Glucoamylase variants and methods of use thereof

Abstract

Described herein, inter alia, are glucoamylase variants and methods for their use in saccharifying starch substrates. In addition, the present disclosure also relates to a process for producing a fermentation product using the glucoamylase variant and a method for increasing starch digestibility in an animal using the glucoamylase variant and a method for producing a fermented beverage using the glucoamylase variant.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to international patent application No. PCT / CN2021 / 124148 filed on October 15, 2021, and the disclosure of that international patent application is incorporated herein by reference in its entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (NB42043-WO-PCT2_sequencelisting.xml; size: 62,000 bytes; and creation date: October 11, 2022) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to compositions comprising glucoamylase variants and methods of using these compositions to saccharify starch substrates and methods of using these compositions to produce fermentation products. Background Art

[0006] 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. Glucoamylase is produced by several filamentous fungi and yeasts.

[0007] The main application of glucoamylase is to saccharify partially processed starch / dextrin into glucose, which is an essential substrate for many fermentation processes. Glucose can then be converted directly or indirectly into fermentation products using fermentation organisms. Examples of commercial fermentation products include alcohols (e.g., ethanol, methanol, butanol, 1,3-propylene glycol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, 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.

[0008] The end product may also be a syrup. For example, the end product may be glucose, but may also be converted, for example, by glucose isomerase to fructose or a mixture consisting of glucose and fructose in almost equal amounts. This mixture, or a mixture further enriched in fructose, is the most commonly used commercial high fructose corn syrup (HFCS) worldwide.

[0009] Although it is reported that a variety of microorganisms produce glucoamylases (because they secrete large amounts of enzymes outside the cell), glucoamylases for commercial purposes are traditionally produced using filamentous fungi. However, commercially used fungal glucoamylases have certain limitations, such as slow catalytic activity or lack of stability, which increases process costs.

[0010] There remains a need for new glucoamylases and glucoamylase variants to improve saccharification efficiency and provide high yield fermentation products. Summary of the invention

[0011] The present disclosure relates to a recombinant host cell, a composition comprising a glucoamylase, and a method for saccharifying a starch substrate using the glucoamylase. In addition, the present disclosure also relates to a process for producing a fermentation product, a method for increasing starch digestibility in animals, and a method for producing a fermented beverage.

[0012] Thus, in some aspects, provided herein is a glucoamylase variant or fragment thereof comprising one or more amino acid substitutions at residue positions corresponding to 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, 234, 236, 237, 238, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265 3, 235, 236, 238, 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 the equivalent position in a parent glucoamylase. In some embodiments, the equivalent position is 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, the equivalent position is determined by sequence identity and wherein 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 glucoamylase.In some embodiments of any of the embodiments disclosed herein, the one or more amino acid substitutions include one or more of the following: 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, X085 092C / I / M, X094A / G / Y, X102G, X119A / D / G, X121G / L / M / P / V, X134Q / S / W, 8A / Y, X164L / T, X165I / T / W, X166A / F / G / H / R, X172G / L, X192F / R, E / G / L / T / Y, X215C / D / F / H / R / V / W, X218F / A / H / K / Q / V / Y, X221M / R / T, / N / S / T / V, X243A / P / S, X252F / H / M / T, X253K / V, X274A / D / K, X278A, / 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.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, 8A / 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, N214 A / 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, G 238H / 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, N310 T / V / Y, N321D, 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, and / or F449L.In some embodiments of any of the embodiments disclosed herein, the variant comprises one or more amino acid substitutions at residue positions corresponding to: SEQ 405, 418, 440, 441 and / or 444 of ID NO:4 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved maltose hydrolysis compared to a parent glucoamylase lacking one or more of these substitutions, or a fragment thereof. In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, X233 M / 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, 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 include one or more of the following: 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 variant comprises one or more amino acid substitutions at residue positions corresponding to the following positions: SEQ ID NO: 4 position 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, 2 74, 281, 290, 302, 321, 341, 350, 351, 352, 370, 390, 403, 404, 405, 418, 430, 440, 444 and / or 445 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved panose hydrolysis compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, X214C, 4A / E, X215D, X218Q / 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, wherein X is the residue corresponding to SEQ 40C, F094Y, X102G, S119G, T121M / L / G / P, M140C / Q / I, L141F, A143G, I164L, Y165W / T, K166R, V172G, R210L, D213 H, 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 comprises one or more amino acid substitutions at residue positions corresponding to the following positions: 40, 416, 418, 434, 440 and / or 445 of NO:4 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved pullulan hydrolysis compared to a parent glucoamylase lacking one or more of these substitutions, or a fragment thereof. In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, 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 include one or more of the following: 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, S215V / 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 variant comprises one or more amino acid substitutions at residue positions corresponding to the following positions: SEQ ID NO: 4 position 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 and / or the equivalent position in a parent glucoamylase or a fragment thereof, and wherein the variant glucoamylase exhibits a higher performance index (PI) for saccharifying soluble starch than a parent glucoamylase lacking one or more of these substitutions.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, X218 Q / F / A, X221R, X222M / 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, X404I / M / K, X405S, X416C / Y, X418E / W, X422A, X440G, X441L, X444S / P, and / or X445M, wherein X is the residue corresponding to SEQ ID NO: 4 and / or any amino acid at the equivalent position in the parent glucoamylase or its fragment. In some embodiments, the one or more amino acid substitutions include one or more 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.In some embodiments of any of the embodiments disclosed herein, the variant comprises one or more amino acid substitutions at 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 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved maltodextrin hydrolysis compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions. In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, 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, wherein X is the residue corresponding to SEQ Any amino acid at the equivalent position in ID NO: 4 and / or the equivalent position in the parent glucoamylase or a fragment thereof.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, N21 4A / E, S215D / C / F, S218V / Y / Q, S221R / T, G222M / C, S233M / P / Y, W235Y, D236 Q / A, V252F, G274K / A / D, E281D, F290M / V, N302P / K / S / V / Q / W, N310T / Y, N321 D, 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. In some embodiments of any of the embodiments disclosed herein, the variant comprises 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 equivalent positions in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved thermostability compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, wherein X is the residue corresponding to SEQ ID NO: Any amino acid at the equivalent position in NO: 4 and / or the equivalent position in the parent glucoamylase or a fragment thereof. In some embodiments, the one or more amino acid substitutions include one or more of the following: 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 variant comprises one or more amino acid substitutions at residue positions 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 an equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits less reversion to sugars having a degree of polymerization (DP) greater than or equal to 2 as compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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 , X235A, 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, 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 include one or more of the following: 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, S2 33T / P, W235F, D236A, G238H / S / N, T243P / S, E253K, G274K / A, N302H / F / P / M / Q / T, F338I, Q403G, H4 05Q / 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 variant comprises 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 equivalent positions in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved saccharification yield of glucose compared to a parent glucoamylase lacking one or more of these substitutions.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, X

[0147] In some embodiments, the present invention relates to an amino acid sequence of the present invention that is a member of the group consisting of: 214H / 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, wherein 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 include one or more of the following: 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, S218H / K / A, S221M / T, G222V, S233T, W235Y / F, D236S / A, G238T / V / M, T243A, E253K, E281P / D, F290 V, 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 variant comprises one or more amino acid substitutions at residue positions corresponding to the following positions: NO: 4 position 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, 4 04, 405, 416, 418, 422, 430, 440, 441, 444 and / or 445 and / or the equivalent position in a parent glucoamylase or a fragment thereof, and wherein the variant glucoamylase exhibits two or more of the following as compared to a parent glucoamylase lacking one or more of these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) improved soluble starch hydrolysis; v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with a DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.In some embodiments, the one or more amino acid substitutions include one or more of the following: 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, , X158A / Y, X164L / T, X165I / T / W, X166G / F / R, X172G, E / Y, X215C / D / F / R, X218A / K / Q / V / Y, X221M / R / T, X222C / M / V, 8H / S, X243A, X252F, X253K, X274A / D / K, X278A, X281D, X350C / E / I, X351E / V, X352D / N / S, X354L / M, 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, 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 include one or more of the following: 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, I084 L / V, V092C / M, F094A / G, P102G, S119A / D / G, T121G / L / M / P, E134Q / S, M140C / I / Q, L141F, F156C / P, T157I, N158A / Y, 1164L / 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, D236 A / Q, G238H / 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, and / or G445M / Y.In some embodiments of any of the embodiments disclosed herein, the variant comprises two or more amino acid substitutions including: 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 X2 90V, j) X290V and X350E, k) X302K and X441W, 1) 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) X243 P 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 X4 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 include 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 G4 45Y, 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, 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 variant comprises three or more amino acid substitutions including: 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) X1 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. In some embodiments, the three or more amino acid substitutions include 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, Y 192F 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, 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 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, wherein 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 five or more amino acid substitutions include 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 compared to a parent glucoamylase lacking these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or its fragments); v) improved hydrolysis of maltodextrin; vi) improved thermal stability; vii) less reversion to sugars with a DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose. In some embodiments of any of the embodiments disclosed herein, the variant comprises substitutions at residue positions corresponding to the following positions: positions a) 66, 67, and 69, b) 102, 119, and 121 of SEQ ID NO: 3, SEQ ID NO: 4, and / or c) 143, 156, 164, 192, and 233 and / or equivalent positions in a parent glucoamylase or its fragments.In some embodiments, the variant comprises amino acid substitutions comprising 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 an equivalent position in a parent glucoamylase. In some embodiments, the one or more amino acid substitutions include X066A / C / F / M / P / T / W and / or X102P / G, wherein 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 include 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 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 the following positions: positions 81, 83, 153, 370 or 372 of SEQ ID NO: 4 and / or the equivalent position in the parent glucoamylase, and wherein the variant glucoamylase exhibits increased glycosylation and increased thermal stability compared to the parent glucoamylase lacking one or more of these substitutions. In some embodiments, the one or more substitutions include one or more substitutions at positions D81N, K83T, A153T, S370N, and / or A372S.

[0013] In other aspects, provided herein is a polynucleotide encoding any of the glucoamylase variant polypeptides disclosed herein.

[0014] In another aspect, provided herein is a vector comprising any of the polynucleotides disclosed herein.

[0015] In other aspects, a host cell is provided herein, comprising any polynucleotide disclosed herein or any vector disclosed herein. In certain embodiments, the host cell is a bacterial or fungal host cell. In some embodiments of any embodiment disclosed herein, the host cell is an Aspergillus species, a Bacillus species, or a Trichoderma species, a Pichia species, a Myceliophthora species, or a Saccharomyces species.

[0016] In other aspects, the present invention provides 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 preparing a fermented beverage.

[0017] In still other aspects, the present invention provides a method for producing a variant glucoamylase in a host cell, the method comprising a) culturing a host cell transformed with any vector 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 or fungal host cell. In some embodiments of any of the embodiments disclosed herein, the host cell is an Aspergillus species, a Bacillus species, or a Trichoderma species, a Pichia species, a Myceliophthora species, or a Saccharomyces species.

[0018] In another aspect, the present invention provides a method for saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises: a) contacting the starch composition with any glucoamylase variant polypeptide 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 to the starch composition one or more of an additional glucoamylase, a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a protease, a pullulanase, a β-amylase, an additional α-amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, an oxidoreductase, an esterase, a transferase, a pectinase, an α-glucosidase, a β-glucosidase, a lyase, a hydrolase, or a combination thereof. In some embodiments of any of the embodiments disclosed herein, the fermentation is a simultaneous saccharification and fermentation (SSF) reaction.

[0019] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of an embodiment or aspect.

[0020] Throughout the specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 provides a multiple amino acid sequence alignment of Mucorales clade glucoamylases.

[0022] Figure 2 A phylogenetic tree of predicted mature Mucorales clade glucoamylases and other fungal glucoamylases is provided.

[0023] Figure 3 An image showing electron density (2fo-fc) at the Asn75 glycosylation site is provided based on the x-ray diffraction structure determined for SvaGA1v2.

[0024] Figure 4 The X-ray diffraction structure determined for SvaGA1v2 provides a picture of the interaction of the glycan chain at residue Asn75 with the protein. Hydrogen bonding interactions Indicated by dotted lines.

[0025] Figure 5 An image is provided based on the x-ray diffraction structure determined for SvaGA1v2 in which the helix containing the conserved Asp63 side chain and the performance improving mutations at positions 66, 67 and 69 are highlighted in dark grey.

[0026] Figure 6 An image is provided based on the x-ray diffraction structure determined for SvaGA1v2 in which the loop at residues 100-129, the expected general acid residues, the conserved Asp side chain, and the amino acid positions of the performance improving mutations are highlighted in black.

[0027] Figure 7 An image based on the x-ray diffraction structure determined for SvaGA1v2 is provided in which the four-helix bundle associated with the glycan chain at residue Asn75 is highlighted in dark grey, while the amino acid positions of several performance-improving mutations are shown as black spheres. The Asn75 glycan is shown as a stick model colored in black. DETAILED DESCRIPTION

[0028] The present disclosure relates to a recombinant host cell, a composition comprising a glucoamylase, and a method for saccharifying a starch substrate using the glucoamylase. In addition, the present disclosure also relates to a process for producing a fermentation product, a method for increasing starch digestibility in animals, and a method for producing a fermented beverage.

[0029] I. Definitions

[0030] Before describing the compositions and methods in detail, the following terms and abbreviations are defined.

[0031] Unless defined otherwise, all technical and scientific terms used have their ordinary meanings in the relevant scientific fields. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, John Wiley and Sons, New York (1994), and Hale and Markham, Harper Collins Dictionary of Biology, Harper Perennial, New York (1991) provide ordinary meanings for many of the terms describing the present invention.

[0032] 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.

[0033] As used herein, the term "glucoamylase variant" means 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 in a given parent glucoamylase amino acid sequence.

[0034] With respect to polypeptides such as glucoamylases, the term "wild type" refers to naturally occurring polypeptides that do not include artificial substitutions, insertions or deletions at one or more amino acid positions. However, in another embodiment, non-limiting examples of wild-type glucoamylases include SEQ ID NOs: 4-37.

[0035] With respect to polypeptides (e.g., glucoamylases), the terms "parent", "parental" or "reference" can refer to a wild-type polypeptide or can also refer to a polypeptide that has had one or more amino acid substitutions introduced therein, which is then used as a reference for comparing the performance properties of polypeptides that have had additional amino acid substitutions therein (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 other embodiments, the parent polypeptide is SEQ ID NO: 4-37.

[0036] The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein" and "peptide", and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". Conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences presented in the standard amino-terminal to-carboxyl-terminal orientation (i.e., N→C).

[0037] The term "mature polypeptide" is defined herein as a polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one aspect, the predicted mature polypeptide is SEQ ID NO: 4 based on analysis by SignalP software version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8: 785-786). In another aspect, the mature polypeptide comprises amino acid positions 20-468 of SEQ ID NO: 2. In another aspect, the mature polypeptide comprises amino acid positions 21-468 of SEQ ID NO: 2. In another aspect, the mature polypeptide comprises amino acid positions 22-468 of SEQ ID NO: 2. In another aspect, the mature polypeptide comprises amino acid positions 23-468 of SEQ ID NO: 2. In another aspect, the mature polypeptide comprises amino acid positions 24-468 ​​of SEQ ID NO: 2. In another aspect, the mature polypeptide comprises amino acid positions 25-468 of SEQ ID NO: 2.

[0038] A "signal sequence" or "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates secretion of the protein outside of the 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 a 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.

[0039] The terms "nucleic acid" or "polynucleotide" are used interchangeably to encompass DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single-stranded or double-stranded, and may be chemically modified. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the invention encompass nucleotide sequences encoding a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5'- to -3' orientation.

[0040] The term "coding sequence" means a polynucleotide sequence that directly specifies the amino acid sequence of a protein product. The boundaries of a coding sequence are usually determined by an open reading frame, which usually begins with an ATG start codon or an alternative start codon (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.

[0041] The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription of a mature spliced ​​mRNA molecule obtained from a eukaryotic cell. The cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to the mRNA that is processed through a series of steps before appearing as a mature spliced ​​mRNA. These steps include removing intron sequences through a process known as splicing. Therefore, the cDNA derived from the mRNA lacks any intron sequences.

[0042] "Synthetic" molecules are produced by in vitro chemical or enzymatic synthesis rather than by biological means.

[0043] A "host strain" or "host cell" is an organism into which an expression vector, bacteriophage, virus or other DNA construct has been introduced, including a polynucleotide encoding a polypeptide of interest (e.g., glucoamylase). Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by a cell.

[0044] As used herein, the term "glycosylation" refers to the attachment of polysaccharides to molecules such as proteins (such as variant glucoamylases). Glycosylation can be an enzymatic reaction. Attachment can be formed by covalent bonds. The phrase "highly glycosylated" refers to molecules (such as enzymes) being glycosylated at many sites and all or nearly all available glycosylation sites (e.g., N-linked glycosylation sites). Alternatively or in addition, the phrase "highly glycosylated" can refer to extensive glycolytic branches at all or substantially all N-linked glycosylation sites (such as, the size and quantity of the glycolytic parts associated with the glycosylation sites connected to a specific N). In some embodiments, engineered glucoamylase polypeptides are glycosylated at all or substantially all consensus sequence N-linked glycosylation sites (i.e., NXS / T consensus sequence N-linked glycosylation sites, wherein X is any amino acid except proline). Glucoamylases can have varying degrees of glycosylation. Such glycosylation is known to improve stability during storage and application. 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, wherein X is any amino acid except proline) can be introduced into a glucoamylase (e.g., a variant glucoamylase) to improve one or more properties of the glucoamylase, such as, but not limited to, improved thermostability, activity or saccharification yield.

[0045] As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide, or the carbohydrate portion of a glycoconjugate (such as a glycoprotein). Glycans can be homopolymers or heteropolymers of monosaccharide residues. They can be straight-chain or branched molecules.

[0046] The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. This process includes both transcription and translation.

[0047] The term "vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0048] "Expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which is operably linked to a suitable control sequence capable of achieving DNA expression in a suitable host. Such control sequences may include a promoter for achieving transcription, an optional operator sequence for controlling transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and a sequence for controlling transcription and translation termination.

[0049] The term "control sequence" is defined herein as including 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 each control sequence may be native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequence includes a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction sites that are conducive to connecting the control sequence to the coding region of the nucleotide sequence encoding the polypeptide, the control sequence may be provided with a plurality of linkers.

[0050] The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that the expression of the coding sequence is controlled by the regulatory sequence.

[0051] The term "sequence motif" is a broad and proven or hypothesized nucleotide or amino acid sequence pattern that has biological significance. In the present invention, the sequence motif is an amino acid sequence motif identified in Mucorales clade glucoamylases.

[0052] "Biologically active" refers to a sequence that possesses a specified biological activity, such as enzymatic activity.

[0053] The term "specific activity" refers to the number of moles of substrate that can be converted into product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is usually expressed as units (U) / mg protein.

[0054] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. In order to determine the percentage of identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the best comparison purpose (for example, a room can be introduced in the first amino acid sequence or nucleic acid sequence so as to be optimally aligned with the second amino acid or nucleic acid sequence). Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the amino acid residue or nucleotide identical to the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (that is, % identity = the number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. The determination of the percentage of identity between the two sequences can also be completed using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performed with the NBLAST nucleotide program parameter settings, e.g., score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches are performed with the XBLAST program parameter settings, e.g., score-50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gap BLAST as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 can be utilized. Alternatively, PSI-BLAST can be used to perform an iterated search to detect distant relationships between molecules (supra). When utilizing BLAST programs, Gap BLAST programs, and PSI-Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing 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 create multiple alignments of protein sequences is MUSCLE. The elements of the MUSCLE algorithm include the rapid distance estimation using k-mer counts, the progressive alignment using a new profile function described as a logarithmic expected score, and the refinement using a restricted segmentation that relies on a tree. This program is described in MUSCLE: multiple sequence alignment with high accuracy and high throughput [MUSCLE: multiple sequence alignment with high accuracy and high throughput] published by Robert C. Edgar (2004) in Nucleic Acids Res. [nucleic acid research] 32: 1792-1797. Can use techniques similar to those described above to determine the identity percentage between two sequences when allowing or not allowing gaps. When calculating the identity percentage, typically only exact matches are counted.

[0055] 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, in Bioinformatics Methods and Protocols, S. Misener and S.A. Krawetz, eds., pp. 185-219) with a glucoamylase, e.g., the glucoamylase of SEQ ID NO: 4.

[0056] As used herein, "equivalent positions" means positions common to both amino acid sequences based on alignment of the amino acid sequences of the parent glucoamylase and the glucoamylase variant and alignment of the three-dimensional structure of the parent glucoamylase with the three-dimensional structure of the variant glucoamylase in three-dimensional space.

[0057] As used herein, with respect to amino acid residue positions, "corresponding to" or "corresponds to" or "corresponds to" refers to the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or identical to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related protein or reference protein.

[0058] As used herein, "performance index" or "PI" refers to the calculated activity per unit of enzyme relative to a parent molecule. In some aspects of any embodiment disclosed herein, the parent molecule used in calculating the performance index is a glucoamylase. In some embodiments, by definition, the parent molecule has a performance index of one. In other embodiments, a performance index greater than one (PI>1.0) indicates an improved activity of a glucoamylase variant compared to the parent molecule.

[0059] As used herein, "reversed sugars" are sugars formed when a monosaccharide condenses with another monosaccharide (occasionally a disaccharide) in the presence of a catalyst (e.g., an acid) to form an oligosaccharide, such as (mostly) a disaccharide or (rarely) a trisaccharide (in a process defined herein as "reversed"). Thus, in many cases, reversed sugars have linkages that are not present in the original starch composition. Examples of reverse sugars include, for example, xylobiose (both the α and β forms of (1,1), (1,2), (1,3) and (1,4) linked xylobiose), O-α-D-xylopyranosyl-α-D-xylopyranosyl, 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, laminaribiose, and any combination thereof. Reverse sugars are typically non-fermentable and are therefore considered a waste product of the saccharification reaction.

[0060] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a biochemical production process in which a microorganism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present in the same process step. SSF involves the simultaneous hydrolysis of a starch substrate (granular, liquefied, or dissolved) to sugars (including glucose) and fermentation of the sugars to alcohols or other biochemicals or biomaterials in the same reaction vessel.

[0061] A "slurry" is an aqueous mixture containing insoluble starch particles in water.

[0062] The term "total sugar content" refers to the total soluble sugar content present in a starch composition including monosaccharides, oligosaccharides and polysaccharides.

[0063] The term "dry solids" (ds) refers to dry solids dissolved in water, dry solids dispersed in water, or a combination of both. Dry solids thus include granular starch and its hydrolysis products, including glucose.

[0064] The term "high DS" refers to an aqueous starch slurry having a dry solids content greater than 38% (wt / wt).

[0065] "Degree of polymerization (DP)" refers to the number (n) of anhydropyranose glucopyranose units in a given saccharide. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP4+ (>DP3) represents a polymer with a degree of polymerization greater than 3.

[0066] The term "contacting" refers to placing the referenced components (including but not limited to enzymes, substrates, and fermenting organisms) in sufficient proximity to affect a desired result, such as the enzyme acting on the substrate or the fermenting organism fermenting the substrate.

[0067] As used herein, the terms "yeast cell", "yeast strain", or simply "yeast" refer to organisms from the phylum Ascomycota and the phylum Basidiomycota. An exemplary yeast is a budding yeast from the order Saccharomycetales. A specific example of a yeast is a species of the genus Saccharomyces, including but not limited to S. cerevisiae. Yeasts include organisms used to produce fuel alcohols as well as organisms used to produce drinkable alcohols, including specialty and proprietary yeast strains used to prepare unique flavored beer, wine, and other fermented beverages.

[0068] "Ethanogenic microorganism" refers to a microorganism that has the ability to convert sugars or other carbohydrates into ethanol.

[0069] The term "biochemical" refers to metabolites of microorganisms, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium isoascorbate, ω3 fatty acids, butanol, isobutanol, amino acids, lysine, itaconic acid, other organic acids, 1,3-propanediol, vitamins, or isoprene, or other biological materials.

[0070] The term "pullulanase" is also known as debranching enzyme (EC 3.2.1.41, Pullulan 6-glucanohydrolase), which is capable of hydrolyzing alpha 1-6 glycosidic linkages in the amylopectin molecule.

[0071] Certain ranges are presented herein with the term "about" preceding a numerical value. The term "about" is used herein to provide textual support for the exact number that follows it and for numbers that are close or approximate to the number that follows the term. When determining whether a number is close to or approximate to a specifically stated number, the close or approximate unstated number may be a number that provides a substantial equivalent of the specifically stated 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 specifically defined in context.

[0072] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0073] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0074] The term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates. It should be further noted that as used herein, the term "comprising" means including but not limited to one or more components following the term "comprising". The one or more components following the term "comprising" are required or mandatory, but the composition comprising the one or more components may further include other non-mandatory or optional one or more components.

[0075] Still, it should be noted that, as used herein, the term "consisting essentially of" refers to a composition in which the component(s) following the term are in the presence of other known component(s), the total amount of the other known component(s) accounting for less than 30% by weight of the total composition and do not affect or interfere with the action or activity of the component(s).

[0076] Still, it should be noted that, as used herein, the term "consisting of" is meant to include and be limited to the one or more components following the term "consisting of." Therefore, the one or more components following the term "consisting of" are required or mandatory, and one or more other components are not present in the composition.

[0077] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0078] 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.

[0079] Additional definitions of terms may appear throughout this specification.

[0080] II. Variant Polypeptides Having Glucoamylase Activity

[0081] In a first aspect, the invention relates to a variant polypeptide comprising an amino acid sequence having glucoamylase activity and having at least one amino acid substitution (such as any substitution shown in Table 1), wherein the amino acid sequence has 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 the 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 another aspect, provided herein is a 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 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.

[0082] In some embodiments, the variant polypeptide comprises an amino acid sequence having at least 60% but less than 100% sequence identity to the 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% but 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, the polypeptide is non-naturally occurring (i.e., does not occur in nature and is a product of human creativity).

[0083] In some embodiments, the glucoamylase variant polypeptides of the invention are homologous polypeptides comprising an amino acid sequence that differs from any of the polypeptides of SEQ ID NO: 2, the polypeptides of SEQ ID NO: 3, the polypeptides of SEQ ID NO: 4, the polypeptides of SEQ ID NO: 5-37, the polypeptide comprising amino acid positions 20-468 of SEQ ID NO: 2, the polypeptide comprising amino acid positions 21-468 of SEQ ID NO: 2, the polypeptide comprising amino acid positions 22-468 of SEQ ID NO: 2, the polypeptide comprising amino acid positions 23-468 of SEQ ID NO: 2, the polypeptide comprising amino acid positions 24-468 ​​of SEQ ID NO: 2, the polypeptide comprising amino acid positions 25-468 of SEQ ID NO: 2 by no more than ten amino acids, no more than nine amino acids, no more than eight amino acids, no more than seven amino acids, no more than six amino acids, no more than five amino acids, no more than four amino acids, no more than three amino acids, no more than two amino acids, and even no more than one amino acid.

[0084] In some embodiments, a variant polypeptide of the invention is a catalytic region comprising amino acids 18 to 449 of SEQ ID NO: 4 predicted by C1ustalX: / / world wide web.ncbi.nlm.nih.gov / pubmed / 17846036.

[0085] In some embodiments, the polypeptides of the invention have pullulan and / or panose and / or maltodextrin hydrolyzing activity.

[0086] In another aspect, the glucoamylase variants disclosed herein may, in some embodiments, comprise one or more conservative substitutions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, any of the amino acid sequences of 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, 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 generated by genetic manipulation while other conservative substitutions are generated by introducing synthetic amino acids into the polypeptide by other means.

[0087]

[0088]

[0089] In some embodiments, the polypeptide of the invention is 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, 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, or a fragment thereof having glucoamylase activity. The variant glucoamylase can comprise a deletion, substitution (such as any of the amino acid substitutions shown in Table 1), insertion or addition of one or more amino acid residues relative 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. 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues.any of the polypeptides of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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, a polypeptide comprising amino acid positions 25-28 of SEQ ID NO: 2, a polypeptide comprising amino acid positions 26-27 of SEQ ID NO: 2, a polypeptide comprising amino acid positions 27-28 of SEQ ID NO: 2, a polypeptide comprising amino acid positions 28-29 of SEQ ID NO: 2, a polypeptide comprising amino acid positions 29-30 of SEQ ID NO: 2 The amino acid substitutions (such as any of the amino acid substitutions shown in Table 1), deletions and / or insertions of the polypeptide at amino acid positions 25-468 of NO:2 may 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.

[0090] 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 a 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 a 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 the substitution S102P of the polypeptide of SEQ ID NO: 4 or at a 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.

[0091] 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 a 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 a 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 of the polypeptide of SEQ ID NO: 4, or at a corresponding position of any one of SEQ ID NOs: 2-37.

[0092] In another embodiment, the variant alteration comprises or consists of a substitution at a position 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 the 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.

[0093] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are changed. For example, amino acid changes can improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0094] In one embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions can be expressed to a greater extent (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%, %, 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 values ​​falling between these percentages, in some embodiments, the present invention can hydrolyze a disaccharide (such as, for example, maltose) to a greater extent than any of the above (including values ​​falling between these percentages). The variant glucoamylase can have one or more amino acid substitutions (such as 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 residue positions corresponding to the following positions: SEQ ID 404, 405, 418, 440, 441 and / or 444 of a parent glucoamylase, such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any one of SEQ ID NOs: 5-37.The one or more substitutions at a particular position may include one or more of the following: 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, / Y, X321D, X341M, X350I / T, X352D, X354L / M, X370M, L (wherein X is any amino acid corresponding to the equivalent position in the parent glucoamylase); or one or more of the following: S020A / P, K021S, A051L / V, V066A / C / F / M / P / T / W, A079C / T, G080K, V092M, P102G, T121G / P / M, M140I / C, M140C, T157I, N158A, Y165W, K166G / R / F, Y192F, D203W, R210L, D213R, N214A / E, S215D / C / F, S22 1R / 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, 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 disaccharide (such as, for example, maltose) hydrolysis, including the assays described in the Examples section.

[0095] In further embodiments, the glucoamylase variants disclosed herein having one or more amino acid substitutions can be expressed to a greater extent (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%, %, 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 values ​​falling between these percentages). Panose is a trisaccharide consisting of a maltose molecule bonded to a glucose molecule by an α-1,6-glycosidic bond, which is often used as a substrate to help characterize the activity of starch degrading enzymes. The variant glucoamylase can have one or more amino acid substitutions (such as 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 residue positions corresponding to the following positions: SEQ ID 404, 405, 418, 430, 440, 444 and / or 445 and / or a parent glucoamylase (such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4). NO: any one of 5-37).The one or more substitutions at a particular position may include one or more of the following: 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, X214C, 4A / E, X215D, X218Q / 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 a or one or more of the following: 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, D21 3H, 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. Any assay known in the art can be used to determine panose hydrolysis, including the assay described in the Examples section.

[0096] In another embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions can be expressed to a greater extent (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%, 111%, , 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 values ​​falling between these percentages)). 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 often used as a substrate to identify glucoamylases that can hydrolyze α1-6 linkages in starch. Variant glucoamylases may have one or more amino acid substitutions (such as 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 any one of 49 amino acid substitutions) at residue positions corresponding to the following positions: SEQ ID 40, 416, 418, 434, 440 and / or 445 of 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.The one or more substitutions at a particular position may include one or more of the following: 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, X354I, X370M, X390D / L / E, X404K, X416Y, X418E / W, X434S, X440G, and / or X445M (where X is a or one or more of the following: 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, N21 4A / Y / L / C, S215V / 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 pullulan hydrolysis, including the assays described in the Examples section.

[0097] In yet another embodiment, the glucoamylase variants disclosed herein having one or more amino acid substitutions can exhibit a higher performance index (PI) for saccharifying soluble starch (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%, 111%, %, 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 values ​​falling between these percentages) in any one of (i.e., improved hydrolysis of soluble starch or fragments thereof). The variant glucoamylase can have one or more amino acid substitutions (such as 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 residue positions corresponding to the following positions: SEQ ID 404, 405, 416, 418, 422, 440, 441, 444 and / or 445 of SEQ ID NO:4 and / or a parent glucoamylase (such as, but not limited to, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4). NO: any one of 5-37).The one or more substitutions at a particular position may include one or more of the following: 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, X215 18Q / F / A, X221R, X222M / 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 a or one or more 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, N214 404M / K, H405S, F416C / Y, R418E / W, Y422A, A440G, Q441L, A444S / P, and / or G445M. The PI can be determined based on the assay described in the Examples section.

[0098] In other embodiments, the glucoamylase variants disclosed herein having one or more amino acid substitutions can exhibit improved maltodextrin hydrolysis (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%, , 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 values ​​falling between these percentages) in any one of improved hydrolysis). Maltodextrin is composed of D-glucose units connected in chains of variable length. Glucose units are mainly connected by α (1→4) glycosidic bonds and maltodextrin is typically composed of a mixture of chains varying from 3 to 17 glucose units in length. Maltodextrin mimics a glucoamylase substrate because it is produced using an alpha amylase. The variant glucoamylase can have one or more amino acid substitutions (such as 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) at residue positions corresponding to the following positions: SEQ ID NO: 4 position 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 and / or a parent glucoamylase (such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3 or the equivalent position in any one of SEQ ID NOs: 5-37).The one or more substitutions at a particular position may include one or more of the following: 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 a or one or more of the following: 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, N2 14A / E, S215D / C / F, S218V / Y / Q, S221R / T, G222M / C, S233M / P / Y, W235Y, D23 6Q / A, V252F, G274K / A / D, E281D, F290M / V, N302P / K / S / V / Q / W, N310T / Y, N32 1D, 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 maltodextrin hydrolysis, including the assay described in the Examples section.

[0099] In still further embodiments, the glucoamylase variants disclosed herein having one or more amino acid substitutions can 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%, %, 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 values ​​falling between these percentages) improved thermal stability). The variant glucoamylase can have one or more amino acid substitutions (such as 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 residue positions corresponding to the following positions: SEQ ID NO: NO:4, position 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 a parent glucoamylase such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any one of SEQ ID NOs: 5-37.The one or more substitutions at a particular position may include one or more of the following: 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 a 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 the assays described in the Examples section.

[0100] In another embodiment, a glucoamylase variant disclosed herein having one or more amino acid substitutions can exhibit improved reduced reversal (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%, %, 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%, 100%, including values ​​falling between these percentages (reversal of reduction). The variant glucoamylase can have one or more amino acid substitutions (such as 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 residue positions corresponding to the following positions: SEQ ID NO:4, position 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 a parent glucoamylase such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any one of SEQ ID NOs: 5-37.The one or more substitutions at a particular position may include one or more of the following: 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 a any amino acid at the equivalent position in the parent glucoamylase); or one or more of the following: 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, W235F, 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. Any assay known in the art can be used to determine the formation of reverse sugars during saccharification, including the assays described in the Examples section.

[0101] In further embodiments, the glucoamylase variants disclosed herein having one or more amino acid substitutions can exhibit improved saccharification yield of glucose (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%, , 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 values ​​falling between these percentages) improved saccharification yield). The variant glucoamylase can have one or more amino acid substitutions (such as 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 residue positions corresponding to the following positions: SEQ ID NO: NO:4, position 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 a parent glucoamylase such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any one of SEQ ID NOs: 5-37.The one or more substitutions at a particular position may include one or more of the following: 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, X 218H / 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 a any amino acid at the equivalent position in the parent glucoamylase); or one or more of the following: 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 the saccharification yield, including the assays described in the Examples section.

[0102] Thus, in still further embodiments, the glucoamylase variants disclosed herein having one or more amino acid substitutions can exhibit improvements (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%, %, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more (including values ​​falling between these percentages)) any of: i) improved hydrolysis of disaccharides (such as, for example, maltose); ii) higher panose hydrolysis; iii) higher pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or its fragments); v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose. The variant glucoamylase can have one or more amino acid substitutions (such as 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, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71 amino acid substitutions) at residue positions corresponding to the positions of SEQ ID NO: 4 position 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, 445 and / or 449 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 one of SEQ ID NOs: 5-37). The one or more substitutions at a particular position may include one or more of the following: 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, X140 C / I / Q, X141F, X156C / P, X157I, X158A / Y, / Y, X215C / D / F / R, X218A / K / Q / V / Y, X221M / R / T, X222C / M / V, / D / K, X278A, X281D, X290M / V, X290V, 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, X416C / Y, X418E / W, X422A, X430A, X440G / H, X441L, X444L / P, and / or X445M / Y (where X is the corresponding or one or more of the following: 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, Y192F, D203C / W, R210L / M / W, D213H / R, N2 14A / 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, 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, Y404I / K / M, H405Q / S / Y, F416C / Y, R418E / W, Y422A, T430A, A440G / H, Q441L, A444L / P, and / or G445M / Y. ,

[0103] Various amino acid substitutions (such as any of those shown in Table 1), deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by relevant 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-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0104] In additional embodiments, provided herein are glucoamylase variants having two or more substitutions at residue positions corresponding to the following positions: 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) X 243P and X290V, j) X290V and X350E, k) X302K and X441W, 1) 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-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 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 (such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any one of SEQ ID NOs: 5-37).In other embodiments, the two or more amino acid substitutions may be 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 G4 45Y, 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, 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 compared to a parent glucoamylase lacking two or more of these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or its fragments); v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.

[0105] In additional embodiments, provided herein are glucoamylase variants having three or more substitutions at residue positions corresponding to the following positions: 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) X067M, X158A, and X281D, ) X156C, X192F and X243P, j) X023L, X066C and X119A, 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, wherein X is the residue corresponding to SEQ 422V, g) A066C, S119A and I164T, h) A066C, S233M and G445Y, i) F156C, Y281D 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, Y281D and Q441W, 192F 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 compared to a parent glucoamylase lacking three or more of these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or its fragments); v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.

[0106] In additional embodiments, provided herein are glucoamylase variants having four or more substitutions at residue positions corresponding to the following positions: 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, wherein X is a residue corresponding to the equivalent position in SEQ ID NO: 4 and / or a parent glucoamylase (such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4). NO: 5-37 in any one). In other embodiments, the four or more amino acid substitutions can be a) S215R, D236S, E281D and Q441W, b) G052N, 1084L, 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 compared to a parent glucoamylase lacking four or more of these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or its fragments); v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.

[0107] In additional embodiments, the present invention still further provides a glucoamylase variant having five or more substitutions at residue positions corresponding to the following: X067M, X157I, X218H, X302H 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 (such as but not limited to SEQ ID NO: 2, SEQ ID NO: 3 or any one of SEQ ID NO: 5-37). In other embodiments, the four or more amino acid substitutions may be S067M, T157I, S218H, N302H and F416Y. The variant glucoamylase exhibits one or more of the following compared to a parent glucoamylase lacking five or more of these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or its fragments); v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.

[0108] In further embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) consensus sequence N-linked glycosylation sites (i.e., NXS / T consensus sequence N-linked glycosylation sites, wherein X can be any amino acid except proline and N(Asp) is glycosylated) can be introduced into any variant glucoamylase disclosed herein or into the glucoamylase of SEQ ID NO: 4 to improve one or more properties of the glucoamylase, such as, but not limited to, i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) higher PI of saccharified soluble starch (i.e., improved hydrolysis of soluble starch or fragments thereof); v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with a DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose compared to a parent glucoamylase lacking one or more additional glycosylation sites. Amino acid substitutions for introducing glycosylation sites into variant glucoamylase polypeptides can be made at residue positions corresponding to positions 81, 83, 153, 370 or 372 of SEQ ID NO: 4, and / or equivalent positions in a parent glucoamylase. In some embodiments, the one or more substitutions include one or more substitutions at positions D81N, K83T, A153T, S370N, and / or A372S.

[0109] As shown in Example 7, it was observed that substitution at the Asn glycosylation site at position 75 resulted in a drastic decrease in catalytic activity. Thus, in some embodiments, the variant glucoamylases disclosed herein have N-linked glycosylation at position N075 of SEQ ID NO: 4 and / or an equivalent position in a parent glucoamylase (such as, but not limited to, SEQ ID NO: 2, SEQ ID NO: 3, or any one of SEQ ID NOs: 5-37).

[0110] III. Production of Glucoamylase

[0111] Variant glucoamylase disclosed herein can be produced in host cells, for example, by secretion or intracellular expression. After the glucoamylase is secreted into the cell culture medium, a culture cell material (e.g., whole cell culture broth) containing the glucoamylase can be obtained. Optionally, the glucoamylase can be separated from the host cell, or even separated from the cell culture broth, depending on the purity required for the final glucoamylase. The gene encoding the glucoamylase 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 reesi) or Myceliomycete species (such as Myceliopthora 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 Schizosaccharomyces species; or species of Saccharomyces, including Saccharomyces cerevisiae; or species belonging to the genus Schizosaccharomyces, such as, for example, S. pombe species. The methylotrophic yeast species strain Pichia pastoris can be used as a host organism.

[0112] In addition, the host can express one or more coenzymes, proteins, peptides. These can be beneficial to liquefaction, saccharification, fermentation, SSF and downstream processes. In addition, in addition to the enzymes for digesting various raw materials, the host cell can also produce ethanol and other biochemicals or biomaterials. Such host cells can be used for fermentation or saccharification and fermentation processes simultaneously to reduce or eliminate the need for adding enzymes.

[0113] A. Carrier

[0114] A DNA construct comprising a nucleic acid encoding a variant glucoamylase polypeptide disclosed herein can be constructed so that it is suitable for expression in a host cell. Due to the known degeneracy in the genetic code, different polynucleotides encoding the same amino acid sequence can be designed and prepared using conventional techniques. It is also known that, depending on the desired host cell, codon optimization may be required before attempting expression.

[0115] The polynucleotide encoding the variant glucoamylase polypeptide of the present disclosure can be incorporated into a vector. The vector can be transferred to a host cell using known transformation techniques (such as those disclosed below).

[0116] Suitable vectors can be vectors that can be transformed into a host cell and / or replicated in a host cell. For example, a vector comprising a nucleic acid encoding a variant glucoamylase polypeptide disclosed herein can be transformed and / or replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector can also be suitably transformed into an expression host so that the encoding polynucleotide is expressed as a functional glucoamylase.

[0117] Representative useful vectors are pTrex3gM (see, published U.S. patent application 20130323798) and pTTT (see, published U.S. patent application 20110020899), which can be inserted into the genome of the host. Both vectors pTrex3gM and pTTT can be modified with conventional techniques so that they contain and express polynucleotides encoding variant glucoamylase polypeptides of the present invention.

[0118] The expression vector generally comprises a control nucleotide sequence, 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, the expression vector may comprise a sequence encoding an amino acid sequence that is capable of targeting the glucoamylase to a host cell organelle (such as a peroxisome) or to a specific host cell compartment. For expression under the guidance of a control sequence, the nucleotide sequence of the variant glucoamylase is operably linked to the control sequence in an appropriate manner for expression.

[0119] The polynucleotide encoding the variant glucoamylase polypeptide disclosed herein can be operably connected to a promoter that allows transcription in a host cell. The promoter can be any DNA sequence that shows transcriptional activity in the selected host cell, and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell. Examples of promoters for directing transcription of a DNA sequence encoding glucoamylase, particularly in a bacterial host, include promoters of the lactose operon of Escherichia coli (E.coli), Streptomyces coelicolor (Streptomyces coelicolor) agarase gene dagA or celA promoters, Bacillus licheniformis (Bacillus licheniformis) amylase gene (amyL) promoters, Bacillus stearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene (amyM) promoters, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) amylase (amyQ) promoters, Bacillus subtilis xylA and xylB gene promoters, etc.

[0120] For transcription in fungal hosts, examples of useful promoters 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, etc. When the gene encoding the glucoamylase is expressed in a bacterial species such as Escherichia coli, a suitable promoter can be selected from phage promoters including, for example, the T7 promoter and the phage lambda promoter. Along these lines, examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. Expression in filamentous fungal host cells typically involves cbhl, an endogenous inducible promoter from T. reesei. See Liu et al. (2008) Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0121] The coding sequence may be operably linked to a signal sequence. The DNA encoding the signal sequence may be a DNA sequence naturally associated with the target variant glucoamylase gene to be expressed, or may be from a genus or species different from the variant glucoamylase (i.e., the species from which the variant is derived). The signal sequence and promoter sequence that make up the DNA construct or vector may be introduced into a fungal host cell and may be derived from the same source. For example, the signal sequence may be a Trichoderma reesei cbhl signal sequence operably linked to a cbhl promoter.

[0122] The expression vector may also comprise a suitable transcription terminator and, in eukaryotes, a polyadenylation sequence operably linked to the DNA sequence encoding the glucoamylase. The termination sequence and polyadenylation sequence may be suitably derived from the same source as the promoter.

[0123] The vector may also comprise a selective marker, e.g., a gene whose product complements a defect in the isolated host cell, such as the dal gene from Bacillus subtilis or Bacillus licheniformis, or a gene that confers antibiotic resistance, such as, for example, ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. In addition, the vector may comprise an Aspergillus (selective marker, such as amdS, argB, niaD, and xxsC, a marker that causes hygromycin resistance, or selection may be achieved by co-transformation, as known in the art. See, e.g., published International PCT Application WO 91 / 17243.

[0124] B. Host cell transformation and culture

[0125] The cell that comprises the separation of DNA construct or expression vector is advantageously used as the host cell of recombinant production variant glucoamylase.By integrating DNA construct (with one or more copies) into host chromosome, can be conveniently transformed cell with the DNA construct of encoding enzyme.It is generally considered that this integration is advantageous because DNA sequence dna is more likely to be stably maintained in cell.Can be according to conventional methods, for example, by homologous or heterologous recombination, DNA construct is integrated into host chromosome.Alternately, can transform cell with the expression vector relevant to different types of host cells.

[0126] Examples of suitable bacterial host organisms are gram-positive bacterial species, such as the Bacillaceae family, including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species, such as Streptomyces murinus; lactic acid bacterial species, including Lactococcus sp., such as Lactococcus lactis; Lactobacillus sp., such as Lactococcus lactis; sp.), including Lactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of gram-negative bacterial species belonging to the family Enterobacteriaceae (including E. coli) or to the family Pseudomonadaceae can be selected as host organisms.

[0127] Suitable yeast host organisms can be selected from biotechnology-related yeast species, such as, but not limited to, yeast species, such as Pichia species, Hansenula species or Kluyveromyces, Yarrowinia, Schizosaccharomyces species; or species of the genus Saccharomyces, including Saccharomyces cerevisiae; or species belonging to the genus Schizosaccharomyces, such as, for example, Schizosaccharomyces pombe species. Methylotrophic yeast species strains Pichia pastoris can be used as host organisms. Alternatively, the host organism can be a Hansenula species.

[0128] Suitable host organisms in filamentous fungi include species of Aspergillus, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori or Aspergillus nidulans. Alternatively, strains of Fusarium species (for example, Fusarium oxysporum) or strains of Rhizomucor species (such as Rhizomucor miehei) can be used as host organisms. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma species can be used as hosts. The glucoamylase expressed by the fungal host cell can be glycosylated, that is, it will contain a glycosyl moiety. The glycosylation pattern can be the same or different as that present in the wild-type glucoamylase. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties.

[0129] It is advantageous to delete genes from an expression host where the gene defect can be cured by a transformed expression vector. Known methods can be used to obtain fungal host cells with one or more inactivated genes. In a non-limiting example, any cloned gene from a Trichoderma species or other filamentous fungal host can be deleted, such as the cbhl, cbh2, egl1, and egl2 genes. 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.

[0130] 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. Patent No. 6,022,725. For transformation of Aspergillus strains, reference is also made to Cao et al. (2000) Science 9: 991-1001. Genetically stable transformants can be constructed with a vector system, whereby the nucleic acid encoding glucoamylase is stably integrated into the host cell chromosome. The transformants are then selected and purified by known techniques. The deletion plasmid is then cut at one or more appropriate restriction enzyme sites within the desired gene coding region, and the gene coding sequence or a portion thereof is replaced with a selective marker. Flanking DNA sequences (preferably between about 0.5 and 2.0 kb) from the locus of the gene to be deleted are retained on either side of the marker gene. Appropriate deletion plasmids will generally have unique restriction enzyme sites present therein so that the fragment containing the deleted gene (including the flanking DNA sequences) and the selective marker gene can be removed as a single linear piece.

[0131] Depending on the host cell used, post-transcriptional and / or post-translational modifications may be performed. A non-limiting example of post-transcriptional and / or post-translational modifications is "chopping" or "truncating" of a polypeptide. In another case, this shearing may result in obtaining a mature glucoamylase polypeptide and further removing the N or C-terminal amino acid (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more N or C-terminal amino acids) to produce a truncated form of a glucoamylase that retains enzymatic activity.

[0132] 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 recognize 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.

[0133] Further sequence modifications may occur to the polypeptide after expression. This includes, but is not limited to, oxidation, deglycosylation, saccharification, etc. It is known that saccharification can affect the activity of glucoamylase when incubated with glucose or other reducing sugars, particularly at temperatures above 30°C and neutral or alkaline pH. Protein engineering to eliminate lysine residues can be used to prevent such modifications. Such examples can be found in US 8,507,240. For example, yeast expression can produce highly glycosylated polypeptides, resulting in a significant increase in molecular weight. In addition, WO 2013 / 119470, with an international publication date of August 15, 2013 (incorporated herein by reference), relates to phytases with increased stability (which is believed to be due to increased glycosylation).

[0134] C. Expression and fermentation

[0135] Methods for producing any of the variant glucoamylases disclosed herein can comprise culturing a host cell under conditions conducive for production of the enzyme, and recovering the enzyme from the cells and / or culture medium.

[0136] The culture medium for culturing cells can be any conventional culture medium suitable for host cell growth and obtaining expression of the variant glucoamylase polypeptide. Suitable culture medium and culture medium components are available from commercial suppliers or can be prepared according to published recipes (e.g., as described in the catalog of the American Type Culture Collection).

[0137] Any fermentation method known in the art may be suitable for fermenting the transformed or derived fungal strains described above. In some embodiments, the fungal cells are grown under batch or continuous fermentation conditions.

[0138] D. Methods for enrichment and purification

[0139] Isolation 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 present invention.

[0140] After fermentation, the fermentation broth is obtained, and the microbial cells and various suspended solids (including the remaining crude fermentation material) are removed by conventional separation techniques to obtain a glucoamylase solution. Typically, filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography, etc. are used.

[0141] Sometimes it may be necessary to concentrate the solution or broth containing the glucoamylase polypeptide to optimize recovery. Using unconcentrated solutions or culture broths typically increases the incubation time in order to collect the enriched or purified enzyme precipitate.

[0142] IV. Composition

[0143] The present invention also relates to compositions comprising polypeptides (such as glucoamylases, e.g., any variant glucoamylase disclosed herein) and / or starch substrates. In some embodiments, variant glucoamylases comprising an amino acid sequence 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 one of SEQ ID NO: 2-30) can also be used in enzyme compositions. Preferably, these compositions are formulated to provide desired characteristics, such as light color, low odor, and acceptable storage stability (at a temperature of about 4° C.-40° C. and a pH of about 3-7).

[0144] The composition can comprise a variant glucoamylase polypeptide of the present invention as a major enzymatic component. Alternatively, the composition can comprise a variety of enzymatic activities, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, perhydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-amylase, isoamylase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, pullulanase, ribonuclease, transglutaminase, xylanase or a combination thereof, which can be added in an effective amount well known to those skilled in the art.

[0145] Peptide compositions can be prepared according to methods known in the art, and these peptide compositions can be in the form of liquid or dry compositions. For example, the composition comprising variant glucoamylase of the present invention can be aqueous or non-aqueous preparations, particles, powders, gels, slurries, pastes, etc., and these compositions can further comprise any one or more of the other enzymes listed herein, as well as buffer, salt, preservative, water, cosolvents, surfactants, etc. Such compositions can work in combination with endogenous enzymes or other ingredients (for example, endogenous plant (including algae) enzymes, residual enzymes from previous processing steps, etc.) already present in slurry, water bath, washing machine, food or beverage products, etc. The polypeptide included in the composition can be stabilized according to methods known in the art.

[0146] The composition can be a cell expressing a polypeptide, including cells capable of producing a product from fermentation. Such cells can be provided in liquid or dry form with a suitable stabilizer. Such cells can further express additional polypeptides, such as the polypeptides mentioned above.

[0147] The dosage of the polypeptide composition of the present invention and other conditions for using the composition can be determined based on methods known in the art.

[0148] The above composition is suitable for use in liquefaction, saccharification and / or fermentation processes, preferably in starch conversion, especially for producing syrups and fermentation products (such as ethanol). The composition is also suitable for use in animal nutrition (such as a component of animal feed) and fermented beverage production.

[0149] V. Application and Methods

[0150] The present invention also relates to the use of variant glucoamylase polypeptides or compositions of the present invention in liquefaction processes, saccharification processes and / or fermentation processes. Variant glucoamylase polypeptides or compositions can be used in a single process, such as in a liquefaction process, a saccharification process, or a fermentation process. Variant glucoamylase polypeptides or compositions can also be used in a combination of processes (e.g., in a liquefaction and saccharification process, in a liquefaction and fermentation process, or in a saccharification and fermentation process (preferably related to starch conversion)).

[0151] A. Saccharification

[0152] Alpha-amylase and variant glucoamylase (optionally in the presence of one or more additional enzymes) can be used to saccharify liquefied starch into a syrup rich in low DP (e.g., DP1+DP2) sugars. The exact composition of the saccharified product depends on the combination of enzymes used and the type of starch processed. Advantageously, the weight percentage of DP1 contained in the syrup obtainable using the variant glucoamylase provided can exceed 90%, such as 90%-98% or 95%-97% of the total oligosaccharides in the saccharified starch. The weight percentage of DP2 in the saccharified starch can be as low as possible, less than about 3%, such as 0%-3% or 0%-2.8%.

[0153] Liquefaction is usually carried out as a continuous process, while saccharification is usually carried out as a batch process. Saccharification conditions depend on the properties of the liquefied material and the types of enzymes available. In some cases, the saccharification process may involve a temperature of about 60°C-65°C and a pH of about 4.0-4.5 (e.g., pH 4.3). Saccharification can be carried out at a temperature of, for example, about 40°C, about 50°C, or about 55°C to about 60°C or about 65°C, and it is necessary to cool the liquefied material. The pH can be adjusted as needed. Saccharification is usually carried out in a stirred tank, which may take several hours to fill or empty. Enzymes are typically added to dry solids at a fixed ratio (when the tank is filled), or added in a single dose (at the beginning of the filling phase). The saccharification reaction for preparing syrup is typically carried out for about 24-72 hours, for example, 24-48 hours. Pre-saccharification can be added before saccharification in simultaneous saccharification and fermentation (SSF), and the pre-saccharification typically lasts 40-90 minutes at a temperature between 30°C-65°C (typically about 60°C).

[0154] B. Raw starch hydrolysis

[0155] The invention provides the use of variant glucoamylases of the invention for producing glucose, etc. from raw starch or granular starch. Typically, the glucoamylases of the invention can be used alone or in the presence of an alpha-amylase in a raw starch hydrolysis (RSH) or granular starch hydrolysis (GSH) process to produce desired sugars and fermentation products. Granular starch is dissolved by enzymatic hydrolysis below the gelatinization temperature. It is reported that such "low temperature" systems (also referred to as "no cooking" or "cold cooking") are able to handle higher concentrations of dry solids (e.g., up to 45%) than conventional systems.

[0156] The "raw starch hydrolysis" process (RSH) is distinct from conventional starch processing processes and involves sequential or simultaneous saccharification and fermentation of granular starch, typically in the presence of at least a glucoamylase and / or an amylase, at or below the gelatinization temperature of the starch substrate.

[0157] Variant glucoamylases of the present invention may also be used in combination with enzymes that hydrolyze only α-(1,6)-glycosidic bonds in molecules comprising at least four glucose residues. Preferably, variant glucoamylases of the present invention are used in combination with pullulanase or isoamylase. The use of isoamylase and pullulanase for starch debranching, the molecular properties of the enzymes, and the potential use of the enzymes together with glucoamylases are described in GMA van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, 101-142.

[0158] C. Fermentation

[0159] Soluble starch hydrolysate (particularly glucose-rich syrup) can be fermented by contacting the starch hydrolysate with a fermenting organism, typically at a temperature of about 32°C (e.g., from 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) sugars (e.g., glucose or maltose) directly or indirectly into a desired fermentation product. Examples of fermenting organisms include yeast (e.g., Saccharomyces cerevisiae) and bacteria (e.g., Zymomonas mobilis) that express ethanol dehydrogenase and pyruvate decarboxylase. Ethanogenic microorganisms can express xylose reductase and xylitol dehydrogenase, both of which convert xylose into xylulose. For example, improved ethanologenic microbial strains that can withstand higher temperatures are known in the art and can be used. See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao [Journal of Bioengineering] 27: 1049-56. Yeasts that can be used for alcohol production include, but are not limited to, Saccharomyces species, including Saccharomyces cerevisiae, and Kluyveromyces, Lachancea, and Schizosaccharomyces species. Many yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high alcohol production, fast growth rates, etc. The temperature and pH of the fermentation will depend on the fermenting organism. Microorganisms that produce other metabolites (such as citric acid and lactic acid) by fermentation are also known in the art. See, for example, Papagianni (2007) Biotechnol. Adv. [Progress in Biotechnology] 25: 244-63; John et al. (2009) Biotechnol. Adv. [Progress in Biotechnology] 27: 145-52.

[0160] Saccharification and fermentation process can be carried out as SSF process.In certain embodiments, SSF process can be carried out with fungal cells that continuously express and secrete variant glucoamylase in whole SSF.The fungal cells expressing variant glucoamylase can also be fermentation microorganisms, such as ethanologenic microorganisms.Therefore, ethanol production can be carried out using fungal cells expressing enough variant glucoamylases, thereby less needing or not needing to add enzymes exogenously.Fungal host cells can be selected from appropriately engineered fungal strains.In addition to variant glucoamylase, fungal host cells expressing and secreting other enzymes can also be used.Such cells can express amylase and / or pullulanase, phytase, alpha-glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, beta-glucosidase, pectinase, esterase, oxidoreductase, transferase or other enzymes.After fermentation, subsequent recovery of ethanol can be carried out.

[0161] D. Fermentation products

[0162] The term "fermentation product" means a product produced by a process comprising a fermentation process using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (e.g., arabitol, butanol, ethanol, glycerol, methanol, ethylene glycol, propylene glycol, butylene glycol, glycerin, sorbitol, and xylitol); organic acids (e.g., acetic acid, acetic 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 xylose); acids); ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); olefins (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); and hormones.

[0163] In preferred aspects, the fermentation product is ethanol, such as fuel ethanol; drinking ethanol, i.e., drinkable neutral alcohol; or industrial ethanol or products used in the consumer alcohol industry (e.g., beer and liquor), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation process preferably used includes alcohol fermentation processes well known in the art. The preferred fermentation process is an anaerobic fermentation method, which is well known in the art.

[0164] E. brewing

[0165] The process for making beer is well known in the art. See, for example, Wolfgang Kunze (2004) "Technology Brewing and Malting" Research and Teaching Institute of Brewing, Berlin (VLB), 3rd edition. Briefly, the process involves: (a) preparing a mash, (b) filtering the mash to prepare wort, and (c) fermenting the wort to obtain a fermented beverage (such as beer).

[0166] 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 of step (a) above, i.e. during the preparation of the mash. Alternatively or in addition, the brewing composition may be added to the mash of step (b) above, i.e. during filtration of the mash. Alternatively or in addition, the brewing composition may be added to the wort of step (c) above, i.e. during fermentation of the wort.

[0167] F Animal Nutrition

[0168] The variant glucoamylases and compositions described herein can be used as feed additives for animals to increase starch digestibility.Described herein is a method for increasing starch digestibility in an animal.

[0169] The term "animal" refers to any organism belonging to the animal kingdom, and includes, but is not limited to, mammals (excluding humans), non-human animals, domestic animals, livestock, farm animals, zoo animals, breeding stock, etc. For example, all non-ruminants and ruminants can be mentioned. In an embodiment, the animal is a non-ruminant, i.e., a monogastric animal. Examples of monogastric animals include, but are not limited to, pigs (pig and swine), such as piglets, growing pigs, sows; poultry, such as turkeys, ducks, chickens, broilers, 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, cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorn antelopes and deer wildebeests.

[0170] The terms "animal feed," "feed," "feedstock," and "fodder" are used interchangeably and may comprise one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and / or large grains, such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, distillers dried grains with solubles (DDGS), particularly corn-based distillers dried grains with solubles (cDDGS), wheat bran, semolina, wheat short meal, rice bran, rice hulls, oat hulls, palm kernels, and citrus pulp; c) proteins obtained from sources such as soy, sunflower, peanut, lupin, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; and / or e) minerals and vitamins.

[0171] The digestibility of starch in feed is highly variable and depends on many factors, including the physical structure of both the starch and the feed matrix. It has been found that the digestibility of starch in animal diets can be improved by using at least one glucoamylase as a feed additive.

[0172] When used as or for preparing feed (e.g., functional feed), the enzyme or feed additive composition of the present invention can 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, a nutritionally active ingredient. For example, at least one component 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 can be mentioned.

[0173] It is also possible that at least one variant glucoamylase described herein (or an enzyme composition comprising at least one variant glucoamylase as described herein) can be homogenized to produce a powder. The powder can be mixed with other components known in the art. Optionally, the feed can also contain additional minerals (such as, for example, calcium) and / or additional vitamins. In some embodiments, the feed is a corn soy flour mixture.

[0174] In alternative preferred embodiments, 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 WO1997 / 016076 or WO1992 / 012645, which are incorporated herein by reference. "TPT" means thermal protection technology. When the feed additive composition is formulated into granules, the granules comprise a hydration barrier salt coated on a protein core. The advantages of such a salt coating are improved heat resistance, improved storage stability and protection from other feed additives that would otherwise have an adverse effect on the enzyme. Preferably, the salt used for the salt coating has a water activity of greater than 0.25 or a constant humidity of greater than 60% at 20°C. In some embodiments, the salt coating comprises Na2SO4.

[0175] Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumable contains one or more of: a buffer, salts, sorbitol and / or glycerol.

[0176] Any glucoamylase used as a feed additive as described herein can be used alone or in combination with at least one direct-fed microorganism. The category of DFM includes bacillus, lactic acid bacteria and yeast. Further, any glucoamylase used as a feed additive as described herein can be used alone or in combination with at least one essential oil (e.g., cinnamaldehyde and / or thymol). Still further, any glucoamylase used as a feed additive as described herein can be used alone or in combination with at least one other enzyme. Examples of such enzymes include, but are not limited to, phytase, xylanase, protease, amylase, glucanase or other glucoamylases.

[0177] Also disclosed is a method for improving the nutritional value of animal feed, wherein an effective amount of any of the variant glucoamylases described herein may be added to the animal feed.

[0178] As used herein, the phrase "effective amount" refers to the amount of an active agent (such as any variant glucoamylase polypeptide disclosed herein) required to confer improved performance on one or more indicators to an animal, either alone or in combination with one or more other active agents (such as, but not limited to, one or more additional enzymes, one or more DFMs, one or more essential oils, etc.).

[0179] As used herein, the term "animal performance" can be determined by any indicator such as, but not limited to, feed efficiency and / or weight gain of the animal, and / or by feed conversion ratio and / or by digestibility of nutrients in the feed and / or digestible or metabolizable energy in the feed, and / or by the ability of the animal to avoid the negative effects of disease or by the immune response of the subject.

[0180] Animal performance characteristics may include, but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; egg size; egg weight; egg quality; 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.

[0181] "Improved animal performance in one or more parameters" means increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio and / or improved digestibility of nutrients or energy in feed and / or improved nitrogen retention and / or improved ability to avoid negative effects of necrotic enteritis and / or improved immune response in a subject as a result of using a feed comprising the feed additive composition as described herein, as compared to a feed not comprising the feed additive composition.

[0182] All references cited herein are hereby incorporated by reference in their entirety for all purposes.To further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative rather than limiting.

[0183] Examples

[0184] Example 1

[0185] Construction of a site evaluation library (SEL) of variant glucoamylases from the Mucorales clade and their transformation into Trichoderma reesei ization and expression

[0186] The polynucleotide encoding the Mucorales clade variant glucoamylase (codon-modified sequence used as expression cassette) was synthesized by Generay (Generay Biotech Co., Ltd, Shanghai, China) and inserted into the pI1c expression vector (a derivative vector from pTTT (see published U.S. Patent Application 20110020899) that lacks the telomeric region and acetoamidase marker). Generay seamless cloning reagents were used as described by the supplier. In these studies, the gene sequence of the parent glucoamylase was SEQ ID NO: 1, and the translation product was SEQ ID NO: 2. The predicted mature sequence of the parent glucoamylase molecule used for this site evaluation study was SEQ ID NO: 3.

[0187] All fungal manipulations (including high-throughput transformation, inoculation, fermentation, and harvesting) were performed in 96-well microtiter plates (MTPs). Plasmids were transformed into appropriate T. reesei host strains using a polyethylene glycol (PEG)-protoplast method that omitted plating on solid agar. Briefly, approximately 0.5-2 μg DNA, 2×10 6 The transformation mixture of 100 protoplasts and 4 pmol ribonucleoprotein (RNP) was treated with 150 μL 25% PEG solution. The protoplasts were regenerated in a liquid selective growth medium containing 0.75M sorbitol to maintain osmotic pressure. The plates with the regenerated fungal cultures were grown in a shaking incubator with a 50 mm amplitude at 200 rpm and 28° C., 80% humidity for 4 days until fungal mycelia were formed. This material was used to inoculate cultures in order to express the glucoamylase variants.

[0188] In order to express the glucoamylase protein, the transformed Trichoderma reesei strain was cultured as follows: 40 μl of the regenerated transformant was used to inoculate 360 ​​μl of a medium containing glucose, (NH4)2SO4, PIPPS buffer (pH5.5), salts and trace elements. The culture was grown in a 96-well MTP incubated at 260 rpm and 28°C, 80% humidity with a 50 mm amplitude in a shaking incubator. After 2 days of pre-culture, the culture was transferred to a culture medium similar to the above-mentioned culture medium, wherein glucose was replaced with a glucose / sophorose mixture. Subsequently, the 96-well MTP was incubated at 260 rpm and 28°C, 80% humidity with a 50 mm amplitude in a shaking incubator. After 5 days of fermentation, the culture was filtered by centrifugation using a hydrophilic PVDF membrane to obtain a clarified supernatant for analysis of recombinant glucoamylase.

[0189] Protein quantification was performed on the supernatant from the conversion screening. A 5 μl aliquot of the clarified culture supernatant from the MTP culture was injected at 80°C on a Zorbax 300 C3 Rapid Resolution HD 2.1x50mm1.8-micron (Agilent) column. 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 a fermentation sample with a known glucoamylase concentration. For the activity assays reported on Table 1 (Example 2 below), samples were normalized to a concentration of 200 ppm for saccharification assays, 40 ppm for reversal 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. A calibration curve of the parent molecule with a concentration range from 0-80 ppm was included in the assay plate for use in performance index (PI) calculations.

[0190] Representative samples of the culture supernatant of the parent glucoamylase were analyzed by mass spectrometry (MS). The results of the MS analysis showed 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 following N-terminal residues: residue 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).

[0191] Example 2

[0192] Evaluation of enzymatic activity of Mucorales clade glucoamylase variants in saccharification-related assays

[0193] The saccharification performance of the variants and the parent molecule (SEQ ID NO: 3) was evaluated at pH 4.5 and 62°C for 48 h using Maltosweet G 120 purchased from Tate & Lyle as substrate (DE = 11-14). The starting DS content of the saccharification blend was 34%. The performance of the glucoamylase was tested at a dose of 28 μg / gds. For this evaluation, for each incubation, the pullulanase OPTIMAX TML 1000 (International Flavors & Fragrances (IFF) product) was dosed at 0.438 ASPU / g DS, and alpha-amylase Aspergillus terreus amylase (described in International Patent Application Publication No. WO2014099415, which is incorporated herein by reference) was dosed at 0.227 SSU / g DS. Reactants consisting of 75 μl maltodextrin and 5 μl saccharification blend (glucoamylase, alpha-amylase, and pullulanase, as described above) were incubated in MTP at pH 4.5, 62° C. with shaking at 900 rpm for 48 h. All reactions were quenched by adding 5 mM H2SO4 at a ratio of 1:40 saccharification syrup to acid solution. The product formation of the quenched aliquots was analyzed by HPLC using an Agilent 1260 series system with a Phenomenex Rezex-RFQ Fast Acid H+ (8%) 100x7.8mm column (Catalog No. 00D-0223-K0) run at 80°C for 5.3 minutes. For this purpose, 5 μL of sample was injected on the column and carbohydrates were separated at a flow rate of 1.0 mL / min using an isocratic 5 mM H2SO4 as the mobile phase. 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 target sugar (where n represents the number of sugars, such as DP3+, DP3, DP2, and DP1). The relative content of each (oligo)saccharide was calculated using the total integrated peak area of ​​the identified saccharification products. The performance of the GA variants in saccharification was compared with that of the parent molecule (SEQ ID NO: 3). To determine the effect of the amino acid substitutions on this GA property, the % increase in glucose yield compared to the parent molecule was determined and the results are shown on Table 1. Values ​​greater than 0.5% were considered a positive effect of the mutation on saccharification.

[0194] Specific activity determination using colorimetric readout

[0195] The specific activity of the glucoamylase variants was determined based on the rate of glucose release from the selected substrate using the coupled glucose oxidase / peroxidase (GOX / HRP) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (described in Anal. Biochem. 105 (1980), 389-397).

[0196] A 5 μL aliquot of the glucoamylase supernatant was combined in MTP with 25 μL of freshly prepared reagent A solution containing 25 mM sodium acetate buffer (pH 4.5) and a final concentration of 25 U / mL HRP (Sigma-Aldrich, Product No.: P8375), 62 U / mL GOX (Sigma-Aldrich, Product No.: G7141) and 0.005% Tween-80. To initiate the reaction, 20 μL of reagent B containing substrate was added, which consisted of 5.4 mg / mL ABTS (Sigma-Aldrich, Product No.: A1888), 25 mM sodium acetate buffer (pH 4.5) and 0.005% Tween-80. The final substrate concentrations for various assays were: 4.6 mM maltose (Sigma-Aldrich, Product No. 47288), 30 mM panose (Megazymes, O-PAN), 1% (w / v) precooked pullulan (Sigma-Aldrich, Product No. P4516), 0.5% (w / v) precooked soluble starch (Sigma-Aldrich, Product No. S9765), and 1% (w / v) maltodextrin (Sigma-Aldrich, Product No. 31410). The reagents were mixed vigorously and the absorbance kinetics were immediately read using a microplate reader from Molecular Devices at room temperature with continuous stirring at 23 second intervals for 6 minutes. The increase in absorbance at 405 nm is proportional to the hydrolytic activity of the enzyme. The reactions were run in triplicate and the Vo of the samples was determined using linear regression. The data generated in this way are used to calculate the performance of each sample on each determination. The activity rate of each variant divided by the predicted activity rate of the parent GA under the equivalent protein concentration is defined as the performance index (PI). The PI value equal to or greater than 1.1 compared to the parent is used as the cutoff value for selecting the mutation that has a beneficial effect on the specific activity of the molecule. The results are shown in Table 1.

[0197] Thermal stability evaluation of glucoamylase variants

[0198] The glucoamylase variant samples set to 15ppm were incubated in a Mastercycler Pro 384 (Eppendorf) in a 384-well PCR MTP at 57°C for 30min. After incubation, the MTP was cooled to room temperature and used to determine the specific activity on maltodextrin substrate using the above-mentioned ABTS method. The residual activity (activity remaining after heat treatment) percentage (%) of the glucoamylase variants was determined by dividing the activity of the heat incubated sample by the activity of the non-incubated sample. The improvement in thermostability greater than 5% over the parent enzyme was used as a cutoff value for selecting mutations that had a beneficial effect on the thermostability of the molecule. The thermostability results are shown in Table 1 as residual activity %.

[0199] Evaluation of the reversal reaction of glucoamylase variants

[0200] The glucose condensation activity (reversal activity) of each GA variant was tested in the following manner: 67 μl of 50% (w / v) glucose in 10 mM sodium acetate buffer (pH 4.5) was combined with 13 μl of enzyme sample in a 384-well MTP and incubated at 50° C. under shaking for 66 hours. The reaction was quenched by mixing 2.5 μl of the reaction mixture with 97.5 μl of 5 mM H2SO4. The reaction products (glucose and condensation products such as DP2, DP3 and DP3+) were determined and quantified by HPLC analysis as described above.

[0201] The performance of the GA variants was compared to that of the parent molecule (SEQ ID NO: 3). To determine the effect of the amino acid substitutions on the properties of this GA, the reduction in the reversion yield (DP2) compared to the parent molecule (expressed as % condensation product) was determined and the results are shown in Table 1. Under this assay condition, a reversion reduction value equal to or greater than 0.4 was considered a positive effect on reversion.

[0202] Note that for the residual activity comparisons, the values ​​used are absolute values ​​(as measured), whereas for the reversal reactions and for the saccharification yields, the values ​​were calculated as differences in percent yield (e.g., variant A51Y had a 93.2% DP1 yield, while the parental GA had a 91.8% DP1, thus giving a 1.4% improved yield in saccharification yield).

[0203] The Mucorales clade GA variants described in Table 1 all show at least one improved property when compared to the parental sequence SEQ ID NO: 3, indicating the benefit of each amino acid substitution. SEQ ID NO: 3 is equivalent to SEQ ID NO: 4 (SvaGA1) with Pro at position 102. Examples where cells on Table 1 appear blank indicate that no benefit (improvement over the parent) was observed for that particular example. For example, PI < 1.0, % residual activity in thermostability is less than 5% improved (compared to the parental control on the assay plate), and the reversion reduction and saccharification improvement results are less than 0.4% or 0.5% higher than the parent, respectively. The asterisk next to the % residual activity of the parental molecule thermostability indicates that this is an average of multiple tests in the evaluation of the variant molecule.

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Example 3

[0213] Comparison of glucoamylase sequences

[0214] A multiple amino acid sequence alignment was constructed for the predicted mature regions of SEQ ID NO: 3 (SvaGA1v2) and SEQ ID NO: 4 (SvaGA1) and the following 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), MciGA3 (SEQ ID NO: 19), 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 used to determine the corresponding amino acids from position 1 to position 449 of SEQ ID NO: 3 and 4 relative to the sequences of these other GAs. The MUSCLE alignment tool (default parameters) within Geneious 10.2 software was used to align these sequences. The multiple sequence alignment is shown in Figure 1 Panels AH. The percentage of sequence identity between these GAs calculated from the MUSCLE alignment is shown in Table 2. Phylogenetic trees of these Mucorales clade GAs and other GAs were generated using Geneious 10.2 software and are shown in Figure 2 superior.

[0215]

[0216]

[0217] Example 4

[0218] Construction of a recombinant library of variant glucoamylases, transformation in Trichoderma reesei and expression

[0219] DNA expression cassettes of glucoamylase variants containing the selected combinations of mutations (Table 1) were obtained by using standard molecular biology techniques using the above-mentioned pI1c vector and the Seamless Cloning and Assembly Kit (Invitrogen) according to the manufacturer's protocol.

[0220] The preparation and transformation of protoplasts are carried out as described in PCT Publication No. WO 2013 / 102674, and the expression cassette is inserted at a specific position in the fungal host genome. As described in Example 1, the RNP complex assembled in vitro is added to the transformation mixture, which is then plated on a 24-well microtiter plate with selective agar minimal medium. Once the transformants are fully grown and sporulated, they are scraped off and re-plated on fresh selective agar plates. The transformants are further cultivated, and the culture supernatant is prepared as described in Example 1 to obtain a protein quantitative sample of the target glucoamylase. Protein quantification is performed as described in Example 1.

[0221] Example 5

[0222] Evaluation of additional Mucorales clade glucoamylase variants for enzymatic activity in saccharification-related assays

[0223] 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 saccharification assays and reversal assays. A 10 ppm GA solution was made for dosing to all colorimetric ABTS 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.

[0224] The saccharification performance of the variants and the parent molecule (SEQ ID NO: 34) was evaluated after 48 h incubation at pH 4.5 and 62°C using Maltosweet G 120 purchased from Tate & Lyle as substrate (DE = 11-14). The starting DS content of the saccharification blend was 34%. The performance of the glucoamylase was tested at a dose of 29 μg / gds. For this evaluation, for each incubation, the pullulanase OPTIMAX TM L 2500 was dosed at 0.454 ASPU / g DS and α-amylase GC626 TMThe reaction mixture was incubated at 384 wells MTP at pH 4.5, 62 ° C under a shaking of 900 rpm for 48 h. All reactions were quenched by adding 5 mM H2SO4 in a ratio of 1:33 saccharification syrup to acid solution. The product formation of the quenched aliquots was analyzed by HPLC using an Agilent 1260 series system, wherein a double philomon Rezex-RFQ fast acid H+ (8%) 100x7.8mm column (catalog number 00D-0223-K0) was run at 80 ° C for 5.3 minutes. For this purpose, 5 μL samples were injected on the column, and carbohydrates were separated at a flow rate of 1.0 mL / min using an isocratic 5 mM H2SO4 as a mobile phase. Oligosaccharide products were detected using a refractive index detector. Standards were run to determine the elution time for each degree of polymerization (DP(n)) of the sugar of interest (where n represents the number of sugars, e.g., DP3+, DP3, DP2, and DPI). The relative content of each (oligo)saccharide was calculated using the total integrated peak area of ​​the identified saccharification products. The improved saccharification yields of the variants compared to the parent are reported in Table 3 as % glucose. A value of at least 0.3% increase in glucose was used as a selection cutoff for mutations that had a positive impact on GA performance in saccharification under the given conditions.

[0225] Specific activity determination using colorimetric readout

[0226] The specific activity of the glucoamylase variants was determined based on the rate of glucose release from the selected substrate using the coupled glucose oxidase / peroxidase (GOX / HRP) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (described in Anal. Biochem. 105 (1980), 389-397).

[0227] An aliquot of the 10 ppm glucoamylase culture sample was combined in a 384-well MTP with 50 μl of freshly prepared Reagent A solution containing 25 mM sodium acetate buffer (pH 4.5) and a final concentration of 25 U / mL HRP (Sigma-Aldrich, Product No.: P8375), 62 U / mL GOX (Sigma-Aldrich, Product No.: 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 initiate the reaction, 40 vl of reagent B containing substrate was added, which consisted of 3.2 mg / mL ABTS (Sigma-Aldrich, product number: A1888), 25 mM sodium acetate buffer (pH 4.5) and 0.01% Tween-80. The final substrate concentrations for each assay were: 4.5 mM maltose (Sigma-Aldrich, product number: 47288), 15 mM panose (Magnase, O-PAN), 1.5% (w / v) precooked pullulan (Sigma-Aldrich, product number: P4516), 0.5% (w / v) precooked soluble starch (Sigma-Aldrich, product number: S9765) and 0.12% (w / v) maltodextrin (Sigma-Aldrich, product number: 31410). The reagents are mixed violently, and the absorbance kinetics are measured at 405nm at room temperature with 17 seconds intervals for 3.5 minutes using an ELISA reader from Molecular Devices. The increase in absorbance at 405nm is proportional to the hydrolytic activity of the enzyme. The reaction is run in triplicate, and the Vo of the sample is determined using linear regression. The data generated in this way are used to calculate the performance of each sample on each substrate. The performance of each variant is calculated by dividing the Vo of the sample by the Vo of the parent GA. This ratio is defined as a performance index (PI). A PI value equal to or greater than 1.1 compared to the parent GA is used as a cutoff value for selecting a mutation that has a beneficial effect on the specific activity of the molecule. The results are shown in Table 3.

[0228] Thermal stability evaluation of glucoamylase variants

[0229] The glucoamylase variant proteins normalized to 10ppm were incubated in a Mastercycler Pro 384 (Eppendorf) in a PCR MTP at 58°C for 30min. After incubation, the MTP was cooled to room temperature and used to determine the specific activity on the maltodextrin-ABTS substrate as described above. The residual activity (activity remaining after heat treatment) percentage (%) of the glucoamylase variants was determined by dividing the activity of the heat incubated samples by the activity of the non-incubated samples. The improvement in thermal stability greater than 3% over the parent enzyme was used as a cutoff value for selecting mutations that had a beneficial effect on the thermal stability of the molecule. The results are shown in Table 3.

[0230] Evaluation of the reversal reaction of glucoamylase variants

[0231] The glucose condensation activity (reversal activity) of each GA variant was tested in the following manner: 70 μl of 45% (w / v) glucose in 20 mM sodium acetate buffer (pH 4.5) was combined with 10 μl of enzyme sample in a 384-well MTP and incubated at 50° C. for 66 hours under shaking. The reaction was quenched by mixing 3 μl of the reaction mixture with 97 μl of 5 mM H2SO4. The reaction products (glucose and condensation products such as DP2, DP3 and DP3+) were determined and quantified by HPLC analysis as described above.

[0232] The performance of the GA variants was compared to that of the parent molecule (SEQ ID NO: 34). In order to determine the effect of the amino acid substitutions on the properties of this GA, the reduction in the reversal yield (expressed as condensation product (DP2) %) was determined and the results are shown in Table 3. Under the given conditions, a reduction value of DP2 equal to or exceeding 0.1% was considered a positive effect on reversal.

[0233] Note that for the reversal reaction and for saccharification yield, these values ​​are calculated as the difference in percent yield (eg, variant x has a 95.2% DP1 yield, while the parental GA has 94.8% DP1, thus giving a 0.4% improved yield in saccharification yield).

[0234] Table 3. Performance of multiply substituted Mucorales clade glucoamylase variants compared to the SEQ ID NO: 34 parent enzyme.

[0235]

[0236]

[0237]

[0238] All SvaGa1 variants described on Table 3 had significantly improved performance in at least one parameter related to GA performance when compared to the parental molecule (SvaGA1+66A / 102P).

[0239] Example 6

[0240] Effect of N-glycosylation on variants of Saksenaea vasiformis B4078 glucoamylase

[0241] To study the effect of N-glycosylation on the performance of glucoamylase 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 mutations with the potential to generate new N-glycosylation sites were evaluated in the variants corresponding to SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 and 45.

[0242] Polynucleotides encoding GA variant sequences (codon-modified sequences used as expression cassettes) were synthesized by Jierui (Jierui Bioengineering Co., Ltd., Shanghai, China) and inserted into pGX256 expression vector (a derivative vector from pTTT (see U.S. Patent Application Publication No. 20110020899)). All plasmids were transformed into appropriate Trichoderma reesei strains using protoplast transformation (Te'o et al., J. Microbiol. Methods [Microbiological Methods Magazine] 51: 393-99, 2002). These transformants were selected and fermented by the method described in WO 2016 / 138315. The supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis.

[0243] Fungal cell cultures were grown in defined media as described by Lv et al. ((2012) Plasmids [plasmid] 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 combined, concentrated and equilibrated to 20 mM sodium acetate (pH 5.0), 150 mM sodium chloride using an Amicon Ultra-15 apparatus with a 10K MWCO. The purified sample was approximately 99% pure (by SDS-PAGE analysis) and stored in 40% glycerol at -80°C until use.

[0244] Evaluation of the thermal stability of glucoamylase

[0245] The thermal stability of the N-glycosylated variants described in Table 4 was compared, wherein the enzyme samples (20 ppm) were pre-incubated at 55° C. for 40 min. The pre-incubation at 4° C. for 40 min was included and set as 100% activity for each glucoamylase sample. The residual activity of the glucoamylase after pre-incubation was then measured using a coupled glucose oxidase / peroxidase (GOX / HRP) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (Anal. Biochem. [Analytical Biochemistry] 105 (1980), 389-397). The substrate solution was prepared by mixing 9 mL of soluble starch (1% aqueous solution, w / w) and 1 mL of 0.5 M sodium acetate buffer at pH 4.5 in a 15 mL conical tube. In 50mM sodium acetate buffer (pH 5.0), prepare the coupled enzyme (GOX / HRP) solution with ABTS, wherein final concentration is 2.74mg / mL ABTS, 0.1U / mL HRP and 1U / mL GOX. The glucoamylase sample (10 μ L) after pre-incubation is transferred to a new microtiter plate (Corning (Corning) 3641) containing 90 μ L substrate solution pre-incubated for 5min at 600rpm at 50 ℃. The reaction is carried out for 10min at 50 ℃ under vibration (650rpm) in a thermal mixer (Eppendorf), then 10 μ L reaction mixtures are quickly transferred to a new microtiter plate (Corning 3641), respectively, and 90 μ L ABTS / GOX / HRP solutions are added afterwards. SoftMax Pro microplate reader (Molecular Devices) is used immediately to measure the absorbance at 405nm for 5min at 11 seconds intervals. The output of reaction rate Vo is used to indicate glucoamylase activity.

[0246] Using the above-mentioned method, the residual activities of the following N-glycosylation variants were determined compared to the corresponding parental sequences SvaGA1v2 (SEQ ID NO: 3) and SvaGA1v3 (SEQ ID NO: 34): SvaGA1v2_D81N_K83T (SEQ ID NO: 38), SvaGA1v2_A153T (SEQ ID NO: 39), SvaGA1v2_S370N_A372S (SEQ ID NO: 40), SvaGA1v2_D81N_K83L_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). NO: 44) and SvaGA1v3_D81N_K83T_A153T (SEQ ID NO: 45). Table 4 shows the results of the thermostability comparison between the variants designed to evaluate the effect of glycosylation on the backbone of the Phiala B4078 glucoamylase. The GA variants were pre-incubated at 55°C for 40 min, followed by residual activity measurements at 50°C, pH 4.5 for 10 min.

[0247]

[0248] As shown in Table 4, the N-glycosylated variants showed higher residual activity compared to their parental molecules, especially the variant with the substitution D81N_K83T_A153T. SvaGA1v2D81N_K83T_A153T retained 33% of its activity, while the SvaGA1V2 parent retained 21%. SvaGA1v3_D81N_K83T_A153T outperformed its parental molecule, retaining 75% of the residual activity, while the parent SvaGA1v3 was 66%.

[0249] Example 7

[0250] Structural elements of Mucorales clade glucoamylases and variants

[0251] It was observed that substitution of the Asn glycosylation site at position 75 resulted in a reduction in catalytic activity. When tested at 55°C for 40 min, the variant SvaGA1v2 N75D showed approximately a 50% reduction in activity compared to the parent SvaGA1v2 protein. At the time of submission, no existing glucoamylase structures in the Protein Data Bank (worldwideweb.rcsb.org) contained an N-glycosylation site at Asn75 or a structurally equivalent position. This glycosylation site was focused in the Mucorales branch of glucoamylases described in Example 3. To identify additional glucoamylases with these stabilizing interactions, the nr database (National Center for Biotechnology Information) of non-redundant protein sequences was searched using all default BLAST parameters with the SvaGA1v2 (SEQ ID NO: 3) sequence as the query. The top 500 sequences containing sequences from various fungal and bacterial phyla were aligned to MOE 2019.0102 (Chemical Computing Group, Montreal, Canada). Sequences containing the N-glycosylation motif NX-(S / T) (Schwarz and Aebi, (2011) Current Opinion in Structural Biology. [Structural Biology New View] 21: 576-582.) at the structural equivalent position to Asn94 in SvaGA1v2 (SEQ ID NO: 3) were identified. The resulting source organisms included 50 species of Mucoromycota fungi and one other type of organism, a single bacterial species.

[0252] To understand the structural basis for the improved activity from glycosylation at Asn 75, a crystallographic structure was determined. A variant of the glucoamylase from SvaGA1v2 (SEQ ID NO: 3) was cloned and expressed using standard methods as described in PCT Application Publication Nos. WO 2011 / 063308 and WO 2016 / 138315. The fermentation broth was concentrated using a VivaFlow 200 ultrafiltration unit (Sartorius Stedim, Goettingen, Germany). After adding (NH4)2SO4 to a final concentration of 1 M, the concentrate was loaded onto a HiPrep TMPhenyl FF 16 / 10 column (GE Healthcare, Pittsburgh, USA), the column was 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 became stable. Protein elution was started with a gradient of 100% buffer A to 100% 20 mM sodium phosphate (pH 7.0) (buffer B) for 20 min at a flow rate of 5 mL / min. The fractions containing the target protein were combined, concentrated, and the buffer was exchanged into buffer B via a 10 kDa Amicon Ultra-15 device (Merck Millipore Ltd., Darmstadt, Germany).

[0253] Crystals were grown by the hanging drop method using a reservoir solution of 2.13 M ammonium sulfate, 0.1 M sodium acetate trihydrate (pH 4.3). The drop contained 2 μL of 11.5 mg / mL protein and 2 μL of the reservoir solution. Cryoprotection included 17% total glycerol in the reservoir solution.

[0254] Native diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 9-2 using the BLU-ICE data collection environment (http: / / pubmed.ncbi.nlm.nih.gov / 12409628 / ) implemented by Accelero Biostructures, California. The data set was collected at 100 K using a Pilatus 6M detector (Dectris AG, Switzerland). The data were processed using XDS (http: / / pubmed.ncbi.nlm.nih.gov / 20124692 / ) to Resolution.

[0255] Molecular replacement was performed with Phaser (McCoy et al., J. Appl. Cryst. (2007). 40, 658-674) using the homology model as the search model. The homology model was constructed with MOE (Chemical Computing Group, Montreal, Canada) using default parameters using glucoamylase from Saccharomycopsis fibuligera (PDB code 2FBA). (Sevcik et al., (2006) FEBS J [Journal of the Federation of European Biochemical Societies] 273: 2161-2171.). Nineteen rounds of reconstruction and refinement with REFMAC 5.8 (Vagin et al. (2004) Acta Crystallogr. D60: 2284-2295) and Coot (Emsley and Cowtan (2004). Acta Crystallogr. D60, 2126-2132) were performed using isotropic temperature factors, followed by the final three rounds of refinement using anisotropic temperature factors. The resolution range of the refinements was to After final refinement using anisotropic temperature factors, Rworking = 0.12 and Rfree = 0.14. At the highest resolution shell ( to ), Rwork = 0.29 and Rfree = 0.31.

[0256] The x-ray diffraction structure determined for SvaGA1v2 (SEQ ID NO: 3) shows clear electron density for the N-glycosylation site at Asn75. At the time of submission, no existing glucoamylase structures in the Protein Data Bank (worldwideweb.rcsb.org) contain an N-glycosylation site at Asn75 or a structurally equivalent position. The electron density for the Asn75 glycan is shown in Figure 3 The structure shows an extensive network of hydrogen-bonded contacts between the glycan and the protein, bridging four helices at the protein surface ( Figure 4 ). The atoms and contact distances are identified in Table 5. Without being bound by theory, it is proposed that this extensive hydrogen bond network between the Asn75 glycan and the protein chain stabilizes the four protein helices, thereby providing a structural anchor for the substrate binding site and interactions that stabilize the substrate and position it within the catalytic active site.

[0257] Table 5. Hydrogen bond contact distances between proteins and Asn75 glycan atoms

[0258]

[0259] The structure of SvaGA1v2 (SEQ ID NO: 3) also illustrates the 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 expected to stabilize the developing positive charge in the catalytic cycle in the well-studied glucoamylase (Aleshin et al., (1996) Biochemistry 35: 8319-8328). Mutations of residues in this helix, including at positions 66, 67, and 69, are shown in Examples 2 and 5 to improve performance in practice. Without being bound by theory, these mutations may alter the position of the helix in the active site and thus modulate the precise position of Asp62 in order to improve the stability of the developing positive charge. The helix, the conserved Asp62 side chain, and the sites of the performance-improving mutations are shown in Figure 5 middle.

[0260] Other performance improvement mutations are located on the substrate binding ring that comprises residue 100-129. Particularly the mutations in positions 102, 119 and 121 are illustrated in Examples 2 and 5 to improve the performance of enzyme. The ring at these residues places is that residue 100-129 constitutes a large part of the active site surface area. This ring is directly adjacent to the spiral that carries the conservative Asp62 side chain, and it further produces hydrogen bond contacts with Glu184, i.e. the general acid of expecting in the glucoamylase catalytic mechanism. Not limited to theory, the change of the ring conformation caused by the mutation in these positions may cause the change of the accurate location of general acid with respect to substrate, thereby improves application performance. Figure 6 The positions of the loops, expected general acid residues, and the positions of performance improving mutations are shown.

[0261] Another set of performance-improving mutations are located on the four helical bundles that the Asn75 glycan contacts. Mutations in positions 143, 156, 164, 192, and 233 result in increased performance, as shown in Example 5. These mutations are shown in Figure 7 In the figure, the four-helix bundle associated with the glycan chain at residue Asn75 is highlighted in dark grey. The four-helix bundle provides a solid structural core that anchors the substrate binding loop in the active site.

Claims

1. A glucoamylase variant or fragment thereof comprising one or more amino acid substitutions at residue positions corresponding to 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, 23 3, 235, 236, 238, 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 the equivalent position in a parent glucoamylase.

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 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.

3. The glucoamylase variant of claim 2, wherein the equivalent positions are determined by sequence identity and wherein 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.

4. The glucoamylase variant of any one of claims 1-3, wherein the parent glucoamylase is a Mucorales clade glucoamylase.

5. The glucoamylase variant of any one of claims 1-4, wherein the one or more amino acid substitutions comprise one or more of the following: 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, X082 4L / V, X092C / I / M, X094A / G / Y, X102G, X119A / D / G, X121G / L / M / P / V, I, X158A / Y, X164L / T, X165I / T / W, X166A / F / G / H / R, X172G / L, A / C / E / G / L / T / Y, X215C / D / F / H / R / V / W, X218F / A / H / K / Q / V / Y, X221M / R / T, H / M / N / S / T / V, X243A / P / S, X252F / H / M / T, X253K / V, X274A / D / K, X278A, / S, 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.

6. The glucoamylase variant of claim 5, wherein the one or more amino acid substitutions comprise one or more of the following: 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, I084 L / V, V092C / I / M, F094A / G / Y, A / 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, V252F / H, E253K, G274A / D / K, P278A, E281D / P, F290M / V, N302F / H / K / M / P / Q / S / T / V / W, N 310T / V / Y, N321D, 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, and / or F449L.

7. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions at residue positions corresponding to the following positions: 404, 405, 418, 440, 441 and / or 444 of NO:4 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved maltose hydrolysis compared to a parent glucoamylase lacking one or more of these substitutions, or a fragment thereof.

8. The glucoamylase variant of claim 7, wherein the one or more amino acid substitutions comprise one or more of the following: 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 / X352C / F, X352K / 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, 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 glucoamylase variant of claim 8, wherein the one or more amino acid substitutions comprise one or more of the following: 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, G 222M / 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.

10. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions at residue positions 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, 254, 74, 281, 290, 302, 321, 341, 350, 351, 352, 370, 390, 403, 404, 405, 418, 430, 440, 444 and / or 445 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved panose hydrolysis compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions.

11. The glucoamylase variant of claim 10, wherein the one or more amino acid substitutions comprise one or more of the following: 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, X211N, X212V / L, X213 13H, X214A / E, X215D, X218Q / 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, wherein X is the residue corresponding to SEQ Any amino acid at the equivalent position in ID NO: 4 and / or the equivalent position in the parent glucoamylase or a fragment thereof.

12. The glucoamylase variant of claim 11, wherein the one or more amino acid substitutions comprise one or more of the following: 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, 1164L, Y165W / T, K166R, V172G, R21 0L, 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, N3 21D, L341M / T, K350C / T, N351E, T352D / N, S370M, S390D / L / E, Q403G, Q4 03R / K, Y404M / K, H405S / Q, R418E / W, T430A, A440G, A444P, and / or G445Y / M.

13. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions at residue positions corresponding to the following positions: 40, 416, 418, 434, 440 and / or 445 of NO:4 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved pullulan hydrolysis compared to a parent glucoamylase lacking one or more of these substitutions, or a fragment thereof.

14. The glucoamylase variant of claim 13, wherein the one or more amino acid substitutions comprise one or more of the following: 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, X211F / Q / W / Y, X212L / M / F / N / A / I, X213G / D, X214L / M / F / N / A / I, X215 14A / 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, wherein X is the residue corresponding to SEQ Any amino acid at the equivalent position in ID NO: 4 and / or the equivalent position in the parent glucoamylase or a fragment thereof.

15. The glucoamylase variant of claim 14, wherein the one or more amino acid substitutions comprise one or more of the following: S020F / A / G, A051K, V069C, K073P / N, T077M / P, A079T, G080K, D081N, 1084V / 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, S215V / C / W / D / H, S218V / K / A / Q, S221R, G222M, S233M / P, W235Y, D236Q, V252F / H, E253K, G274K / D, P278A, E281D, F29 0M, N302P / S / V, N310T, N321D, L341M, K350I / T, T352D / S, V354L, S370M, S390D / L / E, Y404K, F416Y, R418E / W, A434S, A440G, and / or G445M.

16. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions at residue positions 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 and / or the equivalent position in a parent glucoamylase or a fragment thereof, and wherein the variant glucoamylase exhibits a higher performance index (PI) for saccharifying soluble starch than a parent glucoamylase lacking one or more of these substitutions.

17. The glucoamylase variant of claim 16, wherein the one or more amino acid substitutions comprise one or more of the following: 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, 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, X404I / M / K, X405S, X416C / Y, X418E / W, X422A, X440G, X441L, X444S / P, and / or X445M, wherein X is the residue corresponding to SEQ ID Any amino acid at the equivalent position in NO: 4 and / or the equivalent position in the parent glucoamylase or a fragment thereof.

18. The glucoamylase variant of claim 17, wherein the one or more amino acid substitutions comprise one or more 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, D213 H, 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, L341 M / 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.

19. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions at 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 and / or the equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved maltodextrin hydrolysis compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions.

20. The glucoamylase variant of claim 19, wherein the one or more amino acid substitutions comprise one or more of the following: 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, X213 R, X214A / E, X215D / C / F, X218V / Y / Q, X221R / T, X222M / C, 236Q / A, X252F, X274K / A / D, X281D, X290M / V, X302P / K / S / V / Q / W, In some embodiments, the present invention relates to a glucoamylase or a fragment thereof, 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 a fragment thereof.

21. The glucoamylase variant of claim 20, wherein the one or more amino acid substitutions comprise one or more of the following: 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, D2 13R, 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.

22. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises 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 an equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved thermostability compared to a parent glucoamylase or fragment thereof lacking one or more of these substitutions.

23. The glucoamylase variant of claim 22, wherein the one or more amino acid substitutions comprise one or more of the following: 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, 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.

24. The glucoamylase variant of claim 23, wherein the one or more amino acid substitutions comprise one or more of the following: E023M, A051Y, G052N, V066A / C / F / M / W, S067C, T077P, S119A, T121M, E134S, M140I / C, L141F, F156C, T157I, Y16 5W / I, Y192F, D213R, S221R, G222M, S233M, D236Q, V252F, E253F, E281D, F290V, N302 H / Q / W / S / K, K350E / C, T352D, S370M, S390L / D, Q403R, Y404K / M, F416Y, and / or Y422A / V.

25. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions at residue positions 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 an equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits less reversion to sugars having a degree of polymerization (DP) greater than or equal to 2 as compared to a parent glucoamylase or fragment thereof lacking one or more of the substitutions.

26. The glucoamylase variant of claim 25, wherein the one or more amino acid substitutions comprise one or more of the following: 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, 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, 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.

27. The glucoamylase variant of claim 26, wherein the one or more amino acid substitutions comprise one or more of the following: S049W, A051Y, G052P, V66P, V069K / C, T077P / M, G080H, F094A / G, S119D, E134W / Q, N158Y, 1164T / L, Y165W / I, V172G, Y192R / F, D213H, S215R, S218K, G 222C, S233T / P, W235F, 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.

28. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises 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 an equivalent position in a parent glucoamylase, and wherein the variant glucoamylase exhibits improved saccharification yield of glucose compared to a parent glucoamylase lacking one or more of the substitutions.

29. The glucoamylase variant of claim 28, wherein the one or more amino acid substitutions comprise one or more of the following: X021S / W, X023L, X051Y / K, X052N, X066 / A / C / F / M / W, X067M / C / A, X079R, X081S, X092C / M, X119A, X140I, X158A / Y, X1 [0147] In some embodiments, the present invention relates to an amino acid sequence of the present invention that is / are a polypeptide of the invention or a parent glucoamylase, 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.

30. The glucoamylase variant of claim 29, wherein the one or more amino acid substitutions comprise one or more of the following: 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, N214 Y, 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.

31. The glucoamylase variant of any one of claims 1-29, wherein the variant comprises one or more amino acid substitutions at residue positions corresponding to the following positions: NO: 4 position 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, 4 04, 405, 416, 418, 422, 430, 440, 441, 444 and / or 445 and / or the equivalent position in a parent glucoamylase or a fragment thereof, and wherein the variant glucoamylase exhibits two or more of the following as compared to a parent glucoamylase lacking one or more of these substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) improved soluble starch hydrolysis; v) improved maltodextrin hydrolysis; vi) improved thermal stability; vii) less reversion to sugars with a DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.

32. The glucoamylase variant of claim 31, wherein the one or more amino acid substitutions comprise one or more of the following: 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, C / P, X157I, X158A / Y, X164L / T, X165I / T / W, X166G / F / R, , X214A / E / Y, X215C / D / F / R, X218A / K / Q / V / Y, X221M / R / T, Q, X238H / S, X243A, X252F, X253K, X274A / D / K, X278A, X281D, X290M / V, X310A, X310G / H, X311L, X311E / V, X312D / N / S, X312L / M, X313M / T, X314C / E / I, X314E / V, X315D / E / L, X316G / K / R, X316K / M, X318E / 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.

33. The glucoamylase variant of claim 32, wherein the one or more amino acid substitutions comprise one or more of the following: 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, D 081N / 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, Y192F, D203C / W, R210L / M / W, D 213H / 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, 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, and / or G445M / Y.

34. The glucoamylase variant of any one of claims 1 to 6, wherein the variant comprises two or more amino acid substitutions comprising: 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) X 243P and X290V, j) X290V and X350E, k) X302K and X441W, 1) 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-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 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.

35. The glucoamylase variant of claim 34, wherein the two or more amino acid substitutions comprise 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 F2 90V, 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) T 243P 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) G0 52N and Y404K, 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.

36. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises three or more amino acid substitutions comprising: 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, 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.

37. The glucoamylase variant of claim 36, wherein the three or more amino acid substitutions include 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.

38. The glucoamylase variant of any one of claims 1-6, wherein 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, 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.

39. The glucoamylase variant of claim 38, wherein 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.

40. The glucoamylase variant of any one of claims 1-6, wherein the variant comprises five or more amino acid substitutions including X067M, X157I, X218H, X302H, and X416Y, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO: 4 and / or an equivalent position in a parent glucoamylase or fragment thereof.

41. The glucoamylase variant of claim 40, wherein the five or more amino acid substitutions include S067M, T157I, S218H, N302H, and F416Y.

42. The glucoamylase variant of any one of claims 34-41, wherein the variant glucoamylase exhibits one or more of the following compared to the parent glucoamylase lacking the substitutions: i) improved maltose hydrolysis; ii) improved panose hydrolysis; iii) improved pullulan hydrolysis; iv) improved soluble starch hydrolysis; v) improved maltodextrin hydrolysis; vi) improved thermostability; vii) less reversion to sugars with a DP greater than or equal to 2; and / or viii) improved saccharification yield of glucose.

43. The glucoamylase variant of any one of claims 1-6, wherein 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 in a parent glucoamylase or fragment thereof.

44. The glucoamylase of claim 43, wherein the variant comprises amino acid substitutions comprising 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.

45. The glucoamylase of claim 44, wherein the variant comprises amino acid substitutions comprising 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.

46. ​​The glucoamylase variant of any one of claims 1-45, further comprising an amino acid substitution at one or more residue positions corresponding to position 66 and / or 102 of SEQ ID NO: 4 and / or an equivalent position in a parent glucoamylase.

47. The glucoamylase variant of claim 46, wherein the one or more amino acid substitutions comprise X066A / C / F / M / P / T / W and / or X102P / G, wherein X is any amino acid corresponding to the equivalent position in SEQ ID NO: 4 and / or the equivalent position in the parent glucoamylase.

48. The glucoamylase variant of claim 47, wherein the one or more amino acid substitutions comprise V066A / C / F / M / P / T / W and / or S102P / G.

49. The glucoamylase variant of any one of claims 1-48, comprising N-linked glycosylation at position N075 of SEQ ID NO: 4 and / or an equivalent position in a parent glucoamylase.

50. The glucoamylase variant of any one of claims 1-49, 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 in a parent glucoamylase, and wherein the variant glucoamylase exhibits increased glycosylation and increased thermostability compared to a parent glucoamylase lacking one or more of these substitutions.

51. The glucoamylase of claim 50, wherein the one or more substitutions comprise one or more substitutions at positions D81N, K83T, A153T, S370N and / or A372S.

52. A polynucleotide encoding the glucoamylase variant of any one of claims 1-51.

53. A vector comprising the polynucleotide of claim 52.

54. A host cell comprising the polynucleotide of claim 52 or the vector of claim 53.

55. The host cell of claim 54, wherein the host cell is a bacterial or fungal host cell.

56. The host cell of claim 54 or claim 55, wherein the host cell is an Aspergillus species, a Bacillus species, a Trichoderma species, a Pichia species, a Myceliophthora species, or a Saccharomyces species.

57. An enzyme composition comprising the glucoamylase variant of any one of claims 1-51.

58. The enzyme composition of claim 57, wherein the composition is used in a starch conversion process.

59. The enzyme composition of claim 57, wherein the composition is used in an animal feed formulation.

60. The enzyme composition of claim 57, wherein the composition is used in an alcohol fermentation process.

61. The enzyme composition of claim 57, wherein the composition is used in a process for preparing a fermented beverage.

62. A method for producing a variant glucoamylase in a host cell, the method comprising a) culturing a host cell transformed with the vector of claim 53 in a culture medium under conditions suitable for producing the glucoamylase variant; and b) producing the variant.

63. The method of claim 62, further comprising recovering the glucoamylase variant from the culture medium.

64. The method of claim 62 or claim 63, wherein the host cell is a bacterial or fungal host cell.

65. The method of any one of claims 62-64, wherein the host cell is an Aspergillus species, a Bacillus species, or a Trichoderma species, a Pichia species, a Myceliophthora species, or a Saccharomyces species.

66. A method of saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises: a) contacting a starch composition with a glucoamylase variant according to any one of claims 1 to 51; and b) saccharifying the starch composition to produce the glucose composition.

67. The method of claim 67, wherein the composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch.

68. The method of claim 66 or claim 67, wherein the method further comprises c) contacting the starch composition with an alpha-amylase.

69. The method of any one of claims 66-68, wherein the method further comprises d) contacting the starch composition with a pullulanase.

70. The method of any one of claims 66-69, further comprising e) fermenting the glucose composition to produce a fermentation product.

71. The method of claim 70, wherein the fermentation product is alcohol.

72. The method of claim 71, wherein the alcohol is ethanol or butanol.

73. The method of any one of claims 66-72, further comprising f) adding to the starch composition one or more of an additional glucoamylase, a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a protease, a pullulanase, a β-amylase, an additional α-amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, an oxidoreductase, an esterase, a transferase, a pectinase, an α-glucosidase, a β-glucosidase, a lyase, a hydrolase, or a combination thereof.

74. The method of any one of claims 66-73, wherein the fermentation is a simultaneous saccharification and fermentation (SSF) reaction.

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