Glucoamylase variants and polynucleotides encoding them

Abstract

The present invention relates to glucoamylase variants. The present invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201580054636.7, filed on October 23, 2015, entitled "Glucosylamylase variants and polynucleotides encoding them".

[0002] References to sequence lists This application includes a sequence list in computer-readable form, which is incorporated herein by reference. Background of the Invention Invention Field This invention relates to glucosylamylase variants, polynucleotides encoding these variants, methods for producing these variants, and methods for using these variants. The use of the glucosylamylase of the present invention for starch conversion to produce fermentation products, such as ethanol, and syrups (e.g., glucose) is also described. The invention further relates to a composition comprising the glucosylamylase of the present invention.

[0003] Related technical descriptions Glucoamylase (1,4-α-D-glucan glucosylhydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts, with those from the genus *Aspergillus* being the most commercially important.

[0004] Commercially, glucosylamylase is used to convert starch-containing materials that have been partially hydrolyzed by α-amylase into glucose. The glucose can then be converted directly or indirectly into fermentation products using a fermenting organism. Examples of commercial 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, lactic acid, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B vitamins). 12 Fermentation processes also produce edible alcohols (e.g., beer and wine) and dairy products (e.g., in the production of yogurt and cheese).

[0005] The final product can also be a syrup. For example, the final product can be glucose, but it can also be converted into fructose or a mixture of nearly equal amounts of glucose and fructose by glucose isomerase. Such a mixture, or a mixture further enriched with fructose, is the most commonly used commercially available high-fructose corn syrup (HFCS) worldwide.

[0006] The object of this invention is to provide polypeptides with glucosylamylase activity and polynucleotides encoding these polypeptides, and these polypeptides and polynucleotides provide high yields in the production of fermentation products (e.g., ethanol production processes).

[0007] WO 2011 / 068803 discloses glucosyl amylase isolated from the fungus Myxophytes, particularly Myxophytes squarrosa and Myxophytes squarrosa.

[0008] WO 2014 / 177546 discloses a variant of the parental glucosyl amylase disclosed in WO 2011 / 068803.

[0009] This invention provides a glucose amylase variant with improved properties compared to its parent. Invention Overview The present invention relates to a glucosylamylase variant comprising a substitution at position 295 corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0010] The present invention also relates to a glucosylamylase variant comprising substitutions at one or more positions corresponding to positions 271, 410, 72, 77, 145, 219, 303, 224, 318 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0011] In another aspect, the present invention relates to a glucosylamylase variant comprising a substitution at position 271, or 295, or one or more positions corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0012] The present invention also relates to isolated polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells containing these polynucleotides; and methods for producing these variants.

[0013] In another aspect, the present invention relates to a method for producing fermentation products (specifically ethanol) from starch-containing materials, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of α-amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermentation organism; wherein step (b) is performed using at least one variant of a glucosylamylase variant.

[0014] On the other hand, the present invention relates to a method for producing syrup products from starch-containing materials, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of α-amylase; and (b) saccharifying the liquefied material in the presence of a variant of glucosylamylase of the present invention. In another aspect, the present invention relates to compositions including variations of the present invention.

[0015] In another respect, the present invention relates to the use of this variant of glucosylamylase in the production of syrups or fermentation products.

[0016] This application also includes the following implementation schemes.

[0017] 1. A glucosylamylase variant comprising a substitution at position 295 corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0018] 2. The variant as described in embodiment 1 further comprises a substitution at at least one or more positions corresponding to positions 32, 83, 163, 169, 219, 224, 303, or 410 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0019] 3. A variant as described in any one of embodiments 1-2, wherein the number of substitutions is 1-20, for example 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0020] 4. A variant of any of the above embodiments, wherein the variant includes at least one or more of the following substitutions selected from: 295F, 295W, 224A, 224I, 224T, 32V, 83D, 163A, 163W, 169I, 219R, 303N or 410K.

[0021] 5. A variant of any of the above embodiments, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: 295W; 295W + 410K; 224I+295F; 224T+295W+318V; 295F; 224A+295F; 295W+83D+410K; 163A+295W+410K; 163W + 295W + 410K; 303N+295W; 169I+295W; or 32V+219R+295W; The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0022] 6. A variant according to any one of embodiments 1-5, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: 295W+410K; or 163A+295W+410K; The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0023] 7. A variant according to any one of the above embodiments, wherein the substitutions are selected from the following: Y295F, Y295W, L224A, L224I, L224T, A32V, S83D, N163A, N163W, V169I, T219R, S303NR410K, and Q318V.

[0024] 8. A variant as described in any one of embodiments 1-7, wherein the variant has improved properties relative to the parent, wherein such improved property is reduced glucose inhibition.

[0025] 9. A glucosylamylase variant comprising substitutions at one or more positions corresponding to positions 271, 410, 72, 77, 145, 219, 303, 224, 318 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0026] 10. The variant as described in embodiment 8, wherein the number of substitutions is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0027] 11. The variant as described in embodiment 9, wherein the variant includes at least one or more substitutions selected from the following: 72V, 77A, 145A, 219D, 271Q, 271N, 271A, 271S, 271V, 224Q, 303E, 318W, 318F, 410A, 410Q, or 410H.

[0028] 12. The variant according to embodiments 9-11, wherein the variant includes at least one of the following substitutions or combinations of substitutions: 72V + 145A; 271Q; 224Q+271N+410A; 77A; 77A+271A; 271S+318W+410Q; 271V+318F+410H; 77A+219D; 77A+303E; The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0029] 13. The variants according to embodiments 9-12, wherein the substitutions are selected from the following: D72V, L77A, L145A, T219D, L224Q, T271Q, T271N, T271A, T271S, T271V, S303E, Q318W, Q318F, R410Q, R410H and R410A.

[0030] 14. The variant according to embodiments 9-13, which has improved properties relative to the parent, wherein the improved property is increased specific activity.

[0031] 15. A glucosylamylase variant comprising a substitution at position 271, or 295, or one or more positions corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0032] 16. The variant as described in embodiment 15, wherein the number of substitutions is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0033] 17. A variant as described in embodiments 15-16, wherein the variant includes at least one or more substitutions selected from the following: 271Q, 271A, 271V, 295W, 60L, 73A, 77A, 77V, and 318Y.

[0034] 18. A variant according to any one of embodiments 15-17, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: F60L+S73A+T271Q; 271Q; 77V+271V+410A; 77A + 271A; 271V + 318Y; 295W; 271Q+295W; and The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0035] 19. A variant according to any one of embodiments 15-18, wherein the variant has improved properties relative to the parent, wherein when used in SSF, this improved property is increased ethanol yield.

[0036] 20. The variants according to embodiments 18-19, wherein the substitutions are selected from the following: F60L, S73A, T271A, T271V, T271Q, L77A, L77V, R410A, Y295W, and Q318Y.

[0037] 21. A variant according to any one of the above embodiments, wherein the amino acids at positions 95 and 121 are proline.

[0038] 22. A composition comprising the polypeptide as described in any one of embodiments 1-21.

[0039] 23. The composition according to embodiment 22 further comprises a pullulanase.

[0040] 24. The composition according to embodiment 22 or 23 further comprises α-amylase, particularly fungal α-amylase, more particularly α-amylase derived from Rhizopus microsporum or Aspergillus terreus, even more particularly α-amylase selected from the α-amylase disclosed as SEQ ID NO: 4, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N (numbered using SEQ ID NO: 4), and wherein the α-amylase variant has at least 75% similarity to the polypeptide of SEQ ID NO: 4, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, for example even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% similarity.

[0041] 25. Use of a polypeptide as described in any one of embodiments 1-21 for the production of syrups and / or fermentation products.

[0042] 26. A method for producing fermentation products, particularly ethanol, from starch-containing material, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of α-amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermentation organism; wherein step (b) is performed using at least one variant of glucosylamylase as described in any one of embodiments 1-21.

[0043] 27. The method according to implementation scheme 26, wherein steps (b) and (c) are performed simultaneously.

[0044] 28. A method for producing a syrup product from a starch-containing material, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of an α-amylase; and (b) saccharifying the liquefied material in the presence of a variant glucosylamylase as described in any one of embodiments 1-21.

[0045] 29. A polynucleotide encoding a variant as described in any one of embodiments 1-21.

[0046] 30. A nucleic acid construct comprising the polynucleotides as described in embodiment 29.

[0047] 31. An expression vector comprising the polynucleotides as described in embodiment 29.

[0048] 32. A host cell comprising the polynucleotides as described in embodiment 29.

[0049] 33. The host cell according to embodiment 32, wherein the host cell is a yeast cell, particularly of the genus *Saccharomyces*, and more particularly of *Saccharomyces cerevisiae*.

[0050] 34. A method for producing a glucose amylase variant according to any one of embodiments 1-21, comprising: culturing host cells as described in embodiments 32-33 under conditions suitable for expression of the variant; and optionally recovering the variant glucose amylase.

[0051] 35. The method as described in embodiments 26-27, wherein the glucosyl amylase variant described in any one of embodiments 1-21 is expressed from a fermentation organism, preferably a yeast fermentation organism, more preferably a brewer's yeast. Brief description of the attached diagram Figure 1 Ethanol yield is shown as a function of enzyme activity (AGU / gDS) as analyzed by HPLC. Selected variant glucosylamylases were compared with parental glucosylamylase JGA098 and wild-type glucosylamylases from *T. c. A. c.* (Tc-AMG), *G. s.* (Gs-AMG), and *P. s.* (Ps-AMG).

[0052] definition Glucoamylase: The term "glucosylase" (1,4-α-D-glucan glucosylhydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. For the purposes of this invention, glucosylase activity was determined according to the procedure described in the examples. Glucoamylase units (AGU) are defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under standard conditions (37°C, pH 4.3, substrate: 23.2 mM maltose, buffer: 0.1 M acetate, reaction time: 5 min).

[0053] In another embodiment, the polypeptide of the present invention has at least 20%, preferably at least 40%, preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% glucosylamylase activity of the polypeptide of SEQ ID NO: 2.

[0054] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations occur naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (without change in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0055] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. The initial RNA transcript is a precursor to mRNA, which undergoes a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0056] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0057] Control Sequences: The term "control sequence" refers to the nucleic acid sequence required for the expression of the polynucleotide encoding the variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, promoters, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotide encoding the variant.

[0058] Expression: The term “expression” includes any step involved in the generation of variants, including (but not limited to) transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] Expression vector: The term “expression vector” refers to a linear or circular DNA molecule that includes polynucleotides encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0060] Fragment: The term "fragment" refers to a polypeptide in which one or more (e.g., several) amino acids are absent from the amino and / or carboxyl termini of a mature polypeptide, wherein the fragment has glucosylamylase activity. In one aspect, a fragment comprises at least 454 amino acid residues (e.g., amino acids 1 to 454 of SEQ ID NO: 2), includes a catalytic domain, and has one or more substitutions according to the invention.

[0061] Highly stringent conditions: The term "highly stringent conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0062] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors including the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0063] Improved properties: The term "improved properties" refers to characteristics associated with a variant that are improved relative to the parent. These improved properties include, but are not limited to, specific activity, reduced glucose inhibition, reduced isomaltose-forming activity, increased DE11 activity, and increased thermal stability. A further improved property is increased EtOH yield when the variant is applied to saccharification and subsequent fermentation on liquefied mash.

[0064] Isolated: The term “isolated” means a substance that does not exist in nature in a form or environment. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance removed at least partially from one or more naturally occurring components associated with it in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to the kind of substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0065] Low stringency conditions: The term "low stringency conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0066] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. The mature polypeptide is disclosed herein as SEQ ID NO: 2.

[0067] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having glucosylamylase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 52 to 1728 (or 1731, including the stop codon) of SEQ ID NO: 1. Nucleotides 1 to 51 of SEQ ID NO: 1 encode a signal peptide.

[0068] Medium-tough conditions: The term "medium-tough conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0069] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0070] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain segments of nucleic acid, or is synthesized and includes one or more control sequences.

[0071] Operable ligation: The term “operable ligation” means a configuration in which a control sequence is placed in the appropriate position relative to the coding sequence of a polynucleotide so that the control sequence guides the expression of the coding sequence.

[0072] Parent or parental glucosylamylase: The term "parent" or "parental glucosylamylase" refers to a glucosylamylase that has been modified to produce the enzyme variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0073] Sequence consistency: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence consistency".

[0074] For the purposes of this invention, the following methods are used: (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics) Trends Genet. (16: 276-277) (preferably version 5.0.0 or later) The Niederman-Wunsch algorithm implemented in the Niederman program (Needleman and Wunsch, 1970, Journal of Molecular Biology) J. Mol. Biol.(48: 443-453) to determine sequence consistency between two amino acid sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked as "Longest Consistency" (obtained using the -nobrief option) is used as the percentage consistency and is calculated as follows: (Consistent residues x 100) / (Alignment length - total number of vacancies in the alignment) For the purposes of this invention, the Niedle-Onsch algorithm (Niedleman and Onsch, 1970, see above), implemented in the Niedle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later), is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Niedle output labeled "Longest Consistency" (obtained using the -nobrief option) is used as the percentage consistency and is calculated as follows: (Consistent deoxyribonucleotides x 100) / (Alignment length - Total number of vacancies in the alignment) Subsequence: The term "subsequence" refers to a polynucleotide that omits one or more (e.g., several) nucleotides from the 5' and / or 3' end of a mature polypeptide coding sequence, wherein the subsequence encodes a fragment having glucosylamylase activity. In one aspect, a subsequence encodes at least the catalytic domain of the variant according to the invention. For example, it comprises at least 1362 nucleotides (e.g., nucleotides 52 to 1413 of SEQ ID NO: 1).

[0075] Variant: The term "variant" means a polypeptide having glucosylamylase activity that contains a change (i.e., a substitution, insertion, and / or deletion at one or more (e.g., several) positions). Substitution means that an amino acid occupying a position is replaced by a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that an amino acid is added adjacent to or immediately adjacent to an amino acid occupying a position. The variants of the present invention have at least 20%, for example at least 40%, at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% glucosylamylase activity of the polypeptide of SEQ ID NO: 2.

[0076] Very High Tough Conditions: The term "very high tough conditions" means that for probes at least 100 nucleotides in length, standard DNA blotting procedures are followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0077] Very low stringency conditions: The term "very low stringency conditions" means that for probes of at least 100 nucleotides in length, a standard DNA blotting procedure is followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0078] Wild-type glucosylamylase: The term "wild-type" glucosylamylase refers to a type of glucosylamylase expressed by a naturally occurring microorganism (such as a bacterium, yeast, or filamentous fungus) found in nature.

[0079] Variant Naming Rules For the purposes of this invention, the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residues in another glucosylamylase. The amino acid sequence of the other glucosylamylase is compared with the mature polypeptide disclosed in SEQ ID NO: 2, and based on this comparison, the amino acid sequence is determined using a software package such as EMBOSS (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics). Trends Genet.The Niederman-Wunsch algorithm implemented in the Nieder program (preferred version 5.0.0 or later) (Needleman and Wunsch, 1970, Journal of Molecular Biology) 16: 276-277 J. Mol. Biol. (48: 443-453) to determine the amino acid position numbers corresponding to any amino acid residues in the mature polypeptide disclosed in SEQ ID NO: 2. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0080] The identification of corresponding amino acid residues in another glucosylamylase can be determined by using several computer programs that compare multiple polypeptide sequences with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acid Research). Nucleic Acids Research )32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acid Research 30: 3059-3066; Kato et al., 2005, Nucleic Acid Research 33: 511-518; Kazuto Kato, 2007, Bioinformatics ( Bioinformatics 23: 372-374; Kato et al., 2009, Methods in Molecular Biology 537: 39-64 Kazutsu Kato, 2010, Bioinformatics 26: 1899-1900 ) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acid Research 22: 4673-4680).

[0081] When other enzymes diverge from the polypeptide of SEQ ID NO: 2, making conventional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, Journal of Molecular Biology). J. Mol. Biol.(295: 613-615), other pairwise sequence comparison algorithms can be applied. Greater sensitivity in sequence-based searches can be achieved using search procedures that utilize probabilistic representations (profiles) of peptide families to search a database. For example, the PSI-BLAST procedure generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acid Research). Nucleic Acids Res. (25:3389-3402). Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, Journal of Molecular Biology) can be used. J. Mol. Biol .) 287: 797-815; McGuffin and Jones, 2003, Bioinformatics ( Bioinformatics (19: 874-881) This paper utilizes information from various sources (PSI-BLAST, secondary structure prediction, structure alignment spectra, and solvation potential) as input to a neural network that predicts the structural folding of a query sequence. Similarly, Gough et al., 2000, Journal of Molecular Biology ( J. Mol. Biol The method described in 313: 903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0082] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms can be used, such as distance alignment matrices (Holm and Sander, 1998, Protein Alignment Theory). Proteins )33: 88-96) or combined extension (Shindyalov and Bourne, 1998, Protein Engineering) Protein Engineering (11: 739-747) Compare two or more protein structures, and implementations of these algorithms can also be used to query structure databases containing structures of interest in order to discover possible structural homologs (e.g., Olm and Park, 2000, Bioinformatics). Bioinformatics 16: 566-567).

[0083] In the description of variations of the invention, the following nomenclature is used for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are adopted.

[0084] Substitution For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg + Ser411Phe" or "G205R + S411F" represent the substitution of glycine (G) with arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) with phenylalanine (F).

[0085] Deletion For amino acid deletions, use the following nomenclature: original amino acid, position, Therefore, the deletion of glycine at position 195 is named "Gly195". "or "G195 Multiple missing items are separated by plus signs ("+"), for example, "Gly195". + Ser411 "or "G195 +S411 ".

[0086] Insertion For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as "original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2", etc. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0087] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0088] Multiple alterationsVariants with multiple alterations are separated by a plus sign ("+"), such as "Arg170Tyr +Gly195Glu" or "R170Y + G195E", which represent arginine and glycine at positions 170 and 195 being replaced by tyrosine and glutamic acid, respectively.

[0089] Different alterations When different changes can be introduced at a single position, these changes are separated by commas. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala + Arg170Gly,Ala" represents the following variant: “Tyr167Gly + Arg170Gly”, “Tyr167Gly + Arg170Ala”, “Tyr167Ala + Arg170Gly”, and “Tyr167Ala + Arg170Ala”. Invention Details This invention relates to glucosylamylase variants comprising alterations, specifically substitutions, at one or more (e.g., several) positions corresponding to positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73 of the polypeptide of SEQ ID NO: 2, and the variant possesses glucosylamylase activity. Specifically, these variants have improved properties compared to the glucosylamylase disclosed as SEQ ID NO: 2. Specifically, these improved properties are increased specific activity and / or decreased glucose inhibition, and / or increased ethanol yield in SSF (simultaneous saccharification and fermentation). Saccharification and fermentation can also be performed in separate steps. The variants according to the invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0090] In a specific aspect, the present invention relates to a glucosylamylase variant comprising a substitution at position 295 corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0091] In another specific aspect, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions corresponding to positions 271, 410, 72, 77, 145, 219, 303, 224, 318 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0092] In another aspect, the present invention relates to a glucosylamylase variant comprising a substitution at position 271, or 295, or one or more positions of 271 and 295 corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0093] The parental glucosylamylase, SEQ ID NO: 2, contains proline at positions 95 and 121. This combination provides improved thermostability to the parental glucosylamylase. In one specific embodiment, the proline at positions 95 and 121 remains unchanged in all variants of the invention.

[0094] variants This invention provides a variety of glucosylamylase variants comprising alterations, specifically substitutions, at one or more (e.g., several) positions corresponding to positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73, and the variant possessing glucosylamylase activity. Furthermore, the variant exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO: 2.

[0095] In one embodiment, the variant is separated.

[0096] In one embodiment, the variant has 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%, but less than 100%, sequence identity with the parental glucosylamylase.

[0097] In another embodiment, the variant has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[0098] In another aspect, the variant includes changes, specifically substitutions, at one or more (e.g., several) positions corresponding to positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. In another aspect, the variant includes substitutions at two positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. In yet another aspect, the variant includes substitutions at three positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. On the other hand, the variant includes substitutions at four positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. On the other hand, the variant includes changes at five positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. On the other hand, the variant includes substitutions at six positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. In another aspect, the variant includes substitutions at seven positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. In another aspect, the variant includes substitutions at eight positions corresponding to any one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73. In yet another aspect, the variant includes substitutions at each position corresponding to one of positions 295, 224, 410, 271, 72, 77, 145, 318, 60, 32, 83, 163, 169, 303, 219, and 73.

[0099] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 295. In another aspect, the amino acid at the position corresponding to position 295 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, preferably substituted with Phe or Trp. In another aspect, this variant includes, or is composed of, the substituted Y295F or Y295W of the polypeptide of SEQ ID NO: 2.

[0100] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 224. In another aspect, the amino acid at the position corresponding to position 224 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Gln, Ala, Ile, or Thr. In another aspect, this variant includes, or is composed of, the substitution of L224Q, L224A, L224I, or L224T of the polypeptide of SEQ ID NO: 2.

[0101] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 72. In another aspect, the amino acid at the position corresponding to position 72 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In another aspect, this variant includes, or is composed of, the substitution D72V of the polypeptide of SEQ ID NO: 2.

[0102] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 77. In another aspect, the amino acid at the position corresponding to position 77 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala or Val. In another aspect, this variant includes, or is composed of, the substituted L77A or L77V of the polypeptide of SEQ ID NO: 2.

[0103] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 145. In another aspect, the amino acid at the position corresponding to position 145 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala. In another aspect, this variant includes, or is composed of, the substituted L145A of the polypeptide of SEQ ID NO: 2.

[0104] In another aspect, the variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 410. In another aspect, the amino acid at the position corresponding to position 410 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala, Gln, His, or Lys. In another aspect, the variant includes, or is composed of, the substitution of R410A, R410Q, R410H, or R410K of the polypeptide of SEQ ID NO: 2.

[0105] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 318. In another aspect, the amino acid at the position corresponding to position 318 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Val, or Tyr, preferably Trp, Phe, Val, or Tyr. In another aspect, this variant includes, or is composed of, substitutions of the polypeptide of SEQ ID NO: 2, Q318W, Q318F, Q318V, or Q318Y.

[0106] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 271. In another aspect, the amino acid at the position corresponding to position 271 is substituted with Ala, Asn, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Val, or Tyr, preferably substituted with Gln, Asn, Ala, Ser, or Val. In another aspect, this variant includes, or is composed of, substitutions of the polypeptide of SEQ ID NO: 2, T271Q, T271N, T271A, T271S, or T271V.

[0107] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 60. In another aspect, the amino acid at the position corresponding to position 60 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Leu. In another aspect, this variant includes, or is composed of, the substituted F60L of the polypeptide of SEQ ID NO: 2.

[0108] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 73. In another aspect, the amino acid at the position corresponding to position 73 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably substituted with Ala. In another aspect, this variant includes, or is composed of, the substitution S73A of the polypeptide of SEQ ID NO: 2.

[0109] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 32. In another aspect, the amino acid at the position corresponding to position 32 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In another aspect, this variant includes, or is composed of, the substituted A32V of the polypeptide of SEQ ID NO: 2.

[0110] In another aspect, this variant includes, or is composed of, a substitution at the position corresponding to position 83. In another aspect, the amino acid at the position corresponding to position 83 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Asp. In another aspect, this variant includes, or is composed of, the substitution S83D of the polypeptide of SEQ ID NO: 2.

[0111] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 163. In another aspect, the amino acid at the position corresponding to position 163 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala. In another aspect, this variant includes, or is composed of, the substituted N163A or N163W of the polypeptide of SEQ ID NO: 2.

[0112] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 169. In another aspect, the amino acid at the position corresponding to position 169 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably substituted with Ile. In another aspect, this variant includes, or is composed of, the substitution V169I of the polypeptide of SEQ ID NO: 2.

[0113] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 219. In another aspect, the amino acid at the position corresponding to position 219 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably Asp or Arg. In another aspect, this variant includes, or is composed of, the substitution T219R or T219D of the polypeptide of SEQ ID NO: 2.

[0114] In another aspect, this variant includes alterations to or composition thereof, specifically substitutions at the position corresponding to position 303. In another aspect, the amino acid at the position corresponding to position 303 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably substituted with Glu or Asn. In another aspect, this variant includes, or is composed of, the substitution S303E or S303N of the polypeptide of SEQ ID NO: 2.

[0115] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 224, such as those described above.

[0116] On the other hand, these variations include substitutions or components at positions corresponding to positions 295 and 410, such as those described above.

[0117] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271, such as those described above.

[0118] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 72, such as those described above.

[0119] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 77, such as those described above.

[0120] On the other hand, these variations include substitutions or components at positions corresponding to positions 295 and 145, such as those described above.

[0121] On the other hand, these variations include substitutions or components at positions corresponding to positions 295 and 318, such as those described above.

[0122] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 60, such as those described above.

[0123] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 73, such as those described above.

[0124] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410, such as those described above.

[0125] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271, such as those described above.

[0126] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 72, such as those described above.

[0127] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 77, such as those described above.

[0128] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 145, such as those described above.

[0129] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 318, such as those described above.

[0130] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 60, such as those described above.

[0131] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 73, such as those described above.

[0132] On the other hand, these variations include substitutions or components at positions corresponding to positions 410 and 271, such as those described above.

[0133] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 72, such as those described above.

[0134] On the other hand, these variations include substitutions or components at positions corresponding to positions 410 and 77, such as those described above.

[0135] On the other hand, these variations include substitutions or components at positions corresponding to positions 410 and 145, such as those described above.

[0136] On the other hand, these variations include substitutions or components at positions corresponding to positions 410 and 318, such as those described above.

[0137] On the other hand, these variations include substitutions or components at positions corresponding to positions 410 and 60, such as those described above.

[0138] On the other hand, these variations include substitutions or components at positions corresponding to positions 410 and 73, such as those described above.

[0139] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 72, such as those described above.

[0140] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 77, such as those described above.

[0141] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 145, such as those described above.

[0142] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 318, such as those described above.

[0143] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 60, such as those described above.

[0144] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 73, such as those described above.

[0145] On the other hand, these variations include substitutions or components at positions corresponding to positions 72 and 77, such as those described above.

[0146] On the other hand, these variations include substitutions or components at positions corresponding to positions 72 and 145, such as those described above.

[0147] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 72 and 318, such as those described above.

[0148] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 72 and 60, such as those described above.

[0149] On the other hand, these variations include substitutions or components at positions corresponding to positions 72 and 73, such as those described above.

[0150] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145, such as those described above.

[0151] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 318, such as those described above.

[0152] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 60, such as those described above.

[0153] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 73, such as those described above.

[0154] On the other hand, these variations include substitutions or components at positions corresponding to positions 145 and 318, such as those described above.

[0155] On the other hand, these variations include substitutions or components at positions corresponding to positions 145 and 60, such as those described above.

[0156] On the other hand, these variations include substitutions or components at positions corresponding to positions 145 and 73, such as those described above.

[0157] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 318 and 60, such as those described above.

[0158] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 318 and 73, such as those described above.

[0159] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 60 and 73, such as those described above.

[0160] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 410, such as those described above.

[0161] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 271, such as those described above.

[0162] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 72, such as those described above.

[0163] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295, 224, and 77, such as those described above.

[0164] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 145, such as those described above.

[0165] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 318, such as those described above.

[0166] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 60, such as those described above.

[0167] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 224, and 73, such as those described above.

[0168] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271, such as those described above.

[0169] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72, such as those described above.

[0170] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 410 and 77, such as those described above.

[0171] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 145, such as those described above.

[0172] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 318, such as those described above.

[0173] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 60, such as those described above.

[0174] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 410 and 73, such as those described above.

[0175] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72, such as those described above.

[0176] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 271, and 77, such as those described above.

[0177] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 271, and 145, such as those described above.

[0178] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 271, and 318, such as those described above.

[0179] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 60, such as those described above.

[0180] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 73, such as those described above.

[0181] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77, such as those described above.

[0182] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 72 and 145, such as those described above.

[0183] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 318, such as those described above.

[0184] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 60, such as those described above.

[0185] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 73, such as those described above.

[0186] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 77 and 145, such as those described above.

[0187] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 77, and 318, such as those described above.

[0188] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295, 77, and 60, such as those described above.

[0189] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 73, such as those described above.

[0190] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295, 145, and 318, such as those described above.

[0191] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 145 and 60, such as those described above.

[0192] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 145 and 73, such as those described above.

[0193] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 318 and 60, such as those described above.

[0194] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 318 and 73, such as those described above.

[0195] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 60 and 73, such as those described above.

[0196] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271, such as those described above.

[0197] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72, such as those described above.

[0198] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 77, such as those described above.

[0199] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 145, such as those described above.

[0200] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 318, such as those described above.

[0201] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 60, such as those described above.

[0202] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 73, such as those described above.

[0203] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72, such as those described above.

[0204] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77, such as those described above.

[0205] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145, such as those described above.

[0206] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224, 271, and 318, such as those described above.

[0207] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 60, such as those described above.

[0208] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 73, such as those described above.

[0209] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77, such as those described above.

[0210] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 72 and 145, such as those described above.

[0211] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 72 and 318, such as those described above.

[0212] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 72 and 60, such as those described above.

[0213] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 73, such as those described above.

[0214] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 77 and 145, such as those described above.

[0215] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 77 and 318, such as those described above.

[0216] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 77 and 60, such as those described above.

[0217] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 73, such as those described above.

[0218] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 145 and 318, such as those described above.

[0219] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 145 and 60, such as those described above.

[0220] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 145 and 73, such as those described above.

[0221] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 318 and 60, such as those described above.

[0222] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 318 and 73, such as those described above.

[0223] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 60 and 73, such as those described above.

[0224] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72, such as those described above.

[0225] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 271 and 77, such as those described above.

[0226] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 145, such as those described above.

[0227] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 318, such as those described above.

[0228] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 60, such as those described above.

[0229] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 73, such as those described above.

[0230] On the other hand, these variations include substitutions or components at positions corresponding to positions 410, 72, and 77, such as those described above.

[0231] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410, 72, and 145, such as those described above.

[0232] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410, 72, and 318, such as those described above.

[0233] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410, 72, and 60, such as those described above.

[0234] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 73, such as those described above.

[0235] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410, 77, and 145, such as those described above.

[0236] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410, 77, and 318, such as those described above.

[0237] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410, 77, and 60, such as those described above.

[0238] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 73, such as those described above.

[0239] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 145 and 318, such as those described above.

[0240] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 145 and 60, such as those described above.

[0241] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 145 and 73, such as those described above.

[0242] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 318 and 60, such as those described above.

[0243] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 318 and 73, such as those described above.

[0244] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410, 60, and 73, such as those described above.

[0245] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 72, and 77, such as those described above.

[0246] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 72, and 145, such as those described above.

[0247] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 72, and 318, such as those described above.

[0248] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 72, and 60, such as those described above.

[0249] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 72, and 73, such as those described above.

[0250] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271, 77, and 145, such as those described above.

[0251] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 77, and 318, such as those described above.

[0252] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271, 77, and 60, such as those described above.

[0253] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 73, such as those described above.

[0254] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 145 and 318, such as those described above.

[0255] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 145 and 60, such as those described above.

[0256] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 145 and 73, such as those described above.

[0257] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 318 and 60, such as those described above.

[0258] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 318 and 73, such as those described above.

[0259] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 60 and 73, such as those described above.

[0260] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72, 77, and 145, such as those described above.

[0261] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72, 77, and 318, such as those described above.

[0262] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72, 77, and 60, such as those described above.

[0263] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72, 77, and 73, such as those described above.

[0264] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 72 and 145 and 318, such as those described above.

[0265] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 145 and 60, such as those described above.

[0266] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 145 and 73, such as those described above.

[0267] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 318 and 60, such as those described above.

[0268] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 318 and 73, such as those described above.

[0269] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 60 and 73, such as those described above.

[0270] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145 and 318, such as those described above.

[0271] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145 and 60, such as those described above.

[0272] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145 and 73, such as those described above.

[0273] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 77 and 318 and 60, such as those described above.

[0274] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 318 and 73, such as those described above.

[0275] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 77 and 60 and 73, such as those described above.

[0276] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 145 and 318 and 60, such as those described above.

[0277] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 145 and 318 and 73, such as those described above.

[0278] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 145 and 60 and 73, such as those described above.

[0279] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 318 and 60 and 73, such as those described above.

[0280] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271, such as those described above.

[0281] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72, such as those described above.

[0282] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77, such as those described above.

[0283] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 145, such as those described above.

[0284] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 318, such as those described above.

[0285] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 60, such as those described above.

[0286] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 73, such as those described above.

[0287] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72, such as those described above.

[0288] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77, such as those described above.

[0289] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 145, such as those described above.

[0290] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 318, such as those described above.

[0291] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 60, such as those described above.

[0292] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 73, such as those described above.

[0293] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77, such as those described above.

[0294] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145, such as those described above.

[0295] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 318, such as those described above.

[0296] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 60, such as those described above.

[0297] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 73, such as those described above.

[0298] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 145, such as those described above.

[0299] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 318, such as those described above.

[0300] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 60, such as those described above.

[0301] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 73, such as those described above.

[0302] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 145 and 318, such as those described above.

[0303] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 145 and 60, such as those described above.

[0304] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 145 and 73, such as those described above.

[0305] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 318 and 60, such as those described above.

[0306] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 318 and 73, such as those described above.

[0307] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 60 and 73, such as those described above.

[0308] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72, such as those described above.

[0309] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77, such as those described above.

[0310] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 145, such as those described above.

[0311] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 318, such as those described above.

[0312] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 60, such as those described above.

[0313] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 73, such as those described above.

[0314] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77, such as those described above.

[0315] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 145, such as those described above.

[0316] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 410 and 72 and 318, such as those described above.

[0317] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 410 and 72 and 60, such as those described above.

[0318] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 73, such as those described above.

[0319] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 145, such as those described above.

[0320] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 318, such as those described above.

[0321] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 60, such as those described above.

[0322] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 73, such as those described above.

[0323] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 145 and 318, such as those described above.

[0324] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 145 and 60, such as those described above.

[0325] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 410 and 145 and 73, such as those described above.

[0326] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 318 and 60, such as those described above.

[0327] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 295 and 410 and 318 and 73, such as those described above.

[0328] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 60 and 73, such as those described above.

[0329] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77, such as those described above.

[0330] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145, such as those described above.

[0331] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 318, such as those described above.

[0332] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 60, such as those described above.

[0333] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 73, such as those described above.

[0334] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 145, such as those described above.

[0335] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 318, such as those described above.

[0336] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 60, such as those described above.

[0337] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 73, such as those described above.

[0338] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 145 and 318, such as those described above.

[0339] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 145 and 60, such as those described above.

[0340] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 145 and 73, such as those described above.

[0341] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 318 and 60, such as those described above.

[0342] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 318 and 73, such as those described above.

[0343] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 60 and 73, such as those described above.

[0344] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 145, such as those described above.

[0345] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 318, such as those described above.

[0346] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 60, such as those described above.

[0347] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 73, such as those described above.

[0348] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 318, such as those described above.

[0349] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 60, such as those described above.

[0350] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 73, such as those described above.

[0351] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 318 and 60, such as those described above.

[0352] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 318 and 73, such as those described above.

[0353] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 60 and 73, such as those described above.

[0354] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 145 and 318, such as those described above.

[0355] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 145 and 60, such as those described above.

[0356] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 145 and 73, such as those described above.

[0357] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 318 and 60, such as those described above.

[0358] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 318 and 73, such as those described above.

[0359] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 60 and 73, such as those described above.

[0360] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 145 and 318 and 60, such as those described above.

[0361] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 145 and 318 and 73, such as those described above.

[0362] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 145 and 60 and 73, such as those described above.

[0363] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 318 and 60 and 73, such as those described above.

[0364] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72, such as those described above.

[0365] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77, such as those described above.

[0366] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 145, such as those described above.

[0367] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 318, such as those described above.

[0368] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 60, such as those described above.

[0369] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 73, such as those described above.

[0370] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77, such as those described above.

[0371] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 72 and 145, such as those described above.

[0372] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 318, such as those described above.

[0373] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 60, such as those described above.

[0374] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 73, such as those described above.

[0375] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 77 and 145, such as those described above.

[0376] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 318, such as those described above.

[0377] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 60, such as those described above.

[0378] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 73, such as those described above.

[0379] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 145 and 318, such as those described above.

[0380] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 145 and 60, such as those described above.

[0381] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 145 and 73, such as those described above.

[0382] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 318 and 60, such as those described above.

[0383] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 410 and 318 and 73, such as those described above.

[0384] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 60 and 73, such as those described above.

[0385] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77, such as those described above.

[0386] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 145, such as those described above.

[0387] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 318, such as those described above.

[0388] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 60, such as those described above.

[0389] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 73, such as those described above.

[0390] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145, such as those described above.

[0391] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 318, such as those described above.

[0392] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 60, such as those described above.

[0393] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 73, such as those described above.

[0394] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 318, such as those described above.

[0395] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 60, such as those described above.

[0396] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 73, such as those described above.

[0397] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 318 and 60, such as those described above.

[0398] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 318 and 73, such as those described above.

[0399] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 60 and 73, such as those described above.

[0400] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 145, such as those described above.

[0401] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 318, such as those described above.

[0402] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 60, such as those described above.

[0403] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 73, such as those described above.

[0404] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 145 and 318, such as those described above.

[0405] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 145 and 60, such as those described above.

[0406] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 72 and 145 and 73, such as those described above.

[0407] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 72 and 318 and 60, such as those described above.

[0408] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 318 and 73, such as those described above.

[0409] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 60 and 73, such as those described above.

[0410] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 77 and 145 and 318, such as those described above.

[0411] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 145 and 60, such as those described above.

[0412] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 145 and 73, such as those described above.

[0413] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 224 and 77 and 318 and 60, such as those described above.

[0414] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 318 and 73, such as those described above.

[0415] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 60 and 73, such as those described above.

[0416] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 145 and 318 and 60, such as those described above.

[0417] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 145 and 318 and 73, such as those described above.

[0418] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 145 and 60 and 73, such as those described above.

[0419] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 318 and 60 and 73, such as those described above.

[0420] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77, such as those described above.

[0421] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 145, such as those described above.

[0422] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 271 and 72 and 318, such as those described above.

[0423] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 60, such as those described above.

[0424] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 73, such as those described above.

[0425] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 145, such as those described above.

[0426] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 271 and 77 and 318, such as those described above.

[0427] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 60, such as those described above.

[0428] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 73, such as those described above.

[0429] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 145 and 318, such as those described above.

[0430] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 145 and 60, such as those described above.

[0431] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 271 and 145 and 73, such as those described above.

[0432] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 318 and 60, such as those described above.

[0433] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 318 and 73, such as those described above.

[0434] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 60 and 73, such as those described above.

[0435] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 145, such as those described above.

[0436] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 318, such as those described above.

[0437] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 60, such as those described above.

[0438] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 73, such as those described above.

[0439] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 72 and 145 and 318, such as those described above.

[0440] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 145 and 60, such as those described above.

[0441] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 145 and 73, such as those described above.

[0442] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 318 and 60, such as those described above.

[0443] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 318 and 73, such as those described above.

[0444] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 60 and 73, such as those described above.

[0445] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 77 and 145 and 318, such as those described above.

[0446] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 145 and 60, such as those described above.

[0447] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 145 and 73, such as those described above.

[0448] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 318 and 60, such as those described above.

[0449] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 318 and 73, such as those described above.

[0450] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 60 and 73, such as those described above.

[0451] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 145 and 318 and 60, such as those described above.

[0452] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 410 and 145 and 318 and 73, such as those described above.

[0453] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 145 and 60 and 73, such as those described above.

[0454] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 318 and 60 and 73, such as those described above.

[0455] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 145, such as those described above.

[0456] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 318, such as those described above.

[0457] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 60, such as those described above.

[0458] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 73, such as those described above.

[0459] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 72 and 145 and 318, such as those described above.

[0460] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 145 and 60, such as those described above.

[0461] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 145 and 73, such as those described above.

[0462] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 318 and 60, such as those described above.

[0463] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 318 and 73, such as those described above.

[0464] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 60 and 73, such as those described above.

[0465] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 77 and 145 and 318, such as those described above.

[0466] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 77 and 145 and 60, such as those described above.

[0467] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 145 and 73, such as those described above.

[0468] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 318 and 60, such as those described above.

[0469] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 318 and 73, such as those described above.

[0470] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 60 and 73, such as those described above.

[0471] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 271 and 145 and 318 and 60, such as those described above.

[0472] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 145 and 318 and 73, such as those described above.

[0473] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 145 and 60 and 73, such as those described above.

[0474] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 318 and 60 and 73, such as those described above.

[0475] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 145 and 318, such as those described above.

[0476] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 145 and 60, such as those described above.

[0477] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 145 and 73, such as those described above.

[0478] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 318 and 60, such as those described above.

[0479] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 318 and 73, such as those described above.

[0480] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 60 and 73, such as those described above.

[0481] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 72 and 145 and 318 and 60, such as those described above.

[0482] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 145 and 318 and 73, such as those described above.

[0483] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 145 and 60 and 73, such as those described above.

[0484] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 318 and 60 and 73, such as those described above.

[0485] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145 and 318 and 60, such as those described above.

[0486] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145 and 318 and 73, such as those described above.

[0487] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 77 and 145 and 60 and 73, such as those described above.

[0488] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 77 and 318 and 60 and 73, such as those described above.

[0489] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 145 and 318 and 60 and 73, such as those described above.

[0490] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72, such as those described above.

[0491] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77, such as those described above.

[0492] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145, such as those described above.

[0493] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 318, such as those described above.

[0494] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 60, such as those described above.

[0495] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 73, such as those described above.

[0496] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77, such as those described above.

[0497] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145, such as those described above.

[0498] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 318, such as those described above.

[0499] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 60, such as those described above.

[0500] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 73, such as those described above.

[0501] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145, such as those described above.

[0502] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 318, such as those described above.

[0503] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 60, such as those described above.

[0504] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 73, such as those described above.

[0505] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 318, such as those described above.

[0506] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 60, such as those described above.

[0507] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 73, such as those described above.

[0508] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 318 and 60, such as those described above.

[0509] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 318 and 73, such as those described above.

[0510] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 60 and 73, such as those described above.

[0511] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77, such as those described above.

[0512] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145, such as those described above.

[0513] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 318, such as those described above.

[0514] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 60, such as those described above.

[0515] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 73, such as those described above.

[0516] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145, such as those described above.

[0517] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 318, such as those described above.

[0518] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 60, such as those described above.

[0519] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 73, such as those described above.

[0520] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 318, such as those described above.

[0521] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 60, such as those described above.

[0522] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 73, such as those described above.

[0523] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 318 and 60, such as those described above.

[0524] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 318 and 73, such as those described above.

[0525] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 60 and 73, such as those described above.

[0526] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145, such as those described above.

[0527] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 318, such as those described above.

[0528] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 60, such as those described above.

[0529] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 73, such as those described above.

[0530] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 318, such as those described above.

[0531] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 60, such as those described above.

[0532] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 73, such as those described above.

[0533] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 318 and 60, such as those described above.

[0534] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 318 and 73, such as those described above.

[0535] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 60 and 73, such as those described above.

[0536] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 318, such as those described above.

[0537] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 60, such as those described above.

[0538] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 73, such as those described above.

[0539] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 318 and 60, such as those described above.

[0540] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 77 and 318 and 73, such as those described above.

[0541] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 60 and 73, such as those described above.

[0542] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 145 and 318 and 60, such as those described above.

[0543] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 145 and 318 and 73, such as those described above.

[0544] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 145 and 60 and 73, such as those described above.

[0545] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 318 and 60 and 73, such as those described above.

[0546] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77, such as those described above.

[0547] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145, such as those described above.

[0548] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 318, such as those described above.

[0549] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 60, such as those described above.

[0550] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 73, such as those described above.

[0551] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145, such as those described above.

[0552] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 318, such as those described above.

[0553] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 60, such as those described above.

[0554] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 73, such as those described above.

[0555] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 318, such as those described above.

[0556] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 60, such as those described above.

[0557] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 73, such as those described above.

[0558] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 318 and 60, such as those described above.

[0559] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 318 and 73, such as those described above.

[0560] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 60 and 73, such as those described above.

[0561] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145, such as those described above.

[0562] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 318, such as those described above.

[0563] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 60, such as those described above.

[0564] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 73, such as those described above.

[0565] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 318, such as those described above.

[0566] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 60, such as those described above.

[0567] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 73, such as those described above.

[0568] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 318 and 60, such as those described above.

[0569] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 318 and 73, such as those described above.

[0570] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 60 and 73, such as those described above.

[0571] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 318, such as those described above.

[0572] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 60, such as those described above.

[0573] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 73, such as those described above.

[0574] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 318 and 60, such as those described above.

[0575] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 77 and 318 and 73, such as those described above.

[0576] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 60 and 73, such as those described above.

[0577] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 145 and 318 and 60, such as those described above.

[0578] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 145 and 60 and 73, such as those described above.

[0579] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 318 and 60 and 73, such as those described above.

[0580] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145, such as those described above.

[0581] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 318, such as those described above.

[0582] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 60, such as those described above.

[0583] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 73, such as those described above.

[0584] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 318, such as those described above.

[0585] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 60, such as those described above.

[0586] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 73, such as those described above.

[0587] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 318 and 60, such as those described above.

[0588] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 318 and 73, such as those described above.

[0589] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 60 and 73, such as those described above.

[0590] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 318, such as those described above.

[0591] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 60, such as those described above.

[0592] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 73, such as those described above.

[0593] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 318 and 60, such as those described above.

[0594] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 318 and 73, such as those described above.

[0595] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 60 and 73, such as those described above.

[0596] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 145 and 318 and 60, such as those described above.

[0597] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 145 and 318 and 73, such as those described above.

[0598] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 145 and 60 and 73, such as those described above.

[0599] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 318 and 60 and 73, such as those described above.

[0600] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 318, such as those described above.

[0601] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 60, such as those described above.

[0602] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 73, such as those described above.

[0603] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 318 and 60, such as those described above.

[0604] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 318 and 73, such as those described above.

[0605] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 60 and 73, such as those described above.

[0606] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 318 and 60, such as those described above.

[0607] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 318 and 73, such as those described above.

[0608] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 60 and 73, such as those described above.

[0609] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 318 and 60 and 73, such as those described above.

[0610] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 145 and 318 and 60, such as those described above.

[0611] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 145 and 318 and 73, such as those described above.

[0612] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 145 and 60 and 73, such as those described above.

[0613] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 77 and 318 and 60 and 73, such as those described above.

[0614] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 145 and 318 and 60 and 73, such as those described above.

[0615] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77, such as those described above.

[0616] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145, such as those described above.

[0617] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 318, such as those described above.

[0618] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 60, such as those described above.

[0619] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 73, such as those described above.

[0620] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145, such as those described above.

[0621] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 318, such as those described above.

[0622] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 60, such as those described above.

[0623] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 73, such as those described above.

[0624] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 318, such as those described above.

[0625] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 60, such as those described above.

[0626] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 73, such as those described above.

[0627] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 318 and 60, such as those described above.

[0628] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 318 and 73, such as those described above.

[0629] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 60 and 73, such as those described above.

[0630] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145, such as those described above.

[0631] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 318, such as those described above.

[0632] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 60, such as those described above.

[0633] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 73, such as those described above.

[0634] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 318, such as those described above.

[0635] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 60, such as those described above.

[0636] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 73, such as those described above.

[0637] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 318 and 60, such as those described above.

[0638] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 318 and 73, such as those described above.

[0639] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 60 and 73, such as those described above.

[0640] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 318, such as those described above.

[0641] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 60, such as those described above.

[0642] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 73, such as those described above.

[0643] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 318 and 60, such as those described above.

[0644] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 318 and 73, such as those described above.

[0645] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 60 and 73, such as those described above.

[0646] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 145 and 318 and 60, such as those described above.

[0647] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 145 and 318 and 73, such as those described above.

[0648] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 145 and 60 and 73, such as those described above.

[0649] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 318 and 60 and 73, such as those described above.

[0650] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145, such as those described above.

[0651] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 318, such as those described above.

[0652] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 60, such as those described above.

[0653] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 73, such as those described above.

[0654] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 318, such as those described above.

[0655] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 60, such as those described above.

[0656] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 73, such as those described above.

[0657] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 318 and 60, such as those described above.

[0658] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 318 and 73, such as those described above.

[0659] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 60 and 73, such as those described above.

[0660] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 318, such as those described above.

[0661] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 60, such as those described above.

[0662] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 73, such as those described above.

[0663] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 318 and 60, such as those described above.

[0664] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 318 and 73, such as those described above.

[0665] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 60 and 73, such as those described above.

[0666] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 318 and 60, such as those described above.

[0667] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 318 and 73, such as those described above.

[0668] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 60 and 73, such as those described above.

[0669] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 318 and 60 and 73, such as those described above.

[0670] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 318, such as those described above.

[0671] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 60, such as those described above.

[0672] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 73, such as those described above.

[0673] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 318 and 60, such as those described above.

[0674] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 318 and 73, such as those described above.

[0675] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 60 and 73, such as those described above.

[0676] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 145 and 318 and 60, such as those described above.

[0677] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 145 and 318 and 73, such as those described above.

[0678] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 145 and 60 and 73, such as those described above.

[0679] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 318 and 60 and 73, such as those described above.

[0680] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 145 and 318 and 60, such as those described above.

[0681] On the other hand, the variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 77 and 145 and 318 and 73, such as those described above.

[0682] On the other hand, the variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 77 and 145 and 60 and 73, such as those described above.

[0683] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 77 and 318 and 60 and 73, such as those described above.

[0684] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 145 and 318 and 60 and 73, such as those described above.

[0685] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145, such as those described above.

[0686] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 318, such as those described above.

[0687] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 60, such as those described above.

[0688] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 73, such as those described above.

[0689] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 318, such as those described above.

[0690] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 60, such as those described above.

[0691] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 73, such as those described above.

[0692] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 318 and 60, such as those described above.

[0693] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 318 and 73, such as those described above.

[0694] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 60 and 73, such as those described above.

[0695] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 318, such as those described above.

[0696] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 60, such as those described above.

[0697] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 73, such as those described above.

[0698] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 318 and 60, such as those described above.

[0699] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 318 and 73, such as those described above.

[0700] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 60 and 73, such as those described above.

[0701] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 145 and 318 and 60, such as those described above.

[0702] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 145 and 318 and 73, such as those described above.

[0703] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 145 and 60 and 73, such as those described above.

[0704] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 318 and 60 and 73, such as those described above.

[0705] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 318, such as those described above.

[0706] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 60, such as those described above.

[0707] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 73, such as those described above.

[0708] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 318 and 60, such as those described above.

[0709] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 318 and 73, such as those described above.

[0710] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 60 and 73, such as those described above.

[0711] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 72 and 145 and 318 and 60, such as those described above.

[0712] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 145 and 318 and 73, such as those described above.

[0713] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 145 and 60 and 73, such as those described above.

[0714] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 318 and 60 and 73, such as those described above.

[0715] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 145 and 318 and 60, such as those described above.

[0716] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 145 and 318 and 73, such as those described above.

[0717] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 145 and 60 and 73, such as those described above.

[0718] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 77 and 318 and 60 and 73, such as those described above.

[0719] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 145 and 318 and 60 and 73, such as those described above.

[0720] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 318, such as those described above.

[0721] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 60, such as those described above.

[0722] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 73, such as those described above.

[0723] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 318 and 60, such as those described above.

[0724] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 318 and 73, such as those described above.

[0725] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 60 and 73, such as those described above.

[0726] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 145 and 318 and 60, such as those described above.

[0727] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 271 and 72 and 145 and 318 and 73, such as those described above.

[0728] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 145 and 60 and 73, such as those described above.

[0729] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 318 and 60 and 73, such as those described above.

[0730] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 145 and 318 and 60, such as those described above.

[0731] On the other hand, the variant changes include substitutions or composition thereof at positions corresponding to positions 271 and 77 and 145 and 318 and 73, such as those described above.

[0732] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 145 and 60 and 73, such as those described above.

[0733] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 77 and 318 and 60 and 73, such as those described above.

[0734] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 145 and 318 and 60 and 73, such as those described above.

[0735] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 145 and 318 and 60, such as those described above.

[0736] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 145 and 318 and 73, such as those described above.

[0737] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 72 and 77 and 145 and 60 and 73, such as those described above.

[0738] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 77 and 318 and 60 and 73, such as those described above.

[0739] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 72 and 145 and 318 and 60 and 73, such as those described above.

[0740] On the other hand, these variant changes include substitutions or components at positions corresponding to positions 77 and 145 and 318 and 60 and 73, such as those described above.

[0741] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77, such as those described above.

[0742] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145, such as those described above.

[0743] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 318, such as those described above.

[0744] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 60, such as those described above.

[0745] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 73, such as those described above.

[0746] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145, such as those described above.

[0747] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 318, such as those described above.

[0748] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 60, such as those described above.

[0749] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 73, such as those described above.

[0750] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 318, such as those described above.

[0751] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 60, such as those described above.

[0752] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 73, such as those described above.

[0753] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 318 and 60, such as those described above.

[0754] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 318 and 73, such as those described above.

[0755] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 60 and 73, such as those described above.

[0756] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145, such as those described above.

[0757] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 318, such as those described above.

[0758] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 60, such as those described above.

[0759] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 73, such as those described above.

[0760] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 318, such as those described above.

[0761] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 60, such as those described above.

[0762] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 73, such as those described above.

[0763] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 318 and 60, such as those described above.

[0764] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 318 and 73, such as those described above.

[0765] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 60 and 73, such as those described above.

[0766] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 318, such as those described above.

[0767] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 60, such as those described above.

[0768] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 73, such as those described above.

[0769] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 318 and 60, such as those described above.

[0770] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 318 and 73, such as those described above.

[0771] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 60 and 73, such as those described above.

[0772] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 318 and 60, such as those described above.

[0773] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 318 and 73, such as those described above.

[0774] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 60 and 73, such as those described above.

[0775] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 318 and 60 and 73, such as those described above.

[0776] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145, such as those described above.

[0777] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 318, such as those described above.

[0778] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 60, such as those described above.

[0779] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 73, such as those described above.

[0780] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 318, such as those described above.

[0781] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 60, such as those described above.

[0782] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 73, such as those described above.

[0783] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 318 and 60, such as those described above.

[0784] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 318 and 73, such as those described above.

[0785] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 60 and 73, such as those described above.

[0786] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 318, such as those described above.

[0787] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 60, such as those described above.

[0788] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 73, such as those described above.

[0789] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 318 and 60, such as those described above.

[0790] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 318 and 73, such as those described above.

[0791] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 60 and 73, such as those described above.

[0792] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 318 and 60, such as those described above.

[0793] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 318 and 73, such as those described above.

[0794] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 60 and 73, such as those described above.

[0795] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 318 and 60 and 73, such as those described above.

[0796] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 318, such as those described above.

[0797] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 60, such as those described above.

[0798] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 73, such as those described above.

[0799] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 318 and 60, such as those described above.

[0800] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 318 and 73, such as those described above.

[0801] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 60 and 73, such as those described above.

[0802] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 318 and 60, such as those described above.

[0803] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 318 and 73, such as those described above.

[0804] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 60 and 73, such as those described above.

[0805] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 318 and 60 and 73, such as those described above.

[0806] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 318 and 60, such as those described above.

[0807] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 318 and 73, such as those described above.

[0808] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 60 and 73, such as those described above.

[0809] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 318 and 60 and 73, such as those described above.

[0810] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 145 and 318 and 60 and 73, such as those described above.

[0811] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145, such as those described above.

[0812] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 318, such as those described above.

[0813] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 60, such as those described above.

[0814] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 73, such as those described above.

[0815] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 318, such as those described above.

[0816] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 60, such as those described above.

[0817] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 73, such as those described above.

[0818] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 318 and 60, such as those described above.

[0819] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 318 and 73, such as those described above.

[0820] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 60 and 73, such as those described above.

[0821] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 318, such as those described above.

[0822] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 60, such as those described above.

[0823] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 73, such as those described above.

[0824] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 318 and 60, such as those described above.

[0825] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 318 and 73, such as those described above.

[0826] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 60 and 73, such as those described above.

[0827] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 318 and 60, such as those described above.

[0828] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 318 and 73, such as those described above.

[0829] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 60 and 73, such as those described above.

[0830] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 318 and 60 and 73, such as those described above.

[0831] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 318, such as those described above.

[0832] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 60, such as those described above.

[0833] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 73, such as those described above.

[0834] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 318 and 60, such as those described above.

[0835] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 318 and 73, such as those described above.

[0836] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 60 and 73, such as those described above.

[0837] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 318 and 60, such as those described above.

[0838] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 318 and 73, such as those described above.

[0839] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 60 and 73, such as those described above.

[0840] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 318 and 60 and 73, such as those described above.

[0841] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 318 and 60, such as those described above.

[0842] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 318 and 73, such as those described above.

[0843] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 60 and 73, such as those described above.

[0844] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 77 and 318 and 60 and 73, such as those described above.

[0845] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 145 and 318 and 60 and 73, such as those described above.

[0846] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 318, such as those described above.

[0847] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 60, such as those described above.

[0848] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 73, such as those described above.

[0849] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 318 and 60, such as those described above.

[0850] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 318 and 73, such as those described above.

[0851] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 60 and 73, such as those described above.

[0852] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 318 and 60, such as those described above.

[0853] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 318 and 73, such as those described above.

[0854] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 60 and 73, such as those described above.

[0855] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 318 and 60 and 73, such as those described above.

[0856] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 318 and 60, such as those described above.

[0857] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 318 and 73, such as those described above.

[0858] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 60 and 73, such as those described above.

[0859] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 77 and 318 and 60 and 73, such as those described above.

[0860] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 145 and 318 and 60 and 73, such as those described above.

[0861] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 318 and 60, such as those described above.

[0862] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 318 and 73, such as those described above.

[0863] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 60 and 73, such as those described above.

[0864] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 318 and 60 and 73, such as those described above.

[0865] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 145 and 318 and 60 and 73, such as those described above.

[0866] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 77 and 145 and 318 and 60 and 73, such as those described above.

[0867] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145, such as those described above.

[0868] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 318, such as those described above.

[0869] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 60, such as those described above.

[0870] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 73, such as those described above.

[0871] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 318, such as those described above.

[0872] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 60, such as those described above.

[0873] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 73, such as those described above.

[0874] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 318 and 60, such as those described above.

[0875] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 318 and 73, such as those described above.

[0876] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 60 and 73, such as those described above.

[0877] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 318, such as those described above.

[0878] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 60, such as those described above.

[0879] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 73, such as those described above.

[0880] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 318 and 60, such as those described above.

[0881] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 318 and 73, such as those described above.

[0882] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 60 and 73, such as those described above.

[0883] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 318 and 60, such as those described above.

[0884] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 318 and 73, such as those described above.

[0885] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 60 and 73, such as those described above.

[0886] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 318 and 60 and 73, such as those described above.

[0887] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 318, such as those described above.

[0888] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 60, such as those described above.

[0889] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 73, such as those described above.

[0890] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 318 and 60, such as those described above.

[0891] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 318 and 73, such as those described above.

[0892] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 60 and 73, such as those described above.

[0893] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 318 and 60, such as those described above.

[0894] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 318 and 73, such as those described above.

[0895] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 60 and 73, such as those described above.

[0896] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 318 and 60 and 73, such as those described above.

[0897] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 318 and 60, such as those described above.

[0898] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 318 and 73, such as those described above.

[0899] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 60 and 73, such as those described above.

[0900] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 77 and 318 and 60 and 73, such as those described above.

[0901] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 145 and 318 and 60 and 73, such as those described above.

[0902] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 318, such as those described above.

[0903] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 60, such as those described above.

[0904] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 73, such as those described above.

[0905] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 318 and 60, such as those described above.

[0906] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 318 and 73, such as those described above.

[0907] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 60 and 73, such as those described above.

[0908] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 318 and 60, such as those described above.

[0909] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 318 and 73, such as those described above.

[0910] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 60 and 73, such as those described above.

[0911] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 318 and 60 and 73, such as those described above.

[0912] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 318 and 60, such as those described above.

[0913] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 318 and 73, such as those described above.

[0914] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 60 and 73, such as those described above.

[0915] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 77 and 318 and 60 and 73, such as those described above.

[0916] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 145 and 318 and 60 and 73, such as those described above.

[0917] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 318 and 60, such as those described above.

[0918] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 318 and 73, such as those described above.

[0919] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 60 and 73, such as those described above.

[0920] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 72 and 77 and 318 and 60 and 73, such as those described above.

[0921] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 72 and 145 and 318 and 60 and 73, such as those described above.

[0922] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 77 and 145 and 318 and 60 and 73, such as those described above.

[0923] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 318, such as those described above.

[0924] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 60, such as those described above.

[0925] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 73, such as those described above.

[0926] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 318 and 60, such as those described above.

[0927] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 318 and 73, such as those described above.

[0928] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 60 and 73, such as those described above.

[0929] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 318 and 60, such as those described above.

[0930] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 318 and 73, such as those described above.

[0931] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 60 and 73, such as those described above.

[0932] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 318 and 60 and 73, such as those described above.

[0933] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 318 and 60, such as those described above.

[0934] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 318 and 73, such as those described above.

[0935] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 60 and 73, such as those described above.

[0936] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 77 and 318 and 60 and 73, such as those described above.

[0937] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 145 and 318 and 60 and 73, such as those described above.

[0938] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 318 and 60, such as those described above.

[0939] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 318 and 73, such as those described above.

[0940] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 60 and 73, such as those described above.

[0941] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 72 and 77 and 318 and 60 and 73, such as those described above.

[0942] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 72 and 145 and 318 and 60 and 73, such as those described above.

[0943] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 77 and 145 and 318 and 60 and 73, such as those described above.

[0944] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[0945] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[0946] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[0947] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[0948] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[0949] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[0950] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[0951] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145, such as those described above.

[0952] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 318, such as those described above.

[0953] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 60, such as those described above.

[0954] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 73, such as those described above.

[0955] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 318, such as those described above.

[0956] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 60, such as those described above.

[0957] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 73, such as those described above.

[0958] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 318 and 60, such as those described above.

[0959] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 318 and 73, such as those described above.

[0960] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 60 and 73, such as those described above.

[0961] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 318, such as those described above.

[0962] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 60, such as those described above.

[0963] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 73, such as those described above.

[0964] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 318 and 60, such as those described above.

[0965] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 318 and 73, such as those described above.

[0966] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 60 and 73, such as those described above.

[0967] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 318 and 60, such as those described above.

[0968] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 318 and 73, such as those described above.

[0969] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 60 and 73, such as those described above.

[0970] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 318 and 60 and 73, such as those described above.

[0971] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 318, such as those described above.

[0972] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 60, such as those described above.

[0973] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 73, such as those described above.

[0974] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 318 and 60, such as those described above.

[0975] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 318 and 73, such as those described above.

[0976] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 60 and 73, such as those described above.

[0977] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 318 and 60, such as those described above.

[0978] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 318 and 73, such as those described above.

[0979] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 60 and 73, such as those described above.

[0980] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 318 and 60 and 73, such as those described above.

[0981] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 318 and 60, such as those described above.

[0982] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 318 and 73, such as those described above.

[0983] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 60 and 73, such as those described above.

[0984] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 318 and 60 and 73, such as those described above.

[0985] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 410 and 145 and 318 and 60 and 73, such as those described above.

[0986] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 318, such as those described above.

[0987] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 60, such as those described above.

[0988] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 73, such as those described above.

[0989] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 318 and 60, such as those described above.

[0990] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 318 and 73, such as those described above.

[0991] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 60 and 73, such as those described above.

[0992] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 318 and 60, such as those described above.

[0993] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 318 and 73, such as those described above.

[0994] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 60 and 73, such as those described above.

[0995] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 318 and 60 and 73, such as those described above.

[0996] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 318 and 60, such as those described above.

[0997] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 318 and 73, such as those described above.

[0998] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 60 and 73, such as those described above.

[0999] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 318 and 60 and 73, such as those described above.

[1000] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 271 and 145 and 318 and 60 and 73, such as those described above.

[1001] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1002] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1003] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1004] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1005] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1006] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 224 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1007] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 318, such as those described above.

[1008] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 60, such as those described above.

[1009] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 73, such as those described above.

[1010] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 318 and 60, such as those described above.

[1011] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 318 and 73, such as those described above.

[1012] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 60 and 73, such as those described above.

[1013] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 318 and 60, such as those described above.

[1014] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 318 and 73, such as those described above.

[1015] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 60 and 73, such as those described above.

[1016] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 318 and 60 and 73, such as those described above.

[1017] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 318 and 60, such as those described above.

[1018] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 318 and 73, such as those described above.

[1019] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 60 and 73, such as those described above.

[1020] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 318 and 60 and 73, such as those described above.

[1021] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 145 and 318 and 60 and 73, such as those described above.

[1022] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1023] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1024] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1025] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1026] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1027] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1028] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1029] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1030] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1031] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1032] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1033] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1034] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1035] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 318, such as those described above.

[1036] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 60, such as those described above.

[1037] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 73, such as those described above.

[1038] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 318 and 60, such as those described above.

[1039] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 318 and 73, such as those described above.

[1040] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 60 and 73, such as those described above.

[1041] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 318 and 60, such as those described above.

[1042] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 318 and 73, such as those described above.

[1043] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 60 and 73, such as those described above.

[1044] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 318 and 60 and 73, such as those described above.

[1045] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 318 and 60, such as those described above.

[1046] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 318 and 73, such as those described above.

[1047] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 60 and 73, such as those described above.

[1048] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 318 and 60 and 73, such as those described above.

[1049] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 145 and 318 and 60 and 73, such as those described above.

[1050] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1051] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1052] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1053] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1054] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1055] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 410 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1056] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1057] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1058] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1059] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1060] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1061] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1062] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 224 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1063] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1064] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1065] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1066] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1067] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1068] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1069] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 410 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1070] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1071] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 318, such as those described above.

[1072] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 60, such as those described above.

[1073] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 73, such as those described above.

[1074] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 318 and 60, such as those described above.

[1075] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 318 and 73, such as those described above.

[1076] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 60 and 73, such as those described above.

[1077] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 318 and 60, such as those described above.

[1078] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 318 and 73, such as those described above.

[1079] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 60 and 73, such as those described above.

[1080] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 318 and 60 and 73, such as those described above.

[1081] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 318 and 60, such as those described above.

[1082] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 318 and 73, such as those described above.

[1083] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 60 and 73, such as those described above.

[1084] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 318 and 60 and 73, such as those described above.

[1085] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 145 and 318 and 60 and 73, such as those described above.

[1086] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1087] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1088] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1089] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1090] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1091] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1092] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1093] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1094] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1095] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1096] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1097] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1098] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1099] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1100] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1101] On the other hand, these variant changes include substitutions or components thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1102] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1103] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1104] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1105] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1106] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1107] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1108] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1109] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1110] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1111] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1112] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1113] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1114] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1115] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 410 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1116] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 318 and 60, such as those described above.

[1117] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 318 and 73, such as those described above.

[1118] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 60 and 73, such as those described above.

[1119] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 318 and 60 and 73, such as those described above.

[1120] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 145 and 318 and 60 and 73, such as those described above.

[1121] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1122] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1123] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1124] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 410 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1125] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 224 and 410 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1126] On the other hand, these variant changes include substitutions or composition thereof at positions corresponding to positions 295 and 224 and 410 and 271 and 72 and 77 and 145 and 318 and 60 and 73, such as those described above.

[1127] In one aspect, the present invention relates to a glucosylamylase variant comprising a substitution at position 295 corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1128] Furthermore, the variant may further include substitutions at at least one or more positions corresponding to positions 32, 83, 163, 169, 219, 224, 303, or 410 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1129] The exemplary variants disclosed herein are all constructed based on the parental glucosylamylase of SEQ ID NO: 2. Therefore, a starting amino acid to be substituted is included, such as Y295F; however, since other parental glucosylamylases can also be modified according to the invention, the starting amino acid may be different in this case. Therefore, a particular substitution can also be described by specifying only the amino acid to be present at a given position after substitution occurs. In the following, only the amino acid present after substitution is specified.

[1130] Therefore, in one embodiment, these variants of the invention include at least one or more substitutions selected from the following: 295F, 295W, 224A, 224I, 224T, 32V, 83D, 163A, 163W, 169I, 219R, 303N, or 410K.

[1131] On the other hand, this variant includes at least one of the following substitutions for the polypeptide of SEQ ID NO: 2: 295F; 295W; 224A; 224I; 224T; 271Q; 318V; or 410K; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1132] In another embodiment, the variant includes at least one of the following substitutions or combinations of substitutions for the polypeptide of SEQ ID NO: 2: 295W; or 295W+410K; or 224I+295F; or 224T+295W+318V; or 295F; or 224A+295F; or 295W+83D+410K; or 163A+295W+410K; or 163W + 295W + 410K; or 303N+295W; or 169I+295W; or 32V+219R+295W; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1133] In another embodiment, the variant includes at least one of the following substitutions or combinations of substitutions for the polypeptide of SEQ ID NO: 2: 295W; or 295W+410K; or 224I+295F; or 224T+295W+318V; or 295F; or 224A+295F; or 295W+83D+410K; or 163A+295W+410K; or 163W + 295W + 410K; or 303N+295W; or 169I+295W; or 32V+219R+295W; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2; and wherein these variants have reduced glucose inhibition compared to the glucosylamylase of SEQ ID NO: 2.

[1134] In one embodiment, these substitutions are selected from the following: Y295F, Y295W, L224A, L224I, L224T, A32V, S83D, N163A, N163W, V169I, T219R, S303N, R410K, and Q318V.

[1135] In another aspect, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions corresponding to positions 271, 410, 72, 77, 145, 219, 303, 224, 318 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1136] In one embodiment, the variant includes at least one or more substitutions selected from the following: 72V, 77A, 145A, 219D, 271Q, 271N, 271A, 271S, 271V, 224Q, 303E, 318W, 318F, 410A, 410Q, or 410H.

[1137] In the embodiments, the variant includes at least one of the following substitutions for the polypeptide of SEQ ID NO: 2: 72V; 77A; 145A; 219D; 271Q; 271N; 271A; 271S; 271V; 224Q; 303E; 318W; 318F; 410A 410Q; or 410H; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1138] In another embodiment, the variant includes at least one of the following substitutions or combinations of substitutions for the polypeptide of SEQ ID NO: 2: 72V + 145A; or 271Q; or 224Q+271N+410A; or 77A; or 77A+271A; or 271S+318W+410Q; or 271V+318F+410H; or 77A+219D; or 77A+303E; Furthermore, the variant possesses glucosylamylase activity, and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1139] In another embodiment, the variant includes at least one of the following substitutions or combinations of substitutions for the polypeptide of SEQ ID NO: 2: 72V + 145A; or 271Q; or 224Q+271N+410A; or 77A; or 77A+271A; or 271S+318W+410Q; or 271V+318F+410H; or 77A+219D; or 77A+303E; Furthermore, the variant possesses glucosylamylase activity, and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2, and the variant glucosylamylase has increased specific activity compared to the glucosylamylase of SEQ ID NO: 2.

[1140] In one embodiment, these substitutions are selected from the following: D72V, L77A, L145A, T219D, L224Q, T271Q, T271N, T271A, T271S, T271V, S303E, Q318W, Q318F, R410Q, R410H, and R410A.

[1141] Specifically, these variants have improved properties compared to the glucosylamylase disclosed as SEQ ID NO: 2.

[1142] Specifically, the improved properties are reduced glucose inhibition and / or increased specific activity.

[1143] In another specific embodiment, when a variant glucosidase is present during saccharification, and particularly during simultaneous saccharification and fermentation (SSF), the improved characteristic is the increased ethanol yield in the method of the present invention.

[1144] Therefore, in another aspect, the present invention relates to a glucosylamylase variant comprising a substitution at one or more positions corresponding to position 271 or 295 of the polypeptide of SEQ ID NO: 2 or at positions 271 and 295 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1145] In one embodiment, the variant includes at least one or more of the following substitutions: 271Q, 271A, 271V, 295W, 60L, 73A, 77A, 77V, and 318Y.

[1146] In another embodiment, the variant includes at least one of the following substitutions for the polypeptide of SEQ ID NO: 2: 60L; or 73A; or 271Q; or 271A; or 271V; or 295W; or 77V; or 77A; or 318Y; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1147] In another embodiment, the variant comprises at least the following substitutions or combinations thereof for the polypeptide of SEQ ID NO: 2: 60L+73A+271Q; 271Q; 77V+271V+410A; 77A + 271A; 271V + 318Y; 295W; 271Q+295W; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ IDNO: 2.

[1148] In another embodiment, the variant comprises at least the following substitutions or combinations thereof for the polypeptide of SEQ ID NO: 2: 60L+73A+271Q; 271Q; 77V+271V+410A; 77A + 271A; 271V + 318Y; 295W; 271Q+295W; and wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2, and wherein the variant provides increased ethanol yield when used with SSF compared to the glucosylamylase of SEQ ID NO: 2.

[1149] In one embodiment, these substitutions are selected from the following: F60L, S73A, T271A, T271V, T271Q, L77A, L77V, R410A, Y295W, and Q318Y.

[1150] In yet another embodiment, the present invention relates to glucosylamylase variants comprising at least the following substitutions or combinations thereof for the polypeptide comprising SEQ ID NO: 2: F60L+S73A+T271Q; T271Q; L77V+T271V+R410A; L77A+T271A; T271V+Q318Y; Y295W; T271Q+Y295W; and The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2.

[1151] In another aspect, the present invention relates to glucosylamylase variants comprising at least the following substitutions or combinations thereof for the polypeptide comprising SEQ ID NO: 2: F60L+S73A+T271Q; T271Q; L77V+T271V+R410A; L77A+T271A; T271V+Q318Y; Y295W; T271Q+Y295W; and The variant has glucosylamylase activity, and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 2, and the variant provides increased ethanol production when used with SSF compared to the glucosylamylase of SEQ ID NO: 2.

[1152] These variants may further include one or more additional substitutions in one or more (e.g., several) other locations.

[1153] It should be noted that for all the specific variants disclosed, such another change can be introduced without significantly affecting the properties of these glucosylamylase variants. In one respect, apart from the specific substitutions discussed herein, the number of substitutions in the variants of the invention is 1-20, for example 1-10 and 1-5, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[1154] Therefore, the percentage of sequence identity of the variant peptide compared to the parent peptide of SEQ ID NO: 2 can be 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%, for example at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the peptide of SEQ ID NO: 2.

[1155] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 85% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[1156] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 90% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[1157] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 91% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[1158] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 92% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[1159] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 93%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO:2.

[1160] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 94%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO:2.

[1161] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 95% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[1162] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 96%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO:2.

[1163] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 97%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO:2.

[1164] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 98%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO:2.

[1165] In one specific embodiment, the above variant has glucosylamylase activity and the variant has at least 99%, but less than 100%, sequence identity with the polypeptide of SEQ ID NO:2.

[1166] In addition to the specific substitutions described herein, these amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino or carboxyl-terminal extensions, such as methionine residues at the amino terminus; small linker peptides of up to 20–25 residues; or small extensions that are easily purified by altering net charge or another function, such as polyhistidine tracts, antigenic epitopes, or binding domains.

[1167] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[1168] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, amino acid alterations can improve the peptide's thermal stability, change its substrate specificity, change its optimal pH, and so on.

[1169] Essential amino acids in peptides can be identified using methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science). Science (244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the glucosamine activity of the resulting mutant molecule is tested to identify amino acid residues essential to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contract site. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[1170] In one embodiment, this variant glucosylamylase exhibits improved specific activity compared to the parental enzyme. Specific activity was determined using an AGU assay. In one embodiment, this variant glucosylamylase has reduced glucose inhibition compared to the parental glucosylamylase of SEQ ID NO: 2. Glucosylamylase inhibition was defined as the ratio of glucosylamylase activity with and without 30% glucose relative to the wild-type parental enzyme disclosed in SEQ ID NO: 2.

[1171] In another embodiment, this variant glucosylamylase has reduced isomaltose-forming activity compared to the parental glucosylamylase of SEQ ID NO: 2.

[1172] In another embodiment, this variant glucosylamylase has increased DE11 activity compared to the parental glucosylamylase of SEQ ID NO: 2.

[1173] In another embodiment, this variant glucosylamylase has increased thermal stability compared to the parental glucosylamylase of SEQ ID NO: 2.

[1174] In another embodiment, when this variant is applied to saccharification and subsequent fermentation on liquefied mash, the variant glucosyl amylase has an increased EtOH yield compared to the parental glucosyl amylase of SEQ ID NO: 2.

[1175] For details, see the Materials and Methods section included here.

[1176] Parental glucosidase The parental glucosylamylase may be (a) a polypeptide having at least 85% sequence identity with the polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes with the coding sequence of the mature polypeptide of (i) SEQ ID NO: 1 under medium to high stringency conditions, or (ii) with the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide that has at least 70% sequence identity with the coding sequence of the mature polypeptide of SEQ ID NO: 1.

[1177] In one aspect, the parent has 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%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO: 2, and the polypeptide has glucosylamylase activity. In another aspect, the amino acid sequence of the parent differs from the mature polypeptide of SEQ ID NO: 2 by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[1178] On the other hand, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 2.

[1179] On the other hand, the parent is encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[1180] Nucleic acid probes can be designed using the polynucleotide or a subsequence of SEQ ID NO: 1, together with the polypeptide or a fragment of SEQ ID NO: 2, to identify and clone parental DNA encoding strains from different genera or species, according to methods well known in the art. Specifically, such probes can be hybridized with genomic DNA or cDNA of cells of interest, following standard DNA blotting procedures, to identify and isolate the corresponding gene therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15, for example at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe has a length of at least 100 nucleotides, for example at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, these probes are labeled for detecting the corresponding gene (e.g., using...). 32 P, 3 H, 35 S, biotin, or avidin). Such probes are covered in this invention.

[1181] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened for DNA that hybridizes to and encodes the parental DNA as described above. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred to and immobilized on nitrocellulose or other suitable carrier material. The carrier material is used in DNA blotting to identify clones or DNA or their subsequences that hybridize to SEQ ID NO: 1.

[1182] For the purposes of this invention, hybridization indicates that a polynucleotide hybridizes with a labeled nucleic acid probe under very low to very high stringency conditions, the probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) its full-length complement; or (iv) its subsequence. Molecules hybridizing with the nucleic acid probe under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[1183] In one respect, the nucleic acid probe is the coding sequence of the mature polypeptide of SEQ ID NO: 1. In another respect, the nucleic acid probe is nucleotides 52 to 1728 of SEQ ID NO: 1. In another respect, the nucleic acid probe is a polynucleotide encoding a polypeptide or a fragment thereof of SEQ ID NO: 2. In yet another respect, the nucleic acid probe is SEQ ID NO: 1.

[1184] In another embodiment, the parent is encoded by a polynucleotide that has 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%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 1.

[1185] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[1186] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused at the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide with the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they are in a frame and that the expression of the fusion peptide is under the control of one or more promoters and terminators. Fusion peptides can also be constructed using integrin technology, wherein the fusion peptide is generated post-translational (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[1187] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved during the secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[1188] The parent can be a fungal glucosylamylase. For example, the parent can be a glucosylamylase from the genus Myxobolus or Myxobolus.

[1189] On the other hand, the parent is *Clostridium perfringens*, *Clostridium hygroscopicum*, or *Clostridium valvulpis* glucosylamylase.

[1190] On the other hand, the parent is *Cladophora micrantha* glucosylamylase, such as the glucosylamylase of SEQ ID NO: 2.

[1191] It should be understood that, for the foregoing species, this invention covers perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[1192] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Netherlands Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Patent Culture Collection of the Agricultural Research Service.

[1193] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural living environment are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening in a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with one or more probes, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[1194] Preparation of variants These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[1195] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[1196] Site-directed mutagenesis can be achieved in vitro using PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

[1197] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[1198] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[1199] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as those described by Tian et al. (2004, Nature). Nature The technology based on multi-channel microchips described in (432: 1050-1054), and similar technologies for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[1200] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or truncation methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (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, Biochemistry 30: 10832-10837; U.S. Patent 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).

[1201] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology).Nature Biotechnology (17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the peptide.

[1202] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments in conjunction with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenic primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[1203] Polynucleotides The present invention also relates to isolated polynucleotides encoding variants of the invention.

[1204] Nucleic acid constructs The present invention also relates to nucleic acid constructs comprising polynucleotides operably linked to one or more control sequences encoding variants of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[1205] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[1206] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of that variant. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[1207] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters obtained from the genes of: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei lipase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase Mycoplasma β-xylosidase, and the NA2tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-restrictive examples include modified promoters derived from the Aspergillus niger neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and its mutant promoters, truncated promoters and heterozygous promoters.

[1208] In yeast hosts, useful promoters are derived from the genes of the following: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* (… Yeast 8:423-488 describes other useful promoters for yeast host cells.

[1209] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. This terminator sequence is operatively linked to the 3' end of the polynucleotide encoding that variant. Any terminator that is functional in the host cell can be used.

[1210] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[1211] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described above in Romanos et al., 1992.

[1212] Control sequences can also be mRNA stabilizing regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of the gene.

[1213] Examples of suitable mRNA stable regions were obtained from Bacillus thuringiensis. cryIIIA Gene (WO 94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology) Journal of Bacteriology 177: 3465-3471).

[1214] This control sequence can also be a preamble, which is the untranslated region of an mRNA important for translation in the host cell. The preamble sequence is operatively linked to the 5' end of a polynucleotide encoding the variant. Any preamble that functions in the host cell can be used.

[1215] Preferred precursors for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[1216] Precursors suitable for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[1217] The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of the variant-coding sequence and recognized by the host cell during transcription as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[1218] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[1219] Useful polyadenylated sequences for yeast host cells are discussed in Guo and Sherman, 1995, Molecular Cell Biology. Mol. Cellular Biol. It is described in 15:5983-5990.

[1220] The control sequence can also be a signal peptide coding region, encoding a signal peptide linked to the N-terminus of the variant and guiding the variant into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked within the translation reading frame to a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the native signal peptide coding sequence to enhance the variant's secretion. However, any signal peptide coding sequence that guides the expressed variant into the host cell's secretory pathway can be used.

[1221] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens lipase, and Rhizopus oryzae aspartic protease.

[1222] Useful signal peptides for yeast host cells were obtained from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences were described by Romanos et al. (1992, above).

[1223] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. Propeptide-coding sequences can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae sacchari-factor.

[1224] In the presence of both the signal peptide sequence and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[1225] Also desirable could be the addition of regulatory sequences that modulate the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include... lac , tac ,as well as trp Operant systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant is operatively linked to the regulatory sequence.

[1226] expression carrier The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, such that the coding sequence is operatively linked to the suitable control sequence for expression.

[1227] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[1228] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any elements necessary to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[1229] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, heavy metal resistance, or auxotrophic prototrophs, etc.

[1230] Suitable markers for use in yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, those for use in yeast host cells. amdS (acetamase) argB (ornithine carbamoyltransferase) bar (Gluphosphonate acetyltransferase) hph (Hygromycin phosphotransferase) niaD (nitrate reductase) pyrG (orhodoside-5'-phosphate decarboxylase) sC (adenosine sulfate transferase), and trpC (o-aminobenzoic acid synthase), along with its equivalents. Preferred for use in Aspergillus cells are *Aspergillus nidulans* or *Aspergillus oryzae*. amdS and pyrG Genes and Streptomyces hydrophila bar Gene.

[1231] The vector preferably contains one or more elements that allow the vector to integrate into the host cell’s genome or to replicate autonomously in the cell independently of the genome.

[1232] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the variant or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes of the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrative elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to increase the likelihood of homologous recombination. These integrative elements can be any sequence homologous to the target sequence in the host cell genome. Furthermore, these integrative elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[1233] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[1234] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[1235] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene( Gene )98: 61-67; Cullen et al., 1987, Nucleic Acid Research ( Nucleic Acids Res. (15: 9163-9175; WO 00 / 24883). The AMA1 gene can be isolated and plasmids or vec...

Claims

1. A glucosylamylase variant comprising a substitution at position 295 corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:

2.

2. The variant of claim 1, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: 295W； 295W + 410K; 224I+295F; 224T+295W+318V; 295F； 224A+295F; 295W+83D+410K; 163A+295W+410K; 163W + 295W + 410K; 303N+295W; 169I+295W; or 32V+219R+295W; The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:

2.

3. A glucosylamylase variant comprising substitutions at one or more positions corresponding to positions 271, 410, 72, 77, 145, 219, 303, 224, 318 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:

2.

4. The variant of claim 3, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: 72V + 145A; 271Q； 224Q+271N+410A; 77A； 77A+271A; 271S+318W+410Q; 271V+318F+410H; 77A+219D; 77A+303E; The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:

2.

5. A glucosylamylase variant comprising a substitution at position 271, or 295, or one or more positions corresponding to the polypeptide of SEQ ID NO: 2, wherein the variant has glucosylamylase activity, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:

2.

6. The variant of claim 5, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: F60L+S73A+T271Q; 271Q； 77V+271V+410A; 77A + 271A; 271V + 318Y; 295W； 271Q+295W; and The variant has glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:

2.

7. A composition comprising the polypeptide as described in any one of claims 1-6.

8. Use of a polypeptide as described in any one of claims 1-6 for the production of syrups and / or fermentation products.

9. A method for producing fermentation products, particularly ethanol, from starch-containing materials, the method comprising the following steps: (a) liquefying starch-containing material in the presence of α-amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermenting organism; wherein step (b) is performed using at least one variant of glucosylamylase as described in any one of claims 1-6.

10. A method for producing a syrup product from a starch-containing material, the method comprising the following steps: (a) liquefying starch-containing material in the presence of α-amylase; (b) saccharifying the liquefied material in the presence of a variant glucosylase as described in any one of claims 1-6.

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