-based distillers dried grains with solubles (cDDGS), wheat bran, small grains, and combinations thereof); The feed may comprise one or more feed ingredients selected from the group consisting of 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, etc.; d) fats and oils obtained from vegetable and animal sources; and / or e) minerals and vitamins.

[0152] The digestibility of starch in feed is highly variable and depends on several 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 the use of at least one glucoamylase as a feed additive.

[0153] When used as a feed, such as a functional feed, or in the preparation of such a feed, the enzyme or feed additive composition herein may be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, and a nutritionally active ingredient, such as at least one member 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.

[0154] Also, at least one variant glucoamylase described herein (or an enzyme composition comprising at least one variant glucoamylase described herein) can be homogenized to produce a powder. The powder can be mixed with other components known in the art. Optionally, the feed material can also contain additional minerals, such as calcium, and / or additional vitamins. In some embodiments, the feed material is a corn soybean meal mix.

[0155] In an alternative preferred embodiment, the enzyme composition comprising at least one glucoamylase 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 granules, the granules contain 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.

[0156] Alternatively, the composition may be in a liquid formulation suitable for consumption, preferably such liquid consumables contain one or more of the following: a buffer, a salt, sorbitol, and / or glycerol.

[0157] Any of the glucoamylases described herein for use as feed additives may be used alone or in combination with at least one direct-feeding microorganism. DFM categories include Bacillus spp., lactic acid bacteria, and yeast. Additionally, any of the glucoamylases described herein for use as feed additives may be used alone or in combination with at least one essential oil, such as, for example, cinnamaldehyde and / or thymol. Additionally, any of the glucoamylases described herein for use as feed additives may 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.

[0158] Also disclosed are methods of increasing the nutritional value of animal feed, where an effective amount of any of the variant glucoamylases described herein can be added to the animal feed.

[0159] As used herein, the phrase "effective amount" relates to the amount of an active agent (e.g., any of the variant 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.

[0160] 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 the adverse effects of a disease or of the immune response of a subject.

[0161] 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.

[0162] By "improved animal performance of one or more endpoints" is meant 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 as a result of use of a feed comprising the feed additive composition described herein compared to a feed not containing said feed additive composition.

[0163] 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

[0164] Example 1 Construction, transformation, and expression of a site-evaluated library (SEL) of Mucorales clade variant glucoamylases in Trichoderma reesei The polynucleotides encoding the Mucorales clade variant glucoamylase (codon-modified sequences used as expression cassettes) were synthesized by Generay (Generay Biotech Co., Ltd, Shanghai, China) and inserted into pI1c expression vector, a derivative vector of pTTT (see US Patent Publication No. 20110020899), lacking telomeric regions and acetamidase markers. Generay Seamless cloning reagents were used as described by the vendor. In this study, the gene sequence of the parent glucoamylase molecule is SEQ ID NO:1, and the translation product is SEQ ID NO:2. The predicted mature sequence of the parent glucoamylase molecule for this site evaluation study is SEQ ID NO:3.

[0165] All fungal manipulations, including high-throughput transformation, inoculation, fermentation, and harvesting, were carried out in 96-well microtiter plates (MTPs). Plasmids were transformed into the appropriate T. reesei host strain using the polyethylene glycol (PEG)-protoplast method, omitting plating on solid agar. Briefly, approximately 0.5-2 μg of DNA, 2 × 10 6 A total volume of 60 μL of transformation mixture containing 10 protoplasts and 4 pmol of ribonucleoprotein (RNP) was treated with 150 μL of 25% PEG solution. Protoplasts were regenerated in liquid selective growth medium containing 0.75 M sorbitol to maintain osmotic pressure. Plates with regenerated fungal cultures were grown in a shaker incubator with a 50 mm throw at 200 rpm, 28° C., and 80% humidity for 4 days until fungal mycelium formed. This material was used to inoculate cultures for expression of glucoamylase variants.

[0166] To express the glucoamylase protein, the transformed T. reesei strain was cultivated as follows: 40 μl of the regenerated transformant was used to inoculate 360 ​​μl of culture medium containing glucose, (NH4)2SO4, PIPPS buffer (pH 5.5), salts, and trace elements. The culture was grown in a 96-well MTP incubated in a shaker incubator with a 50 mm throw at 260 rpm, 28° C., and 80% humidity. After 2 days of preculture, the culture was transferred to a culture medium similar to that described above, in which glucose was replaced by a glucose / sophorose mixture. The 96-well MTP was then incubated in a shaker incubator with a 50 mm throw at 260 rpm, 28° C., and 80% humidity. After 5 days of fermentation, the culture was filtered by centrifugation using a hydrophilic PVDF membrane to obtain a clear supernatant, which was used for analysis of the recombinant glucoamylase enzyme.

[0167] Supernatants from the transformation screen were subjected to protein quantification. Aliquots of 5 μl of cleared culture supernatant from MTP cultures were injected onto a Zorbax 300 C3 Rapid Resolution HD 2.1×50 mm 1.8-Micron (Agilent) column at 80° C. Glucoamylase was eluted with a 3 min gradient of Buffer A (0.1% TFA in water) and Buffer B (0.07% TFA, 70% acetonitrile, 30% isopropanol). Glucoamylase protein concentration was determined using a calibration curve (0-1024 ppm) of fermentation samples with known glucoamylase concentrations. For the activity assays reported in Table 1 (Example 2 below), samples were normalized to concentrations of 200 ppm for saccharification assays, 40 ppm for conversion assays, and 15 ppm for all colorimetric ABTS assays in 20 mM sodium acetate buffer pH 4.5 containing 0.005% (v / v) Tween-80. Calibration curves of parent molecules ranging in concentration from 0 to 80 ppm were included on the assay plates for performance index (PI) calculations.

[0168] Representative samples of culture supernatants of the parent glucoamylase were analyzed by mass spectrometry (MS). The results of the MS analysis indicate the presence of multiple polypeptides. The predicted C-terminus of SEQ ID NO:3 was confirmed, while several truncations of the predicted N-terminus were detected. Truncations were observed after the N-terminal residues: 1 (SEQ ID NO:5), residue 2 (SEQ ID NO:35), residue 3 (SEQ ID NO:6), residue 5 (SEQ ID NO:36), and residue 6 (SEQ ID NO:37).

[0169] Example 2 Evaluation of enzymatic activity of Mucorales clade glucoamylase variants in saccharification-related assays Saccharification performance of the variant and parent molecule (SEQ ID NO: 3) was evaluated over 48 hours at pH 4.5, 62°C using Maltosweet G 120 (DE=11-14) as substrate purchased from Tate & Lyle. The starting DS content of the saccharification mixture was 34%. Glucoamylase performance was tested at a dosage of 28 μg / dsg. For this evaluation, pullulanase OPTIMAX™ L 1000 (a product of IFF) was dosed at 0.438 ASPU / DSg and alpha-amylase Aspergillus terreus amylase (described in WO2014099415, incorporated herein by reference) was dosed at 0.227 SSU / DSg per incubation. Reactions consisting of 75 μl maltodextrin and 5 μl saccharification mixture (glucoamylase, alpha-amylase, and pullulanase as described above) were incubated in an MTP at pH 4.5, 62° C. for 48 h with shaking at 900 rpm. All reactions were quenched by addition of 5 mM H2SO4 in a ratio of 1:40 saccharification syrup to acid solution. Quenched aliquots were analyzed by HPLC for product formation using an Agilent 1260 series system equipped with dual Phenomenex Rezex-RFQ Fast Acid H+ (8%) 100×7.8 mm columns (cat# 00D-0223-K0) run at 80° C. for 5.3 min. For this purpose, 5 μL of sample was injected onto the column and carbohydrates were separated by an isocratic gradient of 5 mM H2SO4 as mobile phase at a flow rate of 1.0 mL / min. The oligosaccharide products were detected using a refractive index detector. Standards were run to determine the elution time of each degree of polymerization (DP(n)) of the saccharide of interest (n represents the number of saccharides, e.g., DP3+, DP3, DP2, and DP1). The total integrated peak area of ​​the identified glycation products was used to calculate the relative content of each (oligo)saccharide. The performance of the GA variants in glycation was compared to that of the parent molecule (SEQ ID NO: 3). To determine the effect of amino acid substitutions on the GA properties, the % increase in glucose yield relative to the parent molecule was determined. The results are shown in Table 1.A value greater than 0.5% is considered to be a positive effect of the mutation on glycosylation.

[0170] Determination of specific activity using colorimetric readout The specific activity of glucoamylase variants was determined based on the rate of glucose release from selected substrates 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).

[0171] A 5 μl aliquot of glucoamylase supernatant was combined in 25 μl of freshly prepared Reagent A solution in MTP containing 25 mM sodium acetate buffer (pH 4.5) (final concentrations were 25 U / mL HRP (Sigma-Aldrich, Prod.nr: P8375), 62 U / mL GOX (Sigma-Aldrich, Prod.nr: G7141), and 0.005% Tween-80). To start the reaction, 20 μl of substrate-containing Reagent B consisting of 5.4 mg / mL ABTS (Sigma-Aldrich, Prod.nr: A1888), 25 mM sodium acetate buffer (pH 4.5), and 0.005% Tween-80 was added. The final substrate concentrations for the different assays were: 4.6 mM maltose (Sigma-Aldrich, Prod.nr:47288), 30 mM panose (Megazymes, O-PAN), 1% (w / v) pre-boiled pullulan (Sigma-Aldrich, Prod.nr:P4516), 0.5% (w / v) pre-boiled soluble starch (Sigma-Aldrich, Prod.nr:S9765), and 1% (w / v) maltodextrin (Sigma-Aldrich, Prod.nr:31410). The reagents were mixed vigorously and absorbance kinetics were read immediately at 23 s intervals for 6 min at room temperature with continuous stirring using a Molecular Devices plate reader. The increase in absorbance rate at 405 nm is proportional to the hydrolytic activity of the enzyme. Reactions were run in triplicate and the Vo of the samples was determined using linear regression. The data thus generated was used to calculate the performance of each sample for each assay. The percentage activity of each variant divided by the percentage expected activity of the parent GA at an equivalent protein concentration was defined as the performance index (PI). A PI value equal to or 1.1 greater than the parent PI value was used as the cutoff for selection of mutations with beneficial effects on the specific activity of the molecule. The results are shown in Table 1.

[0172] Evaluation of thermostability of glucoamylase variants Glucoamylase variant samples set at 15 ppm were incubated for 30 min at 57°C in a 384-well PCR MTP in a Mastercycler Pro 384 (Eppendorf). After incubation, the MTP was cooled to room temperature and the specific activity on maltodextrin substrate was determined using the ABTS method described above. The percent (%) residual activity (activity remaining after heat treatment) of the glucoamylase variants was determined by dividing the activity of the heat-incubated sample by the activity of the non-incubated sample. An improvement in thermostability of 5% over that of the parent enzyme was used as a cutoff in selecting mutations with beneficial effects on the thermostability of the molecule. Thermostability results are shown in Table 1 as % residual activity.

[0173] Evaluation of conversion reactions of glucoamylase variants The glucose condensation activity (conversion activity) of each GA variant was tested by combining 67 μl of 50% (w / v) glucose in 10 mM sodium acetate buffer pH 4.5 with 13 μl of enzyme sample in a 384-well MTP and incubating with shaking at 50° C. for 66 hours. The reaction was quenched by mixing 2.5 μl of the reaction cocktail with 97.5 μl of 5 mM H2SO4. The reaction products (glucose and condensation products, e.g., DP2, DP3, and DP3+) were determined and quantified by HPLC analysis as described above.

[0174] The performance of the GA variants was compared to that of the parent molecule (SEQ ID NO: 3). To determine the effect of amino acid substitutions on the GA properties, the reduction in conversion yield (DP2) relative to the parent molecule, expressed as % condensate, was determined. The results are shown in Table 1. A value equal to or greater than 0.4 reduced conversion is considered a positive effect on conversion under the conditions of this assay.

[0175] Note that for comparison of residual activity the values ​​used are absolute (as measured), whereas for conversion reactions and for saccharification yields the values ​​are calculated as percent yield difference (e.g. variant A51Y confers a 1.4% yield improvement in saccharification yield as the yield is 93.2% DP1 versus 91.8% DP1 for the parent GA).

[0176] All Mucorales clade GA variants listed in Table 1 exhibit at least one improved property when compared to the parent sequence SEQ ID NO:3, showing a benefit for each amino acid substitution. SEQ ID NO:3 is equivalent to SEQ ID NO:4 (SvaGA1) with Pro at position 102. Examples where cells are blank in Table 1 indicate that no benefit (improvement over parent) was observed for that particular example. For example, PI, thermostability, and residual activity % of <1.0 were less than 5% improved (compared to parent control on assay plate), and the reduction in conversion and improved glycation results were less than 0.4% or more than 0.5% over the parent, respectively. The asterisk next to the residual activity % for thermostability of the parent molecule indicates that this is an average of multiple tests across the evaluations performed on the variant molecules. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0177] Example 3 Comparison of glucoamylase sequences A multiple amino acid sequence alignment was performed with the predicted mature region of SEQ ID NO:3 (SvaGA1v2) and SEQ ID NO:4 (SvaGA1), as well as the related glucoamylases: GAN00808.1 (SEQ ID NO:7), ORE14155.1 (SEQ ID NO:8), RCH88939.1 (SEQ ID NO:9), AelGA1 (SEQ ID NO:31), AosGA3 (SEQ ID NO:30), AtrGA1 (SEQ ID NO:33), AvaGA1 (SEQ ID NO:32), BciGA1 (SEQ ID NO:10), BciGA2 (SEQ ID NO:11), BpoGA1 (SEQ ID NO:12), CcuGA1 (SEQ ID NO:13), CumGA1 (SEQ ID NO:20), DelGA1 (SEQ ID NO:16), FspGA3 (SEQ ID NO:17), GpeGA1 (SEQ ID NO:18), MciGA1 (SEQ ID NO:19), MciGA2 (SEQ ID NO:20), MciGA1 (SEQ ID NO:21), MciGA1 (SEQ ID NO:22), MciGA1 (SEQ ID NO:23), MciGA1 (SEQ ID NO:24), MciGA1 (SEQ ID NO:25), MciGA1 (SEQ ID NO:26), MciGA1 (SEQ ID NO:27), MciGA1 (SEQ ID NO:28), MciGA1 (SEQ ID NO:29), MciGA2 (SEQ ID NO:30), MciGA1 (SEQ ID NO:31), MciGA1 (SEQ ID NO:32), MciGA1 (SEQ ID NO:33), MciGA1 (SEQ ID NO:34), MciGA1 (SEQ ID NO:35), MciGA1 (SEQ ID NO:36), MciGA1 (SEQ ID NO:37), MciGA1 A3 (SEQ ID NO: 19), MciGA5 (SEQ ID NO: 15), McoGA1 (SEQ ID NO: 21), ParGA1 (SEQ ID NO: 22), RmiGA1 (SEQ ID NO: 23), RstGA1 (SEQ ID NO: 14), SfuGA2 (SEQ ID NO: 24), SobGA1 (SEQ ID NO: 29), SraGA1 (SEQ ID NO: 25), SraGA3 (SEQ ID NO: 26), TinGA1 (SEQ ID NO: 27), and ZmeGA1 (SEQ ID NO: 28) were constructed to determine the corresponding amino acids from position 1 to position 449 of SEQ ID NO: 3 and 4 with respect to the sequences of these other GAs. These sequences were aligned using the MUSCLE alignment tool in Geneious 10.2 software with default parameters. The multiple sequence alignment is shown in panels A-H of FIG. 1. The percent sequence identity between these GAs calculated from the MUSCLE alignment is shown in Table 2. Phylogenetic trees for these Mucorales clade GAs and other GAs were generated using Geneious 10.2 software and are shown in FIG. [Table 2]

[0178] Example 4 Construction, transformation, and expression of a combinatorial library of variant glucoamylases in Trichoderma reesei DNA expression cassettes of glucoamylase variants containing the selected mutation combinations (Table 1) were obtained using standard molecular biology techniques using the pI1c vector described above and the Seamless Cloning and Assembly Kit (Invitrogen) according to the manufacturer's protocol.

[0179] Protoplast preparation and transformation were performed as described in WO2013 / 102674 to insert the expression cassette into a specific location in the fungal host genome. The in vitro assembled RNP complex was added to the transformation mixture described in Example 1. It was then plated on a 24-well microtiter plate with selective agar minimal medium. Once the transformants had grown and sporulated sufficiently, they were scraped and repatched on fresh selective agar plates. The transformants were further cultured and culture supernatants were prepared as described in Example 1 to obtain protein quantification samples of the glucoamylase enzyme of interest. Protein quantification was performed as described in Example 1.

[0180] Example 5 Evaluation of the enzymatic activity of additional Mucorales clade glucoamylase variants in saccharification-related assays For activity assays, samples were normalized to a concentration of 80 ppm in 20 mM sodium acetate buffer pH 4.5 containing 0.005% (v / v) Tween-80 for glycation and conversion assays. 10 ppm GA solutions were prepared for all colorimetric ABTS formulation assays. A sample of the parent molecule (SEQ ID NO: 34) was included in the assay plate for comparison. SEQ ID NO: 34 is equivalent to SEQ ID NO: 4 (SvaGA1) with Ala at position 66 and Pro at position 102.

[0181] The saccharification performance of the variant and parent molecule (SEQ ID NO: 34) was evaluated at pH 4.5 and 62°C after 48 hours of incubation with Maltosweet G 120 (DE=11-14) as substrate purchased from Tate & Lyle. The starting DS content of the saccharification mixture was 34%. The glucoamylase performance was tested at a dosage of 29 μg / dsg. For this evaluation, pullulanase OPTIMAX™ L 2500 was dosed at 0.454 ASPU / DSg and alpha-amylase GC626™ was dosed at 0.088 SSU / DSg per incubation. Reactions consisting of 70 μl maltodextrin and 12 μl saccharification mixture (glucoamylase, alpha-amylase, and pullulanase) were incubated in a 384-well MTP at pH 4.5, 62° C. for 48 h with shaking at 900 rpm. All reactions were quenched by addition of 5 mM H2SO4 in a ratio of 1:33 saccharification syrup to acid solution. Quenched aliquots were analyzed by HPLC for product formation using an Agilent 1260 series system equipped with dual Phenomenex Rezex-RFQ Fast Acid H+ (8%) 100×7.8 mm columns (cat# 00D-0223-K0) run at 80° C. for 5.3 min. For this purpose, 5 μL of sample was injected onto the column and carbohydrates were separated by an isocratic gradient of 5 mM H2SO4 as mobile phase at a flow rate of 1.0 mL / min. Oligosaccharide products were detected using a refractive index detector. Standards were run to determine the elution time of each degree of polymerization (DP(n)) of saccharides of interest (n represents the number of saccharides, e.g., DP3+, DP3, DP2, and DP1). The total integrated peak area of ​​the identified glycation products was used to calculate the relative content of each (oligo)saccharide. The improvement in glycation yield of the variants relative to the parent is reported in Table 3 as glucose %. A value of at least 0.3% increase in glucose was used as the selection cutoff for mutations with a positive effect on GA performance of glycation under the given conditions.

[0182] Determination of specific activity using colorimetric readout The specific activity of glucoamylase variants was determined based on the rate of glucose release from selected substrates 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).

[0183] Aliquots of 10 ppm glucoamylase culture samples were combined in 384-well MTPs with 50 μl of freshly prepared Reagent A solution containing 25 mM sodium acetate buffer (pH 4.5) (final concentrations were 25 U / mL HRP (Sigma-Aldrich, Prod.nr: P8375), 62 U / mL GOX (Sigma-Aldrich, Prod.nr: G7141), and 0.01% Tween-80). The final enzyme concentrations in the various substrate assays were: 1.67 ppm in the maltose assay, 0.43 ppm in the maltodextrin assay, 1.43 ppm in the panose assay, 0.72 ppm in the pullulan assay, and 0.32 ppm in the soluble starch assay. To start the reaction, 40 μl of substrate-containing reagent B was added, consisting of 3.2 mg / mL ABTS (Sigma-Aldrich, Prod.nr: A1888), 25 mM sodium acetate buffer (pH 4.5), and 0.01% Tween-80. The final substrate concentrations in the different assays were: 4.5 mM maltose (Sigma-Aldrich, Prod.nr: 47288), 15 mM panose (Megazymes, O-PAN), 1.5% (w / v) preboiled pullulan (Sigma-Aldrich, Prod.nr: P4516), 0.5% (w / v) preboiled soluble starch (Sigma-Aldrich, Prod.nr: S9765), and 0.12% (w / v) maltodextrin (Sigma-Aldrich, Prod.nr: 31410). The reagents were mixed vigorously and absorbance kinetics were immediately measured at 405 nm at 17 second intervals for 3.5 minutes at room temperature using a Molecular Devices plate reader. The increase in absorbance rate at 405 nm is proportional to the hydrolytic activity of the enzyme. Reactions were run in triplicate and the Vo of the samples was determined using linear regression. The data thus generated was used to calculate the performance of each sample on each substrate. The performance of each variant was calculated by dividing the Vo of the sample by the Vo of the parent GA. This ratio was defined as the performance index (PI).A PI value equal to or 1.1 greater than the PI value of the parent GA was used as the cutoff for selection of mutations with beneficial effects on the specific activity of the molecule. The results are shown in Table 3.

[0184] Evaluation of thermostability of glucoamylase variants Glucoamylase variant proteins normalized to 10 ppm were incubated in a PCR MTP at 58° C. for 30 min in a Mastercycler Pro 384 (Eppendorf). After incubation, the MTP was cooled to room temperature and used as described above to determine the specific activity on the maltodextrin-ABTS substrate. The percent (%) residual activity (activity remaining after heat treatment) of the glucoamylase variants was determined by dividing the activity of the heat-incubated sample by the activity of the non-incubated sample. An improvement in thermostability of 3% over that of the parent enzyme was used as a cutoff in selecting mutations with beneficial effects on the thermostability of the molecule. The results are shown in Table 3.

[0185] Evaluation of conversion reactions of glucoamylase variants The glucose condensation activity (conversion activity) of each GA variant was tested by combining 70 μl of 45% (w / v) glucose in 20 mM sodium acetate buffer pH 4.5 with 10 μl of enzyme sample in a 384-well MTP and incubating with shaking at 50° C. for 66 hours. The reaction was quenched by mixing 3 μl of the reaction cocktail with 97 μl of 5 mM H2SO4. The reaction products (glucose and condensation products, e.g., DP2, DP3, and DP3+) were determined and quantified by HPLC analysis as described above.

[0186] The performance of the GA variants was compared to that of the parent molecule (SEQ ID NO: 34). To determine the effect of amino acid substitutions on the GA properties, the reduction in conversion yield (DP2), expressed as % condensate, was determined. The results are shown in Table 3. A value of 0.1% or more reduction in DP2 is considered a positive effect on conversion under the given conditions.

[0187] Note that for the conversion reactions and for the saccharification yields, values ​​are calculated as the percent yield difference (e.g., variant x confers a 0.4% yield improvement in saccharification yield as the yield is 95.2% DP1 versus 94.8% DP1 for the parent GA). [Table 3-1] [Table 3-2]

[0188] All SvaGa1 variants listed in Table 3 showed significantly improved performance when compared to the parent molecule (SvaGA1+66A / 102P) in at least one parameter related to GA performance.

[0189] Example 6 Effect of N-glycosylation on variants of Saksenaea vasiformis B4078 glucoamylase To investigate the effect of N-glycosylation on the performance of glucoamylases SvaGA1v2 (SEQ ID NO: 3) and SvaGA1v3 (SEQ ID NO: 34), several variant sequences were generated using molecular biology techniques known in the art. Amino acid substitutions with the potential to generate new N-glycosylation sites were evaluated in variants corresponding to SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, and 45.

[0190] 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. 20110020899). All plasmids were transformed into the appropriate 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.

[0191] Fungal cell cultures were grown in defined medium as described by Lv et al. ((2012) 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 a 10K MWCO. The purified samples were approximately 99% pure (by SDS-PAGE analysis) and were stored in 40% glycerol at -80°C until use.

[0192] Evaluation of the thermostability of glucoamylase. The thermostability of the N-glycosylation variants listed in Table 4 was compared to pre-incubation of enzyme samples (20 ppm) at 55°C for 40 min. Pre-incubation at 4°C for 40 min was included and set as 100% activity for each glucoamylase sample. The residual activity of glucoamylase after pre-incubation 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). Substrate solution was prepared by mixing 9 mL of soluble starch (1% in water, w / w) and 1 mL of 0.5 M sodium acetate buffer, pH 4.5 in a 15 mL conical tube. A coupled enzyme (GOX / HRP) solution with ABTS (final concentrations were 2.74 mg / mL ABTS, 0.1 U / mL HRP, and 1 U / mL GOX) was prepared in 50 mM sodium acetate buffer (pH 5.0). Preincubated glucoamylase samples (10 μL) were transferred into a new microtiter plate (Corning 3641) containing 90 μL of substrate solution preincubated at 50° C. for 5 min at 600 rpm. The reaction was run for 10 min at 50° C. with shaking (650 rpm) in a thermomixer (Eppendorf), and then 10 μL of the reaction mixture was quickly transferred to a new microtiter plate (Corning 3641) before adding 90 μL of ABTS / GOX / HRP solution. The absorbance at 405 nm was immediately measured at 11 s intervals for 5 min using a SoftMax Pro plate reader (Molecular Device). The reaction velocity output, Vo, was used to indicate glucoamylase activity.

[0193] Using the methods described above, the residual activity of N-glycosylation variants SvaGA1v2_D81N_K83T (SEQ ID NO: 38), SvaGA1v2_A153T (SEQ ID NO: 39), SvaGA1v2_S370N_A372S (SEQ ID NO: 40), SvaGA1v2_D81N_K83T_A153T (SEQ ID NO: 41), SvaGA1v3_D81N_K83T (SEQ ID NO: 42), SvaGA1v3_A153T (SEQ ID NO: 43), SvaGA1v3_S370N_A372S (SEQ ID NO: 44), and SvaGA1v3_D81N_K83T_A153T (SEQ ID NO: 45) was determined in comparison to the respective parent sequences SvaGA1v2 (SEQ ID NO: 3) and SvaGA1v3 (SEQ ID NO: 34). Table 4 shows the results of a thermostability comparison between variants designed to evaluate the effect of glycosylation on the Saksenaea vasiformis B4078 glucoamylase backbone. The GA variants were pre-incubated at 55° C. for 40 min, and then the residual activity was measured at 50° C. for 10 min at pH 4.5. [Table 4]

[0194] As shown in Table 4, the N-glycosylation variants, especially the variant with the substitution D81N_K83T_A153T, showed higher residual activity than their parent molecules. SvaGA1v2_D81N_K83T_A153T retained 33% of its activity, while the SvaGA1V2 parent retained 21%. SvaGA1v3_D81N_K83T_A153T surpassed its parent molecule, maintaining 75% residual activity versus 66% for the parent SvaGA1v3.

[0195] Example 7 Structural elements of Mucorales clade glucoamylases and variants It was observed that substitution of the Asn glycosylation site at position 75 causes a decrease in catalytic activity. The variant SvaGA1v2 N75D shows approximately a 50% decrease in activity compared to the parent SvaGA1v2 protein when tested at 55° C. for 40 min. At the time of submission, existing glucoamylase structures in the Protein Data Bank (worldwideweb.rcsb.org) did not contain an N-glycosylation site at Asn75 or a structurally equivalent position. This glycosylation site is concentrated in the Mucorales clade of glucoamylases described in Example 3. To identify additional glucoamylases with stable interactions, the nr database of non-redundant protein sequences (National Center for Biotechnology Information) was searched with the SvaGA1v2 (SEQ ID NO: 3) sequence as a query, all using default BLAST parameters. The top 500 sequences, containing sequences from various fungal and bacterial phyla, were aligned with MOE 2019.0102 (Chemical Computing Group, Montreal, Canada). Sequences containing the N-glycosylation motif NX-(S / T) (Schwarz & Aebi, (2011) Current Opinion in Structural Biology. 21:576-582.) at a position structurally equivalent to Asn94 in SvaGA1v2 (SEQ ID NO: 3) were identified. The resulting source organisms included 50 species of fungi from the phylum Mucoromycota and only a single bacterial species, one other type of organism.

[0196] To understand the structural basis of the improved activity from glycosylation at Asn 75, the crystallographic structure was determined. A variant of glucoamylase from Saksenaea vasiformis (SvaGA1v2) (SEQ ID NO: 3), described in WO 2011 / 063308 and WO 2016 / 138315, was cloned and expressed using standard methods. The fermentation broth was concentrated by a VivaFlow 200 ultrafiltration device (Sartorius Stedim, Goettingen, Germany). After adding (NH4)2SO4 to a final concentration of 1M, the concentrate was loaded onto a HiPrep™ Phenyl FF 16 / 10 column (GE Healthcare, Pittsburgh, USA) pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.0) supplemented with 1 M (NH4)2SO4) (buffer A). After sample loading, the column was washed with Buffer A until the UV (A280) baseline was stable. Protein elution was started with a gradient from 100% Buffer A to 100% 20 mM sodium phosphate, pH 7.0 (Buffer B) in 20 min at a flow rate of 5 mL / min. Fractions containing the target protein were pooled, concentrated, and buffer exchanged into Buffer B via a 10 kDa Amicon Ultra-15 device (Merck Millipore Ltd., Darmstadt, Germany).

[0197] Crystals were grown by the hanging drop method using a stock solution of 2.13 M ammonium sulfate, 0.1 M sodium acetate trihydrate, pH 4.3. Drops contained 2 μl of 11.5 mg / mL protein and 2 μl of stock solution. Cryo-protection included 17% total glycerol in the stock solution.

[0198] Native diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) at baseline 9-2 using the BLU-ICE data collection environment (Hypertext Transfer Protocol Secure: / / pubmed.ncbi.nlm.nih.gov / 12409628 / ) implemented by Accelero Biostructures, Inc., California. Data sets were collected at 100K using a Pilatus 6M detector (Dectris AG, Switzerland). Data were processed to 0.95 Å resolution using XDS (Hypertext Transfer Protocol Secure: / / pubmed.ncbi.nlm.nih.gov / 20124692 / ).

[0199] Molecular replacement was performed with Phaser (McCoy et al., J. Appl. Cryst. (2007). 40, 658-674) using a homology model as the search model. The homology model was constructed with MOE (Chemical Computing Group, Montreal, Canada) using default parameters with glucoamylase from Saccharomycopsis fibuligera, PDB code 2FBA (Sevcik, et al., (2006) FEBS J 273: 2161-2171). Nineteen rounds of rebuilding and refinement with REFMAC 5.8 (Vagin, et al., (2004) Acta Crystallogr. D60:2284-2295) and Coot (Emsley & Cowtan (2004). Acta Crystallogr. D60,2126-2132) were performed with isotropic temperature factors, followed by a final three rounds of refinement with anisotropic temperature factors. The resolution range for the refinements was 0.97 Å to 40.70 Å. After the final refinement, anisotropic temperature factors, Rwork=0.12 and Rfree=0.14, were used. For the highest resolution shell (0.97 Å to 0.995 Å), Rwork=0.29 and Rfree=0.31.

[0200] The X-ray diffraction structure determined for SvaGA1v2 (SEQ ID NO:3) showed clear electron density for an N-glycosylation site at Asn75. At the time of deposition, existing glucoamylase structures in the Protein Data Bank (worldwideweb.rcsb.org) did not contain an N-glycosylation site at Asn75 or a structurally equivalent position. The electron density for the Asn75 glycan is shown in FIG. 3. The structure shows an extensive network of water-binding contacts of the glycan to the protein, bridging the four helices to the protein surface (FIG. 4). Table 5 identifies the atoms and contact distances. Without wishing to be bound by theory, it is proposed that this extensive hydrogen-bonding network between the Asn75 glycan and the protein chain stabilizes the four protein helices, which in turn provides a structural anchor for the substrate binding site and interactions that stably position the substrate within the catalytic active site. [Table 5]

[0201] The structure of SvaGA1v2 (SEQ ID NO:3) also suggests a rationale for the increased performance of the enzyme variants described in Examples 2 and 5. In particular, the helix spanning residues 60-72 contains the conserved active site residue Asp62, which is predicted to stabilize the developing positive charge in the catalytic cycle by well-studied glucoamylases (Aleshin et al., (1996) Biochemistry 35:8319-8328). Mutations to residues in this helix, for example at positions 66, 67, and 69, to improve performance are shown in Examples 2 and 5. Without wishing to be bound by theory, these mutations may alter the position of the helix in the active site, and thus tune the exact placement of Asp62 for improved stabilization of the developing positive charge. The helix, the conserved Asp62 side chain, and the sites of the performance-enhancing mutations are shown in FIG. 5.

[0202] Additional performance-enhancing mutations are located in the substrate-binding loop, including residues 100-129. Mutations at positions 102, 119, and 121, in particular, that improve enzyme performance are shown in Examples 2 and 5. The loop, with these residues on residues 100-129, forms a significant portion of the surface area of ​​the active site. The loop is directly adjacent to a helix with a conserved Asp62 side chain, which further makes hydrogen bond contacts to Glu184, a predicted general acid in the glucoamylase catalytic mechanism. Without wishing to be bound by theory, the changes in the conformation of the loop caused by mutations at these positions may be responsible for changing the exact positioning of the general acid relative to the substrate, improving performance of use. Figure 6 shows the location of the loop, predicted general acid residues, and the location of the performance-enhancing mutations.

[0203] Another set of performance-enhancing mutations resides on the four-helix bundle contacted by the Asn75 glycan. Mutations at positions 143, 156, 164, 192, and 233 were responsible for increased performance, as shown in Example 5. These mutations are shown in Figure 7, highlighting in dark grey the four-helix bundle associated with the glycan chain of the Asn75 residue. The four-helix bundle provides a solid structural core that anchors the substrate-binding loop in the active site.

Claims

1. Positions 20, 21, 23, 37, 49, 51, 52, 66, 67, 69, 73, 77, 79, 80, 81, 84, 92, 94, 102, 119, 121, 134, 140, 141, 143, 156, 157, 158, 164, 165, 166, 172, 192, 203, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 238, 243, 252, 253, 27 4, 278, 281, 290, 302, 310, 321, 338, 341, 350, 351, 352, 354, 370, 390, 403, 404, 405, 416, 418, 422, 430, 434, 440, 441, 444, 445, and / or 449, and / or glucoamylase variants or fragments thereof comprising one or more amino acid substitutions at residue positions corresponding to the equivalent positions in the parent glucoamylase.

2. 2. The glucoamylase variant of claim 1, wherein the equivalent positions are determined by sequence identity, and the parent glucoamylase has at least 80% sequence identity and less than 100% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37.

3. The one or more amino acid substitutions are X020A / E / F / G / P; X021M / S / T / W; X023L / M; / M;X069A / C / K;X073N / P;X077M / P / S;X079C / R / T;X080H / K / N; L / M / P / V; X134Q / S / W; X140C / I / Q; X141F / K; X143G; 2F / R; X203C / M / Q / W / Y; X210A / F / G / I / L / M / N / W; X213H / R; ;X222C / M / V;X233M / PT / Y;X235F / Y;X236A / Q / S;G238H / M / N / S / T / V; ;F290M / V;X302F / H / K / M / P / Q / S / T / V / W;X310T / V / Y;X321D;X338I; 2. The glucoamylase variant of claim 1, comprising one or more of the following amino acids: X390D / E / L; X403G / K / R; X404K / M; X405Q / S / Y; X416C / Y; X418E / W; X422A / F / V; X430A / Q; X434S; X440G / H / L; X441L / S / W; X444L / P / S; X445M / Y; and / or X449L, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO: 4 and / or the equivalent position in a parent glucoamylase or fragment thereof.

4. The one or more amino acid substitutions are S020A / E / F / G / P; K021M / S / T / W; E023L / M; E037C; S049W; A051K / L / V / Y; G052F / N / P; X66A / C / F / M / P / T / W; S067A / C / M; V069A / C / K; K073N / P; T077M / P / S; A079C / R / T; G080H / K / N; D081N / S; I084L / V; V092C / I / M; F094A / G / Y; X102G; S119A / D / G; T121G / L / M / P / V; E134Q / S / W; M140C / I / Q; L141F / K; A143G; F156C / P; T157A / I; N158A / Y; I164L / T; Y165I / T / W; K166A / F / G / H / R; V172G / L; Y192F / R; D203C / M / Q / W / Y; R210A / F / G / I / L / M / N / W; D213H / R; N214A / C / E / G / L / Y; S215C / D / F / H / R / V / W;A218F / A / H / K / Q / V / Y;S221M / R / T;G222C / M / V;S233M / PT / Y;W235F / Y;D236A / Q / S;G238H / M / N / S / T / V;T243A / P / S;V2 52F / H; E253K; G274A / D / K; P278A; E281D / P; F290M / V; N302F / H / K / M / P / Q / S / T / V / W; N310T / V / Y; N321D; F338I; L341M / T; K3 4. The glucoamylase variant of claim 3, comprising one or more of the following: 50C / E / I; N351E / V; T352D / N / S; V354L / M; S370M / N / R; S390D / E / L; Q403G / K / R; Y404K / M; H405Q / S / Y; F416C / Y; R418E / W; Y422A / F / V; T430A / Q; A434S; A440G / H / L; Q441L / S / W; A444L / P / S; G445M / Y; and / or F449L.

5. The variants are located at positions 20, 21, 23, 37, 51, 52, 66, 67, 69, 73, 77, 79, 80, 81, 84, 92, 94, 102, 119, 121, 134, 140, 141, 156, 157, 158, 164, 165, 166, 172, 192, 203, 210, 213, 214, 215, 218, 221, 222, 233, 235, 236, 238, 243, 252, 253, 274, 278, 281, 290, 302, 310, 341, 350, 351 , 352, 354, 370, 390, 403, 404, 405, 416, 418, 422, 430, 440, 441, 444, and / or 445, and / or equivalent positions within the parent glucoamylase or fragment thereof, wherein the variant glucoamylase, relative to the parent glucoamylase lacking one or more of said substitutions, comprises one or more amino acid substitutions at residue positions corresponding to 403, 404, 405, 416, 418, 422, 430, 440, 441, 444, and / or 445, and / or equivalent positions within the parent glucoamylase or fragment thereof, wherein the variant glucoamylase exhibits, relative to the parent glucoamylase lacking one or more of said substitutions: i) improved hydrolysis of maltose; ii) improved hydrolysis of panose; iii) improved hydrolysis of pullulan; iv) improved hydrolysis of soluble starch; v) improved hydrolysis of maltodextrin; 2. The glucoamylase variant of claim 1, which exhibits two or more of: vi) improved thermostability; vii) less conversion to sugars with a DP of 2 or more; and / or viii) improved saccharification yield of glucose.

6. The one or more amino acid substitutions may be X020A / P; X021M / S / T / W; X023L / M; X037C; X051K / L / V / Y; X052N; X066A / C / F / M / W; X067A / C; X069C; X073N / P; X077M / P / S; X079R / T; X080H / K; X081N / S; X084L / V; X092C / M; X094A / G; X102G; X119A / D / G; X 121G / L / M / P; X134Q / S; X140C / I / Q; X141F; X156C / P; X157I; G; X192F; X203C / W; X210L / M / W; X213H / R; X214A / E / Y; X233M / P / T / Y; X235F / Y; X236A / Q; X238H / S; X243A; ;X302H / K / P / Q / S / V / W;X310T / Y;X341M / T;X350C / E / I;X351E / V;X352D / N / S; 6. The glucoamylase variant of claim 5, comprising one or more of the following amino acids: X403G / K / R; X404K / M; X405Q / S / Y; X416C / Y; X418E / W; X422A; X430A; X440G / H; X441L; X444L / P; and / or X445M / Y, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO: 4 and / or the equivalent position in a parent glucoamylase or fragment thereof.

7. The one or more amino acid substitutions are S020A / E / P; K021M / S / T / W; E023L / M; E037C; A051K / L / V / Y; G052N; V066A / C / F / M / W; S067A / C; V069C; K073N / P; T077M / P / S; A079R / T; G080H / K; D081N / S; I084L / V; V092C / M; F094A / G; P 102G; S119A / D / G; T121G / L / M / P; E134Q / S; M140C / I / Q; L141F; F156C / P; T157I; N158A / Y; I164L / T; Y 165I / T / W; K166G / F / R; V172G; Y192F; D203C / W; R210L / M / W; D213H / R; N214A / E / Y; S215C / D / F / R; S21 8A / K / Q / V / Y; S221M / R / T; G222C / M / V; S233M / P / T / Y; W235F / Y; D236A / Q; G238H / S; T243A; V252F; E25 3K; G274A / D / K; P278A; E281D; F290M / V; F290V; N302H / K / P / Q / S / V / W; N310T / Y; L341M / T; K350C / E / I 7. The glucoamylase variant of claim 6, comprising one or more of: N351E / V; T352D / N / S; V354L / M; S370M; S390D / E / L; Q403G / K / R; Y404K / M; H405Q / S / Y; F416C / Y; R418E / W; Y422A; T430A; A440G / H; Q441L; A444L / P; and / or G445M / Y.

8. The variants are: a) X236S and X281D; b) X215R and X441W; c) X321D and X434S; d) X143G and X434S; e) X079C and X143G; f) X350T and X434S; g) X351E and X403K; h) X052N and X084L; i) X243P and X290V; j) X290V and X350E; k) X302K and X441W; l) X156C and X404K; m) X067M and X404K; n) X052N and X141F; o) X052N and X351E; p) X233M and X445Y; q) X066C and X233M; r) X218H and X290V; s) X067M and X302H; t) X066C and X119A; u) X243P and X445Y; v) X192F and X243P; w) X156C and X243P; x) X023L and X066C; y) X023L-X 119A; z) X020E and X192F; aa) X192F and X310V; bb) X023M and X302H; cc) X192F and X416Y; dd) X119A and X302H; ee) X235Y and X416Y; ff) X052N and X404K; gg) X023M and X449Y; hh) X158A and X172L; ii) X172L and X290V; jj) X023M and X210L; kk) X210L and 11) the glucoamylase variant of claim 1, comprising two or more amino acid substitutions including: 11) X157I and X281D; mm) X164T and X215R; nn) X140C and X422V; oo) X119A and X302K; or pp) X052N and X416Y, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO: 4 and / or the equivalent position in a parent glucoamylase or fragment thereof.

9. The two or more amino acid substitutions are: a) D236S and E281D; b) S215R and Q441W; c) N321D and A434S; d) A143G and A434S; e) A079C and A143G; f) K350T and A434S; g) N351E and Q403K; h) G052N and I084L; i) T243P and F290V; j) F290V and K350E; k) N302K and Q441W; l) F156C and Y404K; m) S067M and Y404K; n) G052N and L141F; o) G052N and N351E; p) S233M and G445Y; q) A066C and S233M; r) S218H and F290V; s) S067M and N302H; t) A066C and S119A; u) T243P and G445Y; v) Y192F and T243P ; w) F156C and T243P; x) E023L and A066C; y) E023L and S119A; z) S020E and Y192F; aa) Y192F and N310V; bb) E023M and N302H; cc) Y192F and F416Y; dd) S119A and N302H; ee) W235Y and F416Y; ff) G052N and Y404K; gg) E023M and F449Y 9. The glucoamylase variant of claim 8, comprising: hh) N158A and V172L; ii) V172L and F290V; jj) E023M and R210L; kk) R210L and F449Y; ll) T157I and E281D; mm) I164T and S215R; nn) M140C and Y422V; oo) S119A and N302K; or pp) G052N and F416Y.

10. The variants are: a) X141F, X281D, and X441W; b) X143G, X321D, and X434S; c) X321D, X350T, and X434S; d) X067M, X158A, and X281D; e) X156C, X192F, and X403K; f) X052N, X140C, and X422V; g) X066C, X119A, and X164T; h) X066C, X233M, and X445Y; i) X156C, X192F, and X243P; j) X023L, X066C, and X119A; k) X023M, X119A, and X404K; 2. The glucoamylase variant of claim 1, comprising three or more amino acid substitutions including: X310V, X416Y, and X445Y; m) X158A, X221R, and X290V; n) X023M, X052N, and X404K; o) X081S, X157I, and X236S; p) X243P, X302K, and X416Y; q) X140C, X302K, and X422V; or r) X052N, X416Y, and X445Y, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO: 4 and / or the equivalent position in a parent glucoamylase or fragment thereof.

11. 2. The glucoamylase variant of claim 1, further comprising an amino acid substitution at one or more residue positions corresponding to positions 66 and / or 102 of SEQ ID NO: 4, and / or equivalent positions in the parent glucoamylase.

12. 2. The glucoamylase variant of claim 1, comprising an N-linked glycosylation at position N075 of SEQ ID NO: 4 and / or an equivalent position in the parent glucoamylase.

13. 2. The glucoamylase variant of claim 1, further comprising one or more amino acid substitutions at residue positions corresponding to positions 81, 83, 153, 370, or 372 of SEQ ID NO: 4 and / or equivalent positions within the parent glucoamylase, wherein the variant glucoamylase exhibits increased glycosylation and increased thermostability compared to the parent glucoamylase lacking one or more of the substitutions.

14. An enzyme composition comprising the glucoamylase variant according to any one of claims 1 to 13.

15. 15. The enzyme composition of claim 14 for use in a starch conversion process.

16. 14. A method for saccharifying a composition comprising starch to produce a composition comprising glucose, the method comprising: a) contacting the starch composition with a glucoamylase variant according to any one of claims 1 to 13; and b) saccharifying the starch composition to produce the glucose composition.

17. 17. The method of claim 16, wherein the composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch.

18. 17. The method of claim 16, further comprising: e) fermenting the glucose composition to produce a fermentation product.

19. 19. The method of claim 18, wherein the fermentation product is alcohol.

20. 17. The method of claim 16, wherein the fermentation is a simultaneous saccharification and fermentation (SSF) reaction.

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