Glucoamylase and methods of use thereof

Abstract

To provide a novel glucoamylase and a method for saccharifying a starch substrate in order to improve saccharification efficiency and provide high yields of fermentation products.SOLUTION: A method comprising contacting a starch substrate with a glucoamylase or 1,4-alpha-D-glucan glucohydrolase (EC3.2.1.3) that is a polypeptide derived from the Mucorales clade, preferably having high sequence identity to the glucoamylase derived from Saksenaea vasiformis.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2020 / 085393, filed April 17, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to recombinant host cells, compositions comprising glucoamylases, and methods for saccharifying starch substrates using glucoamylases. Furthermore, the present disclosure also relates to processes for producing fermentation products and methods for increasing starch digestibility in animals, as well as methods for producing fermented beverages. [Background technology]

[0003] 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 and related oligo- and polysaccharide molecules. Glucoamylases are produced by several species of filamentous fungi and yeasts.

[0004] The primary use of glucoamylase is the saccharification of partially processed starch / dextrins to glucose, an essential substrate for many fermentation processes. The glucose can then be converted directly or indirectly into fermentation products using fermenting organisms. Commercially available examples of fermentation products include alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactate, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2), and more complex compounds.

[0005] The end product can be a syrup. For example, the end product can be glucose, which can be converted, for example, by glucose isomerase, to fructose or to a mixture of approximately equal amounts of glucose and fructose. This mixture, or a more fructose-enriched mixture, is the most commonly used high fructose corn syrup (HFCS) commercially available worldwide. Summary of the Invention [Problem to be solved by the invention]

[0006] Although a diverse group of microorganisms have been reported to produce glucoamylase, glucoamylase for commercial purposes has traditionally been produced using filamentous fungi because they secrete large amounts of the enzyme extracellularly. However, commercially used fungal glucoamylases have certain limitations, such as slow catalytic activity or lack of stability, which increases processing costs.

[0007] Thus, there remains a need for novel glucoamylases to improve the efficiency of saccharification and provide high yields of fermentation products. [Means for solving the problem]

[0008] The present disclosure relates to recombinant host cells, compositions comprising glucoamylases, and methods for saccharifying starch substrates using glucoamylases. Furthermore, the present disclosure also relates to processes for producing fermented products and methods for increasing starch digestibility in animals, as well as methods for producing fermented beverages. 1. In one aspect, a method for saccharifying a starch substrate, comprising: (a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; (b) a polypeptide at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 81; and (c): (i) YX a X b TXXX c X d (SEQ ID NO: 113) wherein X is any amino acid and X a is N or S; X b is T, S, or R; X c is G or N; and X d is D, N, or S); (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii)X a X b X c X c AANXX d (SEQ ID NO: 115) wherein X is any amino acid and X a is S or A; X b is T, N, or V; X c is L or I; and X d is A or G); (iv) STLIAANXA (SEQ ID NO: 116) (wherein X is any amino acid); (v) X a GXGNX b X c (SEQ ID NO: 117) wherein X is any amino acid and X a is N or D; X b is S or G; and X c is Q, K, or E); and (vi) a polypeptide comprising one or more sequence motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO: 61. 2. In some embodiments of the method of paragraph 1, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 3. In some embodiments of the method described in paragraph 2, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 4. In some embodiments of the method of any one of paragraphs 1-3, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 5. In some embodiments of the method described in paragraph 4, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 6. In some embodiments of the method of any one of paragraphs 1-5, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118 Including SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 7. In some embodiments of the method described in any one of paragraphs 1-6, the starch substrate has a dry solids (DS) of about 15%-65%, 15%-60%, or 15%-35%. 8. In some embodiments of the method described in any one of paragraphs 1-7, the starch substrate comprises liquefied starch, gelatinized starch, or granular starch. 9. In some embodiments of the method described in any one of paragraphs 1-8, the method further comprises adding to the starch substrate hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, beta-amylase, alpha-amylase, protease, cellulase, hemicellulase, lipase, cutinase, trehalase, isoamylase, oxidoreductase, esterase, transferase, pectinase, hydrolase, alpha-glucosidase, beta-glucosidase, or a combination thereof. 10. In some embodiments of the method described in any one of paragraphs 1-9, saccharifying the starch substrate produces a high glucose syrup comprising an amount of glucose selected from the group consisting of at least 95.5% glucose, at least 95.6% glucose, at least 95.7% glucose, at least 95.8% glucose, at least 95.9% glucose, at least 96% glucose, at least 96.1% glucose, at least 96.2% glucose, at least 96.3% glucose, at least 96.4% glucose, at least 96.5% glucose, and at least 97% glucose. 11. Some embodiments of the method of any one of paragraphs 1-10 further include fermenting the high glucose syrup into an end product. 12. In some embodiments of the method described in paragraph 11, the saccharifying and fermenting steps are carried out as a simultaneous saccharification and fermentation (SSF) process. 13. In some embodiments of the method described in paragraphs 11 or 12, the end product is an alcohol, optionally ethanol. 14. In some embodiments of the method described in paragraphs 11 or 12, the end product is a biochemical selected from the group consisting of amino acids, organic acids, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega-3 fatty acids, butanol, lysine, itaconic acid, 1,3-propanediol, biodiesel, and isoprene. 15. In yet another embodiment, a process for producing a fermentation product from a starch substrate, comprising: 1) liquefying a starch substrate; 2) saccharifying the liquefied starch substrate; and 3) A process of fermenting using a fermenting organism Step 2) is: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and (c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO: 61. 16. In some embodiments of the process described in paragraph 15, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 17. In some embodiments of the process described in paragraph 16, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 18. In some embodiments of the process of any one of paragraphs 15-17, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 19. In some embodiments of the process described in paragraph 18, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 20. In some embodiments of the process of any one of paragraphs 15-19, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, Including SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 21. In yet another embodiment, a process for producing a fermentation product from a starch substrate, comprising: 1) saccharifying a starch substrate at a temperature below the initial gelatinization temperature of the starch substrate; and 2) fermenting using a fermentation organism; Step 1) is: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO: 61. 22. In some embodiments of the process described in paragraph 21, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 23. In some embodiments of the process described in paragraph 22, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 24. In some embodiments of the process of any one of paragraphs 21-23, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 25. In some embodiments of the process described in paragraph 24, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 26. In some embodiments of the process of any one of paragraphs 21-25, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, Including SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 27. In another embodiment, a method for increasing starch digestibility in an animal, comprising: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO: 61. 28. In some embodiments of the method of paragraph 27, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 29. In some embodiments of the method described in paragraph 28, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 30. In some embodiments of the method of any one of paragraphs 27-29, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 31. In some embodiments of the method described in paragraph 30, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 32. In some embodiments of the method of any one of paragraphs 27-31, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO: Includes sequence number 88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 33. In yet another aspect, a method for producing a fermented beverage, comprising subjecting a mash and / or wort to: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO: 61. 34. In some embodiments of the method of paragraph 33, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 35. In some embodiments of the method described in paragraph 34, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 36. In some embodiments of the method of any one of paragraphs 33-35, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 37. In some embodiments of the method described in paragraph 36, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 38. In some embodiments of the method of any one of paragraphs 33-37, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO: Includes sequence number 88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 39. In another embodiment, a starch substrate and: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); 1. A composition comprising one glucoamylase selected from the group consisting of: wherein the polypeptide has at least 70% identity to the catalytic domain of SEQ ID NO: 61; and wherein the composition is at a temperature of about 4-40°C and a pH of about 3-7. 40. In some embodiments of the composition of paragraph 39, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 41. In some embodiments of the composition of paragraph 40, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 42. In some embodiments of the composition of any one of paragraphs 39-41, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 43. In some embodiments of the composition of paragraph 42, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 44. In some embodiments of the composition of any one of paragraphs 39-43, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, Including SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 45. In another aspect: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 or SEQ ID NO: 142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGNGNSQ (SEQ ID NO: 118); 1. A recombinant host cell comprising a glucoamylase selected from the group consisting of: wherein the polypeptide has at least 70% identity to the catalytic domain of SEQ ID NO:61. 46. ​​In some embodiments of the recombinant host cell described in paragraph 45, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:61. 47. In some embodiments of the recombinant host cell described in paragraph 46, the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H. 48. In some embodiments of the recombinant host cell of any one of paragraphs 45-47, the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:61. 49. In some embodiments of the recombinant host cell described in paragraph 48, the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H. 50. In some embodiments of the recombinant host cell described in any one of paragraphs 45-49, the polypeptide is selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ 7, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:142. 51. In some embodiments of the recombinant host cell described in paragraphs 45-50, it is an ethanol-producing microorganism. 52. In some embodiments of the recombinant host cell described in paragraph 51, it is a yeast cell. 53. In some embodiments of the recombinant host cell of any one of paragraphs 45-52, the host cell is not Saksenaea vasiformis.

[0009] Each of the aspects and embodiments described herein can be used together unless excluded either explicitly or unambiguously from the context of that embodiment or aspect.

[0010] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. All patents, patent applications and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows multiple amino acid sequence alignments of the catalytic domain region of Mucorales-clade glucoamylases and various reference filamentous fungal glucoamylases.

[0012] [Figure 2] FIG. 1 shows a dendrogram of Mucorales-clade glucoamylases and various other filamentous fungal glucoamylases.

[0013] [Figure 3] FIG. 1 shows an alignment of Mucorales-clade GA amino acid sequences (numbered according to the SvaGal catalytic domain region, SEQ ID NO: 81) over the region spanning residues 50-70, showing motif 1: 57Y-58Xa-59Xb-60T-61X-62X-63Xc-64Xd (where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S).

[0014] [Figure 4] FIG. 1 shows an alignment of Mucorales-clade GA amino acid sequences (numbered according to the SvaGal catalytic domain region, SEQ ID NO: 81) over the region spanning residues 240-260, showing motif 2: 244Xa-245Xb-246Xc-247Xc-248A-249A-250N-251X-252Xd (where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G).

[0015] [Figure 5]FIG. 1 shows an alignment of Mucorales-clade GA amino acid sequences (numbered according to the SvaGal catalytic domain region, SEQ ID NO: 81) over the region spanning residues 299-315, showing motif 3: 304Xa-305G-306X-307G-308N-309Xb-310Xc (where X is any amino acid, Xa is N or D; Xb is S or G; and Xc is Q, K, or E).

[0016] [Figure 6] Figure 1 shows multiple amino acid sequence alignments of the catalytic domain region of additional Mucorales-clade glucoamylases and various reference filamentous fungal glucoamylases.

[0017] [Figure 7] FIG. 1 shows a dendrogram of additional Mucorales-clade glucoamylases and other filamentous fungal glucoamylases. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present disclosure relates to recombinant host cells, compositions comprising glucoamylases, and methods for saccharifying starch substrates using glucoamylases. Furthermore, the present disclosure also relates to processes for producing fermented products and methods for increasing starch digestibility in animals, as well as methods for producing fermented beverages.

[0019] I. Definition Before describing the present compositions and methods in detail, the following terms and abbreviations are defined.

[0020] Unless otherwise defined, all technical and scientific terms used have their usual meaning in the relevant scientific field. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, New York (1994) and Hale & Markham, Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide the usual meanings of many of the terms describing this invention.

[0021] 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.

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

[0023] The term "mature polypeptide" is defined herein as a polypeptide in its final form after translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one embodiment, the predicted mature polypeptide is SEQ ID NO: 61, based on analysis with SignalP software version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786), and SEQ ID NO: 41 is the signal peptide. In another embodiment, the mature polypeptide comprises amino acid positions 20-468 of SEQ ID NO: 142. In another embodiment, the mature polypeptide comprises amino acid positions 21-468 of SEQ ID NO: 142. In another embodiment, the mature polypeptide comprises amino acid positions 22-468 of SEQ ID NO: 142. In another embodiment, the mature polypeptide comprises amino acid positions 23-468 of SEQ ID NO: 142. In another embodiment, the mature polypeptide comprises amino acid positions 24-468 ​​of SEQ ID NO: 142. In yet another embodiment, the mature polypeptide comprises amino acid positions 25-468 of SEQ ID NO:142.

[0024] A "signal sequence" or "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside of a cell. The mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process. In some embodiments, SEQ ID NO:41 is the signal peptide. In other embodiments, the signal peptide comprises amino acid positions 1-20 of SEQ ID NO:142. In other embodiments, the signal peptide comprises amino acid positions 1-21 of SEQ ID NO:142. In other embodiments, the signal peptide comprises amino acid positions 1-22 of SEQ ID NO:142. In other embodiments, the signal peptide comprises amino acid positions 1-23 of SEQ ID NO:142. In other embodiments, the signal peptide comprises amino acid positions 1-24 of SEQ ID NO:142.

[0025] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single- or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode specific amino acid sequences. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' direction.

[0026] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. A coding sequence can be a DNA, cDNA, synthetic molecule, or recombinant nucleotide sequence.

[0027] As used herein, the term "cDNA" is defined as a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic cells. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps before appearing as mature, spliced ​​mRNA. These steps include the removal of intron sequences through a process called splicing. Therefore, cDNA derived from mRNA lacks intron sequences.

[0028] A "synthetic" molecule is produced not by a living organism, but rather by in vitro chemical or enzymatic synthesis.

[0029] A "host strain" or "host cell" refers to an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0030] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence, including both transcription and translation.

[0031] The term "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0032] "Expression vector" refers to a DNA construct containing a DNA sequence encoding a polypeptide of interest, the coding sequence operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and sequences to control the termination of transcription and translation.

[0033] As used herein, the term "control sequences" is defined to include all components necessary for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding the polypeptide.

[0034] The term "operably linked" means that certain components are in a relationship, including, but not limited to, juxtaposition, that permits them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0035] The term "sequence motif" refers to a nucleotide or amino acid sequence pattern that is widespread and has proven or predicted biological significance. In the present invention, the sequence motif is an amino acid sequence motif identified within the Mucorales clade glucoamylases.

[0036] "Biologically active" refers to a sequence that has a particular biological activity, such as enzymatic activity.

[0037] The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0038] "Percent sequence identity" means that a particular sequence has at least a certain percentage of identical amino acid residues as in a particular reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are as follows: Gap Start Penalty: 10.0 Gap extension penalty: 0.05 Protein Weight Matrix: BLOSUM Series DNA Weight Matrix: IUB Delay divergence array (%): 40 Gap Separation Distance: 8 DNA transition weight: 0.50 List of hydrophilic residues: GPSNDQEKR Use Negative Matrix: Off Toggle residue-specific penalty: On Toggle Hydrophilic Penalty:On Toggle end gap separation penalty off.

[0039] As used herein, the term "homologous sequence" is defined as a predicted protein having an E-value (i.e., expectation score) of less than 0.001 in a tfasty search (Pearson, WR, 1999, Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) using the glucoamylase of SEQ ID NO: 61.

[0040] As used herein, "corresponding to" or "corresponding to" or "corresponding" in reference to an amino acid residue position refers to the amino acid residue at the recited position in the protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to the recited residue in the protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related or reference protein.

[0041] The terms "wild-type," "parent," or "reference" with respect to a polypeptide refer to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wild-type," "parent," or "reference" with respect to a polynucleotide refer to a naturally occurring polynucleotide that does not contain artificial nucleoside changes. However, it should be noted that a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but encompasses any polynucleotide that encodes a wild-type, parent, or reference polypeptide.

[0042] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microorganism, e.g., an ethanologenic microorganism, and at least one enzyme, e.g., an amylase, are present in the same process step. SSF involves the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) to sugars, including glucose, and the fermentation of the sugars to alcohol or other biochemicals or biological materials in the same reaction vessel.

[0043] A "slurry" is an aqueous mixture containing insoluble starch granules in water.

[0044] The term "total sugar content" refers to the content of all soluble sugars present in a starch composition, including monosaccharides, oligosaccharides, and polysaccharides.

[0045] The term "dry solids" (ds) refers to dry solids dissolved in water, dry solids dispersed in water, or a combination of both. Thus, dry solids include granular starch and the hydrolysis products of this granular starch (e.g., glucose).

[0046] The term "high DS" refers to an aqueous starch slurry having a dry solids content greater than 38% (w / w).

[0047] "Degree of polymerization (DP)" refers to the number of anhydroglucopyranose units (n) in a given sugar. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP4+ (>DP3) refers to polymers with a degree of polymerization greater than 3.

[0048] The term "contacting" refers to placing the referenced components (including, but not limited to, an enzyme, a substrate, and a fermenting organism) in sufficient proximity to affect a desired result, such as an enzyme acting on a substrate or a fermenting organism fermenting a substrate.

[0049] As used herein, the terms "yeast cell," "yeast strain," or simply "yeast" refer to organisms from the phylum Ascomycota and Basidiomycota yeast cells. An exemplary yeast is Saccharomyces cerevisiae, formerly from the order Saccharomycetales. Specific examples of yeast are Saccharomyces spp., including, but not limited to, S. cerevisiae. Yeast includes organisms used for the production of fuel alcohol as well as organisms used for the production of potable alcohol, including specialized and proprietary yeast strains used to produce distinctive tasting beer, wine, and other fermented beverages.

[0050] "Ethanol-producing microorganism" refers to a microorganism with the ability to convert sugars or other carbohydrates into ethanol.

[0051] The term "biochemicals" refers to metabolic products of microorganisms such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega-3 fatty acids, butanol, iso-butanol, amino acids, lysine, itaconic acid, other organic acids, 1,3-propanediol, vitamins, or isoprene, or other biomaterials.

[0052] The term "pullulanase", also called debranching enzyme (EC 3.2.1.41, pullulan 6-glucanohydrolase), is capable of hydrolyzing the alpha 1-6 glucosidic bond in the amylopectin molecule.

[0053] In this specification, certain ranges are indicated by numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the unrecited near or approximately number may be a number that, in the context in which the number is recited, provides a substantial equivalent to the specifically recited number. For example, with respect to a numerical value, the term "about" means a range of -15% to +15% of the numerical value, unless the term is clearly defined otherwise in the context.

[0054] The following abbreviations / acronyms have the following meanings unless otherwise specified: EC Enzyme Committee CAZy carbohydrate-active enzyme w / v weight / volume w / w weight / weight v / v volume / volume wt% weight% ℃ Celsius temperature g or gm grams μg microgram mg milligram kg kilogram μL and μl microliters mL and ml milliliters mm millimeters μm micrometer mol mole mmol millimole M molar concentration mM millimolar concentration μM micromolar nm nanometer U units ppm parts per million hr and h time EtOH ethanol

[0055] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise.

[0056] It should be further noted that the claims may be drafted to exclude any optional element, and as such, this statement is intended to serve as antecedent basis for using exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0057] The term "comprising" and its cognates are used in their inclusive sense, i.e., equivalent to the term "including" and its corresponding cognates. Furthermore, it should be noted that the term "comprising," as used herein, means including, but not limited to, the component listed after "comprising." The component following the term "comprising" is required or essential, but a composition containing this component may further include other non-essential or optional components.

[0058] It should be further noted that the term "consisting essentially of," as used herein, means a composition in which the component following this term is present in the presence of other known components in a total amount of less than 30% by weight of the total composition and does not contribute to or interfere with the action or activity of that component.

[0059] It should also be noted that the term "consisting of," as used herein, means including and limited to the components following the term "consisting of." Thus, the component following the term "consisting of" is required or essential, and no other component is present in the composition.

[0060] It is intended that every numerical upper limit given throughout this specification will include every lower numerical limit, as if such lower numerical limit were expressly stated herein. Every numerical lower limit given throughout this specification will include every higher numerical limit, as if such higher numerical limit were expressly stated herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly stated herein.

[0061] 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.

[0062] Other definitions of terms may appear throughout this specification.

[0063] II. Polypeptides with glucoamylase activity In a first aspect, the present invention relates to a polypeptide comprising an amino acid sequence having preferably 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: 61 or SEQ ID NO: 142, and having glucoamylase activity. In yet another aspect, the present specification provides a polypeptide comprising an amino acid sequence having preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and even at least 99% amino acid sequence identity to a polypeptide comprising amino acid positions 20 to 468 of SEQ ID NO: 142, amino acid positions 21 to 468 of SEQ ID NO: 142, amino acid positions 22 to 468 of SEQ ID NO: 142, amino acid positions 23 to 468 of SEQ ID NO: 142, amino acid positions 24 to 468 of SEQ ID NO: 142, or amino acid positions 25 to 468 of SEQ ID NO: 142.

[0064] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 61 or SEQ ID NO: 142. In other embodiments, the polypeptide comprises an amino acid sequence having at least 70% but less than 100% sequence identity to a polypeptide comprising amino acid positions 20-468 of SEQ ID NO: 142, amino acid positions 21-468 of SEQ ID NO: 142, amino acid positions 22-468 of SEQ ID NO: 142, amino acid positions 23-468 of SEQ ID NO: 142, amino acid positions 24-468 ​​of SEQ ID NO: 142, or amino acid positions 25-468 of SEQ ID NO: 142. In some embodiments, the polypeptide is non-naturally occurring (i.e., not occurring in nature but rather a product of human ingenuity).

[0065] In some embodiments, a polypeptide of the present invention is a homologous polypeptide comprising an amino acid sequence that differs by no more than 10 amino acids, no more than 9 amino acids, no more than 8 amino acids, no more than 7 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid from the polypeptide of SEQ ID NO: 61, the polypeptide of SEQ ID NO: 142, the polypeptide comprising amino acid positions 20 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 21 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 22 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 23 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 24 to 468 of SEQ ID NO: 142, or the polypeptide comprising amino acid positions 25 to 468 of SEQ ID NO: 142.

[0066] In some embodiments, the polypeptide of the invention is a catalytic region comprising amino acids 18-449 of SEQ ID NO: 61 as predicted by the ClustalX Hypertext Transfer Protocol Secure: / / world wide web.ncbi.nlm.nih.gov / pubmed / 17846036.

[0067] In some embodiments, the polypeptides of the invention have pullulan hydrolyzing activity.

[0068] In a second aspect, the glucoamylase of the invention disclosed herein comprises the amino acid sequence of SEQ ID NO:61, the polypeptide of SEQ ID NO:142, the polypeptide comprising amino acid positions 20-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 21-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 22-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 23-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 24-468 ​​of SEQ ID NO:142, or a conservative substitution of one or more amino acid residues relative to amino acid positions 25-468 of SEQ ID NO:142. Exemplary conservative amino acid substitutions are listed below. While some conservative substitutions (i.e., mutations) can be made by genetic engineering, other conservative variations are made by introducing synthetic amino acids into the polypeptide by other means. [Table 1]

[0069] In some embodiments, the polypeptide of the invention is a variant of the polypeptide of SEQ ID NO:61, the polypeptide of SEQ ID NO:142, the polypeptide comprising amino acid positions 20-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 21-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 22-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 23-468 of SEQ ID NO:142, the polypeptide comprising amino acid positions 24-468 ​​of SEQ ID NO:142, or the polypeptide comprising amino acid positions 25-468 of SEQ ID NO:142, or a fragment thereof that has glucoamylase activity. The variant glucoamylase comprises a deletion, substitution, insertion, or addition of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:142, or a homologous sequence thereof. In all cases, the phrase "one or several amino acid residues" refers to 10 or fewer amino acid residues, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid substitutions, deletions, and / or insertions in the polypeptide of SEQ ID NO: 61, the polypeptide of SEQ ID NO: 142, the polypeptide comprising amino acid positions 20 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 21 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 22 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 23 to 468 of SEQ ID NO: 142, the polypeptide comprising amino acid positions 24 to 468 of SEQ ID NO: 142, or the polypeptide comprising amino acid positions 25 to 468 of SEQ ID NO: 142 may be 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, or even at most 1.

[0070] In some embodiments, the variant change comprises or consists of a substitution at a position corresponding to position 102 of the polypeptide of SEQ ID NO: 61. In some embodiments, the amino acid at a position corresponding to position 102 of the polypeptide of SEQ ID NO: 61 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Ile, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably Pro. In some embodiments, the variant change comprises or consists of the substitution S102P in the polypeptide of SEQ ID NO: 61. In yet other embodiments, the variant comprises or consists of the amino acid sequence of SEQ ID NO: 104 or SEQ ID NO: 141.

[0071] In some embodiments, the variant change comprises or consists of a substitution at a position corresponding to position 66 of the polypeptide of SEQ ID NO: 61. In some embodiments, the amino acid at a position corresponding to position 85 of the polypeptide of SEQ ID NO: 61 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 change comprises or consists of the substitution V66A of the polypeptide of SEQ ID NO: 61.

[0072] In another embodiment, the alterations in the variant comprise or consist of substitutions at positions corresponding to positions 66 and 102 of the polypeptide of SEQ ID NO: 61. In some embodiments, the alterations in the variant comprise or consist of the substitutions V66A and S102P of the polypeptide of SEQ ID NO: 61. In yet another embodiment, the variant comprises or consists of the amino acid sequence of SEQ ID NO: 140.

[0073] Alternatively, the amino acid changes may be of such a nature that the physicochemical properties of the polypeptide are altered, for example, the amino acid changes may improve the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0074] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested by known mutagenesis, recombination and / or shuffling methods followed by associated screening procedures such as those disclosed in 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).

[0075] III. Glucoamylase Production The glucoamylase can be produced within host cells, for example, by secretion or intracellular expression. Cultured cell material (e.g., whole cell broth) containing the glucoamylase can be obtained after secretion of the glucoamylase into the cell culture medium. Optionally, the glucoamylase can be isolated from the host cells or even from the cell broth, depending on the desired purity of the final glucoamylase. Genes encoding glucoamylases can be cloned and expressed according to methods 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 niger, Aspergillus oryzae, Trichoderma reesi, or Myceliopthora thermophila. Other host cells include bacterial cells (e.g., Bacillus subtilis or B. licheniformis) and Streptomyces. Suitable yeast host organisms can be selected from Saccharomyces species, including Schizosaccharomyces or Saccharomyces cerevisiae, or species within the Schizosaccharomyces species, such as S. pombe. Strains of the methylotrophic yeast species Pichia pastoris can be used as host organisms.

[0076] Additionally, the host may express one or more accessory enzymes, proteins, or peptides, which may be beneficial in liquefaction, saccharification, fermentation, SSF, and downstream processes. Furthermore, the host cells may produce ethanol and other biochemicals or biomaterials in addition to enzymes used to digest various feedstocks. Such host cells may be useful in fermentation processes or simultaneous fermentation and saccharification processes to reduce or eliminate the need for added enzymes.

[0077] A. Vector A DNA construct containing a nucleic acid encoding a glucoamylase polypeptide can be constructed to be suitable for expression in a host cell. Due to the known degeneracy of the genetic code, various polynucleotides encoding the same amino acid sequence can be designed and produced by routine techniques. It is also known that, depending on the desired host cell, codon optimization may be required before attempting expression.

[0078] A polynucleotide encoding a glucoamylase polypeptide of the present disclosure can be incorporated into a vector, which can be introduced into a host cell using known transformation techniques (e.g., those disclosed below).

[0079] A suitable vector can be one that can be transformed into a host cell and replicated within the host cell. For example, a vector containing a nucleic acid encoding a glucoamylase polypeptide of the present disclosure can be transformed into and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector can be suitably transformed into an expression host such that the encoding polynucleotide is expressed as a functional glucoamylase enzyme.

[0080] Exemplary useful vectors are pTrex3gM (e.g., U.S. Patent Application Publication No. 20130323798) and pTTT (e.g., U.S. Patent Application Publication No. 20110020899), which can be inserted into the genome of a host. Both the pTrex3gM and pTTT vectors can be modified by routine skill so that they contain and express a polynucleotide encoding a glucoamylase polypeptide of the invention.

[0081] Expression vectors typically contain regulatory nucleotide sequences, such as a promoter, operator, ribosome binding site, translation initiation signal, and optionally, a repressor gene or one or more activator genes. Furthermore, expression vectors may contain sequences encoding amino acid sequences capable of targeting the glucoamylase to a host cell organelle, such as peroxisomes, or to a specific cellular compartment. For expression under the direction of the regulatory sequences, the nucleic acid sequence for the glucoamylase is operably linked to the regulatory sequences in a manner appropriate for expression.

[0082] A polynucleotide encoding a glucoamylase polypeptide of the invention can be operably linked to a promoter capable of directing transcription in a host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of promoters for directing transcription of a DNA sequence encoding a glucoamylase, particularly in a bacterial host, include the promoter of the lac operon of E. coli, the promoter of the Streptomyces coelicolor agarose gene dagA or celA, the promoter of the amylase gene of Bacillus licheniformis (amyL), the promoter of the maltogenic amylase gene of Bacillus stearothermophilus (amyM), the promoter of the amylase gene of Bacillus amyloliquefaciens (amyQ), the promoter of the xylA and xylB genes of Bacillus subtilis, and the like.

[0083] 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, and the like. When a glucoamylase-encoding gene is expressed in a bacterial species, such as E. coli, suitable promoters can be selected from bacteriophage promoters, including the T7 promoter and the phage lambda promoter. Along these lines, examples of promoters suitable for expression in yeast species include, but are not limited to, the Gal1 and Gal10 promoters of Saccharomyces cerevisiae and the AOX1 or AOX2 promoters of Pichia pastoris. Expression in filamentous fungal host cells often involves the endogenous inducible promoter cbh1 from T. reesei. See Liu et al. (2008) Acta Biochim. Biophys. Sin (Shanghai) 40(2):158-65.

[0084] The coding sequence can be operably linked to a signal sequence. The DNA encoding this signal sequence can be the DNA sequence naturally associated with the glucoamylase gene of interest to be expressed, or it can be derived from a different genus or species than the glucoamylase. The signal sequence and promoter sequence comprising the DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence can be the Trichoderma reesei cbh1 signal sequence operably linked to the cbh1 promoter.

[0085] The expression vector may also contain a suitable transcription terminator and, in eukaryotes, a polyadenylation sequence operably linked to the DNA sequence encoding the glucoamylase. Termination and polyadenylation sequences may suitably be derived from the same source as the promoter.

[0086] Vectors may also contain, for example, genes whose products complement a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or selectable markers that confer antibiotic resistance (e.g., ampicillin resistance, kanamycin resistance, chloramphenicol resistance, or tetracycline resistance). Additionally, vectors may contain Aspergillus selection markers (e.g., amdS, argB, niaD, and xxsC), markers that confer hygromycin resistance, or selection may be achieved by cotransformation as known in the art. See, e.g., WO 91 / 17243.

[0087] B. Transformation and Cultivation of Host Cells Isolated cells containing either a DNA construct or an expression vector are advantageously used as host cells in the recombinant production of glucoamylase. The cells can be transformed with a DNA construct encoding the enzyme, conveniently by integrating the DNA construct into the host's chromosome (in one or more copies). This integration is generally considered advantageous because the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct into the host's chromosome can be carried out according to conventional methods, for example, by homologous or non-homologous recombination. Alternatively, the cells can be transformed with expression vectors associated with various types of host cells.

[0088] Examples of suitable bacterial host organisms are Gram-positive bacterial species such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus), stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis. Examples of suitable host organisms include Bacillaceae, including Bacillus thuringiensis; Streptomyces species, such as Streptomyces murinus; Lactic acid bacteria, including Lactococcus species, such as Lactococcus lactis; Lactobacillus species, including Lactobacillus reuteri; Leuconostoc species; Pediococcus species; and Streptococcus species. Alternatively, strains of Gram-negative bacteria belonging to the Enterobacteriaceae, including E. coli, or the Pseudomonadaceae family can be selected as host organisms.

[0089] Suitable yeast host organisms can be selected from biotechnologically relevant yeast species, including, but not limited to, yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces, or Saccharomyces species, such as Saccharomyces cerevisiae, or Schizosaccharomyces species, such as S. pombe. Strains of the methylotrophic yeast species Pichia pastoris can be used as host organisms. Alternatively, the host organism may be a Hansenula species.

[0090] Suitable filamentous fungal host organisms include Aspergillus species, such as Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of Fusarium species, such as Fusarium oxysporum, or Rhizomucor species, such as Rhizomucor miehei, can be used as host organisms. Other suitable strains include Thermomyces and Mucor species. Additionally, Trichoderma sp. may be used as a host. The glucoamylase expressed by the fungal host cell may be glycosylated, i.e., will contain glycosyl moieties. The glycosylation pattern may be the same as or different from that present in the wild-type glucoamylase. The type and / or degree of glycosylation may confer altered enzymatic and / or biochemical properties.

[0091] It is advantageous to delete genes from expression hosts where the genetic defect can be cured by the transformed expression vector. Fungal host cells with one or more inactivated genes can be obtained using known methods. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned (e.g., the cbh1, cbh2, egl1, and egl2 genes) can be deleted. Gene deletion can be achieved by inserting a form of the desired gene to be inactivated into a plasmid using methods known in the art.

[0092] General transformation techniques are known in the art. See, for example, Sambrook et al. (2001), supra. Expression of heterologous proteins in Trichoderma is described, for example, in U.S. Pat. No. 6,022,725. For transformation of Aspergillus strains, see also Cao et al. (2000) Science 9:991-1001. Genetically stable transformants can be constructed using a vector system in which the nucleic acid encoding the amylase is stably integrated into the chromosome of the host cell. Transformants are then selected and purified using known techniques.

[0093] C. Expression and Fermentation Methods for producing glucoamylase can include culturing host cells under conditions conducive to production of the enzyme, and recovering the enzyme from the cells and / or culture medium.

[0094] The medium used to culture the cells can be any conventional medium suitable for growing the host cells and obtaining expression of the glucoamylase polypeptide. Suitable media and media components are available from commercial suppliers or can be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0095] Any fermentation method known in the art can be suitably used to ferment the transformed or derived fungal strains as described above, hi some embodiments, the fungal cells are grown under batch fermentation conditions or under continuous fermentation conditions.

[0096] D. Methods for Concentration and Purification Separation and concentration techniques are known in the art, and conventional methods can be used to prepare concentrated solutions or broths containing the glucoamylase polypeptides of the invention.

[0097] After fermentation, a fermentation broth is obtained from which the various suspended solids, including microbial cells and residual raw fermentation materials, are removed to obtain a glucoamylase solution by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, ultrafiltration followed by centrifugation, extraction, or chromatography, which are commonly used.

[0098] At times, it may be desirable to concentrate the solution or broth containing the glucoamylase polypeptide to optimize recovery. Use of an unconcentrated solution or broth will typically result in increased incubation times for collecting the concentrated or purified enzyme precipitate.

[0099] IV. Composition The present invention also relates to compositions comprising a polypeptide and / or a starch substrate. In some embodiments, a polypeptide 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 61 can also be used in the enzyme composition. Preferably, the composition is formulated to provide desirable characteristics such as low color, low odor, and acceptable stability at temperatures between about 4 and 40°C and a pH between about 3 and 7.

[0100] The composition may comprise a polypeptide of the invention as the primary enzyme component, or may comprise multiple enzyme activities such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, beta-amylase, isoamylase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase, xylanase, or combinations thereof, which may be added in effective amounts as would be known to one skilled in the art.

[0101] The polypeptide compositions can be prepared according to methods known in the art and can be in the form of a liquid composition or a dry composition. For example, compositions containing the glucoamylase can be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which can further include any one or more of the additional enzymes listed herein, along with buffers, salts, preservatives, water, cosolvents, surfactants, etc. Such compositions can function in combination with endogenous enzymes or other components already present in slurries, water baths, washing machines, food or beverage products, etc. (e.g., endogenous plant (including algae) enzymes, residual enzymes from prior processing steps, etc.). The polypeptides to be included in the compositions can be stabilized according to methods known in the art.

[0102] The composition can be a cell that expresses a polypeptide, e.g., a cell that can produce a product by fermentation. Such cells can be provided in liquid or dry form, along with suitable stabilizers. Such cells can further express additional polypeptides, such as those described above.

[0103] Preferred examples of the use of the polypeptide or composition of the present invention are given below. The dosage of the polypeptide composition of the present invention and other conditions under which the composition can be used can be determined based on methods known in the art.

[0104] The composition is suitable for use in liquefaction, saccharification and / or fermentation processes, preferably for starch conversion, especially for the production of syrups and fermentation products such as ethanol. The composition is also suitable for use in animal nutrition and fermented beverages.

[0105] V. Use The present invention also relates to the use of a polypeptide or composition of the present invention in a liquefaction process, a saccharification process, and / or a fermentation process. The polypeptide or composition can be used in a single process, such as a liquefaction process, a saccharification process, or a fermentation process. The polypeptide or composition can also be used in a combination of processes, such as a liquefaction and saccharification process, a liquefaction and fermentation process, or a saccharification and fermentation process, preferably in the context of starch conversion.

[0106] A. Glycation Liquefied starch can be saccharified using alpha-amylase and glucoamylase, optionally in the presence of other enzymes, to a syrup rich in low DP (e.g., DP1 + DP2) sugars. The exact composition of the saccharification product depends on the enzyme combination used and the type of starch being processed. Advantageously, the syrup obtained using the provided glucoamylases can contain DP1, with a weight percentage of total oligosaccharides in the saccharified starch of greater than 90%, e.g., 90%-98% or 95%-97%. The weight percentage of DP2 in the saccharified starch can be as low as less than about 3%, e.g., 0-3% or 0-2.8%.

[0107] Liquefaction is generally carried out as a continuous process, while saccharification is often carried out as a batch process. Saccharification conditions depend on the nature of the liquefaction product and the type of enzyme available. In some cases, the saccharification process may include a temperature of about 60-65°C and a pH of about 4.0-4.5, e.g., pH 4.3. Saccharification can be carried out at temperatures of, for example, about 40°C, about 50°C, or about 55°C to about 60°C or about 65°C, requiring cooling of the liquefaction product. The pH may be adjusted as needed. Saccharification is typically carried out in a stirred tank, which may require several hours to fill or empty. Enzymes are generally added in a proportional ratio to the dry solids as the tank is filled, or as a single dose at the beginning of the filling stage. Saccharification reactions to produce syrup are typically carried out for about 24-72 hours, e.g., 24-48 hours. Pre-saccharification can be added prior to saccharification in simultaneous saccharification and fermentation (SSF) at a temperature of 30-65°C, typically about 60°C, for typically 40-90 minutes.

[0108] B. Hydrolysis of raw starch The present invention provides the use of glucoamylases of the present invention to produce glucose and other sugars from raw or granular starch. Generally, glucoamylases of the present invention can be used in raw starch hydrolysis (RSH) or granular starch hydrolysis (GSH) processes, either alone or in the presence of an alpha-amylase, to produce desired sugars and fermentation products. Granular starch is solubilized by enzymatic hydrolysis below the gelatinization temperature. Such "low-temperature" systems (also known as "no-cook" or "cold-cook") have been reported to be capable of processing higher concentrations of dry solids (e.g., up to 45%) than conventional systems.

[0109] The "raw starch hydrolysis" process (RSH) differs from conventional starch processing processes in that it involves sequential or simultaneous saccharification and fermentation of granular starch below the gelatinization temperature of the starch substrate, typically in the presence of at least a glucoamylase and / or an amylase.

[0110] The glucoamylases of the invention can also be used in combination with enzymes that hydrolyze only alpha-(1,6)-glucosidic linkages in molecules containing at least four glucosyl residues. Preferably, the glucoamylases of the invention are used in combination with pullulanases or isoamylases. The use of isoamylases and pullulanases for starch debranching, the molecular properties of the enzymes, and the potential simultaneous use of the enzymes with glucoamylases are described in GM A van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, pp. 101-142.

[0111] C. Fermentation Soluble starch hydrolysates, particularly glucose-rich syrups, can be fermented by contacting the starch hydrolysate with a fermenting organism, typically at a temperature around 32°C, e.g., 30°C to 35°C. The term "fermenting organism" refers to any organism, including bacteria and fungi, 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, such as glucose or maltose, directly or indirectly to the desired fermentation product. Examples of fermenting organisms include yeast (e.g., Saccharomyces cerevisiae) and bacteria (e.g., Zymomonas mobilis) that express alcohol dehydrogenase and pyruvate decarboxylase. Ethanol-producing microorganisms can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. For example, improved strains of ethanol-producing microorganisms that can tolerate high temperatures are known in the art and can be used. See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27:1049-56. Yeasts that can be used for alcohol production include, but are not limited to, Saccharomyces spp., including S. cerevisiae, as well as Kluyveromyces, Lachancea, and Schizosaccharomyces spp. Numerous yeast strains are commercially available, many of which have been selected or genetically modified for desired characteristics, such as high alcohol production and rapid growth rates. The temperature and pH of fermentation will depend on the fermenting organism. Microorganisms that produce other metabolic products, such as citric acid and lactic acid, by fermentation are also known in the art. See, e.g., Papagianni (2007) Biotechnol. Adv. 25:244-63; John et al. (2009), Biotechnol. Adv. 27:145-52.

[0112] The saccharification and fermentation processes can be carried out as an SSF process. The SSF process can be carried out using fungal cells that continuously express and secrete glucoamylase through the SSF. The glucoamylase-expressing fungal cells can also be fermenting microorganisms, e.g., ethanol-producing microorganisms. Thus, ethanol production can be carried out using fungal cells that express sufficient glucoamylase so that exogenous enzyme addition is largely unnecessary. Fungal host cells can be selected from appropriately engineered fungal strains. Fungal host cells that express and secrete other enzymes in addition to glucoamylase can also be used. Such cells can express amylase and / or pullulanase, phytase, alpha-glucosidase, isoamylase, beta-amylase, cellulase, xylanase, other hemicellulase, protease, beta-glucosidase, pectinase, esterase, oxidoreductase, transferase, or other enzymes. Fermentation can be followed by subsequent ethanol recovery.

[0113] D. Fermentation products The term "fermentation product" refers to a product produced by a process involving a fermentation process using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol, ethylene glycol, propylene glycol, butanediol, glycerin, sorbitol, and xylitol); organic acids (e.g., acetic acid, acetone, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); alkenes (e.g., pentene, hexene, heptene, and octene); gases (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO)); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 , beta-carotene); as well as hormones.

[0114] In a preferred embodiment, the fermentation product is ethanol, such as fuel ethanol; potable ethanol, i.e., potable neutral spirit; or industrial ethanol or products used in the potable alcohol industry (e.g., beer and wine), the dairy industry (e.g., fermented dairy products), the leather industry, and the tobacco industry. Preferred fermentation processes used include alcoholic fermentation processes known in the art. Preferred fermentation processes are anaerobic fermentation processes well known in the art.

[0115] E. Brewing The process for producing beer is well known in the art. See, for example, Wolfgang Kunze (2004) "Technology Brewing and Malting," Research and Teaching Institute of Brewing, Berlin (VLB), 3rd edition. Briefly, the process includes (a) preparing a mash, (b) filtering the mash to prepare a wort, and (c) fermenting the wort to obtain a fermented beverage, such as beer.

[0116] The brewing composition comprising glucoamylase in combination with amylase and optionally pullulanase and / or isoamylase may be added to the mash in step (a) above, i.e., during preparation of the mash. Alternatively or additionally, the brewing composition may be added to the mash in step (b) above, i.e., during filtration of the mash. Alternatively or additionally, the brewing composition may be added to the wort in step (c) above, i.e., during fermentation of the wort.

[0117] F. Animal Nutrition The glucoamylases described herein can also be used as feed additives for animals to increase starch digestibility. Described herein are methods for increasing starch digestibility in animals.

[0118] The term "animal" refers to any living organism belonging to the kingdom Animalia, including, without limitation, mammals (excluding humans), non-human animals, domestic animals, livestock, farm animals, zoo animals, breeding stock, etc. For example, all non-ruminant and ruminant animals may be included. In one embodiment, the animal is a non-ruminant, i.e., a monogastric animal. Examples of monogastric animals include, but are not limited to: pigs and swine, such as piglets, growing pigs, and sows; poultry, such as turkeys, ducks, chickens, broilers, and laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In another embodiment, the animal is a cud-chewing animal, including, but not limited to, cows, calves, goats, sheep, giraffes, bison, moose, elk, yak, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns, and nilgai.

[0119] The terms "animal feed," "feed," "feed ingredient," and "livestock feed" are used interchangeably and refer to: a) grains, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and / or large grains (e.g., corn or sorghum); b) grain by-products, such as corn gluten meal, distillers dried grains with solubles (DDGS), particularly corn-based distillers dried grains with solubles (cDDGS), wheat bran, wheat c) proteins obtained from sources such as soybean, sunflower, peanut, lupin, pea, fava bean, cotton, rapeseed, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) fats and oils obtained from plant and animal sources; and / or e) minerals and vitamins.

[0120] Starch digestibility 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 starch digestibility in animal diets can be improved by the use of at least one glucoamylase as a feed additive.

[0121] When used as a feed, such as a functional feed, or when used in the preparation of such a feed, the enzyme or feed additive composition herein may be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, or a nutritionally active ingredient, such as at least one ingredient selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben, and propylparaben.

[0122] At least one glucoamylase described herein (or an enzyme composition comprising at least one glucoamylase described herein) can also be homogenized to produce a powder. The powder can be mixed with other ingredients known in the art. Optionally, the feed can also include additional minerals, such as calcium, and / or additional vitamins. In some embodiments, the feed ingredient is a corn-soybean meal mix.

[0123] In an alternative preferred embodiment, the enzyme composition comprising at least one glucoamylase may be formulated into granules as described in WO 2007 / 044968 (referred to as TPT granules) or WO 1997 / 016076 or WO 1992 / 012645, which are incorporated herein by reference. "TPT" stands for Thermal Protection Technology. When the feed additive composition is formulated into a granule, the granule contains a hydration barrier salt coated onto the protein core. The benefits of such a salt coating are improved heat resistance, improved storage stability, and protection from other feed additives that would otherwise adversely affect the enzyme. Preferably, the salt used in the salt coating has a water activity greater than 0.25 or a constant humidity of greater than 60% at 20°C. In some embodiments, the salt coating comprises Na2SO4.

[0124] Alternatively, the composition is present in a liquid formulation suitable for consumption, preferably such liquid consumables contain one or more of the following: buffer, salt, sorbitol and / or glycerol.

[0125] Any of the glucoamylases for use as feed additives described herein may be used alone or in combination with at least one direct-feed microorganism. Categories of DFM include Bacillus, lactic acid bacteria, and yeast. Additionally, any of the glucoamylases for use as feed additives described herein may be used alone or in combination with at least one essential oil, such as, for example, cinnamaldehyde and / or thymol. Furthermore, any of the glucoamylases for use as feed additives described herein may be used alone or in combination with at least one additional enzyme, examples of which include, without limitation, phytase, xylanase, protease, amylase, glucanase, or other glucoamylase.

[0126] Also disclosed is a method for improving the nutritional value of animal feed, wherein an effective amount of any of the glucoamylases described herein can be added to the animal feed.

[0127] As used herein, the phrase "effective amount" relates to the amount of an active agent (e.g., any of the glucoamylase polypeptides disclosed herein), either alone or in combination with one or more other active agents (e.g., without limitation, one or more additional enzymes, one or more DFMs, one or more essential oils, etc.), required to confer improved performance in an animal in one or more endpoints.

[0128] The term "animal performance" as used herein can be determined by any evaluation index, such as, without limitation, feed efficiency and / or weight gain of the animal and / or feed conversion ratio and / or digestibility of nutrients in the feed and / or digestible or metabolizable energy and / or nitrogen retention in the feed and / or ability of the animal to avoid the adverse effects of necrotic enteritis.

[0129] Animal performance characteristics 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 mass; egg production rate; mineral absorption; mineral excretion, mineral retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake ratio (ADFI); average daily gain (ADG) retention and / or excretion 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.

[0130] By "improved animal performance in one or more endpoints" is meant increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio and / or improved digestibility of nutrients or energy in the feed and / or improved nitrogen retention and / or improved ability to avoid the adverse effects of necrotic enteritis and / or improved immune response in a subject resulting from the use of a feed comprising a feed additive composition as described herein compared to a feed not comprising said feed additive composition.

[0131] All references cited herein are incorporated by reference in their entirety for all purposes. In order to further illustrate the present compositions and methods and their advantages, the following specific examples are provided with the understanding that they are illustrative rather than limiting. [Example]

[0132] Example 1 Identification of Mucorales clade glucoamylase enzymes A search for glucoamylase enzymes in the Zygomycetes phylum was performed by scanning annotated protein sequences of the Zygomycetes phylum using dbCAN (Yin et al. (2012) "dbCAN: a web resource for automated carbohydrate-active enzyme annotation: Nucleic Acids Research 40:W4450-451") to identify all GH15 proteins based on CAZY genealogy analysis. Numerous genes were identified within the genomes of Mucorales order organisms, and the sequences were analyzed in detail. Genes encoding Mucorales-clade glucoamylases were identified from the sequences listed in Table 1 and assigned the SEQ ID NOs shown in Table 1. [Table 2]

[0133] N-terminal signal peptides were predicted using SignalP software version 4.0 (Nordahl Petersen et al. (2011) Nature Methods 8:785-786). Genes encoding glucoamylases from various Mucorales clades were codon-modified for expression in Trichoderma reesei. [Table 3]

[0134] Example 2 Expression of Mucorales-clade glucoamylase in Trichoderma reesei Polynucleotides encoding Mucorales clade glucoamylase genes (codon-modified sequences used as expression cassettes) (SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40) were synthesized by Generay (Generay Biotech Co., Ltd, Shanghai, China) and inserted into the pGX256 expression vector, a derivative vector derived from pTTT (see U.S. Patent Application Publication No. 20110020899).

[0135] A polynucleotide was constructed encoding a variant of SvaGA1 glucoamylase (SEQ ID NO: 61) in which a codon change introduced a mutation at amino acid position 102 to Pro instead of Ser (SvaGA1v2, S102P). An expression cassette encoding SvaGA1v2 was inserted into pGX256 (as described above).

[0136] All plasmids were transformed into suitable Trichoderma reesei strains using the protoplast transformation method (Te'o et al., J. Microbiol. Methods 51:393-99, 2002). Transformants were selected and fermented according to the method described in WO 2016 / 138315. Supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis.

[0137] Fungal cell cultures were grown in defined medium as described by Lv et al. (2012) in "Construction of teo vectors for gene expression in Trichoderma reesei" Plasmids 67:67-71. After 96 hours, the clarified culture broth was collected by centrifugation. The Mucorales clade glucoamylase was purified by methods known in the art. Column chromatography fractions containing the target protein were pooled, concentrated, and equilibrated with 20 mM sodium acetate, pH 5.0, and 150 mM sodium chloride using an Amicon Ultra-15 device 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.

[0138] Example 3 Evaluation of SvaGA1 and SvaGA1v2 glucoamylases in saccharification at pH 4.5 and 60°C. The saccharification performance of the SvaGA1 and SvaGA1v2 variants was evaluated at pH 4.5 and 60°C. A sample of GC126 (a DuPont / IFF product) pretreated with cornstarch liquefaction (38% ds, prepared at pH 3.3) was used as the starting material. Glucoamylase performance was tested at a dose of 30 μg / gds. For comparison, Gloeophyllum trabeum glucoamylase (GtGA), a glucoamylase from EXTENDA® XTRA (a Novozymes product), was included. For this evaluation, pullulanase OPTIMAX™ L 1000 (a DuPont product) was dosed at 10 μg / gds, and alpha-amylase Aspergillus kawachii amylase (AkAA, described in International Publication WO 2013169645, incorporated herein by reference) was dosed at 5 μg / gds for each incubation. The corn starch liquefied substrate and enzymes (glucoamylase, alpha-amylase, and pullulanase) were incubated at pH 4.5 and 60°C for 48 and 65 hours, respectively. All incubations were quenched by heating to 100°C for 15 minutes. An aliquot was removed and diluted 40-fold in 5 mM H2SO4 for product analysis by HPLC using an Agilent 1200 Series system equipped with a Phenomenex Rezex-RFQ Fast Fruit column (product number 00D-0223-K0) run at 80 °C. A 10 L sample was loaded onto the column and separated using an isotonic gradient of 5 mM H2SO4 as the mobile phase at a flow rate of 1.0 mL / min. Oligosaccharide products were detected using a refractive index detector and run against standards (DP3+, DP3, DP2, and DP1) to determine the elution time of each DP(n) of interest. The values ​​shown in Table 3 reflect the percentage of peak area for each DP(n) as a fraction of total DP1–DP3+. The results for DP1 production and DP3+ hydrolysis by SvaGA1 and SvaGA1v2 glucoamylases were superior to those of the reference GtGA enzyme at pH 4.5 and 60 °C. [Table 4]

[0139] Example 4 Specific activity of Mucorales clade glucoamylase enzymes on soluble starch The specific activity of glucoamylase was analyzed using the coupled glucose oxidase / peroxidase (GOX / HRP) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (Anal. Biochem. 105; 1980:389-397), based on the release of glucose from soluble starch. Substrate solution was prepared by mixing 9 mL of soluble starch (1% wt / wt in water) with 1 mL of 0.5 M sodium acetate buffer (pH 5.0) in a 15 mL conical tube. A coupled enzyme (GOX / HRP) solution containing ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0) with final concentrations of 2.74 mg / mL ABTS, 0.1 U / mL HRP, and 1 U / mL GOX. Serial dilutions of each glucoamylase sample and glucose standard to be evaluated were prepared in purified water. Each glucoamylase sample (10 μL) was transferred to a new microtiter plate (Corning 3641) containing 90 μL of substrate solution preincubated at 50°C for 5 minutes at 600 rpm. After 10 minutes of incubation at 50°C with shaking (600 rpm) in a thermomixer (Eppendorf), 10 μL of the reaction mixture and 10 μL of the serial dilutions of glucose standards were quickly transferred to each new microtiter plate (Corning 3641), followed by the addition of 100 μL of ABTS / GOX / HRP solution. Absorbance at 405 nm was immediately measured at 11-second intervals for 5 minutes using a SoftMax Pro plate reader (Molecular Devices). The output was the reaction velocity, Vo, for each enzyme concentration. Linear regression was used to determine the slope of the plot of Vo versus enzyme dosage. The specific activity of glucoamylase was calculated using the following equation: Specific activity (units / m) = slope (enzyme) / slope (standard) × 1000 (1) where 1 unit = 1 μmol glucose / min was used for calculations based on a glucose standard curve.

[0140] Using the methods described above, the specific activities and benchmarks of Mucorales-clade glucoamylases were determined, and the results are shown in Table 4. [Table 5]

[0141] Example 5 Analysis of homologous Mucorales clade glucoamylase sequences A multiple amino acid sequence alignment was constructed for the region encompassing the catalytic domains of Mucorales clade glucoamylases: SvaGA1 SEQ ID NO:81, BciGA1 SEQ ID NO:82, BciGA2 SEQ ID NO:83, BpoGA1 SEQ ID NO:84, CcuGA1 SEQ ID NO:85, RstGA1 SEQ ID NO:86, MciGA5 SEQ ID NO:87, DelGA1 SEQ ID NO:88, FspGA3 SEQ ID NO:89, GpeGA1 SEQ ID NO:90, MciGA3 SEQ ID NO:91, CumGA1 SEQ ID NO:92, McoGA1 SEQ ID NO:93, ParGA1 SEQ ID NO:94, RmiGA1 SEQ ID NO:95, SfuGA2 SEQ ID NO:96, SraGA1 SEQ ID NO:97, SraGA3 SEQ ID NO:98, TinGA1 SEQ ID NO:99, and ZmeGA1 SEQ ID NO:100. In the case of SvaGa1, the catalytic domain is 432 residues long, spanning amino acids 18 to 449 of the predicted mature protein sequence. The aforementioned region overlaps with the catalytic domain of previously defined related glucoamylases, based on studies of the glucoamylase sequences from A. awamori (Aleshin et al., 1994, J. Mol. Bio. 238:575-591) and A. niger (Lee and Paetzel, 2011, Acta Cryst. F67:188-192). These sequences were aligned using the MUSCLE alignment tool in Geneious 10.2 software with default parameters. Additional homologous sequences were identified in the public domain: GAN00808.1 SEQ ID NO:101, ORE14155.1 SEQ ID NO:102, and RCH88939.1 SEQ ID NO:103, and these overlapping sequences were also included in this analysis.Additionally, to identify regions of sequence similarity and differences, the catalytic domains of other glucoamylases that are not members of the fungal order Mucorales were compared: Aspergillus niger glucoamylase (AnGA) SEQ ID NO: 105, Aspergillus fumigatus glucoamylase (AfuGA) SEQ ID NO: 106, Fibroporia radiculosa TFFH 294 glucoamylase (FraGA1) SEQ ID NO: 107, Fusarium verticillioides glucoamylase (FveABC11) SEQ ID NO: 108, Gloeophyllum trabeum glucoamylase (GtGA) SEQ ID NO: 109, Penicillium oxalicum glucoamylase (Penicillium oxalicum) SEQ ID NO: 110, and Aspergillus niger glucoamylase (AnGA) SEQ ID NO: 111. The P. oxalicum glucoamylase (PoxGA) SEQ ID NO: 110, Trichoderma reesei glucoamylase (TrGA) SEQ ID NO: 111, and Wolfiforia cocos MD-104 SS10 glucoamylase (WcoGA1) SEQ ID NO: 112 were also included in this alignment. The multiple sequence alignment is shown in panels A–K of Figure 1. A phylogenetic tree was constructed from the alignment in Figure 1 using Geneious 10.2 software and is shown in Figure 2.

[0142] A series of insertions and deletions, as well as regions of high sequence variability, were observed in the multiple sequence alignments shown in Figures 1A-1K. Several regions of high similarity between Mucorales-clade glucoamylases are evident, and sequence motifs have been identified. Figures 3, 4, and 5 show alignments of three distinct regions of Mucorales-clade glucoamylase catalytic domains, highlighting common sequence motifs. Figure 3 shows the Mucorales-clade GA sequence motif 1 (SEQ ID NO: 113): 57Y-58X. a -59X b-60T-61X-62X-63X c -64X d wherein X is any amino acid; a is N or S; X b is T, S, or R; X c is G or N; and X d FIG. 4 shows an alignment of Mucorales-clade GA amino acid sequences between the region spanning residues 50-70 (numbered according to SEQ ID NO: 81), highlighting the amino acids (A, B, C, D, E, F, G, G, H, I ... a -245X b -246X c -247X c -248A-249A-250N-251X-252X d wherein X is any amino acid; a is S or A; X b is T, N, or V; X c is L or I; and X d Figure 5 shows an alignment of Mucorales-clade GA amino acid sequences (numbered according to SEQ ID NO: 81) over the region spanning residues 240-260, describing the Mucorales clade GA sequence motif 2A (SEQ ID NO: 116): 244S-245T-246L-247I-248A-249A-250N-251X-252A (where X is any amino acid). Figure 5 shows an alignment of Mucorales-clade GA sequence motif 3A (SEQ ID NO: 117): 304X-304X (where X is any amino acid). a -305G-306X-307G-308N-309X b -310X c wherein X is any amino acid;a is N or D; X b is S or G; X c Figure 1 shows an alignment of Mucorales-clade GA amino acid sequences (numbered according to SEQ ID NO:81) over the region spanning residues 299-315, describing the sequence of the Mucorales-clade GA amino acid motif 3A (SEQ ID NO:118): 304N-305G-306N-307G-308N-309S-310Q (where X is any amino acid).

[0143] Example 6 Evaluation of the thermostability of glucoamylase Stability comparison at 60℃ The thermostability of SvaGA1v2 and SvaGA1v3 was compared to pre-incubation of enzyme samples (20 ppm) for 10 min at 60° C. Pre-incubation at 4° C. for 10 min was included and set as 100% activity for each glucoamylase sample. The residual activity of glucoamylase after pre-incubation was measured using the same method as described in Example 4, except that the pH was 4.5 and the incubation temperature was 60° C. As shown in Table 5, SvaGA1v3 retained 55% of its activity under these conditions. [Table 6]

[0144] Measurement of Tm using DSC Differential scanning calorimetry (DSC) was performed using an ultrasensitive MicroCal™ VP-capillary DSC system (GE Healthcare). Purified SvaGA1, SvaGA1v2, and SvaGA1v3 were diluted to a final concentration of 0.4 mg / mL in 100 mM sodium acetate buffer, pH 4.5. 400 μL of enzyme solution and a reference material containing an identical volume of enzyme-free buffer were added to a 96-well plate. The plate was then placed in a thermally controlled autosampler compartment maintained at 10°C. The enzyme samples and buffer reference materials were scanned from 20 to 100°C at a scan rate of 2°C per minute. Tm was determined as the temperature at the peak maximum of the transition from the folded to the unfolded state. The maximum variation in Tm was ±0.2°C. The ORIGIN software package (MicroCal, GE Healthcare) was used for baseline subtraction and graphical presentation of the data. The DSC results in Table 6 show that the Tm of SvaGA1v3 is 3°C higher than the Tm of SvaGA1v2. [Table 7]

[0145] Example 7 Determination of glycation activity The saccharification performance of SvaGA1v2 and SvaGA1v3 was evaluated at pH 4.5 and 60, 62, and 65°C, respectively. All incubation conditions were identical to those in Example 3, except that corn starch liquefaction was purchased from Cargill. Pullulanase OPTIMAX™ L 1000 (a DuPont product) was dosed at 4 μg / gds, and alpha-amylase Aspergillus terreus amylase (AtAA, e.g., as described in WO 2017112635 A1 and WO 2014099415, both incorporated by reference herein) was dosed at 1 μg / gds. The values ​​shown in Table 7 reflect the percentage of peak area for each DP(n) as a fraction of the total DP1–DP3+. The production of DP1 and hydrolysis of DP3+ by SvaGA1v3 shows superior performance compared to the reference GtGA enzyme under all selected conditions. [Table 8]

[0146] Example 8 Identification of Additional Homologous Mucorales-Clade Glucoamylases A number of genes were identified within the genomes of Mucorales order organisms and the sequences were analyzed in detail. Genes encoding Mucorales-clade glucoamylases were identified from the sequences listed in Table 8 and assigned SEQ ID NOs: as shown in Table 9. [Table 9]

[0147] The N-terminal signal peptide was predicted using SignalP software version 4.0 (Nordahl Petersen et al. (2011) Nature Methods 8:785-786). Genes encoding various Mucorales clade glucoamylases were codon-modified for expression in Trichoderma reesei. Based at least in part on this analysis, a novel variant of SvaGA1, designated SvaGA1v3, was generated and assigned SEQ ID NO: 140, as shown in Table 9. [Table 10]

[0148] Example 9 Expression of Mucorales-clade glucoamylase in Trichoderma reesei Polynucleotides encoding Mucorales clade glucoamylase genes (codon-modified sequences used as expression cassettes) (SEQ ID NO: 124, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136) were synthesized by Generay Biotech Co., Ltd., Shanghai, China, and inserted into the pGX256 expression vector, a derivative vector derived from pTTT (see, e.g., U.S. Patent Application Publication No. 20110020899).

[0149] All plasmids were transformed into suitable Trichoderma reesei strains using the protoplast transformation method (Te'o et al., J. Microbiol. Methods 51:393-99, 2002). Transformants were selected and fermented according to the method described in WO 2016 / 138315. Supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis.

[0150] Fungal cell cultures were grown in defined medium as described by Lv et al. (2012) in "Construction of teo vectors for gene expression in Trichoderma reesei." Plasmids 67:67-71. After 96 hours, the clarified culture broth was collected by centrifugation. The Mucorales clade glucoamylase was purified by methods known in the art. Column chromatography fractions containing the target protein were pooled, concentrated, and equilibrated with 20 mM sodium acetate, pH 5.0, and 150 mM sodium chloride using an Amicon Ultra-15 device 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.

[0151] Example 10 Evaluation of additional homologous Mucorales-clade glucoamylases in saccharification The saccharification performance of additional homologous Mucorales-clade glucoamylases was evaluated over 48 hours at pH 4.5, 60°C, 62°C, and 65°C, respectively. All incubation conditions were identical to those described in Example 7, except that the glucoamylase samples were dosed at 25 μg / gds. The values ​​shown in Table 10 reflect the percentage of the peak area of ​​each DP(n) as a fraction of the total DP1-DP3+. The production of DP1 and hydrolysis of DP3+ by Mucorales clade glucoamylases showed superior performance compared to the reference GtGA enzyme when evaluated at pH 4.5 and 60°C for 48 h. When the incubation temperature was increased to 62°C, all Mucorales clade GAs also showed superior saccharification performance compared to the GtGA enzyme. AtrGA1, but not GtGA, maintained its superior performance when the incubation temperature was increased to 65°C. [Table 11]

[0152] Example 11 Sequence analysis of additional homologous Mucorales clade glucoamylase sequences A multiple amino acid sequence alignment was constructed for the region encompassing the catalytic domains of Mucorales clade glucoamylases: SvaGA1 SEQ ID NO:81, SobGA1 SEQ ID NO:119, AosGA3 SEQ ID NO:120, AelGA1 SEQ ID NO:121, AvaGA1 SEQ ID NO:122, and AtrGA1 SEQ ID NO:123, as described in Example 5. These sequences were aligned using the MUSCLE alignment tool in Geneious 10.2 software with default parameters. Additionally, to identify regions of sequence similarity and divergence, the catalytic domains of other glucoamylases that are not members of the fungal order Mucorales were examined: Aspergillus niger glucoamylase (AnGA) SEQ ID NO: 105, Aspergillus fumigatus glucoamylase (AfuGA) SEQ ID NO: 106, Fibroporia radiculosa TFFH294 glucoamylase (FraGA1) SEQ ID NO: 107, Fusarium verticillioides glucoamylase (FveABC11) SEQ ID NO: 108, Gloeophyllum trabeum glucoamylase (GtGA) SEQ ID NO: 109, Penicillium oxalicum glucoamylase (FveABC11) SEQ ID NO: 110, and the catalytic domains of other glucoamylases that are not members of the fungal order Mucorales were examined. Also included in this alignment were the P. oxalicum glucoamylase (PoxGA) SEQ ID NO:110, Trichoderma reesei glucoamylase (TrGA) SEQ ID NO:111, and Wolfiforia cocos MD-104 SS10 glucoamylase (WcoGA1) SEQ ID NO:112. Multiple sequence alignments are shown in panels A-D of Figure 6. Additional novel homologs (SobGA1, AosGA3, AelGA1, AvaGA1, AtrGA1) are contained within the motifs outlined in SEQ ID NO:113, SEQ ID NO:115, and SEQ ID NO:117.The following sequences: SvaGA1 SEQ ID NO:81, BciGA1 SEQ ID NO:82, BciGA2 SEQ ID NO:83, BpoGA1 SEQ ID NO:84, CcuGA1 SEQ ID NO:85, RstGA1 SEQ ID NO:86, MciGA5 SEQ ID NO:87, DelGA1 SEQ ID NO:88, FspGA3 SEQ ID NO:89, GpeGA1 SEQ ID NO:90, MciGA3 SEQ ID NO:91, CumGA1 SEQ ID NO:92, McoGA1 SEQ ID NO:93, ParGA1 SEQ ID NO:94, RmiGA1 SEQ ID NO:95, SfuGA2 SEQ ID NO:96, SraGA1 SEQ ID NO:97, SraGA3 SEQ ID NO:98, TinGA1 SEQ ID NO:99, ZmeGA1 SEQ ID NO:100, GAN00808.1 SEQ ID NO:101, ORE14155.1 SEQ ID NO:102, and RCH88939.1 SEQ ID NO:103, are identified in Aspergillus niger (Aspergillus niger glucoamylase (AnGA) SEQ ID NO: 105, Aspergillus fumigatus glucoamylase (AfuGA) SEQ ID NO: 106, Fibroporia radiculosa TFFH294 glucoamylase (FraGA1) SEQ ID NO: 107, Fusarium verticillioides glucoamylase (FveABC11) SEQ ID NO: 108, Gloeophyllum trabeum glucoamylase (GtGA) SEQ ID NO: 109, Penicillium oxalicum glucoamylase (PoxGA) SEQ ID NO: 110, Trichoderma reesei glucoamylase (TrGA) SEQ ID NO: 111, and Wolfifora cocos A phylogenetic tree was generated from the alignment of WcoGA1 (SEQ ID NO: 112), SobGA1 (SEQ ID NO: 119), AosGA3 (SEQ ID NO: 120), AelGA1 (SEQ ID NO: 121), AvaGA1 (SEQ ID NO: 122), and AtrGA1 (SEQ ID NO: 123) using Geneious 10.2 software and is shown in Figure 7.

Claims

1. 1. A method for saccharifying a starch substrate, comprising: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more sequence motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO:

61.

2. 2. The method of claim 1, wherein the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

3. 3. The method of claim 2, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

4. The method of any one of claims 1 to 3, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:

61.

5. 5. The method of claim 4, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

6. The polypeptides are SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 6. The method of any one of claims 1 to 5, comprising the sequence of SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

7. 7. The method of any one of claims 1 to 6, wherein the starch substrate has a dry solids (DS) of about 15% to 65%, 15% to 60%, or 15% to 35%.

8. The method of any one of claims 1 to 7, wherein the starch substrate comprises liquefied starch, gelatinized starch, or granular starch.

9. 9. The method of any one of claims 1 to 8, further comprising adding to the starch substrate hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, beta-amylase, alpha-amylase, protease, cellulase, hemicellulase, lipase, cutinase, trehalase, isoamylase, oxidoreductase, esterase, transferase, pectinase, hydrolase, alpha-glucosidase, beta-glucosidase, or a combination thereof.

10. 10. The method of any one of claims 1 to 9, wherein saccharifying the starch substrate results in a high glucose syrup comprising an amount of glucose selected from the group consisting of at least 95.5% glucose, at least 95.6% glucose, at least 95.7% glucose, at least 95.8% glucose, at least 95.9% glucose, at least 96% glucose, at least 96.1% glucose, at least 96.2% glucose, at least 96.3% glucose, at least 96.4% glucose, at least 96.5% glucose, and at least 97% glucose.

11. 11. The method of any one of claims 1 to 10, further comprising fermenting the high glucose syrup into an end product.

12. 12. The method of claim 11, wherein the saccharifying and fermenting steps are carried out as a simultaneous saccharification and fermentation (SSF) process.

13. 13. The method of claim 11 or 12, wherein the end product is an alcohol, optionally ethanol.

14. 13. The method of claim 11 or 12, wherein the end product is a biochemical selected from the group consisting of amino acids, organic acids, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega-3 fatty acids, butanol, lysine, itaconic acid, 1,3-propanediol, biodiesel, and isoprene.

15. 1. A process for producing a fermentation product from a starch substrate, comprising: 1) liquefying the starch substrate; 2) saccharifying the liquefied starch substrate; and 3) Fermenting with a fermenting organism; and step 2) comprises: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO:

61.

16. 16. The process of claim 15, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

17. 17. The process of claim 16, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

18. 18. The process of any one of claims 15 to 17, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:

61.

19. 19. The process of claim 18, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

20. The polypeptides are selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, 20. The process of any one of claims 15 to 19, comprising sequence number 92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

21. 1. A process for producing a fermentation product from a starch substrate, comprising: 1) saccharifying the starch substrate at a temperature below the incipient gelatinization temperature of the starch substrate; and 2) Fermenting with a fermenting organism; Including, Step 1) is: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO:

61.

22. 22. The process of claim 21, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

23. 23. The process of claim 22, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

24. 24. The process of any one of claims 21 to 23, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:

61.

25. 25. The process of claim 24, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

26. The polypeptides are selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, 26. The process of any one of claims 21 to 25, comprising sequence number 92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

27. 1. A method for increasing starch digestibility in an animal, comprising: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, and Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO:

61.

28. 28. The method of claim 27, wherein the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

29. 29. The method of claim 28, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

30. 30. The method of any one of claims 27 to 29, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:

61.

31. 31. The method of claim 30, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

32. The polypeptides are selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, 32. The method of any one of claims 27 to 31, comprising SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

33. 1. A method for producing a fermented beverage, said method comprising: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, and Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO:

61.

34. 34. The method of claim 33, wherein the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

35. 35. The method of claim 34, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

36. 36. The method of any one of claims 33 to 35, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:

61.

37. 37. The method of claim 36, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

38. The polypeptides are selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, 38. The method of any one of claims 33 to 37, comprising SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

39. A starch substrate and: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); 1. A composition comprising one glucoamylase selected from the group consisting of: wherein said polypeptide has at least 70% identity to the catalytic domain of SEQ ID NO: 61; and said composition is at a temperature of about 4-40°C and a pH of about 3-7.

40. 40. The composition of claim 39, wherein the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

41. 41. The composition of claim 40, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

42. 42. The composition of any one of claims 39 to 41, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 66 of the polypeptide of SEQ ID NO:

61.

43. 43. The composition of claim 42, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

44. The polypeptides are selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, 44. The composition of any one of claims 39-43, comprising SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

45. 1. A recombinant host cell comprising: a) a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 or SEQ ID NO:142; b) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 81; and c): (i) YXaXbTXXXcXd (SEQ ID NO: 113), where X is any amino acid, and Xa is N or S; Xb is T, S, or R; Xc is G or N; and Xd is D, N, or S; (ii) YNTTXAGD (SEQ ID NO: 114), where X is any amino acid; (iii) XaXbXcXcAANXXd (SEQ ID NO: 115), where X is any amino acid, Xa is S or A; Xb is T, N, or V; Xc is L or I; and Xd is A or G; (iv) STLIAANXA (SEQ ID NO: 116), where X is any amino acid; (v) XaGXGNXbXc (SEQ ID NO: 117), where X is any amino acid, and Xa is N or D; Xb is S or G; and Xc is Q, K, or E; and (vi) a polypeptide comprising one or more signature motifs selected from the group consisting of: NGGNSQ (SEQ ID NO: 118); wherein the polypeptide of c) has at least 70% identity to the catalytic domain of SEQ ID NO:

61.

46. 46. ​​The recombinant host cell of claim 45, wherein the polypeptide comprises a substitution, deletion, or addition at a position corresponding to position 102 of the polypeptide of SEQ ID NO:

61.

47. 47. The recombinant host cell of claim 46, wherein the polypeptide comprises a substitution selected from the group consisting of S102P, S102G, S102A, S102V, S102L, S102I, S102F, S102Y, S102W, S102S, S102T, S102C, S102M, S102N, S102Q, S102D, S102E, S102K, S102R, and S102H.

48. 48. The recombinant host cell of any one of claims 45 to 47, wherein the polypeptide comprises a substitution, deletion or addition at a position corresponding to position 85 of the polypeptide of SEQ ID NO:

61.

49. 49. The recombinant host cell of claim 48, wherein the polypeptide comprises a substitution selected from the group consisting of V66P, VS66G, V66A, V66L, V66I, V66F, V66Y, V66W, V66S, V66T, V66C, V66M, V66N, V66Q, V66D, V66E, V66K, V66R, and V66H.

50. The polypeptides are SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:

50. The recombinant host cell of any one of claims 45-49, comprising SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, or SEQ ID NO:

142.

51. 51. The recombinant host cell of any one of claims 45 to 50, which is an ethanol-producing microorganism.

52. 52. The recombinant host cell of claim 51, which is a yeast cell.

53. 53. The recombinant host cell of any one of claims 45 to 52, wherein the host cell is not Saxenaea vasiformis.

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