Glucoamylase engineered yeast and fermentation method

Abstract

The present invention relates to an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising SEQ ID NO: 1 and SEQ ID NO: 4 or variants thereof. The engineered yeast is capable of providing a glucoamylase to a fermentation medium and causing degradation of starch materials to produce glucose for fermentation into desired bioproducts, such as ethanol. High titers of bioproducts (e.g., 70 g / kg of ethanol) and low residual glucose levels can be achieved. In addition, the yeast exhibits good growth and bioproduct production at temperatures of 32°C or higher.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 636,716, filed on February 28, 2018, entitled “GLUCOAMYLASE ENGINEERED YEAST AND FERMENTATION METHODS,” which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] The ASCII text file named "N00570_Sequence_Listing_ST25.txt" was created on February 27, 2019, is 123 kilobytes in size, and its entire contents are incorporated herein by reference. Technical Field

[0005] The present invention relates to yeast engineered with exogenous glucoamylase nucleic acids and fermentation methods for producing bioproducts such as ethanol. Background Art

[0006] Many fermentation raw materials are derived from plant sources (e.g., corn mash), in which carbohydrates are primarily in the form of starch polymers. The starch polymers in these raw materials must be processed into low molecular weight sugars that can be consumed by yeast and used to grow and produce bioproducts. Typical treatments include acid hydrolysis and / or enzymatic hydrolysis, in which the polymer chains are hydrolyzed to produce sugars that can be used by yeast. Starch degrading enzymes such as alpha amylase and glucoamylase can be added to convert the polymers into simple sugars. However, adding this enzyme can significantly increase the cost and complexity of the fermentation process.

[0007] It may be challenging to realize heterologous expression and functionality of enzymes in yeast to help starch hydrolysis, because it is difficult to know whether nucleic acids will be correctly expressed and whether functional enzymes will be formed, and whether the active form of enzymes will be secreted from cells. It is also challenging to engineer yeast to grow and produce bioproducts under non-optimal conditions such as high temperatures and high bioproduct titers. For example, although it is a well-known industrial process to produce ethanol by fermentation, it is difficult to minimize the formation of byproducts in order to reduce the dependence on supplementary enzymes, so that ethanol rates, titers and productivity are maintained under non-optimal conditions (e.g., temperature), and it may be difficult to minimize byproduct formation in technology. Increased ethanol concentration and unwanted byproduct accumulation may also be unfavorable to cell health. Summary of the invention

[0008] The present invention relates to engineered yeast and fermentation methods, wherein the engineered yeast is capable of secreting heterologous glucoamylase (GA) into the fermentation medium and providing glucoamylase activity (EC 3.2.1.3) to the fermentation substrate. The present invention also relates to a method for producing a product of biological origin such as ethanol by fermentation using the engineered yeast.

[0009] In one aspect, experimental studies related to the present application have identified fungal glucoamylase genes that, when introduced exogenously into yeast, allow them to grow well on raw materials containing low glucose and high starch amounts, and produce high levels of bioproducts. The results show that the engineered yeast is able to secrete glucoamylase into the fermentation medium, and the glucoamylase has enzymatic activity on starch to produce enough glucose for growth and bioproduct production. Other benefits related to the present disclosure include improved fermentation and bioproduct production at elevated fermentation temperatures. Other benefits related to the present disclosure include reducing the amount of glucose at the end of the fermentation period.

[0010] In one aspect, the present invention provides an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, the glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1 (Rhizopus microsporus glucoamylase). In one embodiment, the engineered yeast is capable of producing ethanol at a rate of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 g / L*h or greater during fermentation. In another embodiment, the engineered yeast is capable of producing at least 70 g / kg of ethanol in a fermentation medium made from a feedstock containing glucose polymers having a DE of about 50 or less. In another embodiment, the engineered yeast is capable of producing 90 g / kg or more, 120 g / kg or more, 130 g / kg or more, or 140 g / kg or more of ethanol in a fermentation medium made from a feedstock containing glucose polymers having a DE of about 30. In an embodiment, an amount of 70 g / kg of ethanol can be produced in a fermentation medium within 48 hours of inoculation using a feedstock. In embodiments, at the beginning of the fermentation process (inoculation), the glucose concentration in the fermentation medium may be no greater than 5% by weight. In embodiments, the feedstock may provide a glucose-polymer-containing feedstock sufficient to produce 70 g / kg of ethanol in the culture medium, such as about 20% by weight of the glucose polymer feedstock. In embodiments, the feedstock may have one or more of the following characteristics: the feedstock is a starch hydrolysate; the glucose-polymer has primarily α-1,4 bonds; the feedstock does not substantially include cellulosic material (e.g., less than 20%, 15%, or 10% of cellulosic material).

[0011] In one embodiment, the yeast is capable of producing at least 70 g / kg of ethanol in a fermentation medium made from corn mash with a DE of 30±2, wherein the fermentation medium comprises about 32% by dry weight corn and pH 5.8, 35 ppm CaCl, 1900 ppm urea, 5 ppm ampicillin, wherein the starting yeast concentration is 0.1 (OD600), and the fermentation is carried out at 30° C. with stirring for 48 hours. In a more specific embodiment, the engineered yeast comprises a glucoamylase having a sequence identity of 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more to SEQ ID NO: 1.

[0012] In a related embodiment, the invention provides a fermentation method for producing a bioproduct. The method comprises forming a fermentation medium from a feedstock containing a glucose polymer, and then fermenting the fermentation medium using an engineered yeast, the engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, the glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1. The fermentation is carried out for a period of time to produce a bioproduct, such as ethanol.

[0013] In more specific embodiments, ethanol is produced in the fermentation medium in an amount of 70 g / kg or greater, 90 g / kg or greater, 120 g / kg or greater, 130 g / kg or greater, or 140 g / kg or greater during fermentation. In some embodiments, in the fermentation process, the feedstock containing glucose polymers has a dextrose equivalent (DE) of no greater than 50. In some embodiments, glucose is no greater than 5 weight % of the solid material in the feedstock. Optionally, a supplemental glucoamylase can be introduced into the fermentation process to increase the bioproduct titer.

[0014] In some embodiments, using yeast engineered with a glucoamylase having 81% or greater sequence identity to SEQ ID NO: 1, the fermentation is conducted at a temperature in the range of 31°C to 35°C for most or all of the fermentation period.

[0015] In some embodiments, using yeast engineered with a glucoamylase having 81% or greater sequence identity to SEQ ID NO: 1, the glucose concentration in the fermentation medium is no greater than 1.0 g / L at the end of the fermentation period.

[0016] In another aspect, the present invention provides an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, the glucoamylase comprising a sequence having 97% or greater or 98% or greater sequence identity to SEQ ID NO: 4 (Rhizopus delemar glucoamylase). In one embodiment, the engineered yeast is capable of producing ethanol at a rate of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 g / L*h or greater during fermentation. In another embodiment, the engineered yeast is capable of producing at least 70 g / kg of ethanol in a fermentation medium made from a feedstock containing glucose polymers having a DE of about 30. In an embodiment, an amount of 70 g / kg of ethanol can be produced in the fermentation medium within 48 hours of inoculation using the feedstock. In an embodiment, the glucose concentration in the fermentation medium may be no greater than 5% by weight at the start (inoculation) of the fermentation process. In embodiments, the feedstock can provide a glucose-polymer-containing feedstock sufficient to produce 70 g / kg of ethanol in the culture medium, such as about 20% by weight of the glucose polymer feedstock. In embodiments, the feedstock can have one or more of the following characteristics: the feedstock is a starch hydrolysate; the glucose-polymer has mainly α-1,4 bonds; the feedstock does not substantially include cellulosic material (e.g., less than 20%, 15%, or 10% of cellulosic material).

[0017] In a related embodiment, the invention provides a fermentation method for producing a bioproduct. The method comprises forming a fermentation medium from a feedstock containing a glucose polymer, and then fermenting the fermentation medium using an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO: 4. The fermentation is carried out for a period of time to produce a bioproduct, such as ethanol.

[0018] In more specific embodiments of any of the foregoing fermentations, ethanol is produced in an amount of 70 g / kg or greater, 90 g / kg or greater, 120 g / kg or greater, 130 g / kg or greater, or 140 g / kg or greater in the fermentation medium during the fermentation. In some embodiments, in the fermentation process, the feedstock containing glucose polymers has a dextrose equivalent (DE) of about 30. In some embodiments, glucose is no greater than 5% by weight of the solid material in the feedstock. Optionally, a supplemental glucoamylase can be introduced into the fermentation process to increase the bioproduct titer.

[0019] In some embodiments, fermentation is conducted at a temperature in the range of 31°C to 35°C for most or all of the fermentation period using yeast engineered with a glucoamylase having 97% or greater or 98% or greater sequence identity to SEQ ID NO:4.

[0020] In some embodiments, using yeast engineered with a glucoamylase having 97% or greater sequence identity to SEQ ID NO: 4, the glucose concentration in the fermentation medium is no greater than 2.0 g / L, or no greater than 1.0 g / L at the end of the fermentation period.

[0021] In a more specific embodiment of any of the foregoing yeast embodiments, the engineered yeast is capable of producing 90 g / kg or more, 120 g / kg or more, 130 g / kg or more, or 140 g / kg or more ethanol in a fermentation medium made from a glucose polymer-containing feedstock having a DE of about 30. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 An alignment of SEQ ID NO: 1 with Rhizopus oryzae GA is shown, showing the signal sequence (1-25) and starch binding domain (26-131). DETAILED DESCRIPTION

[0023] The various aspects of the invention described below are not intended to be exhaustive or to limit the invention to the exact forms disclosed in the detailed description below. Instead, the purpose of the various aspects selected and described is to facilitate the understanding and appreciation of the principles and practices of the invention by other persons skilled in the art.

[0024] One aspect of the invention relates to an engineered yeast expressing a glucoamylase comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 1 (Rhizopus microsporus glucoamylase (GA)). Another aspect of the invention relates to an engineered yeast expressing a glucoamylase comprising a sequence having 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 4 (Rhizopus delbrueckii glucoamylase (GA)). The engineered yeast of the present disclosure is capable of expressing and providing a glucoamylase in a culture medium, and the glucoamylase has enzymatic activity on glucose polymer substrates such as starch from various plant sources. The glucoamylase activity within the culture medium can produce monosaccharides and disaccharides that can be consumed by the yeast and can be used as a carbon source for the production of target compounds such as ethanol.

[0025] In embodiments of the present disclosure, fermentation medium can be prepared by raw materials with glucose polymers and minimum glucose. For example, fermentation medium can be prepared by starch-containing raw materials with DE (dextrose equivalent) of 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less or 5 or less. Dextrose equivalent (DE) is a measure of the amount of reducing sugars present in a material (e.g., sugar products or starch-containing raw materials) relative to dextrose (also known as glucose), expressed as a percentage based on dry weight. For example, maltodextrin with a DE of 10 will have 10% of the reducing power of dextrose (DE is 100). Fermentation medium can be prepared by starch-containing raw materials with an amount of glucose not greater than about 5% by weight, about 4% by weight, about 3% by weight, about 2% by weight or about 1% by weight of total solids in the raw material. In some fermentation methods, starch raw materials containing low glucose are added regularly or continuously throughout the fermentation period. The glucoamylase produced by the engineered yeast can have enzymatic activity on starch in the culture medium and produce glucose, which the yeast can use to grow and produce bioproducts. Optionally, the fermentation method can include supplementing the culture medium with a purified glucoamylase, such as a glucoamylase obtained from a commercial source, which can further drive enzymatic hydrolysis of starch and increase growth and the titer of the bioproduct.

[0026] For example, without supplementing any commercial glucoamylase and using a low glucose feedstock, the engineered yeast of the present disclosure can produce an amount of ethanol of about 70 g / kg or greater in the fermentation medium, such as an amount in the range of about 70 g / kg to about 115 g / kg, about 75 g / kg to about 115 g / kg, about 80 g / kg to about 115 g / kg, about 85 g / kg to about 115 g / kg, about 90 g / kg to 115 g / kg, about 95 g / kg to about 115 g / kg, about 100 g / kg to about 115 g / kg, or about 105 g / kg to about 115 g / kg. In embodiments where a supplemental glucoamylase is added to the culture medium, greater amounts of ethanol, such as 110 g / kg or greater, or 125 g / kg or greater, or 140 g / kg or greater, can be produced in the fermentation medium.

[0027] In other embodiments, the yeasts engineered with glucoamylases of the present disclosure can exhibit excellent fermentation performance at temperatures above the standard use temperature in fermentation (i.e., fermentation at 30°C using Saccharomyces cerevisiae as a host organism). For example, in some embodiments using the engineered yeasts of the present disclosure, the fermentation is conducted at a temperature in the range of 31°C to 35°C or 32°C to 34°C for most or all of the fermentation period. Even at higher temperatures, the engineered yeasts are able to produce glucoamylase activity in the culture medium and promote excellent cell growth and bioproduct production.

[0028] In other embodiments, after a period of fermentation, the yeast engineered with glucoamylase of the present disclosure can provide a desired final fermentation medium with high levels of bioproducts and low levels of byproducts. Specifically, under stress fermentation conditions, such as in the presence of low levels of glucose such as 1.0 g / kg, the final fermentation medium can have high levels of ethanol (e.g., 70 g / kg or more).

[0029] As used herein, the term "exogenous" refers to the introduction of molecules such as nucleic acids or activities such as enzymatic activities into a host organism. Exogenous nucleic acids can be introduced into a host organism by well-known techniques, and can be maintained outside the host chromosomal material (e.g., maintained on a non-integrating vector), or can be integrated into the host's chromosome, such as by a recombination event. Exogenous nucleic acids can encode enzymes or portions thereof that are homologous or heterologous to the host organism.

[0030] The term "heterologous" refers to a molecule or activity that is from a different source than the referenced molecule or organism. Thus, a gene or protein that is heterologous to the referenced organism is a gene or protein that is not found in that organism. For example, a specific glucoamylase gene that is found in a first fungal species and that is exogenously introduced into a second fungal species as a host organism is "heterologous" to the second fungal organism.

[0031] The following SEQ ID NOs are related to fungal GA amino acid or protein sequences: SEQ ID NO: 1: Rhizopus microsporus GA amino acid sequence; SEQ ID NO: 2: Rhizopus microsporus GA nucleic acid sequence #1; SEQ ID NO: 3: Rhizopus microsporus GA nucleic acid sequence #2; SEQ ID NO: 4: Rhizopus delemar GA amino acid sequence; SEQ ID NO: 5: Rhizopus delemar GA nucleic acid sequence #1; SEQ ID NO: 6: Rhizopus delemar GA nucleic acid sequence #2.

[0032] Table 1 is a table of sequence identities (global protein alignment) between the amino acid sequence of Rhizopus microsporus GA and the GA sequences of other known GAs. The reference molecule is Rhizopus microsporus GA and the scoring matrix is ​​BLOSUM 62.

[0033] Table 1

[0034]

[0035] Rhizopus microsporus GA and Rhizopus delbrueckii GA are members of the glucoamylase family (EC 3.2.1.3) and are starch hydrolases that continuously hydrolyze 1,4-linked α-D-glucosyl residues from the non-reducing ends of oligosaccharide and polysaccharide chains with the release of D-glucose. Alternative names for glucoamylase include amyloglucosidase; γ-amylase; lysosomal α-glucosidase; acid maltase; exo-1,4-α-glucosidase; glucoamylase; γ-1,4-glucan glucohydrolase; acid maltase; 1,4-α-D-glucan glucohydrolase.

[0036] Glucoamylases such as Rhizopus microsporus GA and Rhizopus delbrueckii GA can also cleave alpha-1,6 bonds at branch points of amylopectin. As used herein, the term "amylolytic activity" pertains to these enzymatic mechanisms.

[0037] The engineered yeast of the present disclosure can include variants of the native sequences of Rhizopus microsporus GA and Rhizopus delbrueckii GA glucoamylase polypeptides, and can include one or more amino acid variations, thereby providing non-natural polypeptides. The polypeptides of the present disclosure can be a portion of the naturally occurring Rhizopus microsporus GA and Rhizopus delbrueckii GA sequences (such as polypeptides truncated at their N-terminus, their C-terminus, or both), while the glucoamylase polypeptides retain amylolytic activity.

[0038] N-terminal truncations can be produced by changing the position of the ATG start codon while ensuring that the sequence downstream remains in frame. C-terminal variants can be produced by inserting an early stop codon in the frame. Random methods can also be used, such as error-prone PCR, and random methods can be combined with growth on starch to ensure peptide function.

[0039] Variation of the Rhizopus microsporus GA (SEQ ID NO: 1) and Rhizopus delbrueckii GA (SEQ ID NO: 4) sequences can be performed using information about the primary sequences of these enzymes, by sequence alignments, and based on information about other glucoamylases known in the art. Most glucoamylases, including Rhizopus microsporus GA and Rhizopus delbrueckii GA, are multidomain enzymes. Based on known crystal structures from similar enzymes, many glucoamylases, including Rhizopus microsporus GA and Rhizopus delbrueckii GA, include a starch binding domain connected to a catalytic domain via an O-glycosylated linker region.

[0040] Glucoamylases may also have a catalytic domain having a distorted (α / α)(6)-barrel configuration with a central funnel-shaped active site. Glucoamylases may have a structurally conserved catalytic domain of approximately 450 residues. In some glucoamylases, the catalytic domain is followed by a linker region of typically 30 to 80 residues that connects to a starch binding domain of approximately 100 residues.

[0041] The properties of glucoamylases can be related to their structural features. Information from the catalytic domain and starch binding domain models was used to construct a structure-based multiple sequence alignment (e.g., see Coutinho, PM, and Reilly, PJ, 1994. Protein Eng. 7: 393-400 and 749-760). It has been shown that the catalytic domain and starch binding domain are functionally independent and that structural similarities exist in microbial glucoamylases based on structure-function relationship studies. Other studies have shown that specific glucoamylase residues are involved in directing protein conformational changes, substrate binding, thermal stability, and catalytic activity (e.g., see Sierks, MR et al., 1993. Protein Eng. 6: 75-79; and Sierks, MR and Svensson, B. 1993. Biochemistry 32: 1113-1117).

[0042] Thus, the correlation between glucoamylase sequence and protein function is known in the art, and a skilled artisan can design and express variants of glucoamylases having amylolytic activity, the variants having one or more amino acid deletions, substitutions and / or additions. For example, in some aspects, the glucoamylase portions of Rhizopus microsporus GA and Rhizopus delbrueckii GA can contain truncated versions of naturally occurring glucoamylases, the truncated versions having at least a catalytic domain and, optionally, a starch binding domain having amylolytic activity as described herein.

[0043] Truncated forms of glucoamylase have been produced and shown to have enzymatic activity. For example, Evans et al. (Gene, 91: 131; 1990) produced a series of truncated forms of glucoamylase to investigate the number of O-glycosylated regions required for activity or stability of GAII (a fully active form of the enzyme lacking the original starch binding domain). It was found that a significant portion of the C-terminus could be deleted from GAII without significant effect on the activity, thermostability or secretion of the enzyme.

[0044] Lin et al. (BMC Biochemistry 8:9, 2007) suggested that there was no loss of glucoamylase activity when the starch binding domain located between positions 26-131 in Rhizopus oryzae glucoamylase was deleted. Furthermore, Mertens and Skory (Enz. Microbiol. Technology 40:874-880, 2007) isolated a native glucoamylase lacking a starch binding domain.

[0045] Various amino acid substitutions associated with causing changes in glucoamylase activity are also known in the art. It has been shown that amino acid substitutions at various positions in the glucoamylase sequence can affect properties such as thermostability, starch hydrolysis activity, substrate usage, and protease resistance. Therefore, the present disclosure contemplates the use of Rhizopus microsporus GA and Rhizopus delbrueckii GA sequences comprising one or more amino acid substitutions in the glucoamylase portion of the polypeptide, wherein the substitutions are different from the wild-type sequence of the glucoamylase.

[0046] For example, U.S. Pat. No. 8,809,023 describes a method for reducing the ratio of isomaltose synthesis to starch hydrolysis activity (IS / SH ratio) during starch hydrolysis. Specifically, a Trichoderma reesei glucoamylase (Tr GA) (632 amino acids in total length with a signal peptide at the N-terminus) is described, which is modified at the following amino acid positions: D44R and A539R; or D44R, N61I and A539R. It is reported that the glucoamylase variant exhibits a reduced IS / SH ratio during starch hydrolysis compared to the parent glucoamylase.

[0047] As another example, U.S. Patent No. 8,592,194 describes a glucoamylase variant having increased thermostability compared to a wild-type glucoamylase variant. Also described in the present disclosure are Trichoderma reesei glucoamylases, but alternatively have one or more amino acid substitutions at the following positions of the native Tr GA sequence: 10, 14, 15, 23, 42, 45, 46, 59, 60, 61, 67, 68, 72, 73, 97, 98, 99, 102, 108, 110, 113, 114, 122, 124, 125, 133, 140, 144, 145, 147, 152, 153, 164, 175, 182, 204, 205, 214, 216, 219, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265 415, 417 and 418. For example, the present disclosure contemplates generating variants at amino acid positions in SEQ ID NO: 1 and SEQ ID NO: 4 that correspond to the corresponding described positions in the TrGA sequence to provide variants with increased thermal stability.

[0048] The determination of "corresponding" amino acids from two or more glucoamylases can be determined by the comparison of all or part of their amino acid sequences. The generation of sequence alignment and sequence identity includes global alignment and local alignment, which are usually calculated using methods. In order to provide a global alignment, a global optimization of forced sequence alignment across the entire length of all query sequences is used. By comparison, in the local alignment, shorter similar regions in the long sequence are identified.

[0049] As used herein, "equivalent positions" refer to positions common to two sequences (e.g., a template GA sequence and a GA sequence with a desired substitution), based on an alignment of the amino acid sequence of a glucoamylase or an alignment of the three-dimensional structure. Thus, equivalence can be determined using sequence alignment or structural alignment or both.

[0050] In some practice modes, the BLAST algorithm is used to compare and determine sequence similarity or identity. In addition, the presence or importance of a gap that can be assigned a weight or score in the sequence can be determined. These algorithms can also be used to determine nucleotide sequence similarity or identity. Parameters for determining correlation are calculated based on methods known in the art for calculating statistical similarity and the importance of determined matching. It is expected that the relevant gene product has a high degree of similarity, such as a sequence identity greater than 50%. Exemplary parameters for determining the correlation of two or more sequences using the BLAST algorithm can be as follows.

[0051] Examination of the nucleic acid or amino acid sequences of two nucleic acids or two polypeptides will reveal sequence identity and similarity between the compared sequences. Generation of sequence alignments and sequence identities includes global alignments and local alignments using computational methods. Alignment can be performed using BLAST (National Center for Biological Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The percentage of amino acid sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: maximum target sequence: 100; short query: parameters that automatically adjust for short input sequences; expected threshold: 10; word length: 6; maximum match within query range: 0; matrix: BLOSUM62; gap cost: (existence: 11, extension: 1); component adjustment: conditional component score matrix adjustment; filter: not selected; mask: not selected. The percent nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: parameters that automatically adjust for short input sequences; Expected threshold: 10; Word length: 28; Maximum match within query range: 0; Match / mismatch score: 1, -2; Gap cost: linear; Filter: low complexity region; Mask: mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently, to have XX% sequence identity with the reference sequence.

[0052] A global alignment can be performed to align sequences with significant identity with, for example, SEQ ID NO: 1 (Rhizopus microsporus GA) or SEQ ID NO: 4 (Rhizopus delbrueckii GA) glucoamylase to determine which corresponding amino acid position(s) in the target sequence (e.g., a glucoamylase ortholog) can be substituted with one or more amino acids if a glucoamylase variant is used.

[0053] In some cases, the substitution may be a conservative substitution, such as one amino acid of a particular type (e.g., polar, nonpolar / aliphatic, positively / basic, negatively / acidic) is replaced by an amino acid of the same type. Exemplary conservative amino acid substitutions of the present disclosure may involve exchanging one aliphatic or hydrophobic amino acid Ala, Val, Leu or Ile for another; exchanging one hydroxy amino acid Ser or Thr for another; exchanging one acidic amino acid Asp or Glu for another; exchanging one amide amino acid Asn or Gln for another; exchanging one basic amino acid Lys, Arg or His for another; exchanging one aromatic amino acid Phe, Tyr or Trp for another; and exchanging one small amino acid Ala, Ser, Thr, Met or Gly for another.

[0054] In embodiments of the present disclosure, SEQ ID NO: 1 has one or more amino acid mutations that make it less than 100% identical to SEQ ID NO: 1. For example, the glucoamylase can have multiple amino acid deletions, substitutions and / or additions that make it about 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to SEQ ID NO: 1. Variants with a single amino acid substitution have 99.8% identity to SEQ ID NO: 1.

[0055] In an exemplary embodiment, more than one position in SEQ ID NO: 1 can be altered to provide a variant that is less than 80% identical to SEQ ID NO: 1. For example, alterations in the signal sequence and deletions in the starch binding domain can provide a variant that is less than 80% identical to SEQ ID NO: 1, such as about 75% to 80% identical to SEQ ID NO: 1.

[0056] Figure 1 An alignment of SEQ ID NO: 1 with Rhizopus oryzae GA is shown, showing the signal sequence (1-25) and starch binding domain (26-131).

[0057] Table 2 is a table of sequence identities (BLAST alignment) of the "core" sequence (lacking the signal sequence and starch binding domain) of the GA sequence of SEQ ID NO:1 to other known GAs. Accession number CEG69155.1 is the same sequence as SEQ ID NO:1.

[0058] Table 2

[0059]

[0060] In embodiments of the present disclosure, SEQ ID NO: 4 has one or more amino acid mutations that make it less than 100% identical to SEQ ID NO: 4. For example, a glucoamylase can have multiple amino acid deletions, substitutions, and / or additions that make it about 98% or more, or 99% or more identical to SEQ ID NO: 4. A variant with a single amino acid substitution has 99.8% identity to SEQ ID NO: 4.

[0061] In an exemplary embodiment, one or more positions in SEQ ID NO: 1 or SEQ ID NO: 4 are altered to provide variant SEQ ID NO: 1 and SEQ ID NO: 4 that also typically include a native "signal sequence". Various other terms may be used to represent "signal sequences" known in the art, such as replacing the word "signal" with "secretion" or "targeting" or "localization" or "transit" or "leader", and replacing the word "sequence" with "peptide" or "signal". In general, a signal sequence is a short stretch of amino acids (usually ranging from 5 to 30 amino acids in length) located at the amino terminus of a newly synthesized protein. Most signal peptides include a basic N-terminal region (n-region), a central hydrophobic region (h-region), and a polar C-terminal region (c-region) (e.g., see von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690).

[0062] In SEQ ID NO: 1 and SEQ ID NO: 4, the predicted signal sequence is from amino acids 1 to 25 of SEQ ID NO: 1 and amino acids 1 to 25 of SEQ ID NO: 4, respectively. The signal sequence can target the protein to a certain part of the cell, or can target the protein for secretion from the cell. For example, it has been shown that the native N-terminal signal sequence of the S. diastaticus glucoamylase STAI gene can target it to the endoplasmic reticulum of the secretion apparatus (e.g., see Yamashita, I. et al., (1985) J. Bacteriol. 161, 567-573).

[0063] The glucoamylase disclosed herein can use the natural signal sequences of SEQ ID NO: 1 and SEQ ID NO: 4 or their variants, or can be modified to include a heterologous signal sequence. In one aspect, the present invention provides a natural signal sequence that partially or completely replaces SEQ ID NO: 1 and SEQ ID NO: 4 with a secretion signal based on the sequence of the N-terminal portion of An aa, Sc FAKS, Sc AKS, Sc MFα1, Sc IV, Gg LZ and Hs SA as described in U.S. Provisional Patent Application 62 / 371,681 (published as WO2018027131) and PCT Application No. PCT / US2016 / 016822 (published as WO2016127083), and the above two applications are hereby incorporated by reference in their entirety.

[0064] These secretion signals can be used as replacements for the natural secretion signals of SEQ ID NO: 1 and SEQ ID NO: 4, or can be used in addition to the natural secretion signals. In view of the addition of heterologous secretion signals, these proteins can be referred to as "fusion proteins" and annotated as follows: [An aa-SS]-[Rm GA], [Sc IV-SS]-[Rd GA], etc.

[0065] Possible heterologous N-terminal replacement sequences for the N-termini of SEQ ID NO: 1 and SEQ ID NO: 4 include the following sequences. Sc-FAKS is a 90 amino acid sequence derived from the N-terminal portion of the Saccharomyces cerevisiae peptide mating pheromone alpha factor (e.g., see Brake, A. et al., Proc. Natl. Acad. Sci., 81:4642-4646, 1984; Kurjan, J. & Herskowitz, I., Cell 30:933-943, 1982). Sc-MFα1 is amino acids 20-89 Sc-FAKS. Sc IV is a 19 amino acid N-terminal signal peptide of sucrose hydrolase (e.g., see Carlson M. et al., (1983) Mol. Cell. Biol. 3:439-447). GgLZ (also known as egg white lysozyme) is the 18 amino acid N-terminal signal peptide of glycoside hydrolase (e.g., see Jigami et al., (1986) Gene 43:273-279). Hs SA is the 18 amino acid N-terminal signal peptide of serum (e.g., see Kober et al., (2013) Biotechnology and Bioengineering; 110:1164-1173). Sc MFα2 is derived from the N-terminus of the Saccharomyces cerevisiae mating factor α2 gene (Sc MFα2). Sc PHO5 is derived from the N-terminus of the Saccharomyces cerevisiae repressible acid phosphatase (Meyhack et al., EMBO J. 6:675-680, 1982).

[0066] Molecular techniques can be used to generate nucleic acid sequences that are templates for expressing genes encoding SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof. Generally, nucleic acids are prepared to encode proteins comprising SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof.

[0067] In other aspects, SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof optionally comprise additional sequences not present in native glucoamylase polypeptides. In some aspects, the additional sequences can provide glucoamylase with functions not present in native polypeptides. Additional functions include, for example, protease sites, or binding sites for other proteins or substances, or linker regions.

[0068] In some modes of practice, nucleic acid sequences encoding SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof, as well as any regulatory sequences (e.g., terminators, promoters, etc.) and vector sequences (e.g., including selection markers, integration markers, replication sequences, etc.) can be prepared using known molecular techniques. General guidance on methods for preparing DNA constructs (e.g., DNA constructs comprising nucleic acids encoding SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof) can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993. PCR techniques can be used to modify nucleic acids encoding SEQ ID NO: 1 or SEQ ID NO: 4 to optionally introduce one or more mutations in the sequence, thereby providing variants.

[0069] PCR technology is described in, for example, Higuchi, (1990) PCR Protocols, pp. 177-183, Academic Press; Ito et al., (1991) Gene 102: 67-70; Bernhard et al., (1994) Bioconjugate Chem. 5: 126-132; and Vallette et al., (1989) Nuc. Acids Res. 17: 723-733. The technology may optionally include site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared DNA encoding a glucoamylase polypeptide.

[0070] Alternatively, nucleic acid molecules can be generated by custom gene synthesis vendors such as ATUM (Menlo Park, CA) or GeneArt (Life Technologies, Thermo Fisher Scientific).

[0071] Expression vectors can be constructed to include a glucoamylase nucleic acid sequence operably connected to an expression control sequence that is functional in a host organism. Expression vectors suitable for use in a host organism include, for example, plasmids, episomes, and artificial chromosomes. The vector may include a selection sequence or a marker that is operable for stable integration into the host chromosome. In addition, the vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that, for example, provide antibiotic or toxin resistance, supplement nutrient deficiency defects, or provide key nutrients that are not present in the culture medium may also be included. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc. that are well known in the art.

[0072] In some aspects, the nucleic acid can be codon optimized. The nucleic acid template can be a native DNA sequence encoding a glucoamylase, or the template can be a codon optimized version optimized for expression in a desired host cell. Databases providing information on desired codon usage in specific host organisms are known in the art.

[0073] The DNA construct comprising the glucoamylase nucleic acid can be operably connected to a promoter sequence, wherein the promoter sequence is functional in the selected host cell. In some respects, the promoter demonstrates transcriptional activity in a fungal host cell, and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell. In some respects, the promoter can be used for expression in yeast saccharomyces cerevisiae. The example of well-known constitutive promoters includes but is not limited to cytochrome c promoter (pCYC), translation elongation factor promoter (pTEF), 3-phosphoglyceraldehyde dehydrogenase promoter (pGPD / TDH3), phosphoglycerate kinase promoter (PGK) and alcohol dehydrogenase promoter (pADH). Optionally, other factors controlling expression, such as enhancers, etc., can also be included on the vector.

[0074] The expression vector comprising the glucoamylase gene may also include any terminator sequence that is functional in the host cell. For example, the terminator sequence and the promoter sequence may be from the same cell, or the terminator sequence may be homologous to the host cell. The terminator sequence may correspond to any promoter used.

[0075] The DNA construct can be introduced into the host cell using a vector. The vector can be any vector that is stably introduced when introduced into the host cell. In some aspects, the vector is integrated into the host cell genome and replicated. The vector includes a cloning vector, an expression vector, a shuttle vector, a plasmid, a phage particle, a box, etc. In some aspects, the vector is an expression vector comprising a regulatory sequence that is operably connected to a glucoamylase encoding sequence. SEQ ID NO as described herein can be assembled in the cell by transforming a plurality of small DNA fragments (such as "SEQ ID NO sub-fragments") with overlapping homology, and the small DNA fragments constitute a specific SEQ ID NO together. For example, the integration of the locus of required SEQ ID NO or its part in the cell can be realized by co-transforming two to five DNA sub-fragments, and the sub-fragments are subjected to recombination with each other and integrated into the locus with homology to the part of the sub-fragments in the cell.

[0076] The DNA construct comprising the glucoamylase gene can also include a selection marker to facilitate selection in the host cell. For example, a selection marker can be used for transformed yeast. The example of a yeast selection marker includes a marker that is generally used to select transformed yeast cells. A nutrient deficiency marker can be used to control the nutrient deficiency type, which means that the gene enables yeast to produce the nutrients required for yeast growth. Example genes for controlling nutrient deficiency include leucine auxotrophy (LEU2), histidine auxotrophy (HIS3), uracil auxotrophy (URA3, URA5) and tryptophan auxotrophy (TRP1).

[0077] The DNA construct can be a construct that is integrated into the genome and replicated with the chromosome into which it has been integrated. For example, a DNA construct encoding glucoamylase can be transformed into a yeast cell, and the DNA construct can be integrated into the host chromosome with one or more copies. This integration is generally considered to be advantageous because the DNA sequence dna is more likely to be stably maintained. The DNA construct can be integrated into the host chromosome according to conventional methods (such as by homologous or heterologous recombination).

[0078] The engineered yeast of the present invention may include multiple copies (two or more) of a gene encoding SEQ ID NO: 1 or SEQ ID NO: 4 or a variant thereof. For example, the engineered yeast may be an engineered yeast having at least a first exogenous nucleic acid, a second exogenous nucleic acid, a third exogenous nucleic acid, and a fourth exogenous nucleic acid, each of which includes a sequence encoding at least one SEQ ID NO: 1 or SEQ ID NO: 4 or a variant thereof. If the engineered yeast includes multiple copies of a gene encoding a glucoamylase, the nucleic acid sequences of the copies may be identical or different from each other. Exemplary methods and yeast strains that have been engineered to include multiple copies of a glucoamylase gene are described in International Application Serial No. PCT / US16 / 24249 (Miller et al.), filed March 25, 2016, which is hereby incorporated by reference in its entirety.

[0079] The engineered yeast may also optionally include the introduction of exogenous nucleic acid sequences, changes in regulatory elements that up-regulate or down-regulate gene expression, an increase in gene copy number, and deletions or mutations that eliminate expression, reduce expression, or increase expression, or an increase in the expression or activity of a gene or gene product. Heterologous modifications may include one or more of the following: using a promoter different from the natural promoter of the desired gene; using a terminator different from the natural terminator of the desired gene; introducing a gene into a location in the genome different from its natural location; introducing multiple copies of a desired gene.

[0080] Other genetic modifications that can be included in engineered yeast are changes or introductions of enzyme activities that convert low molecular weight non-glucose into glucose. For example, an optional other genetic modification affects or introduces the isomaltase activity in engineered yeast during growth on glucose. Isomaltase can convert isomaltose into glucose by hydrolyzing 1,6 ether bonds in isomaltose. Isomaltase can also show cross-activity to hydrolyze 1,4 ether bonds in maltose. Genetic modification can cause isomaltase activity to be introduced into cells, increase the amount of isomaltase in cells, and / or increase isomaltase activity.

[0081] In some embodiments, in addition to the glucoamylase gene, the engineered cell also includes a heterologous isomaltase gene, or an isomaltase gene controlled by a heterologous promoter that provides increased expression in the cell, or is present in multiple copies in the cell. For example, an isomaltase (IMA) gene controlled by a heterologous promoter such as a PDC promoter can be engineered into yeast.

[0082] Examples of isomaltase genes that can be introduced into engineered yeast include, but are not limited to, Saccharomyces cerevisiae IMA1 (P53051), Saccharomyces cerevisiae IMA2 (Q08295), Saccharomyces cerevisiae IMA3 (P0CW40), Saccharomyces cerevisiae IMA4 (P0CW41), Saccharomyces cerevisiae IMA5 (P40884), Bacillus subtilis malL (006994), Bacillus cereus malL (P21332), Bacillus coagulans malL (Q45101), Bacillus sp. malL (P29093), etc. Preferably, the isomaltase gene encodes a polypeptide having a sequence identity of greater than 80%, 85%, 90%, 95%, 98% or 99% to the amino acid sequence of Accession No. NP011803.3 (Saccharomyces cerevisiae IMA1).

[0083] In some embodiments, the engineered yeast can also include a genetic modification that provides a starch degradation polypeptide different from a glucoamylase. For example, the genetic modification can be a modification of a nucleic acid that introduces a different polysaccharide degradation enzyme (such as an exogenous or modified α-amylase, β-amylase, pullulanase, or isoamylase). The genetic modification can also be a genetic modification that increases the amount of an endogenous or exogenous starch degradation polypeptide in the cell, such as by placing the gene under the control of a strong promoter, or providing the gene in the cell with multiple copies (such as multiple copies of a gene integrated into the genome, or multiple copies present on a non-chromosomal construct (e.g., a plasmid)).

[0084] In some embodiments, the engineered yeast may also include a genetic modification that provides an exogenous or modified sugar transporter gene (such as an isomaltose transporter); for example, see commonly assigned U.S. application serial number 62 / 268,932, entitled “Sugar Transporter-Modified Yeast Strains and Methods for Bioproduct Production”, filed on December 17, 2015 (published as WO2017106739), which is hereby incorporated by reference in its entirety.

[0085] Various host cells can be transformed with nucleic acids encoding SEQ ID NO: 1 or SEQ ID NO: 43 or variants thereof. In some aspects, the nucleic acid comprising the glucoamylase gene is present in a bacterial cell. The bacterial cell can be used, for example, to propagate the nucleic acid sequence or to produce large quantities of a polypeptide.

[0086] In other aspects, the host cell is a eukaryotic cell, such as a fungal cell.

[0087] In other aspects, the heterologous glucoamylase can be purified for use in an enzyme composition alone or in combination with other enzymes.

[0088] In some aspects, the host cell is tolerant to higher amounts of biologically derived products such as ethanol in the fermentation medium. In some aspects, the host cell is an "industrial yeast," which refers to any yeast routinely used for ethanol fermentation. Examples include sake yeast, shochu yeast, wine yeast, brewer's yeast, baker's yeast, and the like. Sake yeast exhibits high ethanol fermentation capacity as well as high ethanol tolerance and genetic stability. Typically, industrial yeast has high ethanol tolerance and is preferably viable at 10% or higher ethanol concentrations.

[0089] In exemplary aspects, the yeast comprising the glucoamylase gene is Saccharomyces cerevisiae. Some Saccharomyces cerevisiae strains have high tolerance to ethanol. Various strains of ethanol-tolerant yeast are commercially available, such as RED START and ETHANOL RED yeast (Fermentis / Lesaffre, U.S.), FALI (Fleischmann's Yeast, U.S.), SUPERSTART and THERMOSACC yeast (Ethanol Technology, Wisconsin, U.S.), BIOFERM AFT and XR (NABC-North American Bioproducts Corporation, Georgia, U.S.), GERT STRAND (Gert Strand AB, Sweden), SUPERSTART (Alltech), ANGEL (Angel Yeast Ltd, China) and FERMIOL (DSM Specialties).

[0090] Industrial yeast are generally prototrophic and therefore do not have auxotrophic markers suitable for selecting transformants. If the yeast does not have a genetic background that would otherwise facilitate the retention of a glucoamylase gene of SEQ ID NO: 1 or SEQ ID NO: 4 or a variant thereof within the cell after transformation, the host cell can be engineered to introduce one or more genetic mutations to establish the use of the marker gene in conjunction with the glucoamylase gene in the cell and maintain the glucoamylase gene in the cell. For example, commercially available ethanol-tolerant yeast cells can be genetically modified prior to introducing the glucoamylase gene into the cell.

[0091] Different auxotrophic markers can be provided by destroying the gene that controls the auxotrophic marker. In a practical mode, the ethanol tolerant strain of yeast is engineered to destroy copies of one or more genes (such as LEU2, HIS3, URA3, URA5 and TRP1) that control the auxotrophic marker. With respect to providing uracil auxotrophy, for example, the normal ura3 gene of ethanol tolerant yeast can be replaced with a ura3-fragment obtained from a uracil auxotrophic mutant (e.g., Saccharomyces cerevisiae MT-8 strain) to destroy the normal ura3 gene. With respect to the ura3 gene-destroyed strain, the presence / absence of the marker can be easily identified or selected by utilizing the fact that the ura3 gene-destroyed strain can grow in a culture medium containing 5-fluoroorotic acid (5-FOA) while the normal ura3 strain (wild-type yeast or conventional industrial yeast) cannot grow. With regard to the disrupted strain of lys2 gene, the presence / absence of normal lys2 strain (wild-type yeast or conventional industrial yeast) can be easily identified or selected by utilizing the disrupted strain of lys2 gene to grow in the culture medium containing α-aminoadipic acid and the fact that normal lys2 strain (wild-type yeast or conventional industrial yeast) can not grow. The method of the gene mutant of the gene that destroys the control auxotrophy and selectively separates the control auxotrophy can be used according to the auxotrophy used. Alternatively, dominant selection markers can be used, such as amdS (U.S. Patent number 5,876,988) from Aspergillus nidulans (Aspergillus nidulans), which allows growth on acetamide as the sole nitrogen source; or ARO4-OFP, which allows growth (Fukuda et al.) in the presence of fluorophenylalanine. These marks can be reused using the recyclable cre-loxP system, or alternatively can be used to produce auxotrophy strains that allow the use of other marks.

[0092] After the host cell has been engineered to provide the required genetic background in order to introduce the glucoamylase gene, the gene construct is introduced into the cell to allow expression. The method of introducing the gene construct into the host cell includes conversion, transduction, transfection, cotransfection, electroporation. Specifically, the lithium acetate method, protoplast method, etc. can be used to carry out yeast transformation. The gene construct to be introduced can be combined into the chromosome in the form of a plasmid or by inserting into the host gene or by homologous recombination with the host gene. The transformed yeast that has been introduced into the gene construct can be selected with a selection marker (for example, a nutritional deficiency marker as described above). Further confirmation can be carried out by measuring the activity of the expressed protein.

[0093] The conversion of the exogenous nucleic acid sequence including the glucoamylase gene can be confirmed using methods well known in the art. These methods include, for example, nucleic acid analysis (such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA), or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the exogenous nucleic acid is expressed in an amount sufficient to produce the desired product, and it will also be understood that the expression level can be optimized using methods well known in the art and as disclosed herein to obtain sufficient expression.

[0094] The engineered yeast of the present disclosure can be provided in any suitable form. In some aspects, the non-natural yeast is dehydrated to form a dry yeast composition. The dry yeast composition can have an increased shelf life compared to a wet composition.

[0095] Fermentation using host cells expressing glucoamylase genes can be carried out in a fermentation medium made of raw materials derived from plant materials containing starch and / or sugar, and the plant materials containing starch and / or sugar refer to plant materials containing starch and / or sugar that can be derived from any plant and plant part (such as tuber, root, stem, leaf and seed). Plant materials containing starch and / or sugar can be obtained from cereals (such as barley, wheat, corn, rye, sorghum, millet, barley, potato, cassava or rice, and any combination thereof). Plant materials containing starch and / or sugar can be processed such as by methods such as milling, malting or partial malting. In some aspects, starch material comes from corn flour, milled corn endosperm, sorghum flour, soybean flour, wheat flour, starch from biomass, barley flour and combinations thereof. The raw material containing starch for forming a fermentation medium can be made from any material in these plant materials.

[0096] In some aspects, the raw materials for forming the fermentation medium include treated starch. For example, the fermentation medium may include partially hydrolyzed starch. Partially hydrolyzed starch may include high molecular weight dextrins and high molecular weight maltodextrins. Starch, dextrin, maltodextrin and any other polymerized forms of glucose are collectively referred to as glucose polymers. Partially hydrolyzed starch and its preparation are well known in the art. Partially hydrolyzed starch can be prepared by heating starch with an acid (such as hydrochloric acid or sulfuric acid) at high temperature and then neutralizing the hydrolysis mixture with a suitable alkali (such as sodium carbonate). Alternatively, partially hydrolyzed starch can be prepared by an enzymatic process (such as by adding alpha-amylase to a starch product). Alpha amylase can cause endohydrolysis of (1→4)-α-D-glucosidic bonds in polysaccharides containing three or more (1→4)-α-connected D-glucose units. A partially hydrolyzed starch product with starch and starch degradation product amounts within a desired range can be used. Partially hydrolyzed starches include preparations with minimal hydrolysis (eg, DE of 5, almost no dextrose) to preparations with extensive hydrolysis (eg, DE of 95, mostly dextrose).

[0097] The raw material can be a "liquefact," which is corn starch that has undergone liquefaction, with a dextrose equivalent in the range of about 10 to about 15. A corn wet milling process can be used to provide steepwater, which can be used for fermentation. The corn kernels can be soaked first, then ground and separated into their major component fractions. Light steepwater is a byproduct of the steeping process and contains a mixture of soluble proteins, amino acids, organic acids, carbohydrates, vitamins, and minerals.

[0098] In some aspects, the feedstock can be dry-milled corn, i.e., most or all of the corn kernel components are included in the fermentation feedstock. In the U.S., the dry-milled corn process is the most commonly used technology for converting corn into ethanol. Some aspects of the dry-milling process are different from the wet-milling process (using liquefied material), including but not limited to adding urea to provide enough nitrogen for fermentation. The main aspects of the dry-milling process for producing ethanol are well known in the art.

[0099] The raw materials derived from any of the plant materials described herein generally include "glucose polymers", which refer to those polymers containing two or more glucose residues. Shorter glucose polymers (including glucose dimers (e.g., maltose), trimers (e.g., trisaccharides), and those polymers up to about 10 glucose units) may also be referred to as "glucose oligomers". The raw materials for hydrolyzed starch products with a DE ranging from about 2 to about 20 mainly include maltodextrins, which include DPs ranging from 3 (540 Da) to more than about 5000 (10 6For example, starch preparations with a DE of 2 include most maltodextrins in the range of 200,000 Da to about 1×105 Da, and starch preparations with a DE in the range of about 10 to about 20 have most maltodextrins in the range of about 540 Da to about 100,000 Da. The degree of polymerization (DP) refers to the number of sugar monomer residues in the glucose polymer.

[0100] Based on the DE of the partially hydrolyzed starch, the concentrations (wt %) of glucose (DP 1), maltose (DP 2), trisaccharides (DP 3), and longer glucose polymers (DP 4+) in the composition can be known. Table 3 provides the concentrations (wt %) of various "polymer" forms of glucose at different DE points as understood in the art.

[0101] Table 3

[0102]

[0103]

[0104] The benefits of the engineered yeast of the present disclosure allow the use of fermentation media made from raw materials with low DE (such as a DE of less than about 40, less than about 30, less than about 20, less than about 15, or less than about 10). Such raw materials may require the use of exogenous starch degrading enzymes to produce glucose for cell growth and bioproduct formation. However, starch-containing raw materials with higher DE can be used in various methods with the engineered yeast of the present disclosure, and the engineered yeast can still provide fermentation benefits. For example, the method of the present invention can use a raw material containing partially hydrolyzed starch with a DE of no more than about 75 or no more than about 70 and greater than about 35 or greater than about 40 (and more preferably in the range of about 45 to about 65). DE in the range of about 45 to about 65 means that the amount of glucose present in the raw material composition is in the range of about 19% to about 40% (by weight), maltose is in the range of about 14% to about 35% (by weight), trisaccharides are in the range of about 8% to about 12% (by weight), and glucose polymers with a DP of 4 or higher are in the range of about 17% to about 55%. The percentages are based on the total amount of all "polymer" forms of glucose in the composition. The fermentation and addition of starch-containing feedstock can be performed to provide glucose and glucose polymers within a desired range expressed as a percentage of the total amount of all "polymer" forms of glucose in the composition.

[0105] In various aspects of the present disclosure, given that a glucoamylase is produced and secreted into the fermentation medium from an engineered yeast, the fermentation process can omit the addition of a purified or enriched commercial glucoamylase to the medium, or at least allow the use of significantly less commercial glucoamylase in the fermentation process. For example, the engineered yeast of the present disclosure can allow the elimination or reduction of at least about 50%, 60%, 70%, 80%, 90% or 95% of the commercial glucoamylase addition. A 100% reduction can be achieved using the yeast described herein, particularly if a longer fermentation period, such as 60 hours, is used. Typically, in a fermentation process that does not use an engineered yeast that secretes glucoamylase, an amount of glucoamylase in the range of about .014AGU / g DS to .071AGU / g DS will be used.

[0106] The benefits of using yeast engineered to express a glucoamylase according to SEQ ID NO: 1 or SEQ ID NO: 4 can be understood by fermenting the yeast in a fermentation medium made from liquefied corn mash having a low DE (such as not greater than about 50, not greater than about 35, not greater than about 30, or not greater than about 25, or not greater than about 20, or not greater than about 15, such as in the range of about 2 to about 20, about 2 to about 15, or about 5 to about 5) and fermenting the medium to produce ethanol. The feedstock used to prepare the fermentation medium can optionally be described in terms of glucose concentration as the total percentage of fermentable carbohydrates (glucose and glucose polymers) in the feedstock. For example, the fermentation medium can be made from a starch feedstock having a glucose concentration of about 2% or less, or about 1% or less, such as in the range of about 0.1% to about 2%, 0.1% to about 1.5%, or about 0.1% to about 1%. At the end of the fermentation period, the ethanol concentration in the fermentation medium is 70 g / kg or greater.

[0107] To determine whether yeast expressing glucoamylase is capable of producing 70 g / kg or higher ethanol concentrations in a fermentation medium at the end of the fermentation period, the following test can be performed: First, a fermentation medium using a starch feedstock having a DE of 30+ / -2 is produced by preparing corn mash (or liquefied corn mash) using a predetermined amount of yellow dent #2 corn (milled and passed through a US#20 sieve). The rejects (twice milled corn retained on the US#20 sieve) are added back to the sieved corn at a reject ratio of X:Y (residue / total corn mass ratio is 0.020). The moisture content is measured by the halogen moisture balance method to determine the dry weight of the milled corn. Water is added to produce a 32% slurry (weight / weight, on a dry weight basis). Concentrated sulfuric acid is added to achieve a pH of 5.7 to 5.9. Calcium chloride dihydrate powder is added to achieve a calcium concentration of 35 ppm. Amylase (Liquozyme 2000) is added at a dosage ratio of 2.84 kg / ton dry starch dosage based on the weight of corn dry starch. TMNovozymes Liquozyme Supra 2.2X) and the slurry was transferred to a Buchi Rotovapor R-220 flask equipped with an oil bath (preset at 85°C). The reaction was allowed to proceed for 2 hours and once the dextrose equivalent (DE) reached 30 + / - 2, the reaction was stopped by lowering the temperature to 34°C to 36°C. The pH was adjusted to 5.0 with additional concentrated sulfuric acid. The osmometer (Advanced TM Model 3D3 and Precionsystem Model Osmette XL TM ) to determine DE. Using HPLC and Aminex HPX-87H Column (300 mm × 7.8 mm) Sugar and oligosaccharide content was determined at 60 ° C, 0.01 N sulfuric acid mobile phase, and a flow rate of 0.6 mL / min. 50% urea solution and 10% ampicillin solution were added to the starch feedstock, with target final concentrations of 1900 ppm and 5 ppm, respectively. A single fermentation typically contains 50 g corn mash, 190 ul of 50% urea, and 2.5 ul of 10% ampicillin. The typical fermentation vessel is a baffled 250 ml shake flask equipped with an airlock. The airlock should contain four to five milliliters of canola oil. The flask was inoculated with a cell slurry made by scraping a fresh YPD plate into 1 ml of sterile water to an initial OD 600 0.1. Using an ethanol-tolerant engineered yeast (e.g., ETHANOL ) inoculate the fermentation medium to a concentration of OD 600 The fermentation was carried out in flasks at 30°C in an orbital shaker with shaking at 100 rpm for about 48 hours. At 48 hours, the concentration of ethanol and optionally other compounds (such as glucose) in the samples was analyzed by high performance liquid chromatography with a refractive index detector.

[0108] Without supplementing any commercial glucoamylase and using the above-mentioned raw materials, typical ethanol titers in the range of about 115 g / kg to about 135 g / kg can be observed using engineered yeast expressing glucoamylases of SEQ ID NO: 1 or SEQ ID NO: 4 or their variants. Higher ethanol titers can be achieved by changing yeast and / or fermentation conditions. For example, in embodiments in which supplementary commercial glucoamylases are added to the culture medium, larger amounts of ethanol, such as 110 g / kg or greater, or 125 g / kg or greater, or 140 g / kg or greater, can be produced in the fermentation medium. In addition to higher final ethanol titers, fermentation rates can also be increased because free glucose may no longer limit fermentation. The addition of commercial glucoamylases further acts on starch polymers to produce more glucose in the fermentation medium, resulting in increased cell growth and higher ethanol titers.

[0109] In order to achieve an ethanol concentration of 110 g / kg or greater in the fermentation medium at the end of the fermentation period, the following test can be performed. Prepare the starch feedstock and fermentation medium as described above. Supplement the fermentation medium with a commercial glucoamylase (Spirizyme FuelHS, Novozymes) to provide additional glucoamylase activity in the medium. 0.097 AGU / gDS or 30% of the dose required for the wild type can provide a benefit. Glucoamylase activity (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute using 100 mM acetate buffer (pH 4.3) with 23.2 mM maltose at standard conditions of 37°C and a reaction time of 5 minutes. Ethanol-tolerant engineered yeast (e.g., ETHANOL) with an exogenous nucleic acid expressing SEQ ID NO: 1 or SEQ ID NO: 4 is used. ) was inoculated into the fermentation medium. Fermentation was carried out at 30°C for 48 hours. With the supplementation of commercial glucoamylase and the use of low glucose feedstock, typical ethanol titers ranging from about 110 g / kg to about 160 g / kg were observed.

[0110] Test 1 is the method described in the preceding paragraph performed at 30°C without supplementation of commercial GA, and Test 2 is the method described in the preceding paragraph performed at 30°C with supplementation of 0.097 AGU / g DS GA. Test 3 is the method described in the preceding paragraph performed at 33.3°C without supplementation of commercial GA. Test 4 is the method described in the preceding paragraph performed at 33.3°C with supplementation of 0.0.097 DS GA. The preferred yeast is the yeast that can produce a minimum of 70 g / kg in Test 1 AGU / g (all GA strains) and a minimum of 130 g / kg in Test 4 (2X Rmic and 4X Rdel strains).

[0111] In other embodiments, after a period of fermentation, the yeast engineered with glucoamylase disclosed herein can provide a desired final fermentation medium with high levels of bioproducts (e.g., ethanol) and low levels of byproducts. For example, the final fermentation medium can have high levels of glucose (e.g., 70 g / kg or greater, 90 g / kg or greater, 110 g / kg or greater, 125 g / kg or greater, or 140 g / kg or greater) and low levels of glucose, such as 1.0 g / kg or less (e.g., 0.9 g / kg or less, 0.8 g / kg or less, 0.7 g / kg or less, 0.6 g / kg or less, or 0.5 g / kg or less). In a final fermentation medium with a high ethanol titer, low glucose is beneficial because it improves downstream processes, such as separation of components (e.g., ethanol) in the final fermentation medium.

[0112] The fermentation medium comprises water, and preferably comprises nutrients, such as nitrogen sources (such as proteins), vitamins and salts. A buffer may also be present in the fermentation medium. After a period of fermentation, other components may also be present in the fermentation liquid, such as fermentation products and other metabolites that may accumulate as the fermentation proceeds. Optionally, the fermentation liquid may be buffered with an alkali (such as calcium hydroxide or calcium carbonate, ammonia or ammonium hydroxide, sodium hydroxide or potassium hydroxide) to maintain a pH for the normal functioning of the organism.

[0113] The fermentation medium may optionally contain one or more of the following enzymes different from the glucoamylase of SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof. Exemplary other enzymes include alpha amylase, beta amylase, peptidase (protease, protease, endopeptidase, exopeptidase), pullulanase, isoamylase, cellulase, hemicellulase, endoglucanase and related beta-glucan hydrolysis auxiliary enzymes, xylanases and xylanase auxiliary enzymes, acetolactate decarboxylase, cyclodextrin glycosyltransferase, lipase, phytase, laccase, oxidase, esterase, cutinase, granular starch hydrolyzing enzyme and other glucoamylase. These other enzymes may be optionally added to the fermentation medium or starch-containing raw materials, such as by using a purified commercial preparation of the enzyme. Alternatively, one or more other enzymes may be secreted from an engineered yeast expressing SEQ ID NO: 1 or SEQ ID NO: 4 or from a different engineered cell.

[0114] The engineered yeast of the present disclosure may optionally be described in terms of the specific growth rate of the engineered yeast.The growth rate of yeast may be defined by L=log(number), where number is the number of yeast cells formed per unit volume (mL) versus T (time).

[0115] Fermentation is carried out under conditions where fermentation can occur. Although conditions can vary depending on the specific organism and desired fermentation product, typical conditions include temperatures in the range of about 20°C or higher, more typically about 30°C or higher. During the fermentation process, the reaction mixture can be mixed or stirred. In some practical modes, mixing or stirring can occur by the mechanical action of bubbling gas into the fermented liquid. Alternatively, direct mechanical stirring can be used during the fermentation process, such as by an impeller or by other means.

[0116] The present disclosure also provides non-natural yeasts that have the ability to grow at temperatures higher than the temperatures that yeast (such as Saccharomyces cerevisiae) typically uses for fermentation processes and / or can produce fermentation products at such temperatures. For example, Saccharomyces cerevisiae typically has optimal growth at a temperature of about 30°C. However, the engineered yeasts of the present disclosure can grow at higher temperatures and provide excellent bioproduct (e.g., ethanol) titers, and can also provide low residual glucose. For example, in some embodiments using the engineered yeasts of the present disclosure, fermentation is carried out at a temperature in the range of 31°C to 35°C or 32°C to 34°C for most or all of the fermentation period. Even at higher temperatures, the engineered yeasts are able to produce glucoamylase activity in the culture medium and promote excellent cell growth and bioproduct production.

[0117] In the fermentation process, the fermentation medium can reach the temperature of raising in one or more time periods in the fermentation process, such as about 32 ℃ or about 32 ℃ or higher. Can raise temperature during part of fermentation or during whole fermentation.Can raise temperature 5 minutes or longer, 10 minutes or longer, 30 minutes or longer, 1 hour or longer, 2 hours or longer, 5 hours or longer or 10 hours or longer.The time of the temperature of raising can also be expressed as the summation of whole fermentation period, such as about 0.1% to 100% of fermentation period, about 0.1% to about 75%, about 0.1% to about 50%, about 0.1% to about 25%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2.5%, about 0.1% to about 1% or about 0.1% to about 0.5%.

[0118] The engineered yeast can also provide commercially relevant ethanol titers during or after elevated temperatures. For example, during or after elevated temperatures, for example, the ethanol titer can be in the range of about 110 g / L to about 170 g / L, in the range of about 125 g / L to about 170 g / L, or in the range of about 140 g / L to about 170 g / L. Thus, the engineered yeast described herein can produce ethanol at commercially useful titers during or after elevated temperatures, which would typically cause problems in other currently available yeast strains used in fermentation processes to produce ethanol. These problems include, but are not limited to: a large number of yeast cell deaths; adverse effects on yeast reproduction; and / or reduction or elimination of the ability of yeast to produce fermentation products.

[0119] Miller et al. (WO2016127083, filed February 6, 2015, and PCT / US17 / 045493, filed August 4, 2017, both of which are hereby incorporated by reference in their entirety) described the utility of swapping leader sequences on several glucoamylases, but also emphasized the need for other modifications to the host to achieve acceptable ethanol / glucose titers at elevated temperatures. Specifically, Miller described the effects of expressing Mfalpha2-Rhizopus oryzae GA at two different temperatures, 30°C and 33.3°C, on ethanol and residual glucose titers. Temperature is a well-known antagonist of healthy ethanol fermentation, and producers spend a lot of capital and operating costs on cooling capacity to keep fermenters in a safe zone, typically below 34°C. Heterologous protein production is also a well-described stressor in engineered organisms because energy directed toward cell growth and maintenance is diverted to non-natural production processes, as described by Mattanovich et al. (Journal of Biotechnology 113, 2004). Reducing the burden of heterologous protein production has been an area of ​​focus over the past few decades in various aspects of biotechnology (e.g., pharmaceuticals, industrial enzymes, etc.) and is not limited to Saccharomyces cerevisiae. Classical techniques and targeted pathway engineering, two major approaches to overcome the barriers of producing proteins and maintaining healthy host performance, have achieved some success (Payne et al., 2008; Gasser et al., 2007; Valkonen et al., 2003). These results also show that there is no one solution to the problem and that a solution for one protein may not be effective for another.

[0120] In some cases, fermentation is carried out in an industrial capacity fermentor to allow for commercial scale economies and control. In one aspect, fermentation is carried out in a fermentor having a capacity of about 10,000 liters or greater.

[0121] The pH of the fermentation medium can be adjusted to provide optimal conditions for glucoamylase activity, cell growth and fermentation activity, thereby providing a desired product, such as ethanol. For example, the pH of the solution can be adjusted to within the range of 3 to 5.5. In one practical mode, the pH of the fermentation medium is within the range of 4 to 4.5.

[0122] As described above, the fermentation process of the present invention uses genetically modified yeast that expresses SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof and is capable of secreting the produced enzymes into the fermentation medium. Therefore, these enzymes are directly exposed to the fermentation broth conditions and affect the carbohydrate composition of the fermentation medium. In the fermentation medium, glucoamylase can hydrolyze and release D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules by cleaving α-(1,4) and α-(1,6) glycosidic bonds.

[0123] One or more other amylases (e.g., α-amylases) present in the fermentation medium may also act on the starch. For example, if α-amylase is present in the fermentation medium, it may partially hydrolyze the precursor starch and partially decompose the starch molecules by hydrolyzing internal α-(1,4)-bonds.

[0124] In some practice modes, fermentation is carried out in a single batch until completion. In other practice modes, fermentation is carried out in a fed batch fermentation process. In this practice mode, a first portion of the total amount of starch material to be fermented is added to a fermentation medium, wherein glucoamylase acts on the starch to form glucose, which is used as a fermentation substrate. Additional starch material may be added in one or more portions to provide more substrate for the glucoamylase in the medium. The addition of starch may be regulated, and the formation of glucose may be monitored to provide effective fermentation.

[0125] In some practical modes, fermentation is carried out in a continuous operation mode. In this mode, multiple fermenters are operated in series, wherein starch hydrolysate is provided in a first fermenter, which is sent to a second fermenter, and so on, until the starch hydrolysate is converted into ethanol. Continuous operation can be carried out using 2 to 7 fermenters.

[0126] In some practice modes, a portion of the total amount of starch material is added to the fermented liquid using a variable rate addition system. Examples of such systems include a variable speed pump or a metering valve (such as a throttle valve) operably connected to a pump, which pump or valve can be used to change the amount of starch material introduced into the fermented liquid over time. In some practice modes, during the addition of a portion of starch material, glucose concentration is monitored by a real-time monitoring system.

[0127] Real-time monitoring system comprises the system of directly monitoring glucose concentration and the system of indirectly monitoring glucose concentration.Usually the example of the real-time monitoring system of directly monitoring glucose concentration comprises the system based on infrared (IR) spectrum, near infrared (NIR) spectrum system, Fourier transform infrared (FTIR) system, the system based on refractive index, the automatic measurement system based on enzyme (such as the YSI 2950Biochemistry Analyzer sold by YSI Life Sciences system), the system based on high performance liquid chromatography (HPLC), the system based on gas chromatography (GC) and other real-time monitoring systems well known to those skilled in the art.In addition, can be by determining the typical carbon distribution in the specific fermentation process and the glucose concentration present in the fermentation liquid and fermentation another parameter (for example, the correlation between the measured value of the glucose level present in the fermentation liquid and the carbon dioxide release rate and the carbon dioxide amount present in the waste gas flow from the fermentation vessel) associated, develop the real-time monitoring system of glucose concentration of indirect monitoring / measurement fermentation process.By using mass spectrometer or other suitable instrument technology to measure the component of waste gas flow, carbon dioxide can be easily measured.In a preferred aspect, monitor glucose concentration by using the real-time monitoring system of infrared spectrum.On the other hand, monitor glucose concentration by using the real-time monitoring system of near infrared spectrum. The real-time monitoring system is connected to a device for controlling the introduction of starch material into the fermentation broth to adjust the glucose formation in the fermentation broth to a desired concentration.

[0128] During the fermentation process, a sample of the fermentation medium can be collected to determine the amount of glucoamylase activity in the medium. The amount of glucoamylase activity in the medium can be referred to as extracellular glucoamylase activity because it corresponds to the glucoamylase secreted from the engineered yeast. In some measurement modes, the amount of glucoamylase activity in the medium can be determined by the amount of glucoamylase activity per biomass per volume of medium.

[0129] Measuring glucoamylase activity in the fermentation medium can be another way to reflect the benefits of using yeast engineered to express a glucoamylase according to SEQ ID NO: 1 or SEQ ID NO: 4. This test can be performed using a fermentation medium made from a low DE feedstock, a high DE feedstock, or anything in between. During the fermentation process, samples of the medium are taken and the biomass and enzyme activity are determined. As used herein, "biomass" refers to the weight of the engineered yeast, which can be measured in grams of dry cell weight per liter of culture medium (DCW / L).

[0130] In some modes of practice, the fermentation period is a period of time ranging from about 30 hours or more, about 40 hours or more, about 50 hours or more, or about 60 hours or more, such as about 40 hours to about 120 or 50 hours to about 110 hours.

[0131] The fermentation product (also referred to herein as "product of biological origin" or "bioproduct") can be any product that can be prepared by enzymatic degradation of starch material by glucoamylase, formation of glucose, and fermentation of glucose. In some aspects, the fermentation product is selected from the group consisting of amino acids, organic acids, alcohols, diols, polyols, fatty acids, fatty acid alkyl esters (such as fatty acid methyl esters or ethyl esters (e.g., C6 to C12 fatty acid methyl esters (preferably C8 to C10 fatty acid methyl esters)), monoacylglycerides, diacylglycerides, triacylglycerides, and mixtures thereof. Preferred fermentation products are organic acids, amino acids, fatty acid alkyl esters (such as fatty acid methyl esters (e.g., C8 to C12 fatty acid methyl esters (preferably C8 to C10 fatty acid methyl esters))) and salts thereof, particularly wherein the organic acid is selected from the group consisting of hydroxycarboxylic acids (including monohydroxy and dihydroxy monocarboxylic acids). Examples of fermentation products prepared by the process of the present invention are organic acids or amino acids such as lactic acid, citric acid, malonic acid, hydroxybutyric acid, adipic acid, lysine, ketoglutaric acid, glutaric acid, 3-hydroxy-propionic acid, succinic acid, malic acid, fumaric acid, itaconic acid, muconic acid, methacrylic acid, acetic acid, methyl caproate, methyl octanoate, methyl nonanoate, methyl decanoate, methyl dodecanoate, ethyl caproate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl dodecanoate and mixtures thereof and their derivatives and salts thereof. In a preferred aspect, the fermentation method of the present disclosure produces ethanol as a bioproduct.

[0132] The fermentation product can have an excellent bioproduct (e.g., ethanol) to residual glucose ratio, which is beneficial because it improves downstream processes, such as separation of components (e.g., ethanol) in the final fermentation medium. For example, the amount of glucose in the fermentation medium is 1.0 g / kg or less, 0.9 g / kg or less, 0.8 g / kg or less, 0.7 g / kg or less, 0.6 g / kg or less, 0.5 g / kg or less, 0.4 g / kg or less, 0.3 g / kg or less, or 0.2 g / kg or less, such as an amount of glucose in the range of about 0.05 g / kg to about 1.0 g / kg or about 0.05 g / kg to about 0.5 g / kg. The final fermentation medium may have an ethanol:glucose (weight / weight) ratio of about 70:1 (weight / weight) or greater, about 100:1 (weight / weight) or greater, about 150:1 (weight / weight) or greater, about 200:1 (weight / weight) or greater, about 250:1 (weight / weight) or greater, or about 300:1 (weight / weight) or greater, such as in the range of about 75:1 (weight / weight) to about 750:1 (weight / weight) or about 100:1 (weight / weight) to about 500:1 (weight / weight).

[0133] The fermentation product is recovered from the fermentation broth. The manner in which this process is accomplished will depend on the specific product. However, in some practical modes, the organism is separated from the liquid phase, typically by a filtration step or a centrifugation step, and the product is recovered by, for example, distillation, extraction, crystallization, membrane separation, osmosis, reverse osmosis or other suitable techniques.

[0134] The process of the present invention provides the ability to prepare fermentation products at production scale levels with excellent yield and purity. In one aspect, the process is carried out with at least 25,000 gallons of fermentation broth. In one aspect, a batch process is carried out to produce multiple batches of at least 25,000 gallons of final fermentation broth. In some aspects, the process is a continuous process carried out in a container of at least 200,000 gallons.

[0135] In some aspects, genetically modified yeast expressing SEQ ID NO: 1 or SEQ ID NO: 4 or variants thereof can be used in conversion processes such as for the production of fructose syrups, specialty sugars, and dextrose in alcohols and other end products (e.g., organic acids, ascorbic acid, and amino acids). The production of alcohol from fermentation of starch substrates using the glucoamylases of the present disclosure can include the production of fuel alcohol or drinking alcohol.

[0136] The ethanol mass yield can be calculated by dividing the ethanol concentration by the total glucose consumed. Since glucose can exist in the form of free glucose or be bound together in the form of oligomers, both situations need to be considered at the same time. In order to determine the total glucose present at the beginning and end of the fermentation, the total glucose equivalent is determined. The total glucose equivalent is measured as follows. Glucose is measured using HPLC using RI detection. Separation is completed using a Bio Rad 87H column using a 10mM H2SO4 mobile phase. Glucose is measured in triplicate for each sample. Acid hydrolysis is performed in triplicate at 121°C for 15 minutes in 6% (volume / volume) trifluoroacetic acid. The glucose obtained after hydrolysis is measured by the same HPLC method. The total glucose equivalent present in each sample is the amount of glucose measured after acid hydrolysis. The total glucose consumed is calculated by subtracting the total glucose equivalent present at the end of the fermentation from the total glucose equivalent present at the beginning of the fermentation.

[0137] Use of the engineered yeast of the present disclosure may also provide benefits with respect to increased titer, reduced volatile organic acids (VOCs), and reduced fusel oil compounds (volatile organic acids, higher alcohols, aldehydes, ketones, fatty acids, and esters).

[0138] The fermented product can first be treated with one or more reagents by a treatment system. The treated fermented product can then be sent to a distillation system. In the distillation system, the fermented product can be distilled and dehydrated to ethanol. In some aspects, the components removed from the fermentation medium include water, soluble components, oil and unfermented solids. Some of these components can be used for other purposes, such as for animal feed products. Other by-products, such as syrup, can be recovered from the stillage.

[0139] The present disclosure also provides a method for producing a food, feed or beverage product (such as an alcoholic or non-alcoholic beverage, such as a cereal or malt-based beverage, such as beer or whiskey, such as wine, cider, vinegar, rice wine, soy sauce or juice), the method comprising the step of treating a starch and / or sugar-containing plant material with a composition as described herein. In another aspect, the present invention also relates to a kit comprising a glucoamylase of the present disclosure or a composition contemplated herein; and instructions for use of the glucoamylase or composition. The present invention also relates to a fermented beverage produced by a method using a glucoamylase.

[0140] After fermentation process is completed, the material present in the fermentation medium can be used. In some aspects, after fermentation process is completed or when fermentation process is ongoing, some or all of the biological products can be removed from the fermentation medium to provide a refined composition comprising non-biological product solids. Non-biological product solids can include raw materials and fermentation byproducts that yeast does not utilize in non-natural yeast, culture medium. These materials can provide the source of carbohydrate and protein, can be used as supplements to improve the nutritional content of feed composition. Feed material can be a by-product of fermentation process, such as stillage (full stillage, rare stillage, etc.), or a composition prepared therefrom, including distiller dry grains (DDG), distiller dry grains and solubles (DDGS), distiller wet grains (DWG) and distiller solubles (DS).

[0141] The fermentation medium from which some or all of the target biological product has been optionally removed can be further processed, such as to remove water, or to cause non-biological product solids to precipitate or separate from the medium. In some cases, the medium is processed by freeze drying or oven drying. After processing, the refined composition can be in the form of, for example, a liquid concentrate, a semi-wet cake, or a dried solid. The refined composition itself can be used as a feed composition, or as an ingredient in a preparation of a feed composition. In a preferred preparation, the feed composition is a livestock feed composition, such as for sheep, cattle, pigs, etc.

[0142] The solid in the fermentation medium can provide one or more amino acid sources. Introduced in animal feed, fermentation byproducts can provide an amino acid content increased about one or more essential amino acids. Essential amino acids can include histidine, isoleucine, lysine, methionine, phenylalanine, threonine and tryptophan. These amino acids can be present in the feed composition in the form of free amino acids, or can be derived from amino acid-rich proteins or peptides. The solid in the fermentation medium can provide a source of one or more prebiotics, which are indigestible food substances, such as indigestible oligosaccharides, which selectively stimulate the growth of beneficial bacterial species in the intestinal tract, thereby benefiting the host. The solid in the fermentation medium can provide a source of phytase, beta-glucanase, protease and xylanase.

[0143] Table 4 includes strains used in experimental studies related to the present disclosure.

[0144] Table 4

[0145] Strain No. Strain description strain 1 wild type strain 1-1 Ura3Δ Strain 1-2 2X Mfa2-Rhizopus oryzae GA Strains 1-3 4X Mfa2-Rhizopus oryzae GA Strains 1-4 1X Rhizopus microsporus GA Strains 1-5 2X Rhizopus microsporus GA Strains 1-6 2X Rhizopus microsporus GA Strains 1-7 4X Rhizopus microsporus GA Strains 1-8 2X Rhizopus delbrueckii GA Strains 1-9 4X Rhizopus delbrueckii GA

[0146] Example 1

[0147] Screening of a diverse library of glucoamylases grown on starch

[0148] Heterologous expression of functional glucoamylase in Saccharomyces cerevisiae was first demonstrated in about 1993 using Aspergillus niger glucoamylase. Other uses of glucoamylase in Saccharomyces cerevisiae have been reported, but still represent only a small fraction of the amount of public sequence information for these proteins. To this end, more than 1,000 enzymes have been expressed and tested from a variety of organisms to identify enzymes that confer the desired traits of high glucoamylase expression while maintaining ethanol rate, titer, and yield.

[0149] A DNA library containing 1037 genes encoding glucoamylase, α-amylase, amylopectinase and other starch hydrolyzing enzymes was constructed by cloning synthetically generated open reading frames into Saccharomyces cerevisiae episomal plasmids. The enzymes encoded by these genes are derived from four different categories, including: 1) enzymes annotated with glucoamylase EC numbers (including glucoamylase and α-amylase), 2) enzymes annotated with α-1,6 and α-1,4 glycosidase activity, 3) structural homologs of previously identified functional fungal glucoamylases expressed in Saccharomyces cerevisiae, and finally 4) starch hydrolyzing enzymes from ruminant intestinal microbiota. Each enzyme in the library was screened with its native leader sequence and the leader sequence replaced by the Saccharomyces cerevisiae Mfa2 leader sequence. A total of 1,773 plasmids were successfully transformed into strain 1-1 (described in a previous application). The resulting transformants were tested for growth on starch-containing media using iodine staining to reveal clearing zones. A total of 245 strains were able to produce the clear zone, indicating that they contained plasmids with genes encoding heterologous enzymes that were able to produce starch hydrolysis activity when expressed in yeast. These 245 strains were further screened for ethanol production as described below. The remaining genes encoded by the remaining 1528 plasmids were considered to have insufficient activity to warrant further testing.

[0150] Secondary Screening for Ethanol Production in a Deep Well Microtiter

[0151] Ethanol production was measured using deep well microtiter plates containing 0.5 mL of culture medium. The fermentation medium consisted of 725 g of partially hydrolyzed corn starch in the form of a liquefact, 150 g of filtered light soak water, 125 g of sterile water, 25 g of glucose, and 1 g of urea. The partially hydrolyzed corn starch was provided by a corn wet mill from Cargill (Eddyville, Iowa) (DS 30%-37%, DE 5-15). Light soak water was also provided by a corn wet mill from Cargill (Eddyville, Iowa) (free available nitrogen 2000 ppm-2500 ppm). The light soak water was centrifuged at 8,000 RPM, and the resulting supernatant was filter sterilized using a 0.2 micron filter to produce filtered light soak water. The strain was inoculated and the OD 600The pH was 0.1 and the plates were incubated at 30°C with shaking at 1000 rpm in an orbital shaker. At the end of the fermentation, samples were collected and analyzed by HPLC for the concentration of relevant metabolites.

[0152] Selected results from the 245 strain screens are shown in Table 5. Most strains did not show commercially relevant ethanol titers (e.g., Aspergillus kawachii, Aspergillus terreus, Thermomyces lanuginosus, and Mfa2-Neurospora crassa represent such strains). However, two strains (Rhizopus delbrueckii and Rhizopus microsporus) showed greater ethanol titers than commercially relevant strains known in the art (Mfα2-Rhizopus oryzae and Saccharomycopsis fibuligera).

[0153] Table 5

[0154]

[0155] Example 2

[0156] Construction of strain expressing MFalpha2-R. oryzae GA.

[0157] The generation of the ura3 auxotrophic basic strain was previously described in CAR0233P1 strain 1-3 (referred to herein as strain 1-1). Strain 1-1 was transformed with SEQ ID NO: 15 and SEQ ID NO: 16. SEQ ID NO: 15 contains the following elements: sequences homologous to integration locus A (3986 bp), ScTDH3 promoter (992 bp-1673 bp), Rhizopus oryzae glucoamylase with modified signal sequence (1680 bp-3476 bp), ScCYC1 terminator (3485 bp-3708 bp), loxP recombination site (3717 bp-3750 bp), ScURA3 promoter (3751 bp-4257 bp), upstream portion of ScURA3 (4258 bp-4861 bp). SEQ ID NO: 16 comprises the following elements: the downstream portion of ScURA3 (7 bp-606 bp), ScURA3 terminator (607 bp-927 bp), loxP recombination site (928 bp-961 bp), ScPGK1 promoter (968 bp-1554 bp), Rhizopus oryzae glucoamylase with modified signal sequence (1561-3357 bp), ScGAL10 terminator (3366 bp-3836 bp) and sequence homologous to integration locus A (3838 bp-4748 bp). Transformants were selected on synthetic complete medium lacking uracil (ScD-Ura). The resulting transformants were streaked on ScD-Ura for single colony isolation. Single colonies were selected. SEQ ID NO: 15 and SEQ ID NO: 16 were verified by PCR in single colonies for correct integration into one allele of integration locus A. The PCR-verified isolate was designated strain 1-2 (yNS220).

[0158] Strain 1-2 was transformed with SEQ ID NOs: 17, 18 and 19. SEQ ID NO: 17 contains the following elements: sequences homologous to integration locus A (3 bp-986 bp), ScTDH3 promoter (9921673 bp). SEQ ID NO: 18 contains the following elements: ScTDH3 promoter (6 bp-687 bp), Rhizopus oryzae glucoamylase with modified signal sequence (694 bp-2490 bp), ScCYC1 terminator (2499 bp-2722 bp), loxP recombination site (2731 bp-2674 bp), ScTEF1 promoter (2765 bp-3220 bp) and the upstream portion of Aspergillus nidulans acetamidase (3221 bp-4260 bp). SEQ ID NO: 19 comprises the following elements: the downstream portion of the Aspergillus nidulans acetamidase (7 bp-1032 bp), the ScADH1 terminator (1033 bp-1335 bp), the loxP recombination site (1336 bp-1369 bp), the ScPGK1 promoter (1376 bp-1962 bp), the Rhizopus oryzae glucoamylase with a modified signal sequence (1969 bp-3765 bp), the ScGAL10 terminator (3774 bp-4244 bp), and sequences homologous to integration locus A (4246 bp-5008 bp). Transformants were selected on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. The resulting transformants were streaked for single colony isolation on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. Single colonies were selected. Correct integration of SEQ ID NOs: 17, 18 and 19 into the second allele of locus A was verified by PCR in individual colonies. PCR-verified isolates were designated strains 1-3.

[0159] Example 3

[0160] Construction of strains expressing Rhizopus microsporus GA.

[0161] Strain 1-1 was transformed with SEQ ID NO: 20 and SEQ ID NO: 21. SEQ ID NO: 20 contains the following elements: sequences homologous to integration locus A (3 bp-986 bp), ScTDH3 promoter (992 bp-1673 bp), Rhizopus microsporus glucoamylase (1680 bp-3497 bp), ScCYC1 terminator (3506 bp-3729 bp), loxP recombination site (3738 bp-3771 bp), ScURA3 promoter (3772 bp-4278 bp), upstream portion of ScURA3 (4279 bp-4882 bp). SEQ ID NO: 21 contains the following elements: a portion of the ScURA3 promoter (11 bp-446 bp), ScURA3 (447 bp-1250 bp), the ScURA3 terminator (1251 bp-1570 bp), a loxP recombination site (1571 bp-1604 bp) and sequences homologous to integration locus A (1613 bp-1790 bp). Transformants were selected on synthetic complete medium lacking uracil (ScD-Ura). The resulting transformants were streaked on ScD-Ura for single colony isolation. Single colonies were selected. Correct integration of SEQ ID NO: 20 and SEQ ID NO: 21 into one allele of integration locus A was verified by PCR in single colonies. The PCR-verified isolates were designated strains 1-4.

[0162] Strains 1-4 were transformed with SEQ ID NO: 22 and SEQ ID NO: 23. SEQ ID NO: 22 contains the following elements: sequences homologous to integration locus A (1 bp-193 bp), ScTDH3 promoter (199 bp-880 bp), Rhizopus microsporus glucoamylase (887 bp-2704 bp), ScCYC1 terminator (2713 bp-2936 bp), loxP recombination site (2945 bp-2978 bp), ScTEF1 promoter (2979 bp-3434 bp), and the upstream portion of Aspergillus nidulans acetamidase (3435 bp-4474 bp). SEQ ID NO: 23 contains the following elements: the downstream portion of the Aspergillus nidulans acetamidase (1 bp-1498 bp), the ScTEF1 terminator (1499 bp-1658 bp), the loxP recombination site (1692 bp-1659 bp) and the sequence homologous to the integration locus A (1701 bp-1878 bp). Transformants were selected on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. The resulting transformants were streaked for single colony isolation on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. Single colonies were selected. SEQ ID NO: 22 and SEQ ID NO: 23 were verified by PCR in single colonies for correct integration into the second allele of locus A. The PCR-verified isolates were called strains 1-5.

[0163] Strain 1-1 was transformed with SEQ ID NO: 20 and SEQ ID NO: 24. SEQ ID NO: 24 contains the following elements: the downstream portion of ScURA3 (7 bp-606 bp), ScURA3 terminator (607 bp-927 bp), loxP recombination site (928 bp-961 bp), ScPGK1 promoter (9681554 bp), Rhizopus microsporus glucoamylase (1561 bp-3378 bp), ScGAL10 terminator (3387 bp-3857 bp) and sequence homologous to integration locus A (3859 bp-4823 bp). Transformants were selected on synthetic complete medium lacking uracil (ScD-Ura). The resulting transformants were streaked on ScD-Ura for single colony isolation. Single colonies were selected. Whether SEQ ID NO: 20 and SEQ ID NO: 24 were correctly integrated into one allele of integration locus A was verified by PCR in single colonies. PCR-confirmed isolates were designated strains 1-6.

[0164] Strains 1-6 were transformed with SEQ ID NO: 22 and SEQ ID NO: 25. SEQ ID NO: 25 contains the following elements: the downstream portion of the Aspergillus nidulans acetamidase (7 bp-1032 bp), the ScADH1 terminator (1033 bp-1335 bp), the loxP recombination site (1336 bp-1369 bp), the ScPGK1 promoter (1376 bp-1962 bp), the Rhizopus microsporus glucoamylase (1969 bp-3786 bp), the ScGAL10 terminator (3795 bp-4265 bp), and sequences homologous to integration locus A (4267 bp-4684 bp). Transformants were selected on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. The resulting transformants were streaked for single colony isolation on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. Single colonies were selected. SEQ ID NO: 22 and SEQ ID NO: 25 were correctly integrated into the second allele of locus A by PCR in single colonies. The isolates verified by PCR were called strains 1-7.

[0165] Example 4

[0166] Construction of strains expressing Rhizopus delbrueckii GA.

[0167] Strain 1-1 was transformed with SEQ ID NO: 26 and SEQ ID NO: 27. SEQ ID NO: 26 contains the following elements: sequences homologous to integration locus A (3 bp-986 bp), ScTDH3 promoter (992 bp-1673 bp), Rhizopus delbrueckii glucoamylase (1698 bp-3494 bp), ScCYC1 terminator (3503 bp-3726 bp), loxP recombination site (3735 bp-3768 bp), ScURA3 promoter (3769 bp-4275 bp), upstream portion of ScURA3 (4276 bp-4879 bp). SEQ ID NO: 27 contains the following elements: the downstream portion of ScURA3 (7 bp-606 bp), ScURA3 terminator (607 bp-927 bp), loxP recombination site (928 bp-961 bp), ScPGK1 promoter (968 bp-1554 bp), Rhizopus degeni glucoamylase (1561 bp-3375 bp), ScGAL10 terminator (3384 bp-3854 bp) and sequences homologous to integration locus A (3856 bp-4820 bp). Transformants were selected on synthetic complete medium lacking uracil (ScD-Ura). The resulting transformants were streaked on ScDUra for single colony isolation. Single colonies were selected. SEQ ID NO: 26 and SEQ ID NO: 27 were correctly integrated into one allele of integration locus A by PCR in single colonies. The PCR-verified isolates were designated strains 1-8.

[0168] Strain 1-8 was transformed with SEQ ID NO: 28 and SEQ ID NO: 29. SEQ ID NO: 28 contains the following elements: sequences homologous to integration locus A (1 bp-986 bp), ScTDH3 promoter (992 bp-1673 bp), Rhizopus delbrueckii glucoamylase (1680 bp-3494 bp), ScCYC1 terminator (3503 bp-3726 bp), loxP recombination site (3735 bp-3768 bp), ScTEF1 promoter (3769 bp-4224 bp), and the upstream portion of Aspergillus nidulans acetamidase (4225 bp-5264 bp). SEQ ID NO: 29 comprises the following elements: the downstream portion of the Aspergillus nidulans acetamidase (7 bp-1032 bp), the ScADH1 terminator (1033 bp-1335 bp), the loxP recombination site (1336-1369 bp), the ScPGK1 promoter (1376 bp-1962 bp), the Rhizopus delbrueckii glucoamylase (1969 bp-3783 bp), the ScGAL10 terminator (3792 bp-4262 bp) and a sequence homologous to the integration locus A (4264 bp-5026 bp). Transformants were selected on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. The resulting transformants were streaked for single colony isolation on a yeast nitrogen base (without ammonium sulfate or amino acids) containing 20 g / L glucose and 1 g / L acetamide as the sole nitrogen source. Single colonies were selected. Correct integration of SEQ ID NO: 28 and SEQ ID NO: 29 into the second allele of locus A was verified by PCR in individual colonies. PCR-verified isolates were designated strains 1-9.

[0169] Example 5

[0170] The strains were characterized in 32% DS corn mash at 30°C (Tests #1 and #2).

[0171] Strains 1, 1-3, 1-5, 1-7, 1-9 were scraped onto YPD plates and incubated at 30°C until single colonies were visible (1-2 days). Cells from YPD plates were scraped into phosphate buffer at pH 7.0 and the optical density (OD600) was measured. Optical density was measured at 600nm wavelength and 1cm pathlength using a model Genesys20 spectrophotometer (Thermo Scientific). Shake flasks were inoculated with cell slurry to an initial OD600 of 0.1. Immediately prior to inoculation, the following materials were added to each flask: 50 grams of liquefied corn mash (32% DS, DE 30+ / -2) was added to a 250mL baffled shake flask sealed with an airlock containing 4ml sterilized canola oil, 190ul 500g / L filter-sterilized urea, and 2.5ul 100mg / ml filter-sterilized ampicillin.

[0172] 0.324 AGU / g DS (70 μl of 1:10 dilution) of glucoamylase (Spirizyme Fuel HS, Novozymes) was added to the flasks containing control strain 1, and 0 or 0.097 AGU / g DS (21 μl of 1:10 dilution) of glucoamylase (Spirizyme Fuel HS, Novozymes) were added to the flasks containing control strain 1 according to the "Test" TM , Novozymes) was added to the remaining flasks. Estimated Spirizyme Fuel HS TM With about 769AGU / g enzyme, however, the activity may vary 10% to 20% over time (i.e., the activity of the enzyme generally decreases over time). The replicate flasks of each strain were incubated at 30°C in an orbital shaker at 100rpm for about 48 hours. At 48 hours, 1 ml samples were collected and analyzed for the concentrations of ethanol and glucose in the fermentation broth by high performance liquid chromatography with a refractive index detector. Selected results are shown in Table 6.

[0173] Table 6

[0174]

[0175]

[0176] Example 6

[0177] Strains were characterized in 32% DS corn mash at 33.3°C (Tests #3 and #4).

[0178] Strains 1, 1-3, 1-5, 1-7, 1-9 were scraped onto YPD plates and incubated at 30°C until single colonies were visible (1-2 days). Cells from the YPD plates were scraped into phosphate buffer, pH 7.0, and the optical density (OD 600 The optical density was measured using a Genesys 20 spectrophotometer (Thermo Scientific) at a wavelength of 600 nm and a pathlength of 1 cm. The shake flask was inoculated with the cell slurry to an initial OD of 600 to 0.1. Immediately prior to inoculation, the following materials were added to each flask: 50 grams of liquefied corn mash was added to a 250 mL baffled shake flask sealed with an airlock containing 4 ml sterile canola oil, 190 ul 500 g / L filter-sterilized urea, and 2.5 ul 100 mg / ml filter-sterilized ampicillin. 0.324 AGU / g DS (70 μl 1:10 dilution) of glucoamylase (Spirizyme Fuel HS TM , Novozymes) was added to the flask containing the control strain 1, and glucoamylase (Spirizyme Fuel HS TM , Novozymes) was added to the remaining flasks. Estimated Spirizyme FuelHS TM With about 326AGU / g enzyme. The replicate flasks of each strain were incubated at 33.3°C in an orbital shaker at 100rpm for about 48 hours. At 48 hours, 1 ml samples were collected and analyzed for the concentrations of ethanol and glucose in the fermentation broth by high performance liquid chromatography with a refractive index detector. Selected results are shown in Table 7.

[0179] Table 7

[0180]

[0181]

[0182] Example 7

[0183] Strains were characterized in 34% DS corn mash at 33.3°C.

[0184] Strains 1, 1-3, 1-6, and 1-9 were scraped onto YPD plates and incubated at 30°C until single colonies were visible (1-2 days). Cells from the YPD plates were scraped into phosphate buffer, pH 7.0, and the optical density (OD 600The optical density was measured using a Genesys 20 spectrophotometer (Thermo Scientific) at a wavelength of 600 nm and a pathlength of 1 cm. The shake flask was inoculated with the cell slurry to an initial OD of 600 to 0.1. Immediately prior to inoculation, the following materials were added to each flask: 50 grams of liquefied corn mash was added to a 250 mL baffled shake flask sealed with an airlock containing 4 ml sterile canola oil, 190 ul 500 g / L filter-sterilized urea, and 2.5 ul 100 mg / ml filter-sterilized ampicillin. 0.324 AGU / g DS (70 μl of a 1:10 dilution) of glucoamylase (Spirizyme Fuel HS TM , Novozymes) was added to the flask containing control strain 1, and 0 or 0.032 AGU / g DS, 0.065 AGU / g DS, 0.097 AGU / g DS or 0.162 AGU / g DS (7 μl, 14 μl, 21 μl or 35 μl of 1:10 dilution) was added to the remaining flasks. Estimation of Spirizyme Fuel HS TM With about 326AGU / g enzyme. The replicate flasks of each strain were incubated at 33.3°C in an orbital shaker at 100rpm for about 48 hours. At different time intervals, the flasks were opened and the samples were analyzed by high performance liquid chromatography with a refractive index detector to obtain the concentrations of ethanol and glucose in the fermentation broth. The selected results are shown in Table 8.

[0185] Table 8

[0186]

[0187]

[0188] Example 8

[0189] The strains were characterized in 32% DS corn mash with a DE of 30 + / - 2 at a temperature of 30°C.

[0190] The strain was scraped onto a YPD plate (20 g / L yeast peptone, 10 g / L yeast extract, 20 g / L glucose and 20 g / L agar) and incubated at 30°C until single colonies were visible (1-2 days). Cells from the YPD plate were scraped into a pH 7.0 phosphate buffer to produce a cell slurry, and the optical density (OD 600 The optical density was measured using a Genesys 20 spectrophotometer (Thermo Scientific) at a wavelength of 600 nm and a pathlength of 1 cm. The shake flask was inoculated with the cell slurry to an initial OD of600 to 0.1. Immediately before inoculation, the following materials were added to each flask: 50 grams of liquefied corn mash (32% DS, DE 30) was added to a 250 mL baffled shake flask sealed with an airlock containing 4 ml sterilized canola oil, 190 ul 500 g / L filter-sterilized urea, and 2.5 ul 100 mg / ml filter-sterilized ampicillin. Before adding the fermentation medium and cells, the shake flask, airlock, and canola oil were weighed, and then these weights were subtracted from the total weight of the flask to obtain the starting medium volume. At various time points in the fermentation, the flask was removed and the weight was recorded. The mass loss (grams) at any time point can be calculated by subtracting the mass of T1 from the original mass of T0. The mass loss (grams) at any given time point can be converted to a mass loss percentage by dividing the mass loss at any given time point by the original starting mass. The mass loss percentage (starting mass of the fermentation medium) can be converted to g / kg ethanol by the following formula. Ethanol (g / kg) = (mass loss percentage + 0.0016) / 0.0009 / 1.042.

[0191] Exemplary embodiments

[0192] A. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1, wherein the yeast is capable of producing ethanol at a rate of 1 g / L*h or greater during a fermentation process.

[0193] B. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from (i) a feedstock containing glucose polymers having a DE of no greater than 50.

[0194] C. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from corn mash having a DE of 30+ / -2, wherein the fermentation medium comprises 32% corn by dry weight and a pH of 5.8, 35 ppm CaCl, 1900 ppm urea, 5 ppm ampicillin, wherein the starting yeast concentration is 0.1 (OD600), and the fermentation is carried out with stirring at 30°C for 48 hours.

[0195] D. The engineered yeast of any one of embodiments A to C, wherein the glucoamylase comprises a sequence having 85% or greater sequence identity to SEQ ID NO: 1.

[0196] E. The engineered yeast of embodiment D, wherein the glucoamylase comprises a sequence having 90% or greater sequence identity to SEQ ID NO: 1.

[0197] F. The engineered yeast of embodiment E, wherein the glucoamylase comprises a sequence having 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 1.

[0198] G. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO: 4, wherein the yeast is capable of producing ethanol at a rate of 1 g / L*h or greater during a fermentation process.

[0199] H. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO: 4, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from (i) a feedstock containing glucose polymers having a DE of no greater than 50.

[0200] I. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 4, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from corn mash having a DE of 30, wherein the corn mash is present in a fermentation medium having 32% by weight corn mash and a pH of 5.8, 35 ppm CaCl, 1900 ppm urea, 5 ppm ampicillin, wherein the starting yeast concentration is 0.1 (OD600), and the fermentation is carried out with stirring at 30° C. for 48 hours.

[0201] J. The engineered yeast of any one of embodiments G to I, wherein the glucoamylase comprises a sequence having 98% or greater sequence identity to SEQ ID NO: 4.

[0202] K. The engineered yeast of embodiment J, wherein the glucoamylase comprises a sequence having 99% or greater sequence identity to SEQ ID NO:4.

[0203] L. The engineered yeast according to any one of the above embodiments, wherein there are 2 to 8 copies of the exogenous nucleic acid in the cell.

[0204] M. The engineered yeast according to embodiment L, wherein 2 to 6 copies of the exogenous nucleic acid are present in the cell.

[0205] N. The engineered yeast according to embodiment M, wherein 4 copies of the exogenous nucleic acid are present in the cell.

[0206] O. An engineered yeast according to any one of the above embodiments, wherein the exogenous nucleic acid is controlled by a promoter selected from the group consisting of: a phosphoglycerate kinase (PGK) promoter nucleic acid sequence, a cytochrome c promoter (pCYC) nucleic acid sequence, a translation elongation factor promoter (pTEF) nucleic acid sequence, a 3-phosphoglyceraldehyde dehydrogenase promoter (pGPD / TDH3) nucleic acid sequence, a phosphoglycerate kinase promoter (PGK) nucleic acid sequence, and an alcohol dehydrogenase promoter (pADH) nucleic acid sequence.

[0207] P. The engineered yeast according to any one of the above embodiments, which is a species of the genus Saccharomyces.

[0208] Q. The engineered yeast according to embodiment P, which is Saccharomyces cerevisiae.

[0209] R. The engineered yeast according to any one of the above embodiments, which is tolerant to growth in a fermentation medium having an ethanol concentration greater than 90 g / L.

[0210] S. The engineered yeast according to any one of the above embodiments, which is tolerant to growth at a temperature in the range of greater than 31°C to 35°C.

[0211] T. The engineered yeast according to embodiment S, which is tolerant to growth at a temperature in the range of greater than 32°C to 34°C.

[0212] U. The engineered yeast according to any one of the above embodiments, which produces a greater amount of ethanol than a parent strain that does not contain the exogenous nucleic acid under the same fermentation conditions.

[0213] V. A fermentation method for producing a fermentation product, the method comprising the steps of: forming a fermentation medium from a feedstock containing glucose polymers; and fermenting the fermentation medium using an engineered yeast, the engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, the glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1, wherein the fermentation produces the bioproduct.

[0214] W. A fermentation method for producing a fermentation product, the method comprising the steps of: forming a fermentation medium from a feedstock containing glucose polymers; and fermenting the fermentation medium using an engineered yeast, the engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, the glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO: 4, wherein the fermentation produces the bioproduct.

[0215] X. The fermentation method according to embodiment V or W, wherein at the start of fermentation, the glucose polymer-containing feedstock or the fermentation medium has a DE of about 50 or less.

[0216] Y. The fermentation method according to embodiment V or W, wherein at the start of fermentation, the fermentation medium has a glucose concentration of about 30 g / kg or less.

[0217] Z. The fermentation method according to embodiment V or W, wherein the glucose polymer-containing feedstock comprises glucose polymers having a degree of polymerization of 4 or greater and present in an amount of 75 wt % or greater of the total fermentable carbohydrates in the feedstock.

[0218] AA. The fermentation process according to any one of embodiments V to Z, wherein the glucose polymer-containing feedstock is obtained from corn.

[0219] BB. The fermentation method of embodiment AA, wherein the glucose polymer-containing feedstock is obtained from corn mash.

[0220] CC. The fermentation method according to any one of embodiments V to BB, wherein the fermentation is carried out for a fermentation time of at least 30 hours.

[0221] DD. The fermentation method according to embodiment CC, wherein the fermentation is carried out for a fermentation time ranging from 30 hours to 100 hours.

[0222] EE. The fermentation method according to embodiment DD, wherein the fermentation is carried out for a fermentation time in the range of 40 hours to 60 hours.

[0223] FF. The fermentation process of any one of embodiments V to EE, wherein the fermentation is carried out at a temperature in the range of 31°C to 35°C for most or all of the fermentation period.

[0224] GG. The fermentation method of embodiment FF, wherein the fermentation is carried out at a temperature in the range of 32°C to 34°C for most or all of the fermentation period.

[0225] HH. The fermentation of any one of embodiments V to GG, wherein ethanol is produced in the culture medium at a concentration of 70 g / L or more.

[0226] II. The fermentation according to embodiment HH, wherein ethanol is produced in the culture medium at a concentration of 90 g / L or more.

[0227] JJ. A method according to embodiment II, wherein the fermentation provides ethanol in the range of 90 g / L to 150 g / L.

[0228] KK. A method according to embodiment JJ, wherein the fermentation provides ethanol in the range of 110 g / L to 150 g / L.

[0229] LL. The fermentation process of any one of embodiments V to KK, wherein at the end of the fermentation period, the fermentation medium has an amount of glucose no greater than 1.0 g / L.

[0230] MM. The fermentation method according to embodiment LL, wherein at the end of the fermentation period, the fermentation medium has an amount of glucose no greater than 0.8 g / L.

[0231] NN. The fermentation method of any one of embodiments V to MM, comprising adding a supplemental glucoamylase to the feedstock during the fermentation, or adding a supplemental glucoamylase to the culture medium.

[0232] OO. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1, wherein the yeast is capable of producing at least 70 g / kg of ethanol in the fermentation process of Example 8.

[0233] PP. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO: 4, wherein the yeast is capable of producing at least 70 g / kg of ethanol in the fermentation process of Example 8. Sequence Listing <110> Cargill Christopher Miller Brian Rush Brynne Stanton Joshua Dunn <120> Glucoamylase engineered yeast and fermentation method <130> N00570-WO-PCT <150> 62 / 636,716 <151> 2018-02-28 <160> 29 <170> PatentIn Version 3.5 <210> 1 <211> 605 <212> PRT <213> Rhizopus microsporus <400> 1 Met Lys Leu Met Asn Pro Ser Met Lys Ala Tyr Val Phe Phe Ile Leu 1 5 10 15 Ser Tyr Phe Ser Leu Leu Val Ser Ser Ala Ala Val Pro Thr Ser Ala 20 25 30 Ala Val Gln Val Glu Ser Tyr Asn Tyr Asp Gly Thr Thr Phe Ser Gly 35 40 45 Arg Ile Phe Val Lys Asn Ile Ala Tyr Ser Lys Val Val Thr Val Ile 50 55 60 Tyr Ser Asp Gly Ser Asp Asn Trp Asn Asn Asn Asn Asn Lys Val Ser 65 70 75 80 Ala Ala Tyr Ser Glu Ala Ile Ser Gly Ser Asn Tyr Glu Tyr Trp Thr 85 90 95 Phe Ser Ala Lys Leu Ser Gly Ile Lys Gln Phe Tyr Val Lys Tyr Glu 100 105 110 Val Ser Gly Ser Thr Tyr Tyr Asp Asn Asn Gly Thr Lys Asn Tyr Gln 115 120 125 Val Gln Ala Thr Ser Ala Thr Ser Thr Thr Ala Thr Ala Thr Thr Thr 130 135 140 Thr Ala Thr Gly Thr Thr Thr Thr Ser Thr Gly Pro Thr Ser Thr Ala 145 150 155 160 Ser Val Ser Phe Pro Thr Gly Asn Ser Thr Ile Ser Ser Trp Ile Lys 165 170 175 Asn Gln Glu Glu Ile Ser Arg Phe Ala Met Leu Arg Asn Ile Asn Pro 180 185 190 Pro Gly Ser Ala Thr Gly Phe Ile Ala Ala Ser Leu Ser Thr Ala Gly 195 200 205 Pro Asp Tyr Tyr Tyr Ser Trp Thr Arg Asp Ser Ala Leu Thr Ala Asn 210 215 220 Val Ile Ala Tyr Glu Tyr Asn Thr Thr Phe Thr Gly Asn Thr Thr Leu 225 230 235 240 Leu Lys Tyr Leu Lys Asp Tyr Val Thr Phe Ser Val Lys Ser Gln Ser 245 250 255 Val Ser Thr Val Cys Asn Cys Leu Gly Glu Pro Lys Phe Asn Ala Asp 260 265 270 Gly Ser Ser Phe Thr Gly Pro Trp Gly Arg Pro Gln Asn Asp Gly Pro 275 280 285 Ala Glu Arg Ala Val Thr Phe Met Leu Ile Ala Asp Ser Tyr Leu Thr 290 295 300 Gln Thr Lys Asp Ala Ser Tyr Val Thr Gly Thr Leu Lys Pro Ala Ile 305 310 315 320 Phe Lys Asp Leu Asp Tyr Val Val Ser Val Trp Ser Asn Gly Cys Tyr 325 330 335 Asp Leu Trp Glu Glu Val Asn Gly Val His Phe Tyr Thr Leu Met Val 340 345 350 Met Arg Lys Gly Leu Ile Leu Gly Ala Asp Phe Ala Ala Arg Asn Gly 355 360 365 Asp Ser Ser Arg Ala Ser Thr Tyr Lys Gln Thr Ala Ser Thr Met Glu 370 375 380 Ser Lys Ile Ser Ser Phe Trp Ser Asp Ser Asn Asn Tyr Val Gln Val 385 390 395 400 Ser Gln Ser Val Thr Ala Gly Val Ser Lys Lys Gly Leu Asp Val Ser 405 410 415 Thr Leu Leu Ala Ala Asn Ile Gly Ser Leu Pro Asp Gly Phe Phe Thr 420 425 430 Pro Gly Ser Glu Lys Ile Leu Ala Thr Ala Val Ala Leu Glu Asn Ala 435 440 445 Phe Ala Ser Leu Tyr Pro Ile Asn Ser Asn Leu Pro Ser Tyr Leu Gly 450 455 460 Asn Ser Ile Gly Arg Tyr Pro Glu Asp Thr Tyr Asn Gly Asn Gly Asn 465 470 475 480 Ser Gln Gly Asn Pro Trp Phe Leu Ala Val Asn Ala Tyr Ala Glu Leu 485 490 495 Tyr Tyr Arg Ala Ile Lys Glu Trp Ile Ser Asn Gly Lys Val Thr Val 500 505 510 Ser Asn Ile Ser Leu Pro Phe Phe Lys Lys Phe Asp Ser Ser Ala Thr 515 520 525 Ser Gly Lys Thr Tyr Thr Ala Gly Thr Ser Asp Phe Asn Asn Leu Ala 530 535 540 Gln Asn Ile Ala Leu Gly Ala Asp Arg Phe Leu Ser Thr Val Lys Phe 545 550 555 560 His Ala Tyr Thr Asn Gly Ser Leu Ser Glu Glu Tyr Asp Arg Ser Thr 565 570 575 Gly Met Ser Thr Gly Ala Arg Asp Leu Thr Trp Ser His Ala Ser Leu 580 585 590 Ile Thr Val Ala Tyr Ala Lys Ala Gly Ser Pro Ala Ala 595 600 605 <210> 2 <211> 1818 <212> DNA <213> Rhizopus microsporus <400> 2 atgaagctaa tgaacccatc tatgaaagct tatgtctttt tcatcttatc atacttctct 60 ctcctagttt catctgccgc agtgcctaca tctgctgccg tccaagtgga aagttacaat 120 tacgatggca ctaccttctc cggccgtatc ttcgtgaaaa acattgctta ctcaaaagtt 180 gttacagtga tttactccga tggttctgat aattggaaca acaataacaa caaagtttca 240 gcagcgtact ccgaagccat tagtggatct aactacgaat actggacttt ctccgcaaag 300 ttgtctggta ttaagcaatt ctacgtaaag tacgaagttt caggttcaac atactacgat 360 aacaatggta ctaaaaacta ccaggtacaa gccacaagcg ctacaagtac aactgccaca 420 gctacaacta caactgctac agggacaaca actacatcaa ccggtccaac atcaaccgca 480 tctgtctcct ttccaaccgg taacagtaca atatcatcat ggatcaaaaa ccaagaggaa 540 atctcaagat ttgcaatgtt gagaaacatc aatccaccag ggtcagcgac tggtttcatc 600 gccgcttcat tgtccaccgc tgggccagac tactattact cttggactag agactctgca 660 ttgacagcaa acgttatagc ttacgaatac aacacaacct tcactggtaa cactaccttg 720 ttgaagtatc ttaaagacta cgtcactttt agtgttaagt ctcaatctgt ttctaccgtc 780 tgtaactgtt tagtgaacc aaagtttaat gcagatggct catcatttac tggtccatgg 840 ggcagacctc aaaacgatgg accagcagaa agagcagtca cattcatgtt gatcgctgac 900 tcatacttga cacaaactaa ggatgcttca tacgtgactg gaacactcaa gccagccata 960 ttcaaagacc tggattatgt tgtttctgtg tggtctaatg gttgctacga tttgtgggag 1020 gaagttaatg gcgtacattt ctacacacta atggttatga gaaagggact aattcttggg 1080 gcagatttcg cagctagaaa tggtgattcc tcaagagcat ctacctacaa gcaaacagca 1140 tctacaatgg aatcaaagat cagctctttc tggtctgact ctaacaacta cgttcaagtt 1200 tcacaatctg tgactgctgg tgtaagtaaa aagggtttag atgtttctac tctgttagct 1260 gcaaacattg gttctttacc agatggcttc tttacaccag gatcagaaaa gattttggca 1320 actgccgttg ccttagagaa tgcattcgct tccctttacc ctattaactc taacttacct 1380 tcatatttgg gtaattcaat tggtagatat ccagaggaca catacaacgg aaacggggaat 1440 tcacagggca acccttggtt cttagccgta aacgcgtacg ccgagttata ctacagagcc 1500 attaaggaat ggatctcaaa tggtaaggta acagtctcta atatctctct tcctttcttt 1560 aaaaagttcg attctagcgc cactagcggc aagacctata cagccggaac aagtgatttc 1620 aataacctcg ctcagaacat agctcttgga gcagacagat ttctgtcaac tgttaaattt 1680 cacgcgtaca caaatggcag cttgtctgaa gagtacgatc gttccaccgg gatgagtact 1740 ggtgctagag atctaacctg gtcccatgca tctcttatca cagttgcata cgcaaaagct 1800 ggatctcctg ctgcgtaa 1818 <210> 3 <211> 1818 <212> DNA <213> Rhizopus microsporus <400> 3 atgaaactta tgaatccatc tatgaaggca tacgttttct ttatcttaag ctacttctct 60 ttactcgtta gctcagctgc ggtgccaacc tctgccgccg tacaagttga gtcatacaat 120 tatgacggta ccactttttc aggtagaata ttcgtcaaaa acattgccta ctcaaaggtc 180 gtaacagtta tctactccga tggatcagat aactggaaca ataacaacaa caaagttttct 240 gcagcttact cagaagcaat ttctgggtct aactacgaat actggacatt ctccgcaaag 300 ttatccggaa ttaaacagtt tttgtcaaa tacgaagtttt ctggttcaac atattacgac 360 aacaacggta ccaaaaacta ccaagtccaa gcaacctcag cgacatctac aacagctact 420 gcaaccacaa ctacagctac tggcacaaca actacttcta caggtccaac tagtactgca 480 tccgtatcat tccctaccgg taactcaaca atttcttcct ggataaaaaa tcaagaggaa 540 atcagccgtt ttgctatgtt gagaaatatc aatccacctg ggtctgccac agggttcata 600 gccgcatctc tgtccacagc cggcccagat tactattact cttggactag agattcagca 660 ctaacagcta atgtgatcgc ttacgaatac aacacaacat tcactggaaa caccaccctt 720 cttaagtact tgaaagatta cgttacattt tctgtcaaaa gccaatctgt atctaccgtt 780 tgtaactgtc tgggagaacc aaagttcaac gctgatggta gttcttttac aggtccatgg 840 ggcagaccac aaaacgacgg accagcagag agagctgtta cttttatgtt gattgctgac agctacttga ctcaaacta ggacgcatcc tacgttaccg gtacattaaa gccagcaatc ttcaaagatc ttgattacgt agtttctgtt tggtctaacg gttgctacga tttatgggaa gaggttaatg gtgttcattt ctatactctc atggtcatga gaaagggttt gatcttaggt gccgacttcg ctgctagaaa tggtgactct agtagctt caacctacaa gcaaactgca tcaacaatgg aatcaaagat cagttctttt tggtcagatt ctaacaacta cgtccaagtt tctcaatcag ttaccgccgg agtgtcaaaa aagggactag atgttagtac actattggcg gccaacattg gtagtctgcc tgatggcttt ttcactccag gctccgaaaa gatattggct acagcagtgg cgttagaaaa tgcattcgca tccttgtacc cattaactc taacctacct tcttacttgg gtaactcaat tggaagatat cctgaggata catacaacgg taatggcaac tctcagggga atccatggtt ccttgccgtc aacgcatacg cagaacttta ctacagagct attack ggattagtaa tggcaaggtg acagtatcca atatctcact acctttcttc aaaagtttg attctccgc cactctgga agacataca ctgctggtac atcagatttc 1620 aaacttgg ctcagaacat tgctttaggc gccgatagat tcctgtctac tgttaagttc 1680 cacgcataca ctaacgggag tctatcagaa gagtacgata gatctaccgg tatgagtact 1740 ggggctcgtg atttacatg gtcccatgct tcattgatca cagtggcgta cgcaaggcc 1800 ggtagtccctg cagcttag 1818 <210> 4 <211> 604 <212> PRT <213> Rhizopus delemar <400> 4 Met Gln Leu Phe Asn Leu Pro Leu Lys Val Ser Phe Phe Leu Val Leu 1 5 10 15 To be Tyr Phe To be Leu Leu To be Wing To be Ile Pro To be Wing 20 25 30 Ser Val Gln Leu Asp Ser Tyr Asn Tyr Asp Gly Ser Thr Phe Ser Gly 35 40 45 Lys Ile Tyr Val Lys Asn Ile Ala Tyr Ser Lys Lys Val Thr Val Ile 50 55 60 Tyr Ala Asp Gly Ser Asp Asn Trp Asn Asn Asn Gly Asn Thr Ile Ala 65 70 75 80 Ala Ser Tyr Ser Ala Pro Ile Ser Gly Ser Asn Tyr Glu Tyr Trp Thr 85 90 95 Phe Ser Ala Ser Ile Asn Gly Ile Lys Glu Phe Tyr Ile Lys Tyr Glu 100 105 110 Val Ser Gly Lys Thr Tyr Tyr Asp Asn Asn Asn Ser Ala Asn Tyr Gln 115 120 125 Val Ser Thr Ser Lys Pro Thr Thr Thr Thr Ala Thr Ala Thr Thr Thr 130 135 140 Thr Ala Pro Ser Thr Ser Thr Thr Thr Pro Pro Ser Ser Ser Glu Pro 145 150 155 160 Ala Thr Phe Pro Thr Gly Asn Ser Thr Ile Ser Ser Trp Ile Lys Lys 165 170 175 Gln Glu Gly Ile Ser Arg Phe Ala Met Leu Arg Asn Ile Asn Pro Pro 180 185 190 Gly Ser Ala Thr Gly Phe Ile Ala Ala Ser Leu Ser Thr Ala Gly Pro 195 200 205 Asp Tyr Tyr Tyr Ala Trp Thr Arg Asp Ala Ala Leu Thr Ser Asn Val 210 215 220 Ile Val Tyr Glu Tyr Asn Thr Thr Leu Ser Gly Asn Lys Thr Ile Leu 225 230 235 240 Asn Val Leu Lys Asp Tyr Val Thr Phe Ser Val Lys Thr Gln Ser Thr 245 250 255 Ser Thr Val Cys Asn Cys Leu Gly Glu Pro Lys Phe Asn Pro Asp Gly 260 265 270 Ser Gly Tyr Thr Gly Ala Trp Gly Arg Pro Gln Asn Asp Gly Pro Ala 275 280 285 Glu Arg Ala Thr Thr Phe Ile Leu Phe Ala Asp Ser Tyr Leu Thr Gln 290 295 300 Thr Lys Asp Ala Ser Tyr Val Thr Gly Thr Leu Lys Pro Ala Ile Phe 305 310 315 320 Lys Asp Leu Asp Tyr Val Val Asn Val Trp Ser Asn Gly Cys Phe Asp 325 330 335 Leu Trp Glu Glu Val Asn Gly Val His Phe Tyr Thr Leu Met Val Met 340 345 350 Arg Lys Gly Leu Leu Leu Gly Ala Asp Phe Ala Lys Arg Asn Gly Asp 355 360 365 Ser Thr Arg Ala Ser Thr Tyr Ser Ser Thr Ala Ser Thr Ile Ala Asn 370 375 380 Lys Ile Ser Ser Phe Trp Val Ser Ser Asn Asn Trp Ile Gln Val Ser 385 390 395 400 Gln Ser Val Thr Gly Gly Val Ser Lys Lys Gly Leu Asp Val Ser Thr 405 410 415 Leu Leu Ala Ala Asn Leu Gly Ser Val Asp Asp Gly Phe Phe Thr Pro 420 425 430 Gly Ser Glu Lys Ile Leu Ala Thr Ala Val Ala Val Glu Asp Ser Phe 435 440 445 Ala Ser Leu Tyr Pro Ile Asn Lys Asn Leu Pro Ser Tyr Leu Gly Asn 450 455 460 Ser Ile Gly Arg Tyr Pro Glu Asp Thr Tyr Asn Gly Asn Gly Asn Ser 465 470 475 480 Gln Gly Asn Pro Trp Phe Leu Ala Val Thr Gly Tyr Ala Glu Leu Tyr 485 490 495 Tyr Arg Ala Ile Lys Glu Trp Ile Gly Asn Gly Gly Val Thr Val Ser 500 505 510 Ser Ile Ser Leu Pro Phe Phe Lys Lys Phe Asp Ser Ser Ala Thr Ser 515 520 525 Gly Lys Lys Tyr Thr Val Gly Thr Ser Asp Phe Asn Asn Leu Ala Gln 530 535 540 Asn Ile Ala Leu Ala Ala Asp Arg Phe Leu Ser Thr Val Gln Leu His 545,550,555,560 Ala His Asn Asn Gly Ser Leu Ala Glu Glu Phe Asp Arg Thr Thr Gly 565,570,575 Leu Ser Thr Gly Ala Arg Asp Leu Thr Trp Ser His Ala Ser Leu Ile 580,585,590 Thr Ala Sir Tyr Ala Lys Ala Gly Ala Pro Ala Ala 595,600 <210> 5 <211> 1815 <212> DNA <213> Rhizopus delemar <400> 5 atgcagctgt tcaacttgcc attaaaggtt tcattctttt tggtcctatc atactttagt 60 ttgttggtgt cagccgcatc tattccatct tcagcatctg tacaattaga ctcctacaat 120 tacgacggct ctacattcag cggaaagatt tacgtgaaaa atattgcgta cagcaaaaaa 180 gtaactgtta tctatgccga cggatcagat aactggaaca acaatggaaa cactatcgct 240 gccagttact ctgcaccaat ttcaggttct aactacgaat attggacatt ctcagcctcc 300 atcaatggca ttaaggaatt ctacataaag tacgaagtttt ccggtaagac ttactacgat 360 420. aacaacaatt ctgcaaacta tcaagtatca acatcaaaac ctactaccac caccgccaca gctacaacta caactgcacc ttcaacatct accacaaccc caccatcttc tagcgaacca gctacattcc caactggcaa ttctactatt tctagttgga tcaaaaaaca agagggtatt tccagattcg caatgttgag aaacataaat ccaccaggat cagcaactgg attcatcgca gcttctttgt ccacagcggg gccagattac tactacgcat ggaccagaga tgctgctttg 660 acaagtaacg ttattgttta cgaacaat accactttgt ccggtaacaa gactattctt aacgtcctaa together tacattctct gttaagactc agtctacatc cacagtctgc aattgtttgg gtgaaccaaa gttcaaccca gatggctctg gatacacagg tgcctggggt cgtccacaaa acgatgggcc tgccgagaga gccactacat ttatcctatt tgctgactca 960. aaacaaaaga tgcatcctac gtgactgga cattaagcc tgcaatcttc aaagacctgg attacgttgt caacgtgtgg tctaacggct gtttcgatct atgggaagag gttaacggcg tgcacttcta cactctaatg gtcatgagaa agggtctgtt gttaggtgca gattttgcta agagaaacgg tgattctaca cgtgcttcta cctactcctc aacagcatca 1140 actattgcga acaagatttc ttcattttgg gtttcaagta ataactggat acaagtatct 1200 caaagcgtta cagggggtgt ctcaaaaaag ggtcttgatg tttctacatt actggctgct 1260 aatcttgggt ctgttgatga cggtttcttc acccctggtt ctgaaaagat cctcgctacc 1320 gccgtcgcgg ttgaggatag ttttgcttca ctctatccta taaacaaaaa ccttccttca 1380 tacttaggaa acagtatcgg tagataccca gaggatacat acaatggtaa tggcaattca 1440 cagggaaatc catggttcct tgctgttaca gggtacgcag aactttacta tagagctatt 1500 aaggaatgga tcggcaacgg cggtgtgaca gtttcctcaa tctcattgcc atttttcaaa 1560 aagtttgact ccagcgcgac atctggtaaa aagtatactg tggggacttc tgatttcaac 1620 aatttggctc aaaacattgc cttagctgcc gacagattct tatctaccgt acaactccat 1680 gcacataaca atggtagttt ggcagaggaa tttgatagaa ctacaggact ctctacaggt 1740 gcgagagatt taacttggtc acatgcaagt ttaattacag cctcttacgc aaaggctggt 1800 gctcctgctg cataa 1815 <210> 6 <211> 1815 <212> DNA <213> Rhizopus delemar <400> 6 atgcagttat tcaacttacc acttaaggta tctttctttc tagtcttatc ttacttttca 60 ttgttagtat cagctgcctc tataccaagt tcagcatccg tacaactaga ttcatacaat 120 tacgacggtt caacattctc aggaaagata tacgtgaaaa atattgctta cagcaaaaag 180 gttactgtga tttacgcaga tgggtcagac aactggaata acaatggaaa cacaattgct 240 gcttcctatt ctgcccctat ttctggatct aactacgaat actggacttt ttcagcgagt 300 ataaacggaa ttaaggaatt ctatatcaaa tatgaagtct ctggtaagac ctactacgat 360 aacaacact ccgcaaacta ccaagttagc acatcaaagc caaccacaac aactgctact 420 gcgacaacta caaccgcacc aagcacttct actacaacac ctcctagttc atctgagcca 480 gcaactttcc caactggtaa ttccactatt tcttcttgga tcaaaaaaca agagggtatc 540 tcaagattcg ccatgcttag aaattcaat cctccaggct ctgcaacagg attcattgca 600 gcatctttat caactgcggg gccagactac tactacgcct ggactagaga tgcagctttg 660 acatcaaatg tgattgttta tgaatacaac acaactttgt ccggtaacaa gacaatcttg 720 aacgtcttga aggattatgt gacattctct gtcaagactc aatctacatc aacgtttgt 780 aactgtctcg gcgaaccaaa gttcaaccct gatggtagtg gttacactgg tgcttggggt 840 agaccacaaa acgatggtcc agcagagaga gctacaactt tcatcttgtt tgctgactct 900 tacctaacac aaaccaagga tgcaagctac gttactggaa cactaaagcc tgcaatcttt 960 aaagacctgg actatgttgt aaacgtttgg tcaaatggct gcttcgatct atgggaggaa 1020 gtgaacggtg ttcacttcta cacattaatg gtcatgagaa agggactctt gcttggtgca 1080 gactttgcta agagaaacgg tgattctaca cgtgcctcca cttactcctc cacagcttca 1140 accattgcca acaaaatctc ttctttctgg gtcagctcaa ataactggat tcaagtttct 1200 caatcagtta ctggtggtgt ttctaaaaag ggcctggatg tgtcaacctt gcttgctgcc 1260 aatttgggca gtgttgatga cgggttcttc accccaggtt ctgaaaagat cctcgccacc 1320 gcagttgccg ttgaagattc atttgctagt ttatacccaa tcaacaaaaa tctaccatca 1380 taccttggaa attcaatcgg tagatatcca gaggatacat acaacggtaa tggaaactct 1440 cagggtaacc cttggtttct tgcagttaca gggtacgctg aactgtacta cagagcgatt 1500 aaggaatgga ttggtaatgg cggcgtaact gttagttcta tttctctacc tttcttcaaa 1560 aagttcgata gttctgcaac atctggtaaa aagtacacag tcggcacttc cgattttaac 1620 aatttagctc agaacatagc actggcagct gatcgtttct tgagtacagt ccaattgcat 1680 gcccataaca acggtagttt ggctgaagag tttgatagaa ccaccggttt atcaaccggc 1740 gccagagatt taacatggtc ccatgcgtct ttgataactg cttcttacgc caaggctggg 1800 gcaccagctg cctga 1815 <210> 7 <211> 1815 <212> DNA <213> Rhizopus delemar <400> 7 atgcagttat tcaacttacc acttaaggta tctttctttc tagtcttatc ttacttttca 60 ttgttagtat cagctgcctc tataccaagt tcagcatccg tacaactaga ttcatacaat 120 tacgacggtt cacattctc aggaagata tacgtgaaaa atattgctta cagcaaaaaag gttactgtga tttacgcaga tgggtcagac aactggata acaatggaa cacaattgct gcttcctatt ctgcccctat ttctggatct aactacgaat actggacttt ttcagcgagt ataacggaa ttaaggaatt ctatatcaaa tatgaagtct ctggtaagac ctactacgat aacaacaact ccgcaaacta ccaagttagc acatcaaagc caaccacaac aactgctact 480. gcgacaacta caaccgcacc aagcacttct actacaacac ctcctagttc atctgagcca gcaactttcc caactggtaa ttccactatt tcttcttgga tcaaaaaaca agagggtatc 540 tcaagattcg ccatgcttag aaatcaat cctccaggct ctgcaacagg attcattgca gcatctttat caactgcggg gccagactac tactacgcct ggactagaga tgcagctttg 660 720. acaatcaaatg tgattgttta tgaatacaac acaactttgt ccggtaacaa gacaatcttg aacgtcttga aggattatgt gacattctct gtcaagactc aatctacatc aacagtttgt aactgtctcg gcgaaccaaa gttcaaccct gatggtagtg gttacactgg tgcttggggt 840 agaccacaaa acgatggtcc agcagagaga gctacaactt tcatcttgtt tgctgactct 900 tacctaacac aaaccaagga tgcaagctac gttactggaa cactaaagcc tgcaatcttt 960 aaagacctgg actatgttgt aaacgtttgg tcaaatggct gcttcgatct atgggaggaa 1020 gtgaacggtg ttcacttcta cacattaatg gtcatgagaa agggactctt gcttggtgca 1080 gactttgcta agagaaacgg tgattctaca cgtgcctcca cttactcctc cacagcttca 1140 accattgcca acaaaatctc ttctttctgg gtcagctcaa ataactggat tcaagtttct 1200 caatcagtta ctggtggtgt ttctaaaaag ggcctggatg tgtcaacctt gcttgctgcc 1260 aatttgggca gtgttgatga cgggttcttc accccaggtt ctgaaaagat cctcgccacc 1320 gcagttgccg ttgaagattc atttgctagt ttatacccaa tcaacaaaaa tctaccatca 1380 taccttggaa attcaatcgg tagatatcca gaggatacat acaacggtaa tggaaactct 1440 cagggtaacc cttggtttct tgcagttaca gggtacgctg aactgtata cagagcgatt 1500 aaggaatgga ttggtaatgg cggcgtaact gttagttcta tttctctacc tttcttcaaa 1560 aagttcgata gttctgcaac atctggtaaa aagtacacag tcggcacttc cgattttaac 1620 aatttagctc agaacatagc actggcagct gatcgtttct tgagtacagt ccaattgcat 1680 gcccataaca acggtagttt ggctgaagag tttgatagaa ccaccggttt atcaaccggc 1740 gccagagatt taacatggtc ccatgcgtct ttgataactg cttcttacgc caaggctggg 1800 gcaccagctg cctga 1815 <210> 8 <211> 1818 <212> DNA <213> Rhizopus microsporus <400> 8 atgaagctga tgaatcctag catgaaagca tatgtgtttt tcatcctctc atacttcagc 60 ttgctagtta gttcggccgc agtacccacg tcggccgcgg tccaagtgga gtcgtataat 120 tatgacggaa ctaccttctc agggcgtata ttcgtcaaga atattgcgta ttccaaagtc 180 gtcacagtaa tctattcaga tggctccgat aattggaata ataataacaa caaggttagc 240 gccgcctatt ccgaggctat ttctggatca aactacgaat actggacctt ctcggcgaag 300 ctgtcaggga tcaaacagtt ttacgtcaag tacgaggtca gtggttcaac ttactacgat 360 aataacggaa ccaaaaatta tcaggtccag gcgacgagcg caacgtccac aactgcgaca 420 gccactacca ctactgctac cgggactacg acaacctcga caggaccgac gtccaccgcc 480 tctgtatcct ttcccaccgg aaacagcaca atttcgagct ggataaaaaa ccaagaggaa 540 atctctcgct tcgctatgtt acgtaacatc aatccccccg gttcggcaac tgggtttatt 600 gccgcatcat tatctacagc cgggcccgat tactattata gctggactcg cgacagtgca 660 ctcacggcta atgtcatcgc atatgaatac aacacgactt tcaccggcaa tacgaccctt 720 cttaagtatt tgaaagatta tgtaactttc tcagttaaaa gtcaatcggt tagtacggtc 780 tgtaattgtc tcggtgaacc gaagtttaac gctgacgggt cctcgttcac tggcccttgg 840 gggcgacctc agaacgacgg gcccgcggaa cgcgccgtta cgttcatgct cattgcagac 900 tcatatctca cccagactaa agacgcgtct tatgtcactg ggactttaaa accagctata 960 tttaaagatc tggattacgt tgtttctgtt tggtcaaatg ggtgttatga cttatgggaa 1020 gaagtgaacg gcgttcattt ctacacttta atggtcatgc ggaagggcct cattttaggg 1080 gcggacttcg ccgcacgtaa tggagattca tcgcgcgcgt caacatataa acaaacggca 1140 tcaactatgg aaagtaaaat ttctagcttc tggtcggaca gtaacaacta cgttcaagtg 1200 tctcaatcgg taactgcggg ggtaagcaag aagggacttg acgtgagtac actgcttgcc 1260 gccaatattg ggagcctacc agacgggttt tttacacccg gctcggaaaa gattttagca 1320 acggctgttg ctcttgaaaa cgcttttgcc tcgctgtatc caatcaattc aaatttacca 1380 tcttatctgg gaaatagtat tggaaggtac cccgaggaca cctataatgg gaacggtaat 1440 tcccaaggta acccttggtt cttggctgtg aatgcctatg ccgagttgta ctatcgcgca 1500 attaaggaat ggataagtaa tggcaaagtg acggtttcaa acatctccct accttttttc 1560 aaaaattcg actccagtgc gacttctgga aaaactata cagcgggcac ctccgacttc 1620 aataatctcg cgcagaatat tgcactcgga gcagaccgat ttctttctac agtgaaattc 1680 catgcgtaca cgaacggtag cttgtcagaa gagtacgacc ggtctaccgg catgtcaacg 1740 ggagcacgag atcttacttg gtcccatgca agtttaatta ctgtggcata cgctaaagca 1800 gggtcgccag cggcataa 1818 <210> 9 <211> 1800 <212> DNA <213> Artificial Sequence <220> <223> Mfa2 - Rhizopus oryzae <400> 9 atgaaattca tttctacctt tctcactttt attttagcgg ccgtttctgt cactgctggt 60 gcctcgatcc ccagctcggc cagcgtccaa ctggactcgt ataattatga cggatctacc 120 ttctcaggga aaatatacgt caagaatatt gcgtattcca aaaaagtcac agtagtctat 180 gccgatggct ccgataattg gaataataat ggaaacacaa ttgcggcctc attttccggg 240 cctatttctg gatcaaacta cgaatactgg accttctcgg cgtcggtgaa agggatcaaa 300 gagttttaca ttaagtacga ggtcagtggt aaaacttact acgataataa caactcagct 360 aattatcagg tcagcacgag caagccgact acaactacgg cagccactac cactactact 420 gccccgtcta cgagtaccac aacacggccg agttcctcag aacctgcaac ttttcccacc 480 ggaaacagca caatttcgag ctggataaaa aagcaagagg atatctctcg cttcgctatg 540 ttacgtaaca tcaatccccc cggttcggca actgggttta ttgccgcatc attatctaca 600 gccgggcccg attactatta tgcgtggact cgcgacgcgg cactcacgtc taatgtcatc 660 gtatatgaat acaacacgac tctatcaggc aataagacca tccttaacgt tttgaaagat 720 tatgtaactt tctcagttaa aacgcaatcg acttcgaccg tctgtaattg cttgggcgag 780 ccaaaattca atccggacgg ttcgggatat accggtgctt ggggccgtcc gcagaacgac 840 ggcccggccg aacgagctac gacgttcgta ttattcggg attcatactt gacgcaaact 900 areacgctt cctatgttac gggtacttta aagcctgcaa tttttaaaga ccttgactat 960 gttgttaatg tctggtcaaa cggttgtttc gatttgtggg aagaggtgaa tggtgttcac 1020 ttctatactc tgatggttat gcggaaagga ttattactag gggcagactt tgcaaaacga 1080 aacggtgatt cgaccagagc ctcgacttat tctagcacag cctccacgat cgcaaataaa 1140 attagcagtt tctgggtaag tagtaacaac tgggtccagg tttctcagtc ggttacgggc 1200 ggcgtaagga agaggggct tgatgtctca accctactgg cggcaaacct gggttccgta 1260 gatgatggat tttcactcc tggaagcgaa aaaattcttg ccacagctgt tgccgtagag 1320 gattcgtttg cttcattata tccaattaat aagaacttac cgtcttacct gggaaatgca 1380 ataggccgat atccggagga tacctataat ggtaacggta atagccaagg taacccttgg 1440 tttcttgcgg tcacaggtta tgcagaactg tattacagag cgatcaaaga atggatatct 1500 aatggaggag ttacagtttc atcgatttct ctccctttct tcaaaaagtt tgactcctca 1560 gctacaagtg gcaaaaata cacggtcggt acaagcgact ttaataacct cgcacagaac 1620 atcgcccttg ctgccgatag gttcctgagc actgtgcaac ttcatgcacc gaacaacggt 1680 tcattggcag aagagttcga ccgaactacc ggtttctcca ctggagcgcg tgatttaacg 1740 tggagtcatg cttcgctgat cacagcgtcg tatgctaagg caggcgcccc ggctgcctaa 1800 <210> 10 <211> 1548 <212> DNA <213> Saccharomycopsis fitubogera <400> 10 atgataaggc tcactgtttt cctcaccgca gtcttcgctg cggtcgcatc atgcgtgccg 60 gtcgagttgg acaagcgaaa cacaggccac ttccaagcgt attctgggta cacagttgcc 120 cgttcgaact ttacccagtg gatacatgag cagcccgcag tttcttggta ttatttactt 180 caaaacatcg attatcccga aggccaattt aaatctgcta aacctggagt agtagttgcg 240 tcaccctcaa ccagtgaacc cgattacttt taccagtgga cgagagatac agcaataaca 300 ttccttagtc ttattgcaga agtggaagac cacagttttt cgaacactac cttagcgaag 360 gttgttgagt actatatttc caatacatat acactacaga gggtaagcaa tccatctggg 420 aactttgatt cacctaatca tgatgggtta ggggagccca agttcaacgt ggacgatacc 480 gcctatacag cttcatgggg ccggccacag aacgatggac cggcactacg tgcgtatgcg 540 atttcgcgtt atctcaatgc cgttgctaag cataataatg gcaaactcct attagcgggc 600 caaaatggaa tcccttatag ttcagcgtca gatatatatt ggaagatcat taaaccggac 660 ctacagcacg tttctaccca ctggtccact tcggggttcg atctatggga agaaaaccaa 720 ggcacccact tttttactgc cttagtacaa ctaaaagctc ttagttatgg aatccctctc 780 tccaaaacgt acaatgaccc tgggttcact tcgtggttgg aaaaacaaaa ggatgctcta 840 aacagctaca ttaactcatc cggctttgtg aactcgggaa aaaagcacat cgtcgagtcc 900 ccgcaattaa gtagccgtgg tggcttagac tctgctactt acattgcagc tttgattacg 960 catgacattg gtgatgatga tacctatacg ccttttaacg ttgataattc atacgtgctt 1020 aattctctat actatttact ggtcgataac aagaatcggt ataaaataaa cggaaactat 1080 aaggctggcg cagctgtcgg acgctacccc gaggatgtct ataacggagt cggtacgtca 1140 gagggcaatc cttggcagct agcaacagcg tacgcaggac aaaccttcta tactctcgca 1200 tataacagtc taaagaacaa gaagaatctc gtgataagaga agcttaatta tgatttgtac 1260 aacagtttca tagcggattt gtcgaaaatc gactcctcgt atgcttcgaa ggattcttta 1320 acccttacat atggttcaga caactataag aatgttatca aatctctatt acaattcgga 1380 gatagttttc tcaaggtatt gctagaccat atcgatgaca acggtcaatt gaccgaagag 1440 attaataggt atactgggtt tcaagctggc gcggtcagtt tgacgtggag tagtgggtca 1500 ttgttaagtg cgaatcgagc acggaataag ttaatcgagt tattataa 1548 <210> 11 <211> 1920 <212> DNA <213> Aspergillus kawachii <400> 11 atgagcttcc ggtccttgct ggcattgagt ggcctagttt gctcaggact tgcgagtgtg 60 attagtaaaa gagcaaccct cgactcctgg ctctccaacg aagctacggt agcacgtacc 120 gctatcctaa ataatatcgg cgcggatgga gcgtgggtat ctggtgcgga ttccggtatc 180 gtcgtagcat caccttcaac tgataatccc gattattttt atacttggac acgggatagc 240 ggccttgtca ttaagacttt ggttgacctt ttcagaaatg gggacaccga cctcctgtcg 300 acgatcgagc attatatatc aagccaagcg ataatacaag gagtatctaa tcctagcggg 360 gatttgtcga gtggcggttt gggcgagccc aagttcaacg tggacgagac tgcatatacg 420 ggatcatggg gccgtcctca acgagatgga ccagcgctac gcgccaccgc tatgattggc 480 tttggccagt ggctgctaga taacgggtac acatctgcgg ctacggaaat cgtttggcca 540 ttggtccgta atgacctttc atatgtcgct cagtactgga accaaactgg ctacgattta 600 tgggaggaag tgaatggtag ctcatttttc accattgccg tgcagcaccg ggcgttagtt 660 gaaggatcgg cctttgcaac ggcggttgga tcatcctgta gctggtgtga tagccaagcc 720 cctcaaattc tatgttacct tcaatcattc tggacaggta gttacatcct tgctaatttc 780 gattccagtc ggtccgggaa agacacgaat acactcttgg gctccattca cacctttgac 840 ccggaggcgg gctgcgatga ttcaactttc cagccatgct ctccaagagc ccttgcaaat 900 cacaaagaag tggtggattc ctttcgtagc atttatactc tcaatgacgg tttgtccgac 960 tcagaggctg ttgctgttgg ccgatatccg gaagatagct attataatgg caacccgtgg 1020 tttttatgca cattagctgc ggccgaacag ctctatgacg ctttatacca gtgggacaaa 1080 cagggttcac tagaaatcac agatgtgtca ctcgattttt tcaaggcctt gtactcagga 1140 gccgctactg ggacatactc gtcgagtagt tcgacatatt caagcattgt ttcggcagtt 1200 aagacgttcg cggacgggtt cgtcagcata gtagagacac atgccgccag taacgggtcg 1260 ctaagcgaac aattcgataa atcggatgga gatgaactca gcgctcgtga tcttacctgg 1320 tcttatgccg cactgttaac agccaataat cgtcgtaact ctgttgtgcc acctagttgg 1380 ggagagacat ccgcaagctc agtccccggg acgtgtgccg ccacctccgc atccggtacg 1440 tattccagtg ttaccgttac cagctggcca tctattgtag ctacgggtgg aacaacgacc 1500 accgctacga cgactggcag tggaggtgta acttcaacat ctaaaacaac gaccacagcg 1560 tctaagactt ctaccactac aagttcaacc tcctgcacga cccctacagc ggtggccgtt 1620 acattcgacc tcacggccac caccacttac ggggaaaaca tttacctagt ggggtctata 1680 tcccaattag gagactggga gacttcagat ggaatcgcgc tatcggccga taaatatacg 1740 tcctctaacc cgttatggta tgtgacagta actttaccag cgggagagag cttcgaatat 1800 aagttcatcc gagtggaatc agatgatagc gtggagtggg agtcggaccc aaatcgggag 1860 tatactgtcc ctcaggcttg cggtgagtcc actgctaccg taacggatac atggcgctaa 1920 <210> 12 <211> 1911 <212> DNA <213> Aspergillus terreus <400> 12 atgacgagga tcttgactct ggcattgcac ggcctagctc tggtccagtc agtggtgggg 60 gccccgcaac tagcaccccg ggcaaccacg tcgttggacg cctggctggc atcggaaacc 120 actgtagcat tagatggaat cctggataac gtgggctcgt ctggtgcgta tgctaaatca 180 gccaagtcgg gcattgtcat tgcctccccc tccacttccg atcctgacta ctactatacg 240 tggacccgag atgcggcttt gactgtaaaa gcactaatcg acctatttcg gaacggggag 300 acatcattgc agaccgtgat tatggagtac atctcgtcgc aagcctatct gcaaactgta 360 agtaatcctt ccggctcgct ttcgacgggc ggtctggcag aacctaaata ttatgttgat 420 gagactgcgt acaccggaag ttgggggcgg cctcaacgag atggaccagc gctacgcgca 480 acggcaatga ttgactttgg gaattggtta attgacaatg gctattctac atatgcctca 540 tccattgtat ggcctatagt ccgtaatgac ctttcttatg tagcccagta ttggaaccaa 600 actgggtatg atttgtggga ggaggtaaac ggttcttcat tcttcaccat tgcagtgcag 660 catagagcct tggtcgaggg ctcaaccttc gcttccaaag tcggtgcatc ttgctcatgg 720 780. tgtgactctc aggctccaca ggtcctttgt tttctccaac gcttctggac tgggtcttac ataatggcaa actttggcgg tggtaggtca ggtaaggacg cgaacaccgt gcttggttca attcacacgt tcgaccctaa cgcgggctgt gacgatacga cttttcagcc gtgttccccc 900 cgtgcgttag ctaatcacaa agtatacc gatagctttc ggtccattta ttccattaat tcagggattt ccagcggtaa agctgtcgcg gtggggccgct atccggagga ttcttactat 1020 aatggaaacc cctggttttt aacgacgctc gcggctgccg agcaactata tgacgctatc tatcagtggc aaaaaatagg cagtattact attack tgtcgctcgc gttcttcaag gatctttact cttctgcggc ggtagggaca tatgcaagta gtagtagcgc ctttacctca attgtgagcg cggtaaagac gtatgctgac ggctatatgt ccatcgttca aacacacgct atgactatg gctcgttatc cgaacagttc ggtaagtcag atggcttttc tctatctgct agggacttaa cgtggtcgta cgcagcccta ctgaccgcca atctgaggcg taattccgtc gttccacctt cctggggtga aacgactgcc acttcagtgc cctcggtttg ttcggctacg 1440 tctgcaacag ggacctattc tacggcgact aatacagcat ggccatctac cctcacttct 1500 ggaacgggcg caacgactac gacttctaaa gctacaagtt catcaactac gacgacctct 1560 tcggcgagct caactacagt agagtgcgtg gtccccaccg ctgtagcggt cacatttgac 1620 gaggtggcga ctacgactta tggtgagaac gtatacgttg tcggcagtat cagtcagctc 1680 ggctcttggg atacgtccaa agcagtggct ttatctgcgt cgaagtatac ttcgagcaac 1740 aacctgtggt atgttacggt aacccttccg gctggcacca ctttccagta caagttcatc 1800 cgggtatcat cgtcgggttc cgtcacttgg gaaagcgatc cgaatcgctc ttatacagtg 1860 ccctcggcct gtggtacgtc aacagctgtc gtcaatacca cttggcgcta a 1911 <210> 13 <211> 1854 <212> DNA <213> Thermomyces lanuginosus <400> 13 atgctgttcc agcctactct gtgcgccgca cttgggttag cggccctcat cgtgcagggc 60 ggcgaggcac gcccggagac tactgtgcca cacgctaccg gttcgctaga tgatttcctt 120 gcggctcaga gccccatcgc ttttcagggc attttaaata atataggacc ctccggcgct 180 tattccgaag gtgttaatcc cggtgtagta atcgctagcc cctcaaaaca agaccctgat 240 tatttctata catgggtcag ggacgccgca ctcacggtac agtatctagt cgaagagttg 300 gtggcgggaa atgcatcgtt acaatttctg attcaagact acatctcttc ccaagcacga 360 ctccagacgg tggagaaccc ttcgggtagc ctatcatccg gcggtttagg tgaaccgaaa 420 ttccacgtcg acgagacagc gttcactgat tcctggggac gcccccaacg agatgggcca 480 ccattaaggg ctatagctat gatttccttc gctaattatt taatagataa tggccatcaa 540 tcgacagttg aggatattat ctggcccatt gttagaaatg acctttctta cgtcagccaa 600 cattggaacg agacaacatt cgatatttgg gaggaggtcc attcgagtag ctttttcacc 660 accgccgtgc aatatcgagc tttggttcaa ggtagtgctc tagcctctaa actaggtcac 720 acttgcgaca attgtggttc tcaggctccc cagatcttgt gctttctaca gtcctactgg 780 actggctccc atatacttgc taacacgggt ggcggtagga gcggaaagga tgtaagtacc 840 atcctcggcg tgattggtag tttcgatcct aatgctgact gtgatgatgt aactttccaa 900 ccctgtagcg cccgggcttt agctaatcat aaacaagtgg ttgatagctt tcgctctatc 960 tacgcgatca atgctgggat cccttctggg tcagctgtag cagtcggacg ttatccggag 1020 gacgtgtatc agggtggcca cccttggtac ttgacgaccg cagctgccgc tgagcagctt 1080 tatgacgcca tatatcaatg gaaccacgtg ggacacatag acatcaacgc tgtcaactta 1140 gatttcttca aaagcatata cccgagtgcg gcagaaggaa cgtacacaag cgattcatca 1200 acctttcagg acataatcag tgcggttcgc acatatgccg acggtttctt gtctgtaata 1260 gaaaaatata ccccaccgga taacctttta gccgaacaat tccatcgtga gacaggtata 1320 cctctcagcg cagccagtct gacctggtca tacgcggctt tgaataccgc tgcacaacgc 1380 agggcttcca tagttccctc cccctggaat agtaatagca ctgacctacc ggataaatgt 1440 agtgcgactt cagcaacagg tccatatgcc acacctacta acacggcctg gccgactacg 1500 actcagcctc ccgaacggcc ggcctgtact ccgccgtcag aagtaacgct gacctttaac 1560 gcactcgtag acaccgcctt tggtcagaac atctacttgg tgggaagcat acctgagttg 1620 ggctcttggg acccagcaaa tgccttgcta atgagtgcaa agtcatggac ctctgggaat 1680 cctgtatgga ctttatctat atcgctacct gctggaacgt cgttcgagta caaatttata 1740 cgcaaagacg atggctcatc tgacgtggta tgggagtcgg acccaaatcg gtcgtataat 1800 gtccctaagg attgcggtgc gaatactgct accgtaaact cctggtggcg ctaa 1854 <210> 14 <211> 2045 <212> DNA <213> Artificial Sequence <220> <223> Mfa2 - Neurospora crassa <400> 14 atgaaattca tttctacctt tctcactttt attttagcgg ccgtttctgt cactgctgac 60 gcgatctata atctgaagca agtacttcta tctaaccatg actttacgac gagcttggtg 120 tccaacgcaa ccgacttctc cacgtcgccc ctgacctatt tcgcaatgca tttggtttcg 180 agtttacttg tagtcggggc tgcattccaa gcagttttag ggttgcctga tccccttcac 240 gagaaacgcc attctgatat tatcaaacgt tccgtcgatt cgtacatcca aacggaaacc 300 cccattgcgc aaaagaacct actttgcaac atcggagcaa gcggatgccg agcctccgga 360 gccgcgtccg gggtggtagt cgcatcgcct tcaaaaagca gtcctgatta ctggtatact 420 tggacccggg atgcagcgct agtgacaaag ttgatcgttg acgagtttac taatgattat 480 aataccacgt tacagaacac catccaggca tacgcggctg cccaagctaa gttgcaaggc 540 gtcagtaacc catccggaag cctcagtaat ggggctgggt taggagaacc caagtttatg 600 gttgatctac aacaatttac cggtgcatgg ggtaggcccc aacgagatgg gccccctctg 660 cgtgcgatcg ctttgatagg gtacggaaag tggttagtct cgaacggcta tgcagacacg 720 gctaagtcaa ttatttggcc catcgtcaag aatgatttag cctatacagc tcagtattgg 780 aataacactg gatttgacct atgggaagaa gttaactcct ccagtttctt tacgattgcg 840 gcgtcccacc gggctttggt tgaagggtca gctttcgcga agtccgtggg ctcctcatgt 900 agcgcgtgcg acgctatagc gccccaaatc ttgtgctttc aacagagctt ttggtcaaat 960 agcggttata ttatctccaa ctttgtcaat taccgatcgg gtaaagatat taattcagtc 1020 ctaacttcca tacataattt tgatccggcg gccggttgtg acgtaaatac tttccaacct 1080 tgctcggatc gggctttagc gaaccacaag gtcgtagttg atagtatgcg attctggggt 1140 gtgaactcag gtaggaccgc aggcaaagct gcggcagtgg gacggtacgc ggaggacgtt 1200 tattacaatg gtaacccatg gtacctagca acgctagcgg cagccgagca gctatacgac 1260 gccgtctatg tctggaagaa gcagggaagt ataactgtta catccacctc gttggcattt 1320 tttaaggact tagttccttc agtctcgact ggtacctaca gctctagttc ttctacatat 1380 actgcgatca ttaacgctgt aacgacatat gcagacggct ttgtcgacat agttgcccaa 1440 tatactccat ccgatgggtc cttggcggaa caatttgata aggattcggg tgccccattg 1500 agcgctacac acctaacctg gtcttacgcg tcttttctat cggctgctgc aaggcgcgca 1560 gggattgtgc ctccatcctg gggtgcagcg tcagccaata gtctgcccgg ttcgtgcagc 1620 gcctctacag tagcaggaag ttatgccacc gctacagcga cctcgtttcc ggcaaacctg 1680 actccggctt caactacagt gacaccacct acccaaacag gctgcgcagc agatcatgaa 1740 gttctggtta ctttcaacga aaaagttacg acaagttatg gtcagacggt aaaggtggta 1800 ggttctatag cagcccttgg gaattgggcc cctgcgagcg gagtgacgct ctcggcaaaa 1860 cagtactcct cctcaaatcc attatggtct acgaccattg ctttgcctca agggacctcg 1920 tttaaatata aatatgttgt cgttaatagt gatggatctg taaaatggga gaatgatccc 1980 gatcgcagct atgccgtagg aacagattgt gcttccacag ccactcttga tgatacttgg 2040 aggta 2045 <210> 15 <211> 4863 <212> DNA <213> Artificial Sequence <220> <223> Mfa2 - Rhizopus oryzae GA Integration Construct <400> 15 ctaaattcgg ccttgctcag agactcctgg attttggcta acaacgcagt cccttcgatg 60 catatagcta ggccacaaat tatgccaata acggtccatg ggttgatgtt ttcttgaatt 120 ctttcgtttt tcatgctatt tgcgtcttcc caagtcccag cgttccagta ttcatactgc 180 gcgttagagt ggtagccata agagccggca tattggtaat tttcagtatt aacgttagaa 240 cgtggtgaat acgatgtggt ccagccttgc ctcgttgtgt entaacgat ctttttcttt 300 gggtcacaaa gaatatcata tgcttgagag atgactttaa atctatgtag tttttcgctt 360 gatgttagca gcagcggtga tttactatca ctgttggtaa ccttttctga gctaaatatt 420 tgaatgttat cggaatggtc agggtggtac aattttacat aacgatgata tttttttttt 480 aacgacttct tgtccagttt aggatttcca gatccggcct ttggaatgcc aaaaatatca 540 tagggagttg gatctgccaa ctcaggccat tgttcatccc ttatcgtaag ttttctattg 600 ccatttttat cgttcgctgt agcatactta gctataaaag tgatttgtgg gggacacttt 660 tctacacatg ataagtgcca cttgaataaa aatgggtata cgaacttatg gtgtagcata 720 acaaatatat tgcaagtagt gacctatggt gtgtagatat acgtacagtt agttacgagc 780 ctaaagacac aacgtgtttg ttaattatac tgtcgctgta atatcttctc ttccattatc 840 accggtcatt ccttgcaggg gcggtagtac ccggagaccc tgaacttttc tttttttttt 900 tgcgaatta aaaagttcat tttcaattcg acaatgagat ctacaagcca ttgttttatg ttgatgagag ccagcttaaa gagttctcga gatctcccga gtttatcatt atcaatactg ccatttcaa gathering father gtagtgatt tcctaacttt father aaaattggcc ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 taatggagcc cgcttttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacacaga acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcttg gagtaatga tgacacaagg caattgacct acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaaagctga aaaaaaaggt tgaaaccagt tccctgaat tattccccta tttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt tagtttaaaa caccaagaac ttagtttcga ataaacacac ataaacaaac aaatctagaa 1680 tgaagttcat ttccactttc ttgaccttca ttttggctgc tgtctctgtc accgctgcat 1740 ctattccatc tagtgcatct gtacaattgg actcctacaa ttacgatggt tccacatttt 1800 ccggcaagat ttatgtcaaa aacatcgctt actctaaaaaa ggttactgtt gtgtacgcag 1860 acggttctga caactggaac aataacggca acactattgc tgcatcattt tcaggcccaa 1920 tctctggatc aaattacgaa tactggacat tctcagcatc agtgaagggc ataaaggagt 1980 tctacatcaa atacgaagtt tcaggtaaga catattacga caataacaac tctgcaaact 2040 accaagtctc aacttctaaa cctactacaa ctactgcagc tacaaccaca actacagctc 2100 catcaacttc tacaacaacc cgtccatcta gttcagagcc tgccaccttc cctactggta 2160 attctaccat cagctcttgg atcaaaaagc aggaagatat ttccagattc gctatgctta 2220 gaaaacatcaa cccacctggt tctgcccag ggtttatcgc cgcatcactc tctaccgctg 2280 gtccagatta ctactacgcg tggacaagag atgccgcttt gacatctaac gttatcgttt 2340 acgaatacaa caccacattg tctgggaata agacaattct aaacgtactt aaggattacg 2400 tcacattcag tgttaagaca cagtctactt caacagtttg taattgcctt ggtgaaccaa 2460 agttcaatcc agacggcagt ggttacacag gtgcttgggg tagacctcaa aatgatggtc 2520 ctgcagaaag agcgactaca tttgttctgt ttgccgacag ctacttgact caaactaagg 2580 atgcctcata cgtcactggt acattaaagc cagcaatttt caaagatctc gattacgttg 2640 ttaacgtctg gagtaacgga tgttcgatt tatgggagga ggtgaacgga gttcatttct 2700 acacccttat ggttatgaga aaagggctat tgttgggggc tgatttcgcg aagagaaacg 2760 gtgactcaac tagagcctca acttactctt ctactgcttc cacaattgct aacaagatat 2820 caagtttctg ggttagctca aaactggg tgcaagtatc ccaatctgtc acaggaggtg 2880 tagtaaaaa ggggttagac gttagcaccc tgttagctgc gaatctagga tcagtcgatg 2940 atggatttt cactccaggt tctgaaaaga tattagctac agctgtggca gtcgaagatt 3000 cctttgccag tctataccca atcaacaaaa accttccatc atacttgggg aacgctattg 3060 gaagataccc tgaagataca tacaacggta atggtaactc acaaggcaat ccttggtttc 3120 tggcggttac cggctacgca gagttgtact atagagcaat taaggaatgg atttctaatg 3180 gaggcgttac agtgtcctct atctcattgc catttttcaa aaagttcgat agctctgcaa 3240 catccggtaa aaagtacacc gtaggtactt ctgacttcaa caatttagca caaaacattg 3300 ctcttgctgc agatcgtttc ctatctactg tacaactcca tgcaccaaac aatggttcat 3360 tagcagagga atttgataga acaacaggtt tttctaccgg cgctagagat ttaacatggt 3420 cccacgcctc attgataaca gcatcctatg ccaaagccgg tgctccagct gcataattaa 3480 ttaaacaggc cccttttcct ttgtcgatat catgtaatta gttatgtcac gcttacattc 3540 acgccctcct cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta 3600 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat 3660 ttttcttttt tttctgtaca aacgcgtgta cgcatgtaac gggcagacgg ccggccataa 3720 cttcgtataa tgtatgctat acgaagttat ggcaacggtt catcatctca tggatctgca 3780 catgaacaaa caccagagtc aaacgacgtt gaaattgagg ctactgcgcc aattgatgac 3840 aatacagacg atgataacaa accgaagtta tctgatgtag aaaaggatta gagatgctaa 3900 gagatagtga tgatatttca taaataatgt aattctatat atgttaatta ccttttttgc 3960 gaggcatatt tatggtgaag gataagtttt gaccatcaaa gaaggttaat gtggctgtgg 4020 tttcagggtc cataaagctt ttcaattcat ctttttttt tttgttcttt ttttgattc 4080 cggtttcttt gaaattttt tgattcggta atctccgagc agaaggaaga acgaaggaag 4140 gagcacagac ttagattggt atatacgc atatgtggtg ttgaagaaac atgaaattgc 4200 ccagtattct taacccaact gcacagaaca aaaacctgca ggaaacgaag ataaatcatg 4260 tcgaaagcta catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta 4320 tttatatca tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc 4380 aaggaattac tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat 4440 gtggatatct tgactgattt ttccatggag ggcacagtta agccgctaaa ggcattatcc 4500 gccaagtaca attttttact cttcgaagac agaaaatttg ctgacattgg taatacagtc 4560 aaattgcagt actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca 4620 cacggtgtgg tgggcccagg tattgttagc ggtttgaagc aggcggcgga agaagtaaca 4680 aaggaaccta gaggcctttt gatgttagca gaattgtcat gcaagggctc cctagctact 4740 ggagaatata ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc 4800 tttattgctc aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca 4860 cgc 4863 <210> 16 <211> 4748 <212> DNA <213> Artificial Sequence <220> <223> Mfa2 - Rhizopus oryzae GA Integration Construct <400> 16 ggccgctcca tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt 60 ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct 120 gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc 180 ccaggtattg ttagcggttt gaagcaggcg gcggagaag taacaagga acctagaggc 240 cttttgatgt tagcagaatt gtcatgcaag ggctccctag ctactggaga atactaag 300 ggtactgttg acatgcgaa gagcgacaa gattttgtta tcggctttat tgctcaaga 360 gatagggtg gaagagatga aggttacgat tggttgatta tgacacccgg tgtggtttta 420 gatgacaagg gagacgcatt gggtcacag tatagaaccg tggatgt ggtctctaca 480 ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaggta 540 gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagagatg cggccagcaa 600 aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaat tagagcttca 660 atttaattat atcagttatt acccgggaat ctcggtcgta atgattttta taatgacgaa 720 aaaaaaaaaaaaaaaggaaaaagctcat gggcttata aaaaggaacc atccaatacc 780 tcgccagaac caagcacag tattttacgg ggcacaaatc aagaacaata agacaggact 840 gtaaagatgg acgcattgaa ctccaaagaa siacaaggt tccaaaagt agtgaacaa 900 aagcaaatga aggatttcat gcgtttgata acttcgtata atgtatgcta tacgaagtta 960 tctcgagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt accctcataa 1020 agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt tgcaaaaaga 1080 acaaaactga aaaaacccag acacgctcga cttcctgtct tcctgttgat tgcagcttcc 1140 aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca agcaatcgaa 1200 ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac tgtgatctcc 1260 agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa ttgtccgaat 1320 cgtgtgacaa caacagcctg ttctcacaca ctctttttctt ctaaccaagg gggtggttta 1380 gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat aaattggtca 1440 atgcaagaaa tacatatttg gtcttttcta attcgtagtt tttcaagttc ttagatgctt 1500 tctttttctc ttttttacag atcatcaagg aagtaattat ctacttttta caagtctaga 1560 atgaagttta tctccacgtt tttaaccttt atcctagcag ctgtcagcgt caccgccgca 1620 1680 tcagggaaaa tttatgtgaa aacatagca tatagtaaga aggttaccgt ggtatatgca 1740 gacggttctg atattggaa taataatgga aacactattg ccgccagttt ttccggccca 1800 atttctggtt catcattacga gtattggacc ttttctgt cagtaaagg catcaggaa 1860 ttctatatta agtacgaagt ttcaggtaag acatattacg atacataa ctcagcaat 1920 tatcaagtct ctacatctaa gccacaaca acaactgctg ctaccaccac tacaccgct 1980 ccttctacca gcaccactac cagaccaagc tctagtgac cggctacctt tcctaccgga 2040 aacagtacca tctcaagctg gatcaaaag caaggaca taagtcgtttt tgctatgttg 2100 aggaacatta atcctccagg atccgcgacc ggtttcattg cagcatcact aagtactgcc 2160 gggcctgatt attattatgc ttggactga gacgctgcat taacatcaa cgtgattgtt 2220 tatgaatata atacgaccct ttccggtaat aaacgatct tgaacgtatt aaagactat 2280 gtgaccttta gtgtgaagac ccaatctaca tctacagtgt gtattgttt gggagaacct 2340 aaattcaatc cagacggttc tgggtacact ggtgcctggg gtagacctca aaacgacggt 2400 ccagcagaaa gagcaacaac ctttgttcta tttgctgact cttatttaac gcaaacaaag 2460 gacgcctcat atgttacagg gaccctaaaa ccagcaattt tcaaagactt ggattatgtt 2520 gttaatgttt ggagcaacgg atgttttgac ttgtgggagg aggttaacgg tgtacacttt 2580 tatacattga tggtgatgag aaaagggttg ctattgggag cagatttcgc taaaagaaat 2640 ggtgattcta caagagcgag cacatatagt agcaccgctt caacaatcgc caataaaatc 2700 tcatctttct gggtatctag caacaactgg gtacaagtttt cccaaagtgt taccggcggt 2760 gtgtccaaaa agggtttaga cgttagcaca cttctagctg ctaatttggg tagcgttgat 2820 gacgggtttt ttactccagg tagtgagaag atactggcaa ccgcggtggc ggttgaagac 2880 agctttgctt cattgtatcc tataaataaa aatctgccct cttatctggg taatgcaatt 2940 ggcagatacc cagaagatac ctacaatggt aatggtaatt cccaggggaa cccatggtttt 3000 ttggctgtta caggctacgc agaactttat taccgtgcaa tcaaggaatg gatttcaaat 3060 ggcggcgtca ctgtcagtag tataagtttg ccctttttta agaaatttga ttcctcagca 3120 acgtctggta aaaaatacac cgtaggtact agtgatttca ataatttggc ccaaaatatt 3180 gcgcttgctg ctgacaggtt tcttagtacc gttcagttgc acgctccaaa taatggctca 3240 ttggctgaag aatttgatcg tacgacaggt ttctccactg gtgctaggga tttgacttgg 3300 agtcatgcct cttaatcac agcaagctat gctaaagctg gtgcacctgc tgcttagtta 3360 attaatttac cagcttacta tccttcttga aaatatgcac tctatatctt ttagttctta 3420 attgcaacac atagatttgc tgtataacga attttatgct atttttttaa tttggagttc 3480 ggtgatgaaa gtgtcacagc gaatttcctc acatgtaggg accgaattgt ttacaagttc 3540 tctgtaccac catggagaca tcaaagattg aaaatctatg gaaagatatg gacggtagca 3600 acaagaatat agcacgagcc gcggagttca ttcgttact tttgatatcg ctcacaacta 3660 ttgcgaagcg cttcagtgaa aaaatcataa ggaaaagttg taaatattat tggtagtatt 3720 cgtttggtaa agtagagggg gtaatttttc ccctttattt tgttcataca ttcttaaatt 3780 gctttgcctc tccttttgga aagctatact tcggagcact gttgagcgaa ggctcaggcc 3840 ggcagcacgc agcacgctgt atttacgtat ttaattttat atatttgtgc atacactact 3900 agggaagact tgaaaaaaac ctaggaaatg aaaaaacgac acaggaagtc ccgtatttac 3960 tattttttcc ttccttttga tggggcaggg cggaaataga ggataggata agcctactgc 4020 ttgctgttt ccgtctctac ttcggtagtt gtctcaattg tcgtttcagt attaccttta 4080 gagccgctag acgatggttg agctatttgt tgagggaaaa ctaagttcat gtaacacacg 4140 cataacccga ttaaactcat gaatagcttg attgcaggag gctggtccat tggagatggt 4200 gccttatttt ccttataggc aacgatgatg tcttcgtcgg tgttcaggta gtagtgtaca 4260 ctctgaatca gggagaacca ggcaatgaac ttgttcctca agaaatagc ggccataggc 4320 atggattggt taaccacacc agatatgctt ggtgtggcag aatatagtcc ttttggtggc 4380 gcaatttct tgtacctgtg gtagaaaggg agcggttgaa ctgttagtat atattggcaa 4440 tatcagcaaa tttgaaagaa aattgcggt gaaaaacata cgaaacacaa aggtcgggcc 4500 ttgcaacgtt attcaaagtc attgtttagt tgaggaggta gcagcggagt atatgtattc 4560 cttttttttg cctatggatg ttgtaccatg cccattctgc tcaagctttt gttaaaatta 4620 tttttcagta ttttttcttc catgttgcgc gttacgagaa cagaagcgac agataaccgc 4680 aatcatacaa ctagcgctac tgcggggtgt aaaaagcaca agaactaagc caagatcaca 4740 acagttat 4748 <210> 17 <211> 1674 <212> DNA <213> Artificial Sequence <220> <223> Integration Construct <400> 17 ctaaattcgg ccttgctcag agactcctgg attttggcta acaacgcagt cccttcgatg 60 catatagcta ggccacaaat tatgccaata acggtccatg ggttgatgtt ttcttgaatt 120 ctttcgtttt tcatgctatt tgcgtcttcc caagtcccag cgttccagta ttcatactgc 180 gcgttagagt ggtagccata agagccggca tattggtaat tttcagtatt aacgttagaa 240 cgtggtgaat acgatgtggt ccagccttgc ctcgttgtgt catatacgat ctttttcttt 300 gggtcacaaa gaatatcata tgcttgagag atgactttaa atctatgtag tttttcgctt 360 420. gatgttagca gcagcggtga tttactatca ctgttggtaa ccttttctga gctaaatatt tgaatgttat cggaatggtc agggtggtac aattttacat aacgatgata tttttttttt aacgacttct tgtccagttt aggatttcca gatccggcct ttggaatgcc aaaaatatca 540 tagggagttg gatctgccaa ctcaggccat tgttcatccc ttatcgtaag ttttctattg ccatttttat cgttcgctgt agcatactta gctaaaag tgatttgtgg gggacacttt 660 720. tctacacatg ataagtgcca cttgaataaa aatgggtata cgaacttatg gtgtagcata acaaatat tgcaagtagt gacctatggt gtgtagatat acgtacagtt agttacgagc 780 ctaaagacac aacgtgtttg ttaattatac tgtcgctgta atatcttctc ttccattatc accggtcatt ccttgcaggg gcggtagtac ccggagaccc tgaacttttc tttttttttt 900 tgcgaatta aaaagttcat tttcaattcg acaatgagat ctacaagcca ttgttttatg ttgatgagag ccagcttaaa gagttctcga gatctcccga gtttatcatt atcaatactg ccatttcaa gathering father gtagtgatt tcctaacttt father aaaattggcc ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca 1140 cagaatatat aacatcatag gtgtctgggt gaacagttta ttcctggcat ccactaaata 1200 taatggagcc cgcttttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa 1260 tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacacaga 1320 acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcttg 1380 gagtaaatga tgacacaagg caattgacct acgcatgtat ctatctcatt ttcttacacc 1440 ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt 1500 tccctgaaat tattccccta tttgactaat aagtatataa agacggtagg tattgattgt 1560 aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt 1620 tagtttaaaa caccaagaac ttagtttcga ataaacacac ataaacaaac aaat 1674 <210> 18 <211> 4260 <212> DNA <213> Artificial Sequence <220> <223> Mfa2 - Rhizopus oryzae Integration Construct <400> 18 tcgagatctc ccgagtttat cattatcaat actgccattt caaagaatac gtaataatatt 60 aatagtagtg attttcctaa ctttatttag tcaaaaaatt ggccttttaa ttctgctgta 120 acccgtacat gcccaaaata ggggggcgggt tacacagaat atataacatc ataggtgtct 180 gggtgaacag tttatcctg gcatccacta aatataatgg agcccgcttt ttttaagctg 240 gcatccagaa aaaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt 300 tcataggtcc attctcttag cgcaactaca cagaacaggg gcacaaacag gcaaaaaacg 360 ggcacaacct caatggagtg atgcaacctg cttggagtaa atgatgacac aaggcaattg 420 acctacgcat gtatctatct cattttctta caccttctat taccttctgc tctctctgat 480 ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattatcc cctatttgac 540 taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact 600 tcttaaattc tacttttata gttagtcttt tttttagttt aaaacaccaa gaacttagtt 660 tcgaataaac acacataaac aaacaaatct agaatgaagt tcatttccac tttcttgacc 720 ttcattttgg ctgctgtctc tgtcaccgct gcatctattc catctagtgc atctgtacaa 780 ttggactcct acaattacga tggttccaca ttttccggca agatttatgt caaaaacatc 840 gctactcta aaaaggttac tgttgtgtac gcagacggtt ctgacaactg gaacaataac 900 ggcaacacta ttgctgcatc attttcaggc ccaatctctg gatcaaatta cgaatactgg 960 acattctcag catcagtgaa gggcataaag gagttctaca tcaaatacga agtttcaggt 1020 aagacatatt acgacaataa caactctgca aactaccaag tctcaacttc taaacctact 1080 acaactactg cagctacaac cacaactaca gctccatcaa cttctacaac aacccgtcca 1140 tctagttcag agcctgccac cttccctact ggtaattcta ccatcagctc ttggatcaaa 1200 aagcaggaag atatttccag attcgctatg cttagaaaca tcaacccacc tggttctgcc 1260 acagggttta tcgccgcatc actctctacc gctggtccag attactacta cgcgtggaca 1320 agagatgccg ctttgacatc taacgttatc gtttacgaat acaacaccac attgtctggg 1380 aataagacaa ttctaaacgt acttaaggat tacgtcacat tcagtgttaa gacacagtct 1440 acttcaacag tttgtaattg ccttggtgaa ccaaagttca atccagacgg cagtggttac 1500 acaggtgctt ggggtagacc tcaaaatgat ggtcctgcag aaagagcgac tacatttgtt 1560 ctgtttgccg acagctactt gactcaaact aaggatgcct catacgtcac tggtacatta 1620 aagccagcaa ttttcaaaga tctcgattac gttgttaacg tctggagtaa cggatgtttc 1680 gatttatggg aggaggtgaa cggagttcat ttctacaccc ttatggttat gagaaaaggg 1740 ctattgttgg gggctgattt cgcgaagaga aacggtgact caactagagc ctcaacttac 1800 tcttctactg cttccacaat tgctaacaag atatcaagtt tctgggttag ctcaaacaac 1860 tgggtgcaag tatcccaatc tgtcacagga ggtgtaagta aaaaggggtt agacgttagc 1920 accctgttag ctgcgaatct aggatcagtc gatgatggat ttttcactcc aggttctgaa 1980 aagatattag ctacagctgt ggcagtcgaa gattcctttg ccagtctata cccaatcaac 2040 aaaaaccttc catcatactt ggggaacgct attggaagat accctgaaga tacatacaac 2100 ggtaatggta actcacaagg caatccttgg tttctggcgg ttaccggcta cgcagagttg 2160 tactatagag caattaagga atggattct aatggaggcg ttacagtgtc ctctatctca 2220 ttgccatttt tcaaaaagtt cgatagctct gcaacatccg gtaaaaagta caccgtaggt 2280 acttctgact tcaacaattt agcacaaaac attgctcttg ctgcagatcg tttcctatct 2340 actgtacaac tccatgcacc aaacaatggt tcattagcag aggaatttga tagaacaaca 2400 ggttttcta ccggcgctag agatttaaca tggtcccacg cctcattgat aacagcatcc 2460 tatgccaaag ccggtgctcc agctgcataa ttaattaaac aggccccttt tcctttgtcg 2520 atatcatgta attagttatg tcacgcttac attcacgccc tcctcccaca tccgctctaa 2580 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta 2640 tgttagtatt aagaacgtta tttatattc aaatttttct ttttttctg tacaaacgcg 2700 tgtacgcatg taacgggcag acggccggcc ataacttcgt ataatgtatg ctatacgaag 2760 ttatccttac atcacaccca atcccccaca agtgatcccc cacacaccat agcttcaaaa 2820 tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac 2880 ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta gggtggcgtt 2940 aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg 3000 tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaaatt ttttttttga 3060 tttttttctc tttcgatgac ctcccattga tatttaagtt aataaatggt cttcaatttc 3120 tcaagtttca gtttcgtttt tcttgttcta ttacaacttt ttttacttct tgctcattag 3180 aaagaaagca tagcaatcta atctaagttt taattacaaa atgccacaat cctgggaaga 3240 attggccgcc gacaaacgtg cccgtttggc taaaaccatt cctgacgaat ggaaggttca 3300 aactttgcct gccgaagatt ccgttattga tttcccaaag aagtccggta ttttgtctga 3360 ggctgaattg aagattaccg aagcctctgc tgctgatttg gtctccaagt tggccgctgg 3420 tgagttgact tctgttgaag tcactttggc tttttgtaag agagctgcta ttgctcaaca 3480 attaaccaac tgtgctcacg aattcttccc agatgctgct ttagctcaag ctagagaatt 3540 agatgaatac tacgtaagc ataagagacc agttggtcca ttacacggtt taccaatctc 3600 tttaaaggac caattgcgtg ttaagggtta cgaaacctcc atgggttaca tttcctggtt 3660 aaacaaatac gatgaaggtg attccgtctt aaccaccatg ttgagaaaag ctggtgctgt 3720 tttctacgtt aagacctctg tcccacaaac cttgatggtc tgtgaaaccg tcaacaacat 3780 cattggtaga actgtcaatc caagaaacaa aaattggtcc tgtggtggtt cttctggtgg 3840 tgaaggtgct attgttggta ttagaggtgg tgttattggt gtcggtactg acattggtgg 3900 ttccattaga gtcccagctg ctttcaactt tttatacggt ttgagaccat ctcacggtag 3960 attgccatat gctaaaatgg ctaactctat ggaaggtcaa gaaaccgttc actccgtcgt 4020 tggtcctatc actcactccg tcgaagactt gagattgttc accaaatctg tcttgggtca 4080 agaaccttgg aagtacgact ctaaggtcat ccccatgcca tggagacaat ctgaatctga 4140 catcattgcc tctaagatta agaatggtgg tttgaacatt ggttattaca atttcgacgg 4200 taacgtcttg ccacacccac caattttacg tggtgtcgaa actaccgttg ccgctttggc 4260 <210> 19 <211> 5008 <212> DNA <213> Artificial Sequence <220> <223> Mfa2 - Rhizopus oryzae GA integration construct <400> 19 ggccgcgaag gtgctattgt tggtattaga ggtggtgtta ttggtgtcgg tactgacatt 60 ggtggttcca ttagagtccc agctgctttc aactttttat acggtttgag accatctcac 120 ggtagattgc catatgctaa aatggctaac tctatggaag gtcaagaaac cgttcactcc 180 gtcgttggtc ctatcactca ctccgtcgaa gacttgagat tgttcaccaa atctgtcttg 240 ggtcaagaac cttggaagta cgactctaag gtcatcccaa tgccatggag acaatctgaa 300 tctgacatca ttgcctctaa gattaagaat ggtggtttga acattggtta ttacaatttc 360 gacggtaacg tcttgccaca cccaccaatt ttacgtggtg tcgaaactac cgttgccgct 420 ttggccaagg ctggtcacac cgttactcca tggactccat acaagcatga tttcggtcat 480 gacttgattt cccacatcta tgctgctgat ggttctgccg acgtcatgag agacatttct 540 gcctctggtg agccagccat ccctaacatt aaggacttgt tgaacccaaa tattaaggct 600 gttaacatga acgaattgtg ggacactcat ttacaaaagt ggaactatca aatggaatac 660 ttggaaagt ggcgtgaagc tgaagaaaa gctggtagg aattggacgc tattatcgct 720 ccaattactc ctaccgccgc tgtcagacac gatcaatca gatacgg ttacgcctcc 780 gttattaact tattggatt cacctctgtt gtcgtcccag tcacttcgc tgataagaat 840 attgataaga agaacgaatc ttttaaagct gtttccgaat tggatgcttt ggttcaagaa 900 gatacgacc cagaggctta tcacggtgct cctgttgctg ttcaagttat tggtagaaga 960 ttgtccgaag agagacttt ggctatcgcc gagaagtcg gtaattgtt gggtaacgtc 1020 gtcactccat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa 1080 aaaatagtg tatacaatt ttaagtgac tcttaggtttt taaaacgaaa attcttattc 1140 ttgagtact ctttcctgta ggtcaggttg cttctcagg tagcatga ggtcgctctt 1200 attgaccaca cctctaccgg catgccgagc aaatgcctgc aaatcgctcc ccattcacc 1260 caattgtaga tatgctact ccagcaatga gttgatgaat ctcggtgtgt attttatgtc 1320 ctcagaggac aacacataac ttcgtataat gtatgctata cgaagttatc tcgagggcca 1380 gaaaaaggaa gtgtttccct ccttcttgaa ttgatgttac cctcataaag cacgtggcct 1440 cttatcgaga aagaaattac cgtcgctcgt gatttgtttg caaaaagaac aaaactgaaa 1500 aaacccagac acgctcgact tcctgtcttc ctgttgattg cagcttccaa tttcgtcaca 1560 caacaaggtc ctagcgacgg ctcacaggtt ttgtaacaag caatcgaagg ttctggaatg 1620 gcgggaaagg gtttagtacc acatgctatg atgcccactg tgatctccag agcaaagttc 1680 gttcgatcgt actgttactc tctctctttc aaacagaatt gtccgaatcg tgtgacaaca 1740 acagcctgtt ctcacacact cttttcttct aaccaagggg gtggtttagt ttagtagaac 1800 ctcgtgaaac ttacatttac atatatataa acttgcataa attggtcaat gcaagaaata 1860 catatttggt cttttctaat tcgtagtttt tcaagttctt agatgctttc tttttctctt 1920 ttttacagat catcaaggaa gtaattatct actttttaca agtctagaat gaagtttatc 1980 tccacgtttt taacctttat cctagcagct gtcagcgtca ccgccgcatc aattccgagt 2040 tcagcatctg tacaacttga ctcttacaat tacgatggca gcactttctc agggaaaatt 2100 tatgtgaaaa acatagcata tagtagaag gttaccgtgg tatatgcaga cggttctgat 2160 aattggaata ataatggaaa cactattgcc gccagtttt ccggcccaat ttctggttcc 2220 attack attggacctt ttctgcatca gtaaaaggca tcaggaatt ctatattaag 2280 tacgaagttt caggtaagc atattacgat aacaatact cagcaatta tcaagctct 2340 acatctaagc ccacaac aactgctgct accaccacta acacgctcc ttctaccagc 2400 accactacca gaccaagctc tagtgaccg gctacctttc ctaccggaaa cagtaccac 2460 tcaagctgga tcaaaagca agaggacata agtcgttttg ctatgttgag gaacattaat 2520 cctccaggat ccgcgaccgg ttcattgca gcatcactaa gtactgccgg gcctgattat 2580 tattatgctt ggactagaga cgctgcatta acatcaaacg tgattgttta tgaatataat 2640 acgacccttt ccggtataa aacgatcttg aacgtatta aagactgt gaccttagt 2700 gtgaagaccc aatctacatc tacagtgtgt aattgttttgg gagaacctaa attcaatcca 2760 gacggttctg ggtacactgg tgcctggggt agacctcaa acgacggtcc agcagaaaga 2820 gcaacaacct ttgttctatt tgctgactct tatttaacgc aaacaaagga cgcctcatat 2880 gttacaggga ccctaaaacc agcaattttc aaagacttgg attatgttgt taatgtttgg 2940 agcaacggat gttttgactt gtgggaggag gttaacggtg tacactttta tacattgatg 3000 gtgatgagaa aagggttgct attgggagca gatttcgcta aaagaaatgg tgattctaca 3060 agagcgagca catatagtag caccgcttca acaatcgcca ataaaatctc atctttctgg 3120 gtatctagca acaactgggt acaagtttcc caaagtgtta ccggcggtgt gtccaaaaag 3180 ggttagacg ttagcacact tctagctgct aatttgggta gcgttgatga cgggtttttt 3240 actccaggta gtgagaagat actggcaacc gcggtggcgg ttgaagacag ctttgcttca 3300 ttgtatccta taaataaaaa tctgccctct tatctgggta atgcaattgg cagataccca 3360 gaagatacct acaatggtaa tggtaattcc caggggaacc catggtttt ggctgttaca 3420 ggctacgcag aactttatta ccgtgcaatc aaggaatgga tttcaaatgg cggcgtcact 3480 gtcagtagta taagtttgcc cttttttaag aaatttgatt cctcagcaac gtctggtaaa 3540 aaatacaccg taggtactag tgatttcaat aatttggccc aaaatattgc gcttgctgct 3600 gacaggtttc ttagtaccgt tcagttgcac gctccaaata atggctcatt ggctgaagaa 3660 tttgatcgta cgacaggttt ctccactggt gctagggatt tgacttggag tcatgcctcc 3720 ttaatcacag caagctatgc taaagctggt gcacctgctg cttagttaat taatttacca 3780 gctactatc cttcttgaaa atatgcactc tatatctttt agttcttaat tgcaacacat 3840 agatttgctg tataacgaat tttatgctat tttttaatt tggagttcgg tgatgaaagt 3900 gtcacagcga atttcctcac atgtagggac cgaattgttt acaagttctc tgtaccacca 3960 tggagacatc aaaagattgaa aatctatgga aagatatgga cggtagcaac aagaatatag 4020 cacgagccgc ggagttcatt tcgttacttt tgatatcgct cacaactatt gcgaagcgct 4080 tcagtgaaaa aatcataagg aaaagttgta aatattattg gtagtattcg tttggtaaag 4140 tagagggggt aatttttccc ctttattttg ttcatacatt cttaaattgc tttgcctctc 4200 cttttggaaa gctatacttc ggagcactgt tgagcgaagg ctcaggccgg cagcacgcag 4260 cacgctgtat ttacgtattt aattttatat atttgtgcat acactactag ggaagacttg aaaaaacct aggravation aaaacgacac aggravationccc gtatttacta ttttttcctt 4380 ccttttgatg gggcagggcg cctactgctt agctgtttcc 4440 gtctctactt cggtagttgt ctcaattgtc gtttcagtat tacctttaga gccgctagac 4500. gatggttgag ctattgttg agggaaaact aagttcatgt aacacacgca taacccgatt aaactcatga atagcttgat tgcaggaggc tggtccattg gagatggtgc cttattttcc 4620. ttataggcaa cgatgatgtc ttcgtcggtg ttcaggtagt agtgtacact ctgaatcagg gagaaccagg caatgaactt gttcctcaag aaaatagcgg ccataggcat ggattggtta accacaccag atatgcttgg tgtggcagaa tatagtcctt ttggtggcgc aattttcttg tacctgtggt agaaagggag cggttgaact gttagtatat attggcaata tcagcaaatt tgaaagaaa ttgtcggtga aaaacatacg aaacacaaag gtcgggcctt gcaacgttat tcaaagtcat tgtttagttg aggaggtagc agcggagtat atgtattcct tttttttgcc 4980. tatggatgtt gtaccatgcc cattctga 5008 <210> 20 <211> 4884 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus microsporus GA Integration Construct <400> 20 ctaaattcgg ccttgctcag agactcctgg attttggcta acaacgcagt cccttcgatg 60 catatagcta ggccacaaat tatgccaata acggtccatg ggttgatgtt ttcttgaatt 120 ctttcgtttt tcatgctatt tgcgtcttcc caagtcccag cgttccagta ttcatactgc 180 gcgttagagt ggtagccata agagccggca tattggtaat tttcagtatt aacgttagaa 240 cgtggtgaat acgatgtggt ccagccttgc ctcgttgtgt catatacgat ctttttcttt 300 gggtcacaaa gaatatcata tgcttgagag atgactttaa atctatgtag tttttcgctt 360 gatgttagca gcagcggtga tttactatca ctgttggtaa ccttttctga gctaaatatt 420 tgaatgttat cggaatggtc agggtggtac aattttacat aacgatgata tttttttttt 480 aacgacttct tgtccagttt aggatttcca gatccggcct ttggaatgcc aaaaatatca 540 tagggagttg gatctgccaa ctcaggccat tgttcatccc ttatcgtaag ttttctattg ccatttttat cgttcgctgt agcatactta gctaaaag tgatttgtgg gggacacttt 660 720. tctacacatg ataagtgcca cttgaataaa aatgggtata cgaacttatg gtgtagcata acaaatat tgcaagtagt gacctatggt gtgtagatat acgtacagtt agttacgagc 780 ctaaagacac aacgtgtttg ttaattatac tgtcgctgta atatcttctc ttccattatc accggtcatt ccttgcaggg gcggtagtac ccggagaccc tgaacttttc tttttttttt 900 tgcgaatta aaaagttcat tttcaattcg acaatgagat ctacaagcca ttgttttatg ttgatgagag ccagcttaaa gagttctcga gatctcccga gtttatcatt atcaatactg ccatttcaa gathering father gtagtgatt tcctaacttt father aaaattggcc ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 taatggagcc cgcttttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacacaga 1320 acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcttg 1380 gagtaaatga tgacacaagg caattgacct acgcatgtat ctatctcatt ttcttacacc 1440 ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt 1500 tccctgaaat tattccccta tttgactaat aagtatataa agacggtagg tattgattgt 1560 aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt 1620 tagtttaaaa caccaagaac ttagtttcga ataaacacac ataaacaaac aaatctagaa 1680 tgaaacttat gaatccatct atgaaggcat acgttttctt tatcttaagc tacttctctt 1740 tactcgttag ctcagctgcg gtgccaacct ctgccgccgt acaagttgag tcatacaatt 1800 atgacggtac cactttttca ggtagaatat tcgtcaaaaa cattgcctac tcaaaggtcg 1860 taacagttat ctactccgat ggatcagata actggaacaa taacaacaac aaagtttctg 1920 cagcttactc agaagcaatt tctgggtcta actacgaata ctggacattc tccgcaaagt 1980 2040 acaacggtac caaaaactac caagtccaag caacctcagc gacatctaca acagctactg 2100 caaccaac tacagctact ggcaacaa ctacttctac aggtccaact agtactgcat 2160 ccgtatcatt ccctaccggt aactcaacaa tttcttcctg gataaaaaat caagaggaaa 2220 tcagccgttt tgctatgttg agaaatatca atccacctgg gtctgccaca gggttcatag 2280 ccgcatctct gtccacagcc ggcccagatt actattactc ttggactaga gattcagcac 2340 taacagctaa tgtgatcgct tacaataca acacaacatt cactggaaac accacccttc 2400 ttaagtactt gaaagattac gttacatttt ctgtcaaaag ccaatctgta tctaccgttt 2460 gtaactgtct gggagaacca aagttcaacg ctgatggtag ttcttttaca ggtccatggg 2520 gcagaccaca aaacgagga ccagcagaga gagctgttac ttttatgttg attgctgaca 2580 gctacttgac tcaactaag gacgcatcct acgttaccgg tacattaaag ccagcaatct 2640 tcaaagatct tgattacgta gtttctgttt ggtctaacgg ttgctacgat ttatgggaag 2700 aggttaatgg tgttcatttc tatactctca tggtcatgag aaagggtttg atcttaggtg 2760 ccgacttcgc tgctagaaat ggtgactcta gtagagcttc aacctacaag caaactgcat 2820 caacaatgga atcaaagatc agttcttttt ggtcagattc taacaactac gtccaagtttt 2880 ctcaatcagt taccgccgga gtgtcaaaaa agggactaga tgttagtaca ctattggcgg 2940 ccaacattgg tagtctgcct gatggcttt tcactccagg ctccgaaaag atattggcta 3000 cagcagtggc gttagaaaat gcattcgcat ccttgtaccc aattaactct aacctacctt 3060 cttacttggg taactcaatt ggaagatatc ctgaggatac atacaacggt aatggcaact 3120 ctcaggggaa tccatggttc cttgccgtca acgcatacgc agaactttac tacagagcta 3180 ttaaggaatg gattagtaat ggcaaggtga cagtatccaa tatctcacta cctttcttca 3240 aaaagttga ttcttccgcc acttctggaa agacatacac tgctggtaca tcagatttca 3300 ataacttggc tcagaacatt gctttaggcg ccgatagatt cctgtctact gttaagttcc 3360 acgcatacac taacgggagt ctatcagaag agtacgatag atctaccggt atgagtactg 3420 gggctcgtga tttaacatgg tcccatgctt cattgatcac agtggcgtac gcaaaggccg gtagtcctgc agcttagtta attack ccccttttcc tttgtcgata tcatgtaatt agttatgtca cgcttacatt cacgccctcc tcccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac aaacgcgtgt acgcatgtaa cgggcagacg gccggccata acttcgtata atgtatgcta tacgaagtta tggcaacggt tcatcatctc atggatctgc acatgaacaa acaccagagt caaacgacgt tgaaattgag gctactgcgc caattgatga caatacagac caattgatga aaccgaagtt atctgatgta gaaaaggatt agagatgcta agagatagtg atgatatttc father taattctata tatgttaatt accttttttg cgaggcatat ttatggtgaa ggataagttt tgaccatcaa agaaggttaa tgtggctgtg gtttcagggt ccataaagct tttcaattca tcttttttt 4080. ttttgttctt ttttttgatt ccggtttctt tgaattttt ttgattcggt aatctccgag 4140 cagaaggaag aacgaaggaa ggaggacaga cttagattgg tatatatacg catatgtggt 4200 gttgaagaaa catgaaattg cccagtattc ttaacccac tgcacagaac aaaaacctgc 4260 aggaaacgaa gataaatcat gtcgaaagct acataagg aacgtgctgc tactcatcct 4320 agtcctgttg ctgccaagct atttaatc atgcacgaaa agcaacaaa cttgtgtgct 4380 tcattggatg ttcgtaccac CAggaatta ctggagttag Tgaagcatt aggtcccaaa 4440 atttgtttac taaaaacaca tgtggatatc tgactgattttccatgga gggcacagtt 4500 aagccgctaa agcattatc cgccaagtac aattttac tcttcgaaga cagaaattt 4560 gctgacattg gtaatacagt caattgcag tactctgcgg gtgtatacag atagcagaa 4620 tgggcagaca ttacgaatgc acacggtgtg gtggggcccag gtattgttag cggtttgaag 4680 caggcggcgg agaagtaac aaaggaacct agaggcttt tgatgttagc agaattgtca 4740 tgcaagggct ccctagctac tggagat actaagggta ctgttgacat tgcgaagagc 4800 xaaagatt tgttatcgg ctttattgct caagagaca tggtggag agatgaaggt 4860 tacgattggt tgattatgac acgc 4884 <210> 21 <211> 1790 <212> DNA <213> Artificial Sequence <220> <223> Integration Construct <400> 21 ggaagagctc ctactgcgcc aattgatgac aatacagacg atgataacaa accgaagtta 60 tctgatgtag aaaaggatta gagatgctaa gagatagtga tgatatttca taaataatgt 120 aattctatat atgttaatta ccttttttgc gaggcatatt tatggtgaag gataagtttt 180 gaccatcaaa gaaggttaat gtggctgtgg tttcagggtc cataaagctt ttcaattcat 240 cttttttttt ttgttctttt ttttgattcc ggtttctttg aaattttttt gattcggtaa 300 tctccgagca gaaggaagaa cgaaggaagg agcacagact tagattggta tatatacgca 360 tatgtggtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg cacagaacaa 420 aaacctgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa cgtgctgcta 480 ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag caaacaaact 540 tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt gaagcattag 600 gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt tccatggagg 660 gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc ttcgaagaca 720 gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt gtatacagaa 780 tagcagaatg ggcagacatt acgaatgcgc acggtgtggt gggcccaggt attgttagcg 840 900 aattgtcatg caagggctcc ctagctactg gagatatac taagggtact gttgacattg 960 cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg ggtggagaag 1020 atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac aagggagacg 1080 cattgggtca agagataga gccgtggatg atgtggtctc tacaggatct gacattatta 1140 ttgttggaag aggactattt ccaaagggaa gggatgctaa ggtagagggt gaacgttaca 1200 gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa aaaactgtat 1260 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt 1320 tattacccgg gaatctcggt cgtaatgatt tttataatga cgaaaaaaaa aaattggaaa 1380 gaaaaagctt catggccttt ataaaaagga accatccaat acctcgccag aaccaagtaa 1440 cagtatttta cggggcacaa atcaagaaca ataagacagg actgtaaaga tggacgcatt 1500 gaactccaaa gaacaacaag agttccaaaa agtagtggaa caaaagcaaa tgaaggattt 1560 catgcgtttg ataacttcgt ataatgtatg ctatacgaag ttatgcggcc gccagcacgc 1620 agcacgctgt atttacgtat ttaattttat atatttgtgc atacactact agggaagact 1680 tgaaaaaaac ctaggaaatg aaaaaacgac acaggaagtc ccgtatttac tattttttcc 1740 ttccttttga tggggcaggg cggaaataga ggataggata agcctactgc 1790 <210> 22 <211> 4474 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus microsporus GA integration construct <400> 22 gtgtttgtta attatactgt cgctgtaata tcttctcttc cattatcacc ggtcattcct 60 tgcaggggcg gtagtacccg gagacccctga acttttcttt tttttttttgc gaaattaaaa 120 agttcattt caattcgaca atgagatcta caagccattg ttttatgttg atgagagcca 180 gcttaaagag ttctcgagat ctcccgagtt tatcattatc aatactgcca tttcaaagaa 240 tacgtaaata attaatgta gtgattttcc taactttatt tagtcaaaaa attggccttt 300 taattctgct gtaacccgta catgcccaaa atagggggcg ggttacacag aatatataac 360 atcataggtg tctgggtgaa cagtttattc ctggcatcca ctaaaataa tggagcccgc 420 tttttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat tgttttcttc 480 accaaccatc agttcatagg tccattctct tagcgcaact acacagaaca ggggcacaaa 540 caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcttggag taaatgatga 600 cacaaggcaa ttgacctacg catgtatcta tctcattttc ttacaccttt tattaccttc 660 tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc ctgaaattat 720 tcccctattt gactaataag tatataaaga cggtaggtat tgattgtaat tctgtaaatc 780 840. ttctcttaa acttcttaa ttctactttt atagttagtc tttttttag tttaaaacac caagaactta gtttcgaata aacacacata aacaaacaaa tctagaatga aacttatgaa tccatctatg aaggcatacg ttttctttat cttaagctac ttctctttac tcgttagctc agctgcggtg ccaacctctg ccgccgtaca agttgagtca tacaattatg acggtaccac tttttcaggt agaatattcg tcaaaaacat tgcctactca aaggtcgtaa cagttatcta ctccgatgga tcagataact ggacaata cacaacaa gtttctgcag cttactcaga agcaatttct gggtctaact acgaatactg gacattctcc gcaaagttat ccggaattaa acagttttat gtcaatacg aagtttctgg ttcaacatat tacgacaaca acggtaccaa aaactaccaa gtccaagcaa cctcagcgac atctacaaca gctactgcaa ccacaactac agctactggc acaacaacta cttctacagg tccaactagt actgcatccg tatcattccc 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 cacagccggc ccagattact attactcttg gactagagat tcagcactaa cagctaatgt 1560 gatcgcttac gaatacaaca caacattcac tggaaacacc acccttctta agtacttgaa 1620 agattacgtt acattttctg tcaaaagcca atctgtatct accgtttgta actgtctggg 1680 agaaccaaag ttcaacgctg atggtagttc ttttacaggt ccatggggca gaccacaaaa 1740 cgacggacca gcagagagag ctgttacttt tatgttgatt gctgacagct acttgactca 1800 aactaaggac gcatcctacg ttaccggtac attaaagcca gcaatcttca aagatcttga 1860 ttacgtagtt tctgtttggt ctaacggttg ctacgattta tgggaagagg ttaatggtgt 1920 tcatttctat actctcatgg tcatgagaaa gggtttgatc ttaggtgccg acttcgctgc 1980 tagaaatggt gactctagta gagcttcaac ctacaagcaa actgcatcaa caatggaatc 2040 aaagatcagt tctttttggt cagattctaa caactacgtc caagtttctc aatcagttac 2100 cgccggagtg tcaaaaaagg gactagatgt tagtacacta ttggcggcca acattggtag 2160 tctgcctgat ggctttttca ctccaggctc cgaaaagata ttggctacag cagtggcgtt 2220 agaaaatgca ttcgcatcct tgtacccaat taactctaac ctaccttctt acttgggtaa 2280 ctcaattgga agatatcctg aggatacata caacggtaat ggcaactctc aggggaatcc 2340 atggttcctt gccgtcaacg catacgcaga actttactac agagctatta aggaatggat 2400 tagtaatggc aaggtgacag tatccaatat ctcactacct ttcttcaaaa agttgattc 2460 ttccgccact tctggaaaga catacactgc tggtacatca gatttcaata acttggctca 2520 gaacattgct ttaggcgccg atagattcct gtctactgtt aagttccacg catacactaa 2580 cgggagtcta tcagaagagt acgatagatc taccggtatg agtactgggg ctcgtgattt 2640 aacatggtcc catgcttcat tgatcacagt ggcgtacgca aaggccggta gtcctgcagc 2700 ttagttaatt aaacaggccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc 2760 ttacattcac gccctcctcc cacatccgct ctaaccgaaa aggaaggagt tagacaacct 2820 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata 2880 tttcaaattt ttcttttttt tctgtacaaa cgcgtgtacg catgtaacgg gcagacggcc 2940 ggccataact tcgtataatg tatgctatac gaagttatcc ttacatcaca cccaatcccc 3000 cacaagtgat ccccacaca ccatagcttc aaaatgtttc tactccttt ttactcttcc 3060 agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact 3120 aaatttcccc tctttcttcc tctagggtgg cgttaattac ccgtactaaa ggtttggaaa 3180 agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat 3240 cacgtttctt tttcttgaaa aatttttttt ttgatttttt tctctttcga tgacctccca 3300 ttgatattta agttaataaa tggtcttcaa tttctcaagt ttcagtttcg tttttcttgt 3360 tctattacaa cttttttac ttcttgctca ttagaaagaa agcatagcaa tctaatctaa 3420 gttttaatta caaaatgcca caatcctggg aagaattggc cgccgacaaa cgtgcccgtt 3480 tggctaaaac cattcctgac gaatggaagg ttcaaacttt gcctgccgaa gattccgtta 3540 ttgatttccc aaagaagtcc ggtattttgt ctgaggctga attgaagatt accgaagcct 3600 ctgctgctga tttggtctcc aagttggccg ctggtgagtt gacttctgtt gaagtcactt 3660 tggctttttg taagagagct gctattgctc aacaattaac caactgtgct cacgaattct 3720 tcccagatgc tgctttagct caagctagag aattagatga atactacgct aagcataaga 3780 gaccagttgg tccattacac ggtttaccaa tctctttaaa ggaccaattg cgtgttaagg 3840 gttacgaaac ctccatgggt tacatttcct ggttaaacaa atacgatgaa ggtgattccg 3900 tcttaaccac catgttgaga aaagctggtg ctgttttcta cgttaagacc tctgtcccac 3960 aaaccttgat ggtctgtgaa accgtcaaca acatcattgg tagaactgtc aatccaagaa 4020 acaaaaattg gtcctgtggt ggttcttctg gtggtgaagg tgctattgtt ggtattagag 4080 gtggtgttat tggtgtcggt actgacattg gtggttccat tagagtccca gctgctttca 4140 actttttata cggtttgaga ccatctcacg gtagattgcc atatgctaaa atggctaact 4200 ctatggaagg tcaagaaacc gttcactccg tcgttggtcc tatcactcac tccgtcgaag 4260 acttgagatt gttcaccaaa tctgtcttgg gtcaagaacc ttggaagtac gactctaagg 4320 tcatccccat gccatggaga caatctgaat ctgacatcat tgcctctaag attaagaatg 4380 gtggtttgaa cattggttat tacaatttcg acggtaacgt cttgccacac ccaccaattt 4440 tacgtggtgt cgaaactacc gttgccgctt tggc 4474 <210> 23 <211> 1878 <212> DNA <213> Artificial Sequence <220> <223> Integration Construct <400> 23 gaagattacc gaagcctctg ctgctgattt ggtctccaag ttggccgctg gtgagttgac 60 ttctgttgaa gtcactttgg ctttttgtaa gagagctgct attgctcaac aattaaccaa 120 ctgtgctcac gaattcttcc cagatgctgc tttagctcaa gctagagaat tagatgaata 180 ctacgctaag cataagagac cagttggtcc attacacggt ttaccaatct ctttaaagga 240 ccaattgcgt gttaagggtt acgaaacctc catgggttac atttcctggt taaacaaata 300 cgatgaaggt gattccgtct taaccaccat gttgagaaaa gctggtgctg ttttctacgt 360 taagacctct gtcccacaaa ccttgatggt ctgtgaaacc gtcaacaaca tcattggtag 420 aactgtcaat ccaagaaaca aaaattggtc ctgtggtggt tcttctggtg gtgaaggtgc 480 tattgttggt attagaggtg gtgttattgg tgtcggtact gacattggtg gttccattag 540 agtcccagct gctttcaact tttatacgg tttgagacca tctcacggta gattgccata 600 tgctaaaatg gctaactcta tggaaggtca agaaaccgtt cactccgtcg ttggtcctat 660 cactcactcc gtcgaagact tgagattgtt caccaaatct gtcttgggtc aagaaccttg 720 gaagtacgac tctaaggtca tcccaatgcc atggagacaa tctgaatctg acatcattgc 780 ctctaagatt aagaatggtg gtttgaacat tggttatac aatttcgacg gtaacgtctt 840 gccacaccca ccaattttac gtggtgtcga aactaccgtt gccgctttgg ccaaggctgg 900 tcacaccgtt actccatgga ctccatacaa gcatgatttc ggtcatgact tgatttccca 960 catctatgct gctgatggtt ctgccgacgt catgagagac atttctgcct ctggtgagcc 1020 agccatccct aacattaagg acttgttgaa cccaaatatt aaggctgtta acatgaacga 1080 attgtgggac actcatttac aaaagtggaa ctatcaaatg gaatacttgg aaaagtggcg 1140 tgaagctgaa gaaaaaagctg gtaaggaatt ggacgctatt atcgctccaa ttactcctac 1200 cgccgctgtc agacacgatc aattcagata ctacggttac gcctccgtta ttaacttatt 1260 ggatttcacc tctgttgtcg tcccagtcac tttcgctgat aagaatattg ataagaagaa 1320 cgaatctttt aaagctgttt ccgaattgga tgctttggtt caagaagaat acgacccaga 1380 ggcttatcac ggtgctcctg ttgctgttca agttattggt agaagattgt ccgaagagag 1440 aactttggct atcgccgaag aagtcggtaa attgttgggt aacgtcgtca ctccataagg 1500 agattgataa gacttttcta gttgcatatc tttatattt aaatcttatc tattagttaa 1560 ttttttgtaa tttatcctta tatatagtct ggttattcta aaatatcatt tcagtatcta 1620 aaaattcccc tctttttca gttatatctt aacaggcgat aacttcgtat aatgtatgct 1680 atacgaagtt atgcggccgc cagcacgcag cacgctgtat ttacgtattt aattttatat 1740 atttgtgcat acactactag ggaagacttg aaaaaaaacct aggaaatgaa aaaacgacac 1800 aggaagtccc gtatttacta ttttttcctt ccttttgatg gggcagggcg gaaatagagg 1860 ataggataag cctactgc 1878 <210> 24 <211> 4827 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus microsporus GA Integration Construct <400> 24 ggccgctcca tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt 60 ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct 120 gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc 180 ccaggtattg ttagcggttt gaagcaggcg gcggaagaag taacaaagga acctagaggc 240 cttttgatgt tagcagaatt gtcatgcaag ggctccctag ctactggaga atatactaag 300 ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga 360 gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta 420 gatgacaagg gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca 480 ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta 540 gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa 600 aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca 660 atttaattat atcagttatt acccgggaat ctcggtcgta atgattttta taatgacgaa 720 aaaaaaaaa ttggaaagaa aaagcttcat ggcctttata aaaaggaacc atccaatacc 780 tcgccagaac caagtaacag tattttacgg ggcacaaatc aagaacaata agacaggact 840 gtaaagatgg acgcattgaa ctccaaagaa caacaagagt tccaaaaagt agtggaacaa 900 aagcaaatga aggatttcat gcgtttgata acttcgtata atgtatgcta tacgaagtta 960 tctcgagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt accctcataa 1020 agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt tgcaaaaaga 1080 acaaaactga aaaaacccag acacgctcga cttcctgtct tcctgttgat tgcagcttcc 1140 aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca agcaatcgaa 1200 ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac tgtgatctcc 1260 agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa ttgtccgaat 1320 cgtgtgacaa caacagcctg ttctcacaca ctctttctt ctaaccaagg gggtggttta 1380 gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat aaattggtca 1440 atgcaagaaa tacatatttg gtcttttcta attcgtagtt ttcaagttc ttagatgctt 1500 tctttttctc ttttcag atcatcagg aagtattat ctacttttta caagtctaga 1560 atgaagctaa tgaacccatc tatgaaagct tatgtcttt tcatcttatc attacktctct 1620 ctcctagttt catctgccgc agtgcctaca tctgctgccg tccaagtgga aagttacaat 1680 tacgatggca ctaccttctc cggccgtatc ttcgtgaaa acatgctta ctcaaagtt 1740 gttacagtga tttactccga tggttctgat aattggaaca acaataca caagtttca 1800 gcagcgtact ccgaagccat tagtctct acaccgaat actggacttt ctccgcaag 1860 ttgtctggta ttagcaatt ctacgtaag tacgagttt caggttcac attackacgat 1920 aacaatggta ctaaaaacta cccaggtaca gccacaagcg ctacaagtac aactgccaca 1980 gctacaacta actacaccac ccggtccaac atcacccca 2040 tctgtctcct ttccaaccgg taacagtaca atatcatcat ggatcaaaaa ccaagaggaa 2100 atctcaagat ttgcaatgtt gagaaacatc aatccaccag ggtcagcgac tggtttcatc 2160 gccgcttcat tgtccaccgc tgggccagac tactattact cttggactag agactctgca 2220 ttgacagcaa acgttatagc ttacgaatac aacacaacct tcactggtaa cactaccttg 2280 ttgaagtatc ttaaagacta cgtcactttt agtgttaagt ctcaatctgt ttctaccgtc 2340 tgtaactgtt taggtgaacc aaagtttaat gcagatggct catcatttac tggtccatgg 2400 ggcagacctc aaaacgatgg accagcagaa agagcagtca cattcatgtt gatcgctgac 2460 tcatacttga cacaaactaa ggatgcttca tacgtgactg gaacactcaa gccagccata 2520 ttcaaagacc tggattatgt tgtttctgtg tggtctaatg gttgctacga tttgtgggag 2580 gaagttaatg gcgtacattt ctacacacta atggttatga gaaagggact aattcttggg 2640 gcagatttcg cagctagaaa tggtgattcc tcaagagcat ctacctacaa gcaaacagca 2700 tctacaatgg aatcaaagat cagctctttc tggtctgact ctaacaacta cgttcaagtt 2760 tcacaatctg tgactgctgg tgtaagtaaa aagggtttag atgtttctac tctgttagct 2820 gcaaacattg gttctttacc agatggcttc tttacaccag gatcagaaaa gattttggca 2880 actgccgttg ccttagagaa tgcattcgct tccctttacc ctattaactc taacttacct 2940 tcatatttgg gtaattcaat tggtagatat ccagaggaca catacaacgg aaacgggaat 3000 tcacagggca acccttggtt cttagccgta aacgcgtacg ccgaggtata ctacagagcc 3060 attaaggaat ggatctcaaa tggtaaggta acagtctcta atatctctct tcctttcttt 3120 aaaaagttcg attctagcgc cactagcggc aagacctata cagccggaac aagtgatttc 3180 aataacctcg ctcagaacat agctcttgga gcagacagat ttctgtcaac tgttaaattt 3240 cacgcgtaca caaatggcag cttgtctgaa gagtacgatc gttccaccgg gatgagtact 3300 ggtgctagag atctaacctg gtcccatgca tctcttatca cagttgcata cgcaaaagct 3360 ggatctcctg ctgcgtaatt aattaattta ccagcttact atccttcttg aaaatatgca 3420 ctctatatct tttagttctt aattgcaaca catagatttg ctgtataacg aattttatgc 3480 tattttttta atttggagtt cggtgatgaa agtgtcacag cgaatttcct cacatgtagg 3540 gaccgaattg tttacaagtt ctctgtacca ccatggagac atcaaagatt gaaaatctat 3600 ggaaagatat ggacggtagc aacaagaata tagcacgagc cgcggagttc atttcgttac 3660 ttttgatatc gctcacaact attgcgaagc gcttcagtga aaaaatcata aggaaaagtt 3720 gtaaatatta ttggtagtat tcgtttggta aagtagaggg ggtaattttt cccctttatt 3780 ttgttcatac attcttaaat tgctttgcct ctccttttgg aaagctatac ttcggagcac 3840 tgttgagcga aggctcaggc cggcagcacg cagcacgctg tatttacgta tttaatttta 3900 tatattgtg catacactac tagggaagac ttgaaaaaaa cctaggaaat gaaaaaacga 3960 cacaggaagt cccgtattta ctattttttc cttccttttg atggggcagg gcggaaatag 4020 aggataggat aagcctactg cttagctgtt tccgtctcta cttcggtagt tgtctcaatt 4080 gtcgtttcag tattaccttt agagccgcta gacgatggtt gagctatttg ttgagggaaa 4140 actaagttca tgtaacacac gcataacccg attaaactca tgaatagctt gattgcagga 4200 ggctggtcca ttggagatgg tgccttattt tccttatagg caacgatgat gtcttcgtcg 4260 gtgttcaggt agtagtgtac actctgaatc agggagaacc aggcaatgaa cttgttcctc 4320 aagaaaatag cggccatagg catggattgg ttaaccacac cagatatgct tggtgtggca 4380 gaatatagtc cttttggtgg cgcaattttc ttgtacctgt ggtagaaagg gagcggttga 4440 actgttagta tatattggca atatcagcaa atttgaaaga aaattgtcgg tgaaaaacat 4500 acgaaacaca aaggtcgggc cttgcaacgt tattcaaagt cattgtttag ttgaggaggt 4560 agcagcggag tatatgtatt cctttttttt gcctatggat gttgtaccat gcccattctg 4620 ctcaagcttt tgttaaaatt atttttcagt attttttctt ccatgttgcg cgttacgaga 4680 acagaagcga cagataaccg caatcataca actagcgcta ctgcggggtg taaaaagcac 4740 aagaactaag ccaagatcac aacagttatc gataaaatag cagtgtttgc atggccattg 4800 agaaggacaa cattggcgtg cggcatg 4827 <210> 25 <211> 4684 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus microsporus (Rhizopus microsporus) GA <400> 25 ggccgcgaag gtgctattgt tggtattaga ggtggtgtta ttggtgtcgg tactgacatt 60 ggtggttcca ttagagtccc agctgctttc aactttttat acggtttgag accatctcac 120 ggtagattgc catatgctaa aatggctaac tctatggaag gtcaagaaac cgttcactcc 180 gtcgttggtc ctatcactca ctccgtcgaa gacttgagat tgttcaccaa atctgtcttg 240 ggtcaagaac cttggaagta cgactctaag gtcatcccaa tgccatggag acaatctgaa 300 tctgacatca ttgcctctaa gattaagaat ggtggttga acattggtta ttacaatttc 360 gacggtaacg tcttgccaca cccaccaatt ttacgtggtg tcgaaactac cgttgccgct 420 ttggccaagg ctggtcacac cgttactcca tggactccat acaagcatga ttcggtcat 480 gacttgattt cccacatcta tgctgctgat ggttctgccg acgtcatgag agacatttct 540 gcctctggtg agccagccat ccctaacatt aaggacttgt tgaacccaaa tattaaggct 600 gttaacatga acgaattgtg ggacactcat ttacaaaagt ggaactatca aatggaatac 660 ttggaaagt ggcgtgaagc tgaagaaaa gctggtagg aattggacgc tattatcgct 720 ccaattactc ctaccgccgc tgtcagacac gatcaatca gatacgg ttacgcctcc 780 gttattaact tattggatt cacctctgtt gtcgtcccag tcacttcgc tgataagaat 840 attgataaga agaacgaatc ttttaaagct gtttccgaat tggatgcttt ggttcaagaa 900 gatacgacc cagaggctta tcacggtgct cctgttgctg ttcaagttat tggtagaaga 960 ttgtccgaag agagacttt gggctatcgcc gagaagtcg gtaattgtt gggtaacgtc 1020 gtcactccat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa 1080 aaaatagtg tatacaatt ttaagtgac tcttaggtttt taaaacgaaa attcttattc 1140 ttgagtact ctttcctgta ggtcaggttg cttctcagg tagcatga ggtcgctctt 1200 attgaccaca cctctaccgg catgccgagc aaatgcctgc aaatcgctcc ccattcacc 1260 caattgtaga tatgctact ccagcaatga gttgatgaat ctcggtgtgt attttatgtc 1320 ctcagaggac aacacataac ttcgtataat gtatgctata cgaagttatc tcgagggcca 1380 gaaaaaggaa gtgtttccct ccttcttgaa ttgatgttac cctcataaag cacgtggcct 1440 cttatcgaga aagaaattac cgtcgctcgt gatttgtttg caaaaagaac aaaactgaaa 1500 aaacccagac acgctcgact tcctgtcttc ctgttgattg cagcttccaa tttcgtcaca 1560 caacaaggtc ctagcgacgg ctcacaggtt ttgtaacaag caatcgaagg ttctggaatg 1620 gcgggaaagg gtttagtacc acatgctatg atgcccactg tgatctccag agcaaagttc 1680 gttcgatcgt actgttactc tctctctttc aaacagaatt gtccgaatcg tgtgacaaca 1740 acagcctgtt ctcacacact cttttcttct aaccaagggg gtggtttagt ttagtagaac 1800 ctcgtgaaac ttacatttac atatatataa acttgcataa attggtcaat gcaagaaata 1860 catatttggt cttttctaat tcgtagtttt tcaagttctt agatgctttc tttttctctt 1920 ttttacagat catcaaggaa gtaattatct actttttaca agtctagaat gaagctaatg 1980 aacccatcta tgaaagctta tgtctttttc atcttatcat acttctctct cctagtttca 2040 tctgccgcag tgcctacatc tgctgccgtc caagtggaaa gttacaatta cgatggcact 2100 accttctccg gccgtatctt cgtgaaaaac attgcttact caaaagttgt tacagtgatt 2160 tactccgatg gttctgataa ttggaacaac aatacaaca aagtttcagc agcgtactcc 2220 gaagccatta gtggatctaa ctacgaatac tggactttct ccgcaaagtt gtctggtatt 2280 2340 aaaaactacc aggtacaagc cacaagcgct acaagtacaa ctgccacagc tacaactaca 2400 actgctacag ggaacaac tacatcaacc ggtccaacat caaccgcatc tgtctccttt 2460 ccaaccggta acagtacaat atcatcatgg atcaaaaacc aaggaaat ctcaagattt 2520 gcaatgttga gaaacatcaa tccaccaggg tcagcgactg gtttcatcgc cgcttcattg 2580 tccaccgctg ggccagacta ctattactct tggactagag actctgcatt gacagcaac 2640 2700 aaagactacg tcacttttag tgttaagtct caatctgttt ctaccgtctg taactgttta 2760 ggtgaaccaa agtttaatgc agatggctca tcatttactg gtccatgggg cagacctcaa 2820 aacgatggac cagcaagaag agcagtcaca ttcatgttga tcgctgactc atacttgaca 2880 2940 gattatgttg tttctgtgtg gtctaatggt tgctacgatt tgtgggagga agttaatggc 3000 gtacatttct acacactat ggttatgaga aagggactaa ttcttggggc agatttcgca 3060 gctagaaatg gtgattcctc aagagcatct acctacaagc aaacagcatc tacaatggaa 3120 tcaaagatca gctctttctg gtctgactct aacaactacg ttcaagtttc acaatctgtg 3180 actgctggtg tagtaaaaa gggtttagat gtttctactc tgttagctgc aaacattggt 3240 tctttaccag atggcttctt tacaccagga tcagaaaaga ttttggcaac tgccgttgcc 3300 ttagagaatg cattcgcttc cctttaccct attaactcta acttacctt atatttgggt 3360 aattcaattg gtagatatcc agaggacaca tacaacggaa acgggaattc acagggcaac 3420 ccttggttct tagccgtaaa cgcgtacgcc gagttatact acagagccat tagggaatgg 3480 atctcaaatg gtaaggtaac agtctctaat atctctcttc ctttctttaa aaagttcgat 3540 tctagcgcca ctagcggcaa gacctataca gccggaacaa gtgatttcaa taacctcgct 3600 cagaacatag ctcttggagc agacagattt ctgtcaactg ttaaatttca cgcgtacaca 3660 aatggcagct tgtctgaaga gtacgatcgt tccaccggga tgagtactgg tgctagagat 3720 ctaacctggt cccatgcatc tcttatcaca gttgcatacg caaaagctgg atctcctgct 3780 gcgtaattaa ttaatttacc agcttactat ccttcttgaa aatatgcact ctatatcttt 3840 tagttcttaa ttgcaacaca tagatttgct gtataacgaa ttttatgcta ttttttaat 3900 ttggagttcg gtgatgaaag tgtcacagcg aatttcctca catgtaggga ccgaattgtt 3960 tacaagttct ctgtaccacc atggagacat caaagattga aaatctatgg aaagatatgg 4020 acggtagcaa caagaatata gcacgagccg cggagttcat ttcgttactt ttgatatcgc 4080 tcacaactat tgcgaagcgc ttcagtgaaa aaatcataag gaaaagttgt aaatattatt 4140 ggtagtattc gtttggtaaa gtagaggggg taatttttcc cctttatttt gttcatacat 4200 tcttaaattg ctttgcctct ccttttggaa agctatactt cggagcactg ttgagcgaag 4260 gctcaggccg gcagcacgca gcacgctgta tttacgtatt taattttata tatttgtgca 4320 tacactacta gggaagactt gaaaaaaacc taggaaatga aaaaacgaca caggaagtcc 4380 cgtatttact attttttcct tccttttgat ggggcagggc ggaaatagag gataggataa 4440 gcctactgct tagctgtttc cgtctctact tcggtagttg tctcaattgt cgtttcagta 4500 ttacctttag agccgctaga cgatggttga gctatttgtt gagggaaaac taagttcatg 4560 taacacacgc ataacccgat taaactcatg aatagcttga ttgcaggagg ctggtccatt 4620 ggagatggtg ccttattttc cttataggca acgatgatgt cttcgtcggt gttcaggtag 4680 tagt 4684 <210> 26 <211> 4909 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus delemar GA Integration Construct <400> 26 ctaaattcgg ccttgctcag agactcctgg attttggcta acaacgcagt cccttcgatg 60 catatagcta ggccacaaat tatgccaata acggtccatg ggttgatgtt ttcttgaatt 120 ctttcgtttt tcatgctatt tgcgtcttcc caagtcccag cgttccagta ttcatactgc 180 gcgttagagt ggtagccata agagccggca tattggtaat tttcagtatt aacgttagaa 240 cgtggtgaat acgatgtggt ccagccttgc ctcgttgtgt entaacgat ctttttcttt 300 gggtcacaaa gaatatcata tgcttgagag atgactttaa atctatgtag tttttcgctt 360 gatgttagca gcagcggtga tttactatca ctgttggtaa ccttttctga gctaaatatt 420 tgaatgttat cggaatggtc agggtggtac aattttacat aacgatgata tttttttttt 480 aacgacttct tgtccagttt aggatttcca gatccggcct ttggaatgcc aaaaatatca 540 tagggagttg gatctgccaa ctcaggccat tgttcatccc ttatcgtaag ttttctattg 600 ccatttttat cgttcgctgt agcatactta gctataaaag tgatttgtgg gggacacttt 660 tctacacatg ataagtgcca cttgaataaa aatgggtata cgaacttatg gtgtagcata 720 acaaatatat tgcaagtagt gacctatggt gtgtagatat acgtacagtt agttacgagc 780 ctaaagacac aacgtgtttg ttaattatac tgtcgctgta atatcttctc ttccattatc 840 accggtcatt ccttgcaggg gcggtagtac ccggagaccc tgaacttttc tttttttttt 900 tgcgaatta aaaagttcat tttcaattcg acaatgagat ctacaagcca ttgttttatg ttgatgagag ccagcttaaa gagttctcga gatctcccga gtttatcatt atcaatactg ccatttcaa gathering father gtagtgatt tcctaacttt father aaaattggcc ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 taatggagcc cgcttttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacacaga acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcttg gagtaatga tgacacaagg caattgacct acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaaagctga aaaaaaaggt tgaaaccagt tccctgaat tattccccta tttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt tagtttaaaa caccaagaac ttagtttcga grandfather grandfather aaatctaga tgcagttatt caacttacca cttaaggtat ctttctttct agtcttatct tactttcat tgttagtatc agctgcctct attack cagcatccgt acaactagat tcatacaatt acgacggttc aacattctca ggaagatat acgtgaaaaa tattgcttac agcaaaaagg ttactgtgat ttacgcagat gggtcagaca actggaata caatggaaac acaattgctg cttcctattc tgcccctatt tctggatcta actacgaata ctggactttt tcagcgagta taacggaat taaggaattc tatatcaaat atgaagtctc tggtaagacc tactacgata acaacaactc cgcaaactc caagttagca catcaaagcc aaccacaaca actgctactg cgacaactac aaccgcacca agcacttcta ctacaacacc tcctagttca tctgagccag caactttccc aactggtaat tccactattt cttcttggat caaaaaacaa gagggtatct caagattcgc catgcttaga fathercaatc ctccaggctc tgcaacagga ttcattgcag catctttatc aactgcgggg ccagactact actacgcctg gactagagat gcagctttga 2340 catcaaatgt gattgtttat gaatacaaca caactttgtc cggtaacaag acaatcttga 2400 acgtcttgaa ggattatgtg acattctctg tcaagactca atctacatca acagtttgta 2460 actgtctcgg cgaaccaaag ttcaaccctg atggtagtgg ttacactggt gcttggggta 2520 gaccacaaaa cgatggtcca gcagagagag ctacaacttt catcttgttt gctgactctt 2580 acctaacaca aaccaaggat gcaagctacg ttactggaac actaaagcct gcaatcttta 2640 aagacctgga ctatgttgta aacgtttggt caaatggctg cttcgatcta tgggaggaag 2700 tgaacggtgt tcacttctac acattaatgg tcatgagaaa gggactcttg cttggtgcag 2760 actttgctaa gagaaacggt gattctacac gtgcctccac ttactcctcc acagcttcaa 2820 ccattgccaa caaaatctct tctttctggg tcagctcaaa taactggatt caagtttctc 2880 aatcagttac tggtggtgtt tctaaaaagg gcctggatgt gtcaaccttg cttgctgcca 2940 atttgggcag tgttgatgac gggttcttca ccccaggttc tgaaaagatc ctcgccaccg 3000 cagttgccgt tgaagattca tttgctagtt tatacccaat caacaaaat ctaccatcat 3060 accttggaaa ttcaatcggt agatccag agatacata caacggtaat ggaactctc 3120 agggtaaccc ttggttctt gcagttacag ggtacgctga actgtactac agagcgatta 3180 aggaatggat tggtaatggc ggcgtactg ttagttcttat ttcttacct ttctcaaaa agttcgatag ttctgcaca tctggtaaaa agtacacagt cggcacttcc gattttaca 3300 atttagctca gaacatagca ctggcagctg atcgtttctt gagtacagtc caatgcatg 3360 cccataacaa cggtagtttg gctgaagagt tgatagaac caccggttta tcaccggcg 3420 ccagagattt aacatgtcc catgcgtctt tgatactgc ttcttacgcc aaggctgggg 3480 caccagctgc ctgattaatt aaacaggccc cttttccttt gtcgatatca tgtattagt 3540 tatgtcacgc ttacattcac gccctcctcc cacatccgct ctaccgaaa aggaggt 3600 tagacaacct gaagtctagg tccctattta tttttta gttatgttag tattagaac 3660 gttattatta ttcaattt ttcttttt tctgtacaa cgcgtgtacg catgtaacgg 3720 gcagacggcc ggccataact tcgtataatg tatgctatac gaagttatgg caacggttca 3780 tcatctcatg gatctgcaca tgaacaaaca ccagagtcaa acgacgttga aattgaggct 3840 actgcgccaa ttgatgacaa tacagacgat gataacaaac cgaagttatc tgatgtagaa 3900 aaggattaga gatgctaaga gatagtgatg atatttcata aataatgtaa ttctatatat 3960 gttaattacc ttttttgcga ggcatattta tggtgaagga taagttttga ccatcaaaga 4020 aggttaatgt ggctgtggtt tcagggtcca taaagctttt caattcatct tttttttttt 4080 tgttcttttt tttgattccg gtttctttga aatttttttg attcggtaat ctccgagcag 4140 aaggaagaac gaaggaagga gcacagactt agattggtat atatacgcat atgtggtgtt 4200 gaagaaacat gaaattgccc agtattcttta acccaactgc acagaacaaa aacctgcagg 4260 aaacgaagat aaatcatgtc gaaagctaca tataaggaac gtgctgctac tcatcctagt 4320 cctgttgctg ccaagctatt tatatcatg cacgaaaagc aaacaaactt gtgtgcttca 4380 ttggatgttc gtaccaccaa ggaattactg gagttagttg aagcattagg tcccaaaatt 4440 tgtttactaa aaacacatgt ggatatcttg actgattttt ccatggaggg cacagttaag 4500 ccgctaaagg cattatccgc caagtacaat tttttactct tcgaagacag aaaatttgct 4560 gacattggta atacagtcaa attgcagtac tctgcgggtg tatacagaat agcagaatgg 4620 gcagacatta cgaatgcaca cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag 4680 gcggcggaag aagtaacaaa ggaacctaga ggccttttga tgttagcaga attgtcatgc 4740 aagggctccc tagctactgg agaatatact aagggtactg ttgacattgc gaagagcgac 4800 aaagattttg ttatcggctt tattgctcaa agagacatgg gtggaagaga tgaaggttac 4860 gattggttga ttatgacacg caggttacga ttggttgatt atgacacgc 4909 <210> 27 <211> 4824 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus delemar GA Integration Construct <400> 27 ggccgctcca tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt 60 ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct 120 gcgggtgtat acagaatagc agaatgggca gatacga atgcacacgg tgtggtgggc 180 ccaggtattg ttagcggttt gaagcaggcg gcggagaag taacaagga acctagaggc 240 cttttgatgt tagcagaatt gtcatgcaag ggctccctag ctactggaga atactaag 300 ggtactgttg acatgcgaa gagcgacaa gattttgtta tcggctttat tgctcaaga 360 gatagggtg gaagagatga aggttacgat tggttgatta tgacacccgg tgtggtttta 420 gatgacaagg gagacgcatt gggtcacag tatagaaccg tggatgt ggtctctaca 480 ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaggta 540 gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagagatg cggccagcaa 600 aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaat tagagcttca 660 atttaattat atcagttatt acccgggaat ctcggtcgta atgattttta taatgacgaa 720 aaaaaaaaaaaaaaaggaaaaagctcat gggcttata aaaaggaacc atccaatacc 780 tcgccagaac caagcacag tattttacgg ggcacaaatc aagaacaata agacaggact 840 gtaaagatgg acgcattgaa ctccaaagaa siacaaggt tccaaaagt agtgaacaa 900 aagcaaatga aggatttcat gcgtttgata acttcgtata atgtatgcta tacgaagtta 960 tctcgagggc cagaaaaagg aagtgttcc ctccttctg aattgatgtt accctcataa 1020 agcacgtggc ctcttatcga gaagaaaatt accgtcgctc gtgatttgtt tgcaaaaga 1080 aaaaactga aaaaacccag acacgctcga cttcctgtct tcctgttgat tgcagcttcc 1140 aatttcgtca cacaacagg tcctagcgac ggctcacagg tttgtaaca agcaatcgaa 1200 ggttctggaa tggcgggaa gggtttagta ccacatgcta tgatgcccac tgtgatctcc 1260 agagcaagt tcgttcgatc gtactgttac tctctctt tcaacagaa ttgtccgaat 1320 cgtgtgacaa caacagcctg ttctcacaca ctctttctt ctaaccaagg gggtggttta 1380 gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat aaattggtca 1440 atgcaagaaa tacatatttg gtcttttcta attcgtagtt ttcaagttc ttagatgctt 1500 tctttttctc ttttcag atcatcagg aagtattat ctacttttta caagtctaga 1560 atgcagctgt tcaacttgcc attaaaggtt tcattctttt tggtcctatc atactttagt ttgttggtgt cagccgcatc tattccatct tcagcatctg tacaattaga ctcctacaat 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740 gtaactgtta tctatgccga cggatcagat aactggaaca acaatggaa cactatcgct gccagttact ctgcaccaat ttcaggttct aactacgaat attggacatt ctcagcctcc atcaatggca ttaaggaatt ctacataaag tacgaagttt ccggtaagac ttactacgat aacaacaatt ctgcaaacta tcaagtatca acatcaaaac ctactaccac caccgccaca gctacaacta caactgcacc ttcaacatct accacaaccc caccatcttc tagcgaacca gctacattcc caactggcaa ttctactatt tctagttgga tcaaaaaaca agagggtatt tccagattcg caatgttgag aaacataaat ccaccaggat cagcaactgg attcatcgca gcttctttgt ccacagcggg gccagattac tactacgcat ggaccagaga tgctgctttg 2220 acaagtaacg ttattgttta cgaacaat accactttgt ccggtaacaa gactattctt aacgtcctaa aggattacgt tacattctct gttaagactc agtctacatc cacagtctgc 2340 aattgtttgg gtgaaccaaa gttcaaccca gatggctctg gatacacagg tgcctggggt 2400 cgtccacaaa acgatgggcc tgccgagaga gccactacat ttatcctatt tgctgactca 2460 taccttacac aaacaaaaga tgcatcctac gtgactggaa cattaaagcc tgcaatcttc 2520 aaagacctgg attacgttgt caacgtgtgg tctaacggct gttcgatct atgggaagag 2580 gttaacggcg tgcacttcta cactctaatg gtcatgagaa agggtctgtt gttaggtgca 2640 gattttgcta agagaaacgg tgattctaca cgtgcttcta cctactcctc aacagcatca 2700 actattgcga acaagatttc ttcattttgg gtttcaagta ataactggat acaagtatct 2760 caaagcgtta cagggggtgt ctcaaaaaag ggtcttgatg tttctacatt actggctgct 2820 aatcttgggt ctgttgatga cggttctttc acccctggtt ctgaaaagat cctcgctacc 2880 gccgtcgcgg ttgaggatag ttttgcttca ctctatccta taaacaaaaa ccttccttca 2940 tacttaggaa acagtatcgg tagataccca gaggatacat acaatggtaa tggcaattca 3000 cagggaaatc catggttcct tgctgttaca gggtacgcag aacttacta tagagctatt 3060 aaggaatgga tcggcaacgg cggtgtgaca gtttcctcaa tctcattgcc atttttcaaa 3120 aagtttgact ccagcgcgac atctggtaaa aagtatactg tggggacttc tgatttcaac 3180 aatttggctc aaaacattgc cttagctgcc gacagattct tatctaccgt acaactccat 3240 gcacataaca atggtagttt ggcagaggaa tttgatagaa ctacaggact ctctacaggt 3300 gcgagagatt taacttggtc acatgcaagt ttaattacag cctcttacgc aaaggctggt 3360 gctcctgctg cataattaat taatttacca gcttactatc cttcttgaaa atatgcactc 3420 tatatctttt agttcttaat tgcaacacat agatttgctg tataacgaat tttatgctat 3480 ttttttaatt tggagttcgg tgatgaaagt gtcacagcga atttcctcac atgtagggac 3540 cgaattgttt acaagttctc tgtaccacca tggagacatc aaagattgaa aatctatgga 3600 aagatatgga cggtagcaac aagaatatag cacgagccgc ggagttcatt tcgttacttt 3660 tgatatcgct cacaactatt gcgaagcgct tcagtgaaaa aatcataagg aaaagttgta 3720 aatattattg gtagtattcg tttggtaaag tagagggggt aatttttccc ctttattttg 3780 ttcatacatt cttaaattgc tttgcctctc cttttggaaa gctatacttc ggagcactgt 3840 tgagcgaagg ctcaggccgg cagcacgcag cacgctgtat ttacgtattt aattttatat 3900 atttgtgcat acactactag ggaagacttg aaaaaaacct aggaaatgaa aaaacgacac 3960 aggaagtccc gtatttacta ttttttcctt ccttttgatg gggcagggcg gaaatagagg 4020 ataggataag cctactgctt agctgtttcc gtctctactt cggtagttgt ctcaattgtc 4080 gtttcagtat tacctttaga gccgctagac gatggttgag ctatttgttg agggaaaact 4140 aagttcatgt aacacacgca taacccgatt aaactcatga atagcttgat tgcaggaggc 4200 tggtccattg gagatggtgc cttattttcc ttataggcaa cgatgatgtc ttcgtcggtg 4260 ttcaggtagt agtgtacact ctgaatcagg gagaaccagg caatgaactt gttcctcaag 4320 aaaatagcgg ccataggcat ggattggtta accacaccag atatgcttgg tgtggcagaa 4380 tatagtcctt ttggtggcgc aattttcttg tacctgtggt agaaagggag cggttgaact 4440 gttagtatat attggcaata tcagcaaatt tgaaagaaaa ttgtcggtga aaaacatacg 4560. aaacacaaag gtcggggcctt gcaacgttat tcaaagtcat tgtttagttg aggaggtagc agcggagtat atgtattcct tttttttgcc tatggatgtt gtaccatgcc cattctgctc 4620 aagcttttgt taaaattatt tttcagtatt ttttcttcca tgttgcgcgt tacgagaaca 4740. 4740. 4740. 4740. 4740. 4740. 4740. aactaagcca agatcacaac agttatcgat aaaatagcag tgtttgcatg gccattgaga aggacaacat tggcgtgcgg catg <210> 28 <211> 5264 <212> DNA <213> The snowstorm <220> <223> Honeysuckle(Rhizopus delemar) GA snow leopard <400> 28 ctaaattcgg ccttgctcag agactcctgg attttggcta acaacgcagt cccttcgatg catatagcta ggccacaaat tatgccaata acggtccatg ggttgatgtt ttcttgaatt ctttcgtttt tcatgctatt tgcgtcttcc caagtcccag cgttccagta ttcatactgc 180 gcgttagagt ggtagccata agagccggca tattggtaat tttcagtatt aacgttagaa 240 cgtggtgaat acgatgtggt ccagccttgc ctcgttgtgt entaacgat ctttttcttt 300 gggtcacaaa gaatatcata tgcttgagag atgactttaa atctatgtag tttttcgctt 360 gatgttagca gcagcggtga tttactatca ctgttggtaa ccttttctga gctaaatatt 420 tgaatgttat cggaatggtc agggtggtac aattttacat aacgatgata tttttttttt 480 aacgacttct tgtccagttt aggatttcca gatccggcct ttggaatgcc aaaaatatca 540 tagggagttg gatctgccaa ctcaggccat tgttcatccc ttatcgtaag ttttctattg 600 ccatttttat cgttcgctgt agcatactta gctataaaag tgatttgtgg gggacacttt 660 tctacacatg ataagtgcca cttgaataaa aatgggtata cgaacttatg gtgtagcata 720 acaaatatat tgcaagtagt gacctatggt gtgtagatat acgtacagtt agttacgagc 780 ctaaagacac aacgtgtttg ttaattatac tgtcgctgta atatcttctc ttccattatc 840 accggtcatt ccttgcaggg gcggtagtac ccggagaccc tgaacttttc tttttttttt 900 tgcgaatta aaaagttcat tttcaattcg acaatgagat ctacaagcca ttgttttatg ttgatgagag ccagcttaaa gagttctcga gatctcccga gtttatcatt atcaatactg ccatttcaa gathering father gtagtgatt tcctaacttt father aaaattggcc ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 taatggagcc cgcttttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacacaga acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcttg gagtaatga tgacacaagg caattgacct acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaaagctga aaaaaaaggt tgaaaccagt tccctgaat tattccccta tttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt tagtttaaaa caccaagaac ttagtttcga grandfather grandfather aaatctaga tgcagttatt caacttacca cttaaggtat ctttctttct agtcttatct tactttcat tgttagtatc agctgcctct attack cagcatccgt acaactagat tcatacaatt acgacggttc aacattctca ggaagatat acgtgaaaaa tattgcttac agcaaaaagg ttactgtgat ttacgcagat gggtcagaca actggaata caatggaaac acaattgctg cttcctattc tgcccctatt tctggatcta actacgaata ctggactttt tcagcgagta taacggaat taaggaattc tatatcaaat atgaagtctc tggtaagacc tactacgata acaacaactc cgcaaactc caagttagca catcaaagcc aaccacaaca actgctactg cgacaactac aaccgcacca agcacttcta ctacaacacc tcctagttca tctgagccag caactttccc aactggtaat tccactattt cttcttggat caaaaaacaa gagggtatct caagattcgc catgcttaga fathercaatc ctccaggctc tgcaacagga ttcattgcag catctttatc aactgcgggg ccagactact actacgcctg ccagagat gcagctttga catcaaatgt gattgtttat gaatacaaca caactttgtc cggtaacaag acaatcttga 2400 acgtcttgaa ggattatgtg acattctctg tcaagactca atctacatca acagtttgta 2460 actgtctcgg cgaaccaaag ttcaaccctg atggtagtgg ttacactggt gcttggggta 2520 gaccacaaaa cgatggtcca gcagagagag ctacaacttt catcttgttt gctgactctt 2580 acctaacaca aaccaaggat gcaagctacg ttactggaac actaaagcct gcaatcttta 2640 aagacctgga ctatgttgta aacgtttggt caaatggctg cttcgatcta tgggaggaag 2700 tgaacggtgt tcactttac acattaatgg tcatgagaaa gggactcttg cttggtgcag 2760 actttgctaa gagaaacggt gattctacac gtgcctccac ttactcctcc acagcttcaa 2820 ccattgccaa caaaatctct tctttctggg tcagctcaaa taactggatt caagtttctc 2880 aatcagttac tggtggtgtt tctaaaaagg gcctggatgt gtcaaccttg cttgctgcca 2940 atttgggcag tgttgatgac gggttcttca ccccaggttc tgaaaagatc ctcgccaccg 3000 cagttgccgt tgaagattca tttgctagtt tatacccaat caacaaaaat ctaccatcat 3060 accttggaaa ttcaatcggt agatccag agatacata caacggtaat ggaactctc 3120 agggtaaccc ttggttctt gcagttacag ggtacgctga actgtactac agagcgatta 3180 aggaatggat tggtaatggc ggcgtactg ttagttcttat ttcttacct ttctcaaaa agttcgatag ttctgcaca tctggtaaaa agtacacagt cggcacttcc gattttaca 3300 atttagctca gaacatagca ctggcagctg atcgtttctt gagtacagtc caatgcatg 3360 cccataacaa cggtagtttg gctgaagagt tgatagaac caccggttta tcaccggcg 3420 ccagagattt aacatgtcc catgcgtctt tgatactgc ttcttacgcc aaggctgggg 3480 caccagctgc ctgattaatt aaacaggccc cttttccttt gtcgatatca tgtattagt 3540 tatgtcacgc ttacattcac gccctcctcc cacatccgct ctaccgaaa aggaggt 3600 tagacaacct gaagtctagg tccctattta tttttta gttatgttag tattagaac 3660 gttattatta ttcaattt ttcttttt tctgtacaa cgcgtgtacg catgtaacgg 3720 gcagacggcc ggccatact tcgtataatg tatgctatac gaagttatcc ttacatcaca 3780 cccaatcccc cacaagtgat ccccacaca ccatagcttc aaaatgtttc tactcctttt 3840 ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc 3900 acagcatact aaatttcccc tctttcttcc tctagggtgg cgttaattac ccgtactaaa 3960 ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa 4020 aaatttttat cacgtttctt tttcttgaaa aatttttttt ttgatttttt tctctttcga 4080 tgacctccca ttgatattta agttaataaa tggtcttcaa tttctcaagt ttcagtttcg 4140 tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa 4200 tctaatctaa gttttaatta caaaatgcca caatcctggg aagaattggc cgccgacaaa 4260 cgtgcccgtt tggctaaaac cattcctgac gaatggaagg ttcaaacttt gcctgccgaa 4320 gattccgtta ttgatttccc aaagaagtcc ggtattttgt ctgaggctga attgaagatt 4380 accgaagcct ctgctgctga tttggtctcc aagttggccg ctggtgagtt gacttctgtt 4440 gaagtcactt tggctttttg taagagagct gctattgctc aacaattaac caactgtgct 4500 cacgaattct tcccagatgc tgctttagct caagctagag aattagatga atactacgct 4560 aagcataaga gaccagttgg tccattacac ggtttaccaa tctctttaaa ggaccaattg 4620 cgtgttaagg gttacgaaac ctccatgggt tacatttcct ggttaaacaa atacgatgaa 4680 ggtgattccg tcttaaccac catgttgaga aaagctggtg ctgttttcta cgttaagacc 4740 tctgtcccac aaaccttgat ggtctgtgaa accgtcaaca acatcattgg tagaactgtc 4800 aatccaagaa acaaaaattg gtcctgtggt ggttcttctg gtggtgaagg tgctattgtt 4860 ggtattagag gtggtgttat tggtgtcggt actgacattg gtggttccat tagagtccca 4920 gctgctttca actttttata cggtttgaga ccatctcacg gtagattgcc atatgctaaa 4980 atggctaact ctatggaagg tcaagaaacc gttcactccg tcgttggtcc tatcactcac 5040 tccgtcgaag acttgagatt gttcaccaaa tctgtcttgg gtcaagaacc ttggaagtac 5100 gactctaagg tcatccccat gccatggaga caatctgaat ctgacatcat tgcctctaag 5160 attaagaatg gtggtttgaa cattggttat tacaatttcg acggtaacgt cttgccacac 5220 ccaccaattt tacgtggtgt cgaaactacc gttgccgctt tggc 5264 <210> 29 <211> 5026 <212> DNA <213> Artificial Sequence <220> <223> Rhizopus delemar GA Integration Construct <400> 29 ggccgcgaag gtgctattgt tggtattaga ggtggtgtta ttggtgtcgg tactgacatt 60 ggtggttcca ttagagtccc agctgctttc aactttttat acggtttgag accatctcac 120 ggtagattgc catatgctaa aatggctaac tctatggaag gtcaagaaac cgttcactcc 180 gtcgttggtc ctatcactca ctccgtcgaa gacttgagat tgttcaccaa atctgtcttg 240 ggtcaagaac cttggaagta cgactctaag gtcatcccaa tgccatggag acaatctgaa 300 tctgacatca ttgcctctaa gattaagaat ggtggtttga acattggtta ttacaatttc 360 gacggtaacg tcttgccaca cccaccaatt ttacgtggtg tcgaaactac cgttgccgct 420 ttggccaagg ctggtcacac cgttactcca tggactccat acaagcatga tttcggtcat 480 gacttgattt cccacatcta tgctgctgat ggttctgccg acgtcatgag agacatttct 540 gcctctgtg agccagccat ccctaacatt aaggacttgt tgaacccaaa tattaaggct 600 gttaacatga acgaattgtg ggacactcat ttacaaagt ggaactatca aatggaatc 660 ttggaaagt ggcgtgaagc tgaagaaaa gctggtagg aattggacgc tattatcgct 720 ccaattactc ctaccgccgc tgtcagacac gatcaatca gatacgg ttacgcctcc 780 gttattaact tattggatt cacctctgtt gtcgtcccag tcacttcgc tgataagaat 840 attgataaga agaacgaatc ttttaaagct gtttccgaat tggatgcttt ggttcaagaa 900 gatacgacc cagaggctta tcacggtgct cctgttgctg ttcaagttat tggtagaaga 960 ttgtccgaag agagacttt ggctatcgcc gagaagtcg gtaattgtt gggtaacgtc 1020 gtcactccat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa 1080 aaaatagtg tatacaatt ttaagtgac tcttaggtttt taaaacgaaa attcttattc 1140 ttgagtact ctttcctgta ggtcaggttg cttctcagg tagcatga ggtcgctctt 1200 attgaccaca cctctaccgg catgccgagc aaatgcctgc aaatcgctcc ccattcacc 1260 caattgtaga tatgctaact ccagcaatga gttgatgaat ctcggtgtgt attttatgtc 1320 ctcagaggac aacacataac ttcgtataat gtatgctata cgaagttatc tcgagggcca 1380 gaaaaaggaa gtgtttccct ccttcttgaa ttgatgttac cctcataaag cacgtggcct 1440 cttatcgaga aagaaattac cgtcgctcgt gatttgtttg caaaaagaac aaaactgaaa 1500 aaacccagac acgctcgact tcctgtcttc ctgttgattg cagcttccaa tttcgtcaca 1560 caacaaggtc ctagcgacgg ctcacaggtt ttgtaacaag caatcgaagg ttctggaatg 1620 gcgggaaagg gtttagtacc acatgctatg atgcccactg tgatctccag agcaaagttc 1680 gttcgatcgt actgttactc tctctctttc aaacagaatt gtccgaatcg tgtgacaaca 1740 acagcctgtt ctcacacact cttttcttct aaccaagggg gtggtttagt ttagtagaac 1800 ctcgtgaaac ttacatttac atatatataa acttgcataa attggtcaat gcaagaaata 1860 catatttggt cttttctaat tcgtagtttt tcaagttctt agatgctttc tttttctctt 1920 ttttacagat catcaaggaa gtaattatct actttttaca agtctagaat gcagctgttc 1980 aacttgccat taaaggtttc attctttttg gtcctatcat actttagttt gttggtgtca 2040 gccgcatcta ttccatcttc agcatctgta caattagact cctacaatta cgacggctct 2100 acattcagcg gaaagattta cgtgaaaaat attgcgtaca gcaaaaaagt aactgttatc 2160 tatgccgacg gatcagataa ctggaacaac aatggaaaca ctatcgctgc cagttactct 2220 gcaccaattt caggttctaa ctacgaatat tggacattct cagcctccat caatggcatt 2280 aaggaattct acataaagta cgaagtttcc ggtaagactt actacgataa caacaattct 2340 gcaaactatc aagtatcaac atcaaaacct actaccacca ccgccacagc tacaactaca 2400 actgcacctt caacatctac cacaacccca ccatcttcta gcgaaccagc tacattccca 2460 actggcaatt ctactatttc tagttggatc aaaaaacaag agggtatttc cagattcgca 2520 atgttgagaa acataaatcc accaggatca gcaactggat tcatcgcagc ttctttgtcc 2580 acagcggggc cagattacta ctacgcatgg accagagatg ctgctttgac aagtaacgtt 2640 attgtttacg aatacaatac cactttgtcc ggtaacaaga ctattcttaa cgtcctaaag 2700 gattacgtta cattctctgt taagactcag tctacatcca cagtctgcaa ttgtttgggt 2760 gaaccaaagt tcaacccaga tggctctgga tacacaggtg cctggggtcg tccacaaaac 2820 gatgggcctg ccgagagagc cactacattt atcctatttg ctgactcata ccttacacaa 2880 acaaaagatg catcctacgt gactggaaca ttaaagcctg caatcttcaa agacctggat 2940 tacgttgtca acgtgtggtc taacggctgt ttcgatctat gggaagaggt taacggcgtg 3000 cacttctaca ctctaatggt catgagaaag ggtctgttgt taggtgcaga ttttgctaag 3060 agaaacggtg attctacacg tgcttctacc tactcctcaa cagcatcaac tattgcgaac 3120 aagatttctt cattttgggt ttcaagtaat aactggatac aagtatctca aagcgttaca 3180 gggggtgtct caaaaaaggg tcttgatgtt tctacattac tggctgctaa tcttgggtct 3240 gttgatgacg gtttcttcac ccctggttct gaaaagatcc tcgctaccgc cgtcgcggtt 3300 gaggatagtt ttgcttcact ctatcctata aacaaaaacc ttccttcata cttaggaaac 3360 agtatcggta gatacccaga ggatacatac aatggtaatg gcaattcaca gggaaatcca 3420 tggttccttg ctgttacagg gtacgcagaa cttactata gagctattaa ggaatggatc 3480 ggcaacggcg gtgtgacagt ttcctcaatc tcattgccat tttcaaaaa gtttgactcc 3540 agcgcgacat ctggtaaaaa gtatactgtg gggacttctg atttcaacaa ttggctcaa 3600 aacattgcct tagctgccga cagattctta tctaccgtac aactccatgc acataacaat 3660 ggtagtttgg cagaggaatt tgatagaact acaggactct ctacaggtgc gagagattta 3720 acttggtcac atgcaagttt aattacagcc tcttacgcaa aggctggtgc tcctgctgca 3780 taattaatta atttaccagc tactatcct tcttgaaaat atgcactcta tatcttttag 3840 ttcttaattg caacacatag atttgctgta taacgaattt tatgctattt ttttaatttg 3900 gagttcggtg atgaaagtgt cacagcgaat ttcctcacat gtagggaccg aattgtttac 3960 aagttctctg taccaccatg gagacatcaa agattgaaaa tctatggaaa gatatggacg 4020 gtagcaacaa gaatatagca cgagccgcgg agttcatttc gttacttttg atacgctca 4080 caactattgc gaagcgcttc agtgaaaaaa tcataaggaa aagttgtaaa tattattggt 4140 agtattcgtt tggtaaagta gagggggtaa tttttcccct ttattttgtt catacattct taattgctt tgcctctcct tttggaaagc tatacttcgg agcactgttg agcgaaggct caggccggca gcacgcagca cgctgtattt acgtatttaa ttttatatat ttgtgcatac 4320. actactaggg aagacttgaa aaaaacctag gaatgaaaa aacgacacag gaagtcccgt atttactatt ttttccttcc ttttgatggg gcagggcgga aatagaggat aggataagcc 4440 tactgcttag ctgtttccgt ctctacttcg gtagttgtct caattgtcgt ttcagtatta cctttagagc cgctagacga tggttgagct atttgttgag ggaaaactaa gttcatgtaa cacacgcata acccgataa actcatgaat agcttgattg caggaggctg gtccattgga gatggtgcct tattttcctt ataggcaacg atgatgtctt cgtcggtgtt caggtagtag 4680 tgtacactct gaatcaggga gaccaggca atgaacttgt tcctcaaga aatagcggcc attackcatgg attggttaac cacaccagat atgcttggtg tggcagaata tagtcctttt ggtggcgcaa ttttcttgta cctgtggtag aaagggagcg gttgaactgt tagtatatat 4860. tggcaatatc agcaaatttg aaagaaaatt gtcggtgaaa aacatacgaa acacaaaggt 4920 cgggccttgc aacgttattc aaagtcattg tttagttgag gaggtagcag cggagtatat 4980 gtattccttt tttttgccta tggatgttgt accatgccca ttctga 5026

Claims

1. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, wherein the exogenous nucleic acid encodes the sequence of SEQ ID NO:

1.

2. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, wherein the exogenous nucleic acid is selected from the following sequences: SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:

25.

3. The engineered yeast of claim 1 or 2, wherein the yeast is capable of producing ethanol at a rate of 1 g / L*h or higher during fermentation.

4. The engineered yeast of claim 1 or 2, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from (i) a feedstock containing glucose polymers having a DE of no greater than 50.

5. The engineered yeast of claim 1 or 2, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from corn mash with a DE of 30+ / -2, wherein the fermentation medium comprises 32% by dry weight corn and a pH of 5.8, 35 ppm calcium chloride, 1900 ppm urea, 5 ppm ampicillin, wherein the starting yeast concentration is 0.1 (OD600), and the fermentation is carried out at 30°C with stirring for 48 hours.

6. The engineered yeast of claim 1 or 2, wherein 2 to 8 copies of the exogenous nucleic acid are present in the cell.

7. The engineered yeast according to claim 1 or 2, which is Saccharomyces cerevisiae.

8. The engineered yeast according to claim 1 or 2, which is tolerant to growth in a fermentation medium with an ethanol concentration greater than 90 g / L.

9. The engineered yeast of claim 1 or 2, which is tolerant to growth at a temperature in the range of greater than 31°C to 35°C.

10. A fermentation method for producing a bioproduct, the fermentation method comprising: forming a fermentation medium from a feedstock containing a glucose polymer; and fermenting the fermentation medium using an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, wherein the exogenous nucleic acid encodes the sequence of SEQ ID NO:

1.

11. A fermentation method for producing a bioproduct, the fermentation method comprising: Forming a fermentation medium from a feedstock containing glucose polymers; and fermenting the fermentation medium using an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, wherein the exogenous nucleic acid is selected from the following sequences: SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:

25.

12. The fermentation process of claim 10 or 11, wherein the fermentation produces a bioproduct comprising ethanol produced at a rate of 1 g / L*h or more. 13 . The fermentation method according to claim 10 , wherein the glucose polymer-containing raw material or the fermentation medium has a DE of 50 or less at the start of fermentation.

14. The fermentation method according to claim 10 or 11, wherein the glucose polymer-containing feedstock comprises glucose polymers having a degree of polymerization of 4 or more and present in an amount of 75 wt% or more of total fermentable carbohydrates in the feedstock.

15. The fermentation method according to claim 10 or 11, wherein ethanol is produced in the culture medium at a concentration of 70 g / L or more.

16. The fermentation process of claim 10 or 11, comprising adding a supplemental glucoamylase to the feedstock during the fermentation, or adding a supplemental glucoamylase to the culture medium.

17. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, wherein the exogenous nucleic acid encodes the sequence of SEQ ID NO:

4.

18. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase, wherein the exogenous nucleic acid is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:

29.

19. The engineered yeast of claim 17 or 18, wherein the yeast is capable of producing ethanol at a rate of 1 g / L*h or higher during fermentation.

20. The engineered yeast of claim 17 or 18, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from a glucose polymer-containing feedstock having a DE of no more than 50.

21. The engineered yeast of claim 17 or 18, wherein the yeast is capable of producing (a) at least 70 g / kg of ethanol in a fermentation medium made from corn mash with a DE of 30, wherein the corn mash is present in a fermentation medium having 32 wt% corn mash and pH 5.8, 35 ppm calcium chloride, 1900 ppm urea, 5 ppm ampicillin, wherein the starting yeast concentration is 0.1 (OD600), and the fermentation is carried out at 30° C. with stirring for 48 hours.

22. The engineered yeast of claim 17 or 18, wherein 2 to 8 copies of the exogenous nucleic acid are present in the cell.

23. The engineered yeast of claim 22, wherein 2 to 6 copies of the exogenous nucleic acid are present in the cell.

24. The engineered yeast of claim 17 or 18, wherein four copies of the exogenous nucleic acid are present in the cell.

25. The engineered yeast of claim 17 or 18, wherein the exogenous nucleic acid is controlled by a promoter selected from the group consisting of a phosphoglycerate kinase (PGK) promoter nucleic acid sequence, a cytochrome c promoter (pCYC) promoter nucleic acid sequence, a translation elongation factor promoter (pTEF) promoter nucleic acid sequence, a 3-phosphoglyceraldehyde dehydrogenase promoter (pGPD / TDH3) promoter nucleic acid sequence, and an alcohol dehydrogenase promoter (pADH) promoter nucleic acid sequence.

26. The engineered yeast of claim 17 or 18, which is a species of the genus Saccharomyces.

27. The engineered yeast according to claim 26, which is Saccharomyces cerevisiae.

28. The engineered yeast of claim 17 or 18, which is tolerant to growth in a fermentation medium having an ethanol concentration greater than 90 g / L.

29. The engineered yeast of claim 17 or 18, which is tolerant to growth at a temperature in the range of greater than 31°C to 35°C.

30. The engineered yeast of claim 29, which is tolerant to growth at temperatures in the range of greater than 32°C to 34°C.

31. The engineered yeast of claim 17 or 18, which produces a greater amount of ethanol under the same fermentation conditions than a parent strain that does not contain the exogenous nucleic acid.

Images