Increasing Cellular Lipid Production by Increasing the Activity of Diacylglycerol Acyltransferase and Decreasing the Activity of

Abstract

Disclosed are methods and compositions for increasing the triacylglycerol content of a cell by up-regulating diacylglycerol acyltransferase and down-regulating triacylglycerol lipase. In some embodiments, a DGA1 protein is expressed and a native TGL3 gene is knocked out, thereby increasing the synthesis of triacylglycerol and decreasing its consumption, respectively.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 15/307,518, filed Oct. 28, 2016, now U.S. Pat. No. 10,443,047, which claims the benefit of PCT/US2015/028760 filed May 1, 2015, which claims priority from U.S. Provisional Application No. 61/987,098, filed May 1, 2014, and U.S. Provisional Application No. 62/090,169, filed Dec. 10, 2014, which are incorporated herein by reference in their entireties for all purposes.

BACKGROUND

[0002] Lipids have multiple industrial applications, including in the cosmetic and food industries, as well as serving as precursors for biodiesel and biochemical production. Microbial lipids are produced by many oleaginous organisms, including the well-characterized yeast Yarrowia lipolytica. Lipid yield in oleaginous organisms can be increased by up-regulation or down-regulation or deletion of genes implicated in the lipid pathway. For example, it was reported that up-regulation of native Y. lipolytica DGA1 significantly increased lipid yield and productivity (Metabolic Engineering 15:1-9 (2013)).

[0003] Y. lipolytica DGA1 is a type 2 diacylglycerol acyltransferase encoded by the Y. lipolytica diacylglycerol acyltransferase gene DGAT2. DGA1 is one of the key enzymes in the lipid pathway, and it is involved in the final step of triacylglycerol (TAG) synthesis. Triacylglycerols are the major form of storage lipids in Y. lipolytica. Recent data suggests that DGA1 efficiency may be a significant factor for high levels of lipid accumulation in oleaginous organisms (Metabolic Engineering 15:1-9 (2013)). Additionally, DGA1 genes from other species can be introduced into the host genome and have a significant effect on lipid production and composition. For example, other oleaginous yeast, such as Rhodosporidium toruloides and Lipomyces starkeyi, are able to accumulate significantly more lipids compared to wild type Y. lipolytica strains. It was demonstrated that overexpression in Y. lipolytica of DGA1 from organisms with higher native lipid production levels had a greater effect on Y. lipolytica lipid production than overexpression of native Y. lipolytica DGA1 (U.S. Ser. No. 61/943,664; incorporated by reference). Despite efforts to increase lipid yield in Y. lipolytica by overexpression of DGA1 from Mortierella alpine, no significant effect on lipid production levels has been reported (U.S. Pat. No. 7,198,937; incorporated by reference). The deletion of genes involved in the breakdown of lipids or in pathways that draw flux away from lipid biosynthesis has also been studied. Dulermo et al. demonstrated that the deletion of the triacylglycerol lipase gene TGL3 nearly doubled the total lipid content accumulated by Y. lipolytica (Biochimica Biophysica Acta 1831:1486-95 (2013)). The TGL3 protein is one of two intracellular lipases responsible for the first step of triacylglycerol degradation in Y. lipolytica.

SUMMARY

[0004] In some embodiments, the invention provides a transformed oleaginous cell comprising a first genetic modification, wherein said first genetic modification increases the activity of a native diacylglycerol acyltransferase or encodes at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species, and a second genetic modification, wherein said second genetic modification decreases the activity of a triacylglycerol lipase in the oleaginous cell.

[0005] In some aspects, the invention provides a method of increasing the lipid content of a cell comprising transforming a parent cell with a first nucleic acid and a second nucleic acid, wherein said first nucleic acid increases the activity of a native diacylglycerol acyltransferase or comprises a diacylglycerol acyltransferase gene and said second nucleic acid decreases the activity of a triacylglycerol lipase. Alternatively, the same nucleic acid could embody both elements described above. Thus, the invention also provides a method of increasing the lipid content of a cell comprising transforming a parent cell with a nucleic acid, wherein said nucleic acid decreases the activity of a triacylglycerol lipase in the cell, and said nucleic acid comprises a diacylglycerol acyltransferase gene or increases the activity of a native diacylglycerol acyltransferase.

[0006] Further, in some aspects, the invention provides a method of increasing the triacylglycerol content of a cell comprising: (a) providing a cell, comprising (i) a first genetic modification, wherein said first genetic modification increases the activity of a native diacylglycerol acyltransferase, or encodes at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species; and (ii) a second genetic modification that decreases the activity of a triacylglycerol lipase in the cell; (b) growing said cell under conditions whereby the first genetic modification is expressed, thereby producing a triacylglycerol; and (c) optionally recovering the triacylglycerol.

[0007] One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention. These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, drawings, and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 shows a map of the pNC243 construct used to overexpress the diacylglycerol acyltransferase DGA1 gene NG66 in Y. lipolytica strain NS18 (obtained from ARS Culture Collection, NRRL # YB 392). Vector pNC243 was linearized by PacI/NotI restriction digest before transformation. "2u ori" denotes the S. cerevisiae origin of replication from the 2 .mu.m circle plasmid; "pMB1 ori" denotes the E. coli pMB1 origin of replication from the pBR322 plasmid; "AmpR" denotes the bla gene used as a marker for selection with ampicillin; "PR2" denotes the Y. lipolytica GPD1 promoter -931 to -1; "NG66" denotes the native Rhodosporidium toruloides DGA1 cDNA synthesized by GenScript; "TER1" denotes the Y. lipolytica CYC1 terminator 300 base pairs after stop; "PR22" denotes the S. cerevisiae TEF1 promoter -412 to -1; "NG3" denotes the Streptomyces noursei Nat1 gene used as a marker for selection with nourseothricin; "TER2" denotes the S. cerevisiae CYC1 terminator 275 base pairs after stop; and "Sc URA3" denotes the S. cerevisiae URA3 auxotrophic marker for selection in yeast.

[0009] FIG. 2 is a graph depicting results from a fluorescence-based lipid assay for Y. lipolytica strain NS421, which is a triacylglycerol lipase knockout strain, and Y. lipolytica strain NS377, which is a triacylglycerol lipase knockout strain that overexpresses the diacylglycerol acyltransferase DGA1 gene NG66.

[0010] FIG. 3 is a graph depicting results from a gas chromatography analysis of Y. lipolytica strain NS281, which overexpresses the diacylglycerol acyltransferase DGA1 gene NG66, and Y. lipolytica strain NS377, containing a triacylglycerol lipase knockout and overexpressing the diacylglycerol acyltransferase DGA1 gene NG66.

[0011] FIG. 4 depicts a map of the pNC327 construct used to overexpress the NG112 gene (C. purpurea DGA2) in Y. lipolytica. Vector pNC327 was linearized by a PacI/AscI restriction digest before transformation. "2u ori" denotes the S. cerevisiae origin of replication from the 2 .mu.m circle plasmid; "pMB1 ori" denotes the E. coli pMB1 origin of replication from the pBR322 plasmid; "AmpR" denotes the bla gene used as a marker for selection with ampicillin; "PR3" denotes the Y. lipolytica TEF1 promoter -406 to +125; "NG112" denotes the C. purpurea DGA2 gene synthetized by GenScript; "TER1" denotes the Y. lipolytica CYC1 terminator 300 bp after stop; "PR1" denotes the Y. lipolytica TEF1 promoter -406 to -1; "NG76" denotes the Streptoalloteichus hindustanus BLE gene used as marker for selection with Zeocin; "TER7" denotes the Y. lipolytica TEF1 terminator 400 bp after stop; and "Sc URA3" denotes the S. cerevisiae URA3 auxotrophic marker for selection in yeast.

[0012] FIG. 5 comprises three panels, labeled pane (A), (B), and (C). The figure depicts lipid accumulation measured by a fluorescence-based assay or a percentage of the dry cell weight as determined by gas chromatography for Yarrowia lipolytica strains NS297, NS281, NS450, NS377, and NS432. NS297 expresses an additional copy of Y. lipolytica DGA1; NS281 expresses Rhodosporidium toruloides DGA1; NS450 expresses R. toruloides DGA1 and Claviceps purpurea DGA2; NS377 expresses R. toruloides and carries a deletion of Y. lipolytica TGL3; NS432 expresses R. toruloides DGA1 and C. purpurea DGA2 and carries a deletion of Y. lipolytica TGL3. In panel (A), strains were analyzed by fluorescence assay after 96 hours of fermentation in a 48-well plate where two or three transformants were analyzed for each construct. In panel (B), strains were analyzed by fluorescence assay and gas chromatography after 96 hours of fermentation in 50-mL flasks. In panel (C), strains were analyzed by gas chromatography after 140 hours of fermentation in 1-L bioreactors. Data for NS281, NS377, and NS432 are averages obtained from duplicate bioreactor fermentations. Data for NS450 represents the value obtained from a single bioreactor fermentation.

DETAILED DESCRIPTION

Overview

[0013] Disclosed are methods and compositions for creating transformed oleaginous cells with increased triacylglycerol content. Disrupting the yeast triacylglycerol lipase gene TGL3 eliminates a pathway that depletes triacylglycerol content. Additionally, overexpressing the diacylglycerol acyltransferase DGA1 increases the amount of protein that can synthesize triacylglycerol. Therefore, combining DGA1 overexpression with a TGL3 deletion could be an attractive approach to further increase a cell's triacylglycerol content; however, the manipulation of proteins that affect a metabolic pathway is unpredictable at best. Cells that suppress the effects of a genetic modification frequently possess a selective advantage over those that acquire the desired trait. Thus, cells that display a desired phenotype are often difficult to engineer.

[0014] Disclosed is the successful combination of TGL3 deletion and DGA1 overexpression to increase a cell's triacylglycerol content. TGL3 deletion and the concomitant expression of the R. toruloides DGA1 diacylglycerol acyltransferase in Y. lipolytica strains resulted in a higher triacylglycerol content than strains carrying a single genetic modification.

Definitions

[0015] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0016] The term "activity" refers to the total capacity of a cell to perform a function. For example, a genetic modification that decreases the activity of a triacylglycerol lipase in a cell may reduce the amount of triacylglycerol lipase in a cell or reduce the efficiency of triacylglycerol lipase. A triacylglycerol lipase knockout reduces the amount of triacylglycerol lipase in the cell. Alternatively, a mutation to a triacylglycerol lipase gene may reduce the efficiency of its triacylglycerol lipase protein product with little effect on the amount of cellular triacylglycerol lipase. Mutations that reduce the efficiency of triacylglycerol lipase may affect the active site, for example, by changing one or more active site residues; they may impair the enzyme's kinetics, for example, by sterically blocking substrates or products; they may affect protein folding or dynamics, for example, by reducing the proportion of properly-folded enzymes; they may affect protein localization, for example, by preventing the lipase from localizing to lipid particles; or they may affect protein degradation, for example, by adding one or more protein cleavage sites or by adding one or more residues or amino acid sequences that target the protein for proteolysis. These mutations affect coding regions. Mutations that decrease triacylglycerol lipase activity may instead affect the transcription or translation of the gene. For example, mutation to a triacylglycerol lipase enhancer or promoter can reduce triacylglycerol lipase activity by reducing its expression. Mutating or deleting the non-coding portions of a triacylglycerol lipase gene, such as its introns, may also reduce transcription or translation. Additionally, mutations to the upstream regulators of a triacylglycerol lipase may affect triacylglycerol lipase activity; for example, the over-expression of one or more repressors may decrease triacylglycerol lipase activity, and a knockout or mutation of one or more activators may similarly decrease triacylglycerol lipase activity. A genetic modification that increases the activity of a diacylglycerol acyltransferase in a cell may increase the amount of triacylglycerol acyltransferase in a cell or increase the efficiency of diacylglycerol acyltransferase. For example, the genetic modification may simply insert an additional copy of diacylglycerol acyltransferase into the cell such that the additional copy is transcribed and translated into additional functional diacylglycerol acyltransferase. The added diacylglycerol acyltransferase gene can be native to the host organism or from a different organism. Alternatively, mutating or deleting the non-coding portions of a native diacylglycerol acyltransferase gene, such as its introns, may also increase translation. A native diacylglycerol acyltransferase gene can be altered by adding a new promoter that causes more transcription. Similarly, enhancers may be added to the diacylglycerol acyltransferase gene that increase transcription, or silencers may be mutated or deleted from the diacylglycerol acyltransferase gene to increase transcription. Mutations to a native gene's coding region might also increase diacylglycerol acyltransferase activity, for example, by producing a protein variant that does not interact with inhibitory proteins or molecules. The over-expression of one or more activators may increase diacylglycerol acyltransferase activity by increasing the expression of a diacylglycerol acyltransferase protein, and a knockout or mutation of one or more repressors may similarly increase diacylglycerol acyltransferase activity.

[0017] The term "biologically-active portion" refers to an amino acid sequence that is less than a full-length amino acid sequence, but exhibits at least one activity of the full length sequence. For example, a biologically-active portion of a diacylglycerol acyltransferase may refer to one or more domains of DGA1 or DGA2 having biological activity for converting acyl-CoA and diacylglycerol to triacylglycerol. Biologically-active portions of a DGA1 include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the DGA1 protein, e.g., the amino acid sequence as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61 which include fewer amino acids than the full length DGA1, and exhibit at least one activity of a DGA1 protein. Similarly, biologically-active portions of a DGA2 protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the DGA2 protein, e.g., the amino acid sequence as set forth in SEQ ID NOs: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75, which include fewer amino acids than the full length DGA2, and exhibit at least one activity of a DGA2 protein. Biologically-active portions of a DGA3 protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the DGA3 protein, e.g., the amino acid sequence as set forth in SEQ ID NOs: 83 or 85, which include fewer amino acids than the full length DGA3, and exhibit at least one activity of a DGA3 protein. A biologically-active portion of a diacylglycerol acyltransferase may comprise, for example, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, or 696 amino acids. Typically, biologically active portions comprise a domain or motif having the catalytic activity of converting acyl-CoA and diacylglycerol to triacylglycerol. A biologically active portion of a DGA1 protein can be a polypeptide which is, for example, 278 amino acids in length.

[0018] The term "DGAT1" refers to a gene that encodes a type 1 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA2 protein.

[0019] The term "DGAT2" refers to a gene that encodes a type 2 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA1 protein.

[0020] The term "DGAT3" refers to a gene that encodes a type 2 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA3 protein.

[0021] "Diacylglyceride," "diacylglycerol," and "diglyceride," are esters comprised of glycerol and two fatty acids.

[0022] The terms "diacylglycerol acyltransferase" and "DGA" refer to any protein that catalyzes the formation of triacylglycerides from diacylglycerol. Diacylglycerol acyltransferases include type 1 diacylglycerol acyltransferases (DGA2), type 2 diacylglycerol acyltransferases (DGA1), and all homologs that catalyze the above-mentioned reaction.

[0023] The terms "diacylglycerol acyltransferase, type 1" and "type 1 diacylglycerol acyltransferases" refer to DGA2 and DGA2 orthologs.

[0024] The terms "diacylglycerol acyltransferase, type 2" and "type 2 diacylglycerol acyltransferases" refer to DGA1 and DGA1 orthologs.

[0025] The terms "diacylglycerol acyltransferase, type 3" and "type 3 diacylglycerol acyltransferases" refer to DGA3 and DGA3 orthologs.

[0026] The term "domain" refers to a part of the amino acid sequence of a protein that is able to fold into a stable three-dimensional structure independent of the rest of the protein.

[0027] The term "drug" refers to any molecule that inhibits cell growth or proliferation, thereby providing a selective advantage to cells that contain a gene that confers resistance to the drug. Drugs include antibiotics, antimicrobials, toxins, and pesticides.

[0028] "Dry weight" and "dry cell weight" mean weight determined in the relative absence of water. For example, reference to oleaginous cells as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all water has been removed.

[0029] The term "encode" refers to nucleic acids that comprise a coding region, portion of a coding region, or compliments thereof. Both DNA and RNA may encode a gene. Both DNA and RNA may encode a protein.

[0030] The term "exogenous" refers to anything that is introduced into a cell. An "exogenous nucleic acid" is a nucleic acid that entered a cell through the cell membrane. An exogenous nucleic acid may contain a nucleotide sequence that exists in the native genome of a cell and/or nucleotide sequences that did not previously exist in the cell's genome. Exogenous nucleic acids include exogenous genes. An "exogenous gene" is a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g., by transformation/transfection), and is also referred to as a "transgene." A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from the same or different species relative to the cell being transformed. Thus, an exogenous gene can include a native gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.

[0031] The term "expression" refers to the amount of a nucleic acid or amino acid sequence (e.g., peptide, polypeptide, or protein) in a cell. The increased expression of a gene refers to the increased transcription of that gene. The increased expression of an amino acid sequence, peptide, polypeptide, or protein refers to the increased translation of a nucleic acid encoding the amino acid sequence, peptide, polypeptide, or protein.

[0032] The term "gene," as used herein, may encompass genomic sequences that contain exons, particularly polynucleotide sequences encoding polypeptide sequences involved in a specific activity. The term further encompasses synthetic nucleic acids that did not derive from genomic sequence. In certain embodiments, the genes lack introns, as they are synthesized based on the known DNA sequence of cDNA and protein sequence. In other embodiments, the genes are synthesized, non-native cDNA wherein the codons have been optimized for expression in Y. lipolytica based on codon usage. The term can further include nucleic acid molecules comprising upstream, downstream, and/or intron nucleotide sequences.

[0033] The term "genetic modification" refers to the result of a transformation. Every transformation causes a genetic modification by definition.

[0034] The term "homolog", as used herein, refers to (a) peptides, oligopeptides, polypeptides, proteins, and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived, and (b) nucleic acids which encode peptides, oligopeptides, polypeptides, proteins, and enzymes with the same characteristics described in (a).

[0035] "Inducible promoter" is a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus.

[0036] The term "integrated" refers to a nucleic acid that is maintained in a cell as an insertion into the cell's genome, such as insertion into a chromosome, including insertions into a plastid genome.

[0037] "In operable linkage" refers to a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with a gene if it can mediate transcription of the gene.

[0038] The term "knockout mutation" or "knockout" refers to a genetic modification that prevents a native gene from being transcribed and translated into a functional protein.

[0039] The term "native" refers to the composition of a cell or parent cell prior to a transformation event. A "native gene" refers to a nucleotide sequence that encodes a protein that has not been introduced into a cell by a transformation event. A "native protein" refers to an amino acid sequence that is encoded by a native gene.

[0040] The terms "nucleic acid" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0041] The term "parent cell" refers to every cell from which a cell descended. The genome of a cell is comprised of the parent cell's genome and any subsequent genetic modifications to the parent cell's genome.

[0042] As used herein, the term "plasmid" refers to a circular DNA molecule that is physically separate from an organism's genomic DNA. Plasmids may be linearized before being introduced into a host cell (referred to herein as a linearized plasmid). Linearized plasmids may not be self-replicating, but may integrate into and be replicated with the genomic DNA of an organism.

[0043] The term "portion" refers to peptides, oligopeptides, polypeptides, protein domains, and proteins. A nucleotide sequence encoding a "portion of a protein" includes both nucleotide sequences that can be transcribed and/or translated and nucleotide sequences that must undergo one or more recombination events to be transcribed and/or translated. For example, a nucleic acid may comprise a nucleotide sequence encoding one or more amino acids of a selectable marker protein. This nucleic acid can be engineered to recombine with one or more different nucleotide sequences that encode the remaining portion of the protein. Such nucleic acids are useful for generating knockout mutations because only recombination with the target sequence is likely to reconstitute the full-length selectable marker gene whereas random-integration events are unlikely to result in a nucleotide sequence that can produce a functional marker protein. A "biologically-active portion" of a polypeptide is any amino acid sequence found in the polypeptide's amino acid sequence that is less than the full amino acid sequence but can perform the same function as the full-length polypeptide. A biologically-active portion of a diacylglycerol acyltransferase includes any amino acid sequence found in a full-length diacylglycerol acyltransferase that can catalyze the formation of triacylglycerol from diacylglycerol and acyl-CoA. A biologically-active portion of a polypeptide includes portions of the polypeptide that have the same activity as the full-length peptide and every portion that has more activity than background. For example, a biologically-active portion of a diacylglycerol acyltransferase may have 0.1, 0.5, 1, 2, 3, 4, 5, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, 100, 100.1, 100.2, 100.3, 100.4, 100.5, 100.6, 100.7, 100.8, 100.9, 101, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 percent activity relative to the full-length polypeptide or higher. A biologically-active portion of a polypeptide may include portions of a peptide that lack a domain that targets the polypeptide to a cellular compartment.

[0044] A "promoter" is a nucleic acid control sequence that directs the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0045] "Recombinant" refers to a cell, nucleic acid, protein, or vector, which has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi), or dsRNA that reduce the levels of active gene product in a cell. A "recombinant nucleic acid" is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

[0046] The term "regulatory region" refers to nucleotide sequences that affect the transcription or translation of a gene but do not encode an amino acid sequence. Regulatory regions include promoters, operators, enhancers, and silencers.

[0047] "Transformation" refers to the transfer of a nucleic acid into a host organism or the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "recombinant", "transgenic" or "transformed" organisms. Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or location-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.

[0048] The term "transformed cell" refers to a cell that has undergone a transformation. Thus, a transformed cell comprises the parent's genome and an inheritable genetic modification.

[0049] The terms "triacylglyceride," "triacylglycerol," "triglyceride," and "TAG" are esters comprised of glycerol and three fatty acids.

[0050] The term "triacylglycerol lipase" refers to any protein that can catalyze the removal of a fatty acid chain from a triacylglycerol. Triacylglycerol lipases include TGL3, TLG3/4, and TGL4.

[0051] The term "vector" refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, linear DNA fragments, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.

Microbe Engineering

[0052] A. Overview

[0053] In certain embodiments of the invention, a microorganism is genetically modified to increase its triacylglycerol content.

[0054] Genes and gene products may be introduced into microbial host cells. Suitable host cells for expression of the genes and nucleic acid molecules are microbial hosts that can be found broadly within the fungal or bacterial families. Examples of suitable host strains include but are not limited to fungal or yeast species, such as Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Hansenula, Kluyveromyces, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Yarrowia, or bacterial species, such as members of proteobacteria and actinomycetes, as well as the genera Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherichia, Salmonella, Pseudomonas, and Cornyebacterium. Yarrowia lipolytica and Arxula adeninivorans are suited for use as a host microorganism because they can accumulate a large percentage of their weight as triacylglycerols.

[0055] Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are known to those skilled in the art. Any of these could be used to construct chimeric genes to produce any one of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation techniques to provide high-level expression of the enzymes.

[0056] For example, a gene encoding an enzyme can be cloned in a suitable plasmid, and an aforementioned starting parent strain as a host can be transformed with the resulting plasmid. This approach can increase the copy number of each of the genes encoding the enzymes and, as a result, the activities of the enzymes can be increased. The plasmid is not particularly limited so long as it renders a desired genetic modification inheritable to the microorganism's progeny.

[0057] Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences that direct the transcription and translation of the relevant gene, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene harboring transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

[0058] Promoters, cDNAs, and 3'UTRs, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012); U.S. Pat. No. 4,683,202; incorporated by reference). Alternatively, elements can be generated synthetically using known methods (Gene 164:49-53 (1995)).

[0059] B. Homologous Recombination

[0060] Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic DNA ("donor") containing sequences homologous to the genomic sequences being targeted ("template") is introduced into the organism and then undergoes recombination into the genome at the site of the corresponding homologous genomic sequences.

[0061] The ability to carry out homologous recombination in a host organism has many practical implications for what can be carried out at the molecular genetic level and is useful in the generation of a microbe that can produce a desired product. By its nature, homologous recombination is a precise gene targeting event and, hence, most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events. Homologous recombination also targets gene insertion events into the host chromosome, potentially resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likely impact gene expression, even from exogenous promoters/UTRs, homologous recombination can be a method of querying loci in an unfamiliar genome environment and to assess the impact of these environments on gene expression.

[0062] A particularly useful genetic engineering approach using homologous recombination is to co-opt specific host regulatory elements such as promoters/UTRs to drive heterologous gene expression in a highly specific fashion.

[0063] Because homologous recombination is a precise gene targeting event, it can be used to precisely modify any nucleotide(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of RNA and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities such as substrate specificity, affinities and Km, thereby affecting a desired change in the metabolism of the host cell. Homologous recombination provides a powerful means to manipulate the host genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements such as promoters, enhancers and 3'UTRs.

[0064] Homologous recombination can be achieved by using targeting constructs containing pieces of endogenous sequences to "target" the gene or region of interest within the endogenous host cell genome. Such targeting sequences can either be located 5' of the gene or region of interest, 3' of the gene/region of interest or even flank the gene/region of interest. Such targeting constructs can be transformed into the host cell either as a supercoiled plasmid DNA with additional vector backbone, a PCR product with no vector backbone, or as a linearized molecule. In some cases, it may be advantageous to first expose the homologous sequences within the transgenic DNA (donor DNA) by cutting the transgenic DNA with a restriction enzyme. This step can increase the recombination efficiency and decrease the occurrence of undesired events. Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends homologous to the genomic sequences being targeted.

[0065] C. Vectors and Vector Components

[0066] Vectors for transforming microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell.

[0067] 1. Control Sequences

[0068] Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell. Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence. Another control sequence is a 3' untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal. Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location inside or outside the cell.

[0069] Thus, an exemplary vector design for expression of a gene in a microbe contains a coding sequence for a desired gene product (for example, a selectable marker, or an enzyme) in operable linkage with a promoter active in yeast. Alternatively, if the vector does not contain a promoter in operable linkage with the coding sequence of interest, the coding sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration.

[0070] The promoter used to express a gene can be the promoter naturally linked to that gene or a different promoter.

[0071] A promoter can generally be characterized as constitutive or inducible. Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (heat or cold), lack of nitrogen in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level.

[0072] Inclusion of termination region control sequence is optional, and if employed, then the choice is primarily one of convenience, as the termination region is relatively interchangeable. The termination region may be native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source (See, e.g., Chen & Orozco, Nucleic Acids Research 16:8411 (1988)).

[0073] 2. Genes and Codon Optimization

[0074] Typically, a gene includes a promoter, a coding sequence, and termination control sequences. When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unintegrated (e.g., an episome), in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the vector DNA.

[0075] A common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein. Such a gene, and its corresponding gene product, is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming the organisms of the invention.

[0076] For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons optimally used by the host cell to be transformed. Thus, proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed. The precise mechanisms underlying this effect are many, but include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. When codon usage in the transgene is not optimized, available tRNA pools are not sufficient to allow for efficient translation of the transgenic mRNA resulting in ribosomal stalling and termination and possible instability of the transgenic mRNA.

[0077] D. Transformation

[0078] Cells can be transformed by any suitable technique including, e.g., biolistics, electroporation, glass bead transformation, and silicon carbide whisker transformation. Any convenient technique for introducing a transgene into a microorganism can be employed in the present invention. Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68:326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel & Higa, J. Molecular Biology, 53:159 (1970)), or the like.

[0079] Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowia lipolytica) can be found in the literature (Bordes et al., J. Microbiological Methods, 70:493 (2007); Chen et al., Applied Microbiology & Biotechnology 48:232 (1997)). Examples of expression of exogenous genes in bacteria such as E. coli are well known (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)).

[0080] Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art. In one embodiment, an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism. Alternatively, if the vector does not contain a promoter in operable linkage with the gene of interest, the gene can be transformed into the cells such that it becomes operably linked to a native promoter at the point of vector integration. The vector can also contain a second gene that encodes a protein. Optionally, one or both gene(s) is/are followed by a 3' untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform cells (Protist 155:381-93 (2004)). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.

Exemplary Nucleic Acids, Cells, and Methods

[0081] A. Diacylglycerol Acyltransferase Nucleic Acid Molecules and Vectors

[0082] The diacylglycerol acyltransferase may be a type 1 diacylglycerol acyltransferase, type 2 diacylglycerol acyltransferase, type 3 diacylglycerol acyltransferase, or any other protein that catalyzes the conversion of diacylglycerol into a triacylglyceride. In some embodiments, the diacylglycerol acyltransferase is DGA1. For example, the diacylglycerol acyltransferase may be a DGA1 protein encoded by a DGAT2 gene selected from the group consisting of Arxula adeninivorans, Aspergillus terreus, Aurantiochytrium limacinum, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Pichia guilliermondii, Phaeodactylum tricornutum, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, and Yarrowia lipolytica.

[0083] The DGAT2 gene may have a nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62. In other embodiments, the DGAT2 gene is substantially identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62, and the nucleotide sequence encodes a protein that retains the functional activity of a protein encoded by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62, yet differs in nucleotide sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGAT2 gene comprises an nucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62.

[0084] The DGA1 may have an amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61. In other embodiments, the DGA1 is substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61, and retains the functional activity of the protein of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGA1 protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61.

[0085] In some embodiments, the diacylglycerol acyltransferase is DGA2. For example, the diacylglycerol acyltransferase may be a DGA2 protein encoded by a DGAT1 gene found in an organism selected from the group consisting of Arxula adeninivorans, Aspergillus terreus, Chaetomium globosum, Claviceps purpurea, Lipomyces starkeyi, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricornutum, Pichia guilliermondii, Rhodosporidium toruloides, Rhodotorula graminis, Trichoderma virens, and Yarrowia lipolytica.

[0086] The DGAT1 gene may have a nucleotide sequence set forth in SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76. In other embodiments, the DGAT1 gene is substantially identical to SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76, and the nucleotide sequence encodes a protein that retains the functional activity of a protein encoded by SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76, yet differs in nucleotide sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGAT1 gene comprises an nucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76.

[0087] The DGA2 may have an amino acid sequence set forth in SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75. In other embodiments, the DGA2 is substantially identical to SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75, and retains the functional activity of the protein of SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGA2 protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75.

[0088] In some embodiments, the diacylglycerol acyltransferase is DGA3. For example, the diacylglycerol acyltransferase may be a DGA3 protein encoded by a DGAT3 gene found in an organism selected from the group consisting of Ricinus communis and Arachis hypogaea.

[0089] The DGAT3 gene may have a nucleotide sequence set forth in SEQ ID NO: 84 or 86. In other embodiments, the DGAT3 gene is substantially identical to SEQ ID NO: 84 or 86, and the nucleotide sequence encodes a protein that retains the functional activity of a protein encoded by SEQ ID NO: 84 or 86, yet differs in nucleotide sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGAT3 gene comprises an nucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 84 or 86.

[0090] The DGA3 may have an amino acid sequence set forth in SEQ ID NO: 83 or 85. In other embodiments, the DGA3 is substantially identical to SEQ ID NO: 83 or 85, and retains the functional activity of the protein of SEQ ID NO: 83 or 85, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGA3 protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 83 or 85.

[0091] The DGAT1, DGAT2, and DGAT3 genes may comprise conservative substitutions, deletions, and/or insertions while still encoding a protein that has functional diacylglycerol acyltransferase activity. For example, the DGAT1, DGAT2, or DGAT3 codons may be optimized for a particular host cell, different codons may be substituted for convenience, such as to introduce a restriction site or to create optimal PCR primers, or codons may be substituted for another purpose. Similarly, the nucleotide sequence may be altered to create conservative amino acid substitutions, deletions, and/or insertions.

[0092] The DGA1, DGA2, and DGA3 polypeptides may comprise conservative substitutions, deletions, and/or insertions while still maintaining functional diacylglycerol acyltransferase activity. Conservative substitution tables are well known in the art (Creighton, Proteins (2d. ed., 1992)).

[0093] Amino acid substitutions, deletions and/or insertions may readily be made using recombinant DNA manipulation techniques. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. These methods include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), Quick Change Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis, and other site-directed mutagenesis protocols.

[0094] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes can be at least 95% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions can then be compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0095] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Molecular Biology 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Computer Applications in the Biosciences 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0096] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, MEGABLAST, BLASTX, TBLASTN, TBLASTX, and BLASTP, and Clustal programs, e.g., ClustalW, ClustalX, and Clustal Omega.

[0097] Sequence searches are typically carried out using the BLASTN program, when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is effective for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases.

[0098] An alignment of selected sequences in order to determine "% identity" between two or more sequences is performed using for example, the CLUSTAL-W program.

[0099] A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a protein product, such as an amino acid or polypeptide, when the sequence is expressed. The coding sequence may comprise and/or consist of untranslated sequences (including introns or 5' or 3' untranslated regions) within translated regions, or may lack such intervening untranslated sequences (e.g., as in cDNA).

[0100] The abbreviation used throughout the specification to refer to nucleic acids comprising and/or consisting of nucleotide sequences are the conventional one-letter abbreviations. Thus when included in a nucleic acid, the naturally occurring encoding nucleotides are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Also, unless otherwise specified, the nucleic acid sequences presented herein is the 5'.fwdarw.3'direction.

[0101] As used herein, the term "complementary" and derivatives thereof are used in reference to pairing of nucleic acids by the well-known rules that A pairs with T or U and C pairs with G. Complement can be "partial" or "complete". In partial complement, only some of the nucleic acid bases are matched according to the base pairing rules; while in complete or total complement, all the bases are matched according to the pairing rule. The degree of complement between the nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands as well known in the art. The efficiency and strength of said hybridization depends upon the detection method.

[0102] As used herein, "DGA1" means a diacylglycerol acyltransferase type 2 (DGAT2). DGA1 is an integral membrane protein that catalyzes the final enzymatic step in oil biosynthesis and the production of triacylglycerols in plants, fungi, and mammals. The DGA1 may play a key role in altering the quantity of long-chain polyunsaturated fatty acids produced in oils of oleaginous organisms. DGA1 is related to the acyl-coenzyme A:cholesterol acyltransferase ("ACAT"). This enzyme is responsible for transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol ("DAG") to form triacylglycerol ("TAG") (thereby involved in the terminal step of TAG biosynthesis). DGA1 is associated with membrane and lipid body fractions in plants and fungi, particularly, in oilseeds where it contributes to the storage of carbon used as energy reserves. TAG is believed to be an important chemical for storage of energy in cells. DGA1 is known to regulate TAG structure and direct TAG synthesis.

[0103] The DGA1 polynucleotide and polypeptide sequences may be derived from highly oleaginous organisms having very high, native levels of lipid accumulation. (Bioresource Technology 144:360-69 (2013); Progress Lipid Research 52:395-408 (2013); Applied Microbiology & Biotechnology 90:1219-27 (2011); European Journal Lipid Science & Technology 113:1031-51 (2011); Food Technology & Biotechnology 47:215-20 (2009); Advances Applied Microbiology 51:1-51 (2002); Lipids 11:837-44 (1976)). A list of organisms with a reported lipid content of about 50% and higher is shown in Table 1. R. toruloides and L. starkeyi have the highest lipid content. Among the organisms in Table 1, five have publicly accessible sequence for DGA1, R. toruloides, L. starkeyi, A. limacinum, A. terreus, and C. purpurea (bolded in Table 1).

TABLE-US-00001 TABLE 1 List of oleaginous fungi with reported lipid contents of about 50% and above. Organisms with publicly accessible sequences for a DGA1 gene are in bold. Fungi with reported high lipid content Cryptococcus albidus Cryptococcus curvatus Cryptococcus ramirezgomezianus Cryptococcus terreus Cryptococcus wieringae Cunninghamella echinulata Cunninghamella japonica Leucosporidiella creatinivora Lipomyces lipofer Lipomyces tetrasporus Mortierella isabellina Prototheca zopfii Rhizopus arrhizus Rhodosporidium babjevae Rhodosporidium paludigenum Rhodotorula glutinis Rhodotorula mucilaginosa Tremella enchepala Trichosporon cutaneum Trichosporon fermentans

[0104] Nucleic acid constructs for overexpressing the DGA1 gene were described in U.S. Ser. No. 61/943,664 (hereby incorporated by reference). FIG. 1 shows expression construct pNC243 used for overexpression of the R. toruloides DGA1 gene NG66 (SEQ ID No. 6) in Y. lipolytica. DGA1 expression constructs were linearized before transformation by PacI/NotI restriction digest (FIG. 1). The linear expression constructs each included expression cassette for DGA1 gene and for Nat1 gene, used as marker for selection with nourseothricin (NAT).

[0105] Nucleic acid constructs for overexpressing the DGA2 gene and/or other diacylglycerol acyltransferases may be created using the methods described above and/or other methods known in the art.

[0106] B. Triacylglycerol Lipase Nucleic Acid Molecules and Vectors

[0107] Triacylglycerol lipase depletes a cell's triacylglycerol by removing one or more fatty acid chains. Thus, decreasing the net triacylglycerol lipase activity of a cell may increase the cell's triacylglycerol. This decrease may be accomplished by reducing the efficiency of the enzyme, e.g., by mutating amino acids in its active site, or by reducing the expression of the enzyme. For example, a TGL3 knockout mutation will decrease the activity of a triacylglycerol lipase because it prevents the cell from transcribing TGL3.

[0108] In some embodiments, the triacylglycerol lipase is TGL3, TGL3/4, or TGL4.

[0109] The TGL3 gene in Y. lipolytica encodes the triacylglycerol lipase protein TGL3 (SEQ ID No. 19). SEQ ID No. 20 contains the TGL3 nucleotide sequence, 100 upstream nucleotides, and 100 downstream. Thus, the SEQ ID No. 20 nucleotide sequence may be used to design a nucleic acid capable of recombining with a nucleic acid sequence in the native Y. lipolytica triacylglycerol lipase gene.

[0110] Knockout cassettes SEQ ID Nos. 27 and 28 are capable of recombining with the native TGL3 gene in Y. lipolytica. Thus, in some embodiments, the nucleic acids encoded by SEQ ID Nos. 27 and 28 may be used to generate a triacylglycerol lipase knockout mutation in Y. lipolytica. SEQ ID Nos. 27 and 28 each contain portions of a hygromycin resistance gene hph. Neither isolated sequence encodes a functional protein, but the two sequences are capable of encoding a functional kinase that confers hygromycin resistance upon successful recombination. Further, neither SEQ ID No. 27 nor SEQ ID No. 28 contains a promoter or terminator, and thus, they rely on homologous recombination with the Y. lipolytica TGL3 gene in order for the hph gene to be transcribed and translated. In this way, successfully transformed oleaginous cells may be selected by growing the cells on medium containing hygromycin.

[0111] Knockout cassette SEQ ID NO. 27 may be prepared by amplifying a hygromycin resistance gene hph (SEQ ID NO. 22) with primer NP1798 (SEQ ID NO. 23) and primer NP656 (SEQ ID NO. 24). Knockout cassette SEQ ID NO. 28 may be prepared by amplifying a hygromycin resistance gene hph (SEQ ID NO. 22) with primer NP655 (SEQ ID NO. 25) and primer NP1799 (SEQ ID NO. 26).

[0112] Different approaches may be used to design nucleic acids that knockout the TGL3 gene in Y. lipolytica (Biochimica Biophysica Acta 1831:1486-95 (2013)). The methods disclosed herein and other methods known in the art may be used to knockout triacylglycerol different lipase genes in other species. For example, these methods may be used to reduce the activity of the TGL3 gene of Arxula adeninivorans (SEQ ID NO:78), the TGL3/4 gene of Arxula adeninivorans (SEQ ID NO:80), and/or the TGL4 gene of Arxula adeninivorans (SEQ ID NO:82). Similarly, these methods may be used to reduce the activity of the TGL4 gene in Y. lipolytica (SEQ ID NO:88).

[0113] C. Transformed Oleaginous Cell

[0114] In some embodiments, the transformed oleaginous cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell, a yeast cell, a filamentous fungi cell, a protist cell, an algae cell, an avian cell, a plant cell, or an insect cell.

[0115] The cell may be selected from the group consisting of Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, and Yarrowia.

[0116] In some embodiments, the cell is selected from the group of consisting of Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Tremella enchepala, Trichosporon cutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica.

[0117] In certain embodiments, the transformed oleaginous cell is a high-temperature tolerant yeast cell. In some embodiments the transformed oleaginous cell is Kluyveromyces marxianus.

[0118] In certain embodiments, the cell is Yarrowia lipolytica or Arxula adeninivorans.

[0119] D. Increasing Diacylglycerol Acyltransferase in a Cell

[0120] A protein's activity may be increased by overexpressing the protein. Proteins may be overexpressed in a cell using a variety of genetic modifications. In some embodiments, the genetic modification increases the expression of a native diacylglycerol acyltransferase. A native diacylglycerol acyltransferase may be overexpressed by modifying the upstream transcription regulators of a native diacylglycerol acyltransferase gene, for example, by increasing the expression of a transcription activator or decreasing the expression of a transcription repressor. Alternatively, the promoter of a native diacylglycerol acyltransferase gene may be substituted with a constitutively active or inducible promoter by recombination with an exogenous nucleic acid.

[0121] In some embodiments, the genetic modification encodes at least one copy of a diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a gene native to the cell or from a different species. In certain embodiments, the diacylglycerol acyltransferase gene is the type II diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Aurantiochytrium limacinum, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Pichia guilliermondii, Phaeodactylum tricornutum, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, or Yarrowia lipolytica. In certain embodiments, the diacylglycerol acyltransferase gene is the type I diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Chaetomium globosum, Claviceps purpurea, Lipomyces starkeyi, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricornutum, Pichia guilliermondii, Rhodosporidium toruloides, Rhodotorula graminis, Trichoderma virens, or Yarrowia lipolytica. In certain embodiments, the diacylglycerol acyltransferase gene is the type III diacylglycerol acyltransferase gene from Ricinus communis or Arachis hypogaea. In certain embodiments, diacylglycerol acyltransferase is overexpressed by transforming a cell with a gene encoding a diacylglycerol acyltransferase gene. The genetic modification may encode one or more than one copy of a diacylglycerol acyltransferase gene. In certain embodiments, the genetic modification encodes at least one copy of the DGA1 protein from R. toruloides. In some embodiments, the genetic modification encodes at least one copy of the DGA1 protein from R. toruloides and the transformed oleaginous cell is Y. lipolytica.

[0122] In certain embodiments, the diacylglycerol acyltransferase gene is inheritable to the progeny of a transformed oleaginous cell. In some embodiments, the diacylglycerol acyltransferase gene is inheritable because it resides on a plasmid. In certain embodiments, the diacylglycerol acyltransferase gene is inheritable because it is integrated into the genome of the transformed oleaginous cell.

[0123] E. Decreasing Triacylglycerol Lipase Activity in a Cell

[0124] In some embodiments, the transformed oleaginous cell comprises a genetic modification that decreases the activity of a native triacylglycerol lipase. Such genetic modifications may affect a protein that regulates the transcription of a triacylglycerol lipase gene, including modifications that decrease the expression of a transcription activator and/or increase the expression of a transcription repressor. Modifications that affect a regulator protein may both decrease the expression of triacylglycerol lipase and alter other gene expression profiles that shift the cellular equilibrium toward increased triacylglycerol accumulation. Alternatively, the genetic modification may be the introduction of a small interfering RNA, or a nucleic acid that encodes a small interfering RNA. In other embodiments, the genetic modification consists of the homologous recombination of a nucleic acid and the regulatory region of a native triacylglycerol lipase gene, including an operator, promoter, sequences upstream from the promoter, enhancers, and sequences downstream of the gene.

[0125] In some embodiments the transformed oleaginous cell comprises a genetic modification consisting of a homologous recombination event. In certain embodiments, the transformed oleaginous cell comprises a genetic modification consisting of a homologous recombination event between a native triacylglycerol lipase gene and a nucleic acid. Thus, the genetic modification deletes the triacylglycerol lipase gene, prevents its transcription, or prevents the transcription of a gene that can be transcribed into a fully-active protein. A homologous recombination event may mutate or delete a portion of a native triacylglycerol lipase gene. For example, the homologous recombination event may mutate one or more residues in the active site of a native triacylglycerol lipase, thereby reducing the efficiency of the lipase or rendering it inactive. Alternatively, the homologous recombination event may affect post-translational modification, folding, stability, or localization within the cell. In certain embodiments, the genetic modification is a triacylglycerol lipase knockout mutation. Knockout mutations are preferable because they eliminate a pathway that depletes a cell's triacylglycerol content, thereby increasing the triacylglycerol content of a cell.

[0126] A knockout mutation may delete the triacylglycerol lipase gene. Additionally, the knockout mutation may substitute the triacylglycerol lipase gene with a gene that encodes a different protein. The gene may be operably linked to an exogenous promoter. In certain embodiments, the gene is not linked to an exogenous promoter, and instead, the gene is configured to recombine with the triacylglycerol lipase gene such that the triacylglycerol lipase gene's promoter drives transcription of the gene. Thus, the gene is less likely to be expressed if it randomly integrates into the cell's genome. Methods for creating knockouts are well-known in the art (See, e.g., Fickers et al., J. Microbiological Methods 55:727 (2003)).

[0127] In certain embodiments, the genetic modification comprises two homologous recombination events. In the first event, a nucleic acid encoding a portion of a gene recombines with the triacylglycerol lipase gene, and in the second event, a nucleic acid encoding the remaining portion of the gene recombines with the triacylglycerol lipase gene. The two portions of the gene are designed such that neither portion is functional unless they recombine with each other. These two events further reduce the likelihood that the gene can be expressed following random integration events.

[0128] In certain embodiments, the gene encodes a dominant selectable marker. Thus, knockout cells may be selected by screening for the marker. In some embodiments, the dominant selectable marker is a drug resistance marker. A drug resistance marker is a dominant selectable marker that, when expressed by a cell, allows the cell to grow and/or survive in the presence of a drug that would normally inhibit cellular growth and/or survival. Cells expressing a drug resistance marker can be selected by growing the cells in the presence of the drug. In some embodiments, the drug resistance marker is an antibiotic resistance marker. In some embodiments, the drug resistance marker confers resistance to a drug selected from the group consisting of Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Amorolfin, Butenafine, Naftifine, Terbinafine, Anidulafungin, Caspofungin, Micafungin, Benzoic acid, Ciclopirox, Flucytosine, 5-fluorocytosine, Griseofulvin, Haloprogin, Polygodial, Tolnaftate, Crystal violet, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Streptomycin, Loracarbef, Ertapenem, Doripenem, Imipenem, Meropenem, Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, clavulanate, sulbactam, tazobactam, clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Co-trimoxazole, Sulfonamidochrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin, Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, Trimethoprim, Geneticin, Nourseothricin, Hygromycin, Bleomycin, and Puromycin.

[0129] In some embodiments, the dominant selectable marker is a nutritional marker. A nutritional marker is a dominant selectable marker that, when expressed by the cell, enables the cell to grow or survive using one or more particular nutrient sources. Cells expressing a nutritional marker can be selected by growing the cells under limiting nutrient conditions in which cells expressing the nutritional marker can survive and/or grow, but cells lacking the nutrient marker cannot. In some embodiments, the nutritional marker is selected from the group consisting of Orotidine 5-phosphate decarboxylase, Phosphite specific oxidoreductase, Alpha-ketoglutarate-dependent hypophosphite dioxygenase, Alkaline phosphatase, Cyanamide hydratase, Melamine deaminase, Cyanurate amidohydrolase, Biuret hydrolyase, Urea amidolyase, Ammelide aminohydrolase, Guanine deaminase, Phosphodiesterase, Phosphotriesterase, Phosphite hydrogenase, Glycerophosphodiesterase, Parathion hydrolyase, Phosphite dehydrogenase, Dibenzothiophene desulfurization enzyme, Aromatic desulfinase, NADH-dependent FMN reductase, Aminopurine transporter, Hydroxylamine oxidoreductase, Invertase, Beta-glucosidase, Alpha-glucosidase, Beta-galactosidase, Alpha-galactosidase, Amylase, Cellulase, and Pullulonase.

[0130] Different approaches may be used to knockout the TGL3 gene in Y. lipolytica (See, e.g., Dulermo et al., Biochimica Biophysica Acta 1831:1486 (2013)). The methods disclosed herein and other methods known in the art may be used to knockout different triacylglycerol lipase genes in other species. For example, these methods may be used to knockout the TGL3 gene of Arxula adeninivorans (SEQ ID NO:78), the TGL3/4 gene of Arxula adeninivorans (SEQ ID NO:80), or the TGL4 gene of Arxula adeninivorans (SEQ ID NO:82). Similarly, these methods may be used to knockout the TGL4 gene of Y. lipolytica (SEQ ID NO:88).

[0131] In some embodiments, a genetic modification decreases the expression of a native triacylglycerol lipase gene by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 percent.

[0132] In some embodiments, a genetic modification decreases the efficiency of a native triacylglycerol lipase gene by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 percent.

[0133] In some embodiments, a genetic modification decreases the activity of a native triacylglycerol lipase gene by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 percent.

[0134] F. Decreasing Triacylglycerol Lipase Activity in a Cell with Concomitant Overexpression of Diacylglycerol Acyltransferase

[0135] In some embodiments, the transformed oleaginous cell comprises a triacylglycerol lipase knockout mutation and a genetic modification that increase the expression of a native diacylglycerol acyltransferase. In certain embodiments, the transformed oleaginous cell comprises a triacylglycerol lipase knockout mutation and a genetic modification that encodes at least one copy of a diacylglycerol acyltransferase gene that is either native to the cell or from a different species of cell. In other embodiments, a TGL3 gene is disrupted and a DGA1 protein is overexpressed.

[0136] In some embodiments, one nucleic acid increases the expression of a native diacylglycerol acyltransferase or encodes at least one copy of a diacylglycerol acyltransferase gene and a second nucleic acid decreases the activity of a triacylglycerol lipase in the cell. In some embodiments the same nucleic acid encodes at least one copy of a diacylglycerol acyltransferase gene and decreases the activity of a triacylglycerol lipase in the cell. For example, the nucleic acid designed to knock out a triacylglycerol lipase gene may also contain a copy of a diacylglycerol acyltransferase gene.

[0137] G. Triacylglycerol Production

[0138] In certain embodiments, the transformed oleaginous cell are grown in the presence of exogenous fatty acids, glucose, ethanol, xylose, sucrose, starch, starch dextrin, glycerol, cellulose, and/or acetic acid. These substrates may be added during cultivation to increase lipid production. The exogenous fatty acids may include stearate, oleic acid, linoleic acid, .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid, arachidonic acid, .alpha.-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapenteaenoic acid, docosapentaenoic acid, eicosadienoic acid, and/or eicosatrienoic acid.

[0139] In certain embodiments, the present invention relates to a product produced by a modified host cell described herein. In certain embodiments, the product is an oil, lipid, or triacylglycerol. In some embodiments, the product is palmitic acid, palmitoleic acid, stearic acid, oleic acid, or linoleic acid. In certain embodiments, the product is a saturated fatty acid. Thus, the product may be caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or cerotic acid. In some embodiments, the product is an unsaturated fatty acid. Thus, the product may be myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, .alpha.-linolenic acid, arachidonic acid, eicosapenteaenoic acid, erucic acid, or docosahexaenoic acid.

[0140] In some embodiments, the product comprises an 18-carbon fatty acid. In some embodiments, the product comprises oleic acid, stearic acid, or linoleic acid. For example, the product may be oleic acid.

[0141] In some embodiments, the present invention relates to a transformed oleaginous cell comprising a first genetic modification and a second genetic modification. The first genetic modification may increase the activity of a native diacylglycerol acyltransferase. Alternatively, the first genetic modification may encode at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species. The second genetic modification may decrease the activity of a triacylglycerol lipase in the oleaginous cell.

[0142] In some embodiments, the second genetic modification is a triacylglycerol lipase knockout mutation.

[0143] In some embodiments, the first genetic modification encodes at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species. In some embodiments, a diacylglycerol acyltransferase gene is integrated into the genome of the cell.

[0144] In some embodiments, the transformed oleaginous cell is selected from the group consisting of algae, bacteria, molds, fungi, plants, and yeasts. In certain embodiments, the transformed oleaginous cell is a yeast. The transformed oleaginous cell may be selected from the group consisting of Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, and Yarrowia. In certain embodiments, the transformed oleaginous cell is selected from the group consisting of Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Tremella enchepala, Trichosporon cutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica. In other embodiments, the transformed oleaginous cell is Yarrowia lipolytica or Arxula adeninivorans.

[0145] In some embodiments, the diacylglycerol acyltransferase gene is a type II diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a type II diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Aurantiochytrium limacinum, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Pichia guilliermondii, Phaeodactylum tricornutum, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, or Yarrowia lipolytica.

[0146] In some embodiments, the diacylglycerol acyltransferase gene is a type I diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a type I diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Chaetomium globosum, Claviceps purpurea, Lipomyces starkeyi, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricomuturn, Pichia guilliermondii, Rhodosporidium toruloides, Rhodotorula graminis, Trichoderma virens, or Yarrowia lipolytica.

[0147] In some embodiments, the diacylglycerol acyltransferase gene is a type III diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a type III diacylglycerol acyltransferase gene from Ricinus communis or Arachis hypogaea.

[0148] In some embodiments, the triacylglycerol lipase is TGL3, TGL3/4, or TGL4.

[0149] In some embodiments, the present invention relates to a product derived from a transformed oleaginous cell. In certain embodiments, the product is an oil, lipid, or triacylglycerol.

[0150] In some embodiments, the present invention relates to a method of increasing the lipid content of a cell, comprising transforming a parent cell with a first nucleic acid and a second nucleic acid. The first nucleic acid may increase the activity of a native diacylglycerol acyltransferase or comprise a diacylglycerol acyltransferase gene. The second nucleic acid may decrease the activity of a triacylglycerol lipase. The first nucleic acid and second nucleic acid may be the same. Thus, in some embodiments, the present invention relates to a method of increasing the lipid content of a cell comprising transforming a parent cell with a nucleic acid, wherein the nucleic acid decreases the activity of a triacylglycerol lipase in the cell and either comprises a diacylglycerol acyltransferase gene or increases the activity of a native diacylglycerol acyltransferase.

[0151] In some embodiments, the present invention relates to a method of increasing the triacylglycerol content of a cell, comprising providing a cell and growing the cell. The cell may comprise a first genetic modification and a second genetic modification. The first genetic modification may increase the activity of a native diacylglycerol acyltransferase or encode at least one copy of a diacylglycerol acyltransferase gene either native to the oleaginous cell or from a different species. The second genetic modification may decrease the activity of a triacylglycerol lipase in the cell. The cell may be grown under conditions whereby the first genetic modification is expressed, thereby producing a triacylglycerol. In some embodiments, the triacylglycerol is recovered.

[0152] In some embodiments, the first nucleic acid comprises a diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may encode a type I diacylglycerol acyltransferase polypeptide, a type II diacylglycerol acyltransferase polypeptide, or a type III diacylglycerol acyltransferase polypeptide.

[0153] In some embodiments, the diacylglycerol acyltransferase gene comprises a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 84, or SEQ ID NO: 86, or a complement of any one of them.

[0154] In some embodiments, the diacylglycerol acyltransferase gene comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 84, or SEQ ID NO: 86, or a complement of any one of them.

[0155] In some embodiments, the diacylglycerol acyltransferase polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them.

[0156] In some embodiments, the diacylglycerol acyltransferase polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them.

[0157] In some embodiments, the second nucleic acid is capable of recombining with a nucleic acid sequence in a triacylglycerol lipase gene and/or a nucleic acid sequence in the regulatory region of a triacylglycerol lipase gene. In certain embodiments, the second nucleic acid is capable of recombining with a nucleic acid sequence in a TGL3, TGL3/4, or TGL4 gene and/or a nucleic acid sequence in the regulatory region of a TGL3, TGL3/4, or TGL4 gene. The second nucleic acid may comprise a gene encoding a protein or a portion of a protein. In certain embodiments, the second nucleic acid comprises a gene encoding a protein that confers resistance to a drug. In other embodiments, the second nucleic acid comprises a gene encoding a protein that enables the cell to grow or proliferate more quickly on one or more particular nutrient sources than a native organism of the same species.

[0158] The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications and GenBank Accession numbers as cited throughout this application) are hereby expressly incorporated by reference. When definitions of terms in documents that are incorporated by reference herein conflict with those used herein, the definitions used herein govern.

EXEMPLIFICATION

Example 1: Method to Overexpress DGA1 in Y. lipolytica

[0159] Nucleic acid constructs for overexpressing the DGA1 gene were described in U.S. Ser. No. 61/943,664 (hereby incorporated by reference). FIG. 1 shows expression construct pNC243 used for overexpression of the R. toruloides DGA1 gene NG66 (SEQ ID No. 6) in Y. lipolytica. DGA1 expression constructs were linearized before transformation by a PacI/NotI restriction digest. The linear expression constructs each included an expression cassette for the DGA1 gene and for the Nat1 gene, used as a marker for selection with nourseothricin (NAT).

[0160] DGA1 expression constructs were randomly integrated into the genome of E lipolytica strain NS18 (obtained from ARS Culture Collection, NRRL # YB 392) using a transformation protocol as described in Chen (Applied Microbiology & Biotechnology 48:232-35 (1997)). Transformants were selected on YPD plates with 500 .mu.g/mL NAT and screened for the ability to accumulate lipids by a fluorescent staining lipid assay as described below. For each expression construct, eight transformants were analysed.

[0161] For most constructs, there was significant colony variation between the transformants, likely due to the lack of a functional DGA1 expression cassette in cells that only obtained a functional Nat1 cassette, or due to a negative effect of the site of DGA1 integration on DGA1 expression. Nevertheless, all transformants had a significant increase in lipid content.

[0162] Overexpression of native Y. lipolytica DGA1 (NG15) under a strong promoter increased lipid content as measured by cell fluorescence by about 2-fold compared to the parental strain NS18. Transformants that demonstrated the highest fluorescence (about 3-fold higher compared to NS18) were generated by the overexpression of R. toruloides DGA1 (NG66, NG67) and L. starkeyi DGA1 (NG68).

[0163] In certain experiments, the effect of native R. toruloides DGA1 (NG49) overexpression on lipid production in Y. lipolytica was not as high as the effect of synthetic versions of R. toruloides DGA1 genes that did not contain introns. This result may indicate that the gene splicing of the R. toruloides DGA1 gene in Y. lipolytica was not very efficient. In certain experiments, codon optimization of the R. toruloides DGA1 gene for expression in Y. lipolytica did not have a positive effect on lipid production.

[0164] In order to select strains with the highest lipid production level, Y. lipolytica strain NS18 transformants expressing NG15 (Y. lipolytica DGA1) or NG66 (R. toruloides DGA1) were screened. For NG15, about 50 colonies were screened by lipid assay for the highest lipid accumulation, and the best transformant was named NS249. For NG66, 80 colonies were screened and the 8 best colonies were selected for further analysis. Strain NS249 and the 8 selected NG66 transformants were grown in shake flasks and analysed by the lipid assay for lipid content and by HPLC for glucose consumption. Y. lipolytica strains overexpressing R. toruloides DGA1 have significantly higher lipid content than Y. lipolytica strains with native Y. lipolytica DGA1 gene expressed under the same promoter as R. toruloides DGA1. At the same time, NG66 transformants had significantly less glucose left in the media, demonstrating that NG66 was more efficient in converting glucose to lipids. The difference in efficiency between two DGA1 genes may be attributed to either a higher level of expression of R. toruloides DGA1 in Y. lipolytica or a higher level of R. toruloides DGA1 specific activity, or both. One of the best performing transformants with an integrated NG66 gene was named NS281.

Example 2: Method to Knockout Triacylglycerol Lipase Knockout Gene in Y. lipolytica

[0165] In order to test the idea that combining DGA1 overexpression with a TGL3 deletion leads to higher lipid accumulation in Y. lipolytica, we deleted TGL3 in Y. lipolytica wild-type strain NS18 (obtained from NRLL # YB-392) and its DGA1 overexpressing derivative NS281. NS281 overexpresses the DGA1 gene from Rhodosporidium toruloides as described above. The Y. lipolytica TGL3 gene (YALI0D17534g, SEQ ID NO: 20) was deleted as follows: A two-fragment deletion cassette was amplified by PCR from a plasmid containing the hygromycin resistance gene ("hph," SEQ ID NO: 22) using primer pairs NP1798-NP656 and NP655-NP1799 (SEQ ID NOs: 23-26). The resulting PCR fragments (SEQ ID NOs: 27 & 28) were co-transformed into NS18 and NS281 according to the protocol developed in U.S. Ser. No. 61/819,746 (hereby incorporated by reference). The omission of a promoter and terminator in the hph cassette and the splitting of the hph coding sequence into two PCR fragments reduce the probability that random integration of these pieces will confer hygromycin resistance. The hph gene should only be expressed if it integrates at the TGL3 locus by homologous recombination so that the TGL3 promoter and terminator can direct its transcription. Hygromycin resistant colonies were screened by PCR to confirm the absence of TGL3 and the presence of a tgl3::hyg specific product. Deletion of TGL3 in NS18 resulted in strain NS421. Deletion of TGL3 in NS281 resulted in strain NS377.

Example 3: TGL3 Knockouts that Overexpress DGA1 Accumulate More Lipid than TGL3 Knockouts Alone

[0166] NS421, which contains the TGL3 knockout, and NS377, which contains the TGL3 deletion and also overexpresses R. toruloides DGA1, were grown in a 24-well plate in limiting nitrogen conditions in order to promote lipid accumulation.

[0167] Specifically, each well of an autoclaved, 24-well plate was filled with 1.5 mL of filter-sterilized media containing 0.5 g/L urea, 1.5 g/l yeast extract, 0.85 g/L casamino acids, 1.7 g/L YNB (without amino acids and ammonium sulfate), 100 g/L glucose, and 5.11 g/L potassium hydrogen phthalate (25 mM). Yeast strains that had been incubated for 1-2 days on YPD-agar plates at 30.degree. C. were used to inoculate each well. The 24-well plate was covered with a porous cover and incubated at 30.degree. C., 70-90% humidity, and 900 rpm in an Infors Multitron ATR shaker. After 96 hours, 20 .mu.L of 100% ethanol was added to 20 .mu.L of cells in an analytical microplate and incubated at 4.degree. C. for 30 minutes. 20 .mu.L of cell/ethanol mix was then added to 80 .mu.l of a pre-mixed solution containing 50 .mu.L 1 M potassium iodide, 1 mM .mu.L Bodipy 493/503, 0.5 .mu.L 100% DMSO, 1.5 .mu.L 60% PEG 4000, and 27 .mu.L water in a Costar 96-well, black, clear-bottom plate and covered with a transparent seal. Bodipy fluorescence was monitored with a SpectraMax M2 spectrophotometer (Molecular Devices) kinetic assay at 30.degree. C., and normalized by dividing fluorescence by absorbance at 600 nm. Data was averaged in triplicate growth experiments.

[0168] NS377 accumulated approximately double the amount of lipid as NS421 showing that deletion of TGL3 in combination with DGA1 overexpression increases a cell's lipid content relative to a TGL3 deletion alone (FIG. 2).

Example 4: Cells that Overexpress DGA1 and Contain a TGL3 Deletion Accumulate More TAGs than Cells that Overexpress DGA1 but have No TGL3 Modification

[0169] NS281, which overexpresses R. toruloides DGA1, and NS377, which contains the TGL3 deletion and also overexpresses R. toruloides DGA1, were grown in 1-liter bioreactors using a high cell density fed-batch glucose process. After 118 hours, the cells were analyzed by gas chromatography to measure lipid content as a percentage of dry cell weight. Triplicate chromatography measurements were averaged for the same sample. NS377 reached approximately 4% higher lipid content than NS281, showing that deletion of TGL3 in combination with DGA1 overexpression yields better results than DGA1 overexpression alone (FIG. 3).

Example 5: Cells Comprising Genetic Modifications that Encode a Diacylglycerol Acyltransferase and Decrease the Activity of a Triacylglycerol Lipase Accumulate More TAGs

[0170] In order to test the idea that combining DGA1 and DGA2 overexpression with TGL3 deletion leads to higher lipid accumulation in Y. lipolytica, DGA2 from Claviceps purpurea was overexpressed in strain NS377. Strain NS377 contains a deletion of TGL3 and overexpresses DGA1 from Rhodosporidium toruloides as described in Example 4. DGA2 from Claviceps purpurea was selected based on prior experiments that demonstrate that this gene increases the lipid content of Y. lipolytica in combination with DGA1 from Rhodosporidium toruloides (U.S. Ser. No. 62/004,502, incorporated by reference).

[0171] FIG. 4 shows the map of pNC327, the expression construct used to overexpress C. purpurea DGA2 in NS377. The construct was linearized prior to transformation with a PacI/AscI restriction digest. The linear expression construct included an expression cassette for the C. purpurea DGA2 gene and for the BLE gene used as a marker for selection with Zeocin (ZEO). Transformants were analyzed by the fluorescent lipid assay, and the top lipid producer was designated NS432.

[0172] The lipid production of strains NS297, NS281, NS450, NS377, and NS432 were compared. A subset of these strains were either grown using a batch glucose process (in 48-well plates or 50-mL flasks) or using a high cell density fed-batch glucose process (in 1-L bioreactors). Lipid content was analyzed by fluorescence assay or gas chromatography, and strain NS432 was found to have a higher lipid content than its parent strain NS377 and the strains without the TGL3 knockout (FIG. 5). These results demonstrate the advantage of DGA1 and DGA2 overexpression in a TGL3 knockout.

INCORPORATION BY REFERENCE

[0173] All of the U.S. patents, U.S. patent application publications, foreign patents, foreign patent publications, and non-patent literature cited herein are hereby incorporated by reference.

EQUIVALENTS

[0174] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence CWU 1

1

881514PRTYarrowia lipolytica 1Met Thr Ile Asp Ser Gln Tyr Tyr Lys Ser Arg Asp Lys Asn Asp Thr1 5 10 15Ala Pro Lys Ile Ala Gly Ile Arg Tyr Ala Pro Leu Ser Thr Pro Leu 20 25 30Leu Asn Arg Cys Glu Thr Phe Ser Leu Val Trp His Ile Phe Ser Ile 35 40 45Pro Thr Phe Leu Thr Ile Phe Met Leu Cys Cys Ala Ile Pro Leu Leu 50 55 60Trp Pro Phe Val Ile Ala Tyr Val Val Tyr Ala Val Lys Asp Asp Ser65 70 75 80Pro Ser Asn Gly Gly Val Val Lys Arg Tyr Ser Pro Ile Ser Arg Asn 85 90 95Phe Phe Ile Trp Lys Leu Phe Gly Arg Tyr Phe Pro Ile Thr Leu His 100 105 110Lys Thr Val Asp Leu Glu Pro Thr His Thr Tyr Tyr Pro Leu Asp Val 115 120 125Gln Glu Tyr His Leu Ile Ala Glu Arg Tyr Trp Pro Gln Asn Lys Tyr 130 135 140Leu Arg Ala Ile Ile Thr Thr Ile Glu Tyr Phe Leu Pro Ala Phe Met145 150 155 160Lys Arg Ser Leu Ser Ile Asn Glu Gln Glu Gln Pro Ala Glu Arg Asp 165 170 175Pro Leu Leu Ser Pro Val Ser Pro Ser Ser Pro Gly Ser Gln Pro Asp 180 185 190Lys Trp Ile Asn His Asp Ser Arg Tyr Ser Arg Gly Glu Ser Ser Gly 195 200 205Ser Asn Gly His Ala Ser Gly Ser Glu Leu Asn Gly Asn Gly Asn Asn 210 215 220Gly Thr Thr Asn Arg Arg Pro Leu Ser Ser Ala Ser Ala Gly Ser Thr225 230 235 240Ala Ser Asp Ser Thr Leu Leu Asn Gly Ser Leu Asn Ser Tyr Ala Asn 245 250 255Gln Ile Ile Gly Glu Asn Asp Pro Gln Leu Ser Pro Thr Lys Leu Lys 260 265 270Pro Thr Gly Arg Lys Tyr Ile Phe Gly Tyr His Pro His Gly Ile Ile 275 280 285Gly Met Gly Ala Phe Gly Gly Ile Ala Thr Glu Gly Ala Gly Trp Ser 290 295 300Lys Leu Phe Pro Gly Ile Pro Val Ser Leu Met Thr Leu Thr Asn Asn305 310 315 320Phe Arg Val Pro Leu Tyr Arg Glu Tyr Leu Met Ser Leu Gly Val Ala 325 330 335Ser Val Ser Lys Lys Ser Cys Lys Ala Leu Leu Lys Arg Asn Gln Ser 340 345 350Ile Cys Ile Val Val Gly Gly Ala Gln Glu Ser Leu Leu Ala Arg Pro 355 360 365Gly Val Met Asp Leu Val Leu Leu Lys Arg Lys Gly Phe Val Arg Leu 370 375 380Gly Met Glu Val Gly Asn Val Ala Leu Val Pro Ile Met Ala Phe Gly385 390 395 400Glu Asn Asp Leu Tyr Asp Gln Val Ser Asn Asp Lys Ser Ser Lys Leu 405 410 415Tyr Arg Phe Gln Gln Phe Val Lys Asn Phe Leu Gly Phe Thr Leu Pro 420 425 430Leu Met His Ala Arg Gly Val Phe Asn Tyr Asp Val Gly Leu Val Pro 435 440 445Tyr Arg Arg Pro Val Asn Ile Val Val Gly Ser Pro Ile Asp Leu Pro 450 455 460Tyr Leu Pro His Pro Thr Asp Glu Glu Val Ser Glu Tyr His Asp Arg465 470 475 480Tyr Ile Ala Glu Leu Gln Arg Ile Tyr Asn Glu His Lys Asp Glu Tyr 485 490 495Phe Ile Asp Trp Thr Glu Glu Gly Lys Gly Ala Pro Glu Phe Arg Met 500 505 510Ile Glu21545DNAYarrowia lipolytica 2atgactatcg actcacaata ctacaagtcg cgagacaaaa acgacacggc acccaaaatc 60gcgggaatcc gatatgcccc gctatcgaca ccattactca accgatgtga gaccttctct 120ctggtctggc acattttcag cattcccact ttcctcacaa ttttcatgct atgctgcgca 180attccactgc tctggccatt tgtgattgcg tatgtagtgt acgctgttaa agacgactcc 240ccgtccaacg gaggagtggt caagcgatac tcgcctattt caagaaactt cttcatctgg 300aagctctttg gccgctactt ccccataact ctgcacaaga cggtggatct ggagcccacg 360cacacatact accctctgga cgtccaggag tatcacctga ttgctgagag atactggccg 420cagaacaagt acctccgagc aatcatcacc accatcgagt actttctgcc cgccttcatg 480aaacggtctc tttctatcaa cgagcaggag cagcctgccg agcgagatcc tctcctgtct 540cccgtttctc ccagctctcc gggttctcaa cctgacaagt ggattaacca cgacagcaga 600tatagccgtg gagaatcatc tggctccaac ggccacgcct cgggctccga acttaacggc 660aacggcaaca atggcaccac taaccgacga cctttgtcgt ccgcctctgc tggctccact 720gcatctgatt ccacgcttct taacgggtcc ctcaactcct acgccaacca gatcattggc 780gaaaacgacc cacagctgtc gcccacaaaa ctcaagccca ctggcagaaa atacatcttc 840ggctaccacc cccacggcat tatcggcatg ggagcctttg gtggaattgc caccgaggga 900gctggatggt ccaagctctt tccgggcatc cctgtttctc ttatgactct caccaacaac 960ttccgagtgc ctctctacag agagtacctc atgagtctgg gagtcgcttc tgtctccaag 1020aagtcctgca aggccctcct caagcgaaac cagtctatct gcattgtcgt tggtggagca 1080caggaaagtc ttctggccag acccggtgtc atggacctgg tgctactcaa gcgaaagggt 1140tttgttcgac ttggtatgga ggtcggaaat gtcgcccttg ttcccatcat ggcctttggt 1200gagaacgacc tctatgacca ggttagcaac gacaagtcgt ccaagctgta ccgattccag 1260cagtttgtca agaacttcct tggattcacc cttcctttga tgcatgcccg aggcgtcttc 1320aactacgatg tcggtcttgt cccctacagg cgacccgtca acattgtggt tggttccccc 1380attgacttgc cttatctccc acaccccacc gacgaagaag tgtccgaata ccacgaccga 1440tacatcgccg agctgcagcg aatctacaac gagcacaagg atgaatattt catcgattgg 1500accgaggagg gcaaaggagc cccagagttc cgaatgattg agtaa 15453348PRTRhodosporidium toruloides 3Met Gly Gln Gln Ala Thr Pro Glu Glu Leu Tyr Thr Arg Ser Glu Ile1 5 10 15Ser Lys Ile Lys Phe Ala Pro Phe Gly Val Pro Arg Ser Arg Arg Leu 20 25 30Gln Thr Phe Ser Val Phe Ala Trp Thr Thr Ala Leu Pro Ile Leu Leu 35 40 45Gly Val Phe Phe Leu Leu Cys Ser Phe Pro Pro Leu Trp Pro Ala Val 50 55 60Ile Ala Tyr Leu Thr Trp Val Phe Phe Ile Asp Gln Ala Pro Ile His65 70 75 80Gly Gly Arg Ala Gln Ser Trp Leu Arg Lys Ser Arg Ile Trp Val Trp 85 90 95Phe Ala Gly Tyr Tyr Pro Val Ser Leu Ile Lys Ser Ala Asp Leu Pro 100 105 110Pro Asp Arg Lys Tyr Val Phe Gly Tyr His Pro His Gly Val Ile Gly 115 120 125Met Gly Ala Ile Ala Asn Phe Ala Thr Asp Ala Thr Gly Phe Ser Thr 130 135 140Leu Phe Pro Gly Leu Asn Pro His Leu Leu Thr Leu Gln Ser Asn Phe145 150 155 160Lys Leu Pro Leu Tyr Arg Glu Leu Leu Leu Ala Leu Gly Ile Cys Ser 165 170 175Val Ser Met Lys Ser Cys Gln Asn Ile Leu Arg Gln Gly Pro Gly Ser 180 185 190Ala Leu Thr Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ala His 195 200 205Pro Gly Thr Ala Asp Leu Thr Leu Lys Arg Arg Lys Gly Phe Ile Lys 210 215 220Leu Ala Ile Arg Gln Gly Ala Asp Leu Val Pro Val Phe Ser Phe Gly225 230 235 240Glu Asn Asp Ile Phe Gly Gln Leu Arg Asn Glu Arg Gly Thr Arg Leu 245 250 255Tyr Lys Leu Gln Lys Arg Phe Gln Gly Val Phe Gly Phe Thr Leu Pro 260 265 270Leu Phe Tyr Gly Arg Gly Leu Phe Asn Tyr Asn Val Gly Leu Met Pro 275 280 285Tyr Arg His Pro Ile Val Ser Val Val Gly Arg Pro Ile Ser Val Glu 290 295 300Gln Lys Asp His Pro Thr Thr Ala Asp Leu Glu Glu Val Gln Ala Arg305 310 315 320Tyr Ile Ala Glu Leu Lys Arg Ile Trp Glu Glu Tyr Lys Asp Ala Tyr 325 330 335Ala Lys Ser Arg Thr Arg Glu Leu Asn Ile Ile Ala 340 34541858DNARhodosporidium toruloides 4atgggccagc aggcgacgcc cgaggagcta tacacacgct cagagatctc caagatcaag 60caagtcgagc cagctcttct cctcaccacc ccacaacata ccccgcagcc cacgacagcc 120ctcccacagc acctgcagcc tgctgaccag ctcgagaaca cccacagatt cgcacccttt 180ggcgtcccgc ggtcgcgccg gctgcagacc ttctccgtct ttgcctggac gacggcactg 240cccatcctac tcggcgtctt cttcctcctc tggtgcgtca ggcttggcgt gatctgagag 300tagcgggcgg atcatctgac ctgcttcttc gctgcagctc gttcccaccg ctctggccgg 360ctgtcattgc ctacctcacc tgggtctttt tcattgacca ggcgccgatt cacggtggac 420gggcgcagtc ttggctgcgg aagagtcgga tatgggtctg gtttgcagga tactatcccg 480tcaggtgcgt cctctttcca agcctgcgtc tcgaggcctc gctcacggcc aactcgcccg 540accggctacc tccgaacttt ccgtcaacag cttgatcaag gtcagtctgc gcgtctctcg 600acttcagtgc tctgtggagg agctgcgcca ttgggcccga cctgcggagg gcctcaaagg 660acgatgccgc tgacttcctt tcctccgaca gagcgccgac ttgccgcctg accggaagta 720cgtctttggc taccacccgc acggcgtcat aggcatgggc gccatcgcca acttcgcgac 780cgacgcaacc ggcttctcga cactcttccc cggcttgaac cctcacctcc tcaccctcca 840aagcaacttc aagctcccgc tctaccgcga gttgctgctc gctctcggca tatgctccgt 900ctcgatgaag agctgtcaga acattctgcg acaaggtgag cggtatgcgc aagacgggcg 960gtcaagcgtg aacgcagtga acgagaagag ctgaccttcc gccttactcc atccgtgcag 1020gtcctggctc ggctctcact atcgtcgtcg gtggcgccgc cgagagcttg agtgcgcatc 1080ccggaaccgc cgatcttacg ctcaagcgac gaaaaggctt catcaaactc gcgatccggc 1140aaggcgccga ccttgtgccc gtcttttcgt tcggcgagaa cgacgtgcgc acgctctccg 1200agtctctaaa ccggaagcga atgctgaccg ctgcccaatt ctctctccag atctttggcc 1260agctgcgaaa cgagcgagga acgcggctgt acaagttgca gaagcgtttc caaggcgtgt 1320ttggcttcac cctccgtacg tctcaccgcg ccgtcttgcc gaactgctcg ttcagtcgct 1380cacgcagctt tcactcgcgc agctctcttc tacggccggg gactcttcaa ctgtgcgctc 1440gagttcaccg cttcgccaac agcgaggaat gcctccgagt acagcccagc tgacgcccca 1500tctcttctca tagacaacgt cggattgatg ccgtatcgcc atccgatcgt ctctgtcggt 1560gtgaacccgc tctgtcgctc ctacctgcgt tccttaggct gacaccactc gcgtcaaaca 1620gtcggtcgac caatctcggt agagcagaag gaccacccga ccacggcgga cctcgaagaa 1680gttcaggcgc ggtatatcgc agaactcaag cggtacgttc caagtcgtct gcctccgctt 1740gccgcctcaa ataagctgag gcgtgctgac cgtatctgcc gaaccgtaca gcatctggga 1800agaatacaag gacgcctacg ccaaaagtcg cacgcgggag ctcaatatta tcgcctga 18585348PRTRhodosporidium toruloides 5Met Gly Gln Gln Ala Thr Pro Glu Glu Leu Tyr Thr Arg Ser Glu Ile1 5 10 15Ser Lys Ile Lys Phe Ala Pro Phe Gly Val Pro Arg Ser Arg Arg Leu 20 25 30Gln Thr Phe Ser Val Phe Ala Trp Thr Thr Ala Leu Pro Ile Leu Leu 35 40 45Gly Val Phe Phe Leu Leu Cys Ser Phe Pro Pro Leu Trp Pro Ala Val 50 55 60Ile Ala Tyr Leu Thr Trp Val Phe Phe Ile Asp Gln Ala Pro Ile His65 70 75 80Gly Gly Arg Ala Gln Ser Trp Leu Arg Lys Ser Arg Ile Trp Val Trp 85 90 95Phe Ala Gly Tyr Tyr Pro Val Ser Leu Ile Lys Ser Ala Asp Leu Pro 100 105 110Pro Asp Arg Lys Tyr Val Phe Gly Tyr His Pro His Gly Val Ile Gly 115 120 125Met Gly Ala Ile Ala Asn Phe Ala Thr Asp Ala Thr Gly Phe Ser Thr 130 135 140Leu Phe Pro Gly Leu Asn Pro His Leu Leu Thr Leu Gln Ser Asn Phe145 150 155 160Lys Leu Pro Leu Tyr Arg Glu Leu Leu Leu Ala Leu Gly Ile Cys Ser 165 170 175Val Ser Met Lys Ser Cys Gln Asn Ile Leu Arg Gln Gly Pro Gly Ser 180 185 190Ala Leu Thr Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ala His 195 200 205Pro Gly Thr Ala Asp Leu Thr Leu Lys Arg Arg Lys Gly Phe Ile Lys 210 215 220Leu Ala Ile Arg Gln Gly Ala Asp Leu Val Pro Val Phe Ser Phe Gly225 230 235 240Glu Asn Asp Ile Phe Gly Gln Leu Arg Asn Glu Arg Gly Thr Arg Leu 245 250 255Tyr Lys Leu Gln Lys Arg Phe Gln Gly Val Phe Gly Phe Thr Leu Pro 260 265 270Leu Phe Tyr Gly Arg Gly Leu Phe Asn Tyr Asn Val Gly Leu Met Pro 275 280 285Tyr Arg His Pro Ile Val Ser Val Val Gly Arg Pro Ile Ser Val Glu 290 295 300Gln Lys Asp His Pro Thr Thr Ala Asp Leu Glu Glu Val Gln Ala Arg305 310 315 320Tyr Ile Ala Glu Leu Lys Arg Ile Trp Glu Glu Tyr Lys Asp Ala Tyr 325 330 335Ala Lys Ser Arg Thr Arg Glu Leu Asn Ile Ile Ala 340 34561047DNARhodosporidium toruloides 6atgggccagc aggcgacgcc cgaggagcta tacacacgct cagagatctc caagatcaag 60ttcgcaccct ttggcgtccc gcggtcgcgc cggctgcaga ccttctccgt ctttgcctgg 120acgacggcac tgcccatcct actcggcgtc ttcttcctcc tctgctcgtt cccaccgctc 180tggccggctg tcattgccta cctcacctgg gtctttttca ttgaccaggc gccgattcac 240ggtggacggg cgcagtcttg gctgcggaag agtcggatat gggtctggtt tgcaggatac 300tatcccgtca gcttgatcaa gagcgccgac ttgccgcctg accggaagta cgtctttggc 360taccacccgc acggcgtcat aggcatgggc gccatcgcca acttcgcgac cgacgcaacc 420ggcttctcga cactcttccc cggcttgaac cctcacctcc tcaccctcca aagcaacttc 480aagctcccgc tctaccgcga gttgctgctc gctctcggca tatgctccgt ctcgatgaag 540agctgtcaga acattctgcg acaaggtcct ggctcggctc tcactatcgt cgtcggtggc 600gccgccgaga gcttgagtgc gcatcccgga accgccgatc ttacgctcaa gcgacgaaaa 660ggcttcatca aactcgcgat ccggcaaggc gccgaccttg tgcccgtctt ttcgttcggc 720gagaacgaca tctttggcca gctgcgaaac gagcgaggaa cgcggctgta caagttgcag 780aagcgtttcc aaggcgtgtt tggcttcacc ctccctctct tctacggccg gggactcttc 840aactacaacg tcggattgat gccgtatcgc catccgatcg tctctgtcgt cggtcgacca 900atctcggtag agcagaagga ccacccgacc acggcggacc tcgaagaagt tcaggcgcgg 960tatatcgcag aactcaagcg gatctgggaa gaatacaagg acgcctacgc caaaagtcgc 1020acgcgggagc tcaatattat cgcctga 10477348PRTRhodosporidium toruloides 7Met Gly Gln Gln Ala Thr Pro Glu Glu Leu Tyr Thr Arg Ser Glu Ile1 5 10 15Ser Lys Ile Lys Phe Ala Pro Phe Gly Val Pro Arg Ser Arg Arg Leu 20 25 30Gln Thr Phe Ser Val Phe Ala Trp Thr Thr Ala Leu Pro Ile Leu Leu 35 40 45Gly Val Phe Phe Leu Leu Cys Ser Phe Pro Pro Leu Trp Pro Ala Val 50 55 60Ile Ala Tyr Leu Thr Trp Val Phe Phe Ile Asp Gln Ala Pro Ile His65 70 75 80Gly Gly Arg Ala Gln Ser Trp Leu Arg Lys Ser Arg Ile Trp Val Trp 85 90 95Phe Ala Gly Tyr Tyr Pro Val Ser Leu Ile Lys Ser Ala Asp Leu Pro 100 105 110Pro Asp Arg Lys Tyr Val Phe Gly Tyr His Pro His Gly Val Ile Gly 115 120 125Met Gly Ala Ile Ala Asn Phe Ala Thr Asp Ala Thr Gly Phe Ser Thr 130 135 140Leu Phe Pro Gly Leu Asn Pro His Leu Leu Thr Leu Gln Ser Asn Phe145 150 155 160Lys Leu Pro Leu Tyr Arg Glu Leu Leu Leu Ala Leu Gly Ile Cys Ser 165 170 175Val Ser Met Lys Ser Cys Gln Asn Ile Leu Arg Gln Gly Pro Gly Ser 180 185 190Ala Leu Thr Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ala His 195 200 205Pro Gly Thr Ala Asp Leu Thr Leu Lys Arg Arg Lys Gly Phe Ile Lys 210 215 220Leu Ala Ile Arg Gln Gly Ala Asp Leu Val Pro Val Phe Ser Phe Gly225 230 235 240Glu Asn Asp Ile Phe Gly Gln Leu Arg Asn Glu Arg Gly Thr Arg Leu 245 250 255Tyr Lys Leu Gln Lys Arg Phe Gln Gly Val Phe Gly Phe Thr Leu Pro 260 265 270Leu Phe Tyr Gly Arg Gly Leu Phe Asn Tyr Asn Val Gly Leu Met Pro 275 280 285Tyr Arg His Pro Ile Val Ser Val Val Gly Arg Pro Ile Ser Val Glu 290 295 300Gln Lys Asp His Pro Thr Thr Ala Asp Leu Glu Glu Val Gln Ala Arg305 310 315 320Tyr Ile Ala Glu Leu Lys Arg Ile Trp Glu Glu Tyr Lys Asp Ala Tyr 325 330 335Ala Lys Ser Arg Thr Arg Glu Leu Asn Ile Ile Ala 340 34581047DNARhodosporidium toruloides 8atgggacagc aggctacccc cgaggagctc tacacccgat ccgagatttc taagattaag 60ttcgcccctt ttggagtgcc ccgatcccga cgactccaga ccttctccgt ttttgcctgg 120accactgctc tgcccattct gctcggcgtc ttctttctgc tctgctcttt cccccctctc 180tggcccgccg tcatcgctta cctgacctgg gtgttcttta tcgaccaggc ccctattcac 240ggcggtcgag ctcagtcctg gctgcgaaag tctcgaattt gggtttggtt cgccggttac 300taccccgtct ctctcatcaa gtcggctgac ctgccccctg atcgaaagta cgtgttcggc 360taccaccctc atggtgttat cggtatggga gccattgcta actttgccac cgatgctact 420ggtttctcca ccctctttcc cggactgaac cctcacctgc tcactctcca gtctaacttc 480aagctccccc tgtaccgaga gctgctcctg gccctgggta tctgctccgt ctctatgaag 540tcttgtcaga acattctccg acagggacct ggttcggctc tgaccatcgt cgtgggagga 600gctgctgagt cgctctccgc ccatcctgga accgctgacc tcactctgaa gcgacgaaag 660ggcttcatca agctcgccat tcgacagggt gctgacctgg tgcccgtttt ctcctttgga 720gagaacgata ttttcggcca gctgcgaaac gagcgaggaa cccgactcta caagctgcag 780aagcgatttc agggtgtgtt cggcttcacc ctccctctgt tctacggacg aggcctcttt

840aactacaacg ttggactgat gccctaccga caccctatcg tctcggttgt cggccgaccc 900atttccgtgg agcagaagga ccatcctacc actgccgatc tcgaggaggt gcaggcccga 960tacatcgctg agctgaagcg aatttgggag gagtacaagg acgcctacgc taagtctcga 1020acccgagagc tgaacatcat tgcctaa 10479410PRTLipomyces starkeyi 9Met Ser Glu Lys Ala Glu Ile Glu Val Pro Pro Gln Lys Ser Thr Phe1 5 10 15Pro Arg Ser Val His Phe Ala Pro Leu His Ile Pro Leu Glu Arg Arg 20 25 30Leu Gln Thr Leu Ala Val Leu Phe His Thr Val Ala Leu Pro Tyr Cys 35 40 45Ile Gly Leu Phe Phe Leu Met Leu Ala Phe Pro Pro Phe Trp Pro Leu 50 55 60Leu Val Met Tyr Val Ile Tyr Ala Tyr Gly Phe Asp His Ser Ser Ser65 70 75 80Asn Gly Glu Ile Ser Arg Arg Arg Ser Pro Leu Phe Arg Arg Leu Pro 85 90 95Leu Phe Arg Leu Tyr Cys Asp Tyr Phe Pro Ile His Ile His Arg Glu 100 105 110Val Pro Leu Glu Pro Thr Phe Pro Gly Arg Leu Arg Glu Pro Ser Gly 115 120 125Leu Val Glu Arg Trp Ile Ala Lys Met Phe Gly Val Gln Asp Ala Val 130 135 140Val Glu Gly Asn Glu Ser Asp Val Lys Ala Thr Ala Asn Gly Asn Gly145 150 155 160Thr Thr Lys Glu Ile Gly Pro Thr Tyr Val Phe Gly Tyr His Pro His 165 170 175Gly Ile Val Ser Leu Gly Ala Phe Gly Ala Ile Gly Thr Glu Gly Ala 180 185 190Gly Trp Glu Lys Leu Phe Pro Gly Ile Pro Val Ser Leu Leu Thr Leu 195 200 205Glu Thr Asn Phe Ser Leu Pro Phe Tyr Arg Glu Tyr Leu Leu Ser Leu 210 215 220Gly Ile Ala Ser Val Ser Arg Arg Ser Cys Thr Asn Leu Leu Lys His225 230 235 240Asp Gln Ser Ile Cys Ile Val Ile Gly Gly Ala Gln Glu Ser Leu Leu 245 250 255Ala Glu Pro Gly Thr Leu Asp Leu Ile Leu Val Lys Arg Arg Gly Phe 260 265 270Val Lys Leu Ala Met Ser Thr Ala Arg Val Ser Asp Gln Pro Ile Cys 275 280 285Leu Val Pro Ile Leu Ser Phe Gly Glu Asn Asp Val Tyr Asp Gln Val 290 295 300Arg Gly Asp Arg Ser Ser Lys Leu Tyr Lys Ile Gln Thr Phe Ile Lys305 310 315 320Lys Ala Ala Gly Phe Thr Leu Pro Leu Met Tyr Ala Arg Gly Ile Phe 325 330 335Asn Tyr Asp Phe Gly Leu Met Pro Tyr Arg Arg Gln Met Thr Leu Val 340 345 350Val Gly Lys Pro Ile Ala Val Pro Tyr Val Ala Gln Pro Thr Glu Ala 355 360 365Glu Ile Glu Val Tyr His Lys Gln Tyr Met Asp Glu Leu Arg Arg Leu 370 375 380Trp Asp Thr Tyr Lys Asp Asp Tyr Phe Val Asp His Lys Gly Lys Gly385 390 395 400Val Lys Asn Ser Glu Met Arg Phe Val Glu 405 410101233DNALipomyces starkeyi 10atgagtgaga aggcagagat cgaggttccg ccgcaaaaat cgacattccc tcgcagtgtg 60cacttcgctc cacttcatat tccactggag agacgcctac agactttggc agtcttattc 120cacactgtcg cgctaccata ctgcatcggt ctgttctttc tcatgctcgc gttccctcct 180ttttggccat tattggtaat gtatgtcata tacgcatacg ggttcgacca ctcgagctcg 240aacggagaga tctcccgccg gcgatcgccg ctgtttcgaa gactcccgtt gttcaggctg 300tattgtgatt acttccccat ccacattcac cgggaggttc cgctcgagcc gacgtttcct 360ggtcgccttc gcgaaccgag tggccttgtc gagcggtgga ttgcgaagat gttcggcgtg 420caggacgctg ttgtcgaggg aaatgaatct gacgttaagg ccacggccaa cggcaatggg 480acgacgaaag aaatcggacc gacgtatgtt ttcggctatc atccgcatgg aattgttagc 540ttgggtgcgt ttggtgctat tggtacggaa ggcgctggat gggagaagct ctttcctggg 600atcccggtgt cactgctgac tctcgaaaca aatttcagcc ttccatttta cagagagtat 660ttgctgtcac ttgggattgc ttcagtatct cgacggtctt gtaccaatct cctcaaacac 720gaccaatcca tctgcatcgt tatcggcggc gcccaagagt cgctcttagc ggaaccaggc 780actctagatc tgatcctcgt taaacgtcgc ggttttgtca aacttgcaat gtcaacggcg 840cgggtatctg accaaccgat ttgtcttgtt ccgatcctca gtttcggcga gaacgacgtg 900tacgaccaag tccgcgggga ccgatcgtcg aagttgtata agatccagac ttttatcaag 960aaagcggccg ggtttacgct accattgatg tatgcgcgcg gtatatttaa ttacgacttt 1020gggctgatgc cgtaccgcag gcaaatgacg ctcgtggtcg gcaagccgat tgcagtgccg 1080tacgtggccc agcctacgga ggctgaaatc gaagtgtatc acaagcagta catggatgaa 1140ttgaggaggt tatgggacac gtataaggac gactattttg tagaccacaa gggcaagggg 1200gtcaagaatt ccgagatgcg ttttgtggag taa 123311410PRTLipomyces starkeyi 11Met Ser Glu Lys Ala Glu Ile Glu Val Pro Pro Gln Lys Ser Thr Phe1 5 10 15Pro Arg Ser Val His Phe Ala Pro Leu His Ile Pro Leu Glu Arg Arg 20 25 30Leu Gln Thr Leu Ala Val Leu Phe His Thr Val Ala Leu Pro Tyr Cys 35 40 45Ile Gly Leu Phe Phe Leu Met Leu Ala Phe Pro Pro Phe Trp Pro Leu 50 55 60Leu Val Met Tyr Val Ile Tyr Ala Tyr Gly Phe Asp His Ser Ser Ser65 70 75 80Asn Gly Glu Ile Ser Arg Arg Arg Ser Pro Leu Phe Arg Arg Leu Pro 85 90 95Leu Phe Arg Leu Tyr Cys Asp Tyr Phe Pro Ile His Ile His Arg Glu 100 105 110Val Pro Leu Glu Pro Thr Phe Pro Gly Arg Leu Arg Glu Pro Ser Gly 115 120 125Leu Val Glu Arg Trp Ile Ala Lys Met Phe Gly Val Gln Asp Ala Val 130 135 140Val Glu Gly Asn Glu Ser Asp Val Lys Ala Thr Ala Asn Gly Asn Gly145 150 155 160Thr Thr Lys Glu Ile Gly Pro Thr Tyr Val Phe Gly Tyr His Pro His 165 170 175Gly Ile Val Ser Leu Gly Ala Phe Gly Ala Ile Gly Thr Glu Gly Ala 180 185 190Gly Trp Glu Lys Leu Phe Pro Gly Ile Pro Val Ser Leu Leu Thr Leu 195 200 205Glu Thr Asn Phe Ser Leu Pro Phe Tyr Arg Glu Tyr Leu Leu Ser Leu 210 215 220Gly Ile Ala Ser Val Ser Arg Arg Ser Cys Thr Asn Leu Leu Lys His225 230 235 240Asp Gln Ser Ile Cys Ile Val Ile Gly Gly Ala Gln Glu Ser Leu Leu 245 250 255Ala Glu Pro Gly Thr Leu Asp Leu Ile Leu Val Lys Arg Arg Gly Phe 260 265 270Val Lys Leu Ala Met Ser Thr Ala Arg Val Ser Asp Gln Pro Ile Cys 275 280 285Leu Val Pro Ile Leu Ser Phe Gly Glu Asn Asp Val Tyr Asp Gln Val 290 295 300Arg Gly Asp Arg Ser Ser Lys Leu Tyr Lys Ile Gln Thr Phe Ile Lys305 310 315 320Lys Ala Ala Gly Phe Thr Leu Pro Leu Met Tyr Ala Arg Gly Ile Phe 325 330 335Asn Tyr Asp Phe Gly Leu Met Pro Tyr Arg Arg Gln Met Thr Leu Val 340 345 350Val Gly Lys Pro Ile Ala Val Pro Tyr Val Ala Gln Pro Thr Glu Ala 355 360 365Glu Ile Glu Val Tyr His Lys Gln Tyr Met Asp Glu Leu Arg Arg Leu 370 375 380Trp Asp Thr Tyr Lys Asp Asp Tyr Phe Val Asp His Lys Gly Lys Gly385 390 395 400Val Lys Asn Ser Glu Met Arg Phe Val Glu 405 410121233DNALipomyces starkeyi 12atgtccgaga aggctgagat tgaggtgccc ccccagaagt ctactttccc tcgatccgtt 60catttcgccc ccctgcatat ccccctggag cgacgactcc agaccctggc tgtgctcttc 120cacactgttg ccctgcctta ctgcatcgga ctcttctttc tgatgctcgc tttcccccct 180ttttggcccc tgctcgtgat gtacgttatc tacgcctacg gattcgacca ttcctcttcg 240aacggcgaga tctctcgacg acgatcgcct ctgttccgac gactgcccct ctttcgactc 300tactgtgatt acttccctat ccacattcat cgagaggtcc ccctggagcc tacctttcct 360ggtcgactgc gagagccttc cggactcgtt gagcgatgga ttgctaagat gttcggtgtc 420caggacgccg tcgtggaggg aaacgagtct gatgtgaagg ccaccgctaa cggaaacggc 480accactaagg agatcggccc tacttacgtc ttcggatacc acccccatgg cattgtgtcc 540ctgggagcct ttggcgctat cggtaccgag ggtgctggat gggagaagct cttccctggt 600attcccgtct cgctgctcac cctggagact aacttctccc tcccctttta ccgagagtac 660ctgctctctc tgggaatcgc ctcggtgtcc cgacgatcgt gcaccaacct gctcaagcac 720gaccagtcta tctgtattgt tatcggaggt gctcaggagt ccctgctcgc tgagcctgga 780accctggacc tcattctggt caagcgacga ggcttcgtga agctggccat gtccactgct 840cgagtgtctg atcagcctat ttgcctggtt cccatcctct ctttcggcga gaacgacgtt 900tacgatcagg tccgaggtga ccgatcctct aagctgtaca agattcagac cttcatcaag 960aaggccgctg gctttactct ccctctgatg tacgcccgag gcatcttcaa ctacgacttt 1020ggtctgatgc cctaccgacg acagatgacc ctcgttgtcg gcaagcctat tgccgtcccc 1080tacgtggctc agcccactga ggccgagatc gaggtctacc acaagcagta catggacgag 1140ctgcgacgac tctgggatac ctacaaggac gattacttcg ttgaccataa gggcaagggt 1200gtcaagaact ctgagatgcg atttgtggag taa 123313380PRTAspergillus terreus 13Met Pro Arg Asn Thr His Pro Pro Ala Asn Asn Ala Gly Pro Asn Ala1 5 10 15Ser His Lys Lys Asp Arg Lys Arg Gln Gly Arg Leu Phe Gln His Thr 20 25 30Val Pro Asn Lys Tyr Ser Arg Ile Arg Trp Ala Pro Leu Asn Ile Gly 35 40 45Leu Glu Arg Arg Leu Gln Thr Leu Val Val Leu Cys His Thr Leu Thr 50 55 60Ile Ala Leu Phe Leu Ala Phe Phe Phe Phe Thr Cys Ala Ile Pro Leu65 70 75 80Thr Trp Pro Leu Leu Phe Pro Tyr Leu Val Tyr Ile Thr Leu Phe Ser 85 90 95Thr Ala Pro Thr Ser Gly Thr Leu Lys Gly Arg Ser Asp Phe Leu Arg 100 105 110Ser Leu Pro Ile Trp Lys Leu Tyr Thr Ala Tyr Phe Pro Ala Lys Leu 115 120 125His Arg Ser Glu Pro Leu Leu Pro Thr Arg Lys Tyr Ile Phe Gly Tyr 130 135 140His Pro His Gly Ile Ile Ser His Gly Ala Phe Ala Ala Phe Ala Thr145 150 155 160Asp Ala Leu Gly Phe Ser Lys Leu Phe Pro Gly Ile Thr Asn Thr Leu 165 170 175Leu Thr Leu Asp Ser Asn Phe Arg Ile Pro Phe Tyr Arg Glu Tyr Ala 180 185 190Met Ala Met Gly Val Ala Ser Val Ser Arg Glu Ser Cys Glu Asn Leu 195 200 205Leu Thr Lys Gly Gly Ala Asp Gly Glu Gly Met Gly Arg Ala Ile Thr 210 215 220Ile Val Val Gly Gly Ala Arg Glu Ser Leu Asp Ala Leu Pro His Thr225 230 235 240Met Arg Leu Val Leu Lys Arg Arg Lys Gly Phe Ile Lys Leu Ala Ile 245 250 255Arg Thr Gly Ala Asp Leu Val Pro Val Leu Ala Phe Gly Glu Asn Asp 260 265 270Leu Tyr Glu Gln Val Arg Ser Asp Gln His Pro Leu Ile Tyr Lys Val 275 280 285Gln Met Leu Val Lys Arg Phe Leu Gly Phe Thr Val Pro Leu Phe His 290 295 300Ala Arg Gly Ile Phe Asn Tyr Asp Val Gly Leu Met Pro Tyr Arg Arg305 310 315 320Pro Leu Asn Ile Val Val Gly Arg Pro Ile Gln Val Val Arg Gln Gln 325 330 335Asp Arg Asp Lys Ile Asp Asp Glu Tyr Ile Asp Arg Leu His Ala Glu 340 345 350Tyr Val Arg Glu Leu Glu Ser Leu Trp Asp Gln Trp Lys Asp Val Tyr 355 360 365Ala Lys Asp Arg Ile Ser Glu Leu Glu Ile Val Ala 370 375 380141143DNAAspergillus terreus 14atgccccgaa acacccaccc ccccgccaac aacgccggac ctaacgcctc tcacaagaag 60gaccgaaagc gacagggacg actctttcag cacaccgttc ctaacaagta ctctcgaatc 120cgatgggccc ccctcaacat tggcctggag cgacgactgc agaccctcgt cgtgctgtgc 180cataccctca ctatcgccct gttcctcgct ttctttttct ttacttgtgc cattcccctg 240acctggcctc tgctcttccc ctacctcgtg tacatcaccc tgttttcgac cgctcctact 300tccggtaccc tgaagggacg atctgacttc ctccgatcgc tgcctatttg gaagctctac 360actgcctact ttcccgctaa gctgcaccga tccgagcctc tgctccctac ccgaaagtac 420atcttcggct accaccccca tggtatcatt tcccatggag ccttcgccgc ttttgccact 480gacgctctcg gcttctctaa gctgtttcct ggtatcacca acactctgct caccctggat 540tcgaacttcc gaattccctt ttaccgagag tacgccatgg ctatgggagt ggcttccgtt 600tctcgagagt cgtgcgagaa cctgctcact aagggaggtg ctgacggaga gggaatgggc 660cgagctatca ccattgttgt cggaggcgcc cgagagtccc tcgatgctct gcctcacact 720atgcgactgg tcctcaagcg acgaaagggt ttcatcaagc tggccattcg aaccggagct 780gacctcgttc ccgtcctggc cttcggcgag aacgacctct acgagcaggt gcgatctgat 840cagcaccctc tgatctacaa ggtccagatg ctcgtgaagc gattcctggg ttttaccgtg 900cccctgttcc atgctcgagg aatttttaac tacgacgttg gcctcatgcc ttaccgacga 960cccctgaaca tcgtggttgg tcgacccatt caggtcgtgc gacagcagga ccgagataag 1020atcgacgatg agtacattga ccgactccac gccgagtacg tccgagagct cgagtccctg 1080tgggaccagt ggaaggatgt ttacgccaag gaccgaatct ctgagctgga gattgtcgct 1140taa 114315437PRTClaviceps purpurea 15Met Ala Ala Val Gln Val Ala Arg Pro Val Pro Pro His His His Asp1 5 10 15Gly Ala Gly Arg Glu His Lys Gly Glu Arg Ala His Ser Pro Glu Arg 20 25 30Gly Glu Lys Thr Val His Asn Gly Tyr Gly Leu Ala Glu Thr His Glu 35 40 45Pro Leu Glu Leu Asn Gly Ser Ala Val Gln Asp Gly Lys His Asp Ser 50 55 60Asp Glu Thr Ile Thr Asn Gly Asp Tyr Ser Pro Tyr Pro Glu Leu Asp65 70 75 80Cys Gly Lys Glu Arg Ala Ala His Glu Lys Glu Ala Trp Thr Ala Gly 85 90 95Gly Val Arg Phe Ala Pro Leu Arg Val Pro Phe Lys Arg Arg Met Gln 100 105 110Thr Ala Ala Val Leu Phe His Cys Met Ser Ile Ile Leu Ile Ser Ser 115 120 125Cys Phe Trp Phe Ser Leu Ala Asn Pro Ile Thr Trp Pro Ile Leu Val 130 135 140Pro Tyr Leu Val His Leu Ser Leu Ser Asn Ala Ser Thr Asp Gly Lys145 150 155 160Leu Ser Tyr Arg Ser Glu Trp Leu Arg Ser Leu Pro Leu Trp Arg Leu 165 170 175Phe Ala Gly Tyr Phe Pro Ala Lys Leu His Lys Thr Phe Asp Leu Pro 180 185 190Pro Asn Arg Lys Tyr Ile Phe Gly Tyr His Pro His Gly Ile Ile Ser 195 200 205His Gly Ala Trp Cys Ala Phe Ala Thr Asn Ala Leu Gly Phe Val Glu 210 215 220Lys Phe Pro Gly Ile Thr Asn Ser Leu Leu Thr Leu Asp Ser Asn Phe225 230 235 240Arg Val Pro Phe Tyr Arg Asp Trp Ile Leu Ala Met Gly Ile Arg Ser 245 250 255Val Ser Arg Glu Ser Ile Arg Asn Ile Leu Ser Lys Gly Gly Pro Asp 260 265 270Ser Asn Gly Gln Gly Arg Ala Val Thr Ile Val Ile Gly Gly Ala Arg 275 280 285Glu Ser Leu Glu Ala Gln Pro Gly Thr Leu Arg Leu Ile Leu Gln Gly 290 295 300Arg Lys Gly Phe Ile Lys Val Ala Leu Arg Ala Gly Ala Asp Leu Val305 310 315 320Pro Val Ile Gly Phe Gly Glu Asn Asp Leu Tyr Asp Gln Leu Ser Pro 325 330 335Lys Thr His Pro Leu Val His Lys Ile Gln Met Phe Phe Leu Lys Val 340 345 350Phe Lys Phe Thr Ile Pro Ala Leu His Gly Arg Gly Leu Leu Asn Tyr 355 360 365Asp Val Gly Leu Leu Pro Tyr Arg Arg Ala Val Asn Ile Val Val Gly 370 375 380Arg Pro Ile Gln Ile Asp Glu Thr Tyr Gly Glu Gln Pro Pro Gln Glu385 390 395 400Val Ile Asp Arg Tyr His Glu Leu Tyr Val Gln Glu Val Glu Arg Leu 405 410 415Tyr Ala Ala Tyr Lys Glu Gln Phe Ser Asn Gly Lys Lys Thr Pro Glu 420 425 430Leu Gln Ile Leu Ser 435161314DNAClaviceps purpurea 16atggctgctg ttcaggttgc ccgacccgtt cccccccacc accacgatgg cgctggccga 60gagcacaagg gagagcgagc ccattcccct gagcgaggag agaagaccgt ccacaacggc 120tacggtctgg ccgagactca tgagcccctg gagctcaacg gttctgctgt gcaggacgga 180aagcacgact cggatgagac catcactaac ggtgactact ctccctaccc tgagctcgat 240tgcggaaagg agcgagccgc tcatgagaag gaggcttgga ccgctggagg tgtgcgattc 300gctcctctgc gagttccttt taagcgacga atgcagactg ccgctgtcct cttccactgc 360atgtccatca ttctgatttc ctcttgtttc tggttttctc tcgccaaccc catcacctgg 420cctattctcg ttccctacct ggtccacctg tcgctctcca acgcttctac tgacggcaag 480ctctcctacc gatctgagtg gctgcgatcc ctgcctctct ggcgactgtt cgccggttac 540tttcccgcta agctccacaa gaccttcgat ctgcccccta accgaaagta catctttggt 600taccaccccc atggaatcat ttcccatggc gcctggtgtg ccttcgctac caacgctctg 660ggcttcgttg agaagtttcc tggtattacc aactcgctgc tcactctcga ctccaacttc 720cgagtgccct tttaccgaga ttggatcctg gccatgggca ttcgatctgt ttcgcgagag 780tctatccgaa acattctctc gaagggagga cctgactcca acggacaggg ccgagctgtg 840accatcgtta ttggtggagc ccgagagtct ctggaggctc agcccggaac tctgcgactc 900attctgcagg gccgaaaggg cttcattaag

gtggctctcc gagctggagc tgacctggtt 960cccgtcatcg gtttcggaga gaacgacctc tacgatcagc tgtcccctaa gacccacccc 1020ctcgttcata agatccagat gttctttctg aaggtcttca agtttactat tcctgctctg 1080cacggacgag gtctgctcaa ctacgacgtc ggtctgctcc cttaccgacg agctgtgaac 1140atcgtcgtgg gacgacccat ccagattgac gagacctacg gcgagcagcc ccctcaggag 1200gtcatcgatc gataccacga gctctacgtc caggaggtgg agcgactgta cgccgcttac 1260aaggagcagt tctcgaacgg aaagaagacc cccgagctcc agatcctgtc ctaa 131417351PRTAurantiochytrium limacinum 17Met Leu Ala Trp Met Pro Val Leu Ile Ala Leu Pro Arg Arg Lys Gln1 5 10 15Thr Ala Val Val Leu Leu Phe Val Met Leu Leu Pro Met Ile Met Val 20 25 30Val Tyr Ser Trp Thr Leu Ile Leu Leu Ile Phe Pro Leu Thr Thr Leu 35 40 45Pro Thr Leu Ser Tyr Leu Ile Trp Ile Met Tyr Ile Asp Lys Ser His 50 55 60Glu Thr Gly Lys Arg Lys Pro Phe Met Arg Tyr Trp Lys Met Trp Arg65 70 75 80His Phe Ala Asn Tyr Phe Pro Leu Arg Leu Ile Arg Thr Thr Pro Leu 85 90 95Asp Pro Arg Arg Lys Tyr Val Phe Cys Tyr His Pro His Gly Ile Ile 100 105 110Ser Leu Gly Ala Phe Gly Asn Phe Ala Thr Asp Ser Thr Gly Phe Ser 115 120 125Arg Lys Phe Pro Gly Ile Asp Leu Arg Leu Leu Thr Leu Gln Ile Asn 130 135 140Phe Tyr Cys Pro Ile Ile Arg Glu Leu Leu Leu Tyr Met Gly Leu Cys145 150 155 160Ser Ala Ala Lys Lys Ser Cys Asn Gln Ile Leu Gln Arg Gly Pro Gly 165 170 175Ser Ala Ile Met Leu Val Val Gly Gly Ala Ala Glu Ser Leu Asp Ser 180 185 190Gln Pro Gly Thr Tyr Arg Leu Thr Leu Gly Arg Lys Gly Phe Val Arg 195 200 205Val Ala Leu Asp Asn Gly Ala Asp Leu Val Pro Val Leu Gly Phe Gly 210 215 220Glu Asn Asp Val Phe Asp Thr Val Tyr Leu Pro Pro Asn Ser Trp Ala225 230 235 240Arg Asn Val Gln Glu Phe Val Arg Lys Lys Leu Gly Phe Ala Thr Pro 245 250 255Ile Phe Ser Gly Arg Gly Ile Phe Gln Tyr Asn Met Gly Leu Met Pro 260 265 270His Arg Lys Pro Ile Ile Val Val Val Gly Lys Pro Ile Lys Ile Pro 275 280 285Lys Ile Pro Asp Glu Leu Lys Gly Arg Ala Leu Ser Thr Thr Ala Glu 290 295 300Gly Val Ala Leu Val Asp Lys Tyr His Glu Lys Tyr Val Arg Ala Leu305 310 315 320Arg Glu Leu Trp Asn Leu Tyr Lys Glu Glu Tyr Ala Thr Glu Pro Lys 325 330 335Ala Ala Tyr Leu Glu Pro Asn Ser Ile Arg Lys Asn Gln Asn Val 340 345 350181056DNAAurantiochytrium limacinum 18atgctcgcct ggatgcctgt cctcattgcc ctcccccgac gaaagcagac cgctgttgtt 60ctcctgtttg tgatgctcct ccctatgatc atggtcgtgt actcctggac cctgatcctg 120ctcattttcc ccctcaccac tctgcctact ctctcctacc tgatctggat tatgtacatt 180gacaagtctc acgagaccgg aaagcgaaag ccctttatgc gatactggaa gatgtggcga 240catttcgcca actactttcc tctccgactg atccgaacca ctcccctgga ccctcgacga 300aagtacgtgt tctgctacca cccccatggc atcatttccc tcggagcctt cggcaacttt 360gctaccgact cgactggctt ctcccgaaag tttcccggta tcgatctgcg actgctcacc 420ctccagatta acttctactg tcctatcatt cgagagctgc tcctgtacat gggtctgtgc 480tctgccgcta agaagtcgtg taaccagatc ctccagcgag gacccggctc tgctattatg 540ctggttgtcg gcggtgccgc tgagtccctc gactctcagc ctggcaccta ccgactcact 600ctgggtcgaa agggattcgt gcgagttgcc ctggacaacg gtgctgatct ggtccccgtg 660ctcggtttcg gagagaacga cgtgtttgat accgtttacc tgccccctaa ctcgtgggcc 720cgaaacgtcc aggagttcgt gcgaaagaag ctcggattcg ctacccccat cttttccggc 780cgaggtattt ttcagtacaa catgggtctg atgccccacc gaaagcctat cattgtggtt 840gtcggaaagc ccatcaagat tcccaagatc cctgacgagc tgaagggacg agccctctct 900accactgccg agggcgttgc tctggtcgat aagtaccatg agaagtacgt tcgagccctc 960cgagagctgt ggaacctcta caaggaggag tacgctaccg agcccaaggc cgcttacctc 1020gagcctaact cgattcgaaa gaaccagaac gtctaa 105619666PRTYarrowia lipolytica 19Met Lys Ser Arg Val Ala Val Val Leu Ala Pro Val Leu Ala Pro Phe1 5 10 15Val Ala Ile Leu Lys Asn Leu Trp Val Phe Phe Thr Ala Leu Leu Glu 20 25 30Leu Leu Phe Asp Val Ser Trp His Trp Met Leu Gln Ser Trp His Trp 35 40 45Trp Cys Ser Thr Asp Gln Lys Thr Leu Leu Gln Leu Gln Leu Asp Gln 50 55 60Ala Asp Thr Tyr Glu Glu Trp Glu Ser Ile Ala Ser Glu Leu Asp Glu65 70 75 80Leu Leu Gly Asn Asp Val Trp Arg Gln Thr Ala Ala Ser Lys Arg Tyr 85 90 95Asp Tyr Arg Leu Ile Ala Gly Arg Leu Arg Asp Phe Ile Glu Cys Arg 100 105 110Ala Val Gly Asp Ile Ala Thr Leu Ile Ser Arg Leu Arg Ser Gly Leu 115 120 125Leu Arg Asn Leu Gly Ser Ile Ser Ser Leu Gln Leu Tyr Thr Arg Ser 130 135 140Tyr Leu Gly Ser Lys Leu Leu Ile Glu Glu Tyr Ile Thr Glu Val Ile145 150 155 160Asp Cys Leu Lys Tyr Ile Lys Asp Tyr Gly Thr Thr Gly Gly Leu Asp 165 170 175Thr Lys Gly Val His Phe Phe Pro Lys Ser Glu Gln Arg Gln Leu Asp 180 185 190Ser Glu Gln Leu Thr Arg Gln Lys Lys His Lys Leu Phe Tyr Asp Thr 195 200 205Arg Gln Ser Phe Gly Arg Thr Ala Leu Val Leu Gln Gly Gly Thr Ile 210 215 220Phe Gly Leu Thr His Leu Gly Thr Ile Lys Ala Leu Thr Leu Gln Gly225 230 235 240Leu Leu Pro Gly Ile Val Thr Gly Phe Lys Glu Gly Ala Phe Ile Ala 245 250 255Ala Leu Thr Gly Ile Tyr Val Ser Asp Leu Glu Leu Leu Glu Thr Ile 260 265 270Asp Ser Leu Pro Asp Thr Leu Asn Asp Leu Tyr Gln Lys Tyr Lys Glu 275 280 285Arg Leu Ala Glu Glu Asn Lys His Lys Asp His Ser Phe Ser Asn Ser 290 295 300Asn Ser Asp Tyr Asp Phe Asp Tyr Ala Phe Asp Phe Glu Gln Phe Ala305 310 315 320Asn Thr Tyr Asn Val Thr Phe Ser Ser Val Thr Asp Lys Val Leu Arg 325 330 335Ser Glu Tyr Pro Pro Glu Val Lys Met Tyr Glu Glu Phe Ile Glu Asn 340 345 350Gln Leu Gly Asp Leu Thr Phe Glu Glu Ala Phe Asn Lys Ser Asp Arg 355 360 365Val Leu Asn Ile Val Ala His Ser His Asp Ser Ser Phe Pro Thr Leu 370 375 380Met Asn Tyr Leu Thr Thr Pro Asn Val Leu Ile Arg Ser Ala Cys Arg385 390 395 400Ala Ser Met Val Thr Ala His Asp Glu Pro Gln Thr Lys Lys Ala Cys 405 410 415Ala His Leu Leu Val Lys Asp Asp Asp Asn Ser Val Ile Pro Tyr Asp 420 425 430Ala Cys Lys Ser Arg Arg Gly Ser Ser Thr Asp Val Ile Leu Gly Pro 435 440 445Val Gln Glu Glu Val Asp Pro Leu Asp Ser Thr Ala Asn Gly Thr Asn 450 455 460Ser Ser Gly Pro Pro Lys Leu Glu Ile Thr Thr Asp Thr Trp Lys Arg465 470 475 480Asn Asn Ala Asp Asp Glu Asp His Val Asp Thr Leu Pro Gly Arg Val 485 490 495Ser Ala Leu Pro Thr Pro Ser Tyr Ser Met Ile Asn Gln Gly Lys Ile 500 505 510Val Ser Pro Tyr Ala Arg Leu Ser Glu Leu Phe Asn Val Asn His Phe 515 520 525Ile Val Ser Leu Ser Arg Pro Tyr Leu Ala Pro Leu Leu Ala Ile Glu 530 535 540Gly Arg His Arg Gly Tyr His Gly Trp Arg Val Asn Leu Ile Arg Val545 550 555 560Leu Lys Leu Glu Phe Glu His Arg Leu Ala Gln Phe Asp Tyr Ile Gly 565 570 575Leu Leu Pro Thr Ile Phe Arg Arg Phe Phe Ile Asp Asp Lys Ile Pro 580 585 590Gly Ile Gly Pro Asn Ala Glu Val Leu Ile Val Pro Glu Leu Ala Ala 595 600 605Gly Met Ile Ser Asp Phe Lys Lys Ala Phe Ser Asn His Asp Ile Pro 610 615 620Glu Lys Val Arg Tyr Trp Thr Thr Val Gly Glu Arg Ala Thr Trp Pro625 630 635 640Leu Val Ala Ala Ile Trp Ala Arg Thr Ala Ile Glu Tyr Thr Leu Asn 645 650 655Asp Met Tyr Asn Gln Thr Lys Arg Gln Asn 660 665202201DNAYarrowia lipolytica 20cttttacgag tgtgtatcat cacatgatta tgcagcaaga tcagtatcat ttcggctatc 60cagctctctt cccccgttca gctccttttc taccgcgatt atgaaaagcc gcgtggccgt 120tgtcttggcg ccggttctgg caccatttgt ggcgattttg aaaaacctgt gggtcttctt 180cacagctcta ctggagctct tattcgacgt tagctggcac tggatgttac aatcatggca 240ctggtggtgc tccaccgacc aaaaaacact gctacaactg cagctggacc aggcagacac 300ctacgaggaa tgggaaagca ttgcatcgga gctggacgag ctgctgggca acgacgtgtg 360gcgtcagacc gcagcctcga aacgatacga ctaccggctg attgcaggcc gtctgagaga 420ctttatcgag tgccgggcgg tcggcgacat tgcgacgctg atttctcgtc tgcgaagcgg 480actgctgcgg aatttgggct cgatttcgtc gctccagctg tacactcgct cgtacctcgg 540ctctaaactg ctcatcgaag agtacatcac cgaggtcatt gactgtctca agtacatcaa 600ggactatggg acgacgggcg gactggacac caagggagtg catttcttcc caaagtccga 660acagcgacaa ctggacagtg aacagctgac tcgacaaaag aaacacaagt tattctacga 720cacacgacaa tcttttggcc gaacggccct cgtgttgcag ggaggaacta ttttcggact 780tactcatctc ggaacaatca aggctcttac tctccagggt ctgctaccgg gtattgtcac 840cggtttcaag gagggggcgt ttattgccgc tctcacaggc atctacgtat ccgacctgga 900gctgctcgaa accattgact ctttgccaga cactctcaat gacctgtacc aaaaatacaa 960ggagcgactg gcggaggaaa acaaacacaa ggaccactcg ttcagtaact ccaattcgga 1020ctacgacttt gactacgcat ttgactttga acagtttgca aacacctata atgtgacctt 1080ctcgtctgtc actgacaaag tattgcgatc ggagtacccc ccggaagtca aaatgtacga 1140ggagttcatc gagaatcaac tcggagacct cacgttcgaa gaggccttca acaaaagcga 1200ccgcgtgctc aacattgtcg cccattccca tgactcttcc ttcccgacac tgatgaacta 1260cctcaccact cccaatgtgc tcatcagaag cgcatgtaga gcttccatgg tgaccgccca 1320cgacgagccc caaacgaaaa aggcatgtgc ccatctgctg gtcaaggatg acgacaacag 1380cgtcattccc tatgacgcct gcaaatccag gcgaggaagc tcgaccgacg tgattctggg 1440acctgtccag gaggaggtgg atccattaga ttcaacagct aacggtacta actcttctgg 1500acctcccaaa ctcgaaatca caactgacac ctggaaacga aacaatgcag acgacgagga 1560ccacgtggat actctcccgg gccgcgtgag tgctctacct acaccttcgt actccatgat 1620taaccagggc aagattgtct ctccctacgc tcgcctttcc gaactcttta acgtcaacca 1680cttcatcgtc tctctctcaa gaccctacct ggcgcctctt ctggccatcg aaggccgaca 1740tagaggctac cacggctgga gagtgaacct gatccgagta ctgaaactag aattcgaaca 1800cagactcgcc cagttcgact acataggcct gctgccgacc atcttccgtc ggttcttcat 1860cgacgataag atccctggca tcggtcccaa cgccgaggtg ctcattgttc ctgagctagc 1920ggctggcatg atctccgact tcaaaaaggc cttttcgaac cacgacattc ccgagaaggt 1980ccgctactgg accactgtgg gcgaacgagc cacctggcct ctagtcgccg ccatctgggc 2040cagaacagca atcgagtaca ccctcaacga catgtacaac cagaccaagc gacaaaacta 2100gaccccgagc agagcacata actactaacg atgagactaa agtatgtact gtatgtacta 2160aacatacgct cgtaaacagt tgtatttatt ctttttcagc a 220121341PRTEscherichia coli 21Met Lys Lys Pro Glu Leu Thr Ala Thr Ser Val Glu Lys Phe Leu Ile1 5 10 15Glu Lys Phe Asp Ser Val Ser Asp Leu Met Gln Leu Ser Glu Gly Glu 20 25 30Glu Ser Arg Ala Phe Ser Phe Asp Val Gly Gly Arg Gly Tyr Val Leu 35 40 45Arg Val Asn Ser Cys Ala Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr 50 55 60Arg His Phe Ala Ser Ala Ala Leu Pro Ile Pro Glu Val Leu Asp Ile65 70 75 80Gly Glu Phe Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg Arg Ala Gln 85 90 95Gly Val Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu Pro Ala Val Leu 100 105 110Gln Pro Val Ala Glu Ala Met Asp Ala Ile Ala Ala Ala Asp Leu Ser 115 120 125Gln Thr Ser Gly Phe Gly Pro Phe Gly Pro Gln Gly Ile Gly Gln Tyr 130 135 140Thr Thr Trp Arg Asp Phe Ile Cys Ala Ile Ala Asp Pro His Val Tyr145 150 155 160His Trp Gln Thr Val Met Asp Asp Thr Val Ser Ala Ser Val Ala Gln 165 170 175Ala Leu Asp Glu Leu Met Leu Trp Ala Glu Asp Cys Pro Glu Val Arg 180 185 190His Leu Val His Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn 195 200 205Gly Arg Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe Gly Asp 210 215 220Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg Pro Trp Leu Ala225 230 235 240Cys Met Glu Gln Gln Thr Arg Tyr Phe Glu Arg Arg His Pro Glu Leu 245 250 255Ala Gly Ser Pro Arg Leu Arg Ala Tyr Met Leu Arg Ile Gly Leu Asp 260 265 270Gln Leu Tyr Gln Ser Leu Val Asp Gly Asn Phe Asp Asp Ala Ala Trp 275 280 285Ala Gln Gly Arg Cys Asp Ala Ile Val Arg Ser Gly Ala Gly Thr Val 290 295 300Gly Arg Thr Gln Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly305 310 315 320Cys Val Glu Val Leu Ala Asp Ser Gly Asn Arg Arg Pro Ser Thr Arg 325 330 335Pro Arg Ala Lys Glu 340221026DNAEscherichia coli 22atgaagaagc ccgagctgac cgctacctct gttgagaagt tcctgattga gaagtttgat 60tccgtttccg acctgatgca gctgtccgag ggcgaggagt ctcgagcctt ctcctttgac 120gtgggcggac gaggttacgt tctgcgagtg aactcgtgtg ccgacggctt ctacaaggat 180cgatacgtct accgacactt tgcttctgcc gctctgccca tccctgaggt tctcgacatt 240ggcgagttct ctgagtccct cacctactgc atctctcgac gagctcaggg agtcaccctg 300caggacctcc ctgagactga gctgcctgct gtcctccagc ctgttgctga ggccatggac 360gctatcgctg ctgctgatct gtcccagacc tcgggtttcg gcccctttgg acctcaggga 420attggacagt acaccacttg gcgagacttc atctgtgcta ttgccgatcc tcacgtctac 480cattggcaga ccgttatgga cgatactgtg tcggcttctg tcgctcaggc tctggacgag 540ctgatgctct gggccgagga ttgccccgag gttcgacacc tggtgcatgc tgacttcggt 600tccaacaacg ttctcaccga caacggccga atcactgccg tgattgactg gtccgaggct 660atgtttggcg actcgcagta cgaggtggcc aacatcttct tttggcgacc ctggctggct 720tgtatggagc agcagacccg atacttcgag cgacgacatc ctgagctcgc tggatcccct 780cgactgcgag cttacatgct ccgaattggt ctggaccagc tctaccagtc gctggtggat 840ggcaactttg acgatgctgc ctgggctcag ggacgatgtg acgccatcgt gcgatctggc 900gctggaaccg tcggacgaac tcagattgcc cgacgatccg ctgctgtctg gaccgacgga 960tgcgtggagg tcctggctga ttcgggtaac cgacgaccct ctactcgacc tcgagctaag 1020gagtaa 10262360DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23cagctctctt cccccgttca gctccttttc taccgcgatt atgaagaagc ccgagctgac 602424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24agcatcgtca aagttgccat ccac 242524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25accacttggc gagacttcat ctgt 242660DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26tttagtctca tcgttagtag ttatgtgctc tgctcggggt tactccttag ctcgaggtcg 6027898DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 27cagctctctt cccccgttca gctccttttc taccgcgatt atgaagaagc ccgagctgac 60cgctacctct gttgagaagt tcctgattga gaagtttgat tccgtttccg acctgatgca 120gctgtccgag ggcgaggagt ctcgagcctt ctcctttgac gtgggcggac gaggttacgt 180tctgcgagtg aactcgtgtg ccgacggctt ctacaaggat cgatacgtct accgacactt 240tgcttctgcc gctctgccca tccctgaggt tctcgacatt ggcgagttct ctgagtccct 300cacctactgc atctctcgac gagctcaggg agtcaccctg caggacctcc ctgagactga 360gctgcctgct gtcctccagc ctgttgctga ggccatggac gctatcgctg ctgctgatct 420gtcccagacc tcgggtttcg gcccctttgg acctcaggga attggacagt acaccacttg 480gcgagacttc atctgtgcta ttgccgatcc tcacgtctac cattggcaga ccgttatgga 540cgatactgtg tcggcttctg tcgctcaggc tctggacgag ctgatgctct gggccgagga 600ttgccccgag gttcgacacc tggtgcatgc tgacttcggt tccaacaacg ttctcaccga 660caacggccga atcactgccg tgattgactg gtccgaggct atgtttggcg actcgcagta 720cgaggtggcc aacatcttct tttggcgacc ctggctggct tgtatggagc agcagacccg 780atacttcgag cgacgacatc ctgagctcgc tggatcccct cgactgcgag cttacatgct 840ccgaattggt ctggaccagc tctaccagtc gctggtggat ggcaactttg acgatgct 89828633DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 28accacttggc gagacttcat ctgtgctatt gccgatcctc acgtctacca ttggcagacc 60gttatggacg atactgtgtc ggcttctgtc gctcaggctc tggacgagct gatgctctgg 120gccgaggatt gccccgaggt tcgacacctg gtgcatgctg acttcggttc caacaacgtt 180ctcaccgaca acggccgaat cactgccgtg attgactggt ccgaggctat gtttggcgac 240tcgcagtacg aggtggccaa catcttcttt

tggcgaccct ggctggcttg tatggagcag 300cagacccgat acttcgagcg acgacatcct gagctcgctg gatcccctcg actgcgagct 360tacatgctcc gaattggtct ggaccagctc taccagtcgc tggtggatgg caactttgac 420gatgctgcct gggctcaggg acgatgtgac gccatcgtgc gatctggcgc tggaaccgtc 480ggacgaactc agattgcccg acgatccgct gctgtctgga ccgacggatg cgtggaggtc 540ctggctgatt cgggtaaccg acgaccctct actcgacctc gagctaagga gtaaccccga 600gcagagcaca taactactaa cgatgagact aaa 63329526PRTYarrowia lipolytica 29Met Glu Val Arg Arg Arg Lys Ile Asp Val Leu Lys Ala Gln Lys Asn1 5 10 15Gly Tyr Glu Ser Gly Pro Pro Ser Arg Gln Ser Ser Gln Pro Ser Ser 20 25 30Arg Ala Ser Ser Arg Thr Arg Asn Lys His Ser Ser Ser Thr Leu Ser 35 40 45Leu Ser Gly Leu Thr Met Lys Val Gln Lys Lys Pro Ala Gly Pro Pro 50 55 60Ala Asn Ser Lys Thr Pro Phe Leu His Ile Lys Pro Val His Thr Cys65 70 75 80Cys Ser Thr Ser Met Leu Ser Arg Asp Tyr Asp Gly Ser Asn Pro Ser 85 90 95Phe Lys Gly Phe Lys Asn Ile Gly Met Ile Ile Leu Ile Val Gly Asn 100 105 110Leu Arg Leu Ala Phe Glu Asn Tyr Leu Lys Tyr Gly Ile Ser Asn Pro 115 120 125Phe Phe Asp Pro Lys Ile Thr Pro Ser Glu Trp Gln Leu Ser Gly Leu 130 135 140Leu Ile Val Val Ala Tyr Ala His Ile Leu Met Ala Tyr Ala Ile Glu145 150 155 160Ser Ala Ala Lys Leu Leu Phe Leu Ser Ser Lys His His Tyr Met Ala 165 170 175Val Gly Leu Leu His Thr Met Asn Thr Leu Ser Ser Ile Ser Leu Leu 180 185 190Ser Tyr Val Val Tyr Tyr Tyr Leu Pro Asn Pro Val Ala Gly Thr Ile 195 200 205Val Glu Phe Val Ala Val Ile Leu Ser Leu Lys Leu Ala Ser Tyr Ala 210 215 220Leu Thr Asn Ser Asp Leu Arg Lys Ala Ala Ile His Ala Gln Lys Leu225 230 235 240Asp Lys Thr Gln Asp Asp Asn Glu Lys Glu Ser Thr Ser Ser Ser Ser 245 250 255Ser Ser Asp Asp Ala Glu Thr Leu Ala Asp Ile Asp Val Ile Pro Ala 260 265 270Tyr Tyr Ala Gln Leu Pro Tyr Pro Gln Asn Val Thr Leu Ser Asn Leu 275 280 285Leu Tyr Phe Trp Phe Ala Pro Thr Leu Val Tyr Gln Pro Val Tyr Pro 290 295 300Lys Thr Glu Arg Ile Arg Pro Lys His Val Ile Arg Asn Leu Phe Glu305 310 315 320Leu Val Ser Leu Cys Met Leu Ile Gln Phe Leu Ile Phe Gln Tyr Ala 325 330 335Tyr Pro Ile Met Gln Ser Cys Leu Ala Leu Phe Phe Gln Pro Lys Leu 340 345 350Asp Tyr Ala Asn Ile Ser Glu Arg Leu Met Lys Leu Ala Ser Val Ser 355 360 365Met Met Val Trp Leu Ile Gly Phe Tyr Ala Phe Phe Gln Asn Gly Leu 370 375 380Asn Leu Ile Ala Glu Leu Thr Cys Phe Gly Asn Arg Thr Phe Tyr Gln385 390 395 400Gln Trp Trp Asn Ser Arg Ser Ile Gly Gln Tyr Trp Thr Leu Trp Asn 405 410 415Lys Pro Val Asn Gln Tyr Phe Arg His His Val Tyr Val Pro Leu Leu 420 425 430Ala Arg Gly Met Ser Arg Phe Asn Ala Ser Val Val Val Phe Phe Phe 435 440 445Ser Ala Val Ile His Glu Leu Leu Val Gly Ile Pro Thr His Asn Ile 450 455 460Ile Gly Ala Ala Phe Phe Gly Met Met Ser Gln Val Pro Leu Ile Met465 470 475 480Ala Thr Glu Asn Leu Gln His Ile Asn Ser Ser Leu Gly Pro Phe Leu 485 490 495Gly Asn Cys Ala Phe Trp Phe Thr Phe Phe Leu Gly Gln Pro Thr Cys 500 505 510Ala Phe Leu Tyr Tyr Leu Ala Tyr Asn Tyr Lys Gln Asn Gln 515 520 525301581DNAYarrowia lipolytica 30atggaagtcc gacgacgaaa aatcgacgtg ctcaaggccc agaaaaacgg ctacgaatcg 60ggcccaccat ctcgacaatc gtcgcagccc tcctcaagag catcgtccag aacccgcaac 120aaacactcct cgtccaccct gtcgctcagc ggactgacca tgaaagtcca gaagaaacct 180gcgggacccc cggcgaactc caaaacgcca ttcctacaca tcaagcccgt gcacacgtgc 240tgctccacat caatgctttc gcgcgattat gacggctcca accccagctt caagggcttc 300aaaaacatcg gcatgatcat tctcattgtg ggaaatctac ggctcgcatt cgaaaactac 360ctcaaatacg gcatttccaa cccgttcttc gaccccaaaa ttactccttc cgagtggcag 420ctctcaggct tgctcatagt cgtggcctac gcacatatcc tcatggccta cgctattgag 480agcgctgcca agctgctgtt cctctctagc aaacaccact acatggccgt ggggcttctg 540cataccatga acactttgtc gtccatctcg ttgctgtcct acgtcgtcta ctactacctg 600cccaaccccg tggcaggcac aatagtcgag tttgtggccg ttattctgtc tctcaaactc 660gcctcatacg ccctcactaa ctcggatctc cgaaaagccg caattcatgc ccagaagctc 720gacaagacgc aagacgataa cgaaaaggaa tccacctcgt cttcctcttc ttcagatgac 780gcagagactt tggcagacat tgacgtcatt cctgcatact acgcacagct gccctacccc 840cagaatgtga cgctgtcgaa cctgctgtac ttctggtttg ctcccacact ggtctaccag 900cccgtgtacc ccaagacgga gcgtattcga cccaagcacg tgatccgaaa cctgtttgag 960ctcgtctctc tgtgcatgct tattcagttt ctcatcttcc agtacgccta ccccatcatg 1020cagtcgtgtc tggctctgtt cttccagccc aagctcgatt atgccaacat ctccgagcgc 1080ctcatgaagt tggcctccgt gtctatgatg gtctggctca ttggattcta cgctttcttc 1140cagaacggtc tcaatcttat tgccgagctc acctgttttg gaaacagaac cttctaccag 1200cagtggtgga attcccgctc cattggccag tactggactc tatggaacaa gccagtcaac 1260cagtacttta gacaccacgt ctacgtgcct cttctcgctc ggggcatgtc gcggttcaat 1320gcgtcggtgg tggttttctt tttctccgcc gtcatccatg aactgcttgt cggcatcccc 1380actcacaaca tcatcggagc cgccttcttc ggcatgatgt cgcaggtgcc tctgatcatg 1440gctactgaga accttcagca tattaactcc tctctgggcc ccttccttgg caactgtgca 1500ttctggttca cctttttcct gggacaaccc acttgtgcat tcctttatta tctggcttac 1560aactacaagc agaaccagta g 158131697PRTRhodosporidium toruloides 31Met Thr Glu Arg Ser Leu Pro Val Thr Leu Pro Leu Pro Arg Asn Phe1 5 10 15Ala Leu Thr Pro His Gln Met Ala Ser Pro Asp Pro Pro Leu Pro Gly 20 25 30Pro Ala Asn Leu Val Asp Asp Ala Leu Arg His Pro Asp Ser Ala Pro 35 40 45Pro Ile Ser Pro Asp Ser Ala Pro Pro Ser Thr Ala Thr Arg Pro Ser 50 55 60Ala Leu Ser Arg Gly Glu Leu Ser Thr Ala Ser Ser Tyr Ala Ser Glu65 70 75 80Val Ser Thr Arg Glu Gly Thr Pro Asp Leu Ala Asn Gly Gln Gly Val 85 90 95Thr Thr Thr Ile Thr Thr Val Thr Gly Lys Gly Gly Lys Ala Val Thr 100 105 110Gln Thr Leu Thr His Val Gly Ala Ala Ser Val Asp Ala Arg Phe Ser 115 120 125Ser Thr Thr Asn Ser Ile Thr Leu Arg Pro Ile Pro Ala Arg Gly Gly 130 135 140Asp Pro Lys Lys Ile Lys Val Leu Arg Ser Arg Arg Thr His Phe Ala145 150 155 160Pro Arg Thr Ser His Phe Asp Arg His Asn Leu Thr Ser Ala Ser Asp 165 170 175Pro Phe Arg Gly Leu Tyr Thr Leu Phe Trp Ile Val Ile Phe Val Gly 180 185 190Ala Leu Lys Thr Val Tyr His Arg Phe Ala Glu Gln Gly Gly Trp Gly 195 200 205Gly Glu Trp Arg Phe Ala Ala Leu Ile Ser Arg Asp Gly Trp Val Leu 210 215 220Ala Val Ser Asp Ala Val Leu Val Ser Ala Ser Leu Leu Cys Val Pro225 230 235 240Tyr Ala Lys Leu Leu Val His Gly Trp Ile Arg Tyr His Gly Ala Gly 245 250 255Val Ile Ile Gln His Ile Cys Gln Thr Leu Tyr Leu Ala Ile Ala Ile 260 265 270Arg Trp Thr Phe His Arg Asn Trp Pro Trp Val Gln Ser Gly Phe Met 275 280 285Thr Leu His Ala Leu Ser Met Leu Met Lys Ile His Ser Tyr Cys Ser 290 295 300Leu Asn Gly Glu Leu Ser Glu Arg Arg Arg Gln Leu Lys Lys Asp Glu305 310 315 320Lys Arg Leu Glu Glu Val Leu Glu Glu Met Gly Gly Arg Arg Lys Ala 325 330 335Glu Arg Glu Ala Arg Glu Glu Trp Glu Arg Gln Cys Gly Glu Ala Ala 340 345 350Arg Ala Lys Glu Gly Glu Ala Gly Val Ser Glu Gly Glu Lys Glu Ala 355 360 365Ala Ala Thr Leu Ser Ser Thr Asp Ala Ser Asn Ser Ala Leu Ser Ser 370 375 380Glu Asp Glu Ala Ala Ala Ala Leu Leu Arg His Arg Gln Pro Thr Ala385 390 395 400Arg Arg Arg Ser Ile Ser Pro Ser Ala Ser Arg Thr Gly Ser Ser Ser 405 410 415Ala Pro Ser Ala Thr Leu Ala Pro Ser Arg Ala Glu Glu Pro Gln Glu 420 425 430Gly Val Glu Thr Leu Thr Trp His Pro Ser Asp Gln Val Ser Lys Leu 435 440 445Ala Ile Ala Ile Cys Glu Ala Lys Asp Leu Leu Thr Ser Asn Gly Lys 450 455 460Lys Pro Val Thr Phe Pro Glu Asn Val Thr Phe Ala Asn Phe Ile Asp465 470 475 480Tyr Leu Leu Val Pro Thr Leu Val Tyr Glu Leu Glu Tyr Pro Arg Thr 485 490 495Asp Ser Ile Arg Pro Leu Tyr Ile Leu Glu Lys Thr Leu Ala Thr Phe 500 505 510Gly Thr Phe Ser Ile Leu Val Leu Ile Val Asp Ser Phe Ile Leu Pro 515 520 525Val Thr Ser Arg Thr Asp Thr Pro Leu Phe Gly Phe Val Leu Asp Leu 530 535 540Ala Leu Pro Phe Thr Leu Ala Tyr Leu Leu Ile Phe Tyr Val Ile Phe545 550 555 560Glu Gly Val Cys Asn Gly Phe Ala Glu Leu Thr Arg Phe Ala Asp Arg 565 570 575Asn Phe Phe Asp Asp Trp Trp Asn Ser Cys Thr Phe Asp Glu Phe Ser 580 585 590Arg Lys Trp Asn Arg Pro Val His Ala Phe Leu Leu Arg His Val Tyr 595 600 605Ala Glu Thr Met Ala Ser Tyr Lys Leu Ser Lys Leu Ser Ala Ala Phe 610 615 620Val Thr Phe Leu Phe Ser Ala Cys Val His Glu Leu Val Met Ala Val625 630 635 640Val Thr Lys Lys Leu Arg Leu Tyr Leu Phe Ser Met Gln Met Ala Gln 645 650 655Leu Pro Leu Ile Met Val Gly Arg Ala Lys Ile Phe Arg Gln Tyr Pro 660 665 670Ala Leu Gly Asn Leu Phe Phe Trp Leu Ala Leu Leu Ser Gly Phe Pro 675 680 685Leu Leu Gly Thr Leu Tyr Leu Arg Tyr 690 695322094DNARhodosporidium toruloides 32atgacggagc gatcccttcc agtgacgctc cctcttcctc gaaactttgc gctcacaccg 60caccagatgg cctcgccaga cccgccactc ccaggcccag ccaacctcgt cgacgacgca 120ctccgacacc cagactcggc gccgcccatc tcgcccgact ccgcgcctcc ttcgactgcg 180actcggccct ctgctctctc gcgcggagag ctctcgaccg cttcgagcta cgcgagcgag 240gtgtcgacga gggaggggac accggatctg gcgaatgggc aaggggttac gacgaccatc 300acgactgtca caggcaaagg cggaaaggcc gtcacccaga ccctcaccca cgtcggcgcc 360gcctccgtcg acgcccgctt ctcctccacc acaaactcca tcactctccg ccctatcccc 420gcccgtggcg gcgacccgaa aaagatcaaa gtcctccgct ctcgtcggac ccacttcgcc 480ccacgcacct cacacttcga ccgtcacaac ctcacctccg cctctgaccc gttccgcgga 540ctgtacacgt tgttctggat cgtgatcttc gttggggcac tcaagactgt gtatcatcgg 600tttgcggaac agggtgggtg gggtggagaa tggaggtttg cggcgttgat tagtcgcgat 660gggtgggttc tggcggttag tgatgcggtg ttggttagcg cgtcgttgtt gtgcgtgccg 720tatgcaaagc tcctcgtaca cggctggatc cggtaccacg gcgcaggcgt catcatccaa 780cacatctgtc aaacgctcta cctcgccatc gcgatccgct ggaccttcca ccgcaactgg 840ccctgggtcc aaagcggttt catgaccctc cacgccctct cgatgctcat gaagatccat 900agctactgtt ctctgaacgg cgagctttcg gagcggcgga gacagttgaa gaaggacgag 960aagcggttgg aggaggtgct ggaggagatg ggtggacgga ggaaggcgga gagggaggcg 1020agggaggagt gggagaggca gtgtggggag gcggcgaggg ccaaggaggg tgaggcggga 1080gtgagcgagg gggagaagga ggcggcggcg actctatctt cgacggatgc gtcgaattcg 1140gccctttcgt cggaggacga ggcggctgcg gcgctgttgc ggcatcgaca gccgactgct 1200cgacgacgat ccatctcgcc ctctgcctca cgcaccggtt cctcctccgc cccctccgct 1260accctcgccc cctctcgcgc cgaagaaccc caagaaggcg ttgagacgct cacctggcac 1320ccatccgacc aagtcagcaa actcgctatc gccatctgcg aggcaaagga cctcctcacg 1380agtaacggca agaagcccgt cacgttcccc gagaacgtca cctttgcgaa ctttatcgac 1440tacttgcttg tgccgacgtt ggtgtacgag ttggagtacc ctcggacgga ttccatccgg 1500cccctctaca tcctcgaaaa gaccctcgca accttcggca ccttctccat tctcgtcctc 1560atcgtcgact cgttcatcct ccccgtcacc tcgcgcaccg acacgcccct cttcgggttc 1620gtcctcgacc tcgccctgcc gttcacgctc gcgtacctcc tcatcttcta cgtcatcttt 1680gagggcgtgt gcaatgggtt tgcggagttg acgaggtttg cggatcggaa tttcttcgac 1740gattggtgga actcgtgcac gttcgacgag ttctcgcgca agtggaatcg ccccgtccac 1800gccttcctcc tccgccacgt ttacgccgaa acgatggctt cttacaagct ctcgaagctc 1860tcggctgcgt tcgtcacgtt cttgttcagc gcctgcgtgc acgaactcgt catggcggtc 1920gtgacgaaga agcttcggct gtacctgttc tcgatgcaga tggcccagct cccgctcatc 1980atggtgggcc gcgccaagat cttccgacag tatccagcgc tcggcaacct cttcttctgg 2040ctcgcccttc tctcgggatt cccgcttctc gggacgctgt atctgcggta ctga 209433555PRTLipomyces starkeyi 33Met Ser Thr Ala Ala Gln Ser Asp Thr Asp Asn Glu Asp Ile Ser Thr1 5 10 15Val Asp Leu Val Asp Ser Arg Ala Asp Thr His Thr Ser Ser Asn Val 20 25 30Met Leu Gln Gln Gln Lys Ser Arg Arg Arg Leu Ile Gly Lys Asp Ala 35 40 45Glu Pro Arg Thr Gln His Pro Ser Gly Gly Lys Ser Glu Lys Glu Glu 50 55 60Leu Thr Lys Pro Asp Asp Ser Lys Gly Pro Ile Lys Leu Ser His Ile65 70 75 80Tyr Pro Ile His Ala Val Ser Arg Gly Ser Ile Leu Ser Arg Glu Ser 85 90 95Thr Thr Pro Thr Pro Ser Phe Val Gly Phe Arg Asn Leu Ala Met Ile 100 105 110Val Leu Gly Lys Leu Gln Tyr Ser Leu Phe Phe Trp Cys Asp Arg Ala 115 120 125Asn Ile Pro Thr Ala Val Ser Asn Leu Arg Leu Val Ile Glu Asn Tyr 130 135 140Ser Lys Tyr Gly Val Leu Ile Arg Phe Ala Arg Leu Gly Ile Ser Gln145 150 155 160Lys Asp Ile Leu Tyr Cys Ile Phe Leu Thr Ala Thr Ile Pro Leu His 165 170 175Leu Phe Ile Ala Ile Val Ile Glu Arg Leu Val Ala Ile Pro Thr Val 180 185 190Asn Tyr Val Ala Ser Leu Ser Glu Ser Glu Asp Lys Lys Arg Ser Asn 195 200 205Pro Lys Met Gly Arg Lys Gly Gly Ser Ile Ser Ile Leu Arg Pro Lys 210 215 220Pro Lys Tyr Met Trp Arg Leu Ile Val Leu Leu His Ser Ile Asn Ala225 230 235 240Met Ala Cys Leu Trp Val Thr Thr Val Val Val Tyr Asn Ser Ile Tyr 245 250 255His Pro Leu Ile Gly Thr Ala Cys Glu Phe His Ala Val Ile Val Cys 260 265 270Leu Lys Val Ala Ser Phe Ala Leu Thr Asn Arg Asp Leu Arg Glu Ser 275 280 285Met Leu Asn Ser Gln Pro Val Pro Ala Ile Tyr Asn Leu Ala Pro Tyr 290 295 300Pro Lys Asn Leu Thr Leu Lys Asn Leu Ser Tyr Phe Trp Trp Ala Pro305 310 315 320Thr Leu Val Tyr Gln Pro Val Tyr Pro Arg Ser Pro Ser Phe Arg Pro 325 330 335Leu Phe Phe Val Lys Arg Ile Leu Glu Met Val Gly Leu Ser Phe Leu 340 345 350Ile Trp Phe Leu Ser Ala Gln Tyr Ala Val Pro Thr Leu Glu Asn Ser 355 360 365Leu Val His Phe His Ser Leu Gln Phe Met Gly Ile Met Glu Arg Leu 370 375 380Met Lys Leu Ala Ser Ile Ser Met Ala Ile Trp Leu Ala Gly Phe Phe385 390 395 400Cys Ile Phe Gln Ser Gly Leu Asn Ala Leu Ala Glu Val Met Arg Phe 405 410 415Gly Asp Arg Ala Phe Tyr Asp Asp Trp Trp Asn Ser Lys Ser Val Gly 420 425 430Glu Tyr Trp Arg Leu Trp Asn Lys Pro Val Thr Asn Tyr Phe Arg Arg 435 440 445His Ile Tyr Val Pro Leu Val Arg Arg Gly Trp Asn Ser Ala Thr Ala 450 455 460Ser Val Met Val Phe Phe Val Ser Ala Val Leu His Glu Leu Val Val465 470 475 480Gly Val Pro Thr His Asn Val Ile Gly Val Ala Phe Ser Ser Met Ile 485 490 495Leu Gln Ile Pro Leu Ile Gln Val Thr Ala Pro Leu Glu Lys Met His 500 505 510Gly Pro Thr Ser Gly Ile Ile Gly Asn Cys Ile Phe Trp Phe Ser Phe 515 520 525Phe Ile Gly Gln Pro Leu Gly Val Leu Leu Tyr Tyr Phe Ala Trp Asn 530 535 540Val Ser Met Ser Lys Val Lys Met Val Glu Ser545 550

555341668DNALipomyces starkeyi 34atgtcgaccg ctgcacaatc tgatacagac aacgaggata tatcgactgt cgatttggtt 60gactctcgtg cagatactca cacatcttca aatgttatgt tgcaacagca aaaatcgcgt 120cggagactaa tcgggaaaga cgccgagcca agaacacagc atccgtctgg aggcaaatcg 180gagaaggagg agttgacgaa gccggatgac tcaaagggac ccataaaatt aagtcacata 240tacccgatac atgccgttag ccgaggcagt attctgtcac gagagtcgac aactcctaca 300ccgagttttg ttgggtttcg aaacttagcc atgatagtgc tagggaagtt acagtattca 360ttattctttt ggtgcgatcg ggctaacatt ccgacagccg tcagcaatct tcgattggtg 420attgaaaatt actcaaagta cggcgttctg atccgattcg cccgactcgg tatttcacaa 480aaggacattc tgtattgcat attcttgacc gctaccatcc cgctgcacct atttattgct 540attgtcattg aaagactagt tgcgattccg acggtaaact acgtcgcttc gctcagcgag 600agcgaggata aaaaacgctc caaccccaaa atgggacgga aggggggcag tatatcgatt 660ttgcgtccta agccaaaata tatgtggcgc ctgatcgtcc tattgcattc aataaacgca 720atggcttgct tgtgggttac gactgttgtt gtttacaatt ctatttatca tccccttatt 780gggacagctt gtgaatttca tgcagtgatt gtgtgtctta aggtcgcatc gtttgcgctt 840accaatcgcg atcttcggga gtcgatgctg aactctcaac ctgtgccagc catatacaac 900ttggcccctt atccaaaaaa cttaaccctc aagaacttgt catacttttg gtgggcgccg 960actcttgttt atcaacctgt ctatccgcga tcgccttcat tccggccttt gttttttgtc 1020aagcggattc tggagatggt gggcctatca tttttaatat ggttcttgtc agctcaatat 1080gctgtgccga cgctagaaaa tagtttggtg cattttcaca gtttgcaatt catgggaatt 1140atggagcgac tcatgaagct tgctagcatt agcatggcta tttggcttgc tggttttttc 1200tgcatttttc agtctggact caatgcgctt gcggaggtaa tgcggtttgg tgacagagcc 1260ttttacgacg actggtggaa cagcaaatct gtgggagagt attggcgtct gtggaataag 1320ccggttacga attacttccg gcgtcatatt tacgtaccgc ttgtgcgccg cgggtggaat 1380tctgcgacag ccagtgtcat ggtatttttc gtcagcgcgg tgttgcatga gctagttgtt 1440ggagttccga cgcataacgt aattggagtt gcattctcgt cgatgattct acaaatccca 1500ctcatacaag taaccgcgcc tctggagaag atgcatggac ctacatctgg aataataggg 1560aactgtatct tttggtttag cttcttcatc ggtcagcctc tgggcgtgct actttactat 1620tttgcgtgga acgttagtat gagcaaagta aagatggtcg agagctag 166835517PRTAspergillus terreus 35Met Val Met Asp Thr Gln Thr Thr Ala Ser Ala Thr Ser Thr Ala Leu1 5 10 15Thr Thr Asp His Thr Val Ala Ser Arg Thr Ser Arg Ser Glu Pro Asn 20 25 30Gly Gly Val His Asn Val Ser Ser Pro Pro Thr Ser Glu Pro Thr Gly 35 40 45Gly Asn Gly Gly Gly Arg Arg Arg Ser Lys Tyr Arg His Val Ala Ala 50 55 60Tyr His Ser Glu Val Arg His Ser Ser Leu Ser Arg Glu Ser Asn Thr65 70 75 80Ser Pro Ser Phe Leu Gly Phe Arg Asn Leu Met Val Ile Val Leu Gly 85 90 95Glu Cys Pro Ser Ala Leu Leu Arg Phe Val Asn Pro Thr Glu Asn Ser 100 105 110Tyr Gly Ser Arg Leu Val Ala Met Asn Leu Arg Leu Val Ile Glu Asn 115 120 125Tyr Val Lys Tyr Gly Val Leu Ile Cys Ile Arg Cys His Asp Tyr Arg 130 135 140Lys Gln Asp Val Val Leu Gly Ser Met Leu Phe Ala Leu Val Pro Cys145 150 155 160Gln Leu Phe Ile Ala Tyr Leu Leu Glu Leu Ala Ala Ala Gly Arg Ala 165 170 175Lys Gln Thr Val Gly Arg Lys Lys Lys Asp Gly Ser Ala Glu Glu Gly 180 185 190Glu Arg Glu Ala Arg Ala Phe Arg His Ile Trp Arg Phe Ala Leu Ser 195 200 205Phe His Ile Leu Asn Ile Val Leu Asn Leu Ala Val Thr Ser Phe Val 210 215 220Val Tyr Tyr Tyr Ile His His Pro Gly Ile Gly Thr Leu Cys Glu Val225 230 235 240His Ala Ile Val Val Ala Leu Lys Asn Trp Ser Tyr Ala Phe Thr Asn 245 250 255Arg Asp Leu Arg Glu Ala Met Leu Asn Pro Ser Ala Glu Ser Ala Leu 260 265 270Pro Glu Ile Tyr Ser Ser Leu Pro Tyr Pro Lys Asn Ile Thr Leu Gly 275 280 285Asn Leu Thr Tyr Phe Trp Leu Ala Pro Thr Leu Leu Tyr Gln Pro Val 290 295 300Tyr Pro Arg Ser Pro Ser Ile Arg Trp Pro Phe Val Ala Lys Arg Leu305 310 315 320Ser Glu Phe Ala Cys Leu Ser Val Phe Ile Trp Leu Leu Ser Ala Gln 325 330 335Tyr Ala Ala Pro Val Leu Arg Asn Ser Ile Asp Lys Ile Arg Asp Met 340 345 350Ala Tyr Ala Ser Ile Phe Glu Arg Val Met Lys Leu Ser Thr Ile Ser 355 360 365Leu Val Ile Trp Leu Ala Gly Phe Phe Ala Ile Phe Gln Ser Leu Leu 370 375 380Asn Ala Leu Ala Glu Ile Met Lys Phe Gly Asp Arg Glu Phe Tyr Thr385 390 395 400Asp Trp Trp Asn Ser Pro Ser Leu Gly Val Tyr Trp Arg Ser Trp Asn 405 410 415Arg Pro Val Tyr Gln Phe Met Lys Arg His Val Tyr Ser Pro Leu Ile 420 425 430Gly Arg Gly Tyr Ser Pro Phe Val Ala Ser Thr Val Val Phe Thr Ile 435 440 445Ser Ala Leu Leu His Glu Leu Leu Val Gly Ile Pro Thr His Asn Met 450 455 460Ile Gly Val Ala Leu Val Gly Met Leu Phe Gln Leu Pro Leu Ile Ala465 470 475 480Ile Thr Ala Pro Leu Glu Lys Met Lys Asp Pro Leu Gly Lys Pro Leu 485 490 495Gly Ala Leu Leu Tyr Phe Phe Ala Trp Gln Ala Lys Tyr Gly Ser Val 500 505 510Ser Arg Met Gly Asn 515361554DNAAspergillus terreus 36atggtgatgg acacacaaac cacagcatcc gccaccagca cggcgctcac gaccgaccac 60actgttgcct ctcggacgtc ccgctctgag ccgaacggtg gtgtgcataa tgtatcgtca 120cctccaacga gcgaaccgac tgggggaaat ggcggaggcc ggcgaaggag taaataccgg 180catgtcgcag cgtaccattc cgaagtgcgc cattccagtc tcagtcggga atcgaatact 240tctccgagtt tcctcggatt ccggaacctc atggtaatcg tattaggtga gtgccctagt 300gctctcctac gttttgtgaa cccgacggag aactcatacg ggtcgcgact agttgctatg 360aatcttcgat tggttatcga gaattacgtg aagtatgggg tcttgatctg catcagatgc 420cacgattatc gaaagcagga cgttgtcctg ggctcaatgt tatttgctct cgtcccatgc 480cagctattca tcgcctacct cctggaattg gccgcagcgg gtagggccaa acagactgtg 540ggccgaaaga aaaaggacgg atcagccgag gagggcgaac gtgaagcacg tgcttttcga 600cacatctggc ggtttgcatt gtcctttcac atcctcaaca ttgttctcaa tctcgccgtc 660acgagcttcg ttgtgtatta ctacatccac catcccggca ttggtacgct ctgtgaagtg 720catgcgatcg ttgtcgcgtt gaaaaactgg tcctatgcgt tcaccaatcg ggatctgcga 780gaggcgatgc ttaatccctc ggcggagtcg gcgcttcccg agatctattc cagcctcccg 840tacccgaaaa acatcacgtt aggaaatcta acgtacttct ggcttgcacc gacactgttg 900tatcagccag tataccccag gtcgccttcc atccgatggc cattcgtggc caaacgcttg 960tcggaatttg cgtgcttgtc ggtgttcatt tggctacttt cggcccaata cgctgcgcca 1020gttttgcgca actccattga caagattcgt gatatggcat atgcatccat ttttgagcgc 1080gttatgaagc tatccaccat ctctctcgtc atttggctgg ctgggttctt tgcgattttc 1140caatcactct tgaatgcttt ggcggagatc atgaagtttg gcgatcggga attctacacc 1200gattggtgga atagcccaag tctcggtgtt tactggcggt catggaatcg gccagtgtac 1260cagttcatga agcggcacgt atattctccg ttgatagggc gggggtacag cccgtttgtg 1320gcaagcactg tcgtattcac catctccgct ctccttcatg agctcctcgt ggggataccc 1380acgcacaaca tgataggcgt cgcgcttgtt ggaatgctgt tccagctccc gttgatcgcc 1440atcactgccc cattggaaaa gatgaaagat ccattgggta agcccctggg agcactgctg 1500tatttctttg cctggcaggc aaaatatggc agtgtgagca ggatgggcaa ctga 155437506PRTClaviceps purpurea 37Met Ser Ala Thr Gly Val Asp Val Ala Asn Gly Arg Ser Gly Ala Arg1 5 10 15Arg Arg Asn Asp Thr Ala Val Asp Glu Thr Ile Ser Ala Val Thr Ala 20 25 30Glu Met Arg Ser Ser Ser His Pro Thr Tyr Arg His Val Ser Ala Val 35 40 45His Ser Thr Ser Arg Pro Ser Cys Leu Ser His Asp Ser Asp Ala Ala 50 55 60Pro Ser Phe Ile Gly Phe Arg Asn Leu Met Val Ile Val Leu Val Val65 70 75 80Gly Asn Val Arg Leu Met Ile Glu Asn Leu Lys Lys Tyr Gly Val Leu 85 90 95Ile Cys Leu Arg Cys His Ser Tyr Lys Asn Glu Asp Ile Ile Ile Gly 100 105 110Gly Leu Leu Tyr Phe Leu Ile Pro Cys His Leu Leu Val Ala Tyr Gly 115 120 125Ile Glu Leu Ala Ala Ala Arg Gln Ala Arg Glu Ser Arg Thr Arg Pro 130 135 140Pro Gly Gln Ser Asp Thr Ala Ser Lys Ser Thr Glu Asp Asp Asn Lys145 150 155 160His Phe His Ser Thr Trp Val Leu Ala Ala Trp Ala His Ile Ile Asn 165 170 175Met Thr Leu Ser Phe Ile Leu Thr Thr Phe Val Val Tyr Tyr Tyr Val 180 185 190His His Pro Leu Val Gly Thr Leu Thr Glu Met His Ala Val Ile Val 195 200 205Ser Leu Lys Thr Ala Ser Tyr Ala Phe Thr Asn Arg Asp Leu Arg His 210 215 220Ala Tyr Leu His Pro Asp Lys Arg Lys His Ile Pro Glu Leu Tyr Leu225 230 235 240Glu Cys Pro Tyr Pro Gln Asn Leu Thr Phe Gly Asn Leu Val Tyr Phe 245 250 255Trp Trp Ala Pro Thr Leu Val Tyr Gln Pro Val Tyr Pro Arg Thr Asp 260 265 270Lys Ile Arg Trp Val Phe Val Phe Lys Arg Leu Gly Glu Val Cys Cys 275 280 285Leu Ser Ala Phe Ile Trp Phe Ala Ser Phe Gln Tyr Ala Ala Pro Val 290 295 300Leu Arg Asn Ser Leu Asp Lys Ile Ala Ser Leu Asp Phe Ile Met Ile305 310 315 320Phe Glu Arg Leu Leu Lys Leu Ser Thr Ile Ser Leu Val Ile Trp Leu 325 330 335Ala Gly Phe Phe Ala Leu Phe Gln Ser Phe Leu Asn Ala Leu Ala Glu 340 345 350Val Leu Arg Phe Gly Asp Arg Cys Phe Tyr Asp Asp Trp Trp Asn Ser 355 360 365Glu Ser Leu Gly Ala Tyr Trp Arg Thr Trp Asn Arg Pro Val Tyr Thr 370 375 380Tyr Phe Lys Arg His Val Tyr Val Pro Met Ile Gly Arg Gly Trp Ser385 390 395 400Pro Trp Thr Ala Ser Cys Thr Val Phe Phe Val Ser Ala Val Leu His 405 410 415Glu Val Leu Val Gly Val Pro Thr His Asn Ile Ile Gly Val Ala Phe 420 425 430Val Gly Met Phe Leu Gln Leu Pro Leu Ile Ala Leu Thr Ala Pro Met 435 440 445Glu Lys Lys Lys Trp Gly His Thr Gly Arg Val Met Gly Asn Val Ile 450 455 460Phe Trp Val Ser Phe Thr Ile Phe Gly Gln Pro Phe Ala Ala Leu Met465 470 475 480Tyr Phe Tyr Ala Trp Gln Ala Lys Tyr Gly Ser Val Ser Arg Gln Ile 485 490 495Val Leu Val Asn Pro Val Glu Glu Ala Ser 500 505381521DNAClaviceps purpurea 38atgtccgcca cgggcgttga tgtggccaac ggccgcagcg gcgcgcgacg acgcaacgat 60actgccgtcg acgagactat atccgccgtc acggccgaga tgcgttcctc gtcgcatcca 120acataccgcc atgtgtctgc tgtgcactcc acgagccggc cctcgtgtct gagccatgat 180tctgacgctg cgccgagctt cattggcttt cgaaatctca tggtcattgt tctggtcgtt 240ggcaatgttc gattaatgat tgaaaatcta aaaaagtacg gcgtactgat atgcctccga 300tgtcactcgt ataaaaacga agacatcatt atcggcggac tgctctactt cctgatcccc 360tgccacttgc ttgtcgccta cggaatcgag ttagccgccg ccagacaagc acgcgaatct 420cgaactcgtc caccaggcca gtccgacacg gcgtcgaaat caacagaaga tgacaacaag 480cacttccact caacatgggt gctcgctgcc tgggcacaca tcatcaacat gacactttcc 540ttcatcctca ccaccttcgt cgtctactac tacgtgcacc atcccctcgt cggcaccctg 600accgagatgc acgccgtcat cgtctctctc aaaacagctt cctacgcatt caccaaccga 660gatcttcgcc acgcatacct ccatcctgac aagcgcaagc acatccccga gctatatctc 720gaatgtccct acccccagaa cctcaccttt ggcaatctcg tgtatttctg gtgggccccc 780acgctggtat accagcccgt gtatccgcgc accgacaaga tcagatgggt ttttgttttt 840aaaagactag gcgaagtctg ctgtctcagc gcattcatct ggttcgccag cttccagtac 900gccgcgcccg tgttgcggaa ctccctggac aagattgcgt ctctcgactt catcatgatc 960tttgagcgcc ttctcaagct atccaccatt tctctcgtca tctggctcgc cggcttcttc 1020gccctgttcc agtctttcct gaatgccctg gctgaggtat tgcgctttgg ggaccggtgc 1080ttctacgacg attggtggaa tagcgagagt ctgggggcgt attggaggac gtggaacagg 1140cctgtgtata cctacttcaa gcgccatgtg tatgtgccca tgattgggag gggatggagt 1200ccctggactg ctagttgtac tgtttttttt gtgtcggcgg tgctgcacga ggttcttgtt 1260ggggtgccca cccacaatat cattggtgtc gcctttgtgg gcatgtttct gcagcttccc 1320ctaatagccc tcaccgctcc catggaaaag aagaaatggg gccacaccgg ccgtgtgatg 1380ggcaatgtta ttttctgggt gtcctttaca atctttgggc agccctttgc agcgctcatg 1440tacttttatg cctggcaggc caagtacggg agcgtgagtc ggcaaattgt gctggtgaat 1500ccggtggagg aggcgtcttg a 152139551PRTChaetomium globosum 39Met Lys Ala Glu Thr Gly Thr Thr Met Ala Thr Ser Thr Ser Leu Glu1 5 10 15Thr Ser Gln Val Asn Gly Val Thr Asn Arg Ala Pro Val Gly Pro Ser 20 25 30His Asp Pro His Ala Thr Thr Pro Thr His Glu Thr Thr Thr Thr Ile 35 40 45Pro Ser Asp Val Leu Ala Asn Gly Ser Thr Asn Gly Thr Thr Asn Gly 50 55 60Thr Thr Asp Asp Ser Leu Asp Ile Ser Glu Leu Arg Lys Ala Phe Arg65 70 75 80Asn Lys Tyr Arg His Val Glu Ala Val His Ser Glu Ser Lys Pro Ser 85 90 95Cys Leu Ser His Asp Ala Thr Glu Thr Pro Ser Phe Ile Gly Phe Arg 100 105 110Asn Leu Met Val Ile Val Leu Val Ala Ala Asn Leu Arg Leu Val Ile 115 120 125Glu Asn Ile Gln Lys Tyr Gly Val Leu Ile Cys Ile Lys Cys His Asp 130 135 140Phe Arg Pro Asn Asp Val Arg Leu Gly Leu Leu Leu Tyr Ile Leu Ile145 150 155 160Pro Trp His Leu Met Leu Ala Tyr Leu Ile Glu Leu Val Ala Ala Ala 165 170 175Asn Ala Arg Asn Ser Arg Ala Lys Ala Lys Lys Arg Asp Gly Ser Thr 180 185 190Ser Pro Thr Glu Asp Glu Ser Lys Gln Phe Leu Gln Thr Trp Arg Met 195 200 205Leu Arg Ile Leu His Ala Val Asn Val Thr Ala Ala Leu Ala Val Thr 210 215 220Ser Tyr Val Val Tyr Tyr Tyr Ile His His Pro Leu Ile Gly Thr Leu225 230 235 240Ser Glu Leu His Ala Ile Ile Val Trp Leu Lys Thr Ala Ser Tyr Ala 245 250 255Leu Thr Asn Arg Asp Leu Arg His Ala Tyr Leu His Pro Val Arg Gly 260 265 270Glu Arg Asp Ala Leu Pro Glu Ile Tyr Ala Gln Cys Pro Tyr Pro Ala 275 280 285Asn Val Thr Phe Ser Asn Leu Thr Tyr Phe Trp Trp Ala Pro Thr Leu 290 295 300Val Tyr Gln Pro Ala Tyr Pro Arg Thr Gln Arg Ile Arg Trp Val Phe305 310 315 320Val Ala Lys Arg Leu Gly Glu Val Val Cys Leu Ser Ala Phe Ile Trp 325 330 335Phe Ala Ser Ala Gln Tyr Ala Thr Pro Val Leu Arg Asn Ser Leu Asp 340 345 350Lys Ile Ala Thr Leu Asp Tyr Met Ser Ile Val Glu Arg Leu Leu Lys 355 360 365Leu Ser Thr Ile Ser Leu Val Ile Trp Leu Ala Gly Phe Phe Ala Leu 370 375 380Phe Gln Ser Phe Leu Asn Ala Leu Ala Glu Val Met Arg Phe Gly Asp385 390 395 400Arg Glu Phe Tyr Glu Ala Trp Trp Asn Ser Glu Ser Leu Gly Ala Tyr 405 410 415Trp Arg Thr Trp Asn Lys Pro Val Tyr Gln Phe Phe Arg Arg His Val 420 425 430Tyr Ser Pro Met Arg Ser Arg Gly Trp Ser His Leu Ser Ala Ser Leu 435 440 445Ala Val Phe Leu Leu Ser Ala Val Leu His Glu Leu Leu Val Gly Val 450 455 460Pro Thr His Asn Ile Ile Gly Val Ala Phe Leu Gly Met Phe Leu Gln465 470 475 480Leu Pro Leu Ile Ala Met Thr Ala Arg Leu Gly Gly Arg Arg Gly Asn 485 490 495Thr Ala His Gly Arg Leu Leu Gly Asn Thr Ile Phe Trp Val Ser Phe 500 505 510Thr Ile Phe Gly Gln Pro Phe Ala Ala Leu Met Tyr Phe Tyr Ala Trp 515 520 525Gln Ala Lys Tyr Gly Ser Val Ser Lys Met Pro Leu Ala Gln Pro Gly 530 535 540Thr Cys Pro Ala Val Val Val545 550401656DNAChaetomium globosum 40atgaaggcag aaacgggcac aacgatggca acgtcgacta gtctcgagac ttcccaagtc 60aatggcgtca ccaaccgggc ccctgttggc cctagtcacg acccccacgc tacaactccg 120actcatgaga cgacaaccac cataccgtcc gacgtcctcg ccaatggttc tacaaatggg 180actacgaatg ggacgacaga tgattcattg gacatatccg aattgcgcaa agcgttccgc 240aacaagtatc gccatgtcga ggctgtccac tccgaatcga aaccatcctg tctgagccat 300gacgctacag agacacccag tttcatcggt tttaggaatc tcatggtgat tgtgttggtt 360gctgccaatc ttcgcctggt catcgagaac attcaaaagt atggagttct gatctgcatc 420aaatgccacg actttcgccc caacgatgta cgcctggggc tcctcctcta

catcctgatc 480ccatggcacc tcatgctcgc ctacctcatt gagctggtcg ccgccgccaa tgcccgcaac 540tcccgggcca aggcgaagaa gcgggacggc agtaccagcc cgaccgaaga cgagtccaag 600caattcctgc agacctggcg gatgctccgc attctccacg ccgtcaacgt cacggccgcc 660ctggccgtca cctcctacgt ggtctactac tacattcacc acccgctgat cggcacgctc 720tcggagctgc acgccatcat cgtgtggctc aagacggcgt cgtacgcgct caccaaccgc 780gacctgcgcc acgcctacct acacccggtg cgcggcgagc gcgacgctct gcccgagatc 840tacgcccagt gcccctaccc ggccaacgtg accttctcca acttgaccta cttctggtgg 900gcgcccaccc tggtgtacca gccggcgtac ccgcgcactc agcgcatccg ctgggtcttt 960gtggctaagc gcctcggcga ggtcgtctgc ttgagcgcct tcatctggtt cgccagcgcc 1020cagtacgcta cccccgtgct gcgaaactcg ctcgacaaga tcgctaccct ggattacatg 1080tccattgtcg agcgtctgtt gaagctgtcg accatctcgc tggtcatctg gctggcgggc 1140ttctttgcgc tgtttcagag tttcctgaat gccttggccg aggtgatgcg gtttggagac 1200cgcgagttct acgaagcatg gtggaacagc gaaagcctcg gcgcctactg gcgcacctgg 1260aacaaacccg tgtaccaatt cttccggcgg cacgtctact cgccgatgcg gtcgcgcggg 1320tggagccact tgtcggccag cctcgccgtg tttctgctct cggccgtgct acacgagctg 1380ctggtggggg tgccgacgca caacatcatc ggcgtcgcct tcctgggcat gttcctgcag 1440ctgccgctca tcgccatgac ggcgcgcctg ggcggccgcc gcgggaacac cgcccacggc 1500cgcctgctcg gcaacactat cttttgggtg tcatttacca tttttggcca gccgtttgcc 1560gcgctgatgt atttttatgc atggcaggcc aagtatggta gtgtgagcaa gatgccgctg 1620gcgcagccgg ggacgtgtcc ggctgtggtt gtttga 165641388PRTArxula adeninivorans 41Met Val Arg Phe Ala Pro Leu Asn Val Pro Leu His Arg Arg Leu Glu1 5 10 15Thr Phe Ala Leu Thr Tyr His Ile Leu Ser Ile Pro Val Trp Met Ser 20 25 30Phe Phe Leu Leu Cys Cys Ala Ile Pro Leu Met Trp Pro Leu Val Ile 35 40 45Ile Tyr Leu Leu Tyr Tyr Ala Ser Asp Asn Ser Ser Glu Asn Gly Gly 50 55 60Val Ala Ser Arg Tyr Ser Pro Lys Phe Arg Ser Val Pro Leu Trp Lys65 70 75 80Tyr Phe Ala Asn Tyr Phe Pro Ile Thr Leu His Arg Thr Gln Glu Leu 85 90 95Pro Pro Ala Phe Val Tyr Gln Gly Glu Asp Leu Asp Pro Glu Thr Pro 100 105 110Asp Asp Ser Asp Asp Gly His Ala Lys Ser Lys Ser Ile Val Leu Lys 115 120 125Leu Trp Lys Val Ala Phe Trp Trp Tyr Tyr Leu Pro Lys His Phe Leu 130 135 140Arg Lys Pro Glu Val Arg Pro Thr Gly Arg Arg Tyr Ile Phe Gly Tyr145 150 155 160His Pro His Gly Ile Ile Gly Met Gly Ala Ile Gly Ala Ile Ala Thr 165 170 175Glu Gly Ala Gly Trp Ser Lys Leu Phe Pro Gly Ile Pro Val Ser Leu 180 185 190Leu Thr Leu Ala Asn Asn Phe Arg Ile Pro Leu Tyr Arg Glu Tyr Leu 195 200 205Met Ser Leu Gly Ile Ala Ser Val Ser Arg Arg Ser Cys Glu Ala Leu 210 215 220Leu Lys Arg Gly Gln Ser Ile Cys Ile Val Ile Gly Gly Ala Gln Glu225 230 235 240Ser Leu Leu Ala His Pro Gly His Met Asp Leu Val Leu Lys Arg Arg 245 250 255Lys Gly Phe Ile Lys Leu Ala Leu Glu Val Gly Asn Thr Asp Leu Val 260 265 270Pro Val Met Ala Phe Gly Glu Asn Asp Leu Tyr Gln Gln Val Asn Ser 275 280 285Ser Lys Ser Ser Arg Leu Tyr Lys Leu Gln Ser Leu Val Lys Asn Ala 290 295 300Leu Gly Phe Thr Leu Pro Leu Met His Ala Arg Gly Val Phe Asn Tyr305 310 315 320Asp Val Gly Ile Ile Pro Tyr Arg Arg Pro Ile Asn Val Val Val Gly 325 330 335Lys Pro Ile Pro Ile Pro His Ile Pro Asn Pro Ser Ala Asp Gln Val 340 345 350Asn Arg Tyr Gln Ile Gln Tyr Met Thr Glu Leu Lys Glu Leu Tyr Asp 355 360 365Lys Tyr Lys Asp Lys Cys Ser Asn Lys Asp Leu Pro Val Pro Glu Leu 370 375 380Thr Phe Val Glu385421167DNAArxula adeninivorans 42atggttcggt tcgctccttt aaatgttcct cttcatcgga ggttagagac gttcgcgctc 60acctaccata tcctgtcgat tccagtatgg atgtccttct ttttgctatg ctgtgccatt 120cctttaatgt ggccgttggt tatcatctac ctgctgtact atgcttccga caacagctct 180gagaatggag gggttgcgag caggtattcg ccaaagttca ggtccgtgcc tctttggaag 240tactttgcaa actactttcc aatcaccctt caccgtactc aagagctacc gcccgcattc 300gtgtaccaag gcgaagactt ggaccctgag acgcccgatg acagtgacga cgggcatgca 360aagtcaaagt ctattgtatt aaagctgtgg aaagttgcat tctggtggta ctacttgccc 420aagcattttc ttcgcaaacc agaggttcgt cctacgggtc gaagatacat ctttggatat 480cacccccatg gaatcattgg catgggtgcc attggcgcaa ttgctactga aggtgcgggg 540tggtccaagc tcttccccgg gatccctgtc agtttgctca ctctggcaaa caactttcga 600atccccctgt accgggaata tctcatgtct ctgggcattg cctcggtatc tagacggtcc 660tgtgaagctt tattaaaaag aggacagtca atttgcattg taattggagg cgctcaggaa 720agtcttcttg cacatccagg gcacatggat ttggtgctca agcgacgcaa gggattcatt 780aaactagctc ttgaagttgg caacaccgac ttggtgccag ttatggcatt tggagaaaac 840gatctctacc agcaagtgaa cagtagcaaa tcctcccgtc tatacaagct ccagagccta 900gttaagaatg ccttgggatt cacgcttccg ctgatgcacg ctcgaggagt gttcaattat 960gacgtgggca taatacccta tcgaagacca attaacgttg tagtgggcaa gcccatcccc 1020attccacaca ttccaaaccc atctgccgac caggtcaatc ggtaccagat ccagtacatg 1080actgaactca aagaattgta cgacaagtac aaagacaagt gcagtaacaa ggatcttcca 1140gttccggagc ttacatttgt agagtag 116743346PRTRhodotorula graminis 43Met Gly Ala Gln Glu Glu Val Asp Tyr Asp Gln Ser Asp His Thr Lys1 5 10 15Ile Lys Phe Val Pro Phe Val Val Pro Arg His Arg Arg Leu Gln Thr 20 25 30Phe Ser Val Phe Leu Trp Thr Thr Ala Leu Pro Ile Ser Leu Gly Ile 35 40 45Phe Cys Ile Leu Cys Ser Phe Pro Pro Leu Trp Pro Leu Val Ile Gly 50 55 60Tyr Leu Thr Trp Val Phe Leu Ile Asp Gln Ala Pro Met Arg Gly Gly65 70 75 80Arg Pro Gln Ala Trp Leu Arg Lys Ser Arg Val Trp Glu Trp Phe Ala 85 90 95Gly Tyr Tyr Pro Val Ser Leu Ile Lys Ser Ala Asp Leu Pro Pro Asp 100 105 110Gln Arg Tyr Val Phe Gly Tyr His Pro His Gly Val Ile Gly Met Gly 115 120 125Ala Ile Ala Asn Phe Gly Thr Asp Ala Thr Gly Phe Ser Arg Leu Phe 130 135 140Pro Gly Ile Thr Pro His Leu Leu Thr Leu Ala Ser Asn Phe Lys Leu145 150 155 160Pro Val Tyr Arg Glu Leu Leu Leu Ala Leu Gly Ile Ser Ser Val Ser 165 170 175Met Lys Ser Cys Gln Asn Ile Leu Arg Gln Gly Pro Gly Ser Ser Ile 180 185 190Thr Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ala His Pro Gly 195 200 205Thr Ala Asp Leu Thr Leu Lys Arg Arg Lys Gly Phe Ile Lys Leu Ala 210 215 220Ile Arg Thr Gly Ala Ser Leu Val Pro Val Phe Ser Phe Gly Glu Asn225 230 235 240Asp Ile Phe Asn Gln Leu Ser Asn Glu Arg Gly Thr Arg Leu Tyr Lys 245 250 255Leu Gln Lys Arg Phe Gln Ala Val Phe Gly Phe Thr Leu Pro Ile Phe 260 265 270Phe Gly Arg Gly Leu Phe Asn Tyr Asn Met Gly Leu Met Pro Tyr Arg 275 280 285His Pro Ile Val Ser Val Val Gly Arg Pro Ile Lys Val Lys Gln Lys 290 295 300Asp His Pro Ser Thr Ala Asp Leu Glu Glu Val Gln Glu Arg Tyr Ile305 310 315 320Ala Glu Leu Lys Arg Ile Trp Glu Asp Tyr Lys Glu Val Tyr Ala Lys 325 330 335Ser Arg Thr Lys Glu Leu Thr Ile Ile Ala 340 345441041DNARhodotorula graminis 44atgggcgcac aagaagaggt cgactacgac cagtcggacc acaccaagat caagttcgtg 60ccctttgtcg tcccgcggca ccgtcgcctc cagacgttct cggtcttcct gtggacgacg 120gccctcccta tctcgctcgg catcttctgc atcctgtgct ccttccctcc tctttggccg 180ctcgtcatcg ggtacctcac ctgggtcttc ctcattgacc aggcgccgat gcgcggcggg 240aggccacaag cctggctgcg aaagtcgcgc gtgtgggagt ggttcgccgg ctactatccc 300gtcagcctca tcaagagcgc cgacctcccg cccgaccagc gttacgtctt tggctaccac 360cctcacggcg tcatcggcat gggcgccatc gccaactttg gcaccgacgc gaccgggttc 420tcgcgcctgt tcccgggcat cacgccgcac ctcctcacgc tcgcgagcaa cttcaagctc 480ccagtctacc gagagctcct cctcgccctc ggcatctcgt ccgtctcgat gaagagctgc 540cagaacatcc tgcggcaagg tcccggctcg tccatcacga tcgtcgtcgg cggcgccgcc 600gagagcctga gcgcgcaccc tggcacggcc gacctgacgc tcaagcgccg caagggcttc 660atcaagctcg ccatccgcac cggcgcctcg ctcgtgcccg tcttttcctt tggcgagaac 720gacatcttca accagctgtc gaacgagcga gggacgcgcc tgtacaagct gcagaagcgg 780ttccaggccg tctttggctt cacattgccc atcttcttcg gccgaggcct gttcaactac 840aacatgggct tgatgccgta ccgacacccg atcgtctcgg tcgtcggccg cccgatcaag 900gtcaagcaga aggaccaccc gtcgactgcc gacctcgaag aagtccagga gcggtacatc 960gccgagctca aaaggatctg ggaggactac aaggaggtgt acgccaagag tcgcaccaag 1020gagctcacca tcatcgcctg a 104145635PRTPichia guilliermondii 45Met Thr Lys Glu Val Asp Glu Ser Thr Gly Gly Ala Ser Asp Ile Pro1 5 10 15Asn Met Val Glu Glu Ala Lys Ser Ser Ser Phe Asp Arg Glu Thr Glu 20 25 30Glu Asn Leu Leu Leu Glu Thr Thr Lys Pro Asp Glu Asn Leu Val Pro 35 40 45Glu Ser Thr Lys His Asp Glu Lys Leu Val Pro Glu Ile Thr Lys His 50 55 60Glu Asp Asn Pro Met Glu Asn Asp Gln Val Ser Gln Asn Thr Ala Thr65 70 75 80Ser Pro Met Thr Gly Ala Gly Ser Glu Glu Thr Arg Asp Leu Ile Thr 85 90 95Glu Asn Ile Glu Lys Pro Asp Glu Gly Asp Leu Leu Ile Glu Leu Ile 100 105 110Ser Lys Asp Asn Asp Gly Asp Gly Asp Asp Gly Leu Lys Asn Arg Lys 115 120 125Gln Lys Arg Ser Ser Ser Glu Val Lys Arg Leu Arg Met Ser Ser Leu 130 135 140Ala Pro Lys Gly Pro Thr Pro Gln Lys His Glu Arg Pro Lys Tyr Ile145 150 155 160Asn Val Ala Pro Leu Asn Ile Pro Ile Arg Arg Arg Leu Glu Met Val 165 170 175Gly Ile Ile Trp His Thr Ile Cys Ile Pro Thr Phe Val Ser Leu Phe 180 185 190Phe Leu Thr Leu Ser Leu Gly Pro Phe Ala Trp Val Gly Val Ile Leu 195 200 205Pro Tyr Phe Leu Trp Trp Tyr Leu Ile Asp Leu His Thr Pro Thr Asn 210 215 220Gly Lys Val Ala Tyr Arg Ser Arg Asp Trp Met Lys Asn Phe Ile Val225 230 235 240Trp Asp Trp Phe Val Asp Tyr Phe Pro Ile Arg Val His Lys Ser Cys 245 250 255Glu Leu Glu Pro Thr Phe Ser Asp Val Ile Ile Glu Asp Asp Val Val 260 265 270Pro Asp Asp Glu Glu Asp Leu Ile Ser Glu Gln Ser Arg Thr Gly Val 275 280 285Asp Lys Leu Phe Lys Phe Leu Gly Leu Arg Lys Arg Leu Asn Asp Asp 290 295 300Ser Asp Ala Ser Ser Gln Cys Ser Leu Leu Gln Glu Ser Leu Ser Thr305 310 315 320Arg Arg Lys Val Lys Arg Met Ser Thr Gly Pro Arg Tyr Ile Phe Gly 325 330 335Tyr His Pro His Gly Val Ile Ser Met Gly Val Phe Gly Thr Phe Ala 340 345 350Thr Asn Ala Leu Arg Asn Glu Pro Tyr Glu Pro Pro Leu Arg Leu Leu 355 360 365Lys Pro Phe Phe His Asp Ser Ser Lys Gly Glu Arg Leu Phe Pro Gly 370 375 380Ile Gly Thr Val Phe Pro Leu Thr Leu Thr Thr Gln Phe Ile Val Pro385 390 395 400Tyr Tyr Arg Asp Tyr Ile Leu Gly Met Gly Leu Thr Ser Ala Ser Ala 405 410 415Lys Asn Ile Lys Ser Leu Ile Ser Asn Gly Asp Asn Ser Ile Cys Val 420 425 430Val Val Gly Gly Ala Gln Glu Ser Leu Leu Asn Asp Met Val Ala Ala 435 440 445Thr Thr Val Pro Gly Arg Tyr Gly Lys Ser Asn Leu Pro Asn Asp Ser 450 455 460Asp Thr Asp Ser Glu Phe Asp Pro Gln Arg Lys Ile Glu Glu Asn Lys465 470 475 480Glu Glu Thr Gly Val Lys Lys Ile Glu Leu Val Leu Asn Lys Arg Lys 485 490 495Gly Phe Val Lys Ile Ala Ile Glu Leu Gly Asn Val Ser Leu Val Pro 500 505 510Thr Phe Gly Phe Gly Glu Ala Asp Ile Tyr Arg Ile Thr Lys Pro Lys 515 520 525Pro Gly Ser Phe Gly Glu Met Phe Gln Ser Trp Met Lys Arg Thr Phe 530 535 540Gln Phe Thr Val Pro Phe Phe Ser Ala Arg Gly Val Phe Ile Tyr Asp545 550 555 560Phe Gly Phe Leu Pro Tyr Arg Asn Pro Ile Asn Val Cys Phe Gly Arg 565 570 575Pro Ile His Ile Pro Ala Gly Leu Leu Asp Gln Tyr Lys Glu Pro Glu 580 585 590Thr Glu Lys Asp Glu Lys Glu Lys Glu Lys Asn Val Phe Gln Phe Thr 595 600 605Gln Asp Lys Gln Ala Pro Ala Phe Asn Ile Gln Ser Ile Gln Val Phe 610 615 620Gln Gly Glu Ala Thr Ile Lys Glu Glu Thr Ser625 630 635461908DNAPichia guilliermondii 46atgaccaagg aggttgatga aagcactggg ggtgccagtg atataccaaa tatggttgaa 60gaagcgaaat catcgagttt tgaccgtgaa actgaagaga atctgctact ggagaccact 120aaacctgacg agaatctggt accggagagt actaaacatg acgagaaact tgtaccggag 180atcactaaac atgaagacaa tcccatggaa aatgaccaag tttcccaaaa cacagccacc 240agtcctatga caggagctgg ttccgaagaa acccgtgatt tgattacaga gaacattgag 300aaaccagatg agggtgatct gctaattgag cttatttcca aagataacga tggtgatgga 360gatgatgggt tgaaaaatag aaaacaaaaa cgatcttctt ctgaagtgaa aaggctgcgc 420atgtcgtctc tggctcctaa aggtccaact cctcaaaagc atgaacgtcc caagtatata 480aatgtggcac ctcttaatat ccccattcga cggcgcttgg agatggtggg gataatctgg 540cacaccattt gtattcccac gtttgtcagt ttgtttttct tgactttgtc gttgggtccg 600tttgcttggg taggggtgat attgccgtac tttttatggt ggtatcttat cgatttacat 660actcctacaa acggtaaggt tgcgtatcgg tctcgcgact ggatgaagaa tttcattgtg 720tgggattggt tcgttgacta ttttcctatc agggtccaca agtcttgtga gttggagcct 780acctttagcg atgttattat tgaagacgat gtggtgcccg atgatgaaga agaccttatc 840tcagagcaat cacgaactgg agtcgataaa cttttcaaat ttttggggct tcgaaaacgc 900ttaaatgacg actcggatgc ttcgtcgcag tgctcactgc tgcaagagtc tttaagcaca 960agacgtaaag tgaaacgtat gtctactggt cctcgctaca tctttggata ccatccccat 1020ggagtaattt cgatgggtgt ttttggaact ttcgctacca atgcgttgcg taacgagccg 1080tacgaacctc ccttgcgttt gctaaagcca tttttccacg actcttccaa gggagaacgg 1140ttgtttcccg gtattggcac cgtctttcca ttgacattga caacccaatt tattgtgccg 1200tactaccgtg actatatctt gggcatggga ctcaccagtg cttcggctaa aaacatcaag 1260agccttataa gcaacggaga caactcgata tgtgtcgttg ttggaggtgc tcaggaatcg 1320ctcctaaacg atatggtagc cgcaaccaca gttcccggtc gttacggaaa gagcaatttg 1380cccaatgaca gtgataccga tagcgagttt gatcctcagc gtaagattga agaaaacaag 1440gaagaaaccg gcgtaaagaa aattgaactt gtacttaata agagaaaggg tttcgtcaag 1500atagcgattg agttgggcaa cgtttcactc gtgcctacgt ttggttttgg agaagctgac 1560atctacagaa tcaccaaacc caaaccaggt tcatttggag aaatgttcca atcttggatg 1620aaacgcacat ttcaattcac ggttccattt ttcagcgcta gaggtgtgtt catttacgac 1680tttgggtttc ttccttacag aaatcccatc aatgtctgct ttggacggcc cattcatatt 1740ccagccggct tattggatca atacaaagag cccgaaactg agaaagatga aaaagaaaag 1800gaaaaaaacg tcttccagtt cactcaagac aaacaagcgc cagccttcaa tatccaatct 1860attcaagttt tccaagggga agcaaccatc aaagaggaaa cgagttag 190847370PRTMicrobotryum violaceum 47Met Thr Ser Leu Arg Asp Val Asn Pro Thr Ser Thr Gln Ala Ser Leu1 5 10 15Tyr Lys Asp Glu Gly Lys Asp Lys Glu Asp Val Ala Pro Gln Glu Lys 20 25 30Tyr Thr Gln Ser Leu Arg Thr Asn Ile Lys Phe Ala Pro Leu Ala Val 35 40 45Pro Arg His Arg Arg Leu Gln Thr Met Ala Val Leu Gly Trp Thr Thr 50 55 60Ala Leu Pro Leu Met Leu Gly Leu Phe Phe Leu Leu Cys Ser Ile Pro65 70 75 80Leu Leu Trp Pro Ile Ile Val Pro Tyr Leu Phe Trp Ile His Leu Ile 85 90 95Asp Asn Ser Pro Thr Gln Gly Gly Arg Ala Ser Lys Trp Leu Arg Gln 100 105 110Ser Arg Phe Trp Val Trp Phe Thr Gly Tyr Tyr Pro Ile Ser Leu Val 115 120 125Lys Thr Val Asp Leu Pro Pro Asp Arg Lys Tyr Val Phe Gly Tyr His 130 135 140Pro His Gly Ile Ile Gly Met Gly Ala Ile Ala Asn Phe Gly Thr Asp145 150 155 160Ala Thr Gly Phe Ser Glu Leu Phe Pro Gly Leu Asn Pro His Leu Leu 165 170 175Thr Leu Ala Ser Asn Phe Lys Leu Pro Ile Tyr Arg

Asp Phe Leu Leu 180 185 190Ala Leu Gly Ile Cys Ser Val Ser Met Lys Ser Cys Gln Asn Ile Leu 195 200 205Lys Gln Gly Pro Gly Ser Ala Leu Thr Ile Val Val Gly Gly Ala Ala 210 215 220Glu Ser Leu Ser Ala His Pro Gly Thr Ala Asn Leu Thr Leu Arg Arg225 230 235 240Arg Met Gly Phe Ile Lys Leu Ala Met Arg Gln Gly Ala Asp Leu Val 245 250 255Pro Val Phe Ser Phe Gly Glu Asn Asp Ile Phe Glu Gln Met Pro Asn 260 265 270Glu Arg Gly Thr Lys Leu Tyr Lys Met Gln Lys Lys Phe Gln Thr Ala 275 280 285Phe Gly Phe Thr Leu Pro Ile Phe His Gly Arg Gly Ile Phe Asn Tyr 290 295 300Asn Leu Gly Ile Leu Pro Tyr Arg His Pro Ile Val Ser Val Val Gly305 310 315 320Arg Pro Ile Arg Val Ser Gln Arg Asp Asn Pro Thr Lys Glu Glu Leu 325 330 335Glu Glu Val Gln Glu Arg Tyr Ile Glu Glu Leu Lys Arg Ile Trp Asp 340 345 350Asp Tyr Lys Asn Gln His Ala Ile Lys Arg Lys Gly Glu Leu Arg Ile 355 360 365Ile Ala 370481113DNAMicrobotryum violaceum 48atgacgtcgc tgcgagacgt gaacccgacc tcgacccaag catcgttgta caaagacgag 60ggcaaggaca aggaggatgt cgcaccgcag gagaaataca cgcagtcgct ccggaccaac 120atcaaatttg cacctctagc tgtaccacgc catcgacgac tgcagaccat ggcagtgttg 180ggatggacga ccgcactgcc actcatgctt ggtttgttct ttctattgtg ctcaatcccc 240cttctatggc ccatcatcgt gccctatctc ttctggatcc acctcatcga caactcgccg 300acgcagggag gacgagcgag caaatggctt cgccaaagtc ggttctgggt gtggttcaca 360gggtactatc ctatcagtct cgtcaagacg gtcgatttac ctccagatcg gaaatacgtc 420ttcggttacc acccccatgg cataattgga atgggtgcaa ttgccaattt tgggaccgac 480gccactgggt tctctgagct cttcccagga ctcaaccctc atctcctcac acttgccagc 540aacttcaaat tgccgatata tcgagacttc ttgcttgcgc tcggcatctg ctcagtcagt 600atgaaatctt gccaaaacat cctcaaacag ggcccggggt ctgctttgac cattgtcgtc 660ggaggagctg cggaatccct ttcggcgcat cctggcacag ccaacttgac actccgtcgc 720cgaatgggct tcatcaagct ggcgatgcgt caaggcgcgg atcttgtacc cgtcttttca 780ttcggagaga acgatatctt cgaacagatg ccgaacgaga gagggacgaa gctgtacaag 840atgcaaaaga agtttcagac cgcttttgga ttcactctac cgatcttcca cggccgagga 900atttttaact ataaccttgg catcttgccg taccgtcatc cgatcgtgtc ggtcgtcggt 960cggcccatcc gcgtttcgca gcgtgacaac cctactaagg aggaactcga ggaggtgcag 1020gaacgataca tcgaggagtt gaagagaatc tgggacgatt acaaaaatca acatgccatc 1080aagcgaaagg gcgaacttcg tattattgcc tga 111349365PRTPuccinia graminis 49Met Lys Asp Asp Ser Arg Ser Pro Ser Gly Ser Glu Pro Glu Gly Asp1 5 10 15Asn His Lys Lys Glu Lys Arg Pro Ile Trp Ala Pro Ile Arg Val Pro 20 25 30Pro Tyr Arg Arg Ile Gln Thr Ala Ala Val Leu Leu Trp Thr Ser Gln 35 40 45Leu Ser Leu Cys Ile Ser Leu Phe Phe Phe Leu Met Ser Tyr Pro Ile 50 55 60Thr Trp Pro Ile Leu Leu Pro Tyr Val Ile Trp Ile Leu Val Ile Asp65 70 75 80Pro Ala Pro Glu Lys Gly Gly Arg Leu Asn Gln Ser Val Arg Thr Trp 85 90 95Lys Phe Trp Asn Leu Phe Ala Ser Tyr Phe Pro Ile Ser Leu Ile Lys 100 105 110Thr Val Asp Leu Pro Ser Asp Arg Lys Tyr Val Phe Gly Tyr His Pro 115 120 125His Gly Ile Ile Gly Met Gly Ala Val Ala Asn Phe Gly Thr Glu Ala 130 135 140Thr Gly Phe Ser Glu Lys Phe Pro Gly Leu Asn Pro His Leu Leu Thr145 150 155 160Leu Ser Thr Asn Phe Ile Ile Pro Phe Tyr Arg Asp Leu Ile Leu Ser 165 170 175Leu Gly Ile Cys Ser Val Ser Ile Lys Ser Cys Ile Ser Ile Leu Lys 180 185 190Ser Lys Asn Lys Arg Ser Ala Asp Val Lys Asn Asn Lys Gly Glu Gly 195 200 205Asn Cys Leu Val Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ala 210 215 220His Pro Gly Thr Ala Asp Leu Thr Leu Lys Arg Arg Leu Gly Phe Ile225 230 235 240Lys Leu Ala Ile Arg Glu Gly Ala Asp Leu Val Pro Val Phe Ser Phe 245 250 255Gly Glu Asn Asp Ile Tyr Ala Gln Leu Ser Asn Ser Lys Gly Thr Ala 260 265 270Leu Tyr Ser Leu Gln Lys Arg Phe Gln Ala Val Phe Gly Phe Thr Leu 275 280 285Pro Val Phe His Gly Arg Gly Ile Phe Asn Tyr Ser Leu Gly Leu Leu 290 295 300Pro Tyr Arg His Pro Ile Val Ser Val Val Gly Lys Pro Ile Arg Val305 310 315 320Glu Gln Asn Lys Asn Pro Gly Leu Glu Glu Ile Glu Lys Val Gln Lys 325 330 335Glu Tyr Ile Ala Glu Leu Thr Ala Val Trp Asp Gln Tyr Lys Asp Leu 340 345 350Tyr Ala Arg Asn Arg Lys Ser Glu Leu Thr Leu Ile Ala 355 360 365501098DNAPuccinia graminis 50atgaaggatg actccagaag cccgtctggg tccgaacccg agggcgataa tcacaagaag 60gagaaaaggc caatctgggc tccgattcgt gtacctcctt acaggcgcat ccaaacggcc 120gcagtactct tatggacttc tcaactctca ctatgcattt ccttattctt tttcttaatg 180tcttacccga tcacctggcc gatcctcctt ccatacgtta tttggatctt ggtcatagat 240cctgctcccg agaagggtgg ccggttgaat caatctgttc ggacctggaa gttttggaat 300ctatttgcgt cgtatttccc aatcagttta atcaaaactg ttgatttgcc cagtgaccgc 360aaatatgtct ttggttacca ccctcatggt atcatcggaa tgggcgcggt ggccaacttt 420ggaacggaag cgacaggatt ttcggaaaaa ttccctggtc tcaatccaca tctactcaca 480ttgagcacga actttatcat cccattctat cgagacctga tcctcagtct tggaatctgt 540tcggtgtcga tcaaatcatg catctcgatc ctcaaatcca aaaacaaacg ctcagctgat 600gtcaagaaca ataagggcga aggaaattgt ttggttatcg ttgtcggtgg ggctgcggaa 660agtttgtctg ctcatcctgg aacagccgat ctcactctaa aacgacggct gggtttcatc 720aaactggcca ttcgagaagg agccgatctc gtccctgtgt tctcctttgg agagaatgac 780atttacgccc aattatcaaa ctcaaaaggc acggcactct actctcttca aaaacgattt 840caagctgtat ttggctttac cttacctgtt ttccatggcc gaggtatctt caactactct 900ctcggcttgc ttccctatcg acacccgatt gtttcagtag ttggtaaacc tattcgagtc 960gagcaaaata aaaaccccgg gctcgaagaa atcgaaaagg ttcagaaaga atacattgct 1020gaacttaccg cagtatggga tcagtataaa gatttatacg ctagaaatcg gaagagtgaa 1080ttgactttga ttgcttag 109851373PRTGloeophyllum trabeum 51Met Asp Ala Gly Arg Ala Phe Ser Ser Ala Ser Arg Ser Leu Ser Ser1 5 10 15Ser Ser Leu Lys Asp Lys Leu Ser Lys Val Ser Lys Leu Ser Thr Thr 20 25 30Pro Leu Arg Pro Val Ala Ala His Val Lys Asn Ile Asp Phe Val Pro 35 40 45Ser Lys Ile Pro Arg Lys Arg Arg Leu Gln Met Leu Ala Val Ala Val 50 55 60Trp Ala Leu Leu Ile Pro Ile Thr Thr Phe Leu Phe Leu Ile Leu Cys65 70 75 80Ser Phe Pro Pro Leu Trp Pro Phe Leu Ala Ala Tyr Leu Ile Trp Ile 85 90 95Arg Trp Ile Asp Arg Ser Pro Glu His Gly Gly Arg Ile Ser Pro Trp 100 105 110Phe Arg Ser Met Arg Phe Trp Arg Tyr Phe Ala Asp Tyr Tyr Pro Ala 115 120 125Ser Phe Leu Lys Glu Cys Asp Leu Pro Pro Asp Arg Pro Tyr Val Phe 130 135 140Gly Tyr His Pro His Gly Ile Ile Gly Met Gly Ala Met Ala Thr Phe145 150 155 160Ala Thr Glu Ala Thr Gly Phe Ser Glu Gln Phe Pro Gly Leu Thr Pro 165 170 175His Leu Leu Thr Leu Ala Thr Asn Phe Thr Met Pro Ile Tyr Arg Asp 180 185 190Ile Ile Leu Ala Leu Gly Ile Cys Ser Val Ser Lys Gln Ser Cys Ser 195 200 205Asn Ile Leu Ser Ser Gly Pro Gly Gln Ala Ile Thr Ile Val Val Gly 210 215 220Gly Ala Ala Glu Ser Leu Ser Ala Arg Pro Gly Thr Ala Asp Leu Thr225 230 235 240Leu Lys Arg Arg Leu Gly Phe Ile Lys Ile Ala Ile Gln His Gly Ala 245 250 255Ala Leu Val Pro Val Phe Ser Phe Gly Glu Asn Asp Ile Tyr Gln Gln 260 265 270Met Pro Asn Glu Lys Gly Thr Thr Ile Tyr Ala Leu Gln Lys Lys Phe 275 280 285Gln Ser Val Phe Gly Phe Thr Leu Pro Leu Phe His Gly Arg Gly Met 290 295 300Leu Asn Tyr Asn Leu Gly Leu Met Pro Tyr Arg Arg Arg Ile Val Ser305 310 315 320Val Ile Gly Arg Pro Ile Leu Cys Glu Lys Cys Glu Lys Pro Ser Met 325 330 335Glu Glu Val Thr Arg Val Gln Gln Glu Tyr Ile Ala Glu Leu Leu Arg 340 345 350Ile Trp Asp Thr Tyr Lys Asp Gln Phe Ala Arg Ser Arg Lys Arg Glu 355 360 365Leu Ser Ile Ile Asp 370521122DNAGloeophyllum trabeum 52atggacgctg gtcgcgcctt ctcctctgca tcccgctcgt tatcgtcctc gtccctgaag 60gacaagctgt caaaggtctc gaagctcagc accactcctc tgcgaccggt cgctgcccat 120gtcaagaata tcgacttcgt cccgtccaag atcccccgga aacggaggct gcagatgctc 180gctgttgcag tatgggcgct cctgataccc atcacgacgt ttttgttcct catactatgt 240tcttttccac cgctgtggcc atttttagcg gcgtatctta tatggataag atggatagac 300cggagtcctg agcatggcgg gaggataagt ccgtggttcc gctcgatgag gttctggaga 360tactttgccg actactaccc tgcatcgttc ttgaaggaat gcgacctccc cccagaccga 420ccttacgtct tcgggtatca ccctcatggc atcattggca tgggtgccat ggccactttc 480gccaccgaag ccactggatt cagcgaacag ttccctgggc tcactcccca cctgctcacc 540ctagccacaa atttcaccat gcccatatac agagacatca tcctcgccct gggcatatgc 600tccgtcagca agcagtcctg ctcgaacatc ctcagcagcg gccccgggca ggctatcaca 660atcgtagtag gaggcgcagc agagagtctt agcgctcggc cgggcacggc cgacctcacg 720ctcaaacgga ggcttggctt catcaagatt gctatacaac acggagcggc actggtccct 780gtattttctt tcggcgagaa tgatatttat caacaaatgc ccaacgaaaa gggaaccaca 840atatatgccc tacagaagaa attccagagc gtcttcggct tcacgttgcc cttgttccac 900ggtcggggca tgctaaatta taaccttggt ttgatgccgt atcgacggcg gatcgtgtct 960gtcatcggtc ggcccatatt atgcgagaag tgcgagaagc caagcatgga ggaggttacg 1020cgggtgcaac aggagtacat cgcagagctg ctcagaatat gggacacgta caaagatcaa 1080tttgctcggt cgcggaagag agaactgagt attattgatt ga 112253346PRTRhodosporidium diobovatum 53Met Gly Ala Leu Asp Ala Gly Asp His Glu Gly Thr Glu His Pro Lys1 5 10 15Ile Lys Phe Val Pro Phe Val Val Pro Arg His Arg Arg Leu Gln Thr 20 25 30Phe Ser Val Phe Leu Trp Thr Thr Ala Leu Pro Leu Ser Leu Gly Ile 35 40 45Phe Cys Ile Leu Cys Ser Phe Pro Pro Leu Trp Pro Leu Val Ile Gly 50 55 60Tyr Leu Thr Trp Val Phe Leu Ile Asp Gln Ala Pro Met Arg Gly Gly65 70 75 80Arg Pro Gln Ala Trp Leu Arg Lys Ser Arg Val Trp Glu Trp Phe Ala 85 90 95Gly Tyr Tyr Pro Val Ser Leu Ile Lys Ser Ala Asp Leu Pro Pro Asp 100 105 110Gln Arg Tyr Val Phe Gly Tyr His Pro His Gly Val Ile Gly Met Gly 115 120 125Ala Ile Ala Asn Phe Gly Thr Asp Ala Thr Gly Phe Ser Arg Leu Phe 130 135 140Pro Gly Ile Lys Pro His Leu Leu Thr Leu Ala Ser Asn Phe Lys Leu145 150 155 160Pro Leu Tyr Arg Glu Leu Leu Leu Ala Leu Gly Ile Ser Ser Val Ser 165 170 175Met Lys Ser Cys Gln Asn Ile Leu Arg Gln Gly Pro Gly Ser Ser Ile 180 185 190Thr Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ala His Pro Gly 195 200 205Thr Ala Asp Leu Thr Leu Lys Arg Arg Lys Gly Phe Ile Lys Leu Ala 210 215 220Ile Arg Ser Gly Ala Tyr Leu Val Pro Val Phe Ser Phe Gly Glu Asn225 230 235 240Asp Ile Phe Asn Gln Leu Ser Asn Glu Arg Gly Thr Arg Leu Tyr Lys 245 250 255Leu Gln Lys Arg Phe Gln Ala Val Phe Gly Phe Thr Leu Pro Ile Phe 260 265 270Phe Gly Arg Gly Leu Phe Asn Tyr Asn Met Gly Leu Met Pro Tyr Arg 275 280 285His Pro Ile Val Ser Val Val Gly Arg Pro Ile Lys Val Thr Gln Lys 290 295 300Asp His Pro Ser Thr Ala Asp Leu Glu Glu Val Gln Asp Arg Tyr Ile305 310 315 320Ala Glu Leu Lys Arg Ile Trp Glu Asp Tyr Lys Glu Val Tyr Ala Lys 325 330 335Ser Arg Thr Lys Glu Leu Thr Ile Ile Ala 340 345541041DNARhodosporidium diobovatum 54atgggagcac tagatgcggg cgaccacgag gggaccgaac accccaagat caagttcgtt 60cctttcgttg tgccgcgaca ccgcaggctg cagacctttt cggtgtttct gtggacgacc 120gcgctgcctc tgtcgctcgg catcttctgc attctctgct ccttcccccc actctggccc 180ctcgtcatag ggtatctcac gtgggtattc ctcatcgacc aggcgcccat gcggggtggc 240aggcctcagg cctggttgcg caagtcgcgt gtgtgggagt ggttcgccgg ctactaccct 300gtcagcttga tcaagagcgc cgacctcccg cccgaccagc gctacgtctt tggctaccac 360ccacacggcg tcattgggat gggcgccatc gccaactttg gtaccgacgc gaccggcttc 420tcgcggctgt tccccggcat caagccgcac ctcctcacgc tcgccagcaa cttcaagctg 480ccgctctacc gagaactgct cctcgccttg ggcatttcgt ccgtgtcgat gaagagctgc 540cagaacatcc tgcgccaagg tcccggctcg tcgatcacga ttgtcgtcgg aggggcagca 600gaaagcctca gcgcgcaccc gggaacggca gacctgacgc tcaagcggcg gaaggggttc 660atcaagctcg cgatccgctc aggggcctac ctcgtcccgg tattttcctt tggcgagaat 720gacatcttca accagctgtc gaatgagcgc ggcacccgac tctacaagct gcaaaagcgg 780ttccaggccg tctttggctt caccttgccc atcttcttcg gtcgcggcct cttcaactac 840aacatgggct tgatgccata tcgacacccg atcgtctcgg tcgtcggacg ccccatcaag 900gtcacgcaga aggatcaccc gtcgacggcc gacctcgaag aggtacagga ccgctacatt 960gccgagttga agaggatctg ggaggactac aaagaggtgt acgccaagag ccgcaccaag 1020gagctcacca tcatcgcatg a 104155359PRTPhaeodactylum tricornutum 55Met Lys Glu Arg Arg Ser Gly Leu Asn Pro Ser Gly Ser Ser Val Tyr1 5 10 15Pro Leu His Pro Pro Asp Ser Arg Val Leu Val Arg Val Pro Ser Asp 20 25 30Ile Ser Phe Leu Asp Arg Leu Ile Val Ala Gly Ser Ser Ile Phe Ile 35 40 45Val Gly Ser Leu Val Trp Val Pro Leu Thr Ala Arg Trp Val Tyr Arg 50 55 60Arg Trp Lys Gln Ala Lys Asp Lys Arg Lys Arg Ala Met Tyr Ala Ser65 70 75 80Leu Leu Val Ile Leu Ala Val Leu Val Ile Gly Gly Pro His Arg Ser 85 90 95Pro Arg Val Gly Lys Trp Leu Gln Val Arg Lys Trp Ser Leu Phe Gln 100 105 110Ala Trp Val Lys Phe Ile Ala Met Glu Val Ile Leu Asp Gln Pro Lys 115 120 125Gly Ile Thr Met Asp Val Gln Gln Asp Lys Ala Ile Phe Ala Phe Ala 130 135 140Pro His Gly Ile Phe Pro Phe Ala Phe Ala Phe Gly Val Leu Pro Asp145 150 155 160Ile Ala Thr Gln Ser Phe Gly Tyr Val Arg Pro Val Val Ala Thr Ala 165 170 175Thr Arg Leu Phe Pro Val Val Arg Asp Phe Ile Ser Trp Ala Asn Pro 180 185 190Val Asp Ala Ser Lys Asp Ser Val Glu Arg Ala Leu Ala Leu Gly Asp 195 200 205Arg Ile Ala Val Ile Pro Gly Gly Ile Ala Glu Ile Phe Glu Gly Tyr 210 215 220Pro Lys Pro Asn Thr His Pro Asp Glu Glu Tyr Ala Ile Val Arg Ser225 230 235 240Gly Phe Leu Arg Leu Ala Ile Lys His Gly Ile Pro Val Ile Pro Val 245 250 255Tyr Cys Phe Gly Ala Thr Lys Met Leu Lys Arg Leu Glu Leu Pro Gly 260 265 270Leu Glu Gln Leu Ser Leu Phe Leu Arg Val Ser Ile Cys Leu Phe Phe 275 280 285Gly Val Gly Gly Leu Pro Ile Pro Phe Arg Gln Arg Leu Ser Tyr Val 290 295 300Met Gly Gln Pro Ile Leu Pro Pro Val Arg Thr Thr Gly Ser Asp Ile305 310 315 320Ser Asp Ala His Val Lys Glu Met Gln Asp Arg Phe Cys Ala Glu Val 325 330 335Gln Arg Leu Phe Asp Arg His Lys Glu Ala Tyr Gly Trp Ser Tyr Lys 340 345 350Thr Leu Lys Leu Leu Glu Gln 355561080DNAPhaeodactylum tricornutum 56atgaaagaaa gaagatctgg cctaaatccg tcaggatcct ccgtgtatcc attgcaccct 60cctgacagtc gcgttctcgt tcgagtcccc tccgatattt cctttcttga tcgtctcatc 120gtcgctggca gcagtatctt tattgtcggt tcgctagtat gggttccatt gaccgcaaga 180tgggtctaca ggcggtggaa gcaagctaaa gataaacgaa agcgggctat gtatgcctct 240ctactcgtga ttctggcagt tctcgttatt ggcggacccc accgatctcc tcgtgtcggc 300aaatggctcc aagtacgaaa gtggtccctc ttccaagcgt gggtaaagtt tattgccatg 360gaagtgattt tggatcaacc gaaaggcatt

actatggacg tccaacaaga caaggcgatt 420tttgcattcg cgccacatgg aatctttccg tttgcgttcg cctttggagt gcttcccgat 480attgccacac aatcgtttgg ctacgttcgt ccggtcgtgg caaccgccac aaggttgttt 540cctgtagtcc gggatttcat ctcttgggcg aatccggtag acgcttccaa agattccgtt 600gaacgtgctt tagcattggg cgatcgcatt gctgtaatac ctggaggaat tgcagaaatt 660ttcgaaggat atccgaaacc gaacacgcat ccggatgaag agtacgctat cgtacggagt 720ggatttttgc gtttggcaat aaaacacggt atcccagtga ttcccgtata ctgtttcggc 780gctaccaaaa tgttgaagcg tctggagctt cccggcctgg agcaactgtc cctgtttcta 840cgcgtgagca tttgcctctt ttttggagtc ggcgggttgc ccatcccttt ccgacaacga 900ttgtcgtacg taatgggaca accaattttg ccacccgtaa ggacaacggg cagcgatatt 960tcggacgcac acgtcaaaga aatgcaagat cgcttttgtg ctgaggtcca gcggctcttt 1020gatcgacata aggaagctta tggttggtca tacaaaacgc tgaaactatt ggaacagtga 108057329PRTPhaeodactylum tricornutum 57Met Glu Arg Thr Lys Ile Gln Asp Glu His Lys Ser Pro Pro Asn Pro1 5 10 15Ser Thr Phe Arg Trp Phe Leu Gly Leu Leu Val Ala Ser Thr Phe Ser 20 25 30Met Val Tyr Phe Val Ala Pro Phe Tyr Met Leu Thr Val Val Phe Ala 35 40 45Leu Val Phe Lys Tyr Pro Ser Val Glu Ile Ala Trp Met Tyr Ala Ile 50 55 60Pro Met Ile Val Ser Ala Ile Leu Pro Pro Met Ala Ser Pro Leu Ala65 70 75 80Leu Arg Leu Ile Ser Pro Leu Ile Asp Tyr Phe Asp Tyr Glu Glu Ile 85 90 95His Glu Thr Ser Pro Val Asp Val Gln Lys Glu Ile Leu Ser Asn Asn 100 105 110Lys Asn Tyr Leu Leu Val Phe Gln Pro His Gly Ala Leu Ser Phe Thr 115 120 125Gly Ile Thr Ser Met Val Thr Ala Pro Gln Ala Met Lys Gly Lys Leu 130 135 140Pro Thr Ala Val Ala Asp Ala Leu Leu Tyr Thr Pro Ile Leu Lys His145 150 155 160Val Leu Gly Ile Phe Gly Leu Ile Ser Ala Ser Lys Ser Ser Met Ile 165 170 175Arg Thr Leu Lys Lys Lys Gly Val Glu Gly Thr Ile Val Leu Tyr Val 180 185 190Gly Gly Ile Ala Glu Leu Phe Leu Thr Asp Glu Thr Asp Glu Arg Leu 195 200 205Tyr Leu Arg Lys Arg Lys Gly Phe Ile Lys Leu Ala Leu Gln Gln Gly 210 215 220Val Asp Val Val Pro Val Tyr Leu Phe Gly Asn Thr Asn Ala Leu Ser225 230 235 240Val Leu Lys Thr Gly Phe Leu Ala Ala Ile Ser Arg Lys Leu Gln Ile 245 250 255Ser Leu Thr Tyr Ile Trp Gly Lys Trp Tyr Leu Pro Ile Pro Arg Asp 260 265 270Cys Lys Leu Leu Tyr Ala Ser Gly Gln Pro Leu Gly Met Pro His Ile 275 280 285Leu Asp Pro Ser Gln Ala Asp Ile Asp Lys Trp His Glu Lys Tyr Cys 290 295 300Ser Glu Val Met Arg Ile Phe Glu Lys Tyr Lys Glu Lys Val Pro Glu305 310 315 320Tyr Lys His Lys Lys Leu Glu Ile Ile 32558990DNAPhaeodactylum tricornutum 58atggagagaa caaagataca agacgagcac aaaagtcccc ctaatccgtc gacatttcga 60tggttcctcg gccttctagt ggcgtcgacg ttttccatgg tctattttgt ggctcccttt 120tacatgctta cagtcgtgtt tgcactagtt ttcaaatatc cttcggtaga aattgcatgg 180atgtacgcta ttccgatgat tgtctcggcc attttgccac caatggcttc tccactggcc 240ttgcgactca tctccccgct cattgactac ttcgattacg aagagatcca cgaaacctca 300ccggtggacg tccagaagga aatactaagc aacaacaaaa actatttgct agtctttcaa 360ccgcatggag cactgtcgtt tacaggaatc acttcaatgg tgacagctcc acaagcaatg 420aaaggcaaat tgccaacagc tgtggctgac gcactcttgt acacacctat actgaaacat 480gtcttaggaa ttttcgggct gattagtgcc tccaaaagca gcatgatccg aactttaaaa 540aagaagggtg tggaaggaac cattgttttg tacgttggtg ggattgccga gctctttttg 600accgacgaga cggacgagcg cctctatctg cgaaagcgaa aagggtttat caaattagct 660ctacaacagg gtgtcgatgt tgtacctgtg tatctatttg ggaacacaaa cgcgctgtcg 720gtactaaaga cgggatttct cgcggcaatt tcgcgaaaat tacagatatc tctgacgtac 780atttggggaa agtggtatct tccgattccc cgtgattgca aattgctgta tgcttccggt 840cagccattag gaatgcctca tattttagac ccaagccaag ccgacattga taaatggcac 900gaaaagtact gctccgaggt catgcggatc ttcgaaaaat acaaggaaaa ggttccggaa 960tacaagcaca agaaattaga aattatttga 99059392PRTPhaeodactylum tricornutum 59Met Arg Glu Arg Ser Cys Ala Asn Ala Ser Asp Asp Asp Ser Ile His1 5 10 15Lys Gln Ser Pro Glu Leu Glu Ala Glu Phe Leu His Thr Ser Lys Leu 20 25 30Thr Leu Ala Asp Met Arg Arg Leu Ala His Asp Pro Lys Asp Arg Gly 35 40 45Leu Ala Thr Lys Pro Ala Ala Gln Ala Thr Lys Glu Asp Val Leu Thr 50 55 60Val Gln Pro Met Ser Phe Val Glu His Thr Ala Cys Cys Leu Phe Leu65 70 75 80Ala Phe Gly Val Pro Asn Gly Ala Leu Thr Ile Pro Ile Ala Thr Trp 85 90 95Leu Ile Gly Lys Phe Val Leu Arg Asn Val Phe Leu Ala Phe Leu Leu 100 105 110Ala Gly Cys Ile Leu Leu Pro Leu Ala Ile Leu Pro Gln Glu Tyr Val 115 120 125Pro Ala Arg Leu Gln Ser Trp Leu Ala Leu Gln Ile Leu Lys Tyr Phe 130 135 140Ser Phe Ser Leu Val Met Glu Glu Arg Pro Pro Thr Met Cys Thr Gly145 150 155 160Lys Gln Leu Ile Glu Gln Pro Ala Arg Pro Arg Ile Val Thr Ala Tyr 165 170 175Pro His Gly Val Phe Pro Tyr Gly Asn Ala Leu Thr Val Val Thr Trp 180 185 190Pro Leu Leu Thr Gly His His Ile Val Gly Leu Ala Ala Asn Ala Ala 195 200 205Leu Arg Thr Pro Ile Phe Lys Gln Ile Leu Arg Ser Ile Gly Val Lys 210 215 220Asp Ala Ser Arg Ala Ser Val Arg Asn Ala Leu Glu Thr Trp Pro Phe225 230 235 240Thr Val Gly Ile Ser Pro Gly Gly Val Ala Glu Val Phe Glu Thr Asn 245 250 255His Phe Asn Glu His Ile Leu Leu Lys Glu Arg Ile Gly Val Ile Lys 260 265 270Met Ala Ile Arg Thr Gly Ala Asp Leu Val Pro Gly Tyr Met Tyr Gly 275 280 285Asn Thr Asn Leu Tyr Trp Cys Trp Thr Gly Glu Gly Ile Pro Gly Ala 290 295 300Arg Trp Leu Leu Glu Tyr Val Ser Arg Lys Ile Leu Gly Phe Ala Leu305 310 315 320Val Pro Ile Ala Gly Arg Trp Arg Leu Pro Ile Pro Tyr Arg Thr Pro 325 330 335Ile Leu Cys Val Val Gly Lys Pro Ile Pro Thr Ile His Leu Gln Thr 340 345 350Glu Glu Pro Ser Met Glu Gln Ile Val Asp Ile Gln Glu Gln Leu Ser 355 360 365Thr Glu Leu Lys Ser Met Phe Asp Arg Tyr Lys His Leu Tyr Gly Trp 370 375 380Glu Asp Arg Met Leu Val Ile Thr385 390601179DNAPhaeodactylum tricornutum 60atgcgtgagc gaagctgcgc caacgcttct gacgatgaca gcattcacaa gcagtcgcca 60gaattggagg ctgagtttct tcataccagc aagttgacct tagccgacat gcgacgattg 120gcgcacgatc cgaaggatcg ggggttggca acaaaacctg cggcgcaagc tacgaaagaa 180gacgtcttga cggtacaacc catgagtttc gtagaacaca ctgcttgctg tctgtttctc 240gcgtttggag tgcccaatgg cgctctgacg attcccatag caacgtggct gatcggaaaa 300ttcgtgttgc gcaacgtttt cttggcgttt ctgttagcag gctgtatact tctaccgctt 360gcgatactgc cgcaagaata tgtgcccgcc cgattgcaat cgtggcttgc tttgcagata 420ctgaaatatt tttctttctc tttggtcatg gaggaacgcc ctccgacaat gtgtactggc 480aagcagctga tcgagcagcc cgctcggcca cgaatcgtca cagcctatcc gcacggagtt 540ttcccatacg gaaacgcgtt gactgtagtc acatggccgt tgttgacggg acaccatatt 600gtgggtttgg cagcaaatgc cgctttgcgg acaccgatct ttaaacaaat cttgcggagc 660attggcgtca aggacgcctc tcgagcgtcg gtacggaatg cgctggaaac atggcctttc 720accgtcggga tttcgccagg tggcgtggcg gaagtttttg aaacaaacca cttcaatgag 780cacattctgt tgaaagaacg tattggtgtc atcaagatgg ccattcgcac cggtgcggat 840cttgtaccag gctatatgta tggtaatact aatctgtact ggtgctggac aggggaaggt 900attcctggag ctcggtggct attggagtat gtttcgcgta aaatcctagg ttttgccctc 960gtgcctatag cgggtagatg gagactacca ataccgtaca ggactccgat attgtgtgtc 1020gtgggcaagc caataccaac cattcatttg caaaccgaag aaccatcaat ggagcaaatc 1080gtggacattc aggaacaatt gtcaacagaa ttgaaatcaa tgttcgaccg ctataagcac 1140ctgtacggat gggaagatcg aatgctagtg atcacataa 117961320PRTPhaeodactylum tricornutum 61Met Thr Arg Ser Lys Phe Ile Gly Ser Ala Gly Ala Ile Gly Leu Phe1 5 10 15Cys Leu Met Ile Ile Pro Asn Val Gly Ile Leu Ile Ala Thr Phe Leu 20 25 30Tyr Pro Lys Val Leu Gly Leu Tyr Phe Leu Ile Pro Tyr Tyr Ala Tyr 35 40 45Asn Leu Ser Ile Gly Lys His Glu Ala Arg Asp Gly Asn Gly Trp Asn 50 55 60Trp Phe Ser Glu Asn Phe Phe Val Phe Asn Ile Val Arg Gly Tyr Leu65 70 75 80Asn Leu Lys Ile Glu Ala Asp Ser Glu Leu Lys Glu Ala Glu Ala Lys 85 90 95Glu Gly Ala Gln Phe Val Phe Ala Val Ser Pro His Gly Thr Asn Ala 100 105 110Asp Tyr Arg Val Phe Ile Asp Gly Met Leu His Glu Ala Leu Pro Gln 115 120 125Thr Ala Ser Lys Ile Arg Thr Leu Ala Ala Thr Val Leu Phe His Ile 130 135 140Pro Leu Val Arg Glu Ile Ala Leu Trp Thr Gly Cys Val Asp Ala Ser145 150 155 160Arg Ala Val Ala Val Glu Arg Leu Lys Glu Glu Gly Gly Ser Leu Leu 165 170 175Val Ile Pro Gly Gly Gln Ala Glu Gln Met Tyr Thr Gln Tyr Gly Arg 180 185 190Glu Arg Val Tyr Leu Lys Arg Arg Lys Gly Phe Leu Lys Leu Cys Leu 195 200 205Lys Tyr Glu Ile Pro Val Val Pro Ala Tyr Val Phe Gly Val Ser Asp 210 215 220Tyr Tyr Phe Thr Ser Ala Lys Leu Phe Gly Leu Arg Met Trp Leu Val225 230 235 240Gln Asn Leu Gly Ile Ala Leu Pro Leu Cys Trp Gly Arg Tyr Gly Leu 245 250 255Pro Ile Cys Pro Arg Pro Val Asp Thr Thr Leu Val Phe Asp Lys Pro 260 265 270Leu Tyr Leu Ser Cys Gln Asn Pro Ser Asn Pro Ser Glu Asp Glu Val 275 280 285Asp Lys Ala His Leu Gln Phe Cys Gln Ala Leu Glu Lys Leu Phe Asp 290 295 300Thr His Lys Glu Arg Leu Gly Tyr Gly Asp Arg Lys Leu Glu Ile Ile305 310 315 32062963DNAPhaeodactylum tricornutum 62atgaccagat cgaagtttat aggaagtgct ggagctattg gcttattttg tttgatgatc 60ataccgaatg tgggaattct gatcgcaaca tttctttatc ccaaagtact tgggctctac 120tttctgattc cgtactacgc atacaacttg tccattggca aacacgaagc tcgagacggc 180aacggctgga attggttcag cgagaatttc tttgtcttta acattgtgag gggatatcta 240aatcttaaga ttgaagctga ctccgagctc aaggaagccg aagcgaaaga aggcgcccaa 300tttgtgttcg ccgttagccc tcacggaacg aacgcagact atcgagtttt tattgacggt 360atgctacatg aggcactccc acagactgca agcaagatca gaacactagc ggcgacagta 420ctgttccaca ttcccttggt tcgtgaaatc gcactttgga caggatgtgt cgatgccagc 480cgcgcagttg ctgtcgagag attaaaagaa gaaggtggtt cactgcttgt gattcccggt 540ggccaagcag aacaaatgta cacccaatat ggacgtgaaa gagtatatct gaaacggcgc 600aaaggatttt tgaagctttg cttgaagtac gagattccgg tcgtcccagc ttatgttttt 660ggcgtatctg actattactt cacgtccgca aagctctttg gtctgcgaat gtggctcgtt 720cagaatcttg gcattgctct tccactgtgc tggggaagat atggtctacc aatctgtcct 780agaccagtcg ataccaccct tgtctttgac aaacctttat acctatcctg ccagaatccg 840tcgaatccct cggaagacga ggttgacaag gctcatctgc aattttgcca agccctcgag 900aagctgtttg atacacacaa agagaggctt gggtacggcg atcgaaagct ggaaataatt 960tag 96363663PRTRhodotorula graminis 63Met Ser Thr Ala Asp Leu Pro Pro Gly Pro Ala Gln Leu Leu Glu Asp1 5 10 15Ala Leu Arg Gln Pro Asp Gly Pro Pro Leu Leu Ser Thr Ser Ala Ala 20 25 30Asp Pro Ser Ser Pro Leu Gln Leu Asp His Asp His Arg Pro Gly Met 35 40 45Ala Ala Asp Ala Ala Ser Ser Ala Ser Asp Ser Ser Ile Ser Thr Val 50 55 60Ser Ser Val Leu Arg Gly Gln Gln Ala Thr Thr Thr Val Thr Thr Asn65 70 75 80Arg Gly Glu Gly Gly Arg Glu Thr Thr Glu Thr Phe Thr His Val Gly 85 90 95Ala Ala Asn Val Asp Ala Glu Tyr Ser Ser Ser Thr Gly His Ile Thr 100 105 110Leu Arg Pro Val Val Ala Lys Gly Gly Asp Pro Arg Arg Ile Arg Leu 115 120 125Val Arg Ser Arg Arg Thr His Phe Glu Pro Arg Ile Ser His Phe Asp 130 135 140Arg His Asn Lys Thr Ser Ala Glu Asp Thr Phe Arg Gly Phe Phe Ser145 150 155 160Leu Phe Trp Ile Val Ile Ala Val Gly Gly Thr Arg Thr Ile Tyr Asn 165 170 175Arg Val Ala Glu Thr Gly Gly Leu Leu Gly Gly Trp Gln Phe Ala Ala 180 185 190Leu Ile Ser Glu Asp Ala Trp Ala Leu Ala Leu Ser Asp Ala Val Leu 195 200 205Val Gly Ser Thr Ile Leu Cys Val Pro Phe Val Lys Leu Ile Val Asn 210 215 220Gly Trp Val Arg Tyr Tyr Tyr Thr Gly Leu Val Leu Gln His Leu Ala225 230 235 240Gln Thr Leu Tyr Leu Gly Ile Ala Val Arg Trp Thr Phe His Arg His 245 250 255Trp Pro Trp Val Gln Ser Gly Phe Met Thr Leu His Ala Leu Ser Met 260 265 270Leu Met Lys Ile His Ser Tyr Cys Ser Leu Asn Gly Glu Leu Ser Glu 275 280 285Arg Val Arg Gln Leu Glu Lys Asp Glu Arg Lys Leu His Glu Ala Val 290 295 300Glu Glu Leu Gly Gly Gln Asp Ala Leu Glu Arg Glu Gly Arg Val Ala305 310 315 320Trp Glu Lys Ala Cys Ala Glu Ala Ala Glu Gln Lys Ala Ala Glu Glu 325 330 335Ala Ala Gly Gly Arg Gly Lys Ala Ser Ala Ser Ser Leu Ala Pro Pro 340 345 350Pro Ala Thr Gly Pro Gln Pro Ser Ser Asp Glu Glu Ala Val Ser Thr 355 360 365Thr Leu Arg Gln Arg Pro Ser Ala Ala Arg Arg Arg Ser Leu Ser Pro 370 375 380Ser Ala Ala Arg Thr His Val Thr Pro Pro Ser Arg Lys Ala Glu Pro385 390 395 400His Asp Val Glu Thr Leu Thr Trp Ser Pro Asn Glu Arg Val Ser His 405 410 415Leu Ala Ile Ala Ile Cys Glu Ala Arg Glu Ala Leu Ser Ser Ser Gly 420 425 430Ala Ala Lys Val Ser Phe Pro Asp Asn Val Thr Val Leu Asn Phe Val 435 440 445Asp Tyr Leu Leu Val Pro Thr Leu Val Tyr Glu Leu Glu Tyr Pro Arg 450 455 460Thr Asp Ser Ile Arg Pro Leu Tyr Ile Leu Glu Lys Thr Leu Ala Thr465 470 475 480Phe Gly Thr Phe Ser Val Leu Leu Leu Ile Val Glu His Phe Ile Tyr 485 490 495Pro Val Met Pro Gly Pro Asp Ser Ser Phe Ile Ser Ser Leu Leu Asp 500 505 510Leu Ala Leu Pro Phe Thr Ile Cys Tyr Leu Leu Ile Phe Tyr Ile Ile 515 520 525Phe Glu Cys Ile Cys Asn Ala Phe Ala Glu Ile Thr Arg Phe Ser Asp 530 535 540Arg Ala Phe Tyr Ser Asp Trp Trp Asn Ser Ile Ser Phe Asp Glu Phe545 550 555 560Ser Arg Lys Trp Asn Arg Pro Val His Thr Phe Leu Leu Arg His Val 565 570 575Tyr Ala Thr Thr Ile Ser Thr Tyr Lys Leu Ser Lys Phe Ser Ala Ala 580 585 590Phe Val Thr Phe Leu Leu Ser Ala Leu Val His Glu Leu Val Met Val 595 600 605Val Val Thr His Lys Ile Arg Met Tyr Leu Phe Met Ala Gln Leu Pro 610 615 620Leu Ile Met Leu Gly Arg Ala Ser Ile Phe Lys Arg His Pro Ala Leu625 630 635 640Gly Asn Leu Phe Phe Trp Phe Gly Leu Leu Ser Gly Phe Pro Leu Leu 645 650 655Ala Val Ala Tyr Leu Lys Phe 660641992DNARhodotorula graminis 64atgagcaccg ccgatcttcc accaggtcct gcccagctgc tcgaagacgc cctgcgccag 60ccagacggcc cccctctcct gtcgacctcc gccgccgatc cctcctcccc acttcaactc 120gaccacgacc accgccccgg catggctgca gacgccgcca gctcagcttc agacagctct 180atcagcacgg tgtccagtgt cctgcgcggt cagcaagcca cgacaacggt gacgaccaac 240aggggagaag gcgggcgaga aacgaccgag accttcaccc acgtcggcgc cgccaatgtc 300gacgccgagt actcgtcctc gaccggccac atcacgctcc gacccgtcgt ggcaaagggc 360ggtgaccctc gccggatccg cctcgtccgc tcgcgccgca cccacttcga gccgcgcatc 420tcgcacttcg accgccacaa caagacgtcg gccgaggaca cgttccgcgg cttcttctcg 480ctcttctgga tcgtcatcgc cgtcggcggc acgaggacca tctacaaccg cgtcgccgag 540acgggcggtc tcctcggcgg

gtggcagttt gcggcgctca tctccgagga cgcatgggct 600ctggcgctga gcgatgcggt cctcgtcggg tcgacgatac tctgcgtccc gttcgtcaag 660ctcatcgtca acggctgggt ccggtactac tacacgggcc tcgtcctcca gcacctcgcc 720cagacgctct acctcggcat cgccgtccga tggacgttcc accgtcactg gccctgggtc 780cagagcggct tcatgacgct gcacgccctg agcatgctca tgaagatcca ctcgtactgc 840tcgctcaacg gcgagctgtc cgagcgcgtg cggcagctcg agaaggacga gcgcaagctg 900cacgaggcgg tcgaggagct tggcggccag gacgcgctcg agcgcgaggg gcgcgtggcg 960tgggagaagg cgtgcgccga ggcggccgag cagaaggcgg ccgaggaggc ggcaggcggt 1020cgcggcaaag cttcggcgtc ctcgctcgcc ccgccgccgg cgacagggcc gcagccctcg 1080tccgacgagg aggccgtctc gacgacgctc cgacagcgac cgtcggccgc tcgccgccgc 1140tcgctctcgc cgtcggccgc acggacccac gtcacgccgc cgtcgcgcaa ggccgagccg 1200cacgacgtcg agacgctcac ctggtcgccc aacgagcgcg tgtcgcacct cgccatcgcc 1260atctgcgagg cacgcgaggc cctgtcgtcg agcggcgccg ccaaggtctc gttcccggac 1320aacgtcacgg tcctcaactt tgtcgactac cttctcgtcc cgacgctcgt gtacgagctt 1380gagtacccga ggaccgactc tatccgaccc ttgtacatcc tcgagaagac cctcgccacg 1440ttcggcacat tctcggtcct cctcctcatc gtcgagcact tcatctaccc ggtcatgccc 1500gggcccgaca gctcgttcat ctcgtccctc ctcgacctcg ccctcccatt caccatctgc 1560tacctcctca tcttctacat catcttcgag tgtatctgca acgccttcgc cgagatcacg 1620cgcttctcgg accgggcctt ctacagcgac tggtggaact cgatctcgtt cgacgagttc 1680tcgcgcaagt ggaaccggcc cgtgcacacg ttcctcctgc gccacgtgta cgcgacgacc 1740atctcgacct acaagctcag caagttctcg gccgcctttg tcacgttcct cctgagcgcg 1800ctcgtgcacg agctcgtcat ggtagtcgtg acgcacaaga tccgcatgta tctctttatg 1860gcgcagctcc ccctcatcat gctcggccga gcaagcatct tcaagcgtca ccctgcgctc 1920ggcaacctct tcttctggtt cggcctcttg agcggtttcc ctctgctagc tgtagcgtac 1980ctcaagttct ag 199265597PRTPichia guilliermondii 65Met Ser Lys Glu Asn Leu Leu Lys Ile Ser Gln Tyr Asn Thr Glu Arg1 5 10 15Arg Pro Ser Leu Ala Thr Asp Val Asp Tyr Ser Ser Thr Asp Leu Ser 20 25 30Ser Arg Leu Asp Ser Ala Asn Thr Thr Asn Gly Thr Pro Thr Val Thr 35 40 45Leu His Lys Arg Gln Ser Ser Thr Glu Leu Leu Ser Glu Ser Pro Glu 50 55 60Gln Lys Arg Phe Leu Lys Thr Ile Asp Thr Leu Asn Arg Thr Thr Asn65 70 75 80Ser Arg Leu Arg Gln Arg Leu Asn Arg Glu Gly Asp Lys His Lys Lys 85 90 95Glu His Lys Glu His Glu Lys His Lys Asp Asp His Ser Lys Tyr Lys 100 105 110Ser Arg Phe Gly Asp Ile His Phe Tyr Ser Asn Met Thr Thr Ile Phe 115 120 125Asp Ala Asp Tyr Phe Lys Glu Ser Gln Phe Phe Gly Val Tyr Ile Leu 130 135 140Phe Trp Leu Gly Thr Ala Phe Leu Ile Leu Asn Asn Leu Val His Thr145 150 155 160Phe Leu Glu Asn Gly Asp Asn Leu Leu Asp Gly Pro Val Val Arg Thr 165 170 175Phe Lys Lys Asp Leu Leu Lys Ile Ala Leu Thr Asp Leu Gly Met Tyr 180 185 190Leu Thr Met Tyr Val Ser Val Phe Ile Gln Leu Gly Ile Arg Lys Gly 195 200 205Trp Tyr Ser Trp Ser Ser Thr Gly Ala Thr Leu Gln Asn Ile Tyr Ser 210 215 220Phe Val Tyr Phe Phe Ala Trp Ser Tyr Phe Ala Ser Pro Lys Tyr Met225 230 235 240Asp Tyr Pro Trp Ile Gly Lys Val Phe Leu Ala Leu His Ser Leu Val 245 250 255Phe Leu Met Lys Met His Ser Tyr Ala Thr Tyr Asn Gly Tyr Leu Trp 260 265 270Asn Ile Phe Asn Glu Leu Gln Val Ser Arg Lys Tyr Leu Lys Ile Leu 275 280 285Asp Glu Thr Asp Glu Ser Met Ile Glu Gly Lys Ser Val Ser Asp Leu 290 295 300Arg Lys Ala Leu Val Asp Ser Ile Gly Phe Cys Ser Tyr Glu Leu Glu305 310 315 320Tyr Gln Ser Lys Ser Thr Ser Val Asn Thr Asp Val Glu Ile Thr Gly 325 330 335Asp Lys Asn Lys Leu Asn Thr Thr Lys Ser Thr Ser Ser Leu Asp Asp 340 345 350Asp Tyr Val Ser Phe Pro Asn Asn Ile Thr Phe Phe Asp Phe Phe Arg 355 360 365Tyr Ser Met Phe Pro Thr Val Val Tyr Ser Leu Lys Phe Pro Arg Thr 370 375 380Lys Arg Ile Arg Trp Gly Tyr Val Met Glu Lys Ser Phe Ala Val Phe385 390 395 400Gly Ile Ile Phe Leu Met Ile Thr Val Ala Gln Asn Trp Met Tyr Pro 405 410 415Ile Val Val Arg Ala Gln Glu Ala Ser Lys Leu Pro Met Ser Arg Glu 420 425 430Lys Val Leu Gln Tyr Cys Leu Val Leu Leu Asp Met Ile Pro Pro Phe 435 440 445Leu Met Glu Tyr Leu Phe Thr Phe Phe Leu Ile Trp Asp Val Ile Leu 450 455 460Asn Ala Ile Ala Glu Leu Ser Arg Phe Ala Asp Arg Asp Phe Tyr Gly465 470 475 480Pro Trp Trp Ser Cys Thr Asp Trp Ser Glu Phe Ala Arg Ile Trp Asn 485 490 495Arg Pro Val His Lys Phe Leu Leu Arg His Val Tyr Gln Ser Thr Ile 500 505 510Ser Thr Phe Lys Leu Asn Lys Asn Gln Ala Ser Leu Val Thr Phe Ile 515 520 525Ile Leu Ser Phe Val His Glu Phe Val Met Phe Val Ile Phe Arg Lys 530 535 540Val Arg Phe Tyr Met Leu Ala Leu Gln Met Ser Gln Leu Pro Leu Ile545 550 555 560Met Ile Ser Arg Thr Lys Phe Met Arg Asp Lys Lys Val Leu Gly Asn 565 570 575Val Ile Cys Trp Val Gly Phe Ile Ser Gly Pro Ser Met Ile Cys Thr 580 585 590Leu Tyr Leu Val Phe 595661794DNAPichia guilliermondii 66atgtccaagg aaaacttact taagatcagc cagtataata ctgagagaag accgtcgttg 60gccacagacg ttgactactc ttccaccgat ttatccagtc gtctggattc ggccaacacg 120acaaacggaa caccgaccgt aactcttcac aagaggcaat cgtctacaga gctcttgtct 180gagtcacctg aacagaaaag gttcttgaaa acgatagaca ctttgaatcg aaccacaaat 240tctagattac gccagaggtt aaaccgtgag ggcgataagc ataaaaagga acacaaagaa 300catgaaaaac ataaagatga ccattctaaa tacaagtctc ggtttggaga tatccatttc 360tactcaaaca tgacaaccat cttcgatgct gattacttta aggaatcgca gttctttgga 420gtttacattc tcttttggct cggaacggca ttcttaattc tcaacaactt ggtccataca 480tttttggaga acggagacaa tcttctcgat ggaccagttg tcagaacgtt taaaaaggac 540ttacttaaaa ttgctcttac agacttggga atgtacttga cgatgtacgt ctctgtcttt 600attcaattgg gcatccgcaa aggatggtat agctggagct caacaggagc caccttgcaa 660aacatatact cattcgtgta cttctttgcc tggagttact ttgcgtcgcc aaagtacatg 720gactaccctt ggattggaaa ggtgtttctt gcacttcaca gcttggtgtt tctcatgaaa 780atgcattctt atgccacata caacggctat ctttggaaca tcttcaacga gcttcaagtg 840tcacgaaagt acttgaagat attggacgag accgatgaat ccatgattga gggtaagagt 900gtttccgatt tgcgaaaggc tttggtagac agcattggtt tctgctcata cgagttggag 960taccagtcca aatcaacgag cgtgaacacg gatgtcgaaa tcaccggcga caagaacaaa 1020ttgaacacaa ccaagtctac cagttcactc gatgacgact atgtgagttt ccccaataac 1080attacgtttt tcgatttttt caggtattca atgtttccaa cagtggtgta ttctctcaag 1140ttcccacgta caaagcgtat tagatggggt tacgtcatgg aaaagtcatt tgcagtgttt 1200ggcatcatct tcttgatgat caccgtcgct caaaactgga tgtatcctat cgttgtacga 1260gcacaagagg ctagcaaact cccaatgtca agagaaaagg tattgcagta ctgtttggtt 1320ttactagaca tgattccacc atttctcatg gaatatcttt tcaccttttt cttgatttgg 1380gacgtgatcc taaatgcgat agccgaattg agtaggtttg cagatcggga cttttatggt 1440ccttggtggt cttgtaccga ttggtcggaa tttgcaagaa tttggaatcg tcctgttcac 1500aaatttttgc ttcgtcatgt gtaccagtca actatcagta ctttcaaact caataaaaac 1560caagcgtcgt tggtgacgtt tatcattctg agttttgttc atgagtttgt catgtttgtc 1620atttttagaa aggtgagatt ctacatgttg gcgctccaga tgtctcagct tccattgata 1680atgattagtc gaacaaaatt catgagagac aaaaaagtgt tgggaaatgt tatctgctgg 1740gtaggattca tttctggacc atcgatgatc tgtactttgt atttagtatt ttaa 179467515PRTArxula adeninivorans 67Met Ala Thr Ala Thr Ala Ile Ala Thr Val Thr Glu Gly Leu Gly Leu1 5 10 15Asp Lys Val Leu Ser Lys Glu Gln Pro Gly Leu Ser Lys Leu Ala Pro 20 25 30Arg Ala Asn Thr Asn Val Gln Pro Thr Gln Leu Gln Ser Pro Ser Pro 35 40 45Pro Gln Ser Arg Ser Ser Ser Pro Ile Ser Ala Ser Ser Ser Ser Glu 50 55 60Ser Leu Glu Leu Lys Val Pro Lys Ala Lys Ser Pro Ser Ser Ser Lys65 70 75 80His Lys Pro His Tyr Arg Pro Val His Val Arg Ser Thr Ala Ser Ile 85 90 95Leu Ser Arg Asp Pro Ala Ala Arg Thr Glu Pro Pro Ser Tyr Ser Gly 100 105 110Phe Arg Asn Leu Ala Met Ile Ala Leu Ala Val Ser Asn Met Arg Leu 115 120 125Leu Leu Glu Asp Tyr Gln Asn Tyr Gly Val Phe His Thr Leu Asn Ile 130 135 140Met Gly Leu Ser Ala His Asp Val Arg Leu Thr Leu Ala Leu Thr Ala145 150 155 160Ser Val Pro Phe His Leu Phe Val Ala Leu Ala Ile Glu Arg Ile Ala 165 170 175Val Leu Thr Met Pro Ser Lys Ser Thr Ala His Asn His Arg Ser Lys 180 185 190His Leu Trp Gly Leu Phe Ala Val Leu His Ala Leu Asn Ala Ala Ala 195 200 205Val Leu Ala Ile Ser Ser Tyr Thr Val Tyr Ser Arg Met Trp Ser Pro 210 215 220Ala Val Gly Thr Leu Cys Glu Cys His Ala Ile Val Val Cys Phe Lys225 230 235 240Val Ala Ser Tyr Ala Leu Thr Asn Arg Asp Leu Arg Asp Ala Ala Ile 245 250 255Asp Gly Leu Glu Thr Thr Asp Pro Leu Leu Ser Lys Leu Pro Tyr Pro 260 265 270Ser Asn Leu Thr Leu Ser Asn Leu Val Tyr Phe Trp Trp Ala Pro Thr 275 280 285Leu Val Tyr Gln Pro Ile Tyr Pro Arg Trp Pro Leu His Arg Arg Trp 290 295 300Gly Phe Ile Phe Ser Arg Leu Leu Glu Ile Met Gly Ser Met Val Leu305 310 315 320Ile Trp Phe Ile Ser Thr Gln Tyr Ala Asn Pro Ile Leu Glu Ser Ser 325 330 335Leu Gly His Phe Glu Gln Phe Asn Val Val Lys Ile Ser Glu Cys Leu 340 345 350Leu Lys Leu Ala Ser Val Ser Met Ala Ile Trp Leu Leu Gly Phe Phe 355 360 365Cys Leu Phe Gln Ser Phe Leu Asn Leu Leu Ala Glu Leu Val Arg Phe 370 375 380Gly Asp Arg Glu Phe Tyr Gln Asp Trp Trp Asn Ala Gly Ser Val Gly385 390 395 400Thr Tyr Trp Arg Lys Trp Asn Arg Pro Val His Asn Tyr Phe Leu Arg 405 410 415His Phe Tyr Ile Pro Met Leu Lys Arg Gly Tyr Ser Gln Arg Thr Ala 420 425 430Ser Val Ile Val Phe Phe Leu Ser Ala Ile Leu His Glu Val Ala Val 435 440 445Gly Val Pro Thr Gln Ser Leu Ile Gly Val Ala Phe Val Gly Met Gly 450 455 460Ala Gln Ile Pro Leu Val Leu Ala Thr Ser Pro Leu Glu Lys Met Gly465 470 475 480Glu Thr Gly Ala Thr Ile Gly Asn Cys Ile Phe Trp Leu Ser Phe Phe 485 490 495Leu Gly Gln Pro Met Gly Val Leu Leu Tyr Tyr Phe Ala Trp Asn Met 500 505 510Lys His Gln 515681548DNAArxula adeninivorans 68atggccaccg ctactgctat cgctacggtc acggagggcc tgggactaga taaggtgcta 60tccaaggagc agccaggctt gtcgaagcta gctcctcgag cgaatacaaa tgtacaaccg 120acccagttgc agtccccgtc tccaccacaa tctcgatctt cgtctccaat ttcggcctcc 180tcatcatcag agtccctgga gctcaaggtg cccaaggcca aatcgccatc atcttccaaa 240cacaaaccac actaccgccc cgtgcatgtg cggtcaacag catccatcct gtccagagac 300ccggccgcca gaaccgagcc tccctcttac tctgggttca ggaacctagc catgattgca 360ttggcggttt ctaatatgcg cctccttctc gaggactatc aaaactatgg cgtgttccac 420actctcaaca ttatgggctt gagcgcacac gacgttcgcc tcacactggc attgacagct 480tcggttccgt tccatctgtt tgtggccctg gccattgagc gcatcgcagt cctcactatg 540ccctccaaat ctacagcaca caaccaccgc tcaaagcatc tctggggctt gtttgcagtt 600ctgcatgctc tcaacgccgc tgctgtgcta gcaatcagct catacaccgt atacagtcgc 660atgtggagtc ctgctgtggg aacattgtgc gaatgccacg caatcgtggt atgctttaag 720gtggcatcgt atgcgcttac caaccgagac ttacgagatg ctgccattga tgggctagag 780acaactgacc ctctgttgtc caagttgccc tacccatcca accttacctt gtcaaatctc 840gtgtatttct ggtgggcccc aaccctagtg tatcagccaa tttaccctcg atggcccctg 900catcgacgat ggggcttcat cttttctcgc ctgctcgaga ttatgggatc tatggtacta 960atctggttca tttccaccca atacgccaac cccattttgg aatcatcctt ggggcacttt 1020gaacagttta acgtggttaa aatctcagaa tgtctcctca aattagcatc ggtctccatg 1080gccatctggc ttttgggttt cttttgtctc tttcaatcgt ttttgaactt gctggcagaa 1140ttggttcgtt ttggcgaccg cgagttctac caagactggt ggaacgccgg ctcagtaggt 1200acctactggc gcaaatggaa ccgaccagtg cacaactatt tcttgcgcca tttctacatc 1260ccaatgctca agcgaggtta ttcacagcgc actgcctcgg tcattgtatt ctttttatct 1320gccattctcc atgaagttgc tgttggcgtg cctactcagt ccttgattgg agttgcgttt 1380gtaggcatgg gtgcccagat tcctctagtg ctggccacta gtcctttgga aaagatgggc 1440gaaactggcg caactattgg caactgcatc ttttggctct ctttcttcct gggccagcca 1500atgggggtac tgctttacta ctttgcgtgg aatatgaagc accagtag 154869564PRTPhaeodactylum tricornutum 69Met Asp Glu Thr Glu Ile Thr Pro Leu Leu Arg Phe Ser Thr Pro Ser1 5 10 15Arg Ala Glu His Ser Ser Trp Ile Lys Leu Ala Ser Glu Ser Cys Ala 20 25 30Tyr Ser Glu Thr Asp Glu Phe Leu Ala Asp Glu Ala Ala Arg Ala Thr 35 40 45Gln Arg Ala Leu Gln His Gln Glu Ala Leu Gln Met Ala Gln Ala Met 50 55 60Pro Gly Ala Lys Pro Gly Thr Leu Pro Pro Leu Tyr Phe Ala Pro Thr65 70 75 80Ile Lys Arg Ser Arg Ser Phe Ala Lys Leu Gln Glu His His Gly Asp 85 90 95Gly Met Pro Arg Val Asn Met Arg Arg Thr Lys Ser Arg Asp Phe Asn 100 105 110Ala Asp Lys Leu Asp Ala Arg Ser Thr Lys Gly Tyr Pro Pro Ser Lys 115 120 125Pro Met His Arg Ala Ala Glu Pro Ser Tyr Leu Ser Ala Asp Ala Pro 130 135 140Ile Gln Asn Tyr Arg Gly Phe Leu Asn Leu Gly Val Ile Ile Leu Ile145 150 155 160Val Ser Asn Phe Arg Leu Ile Leu Gly Thr Ile Arg Ser Asn Gly Phe 165 170 175Val Leu Thr Thr Ala Val Lys His Tyr Lys Asn Leu Asn His Leu Lys 180 185 190Glu Asp Pro Trp Gln Glu Phe Pro Phe Val Ser Gly Phe Leu Leu Gln 195 200 205Leu Val Phe Val Ser Ile Ala Phe Gly Ile Glu Trp Met Leu Cys Arg 210 215 220Lys Tyr Phe Asn Glu Asn Phe Gly Met Ile Leu His His Phe Asn Ala225 230 235 240His Ser Ala Leu Leu Ile Pro Leu Gly Ile Val Trp Asn Leu Ile Asp 245 250 255Arg Pro Ala Val Gly Ala Ile Leu Leu Leu His Ala Thr Ile Thr Trp 260 265 270Met Lys Leu Ile Ser Tyr Met Leu Ala Asn Glu Asp Tyr Arg Leu Ser 275 280 285Ser Arg Arg Val Gly Gly Asn Pro His Leu Ala Thr Leu Ala Leu Val 290 295 300Glu Asn Leu Asp Ser Asp Glu Ala Asn Ile Asn Tyr Pro Gln Asn Val305 310 315 320Thr Leu Arg Asn Ile Phe Tyr Phe Trp Cys Ala Pro Thr Leu Thr Tyr 325 330 335Gln Ile Ala Phe Pro Lys Ser Pro Arg Val Arg Tyr Trp Lys Ile Ala 340 345 350Asp Ile Leu Met Arg Met Thr Val Ser Ile Ala Leu Phe Thr Phe Leu 355 360 365Leu Ala Gln Ile Val Gln Pro Ala Leu Glu Glu Leu Val Ser Asp Leu 370 375 380Asp Glu Thr Asn Gly Ser Tyr Thr Ala Ala Ile Phe Ala Glu Tyr Trp385 390 395 400Leu Lys Leu Ser Ile Ala Asn Thr Tyr Leu Trp Leu Leu Met Phe Tyr 405 410 415Thr Tyr Phe His Leu Tyr Leu Asn Leu Phe Ala Glu Leu Leu Arg Phe 420 425 430Gly Asp Arg Val Phe Tyr Lys Asp Trp Trp Asn Ser Ser Glu Val Ser 435 440 445Ala Tyr Trp Arg Leu Trp Asn Met Pro Val His Tyr Trp Leu Ile Arg 450 455 460His Val Tyr Phe Pro Cys Val Arg Leu Lys Met Pro Lys Val Ala Ala465 470 475 480Thr Phe Val Val Phe Phe Leu Ser Ala Val Met His Glu Val Leu Val 485 490 495Ser Val Pro Phe His Ile Ile Arg Pro Trp Ser Phe Ile Gly Met Met 500 505 510Met Gln Ile Pro Leu Val Ala Phe Thr Lys Tyr Leu Tyr Arg Lys Phe 515 520 525Pro Gly Gly Ser Ile Gly Asn Val Leu Phe Trp Met Thr Phe Cys Val 530 535

540Ile Gly Gln Pro Met Ala Ile Leu Leu Tyr Tyr His Asp Ile Met Asn545 550 555 560Arg Lys Gly Asn701695DNAPhaeodactylum tricornutum 70atggatgaga ccgaaattac acctttgttg cgtttttcga caccttcccg agccgaacac 60tcgtcctgga taaagcttgc ctcggaatcc tgtgcttaca gcgaaacgga cgagtttctc 120gctgacgagg ccgctcgcgc aacccagcgt gctttgcaac atcaagaagc gctgcaaatg 180gcccaagcca tgcctggggc aaagccagga acgctgccgc cactctactt cgcgcctacc 240ataaagcgtt cgcgttcctt tgctaagcta caagaacatc atggagatgg gatgcctcgg 300gtaaatatgc gtcggaccaa atcgcgagat tttaacgcgg ataagttgga tgcgcgaagt 360accaagggct atcccccttc caagccgatg catcgtgcgg cagagccctc atacctcagc 420gcggatgctc ccattcaaaa ctaccgagga tttctgaatt taggcgttat tattttgatt 480gtttctaact ttcggctgat cttgggcaca atccgtagca acggatttgt cttgacgact 540gcagtgaagc actacaagaa cctaaatcac ctcaaggaag atccctggca ggaatttcct 600tttgtatcag gatttcttct ccagctcgtc tttgtttcga ttgcgtttgg gatcgaatgg 660atgttgtgcc ggaaatactt caacgaaaac ttcggcatga tccttcatca cttcaatgcc 720cactcagcct tgctgatacc tttaggtatt gtttggaatc tcatcgatag acctgcggtt 780ggtgcaattt tgcttttaca cgctacgata acatggatga aactcatttc ttacatgttg 840gcgaacgaag attaccggct atcatcgcgt cgcgttgggg gcaacccaca cctagctacg 900ctcgcattag tcgaaaatct agattcagat gaggcgaaca ttaactaccc ccaaaatgtt 960actctccgca acatttttta tttttggtgt gctccgacgt tgacttacca gattgccttc 1020ccgaagtccc cgcgagttcg ctattggaaa atcgcggata tcctgatgcg catgacggtg 1080tccatcgcac tattcacctt tttgctggca caaattgttc agcctgcatt ggaagagcta 1140gtgagcgacc tggacgagac caatggatcc tacaccgcag caatatttgc cgagtactgg 1200ctgaaacttt cgattgctaa cacatattta tggcttctta tgttctatac atatttccat 1260ttgtatctga acctctttgc tgagcttctg cgatttggag atcgtgtgtt ctacaaagat 1320tggtggaatt cgtcggaagt atctgcatat tggaggcttt ggaatatgcc tgttcactat 1380tggttgatcc gacatgtgta tttcccctgc gtgcgactga agatgccgaa ggtcgctgca 1440acctttgtcg tttttttcct ctccgccgtt atgcacgagg tgcttgtcag cgtacccttt 1500catattattc gtccgtggtc ttttatcggg atgatgatgc agattccttt ggttgcgttc 1560acaaagtatc tctatcgcaa attcccgggc ggctcgattg gtaatgtcct gttctggatg 1620acattttgcg tcattggcca gccaatggcg attctcttgt actatcatga tattatgaat 1680cgaaaaggaa attga 169571503PRTMetarhizium acridum 71Met Ser Thr Ala Thr Thr Thr Ser Val Ser Pro Ala Asn Gly Thr Val1 5 10 15Ser Lys Arg Asn Ala Thr Lys Arg Arg Asn Gly Asn Ala Ser Pro Gly 20 25 30Pro Val Glu Glu Glu Ser Glu Asp Ala Ala Ala Ala Glu Lys Pro Arg 35 40 45Ala Ser Val Ala Gln Lys Asn Tyr Arg His Val Ala Ala Val His Ser 50 55 60Lys Ser Arg Pro Ser Cys Leu Ser His Asp Ser Asp Ala Thr Pro Ser65 70 75 80Phe Ile Gly Phe Arg Asn Leu Met Val Ile Val Leu Asp Val Leu Ile 85 90 95Gly Gly Leu Leu Tyr Phe Leu Ile Pro Cys His Leu Leu Val Ala Tyr 100 105 110Leu Ile Glu Leu Ala Ala Ala Lys Gln Ala Arg Gly Ser Arg Lys Arg 115 120 125Leu Lys Pro Gly Ser Thr Val Pro Ser Glu Gln Asp Asn Ser Lys Phe 130 135 140His Ser Thr Trp Val Leu Val Ala Trp Ala His Gly Ile Asn Met Thr145 150 155 160Leu Ala Leu Ala Leu Thr Thr Phe Met Val Tyr Phe Tyr Ile His His 165 170 175Pro Leu Val Gly Thr Leu Thr Glu Met His Ala Val Ile Val Ser Leu 180 185 190Lys Thr Ala Ser Tyr Ala Phe Thr Asn Arg Asp Leu Arg His Ala Tyr 195 200 205Leu His Pro Val Lys Gly Glu Phe Ile Pro Glu Leu Tyr Ser Lys Cys 210 215 220Pro Tyr Pro Asn Asn Ile Thr Phe Gly Asn Leu Ala Tyr Phe Trp Trp225 230 235 240Ala Pro Thr Leu Val Tyr Gln Pro Val Tyr Pro Arg Thr Asp Lys Ile 245 250 255Arg Trp Val Phe Val Phe Lys Arg Leu Gly Glu Val Cys Cys Leu Ser 260 265 270Ala Phe Ile Trp Phe Ala Ser Phe Gln Tyr Ala Ala Pro Val Leu Gln 275 280 285Asn Ser Leu Asp Lys Ile Ala Ser Leu Asp Leu Leu Met Ile Leu Glu 290 295 300Arg Leu Leu Lys Leu Ser Thr Ile Ser Leu Val Ile Trp Leu Ala Gly305 310 315 320Phe Phe Ala Leu Phe Gln Ser Phe Leu Asn Ala Leu Ala Glu Val Leu 325 330 335Arg Phe Gly Asp Arg Ser Phe Tyr Asp Asp Trp Trp Asn Ser Glu Ser 340 345 350Leu Gly Ala Tyr Trp Arg Thr Trp Asn Arg Pro Val Tyr Thr Tyr Phe 355 360 365Lys Arg His Val Tyr Val Pro Met Ile Gly Arg Gly Trp Ser Pro Trp 370 375 380Ala Ala Ser Cys Ala Val Phe Phe Val Ser Ala Val Leu His Glu Val385 390 395 400Leu Val Gly Val Pro Thr His Asn Ile Ile Gly Thr Leu Ser Ser Val 405 410 415Leu Ser Ile Val Leu Thr Leu Val Pro Asn Leu Tyr Ser Gly Val Ala 420 425 430Phe Leu Gly Met Phe Leu Gln Leu Pro Leu Ile Ala Ile Thr Ala Pro 435 440 445Leu Glu Lys Met Lys Trp Gly His Thr Gly Arg Val Met Gly Asn Val 450 455 460Ile Phe Trp Val Ser Phe Thr Ile Phe Gly Gln Pro Phe Ala Ala Leu465 470 475 480Met Tyr Phe Tyr Ala Trp Gln Ala Lys Tyr Gly Ser Val Ser Lys Glu 485 490 495Pro Ile Leu Ala Leu Gln Thr 500721512DNAMetarhizium acridum 72atgagcacgg ccaccaccac cagtgtcagc ccagcgaatg gcaccgtgag caagagaaat 60gccaccaagc gtcgcaacgg caatgcatct cccggcccgg tggaagaaga atccgaagac 120gcagccgcag ccgagaagcc cagagcctct gtcgcccaga agaactatcg ccacgtagca 180gcagtgcatt ccaagagccg cccgtcgtgc ctaagccacg actccgatgc cacgccaagc 240tttatcgggt ttcgaaatct catggtcatt gttttggatg tcctcatcgg cggacttctc 300tactttctca ttccctgcca tctgttggtt gcctacttga tcgaattggc cgccgcaaaa 360caggctcgag gatcccgaaa gcgcctcaaa ccaggctcta ctgtaccgtc ggaacaagac 420aattccaagt tccattcaac atgggttctg gtggcctggg ctcatggtat caatatgacg 480cttgctttag ccctcacaac ctttatggtt tacttttaca tccaccaccc gctcgttggg 540accctgaccg agatgcatgc cgtcattgtg tcgttgaaga cagcctcgta cgcattcacc 600aaccgagatc ttcgccacgc ttacctgcac cccgttaaag gagagtttat tcctgaactc 660tactcgaaat gcccgtaccc gaataacatc acctttggca acctcgccta cttctggtgg 720gcgccgacgc tggtctatca gcccgtatac ccgcgcaccg acaagatcag atgggtcttt 780gtttttaaga ggctgggcga agtatgctgt ttgagcgcat tcatctggtt cgccagcttc 840caatacgccg cgccggttct gcagaattcg ctcgacaaga ttgcttcgtt ggacttactc 900atgatcctag agcggctgct gaagctgtca accatttctc tggttatttg gctggcagga 960ttctttgccc tattccagtc cttcttaaac gcacttgccg aagtgctgcg gttcggcgac 1020cgatcatttt acgacgactg gtggaacagc gagagtctcg gagcctactg gagaacgtgg 1080aacaggcccg tatatacgta ctttaagcgc catgtgtatg tacccatgat tgggcgtgga 1140tggagcccat gggctgcaag ttgcgccgtc ttttttgtgt ctgccgtgtt acacgaggtt 1200cttgttggtg ttcccaccca caacattatc ggtacgctat cctccgtctt atccatcgtc 1260ttgaccctcg ttcctaacct atattcaggc gttgcttttc taggcatgtt cttgcagctt 1320cctctcatcg ccatcacggc ccctctagag aaaatgaaat gggggcatac cggcagagta 1380atgggaaacg taatcttttg ggtgtccttt accatcttcg gtcagccatt tgcggcattg 1440atgtactttt acgcatggca ggccaagtac ggtagcgtca gtaaagaacc gattcttgcg 1500ttgcagacat ga 151273516PRTOphiocordyceps sinensis 73Met Ala Ala Thr Gly Thr Ser Val Glu Pro Ser Thr Gly Thr Ala Thr1 5 10 15Gln Arg His Ser Gly Lys Asp Gln Thr Gly Val Glu Pro Arg Thr Gly 20 25 30Thr Val Lys Thr Ser Gln Lys Lys Tyr Arg His Val Val Val Val His 35 40 45Ser Gln Val Arg Pro Ser Cys Leu Ser His Asp Ser Asp Ala Ala Pro 50 55 60Ser Phe Ile Gly Phe Arg Asn Leu Met Val Ile Val Leu Val Val Gly65 70 75 80Asn Leu Arg Leu Met Ile Glu Asn Ile Gln Lys Ala Arg Ser Tyr Leu 85 90 95Ser Phe Ile Pro Gly Gln Cys Ala Pro Gly Tyr Gly Val Leu Ile Cys 100 105 110Ile Arg Cys His Ala Tyr Ser Arg Gln Asp Ile Leu Val Gly Gly Leu 115 120 125Leu Tyr Ile Leu Ile Pro Cys His Leu Leu Ala Ala Tyr Leu Ile Glu 130 135 140Leu Ala Ala Ala Gln Gln Ala Leu Gly Ser Arg Lys Arg Leu Lys Asp145 150 155 160Gly Ala Ala Ser Pro Glu Glu Glu Asp Arg Asn Ser Asn Lys Phe His 165 170 175Ala Thr Trp Leu Ile Val Ala Trp Val His Ala Val Asn Ile Thr Leu 180 185 190Ala Leu Val Val Thr Ser Ala Val Val Tyr Phe Tyr Ile His His Pro 195 200 205Leu Ile Gly Thr Leu Thr Glu Met His Ala Ile Ile Val Trp Leu Lys 210 215 220Thr Ala Ser Tyr Ala Phe Thr Asn Arg Asp Leu Arg His Ala Tyr Leu225 230 235 240His Pro Val Glu Gly Glu Leu Val Pro Asp Met Tyr Ala Lys Cys Pro 245 250 255Tyr Pro Gln Asn Ile Thr Phe Gly Asn Leu Val Tyr Phe Trp Trp Ala 260 265 270Pro Thr Leu Val Tyr Gln Pro Val Tyr Pro Arg Thr Asp Lys Ile Arg 275 280 285Trp Leu Phe Val Ala Lys Arg Leu Gly Glu Val Phe Cys Leu Ser Ala 290 295 300Phe Ile Trp Phe Ala Ser Phe Gln Tyr Ala Ala Pro Val Leu Arg Asn305 310 315 320Ser Leu Asp Lys Ile Ala Ser Leu Asp Phe Ala Ser Ile Phe Glu Arg 325 330 335Leu Val Lys Leu Ser Thr Ile Ser Leu Val Ile Trp Leu Ala Gly Phe 340 345 350Phe Ala Leu Phe Gln Ser Phe Leu Asn Ala Leu Ala Glu Val Leu Arg 355 360 365Phe Gly Asp Arg Ala Phe Tyr Asp Asp Trp Trp Asn Ser Glu Ser Leu 370 375 380Gly Ala Tyr Trp Arg Thr Trp Asn Lys Pro Val Tyr Thr Tyr Phe Lys385 390 395 400Arg His Val Tyr Met Pro Met Ile Gly Arg Gly Trp Ser Pro Arg Val 405 410 415Ala Ser Leu Val Val Phe Phe Ile Ser Ala Val Leu His Glu Ile Leu 420 425 430Val Gly Leu Pro Thr His Asn Val Ile Gly Val Ala Phe Leu Gly Met 435 440 445Phe Leu Gln Leu Pro Leu Ile Ala Ile Thr Ala Pro Met Glu Lys Met 450 455 460Arg Leu Gly Lys Gly Gly Lys Leu Val Gly Asn Val Ile Phe Trp Val465 470 475 480Ser Phe Thr Ile Phe Gly Gln Pro Phe Ala Thr Leu Met Tyr Phe Tyr 485 490 495Ala Trp Gln Ala Lys Tyr Gly Ser Val Ser Arg Glu Met Gln Gln Ala 500 505 510Ala Ser Ile Lys 515741551DNAOphiocordyceps sinensis 74atggcggcta cggggaccag cgtcgagccc tcgactggta ccgcgacaca acgccactcc 60ggcaaggatc agactggggt cgagccacgc accggcacgg tcaagacatc ccagaaaaag 120tatcgccatg tcgttgtcgt ccactcccag gtccggccct cgtgcctcag ccacgattca 180gatgccgccc ccagcttcat tggcttccgc aatctcatgg ttattgtcct ggtcgtcggc 240aacttgcgat tgatgattga aaacatccaa aaggctcgtt catacctgtc gttcatacct 300ggccaatgcg cccccggcta cggagtcttg atctgcatcc gctgccacgc ctacagccgc 360caagacattc tcgtcggcgg gctgctgtac atcctcattc cctgccatct cctggccgcc 420tatctcatcg agctcgccgc cgcccagcag gcactggggt cgagaaagcg cctcaaggat 480ggcgccgcca gcccggagga ggaggaccgc aacagcaaca agtttcacgc gacatggctc 540atcgtcgcct gggtccatgc cgtcaacatc accctggccc tggtcgtgac ctcggccgtc 600gtctactttt acatccacca cccactcatc ggcaccctca ccgaaatgca cgccatcatc 660gtctggctca agacggcctc gtacgccttt actaaccgcg acctgcgcca cgcgtacctg 720caccccgtcg agggcgagct cgtcccggac atgtacgcca agtgcccgta tccgcaaaac 780atcacctttg gcaacctcgt ctacttctgg tgggccccga cgctcgtcta ccagcccgtc 840tatccccgga ccgacaagat caggtggctc tttgtcgcca agcggctggg agaggtcttt 900tgcttgagcg ccttcatctg gttcgccagc ttccagtatg ccgcgcccgt cctgcgcaac 960tctctcgaca aaattgcttc gctcgacttt gcctccatct ttgagcggct ggtgaagctg 1020tccaccatct ccctcgtcat ctggctcgcc ggcttcttcg ccctcttcca gtcctttctc 1080aacgccctcg ccgaggtgct tcggttcggc gaccgggctt tctacgatga ctggtggaac 1140agcgagagcc taggcgccta ctggcggacc tggaacaagc ccgtctacac ctacttcaag 1200cgccacgtgt acatgcccat gatcgggcgt ggctggagtc ccagggtggc cagtctggtc 1260gtcttcttca tctcagccgt cctccacgag atccttgtcg ggctacccac tcacaacgtc 1320atcggcgtcg cctttctcgg catgtttctc cagctgcctc tcatcgccat cacggcgccc 1380atggagaaga tgaggctcgg caaaggcggc aagctcgtag gcaacgtcat cttctgggtg 1440tcgtttacca tctttggcca gccctttgcg acattgatgt acttttatgc ttggcaggcc 1500aaatacggga gcgtgagcag ggagatgcag caagcggcaa gcatcaagta a 155175510PRTTrichoderma virens 75Met Ala Pro Pro Ala Glu Ser Ser Thr Thr Thr Ser Val Glu Ala Ser1 5 10 15Thr Gly Ser Val Ser Arg Arg His Ala Ser Gln Ser Glu Ala Asp Leu 20 25 30Thr Ser Val Glu Pro Val Asn Gly Thr Thr Lys Asn Arg Leu Ser Lys 35 40 45Thr Pro Pro Lys Lys Tyr Arg His Val Ala Ala Val His Ser Gln Thr 50 55 60Arg Pro Ser Cys Leu Ser His Asp Ser Pro Ala Ala Pro Ser Phe Leu65 70 75 80Gly Phe Arg Asn Leu Met Val Ile Val Leu Val Val Gly Asn Leu Arg 85 90 95Leu Met Ile Glu Asn Ile Gln Lys Tyr Gly Val Leu Ile Cys Ile Arg 100 105 110Cys His Asp Tyr Arg Arg Gln Asp Val Leu Leu Gly Leu Leu Leu Tyr 115 120 125Phe Leu Ile Pro Cys His Leu Phe Ala Ala Tyr Leu Ile Glu Leu Val 130 135 140Ala Ala Lys Gln Ala Glu Gly Ser Arg Lys Arg Ile Lys Asp Asn Asn145 150 155 160Ser Gly Pro Ser Glu Ala Glu Arg Lys Lys Phe His Ser Ile Trp Val 165 170 175Leu Ala Ala Leu Ala His Gly Ile Asn Ile Thr Leu Ala Leu Ala Ile 180 185 190Thr Thr Val Val Val Tyr Phe Tyr Val Tyr His Pro Leu Ile Gly Thr 195 200 205Leu Thr Glu Met His Ala Ile Ile Val Trp Leu Lys Thr Ala Ser Tyr 210 215 220Ala Phe Thr Asn Arg Asp Leu Arg His Ala Tyr Leu His Pro Val Glu225 230 235 240Gly Glu Glu Val Pro Asp Leu Tyr Lys Ser Cys Pro Tyr Pro Gln Asn 245 250 255Val Thr Met Lys Asn Leu Val Tyr Phe Trp Trp Ala Pro Thr Leu Val 260 265 270Tyr Gln Pro Val Tyr Pro Arg Thr Asp Lys Ile Arg Trp Val Phe Val 275 280 285Phe Lys Arg Leu Gly Glu Ile Phe Cys Leu Ala Val Phe Ile Trp Val 290 295 300Ala Ser Ala Gln Tyr Ala Thr Pro Val Leu Arg Asn Ser Leu Asp Lys305 310 315 320Ile Ala Ser Leu Asp Leu Pro Asn Ile Leu Glu Arg Leu Met Lys Leu 325 330 335Ser Thr Ile Ser Leu Val Ile Trp Leu Ala Gly Phe Phe Ala Leu Phe 340 345 350Gln Ser Phe Leu Asn Ala Leu Ala Glu Ile Met Arg Phe Gly Asp Arg 355 360 365Ser Phe Tyr Asp Asp Trp Trp Asn Ser Glu Ser Leu Gly Ala Tyr Trp 370 375 380Arg Thr Trp Asn Lys Pro Val Tyr Thr Tyr Phe Lys Arg His Val Tyr385 390 395 400Met Pro Met Ile Gly Arg Gly Trp Ser Pro Ala Ala Ala Ser Phe Ala 405 410 415Val Phe Phe Val Ser Ala Val Leu His Glu Ile Leu Val Gly Val Pro 420 425 430Thr His Asn Ile Ile Gly Val Ala Phe Phe Gly Met Phe Leu Gln Leu 435 440 445Pro Leu Ile Ala Ile Thr Thr Pro Leu Glu Lys Met Lys Leu Gly His 450 455 460Gly Gly Arg Ile Leu Gly Asn Val Ile Phe Trp Val Ser Phe Thr Ile465 470 475 480Phe Gly Gln Pro Phe Ala Ala Leu Met Tyr Phe Tyr Ala Trp Gln Ala 485 490 495Lys Tyr Gly Ser Val Ser Arg Leu Pro Gln Met Val His His 500 505 510761533DNATrichoderma virens 76atggcgcctc ctgcagagtc ctccacgacg acaagcgtcg aggcctctac cggctccgtg 60tctcgccgcc acgcctcaca aagtgaagca gatctaacgt cggtggagcc cgtcaacggc 120acgaccaaga accggctctc caagacaccg ccgaagaaat atcgccatgt cgctgcggtg 180cattcccaga cgcggccgtc gtgcctgagc catgattccc ctgcggctcc cagctttctc 240ggattccgca atctcatggt cattgtgctg gttgttggca atctccgatt gatgattgag 300aatattcaaa agtacggcgt cttaatttgc atcaggtgtc acgactacag acgtcaagat 360gtgctcttgg gtcttttgct ttattttctt atcccctgcc atttgtttgc agcatacctg 420atagagctgg tcgctgccaa gcaggctgag ggatccagga agcgaatcaa ggacaacaac 480tctggcccgt cagaggcaga gcgcaagaag ttccactcaa tctgggttct tgcggctttg 540gcccatggaa

tcaacatcac tcttgccctt gcaattacca ccgttgtggt ctacttttac 600gtctatcatc cgctgattgg cactttgacc gagatgcatg ccatcattgt gtggctcaag 660acggcatcat atgcattcac caaccgagat cttcgtcacg cctatctgca tccagttgag 720ggagaggaag tgcctgattt gtacaaatcc tgcccctatc cacaaaacgt gacgatgaag 780aacttggtat acttctggtg ggctccgact ctggtgtacc aacctgttta tccgcggacc 840gacaagattc gatgggtgtt cgtgtttaag cgactaggag agatcttttg ccttgctgtg 900ttcatttggg ttgccagtgc ccaatatgcc acccccgttt tgcgcaactc tctcgacaag 960attgcctctc ttgatttgcc caacatcttg gagcggctta tgaaactctc gacaatctct 1020ttggtcatct ggctggccgg cttctttgcg ctcttccaat ctttcttaaa cgcccttgcc 1080gagataatga ggtttggcga taggtcattc tacgacgact ggtggaacag tgagagcttg 1140ggcgcctact ggaggacgtg gaacaagcct gtttatactt acttcaagcg ccatgtctat 1200atgcccatga tcggacgagg ctggagcccg gccgctgcca gtttcgcagt cttttttgtt 1260tctgccgttc ttcatgaaat tcttgttggt gttccaacac ataacattat cggcgtcgct 1320ttcttcggca tgttccttca gcttcctctc atcgccatta ctactccgct ggagaagatg 1380aaactcggtc atggtggccg cattcttgga aatgtcatat tttgggtttc gtttacaatc 1440tttggacagc cattcgcggc cctgatgtat ttctacgctt ggcaggccaa gtatggcagc 1500gtgagtaggt tacctcagat ggtgcaccac taa 153377579PRTArxula adeninivorans 77Met Ile Arg Ala Ala Tyr Gly Ser Val Ser Arg Ala Arg Asp Ser Leu1 5 10 15Thr Leu Arg Ala Pro Ser Phe Pro Thr Thr Ala Val Glu Val Arg Asp 20 25 30Lys Ile Leu Trp Ile Leu Tyr Ala Trp Ile Glu Met Phe Thr Asp Val 35 40 45Phe Ser Phe Trp Thr Glu Lys Val Trp Gly Tyr Val Ser Thr Pro Thr 50 55 60Lys Glu Ser Ile Leu Arg Lys Gln Leu Asp Glu Ala Lys Ser Tyr His65 70 75 80Glu Trp Glu Glu Leu Ser Tyr Lys Leu Asp Ser Ile Leu Gly Asn Asp 85 90 95Ile Trp Arg Gln Asn Pro Val Ser Arg Lys Tyr Asp Tyr Arg Leu Ile 100 105 110Ser Thr Arg Leu Lys Glu Leu Val Ala Ala Arg Asp Asn Arg Asn Ile 115 120 125Glu Leu Leu Met Asp Arg Leu Arg Ser Gly Leu Leu Arg Asn Ile Gly 130 135 140Ser Ile Ala Ser Thr His Leu Tyr Asn Arg Ala Tyr Ser Gly Thr Lys145 150 155 160Leu Leu Ile Glu Asp Tyr Ile Asn Val Val Ile Gln Cys Leu Glu Tyr 165 170 175Val Glu Arg Gly Gly Arg Pro Leu Thr Ala Ser Ala Ser Lys Ile Pro 180 185 190Asn Gly Gly Glu Pro Pro Ser Pro Arg Thr Tyr His Lys Pro Met Ile 195 200 205Thr Arg Gln Arg Lys Leu Asn Phe Phe Asn Asp Thr Arg Gln Ser Phe 210 215 220Gly Ser Thr Ala Val Val Leu His Gly Gly Ser Leu Phe Gly Leu Cys225 230 235 240His Ile Gly Met Ile Lys Thr Leu Phe Asn Gln Gly Leu Leu Pro Arg 245 250 255Ile Val Cys Gly Ser Thr Val Gly Ala Leu Val Ala Ser Leu Val Cys 260 265 270Ser Cys Val Asp Glu Glu Val Tyr Glu Thr Leu Asp Asn Val Ser Ser 275 280 285Glu Met Ser Pro Leu Arg Gln Gly Tyr Thr Asp Ile Lys Tyr His Ser 290 295 300Val Ala Glu Gly Val Ile Ser Ser Met Cys Pro Pro Glu Ile Leu Ile305 310 315 320Phe Glu Gln Tyr Ile Arg Glu Lys Leu Gly Asp Leu Thr Phe Glu Glu 325 330 335Ala Tyr Gln Arg Thr Gly Arg Ile Leu Asn Ile Pro Val Thr Pro Lys 340 345 350Ala Lys Pro Gly Gln Val Ala Pro Pro Val Pro Thr Leu Leu Asn Tyr 355 360 365Leu Ser Ser Pro Asn Val Val Val Trp Ser Ala Ala Gln Cys Ser Ile 370 375 380Gly Thr Gly Ile Ile His Lys Lys Val Glu Leu Leu Val Lys Gly Leu385 390 395 400Asp Gly Gln Leu Lys Pro Tyr Leu Asp Ala Asp Asp Ile Glu Tyr Thr 405 410 415Pro Ala Asn Gln Ala Val Tyr Ala Ala Asp Arg Glu Ser Pro Tyr Thr 420 425 430Arg Leu Ser Glu Leu Phe Asn Val Asn Asn Tyr Ile Val Ser Val Ala 435 440 445Arg Pro Tyr Phe Ala Pro Ile Leu Leu Ser Asp Phe Lys Tyr Arg Ala 450 455 460Ala Lys Ser Phe Lys Thr Arg Phe Leu Lys Leu Thr Arg Leu Glu Leu465 470 475 480Gln Tyr Arg Leu Asn Gln Leu Ser Gln Leu Gly Leu Val Pro Pro Met 485 490 495Ile Gln Gln Trp Phe Val Asp Gly Asn Ile Pro Ala Gly Phe Gln Val 500 505 510Thr Val Val Pro Glu Leu Pro Ser Leu Ile Arg Asp Ile Gly Lys Val 515 520 525Phe Asp Ser Asp Asn Ile Lys Glu Lys Val Asp Tyr Trp Ile Lys Ile 530 535 540Gly Glu Arg Ser Val Trp Pro Val Leu Asn Ile Ile Trp Ala Arg Cys545 550 555 560Ala Ile Glu Phe Val Leu Asp Asp Leu Tyr His Ser Arg Arg Lys Asp 565 570 575Glu Leu Asp781740DNAArxula adeninivorans 78atgattaggg ctgcctacgg gtcagtgtcc agggcccgag attctttaac gttgagggct 60ccatcttttc ctaccactgc tgtggaggtc cgtgacaaga ttctatggat tctgtatgcc 120tggattgaaa tgttcacgga cgtctttagc ttctggacgg agaaggtgtg gggttatgtt 180tctactccta ctaaagaaag cattcttaga aagcaactcg acgaggcaaa atcataccat 240gaatgggagg agctcagcta caaactagac tcaattttag ggaacgatat ttggcgacag 300aaccctgtta gccgaaagta tgactatcgc ctgatttcta cccgcctcaa ggaattggtt 360gctgctaggg ataatcgcaa cattgaattg ctaatggatc ggctaaggtc aggcctgctt 420cgtaatattg gatcgattgc aagtactcat ctctacaacc gagcgtattc gggcacaaaa 480ctgttaattg aggattacat taatgtagtg attcaatgcc tggagtatgt tgaacggggc 540ggcaggccat tgactgcttc agcatccaag attcccaatg gcggtgaacc cccttctcca 600cgaacctacc ataagcccat gattaccaga cagcgcaagc tcaacttctt caatgataca 660cgccagtcgt ttggaagtac agctgtggta cttcacggcg ggtccttgtt tggactttgc 720catattggca tgattaaaac attgttcaac cagggtctac ttcctcgcat agtctgtggc 780tccacagtgg gagcactagt agcgagtcta gtatgctcct gtgtggatga agaggtgtat 840gagactttgg ataatgtgtc ttcggaaatg tctcctctcc gccaaggata cactgatata 900aagtaccatt cggtagccga aggggtcatt tcatcaatgt gtccgccaga gattttgatt 960tttgaacagt acatccgaga aaaactcgga gacctgacat ttgaagaagc atatcaacgc 1020accggccgca ttcttaatat cccagtgaca ccaaaggcaa aaccaggtca ggtagcacca 1080ccagtcccga cgctcctgaa ttatttgtcg agcccgaatg ttgtagtatg gtcagcagcg 1140caatgcagca ttggaacggg gattattcac aagaaggttg aacttttagt aaaaggtctg 1200gatggtcaat taaaacctta tttggatgcg gatgatattg aatacactcc tgcaaatcaa 1260gctgtatacg ctgctgatcg cgagagtccc tatacaagat tgtctgagct gttcaatgtg 1320aacaattaca ttgtatcagt agctcgcccc tactttgccc caattctgct ttcggatttc 1380aagtaccgtg cagctaaaag cttcaagacc cggttcctca aactaacccg tctggagtta 1440cagtatcgtc tcaatcagct gtctcaattg gggctggttc cgcccatgat tcaacaatgg 1500tttgtggacg gtaacattcc cgccgggttc caagttaccg tggtgcctga attaccctca 1560cttattagag acatcggcaa ggttttcgat tcggataata taaaggagaa ggtcgactac 1620tggattaaga tcggtgagcg cagtgtgtgg ccagtgctga atattatctg ggcaaggtgc 1680gcaattgagt ttgtgctcga cgatctatat cacagccgac gtaaagacga actcgactag 174079633PRTArxula adeninivorans 79Met Asn Pro Phe Asp Val Asp Tyr Thr Asn Arg Asp His Leu Val Asp1 5 10 15Phe Glu Arg Ala Leu His Glu Asp Glu Ala Ser His Ile Ile Ser Val 20 25 30Asn Asp Trp Ala Pro Val His Ala Pro Leu Lys Arg Arg Leu Arg Arg 35 40 45Lys Pro Thr Asp Ser Asp Pro Gly Thr Gly Leu Gly Tyr Thr Leu Leu 50 55 60Arg Trp Pro Ile Leu Val Ala Ile Ala Leu Trp Leu Ala Leu Leu Ala65 70 75 80Phe Val Tyr Ala Ile Val Arg Phe Trp Val Ala Leu Phe Glu Tyr Phe 85 90 95Val Thr Trp Arg Gly Pro Arg Arg Asn Leu Arg Glu Lys Leu Arg Ser 100 105 110Ala Arg Ser Tyr Glu Glu Trp Ile Ser Ala Ala Lys Val Leu Asp Asp 115 120 125His Leu Gly Asn Thr Ser Trp Lys His Asn Pro Lys Phe Ser Arg Tyr 130 135 140Asp Tyr Arg Thr Ile Asp Arg Ile Thr Asn Ser Leu Arg Gln Leu Arg145 150 155 160Asn Gln Asn Lys Ala Glu Glu Val Gly Ser Ile Leu Gln Gly Cys Val 165 170 175Lys His Asn Phe Ala Gly Thr Gln Gly Gln Pro Leu Tyr Ser Gln Cys 180 185 190Tyr Tyr Gly Thr Lys Asp Leu Val Glu Glu Phe Asn Ser Glu Ile Val 195 200 205Lys Ser Leu Asp Tyr Leu Ala Thr His Pro Asp Leu Ser Pro Gln Ser 210 215 220Arg Arg Leu Leu Phe Lys Met Phe Ser Lys Asn Phe Gly Lys Thr Ala225 230 235 240Leu Cys Leu Ser Gly Gly Ala Thr Phe Ala Tyr Arg His Phe Gly Val 245 250 255Val Lys Ala Leu Leu Glu Gln Gly Leu Leu Pro Asn Ile Ile Ser Gly 260 265 270Thr Ser Gly Gly Gly Leu Val Ala Ala Leu Val Gly Thr Arg Thr Asn 275 280 285Ser Glu Leu Arg Glu Leu Leu Thr Pro Gln Leu Ala Asp Lys Ile Thr 290 295 300Ala Cys Trp Glu Lys Phe Pro Lys Trp Val Tyr Arg Phe Tyr Ser Thr305 310 315 320Gly Ala Arg Phe Asp Ala Val Asp Trp Ala Glu Arg Ser Cys Trp Phe 325 330 335Thr Leu Gly Ser Leu Thr Phe Arg Glu Ala Tyr Asp Arg Thr Gly Lys 340 345 350Ile Leu Asn Ile Ser Thr Val Pro Ala Asp Pro Asn Ser Pro Ser Ile 355 360 365Leu Cys Asn Tyr Ile Thr Ser Pro Asp Cys Val Ile Trp Ser Ala Leu 370 375 380Leu Ala Ser Ala Ala Val Pro Gly Ile Leu Asn Pro Val Val Leu Met385 390 395 400Met Lys Thr Lys Lys Gly Asn Leu Val Pro Tyr Ser Phe Gly Asn Lys 405 410 415Trp Lys Asp Gly Ser Leu Arg Thr Asp Ile Pro Val His Ala Leu Asn 420 425 430Val Tyr Phe Asn Val Asn Phe Thr Ile Val Ser Gln Val Asn Pro His 435 440 445Ile Ser Leu Phe Met Tyr Ala Pro Arg Gly Thr Val Gly Arg Pro Val 450 455 460Ser His Arg Gln Gly Lys Gly Trp Arg Gly Gly Phe Leu Gly Ser Ala465 470 475 480Leu Glu Asp Met Leu Lys Leu Glu Ile Arg Lys Trp Leu Lys Leu Met 485 490 495Lys Asn Leu Ser Leu Met Pro Arg Phe Phe Asn Gln Asp Trp Ser Ser 500 505 510Val Trp Leu Gln Thr Phe Glu Gly Ser Val Thr Leu Trp Pro Arg Ile 515 520 525Arg Leu Lys Asp Phe Tyr Tyr Ile Leu Ser Asp Pro Thr Arg Glu Gln 530 535 540Met Glu Thr Met Ile Ile Ser Gly Gln Arg Cys Thr Phe Pro Lys Leu545 550 555 560Leu Phe Ile Lys His Gln Val Asn Ile Glu Arg Ala Ile Asp Arg Gly 565 570 575Arg Lys His Asn Ala Lys Ala Arg Glu Glu Asn Gly Pro Gln Leu Arg 580 585 590Arg Val Asn Pro Phe Leu His Asp Leu Asp Asp Arg Val Tyr His Ser 595 600 605Ser Ser Ser Val Asp Pro Arg Glu Phe Gln Asp Asp His Asp Asp Glu 610 615 620Asp Asp Asp Ser Thr Asp Ser Ser Met625 630801902DNAArxula adeninivorans 80atgaacccgt ttgatgtaga ttacacaaac agggaccatc tggtcgactt tgaacgagct 60ttgcacgaag atgaggcttc ccatattata tcggtaaacg actgggctcc agtgcatgct 120cctctcaagc gacggttgag acgcaagccg acagattcgg atcctgggac aggattagga 180tacactttgc ttagatggcc tattctggtg gcaattgcgc tgtggctggc cctgttagca 240tttgtgtacg ccatagtgag gttttgggtc gctctgtttg agtactttgt tacctggcga 300ggaccccggc gcaatcttcg tgaaaagcta cgcagcgctc gtagttacga ggaatggatt 360agtgctgcca aagttcttga tgaccatcta ggaaatactt cttggaagca caacccaaag 420ttctctcgat acgactaccg tactattgat cgcatcacta actcactgcg gcaactgcga 480aaccagaaca aggccgagga ggttggctct attctacaag gatgcgtcaa gcacaacttt 540gctggaactc agggccaacc tttgtactct cagtgctact atggcacaaa ggacctggta 600gaggagttca attctgaaat tgtgaaatcg ctcgattacc tggcaaccca tccagacctg 660agtcctcaat ctagacgtct tttgttcaaa atgttttcca agaattttgg aaagacggca 720ttgtgcctct ctggaggggc aacatttgcc tatagacatt tcggagttgt taaagcgctc 780ttggaacagg gcttgctgcc taatattatt tctggtactt ctggcggagg attggtagct 840gcgctagttg gtaccagaac aaatagtgaa ctccgtgagc ttctcactcc tcaactggcc 900gacaagatca ccgcctgctg ggaaaagttc ccaaaatggg tttatagatt ctacagcacc 960ggcgctcgat tcgatgccgt cgactgggct gaacggtctt gctggtttac actaggaagc 1020ctgactttta gagaggccta cgatcgaact ggaaagatcc tcaacatttc cactgttcct 1080gctgacccta attccccttc aatcctctgc aattacatta cttctcccga ctgtgtcatc 1140tggtcggctt tacttgcttc tgctgcagta ccgggaattc tgaacccagt ggtgctcatg 1200atgaagacga aaaagggcaa tctggtacct tacagctttg gtaacaagtg gaaggatggt 1260tctctccgaa ctgatattcc tgtccacgca ctcaacgtgt actttaacgt caacttcacc 1320atcgtgtccc aggtcaaccc tcacatttct ctgttcatgt atgccccgcg gggaactgtg 1380ggtaggccag tatctcaccg tcagggtaaa ggctggcgag gtgggttcct aggctcagct 1440ttggaagaca tgctgaagct ggaaattcgt aaatggctca aactcatgaa aaaccttagt 1500cttatgccac ggtttttcaa tcaagattgg tcttcagtat ggcttcaaac gttcgaggga 1560tccgtcacct tgtggccaag gatcaggcta aaggactttt attatattct gtctgatccc 1620actcgggaac aaatggaaac catgatcatt agtggacagc gatgcacatt cccaaagctc 1680ttgttcatca agcaccaagt caacatagag cgggcaattg accgtggaag aaagcacaat 1740gcaaaagcca gggaggaaaa tggtccccag cttagacggg taaacccatt cctgcacgac 1800ttggatgacc gtgtatacca ttccagctct agcgtggacc ctcgcgagtt tcaggatgat 1860cacgatgatg aagacgacga cagcactgat tctagcatgt aa 190281662PRTArxula adeninivorans 81Met Gln Ser Leu Asp Leu Leu Asp Asp Arg Ser Trp Val Pro Asn Tyr1 5 10 15Ala Arg Val Gly Leu Lys Ser Leu Lys Glu Tyr Leu Val Ser His Arg 20 25 30Tyr Gln Ser Glu Glu Ala Arg Lys His Ala Glu Ala Leu Glu Arg Trp 35 40 45Thr Lys Ser Gln Ala Gln Ala Glu Thr Tyr Glu Gln Trp Leu Phe Ala 50 55 60Ser Glu Gln Leu Asp Lys Leu Ser Gly Asn Asp Lys Trp Lys Glu Asp65 70 75 80Pro Val Ser Pro Tyr Tyr Asp Ser Val Leu Val Gln Gln Arg Leu Gln 85 90 95Gln Leu Arg Asp Ala Arg Val Asn Ser Asn Met Asp Glu Leu Leu Tyr 100 105 110Leu Val Arg Thr Ser Leu Gln Arg Asn Leu Gly Asn Met Gly Asp Pro 115 120 125Arg Leu Tyr Val Arg Thr His Thr Gly Ser Lys Thr Leu Ile Glu Gln 130 135 140Tyr Ile Ala Glu Val Glu Leu Ala Leu Asp Thr Leu Leu Ser Cys Gly145 150 155 160Pro Gly Thr Phe Ser Pro Lys Val Leu Leu Ser Asn Leu Ile Gln Thr 165 170 175Arg Lys Ala Phe Gly Arg Thr Ala Leu Val Leu Ser Gly Gly Ser Thr 180 185 190Phe Gly Ile Leu His Ile Gly Val Met Arg Glu Leu His Arg Ala His 195 200 205Leu Leu Pro Gln Val Ile Ser Gly Ser Ser Ala Gly Ser Ile Phe Ala 210 215 220Ser Met Leu Cys Ile His Leu Glu Asp Glu Ile Glu Glu Leu Leu Gln225 230 235 240Leu Pro Leu His Lys Glu Ser Phe Glu Ile Phe Glu Pro Ala Gly Glu 245 250 255Arg Glu Gly Leu Met Val Arg Leu Ala Arg Phe Leu Lys His Gly Thr 260 265 270Trp Phe Asp Asn Lys Tyr Leu Ser Thr Thr Met Arg Glu Leu Leu Gly 275 280 285Asp Leu Thr Phe Gln Glu Ala Tyr Tyr Arg Thr Gln Arg Ile Leu Asn 290 295 300Val Thr Val Ser Pro Ser Ser Met His Glu Met Pro Lys Ile Leu Asn305 310 315 320Tyr Leu Thr Ala Pro Asn Val Leu Ile Trp Ser Ala Val Cys Ala Ser 325 330 335Cys Ser Val Pro Phe Val Phe Asp Ser His Asp Ile Leu Ala Lys Asn 340 345 350Pro Arg Thr Gly Glu Phe Tyr Ser Trp Asn Ala Ser Thr Phe Ile Asp 355 360 365Gly Ser Val Tyr Asn Asp Leu Pro Leu Ser Arg Leu Ala Glu Met Phe 370 375 380Asn Val Asn His Phe Ile Ala Cys Gln Val Asn Pro His Val Val Pro385 390 395 400Phe Val Lys Phe Ala Glu Thr Met Ser Leu Val Glu Ala Arg Pro Thr 405 410 415Thr Thr Glu Pro Gly Ser Leu Thr Lys Leu Trp His Ser Thr Gln Leu 420 425 430Ala Leu Ser Ser Glu Ile Ser His Tyr Leu Asp Leu Ala Ala Glu Met 435 440 445Gly Leu Phe Lys Asn Ile Ser Ser Lys Leu Arg Ser Val Leu Asp Gln 450 455 460Gln Tyr Ser Gly Asp Ile Thr Ile Leu Pro Glu Leu Tyr Leu Ser Glu465

470 475 480Phe Gly Gln Ile Phe Lys Asn Pro Ser Lys Glu Phe Phe Gln Lys Ala 485 490 495Glu Leu Arg Ala Ala Arg Ala Thr Trp Pro Lys Met Ser His Ile His 500 505 510Asn Arg Val Ala Ile Glu Leu Ala Leu Val Lys Ala Ile His Lys Leu 515 520 525Arg Ala Arg Ile Val Ser Gln Ser Val His Glu Pro Gly Ser Ser Leu 530 535 540Gln Val His Ala Ala Asn Asp Glu Gly Thr Leu Ala Pro Ile Arg Arg545 550 555 560Arg His Ser Ser Thr Lys Leu His His Arg Arg Gln Arg Ser Asp Gly 565 570 575Met Ala Val Lys Tyr Leu Val Arg Arg His Ser Leu Gln Tyr Phe Gly 580 585 590Thr Glu Gly Pro Gly Pro Ala Ala Leu Ser Arg Lys Lys Ser Ser Ala 595 600 605Gly Leu Thr Gln Ala His Thr Pro Thr Pro Ser Leu Thr Asn Ser Val 610 615 620Ser Val Gly Gly Ser Pro Arg His Arg Arg Phe Thr Thr Ser Ser Arg625 630 635 640Gln Ser Ser Gly Asp His Leu Glu Met Phe Ser Gln Asn His Pro Leu 645 650 655Glu Arg Ile Ser Thr Gly 660821989DNAArxula adeninivorans 82atgcaatccc tggacctatt agacgacagg tcctgggtcc ccaattatgc gcgtgtgggc 60ctgaaatcgc taaaagaata cttggttagc catagatatc agtctgaaga agctcgaaag 120catgccgaag cgttagaaag atggacaaag tctcaggctc aggcggagac atacgaacag 180tggctatttg cttcggagca gctcgacaag ctgtctggga acgacaagtg gaaagaggac 240ccggtgtccc catattatga cagtgtgcta gtacaacagc ggttacagca gctccgagat 300gctagggtga atagtaacat ggacgagctg ctgtatttgg tccgcactag cttgcaaaga 360aacttgggta acatgggtga tcctcgacta tacgtgagga cccatactgg ctctaagacg 420ctcattgaac aatatattgc tgaggtagaa ctggcattag acactctgct gagctgcgga 480ccggggacgt tttcacccaa agttctgtta tccaatctta ttcagacaag aaaggcgttt 540ggacgaacag ccctggtgct ttctggaggt agtacgtttg gaattttaca tattggtgta 600atgcgagagc ttcaccgagc ccatctgtta ccgcaggtca tttctggatc gtcggccgga 660tccatctttg cgtccatgct atgtattcac ttagaagacg agattgaaga actactgcaa 720ctgcctctac acaaggaaag ctttgaaatc ttcgaacctg ctggagaacg agaaggacta 780atggttcggc tggcacggtt cctcaaacat ggcacttggt tcgacaacaa gtatcttagc 840acaactatgc gagagcttct aggagacctc actttccagg aggcctacta ccgaacgcag 900cgaattctaa atgtcactgt gtctccttcg agtatgcacg aaatgccgaa gattctcaac 960tatctgaccg ctcctaacgt gctcatttgg tcggcagtgt gtgcatcgtg ctcagtacca 1020tttgtgtttg attctcacga cattctggca aaaaaccctc gaactgggga gttttattca 1080tggaacgctt ctactttcat cgacgggagt gtgtataatg atctgccatt gtctcgacta 1140gcggaaatgt ttaacgtgaa ccattttatt gcgtgccagg taaacccgca tgtggttcca 1200ttcgtcaaat ttgccgagac aatgtcattg gtggaagctc gtcccactac tactgaaccg 1260ggatcgttga caaagctatg gcacagtact cagctcgcgc tttctagtga gatctcacac 1320tatctggatt tggctgctga aatgggcttg ttcaagaaca ttagttccaa gctgcgatcg 1380gtgctagatc aacaatattc cggcgacatt actattcttc ccgaattata cctgtctgag 1440tttggtcaga ttttcaaaaa cccatcaaag gagttcttcc agaaggcaga gcttcgagct 1500gccagagcga catggcccaa gatgtcccac attcacaacc gtgtggccat cgagttggct 1560ttagtaaagg caattcacaa gcttcgtgcc cgtattgtat ctcagagcgt ccatgagcct 1620ggcagttctc tacaagtaca tgctgctaat gacgaaggca ccctagcacc tattcgccgt 1680cgccattctt cgaccaagct tcaccataga cgacaacggt ccgatggaat ggccgtgaaa 1740tacttggtcc gcagacattc gctacagtac tttggcactg agggccctgg tcccgctgcg 1800ctatctcgta aaaagagttc ggccgggctt acccaggctc atactcctac gccttcactg 1860accaacagcg ttagtgtagg gggcagtcca aggcaccgtc gcttcactac tagctctaga 1920cagtcctcag gagaccattt ggaaatgttc tctcaaaatc atccgctaga acgtatctct 1980accggctga 198983332PRTRicinus communis 83Met Glu Val Ser Gly Leu Gly Cys Phe Ser Ser Ala Ala Thr Pro Ser1 5 10 15Leu Cys Gly Ala Val Asp Ser Gly Gly Val Ser Ser Leu Arg Pro Arg 20 25 30Lys Ala Phe His Arg Val Ser Asp Ser Cys Leu Gly Phe Arg Asp Asn 35 40 45Gly His Leu Gln Tyr Tyr Cys Gln Gly Gly Phe Val Arg Cys Gly Gly 50 55 60Gly Asn Lys Lys Ser Ile Lys Lys Lys Leu Lys Leu Val Lys Ser Leu65 70 75 80Ser Glu Asp Phe Ser Met Phe Pro His Asn Asn Ala Leu Leu His Gln 85 90 95Pro Gln Ser Ile Ser Leu Gln Glu Ala Ala Gln Gly Leu Met Lys Gln 100 105 110Leu Gln Glu Leu Arg Ala Lys Glu Lys Glu Leu Lys Arg Gln Lys Lys 115 120 125Gln Glu Lys Lys Ala Lys Leu Lys Ser Glu Ser Ser Ser Ser Ser Ser 130 135 140Ser Glu Ser Ser Ser Asp Ser Glu Arg Gly Glu Val Ile His Met Ser145 150 155 160Arg Phe Arg Asp Glu Thr Ile Pro Ala Ala Leu Pro Gln Leu His Pro 165 170 175Leu Thr His His His Pro Thr Ser Thr Leu Pro Val Ser Pro Thr Gln 180 185 190Glu Cys Asn Pro Met Asp Tyr Thr Ser Thr His His Glu Lys Arg Cys 195 200 205Cys Val Gly Pro Ser Thr Gly Ala Asp Asn Ala Val Gly Asp Cys Cys 210 215 220Asn Asp Arg Asn Ser Ser Met Thr Glu Glu Leu Ser Ala Asn Arg Ile225 230 235 240Glu Val Cys Met Gly Asn Lys Cys Lys Lys Ser Gly Gly Ala Ala Leu 245 250 255Leu Glu Glu Phe Gln Arg Val Leu Gly Val Glu Ala Ala Val Val Gly 260 265 270Cys Lys Cys Met Gly Asn Cys Arg Asp Gly Pro Asn Val Arg Val Arg 275 280 285Asn Ser Val Gln Asp Arg Asn Thr Asp Asp Ser Val Arg Thr Pro Ser 290 295 300Asn Pro Leu Cys Ile Gly Val Gly Leu Glu Asp Val Asp Val Ile Val305 310 315 320Ala Asn Phe Phe Gly Leu Gly Leu Ala Pro Ala Ser 325 33084999DNARicinus communis 84atggaagtct caggcctggg ctgcttctcc tcggctgcaa cgccatcttt gtgtggggcg 60gtggattcag gcggagtatc ctctttgaga ccgaggaagg cattccatag ggtttctgat 120tcttgtttag ggtttagaga taatggacat ctgcagtatt attgtcaagg aggatttgtc 180aggtgcggag gagggaacaa gaaatctatc aagaaaaagt tgaaattagt gaagtccttg 240tctgaggact tttccatgtt tcctcataac aatgctttgc tccatcaacc tcaatccatc 300tccctccagg aagctgcaca aggattaatg aaacagctcc aagaattgcg agcaaaggag 360aaggaattaa agaggcagaa gaaacaagag aaaaaagcca agctaaaatc tgaatcatcc 420tcatcctcat cctctgaatc cagtagtgat agcgaacgtg gggaggttat tcacatgagc 480cgcttcagag atgaaactat tcctgccgca ctacctcaat tgcacccact tactcatcac 540cacccaactt ccaccctacc agtctcccca acccaagaat gcaacccgat ggattacact 600tcaacacatc atgaaaaacg atgctgcgtt ggaccaagca ccggtgccga taacgcagtc 660ggtgactgtt gcaatgatag gaatagctcg atgacagagg aattgtcagc aaacagaatt 720gaggtgtgca tgggtaataa gtgcaagaag tcgggaggtg cagcgttatt ggaggaattt 780cagagggttt tgggtgtaga ggctgcagtt gttgggtgca agtgcatggg gaactgcagg 840gacggtccta atgtaagggt caggaattct gtccaagaca gaaacacaga tgactctgtt 900cgaaccccct ccaatcctct ctgcattggt gttggtttgg aggatgtgga tgttattgtg 960gccaatttct ttgggttggg tctggcccct gcatcttaa 99985345PRTArachis hypogaea 85Met Glu Val Ser Gly Ala Val Leu Arg Asn Val Thr Cys Pro Ser Phe1 5 10 15Ser Val His Val Ser Ser Arg Arg Arg Gly Gly Asp Ser Cys Val Thr 20 25 30Val Pro Val Arg Met Arg Lys Lys Ala Val Val Arg Cys Cys Cys Gly 35 40 45Phe Ser Asp Ser Gly His Val Gln Tyr Tyr Gly Asp Glu Lys Lys Lys 50 55 60Glu Asn Gly Thr Ala Met Leu Ser Thr Lys Lys Lys Leu Lys Met Leu65 70 75 80Lys Lys Arg Val Leu Phe Asp Asp Leu Gln Gly Asn Leu Thr Trp Asp 85 90 95Ala Ala Met Val Leu Met Lys Gln Leu Glu Gln Val Arg Ala Glu Glu 100 105 110Lys Glu Leu Lys Lys Lys Arg Lys Gln Glu Lys Lys Glu Ala Lys Leu 115 120 125Lys Ala Ser Lys Met Asn Thr Asn Pro Asp Cys Glu Ser Ser Ser Ser 130 135 140Ser Ser Ser Ser Glu Ser Glu Ser Glu Ser Ser Glu Ser Glu Cys Asp145 150 155 160Asn Glu Val Val Asp Met Lys Lys Asn Ile Lys Val Gly Val Ala Val 165 170 175Ala Val Ala Asp Ser Pro Arg Lys Ala Glu Thr Met Ile Leu Tyr Thr 180 185 190Ser Leu Val Ala Arg Asp Val Ser Ala Asn His His His His Asn Ala 195 200 205Val Glu Leu Phe Ser Arg Asn Asn Asp Ile Ser Val Gly Ser Ile Asn 210 215 220Gly Gly Leu Lys Asn Glu Asn Thr Ala Val Ile Thr Thr Glu Ala Ile225 230 235 240Pro Gln Lys Arg Ile Glu Val Cys Met Gly Asn Lys Cys Lys Lys Ser 245 250 255Gly Ser Ile Ala Leu Leu Gln Glu Phe Glu Arg Val Val Gly Ala Glu 260 265 270Gly Gly Ala Ala Ala Ala Val Val Gly Cys Lys Cys Met Gly Lys Cys 275 280 285Lys Ser Ala Pro Asn Val Arg Ile Gln Asn Ser Thr Ala Asp Lys Ile 290 295 300Ala Glu Gly Phe Asn Asp Ser Val Lys Val Pro Ala Asn Pro Leu Cys305 310 315 320Ile Gly Val Ala Trp Arg Met Leu Lys Pro Leu Trp Leu Arg Phe Leu 325 330 335Gly Glu Asn Gln Glu Ser Thr Asn Glu 340 345861038DNAArachis hypogaea 86atggaggttt caggcgccgt tctaaggaat gtcacgtgcc cttccttttc tgtgcacgtg 60agttcccgtc gtcgtggtgg tgatagttgt gttacagtgc cggtgaggat gagaaaaaag 120gcggtggtgc gttgttgctg cgggttcagt gattcggggc atgtgcagta ttacggggac 180gagaagaaga aggagaatgg aaccgctatg ttgagcacca agaagaagct caagatgctg 240aagaaacgtg tccttttcga tgatcttcaa ggaaacctga cttgggatgc tgctatggtt 300ttgatgaagc agctagagca agtaagggca gaggagaagg aattgaagaa aaaaaggaag 360caagagaaga aggaggcaaa actcaaagcc tctaagatga acaccaatcc tgattgcgaa 420tcgtcatcgt catcgtcatc atctgaatct gaatctgaat caagtgagag tgaatgtgac 480aatgaggtgg ttgacatgaa gaagaacatt aaggttggtg ttgccgttgc tgttgccgat 540tccccacgaa aggcggaaac catgattcta tacacctccc ttgttgcccg agatgttagt 600gctaatcatc atcatcataa tgccgtggaa ttattctcta gaaacaatga catatcagtt 660ggaagcatta atggtggcct taagaatgag aatactgcgg ttattaccac tgaagctatt 720cctcagaaga ggattgaggt atgcatggga aacaagtgca agaaatccgg atctattgca 780ttgttgcaag aatttgagag agtggttggt gctgaaggag gtgctgctgc tgcagttgtt 840ggatgcaagt gcatggggaa gtgcaagagt gcacctaatg tgaggattca gaactctact 900gcagataaaa tagctgaggg gttcaatgat tcagttaagg ttccagctaa ccctctttgc 960attggggttg catggaggat gttgaaacca ttgtggctta gattcttggg cgagaatcag 1020gaaagtacta atgaataa 103887816PRTYarrowia lipolytica 87Met Phe Thr Ser Arg Val Ser Glu Ala Ser Thr Thr Asn Phe Ile Arg1 5 10 15Pro Thr Ala Arg Ser His Ile His Phe Phe Phe Ala Phe Ile Ala Ala 20 25 30Thr Val His Gln Leu Leu Leu Met Leu Tyr Gln Leu Leu Gly Asp Gly 35 40 45Tyr Leu Lys Ser Phe Val Asp Thr Gly Ile Thr Leu Ala Gln Gln Ser 50 55 60Gly Leu Ser Gly Ile Val Asn Ala Leu Thr Ser Glu Ala Lys Leu Arg65 70 75 80Ile Asp Lys Arg Ser Ile Ile Lys Lys Leu Leu Glu Asp Gln Glu Asn 85 90 95Ala Glu Ser Tyr Phe Asp Trp Leu Lys Ala Ser Ser Glu Leu Asp Tyr 100 105 110Leu Leu Gly Asn Gln Glu Trp Lys Glu Arg Asp Glu Cys Pro Ala Tyr 115 120 125Asp Tyr Glu Tyr Val Arg Leu Arg Leu Asp Glu Leu Arg His Ala Arg 130 135 140Thr Asn Asn Asp Thr Thr Arg Leu Leu Tyr Leu Val Arg Thr Thr Trp145 150 155 160Ser Arg Asn Leu Gly Asn Leu Gly Asp Val Lys Leu Tyr His Asn Ser 165 170 175Phe Thr Gly Thr Lys Arg Leu Ile Glu Asp Tyr Ile Leu Glu Cys Glu 180 185 190Leu Ala Leu Asn Ala Leu Leu Ala Ala Gly Asn Asp Lys Ile Pro Asp 195 200 205Gln Glu Leu Leu Thr Glu Leu Leu Asn Thr Arg Lys Ala Phe Gly Arg 210 215 220Thr Ala Leu Leu Leu Ser Gly Gly Gly Cys Leu Gly Leu Leu His Thr225 230 235 240Gly Val Leu Gln Ala Leu Ser Asp Thr Ser Leu Leu Pro His Val Ile 245 250 255Ser Gly Ser Ser Ala Gly Ser Ile Met Ala Ala Gly Leu Cys Ile His 260 265 270Lys Asp Glu Glu His Glu Ala Phe Ile Thr Glu Leu Met Glu Arg Asp 275 280 285Phe Asp Ile Phe Glu Glu Ser Gly Asn Glu Asp Thr Val Leu Glu Arg 290 295 300Val Ser Arg Met Leu Lys His Gly Ser Leu Leu Asp Asn Arg Tyr Met305 310 315 320Gln Asp Thr Met Arg Glu Leu Phe Gly Asp Met Thr Phe Leu Glu Ala 325 330 335Tyr Asn Arg Thr Arg Arg Ile Leu Asn Val Thr Val Ser Ser Ala Gly

References

• gcg.com

Claims

1.-13. (canceled)

14. A method of increasing the lipid content of a cell, comprising transforming a parent cell with a first nucleic acid and a second nucleic acid; wherein said first nucleic acid increases the activity of a native diacylglycerol acyltransferase or comprises a diacylglycerol acyltransferase gene; and said second nucleic acid decreases the activity of a triacylglycerol lipase.

15. The method of any one of claim 14, wherein said first nucleic acid comprises a diacylglycerol acyltransferase gene.

16. The method of claim 15, wherein said diacylglycerol acyltransferase gene encodes a type I diacylglycerol acyltransferase polypeptide, a type II diacylglycerol acyltransferase polypeptide, or a type III diacylglycerol acyltransferase polypeptide.

17. The method of claim 15, wherein said diacylglycerol acyltransferase gene encodes an amino acid sequence selected from the group consisting of: (a) an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them; and (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them.

18. The method of claim 14, wherein said second nucleic acid is capable of recombining with a nucleic acid sequence in a triacylglycerol lipase gene and/or a nucleic acid sequence in the regulatory region of a triacylglycerol lipase gene.

19. The method of claim 14, wherein said second nucleic acid is capable of recombining with a nucleic acid sequence in a TGL3, TGL3/4, or TGL4 gene and/or a nucleic acid sequence in the regulatory region of a TGL3, TGL3/4, or TGL4 gene.

20. The method of claim 14, wherein said cell is Yarrowia lipolytica or Arxula adeninivorans.

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