US20240026395A1

US20240026395A1 - Oleic acid production in yeast

Info

Publication number: US20240026395A1
Application number: US18/138,472
Authority: US
Inventors: Vasiliki Tsakraklides; Elena E. Brevnova; Jonathan FRIEDLANDER; Annapurna KAMINENI; Arthur J. Shaw, IV
Original assignee: Ginkgo Bioworks Inc
Current assignee: Ginkgo Bioworks Inc
Priority date: 2014-12-10
Filing date: 2023-04-24
Publication date: 2024-01-25
Also published as: US11634737B2; CN107208088A; EP3230446A1; US20200109377A1; CN114395500A; CN107208088B; EP3230446A4; US20170369910A1; WO2016094520A1; BR112017011542A2

Abstract

Disclosed are transformed cells comprising one or more genetic modifications that affect the lipid content of the cell, e.g., by increasing the concentration of oleic acid in the cell relative to an unmodified cell of the same type. Also disclosed are methods for modifying the lipid content of a cell by increasing the activity of one or more proteins in the cell and/or by decreasing the activity of one or more proteins in the same cell.

Description

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 15/534,818, filed on Jun. 9, 2017, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2015/064710, filed Dec. 9, 2015, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/090,169, filed Dec. 10, 2014.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Jul. 24, 2023, is named GBB-73702_SL.xml and is 310,925 bytes in size.

BACKGROUND

Lipids are indispensable ingredients in the food and cosmetics industries, and they are important precursors in the biodiesel and biochemical industries. Many oleaginous microorganisms, including the well-characterized yeast Yarrowia lipolytica, produce lipids.
Microorganisms synthesize lipids with distinct carbon chain lengths and degrees of unsaturation. These fatty acids can be stored in organelles, termed lipid bodies or lipid droplets, as storage lipids, for example, as triacylglycerides (TAG). The lipid profile of a cell, i.e., the relative amounts of fatty acid species that make up the total lipids in the cell, is determined by the activities and substrate specificities of various enzymes that synthesize fatty acids (fatty acid synthase, elongase, desaturase), various enzymes that stabilize fatty acids by incorporating them into storage lipids (acyltransferases), and various enzymes that degrade fatty acids and storage lipids (e.g., lipases).
The ability to tailor the lipid profile of a cell to increase the concentration of a particular fatty acid is desirable when targeting the lipid product to a specific market/application. Specifically, increasing the oleic acid content of an oleaginous yeast, like Yarrowia lipolytica, increases the value of the TAG produced in the organism.
The lipid yield of oleaginous organisms can be increased by the up-regulation, down-regulation, or deletion of genes implicated in a lipid pathway. The successful modulation of enzymes, however, is unpredictable, at best. For example, overexpressing in Y. lipolytica the DGA1 from Mortierella alpine has no significant effect on lipid content (U.S. Pat. No. 7,198,937; incorporated by reference); likewise, overexpressing DGA2 has no significant effect on the lipid content in the absence of other genetic modifications.

SUMMARY

In some aspects, the invention relates to a transformed cell, wherein the cell is selected from the group consisting of algae, bacteria, molds, fungi, plants, and yeasts. The cell may be a yeast. For example, the cell may be a yeast selected from the group consisting of Arxula adeninivorans, Saccharomyces cerevisiae, and Yarrowia lipolytica.
In some embodiments, the transformed cell comprises one or more genetic modifications that increase the activity of one or more proteins in the cell. For example, the transformed cell may comprise one or more genetic modifications that increase the activity of a Δ9 desaturase protein; an elongase protein; a type 1 diacylglycerol acyltransferase protein; a type 2 diacylglycerol acyltransferase protein; a type 3 diacylglycerol acyltransferase protein; a glycerol-3-phosphate acyltransferase protein; a sn-2 acylglycerol fatty acyltransferase protein; a lysophosphatidic acid acyltransferase protein; a phosphatidate phosphatase protein; a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein; and/or a phospholipid:diacylglycerol acyltransferase protein. The one or more genetic modifications may be transformation with one or more nucleic acids that encode a Δ9 desaturase protein; an elongase protein; a type 1 diacylglycerol acyltransferase protein; a type 2 diacylglycerol acyltransferase protein; a type 3 diacylglycerol acyltransferase protein; a glycerol-3-phosphate acyltransferase protein; a sn-2 acylglycerol fatty acyltransferase protein; a lysophosphatidic acid acyltransferase protein; a phosphatidate phosphatase protein; a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein; and/or a phospholipid:diacylglycerol acyltransferase protein.
In some embodiments, the transformed cell comprises one or more genetic modifications that decrease the activity of a native protein in the cell. For example, the transformed cell may comprise one or more genetic modifications that decrease the activity of a native Δ9 desaturase protein; a native Δ12 desaturase protein; a native diacylglycerol acyltransferase protein; a native triacylglycerol lipase protein; a native sn-2 acylglycerol fatty acyltransferase protein; a native lysophosphatidic acid acyltransferase protein; a native phosphatidate phosphatase protein; a native glycerol-3-phosphate acyltransferase protein; a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein; and/or a native phospholipid:diacylglycerol acyltransferase protein. The one or more genetic modifications may be, for example, knockout mutations.
In some aspects, the invention relates to a product derived from a transformed cell of the invention. In some embodiments, the product comprises an oil, lipid, or triacylglycerol. The product may comprise stearic acid, oleic acid, or linoleic acid. For example, the product may be oleic acid.
In some aspects, the invention relates to methods of modifying the lipid content of a cell, comprising transforming the cell. The cell may be selected from the group consisting of algae, bacteria, molds, fungi, plants, and yeasts, e.g., the cell may be a yeast. For example, the cell may be a yeast selected from the group consisting of Arxula adeninivorans, Saccharomyces cerevisiae, and Yarrowia lipolytica.
In some embodiments, the method comprises transforming the cell with one or more nucleic acids that increase the activity of one or more proteins in the cell. For example, the one or more nucleic acids may increase the activity of a Δ9 desaturase protein; an elongase protein; a type 1 diacylglycerol acyltransferase protein; a type 2 diacylglycerol acyltransferase protein; a type 3 diacylglycerol acyltransferase protein; a glycerol-3-phosphate acyltransferase protein; a sn-2 acylglycerol fatty acyltransferase protein; a lysophosphatidic acid acyltransferase protein; a phosphatidate phosphatase protein; a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein; and/or a phospholipid:diacylglycerol acyltransferase protein. The one or more nucleic acids may encode a Δ9 desaturase, elongase, type 1 diacylglycerol acyltransferase, type 2 diacylglycerol acyltransferase, type 3 diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase genes.
In some embodiments, the method comprises transforming the cell with a nucleic acid that decreases the activity of a native protein in the cell. For example, the nucleic acid may decrease the activity of a native Δ9 desaturase protein; a native Δ12 desaturase protein; a native diacylglycerol acyltransferase protein; a native triacylglycerol lipase protein; a native sn-2 acylglycerol fatty acyltransferase protein; a native lysophosphatidic acid acyltransferase protein; a native phosphatidate phosphatase protein; a native glycerol-3-phosphate acyltransferase protein; a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein; and/or a native phospholipid:diacylglycerol acyltransferase protein. The nucleic acid may decrease the activity of a native protein by knocking out the gene that encodes the protein, e.g., the nucleic acid may recombine with the gene and/or a nucleotide sequence in the regulatory region of the gene, thereby disrupting the transcription or translation of the gene into a protein with the same level of activity as the native protein.
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

FIG. 1 depicts various biosynthetic pathways that may be manipulated to modify the lipid content or lipid composition of a cell.

FIG. 2 depicts 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 a PacI/NotI restriction digest before transformation. “2u ori” denotes the S. cerevisiae origin of replication from the 2 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 Natl 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.

FIG. 3 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for a Y. lipolytica strain before (NS18) and after (NS419) deletion of a native Δ12 desaturase gene.

FIG. 4 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for a Y. lipolytica strain before (NS18) and after (NS441) transformation with a nucleic acid that encodes an additional copy of the Y. lipolytica Δ9 desaturase gene.

FIG. 5 (consisting of panels A-C) depicts experiments on Y. lipolytica cells comprising genetic modifications that increase or decrease the activity of an elongase protein. (A) The percentage of fatty acids that are either C16 or C18 fatty acids for a Y. lipolytica strain before (NS18) and after (NS276) deletion of a native ELO1 gene. (B) The percentage of fatty acids that are either C16 or C18 fatty acids for a Y. lipolytica strain before (NS452) and after (NS477) transformation with a nucleic acid that encodes an additional copy of the Y. lipolytica ELO1 gene. (C) The percentage of fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for a Y. lipolytica strain before (NS452) and after (NS477) transformation with a nucleic acid that encodes an additional copy of the Y. lipolytica ELO1 gene. Strain NS452 comprises an additional copy of the Y. lipolytica DGAT2 gene, which encodes a DGA1 protein, and a copy of the Claviceps purpurea DGAT1 gene, which encodes a DGA2 protein, and a deletion of the native J12 desaturase gene.

FIG. 6 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for Y. lipolytica strain NS418, which comprises a deletion of a native z9 desaturase gene, after transforming the strain with nucleic acids comprising a Δ9 desaturase gene from other organisms.

FIG. 7 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for a Y. lipolytica strain before (NS18) and after (NS563) deletion of a native glycerol acyltransferase gene (SCT1).

FIG. 8 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for Y. lipolytica strain NS18 after transforming the strain with nucleic acids comprising a DGAT2 gene from various species. The DGAT2 gene encodes the DGA1 protein.

FIG. 9 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for Y. lipolytica strain NS281, which comprises a nucleic acid that encodes the DGA1 protein from R. toruloides, after transforming the strain with nucleic acids comprising a DGAT1 gene from various species. The DGAT1 gene encodes the DGA2 protein.

FIG. 10 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for Y. lipolytica strain NS564, which comprises a deletion of a native J12 desaturase gene and a native SCT1 gene, after transforming the strain with a nucleic acid comprising a SCT1 gene from various species.

FIG. 11 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for an A. adeninivorans strain before (NS252) and after (NS478) deletion of a native J12 desaturase gene.

FIG. 12 depicts the percentage of C16 and C18 fatty acids that are palmitate, palmitoleate, stearate, oleate, and linoleate for A. adeninivorans strain NS252 after transforming the strain with nucleic acids comprising a DGAT2 gene from various species. The DGAT2 gene encodes the DGA1 protein.

FIG. 13 depicts the strategy for engineering Y. lipolytica strain NS551.

FIG. 14 depicts the percentage of C16 and C18 fatty acids that are either C16 or C18 fatty acids for an A. adeninivorans strain comprising a Δ12 desaturase knockout and the addition of various elongase genes. Each elongase gene was added to A. adeninivorans strain NS554, which comprises a Δ12 desaturase knockout and is shown as a control.

FIG. 15 depicts the percentage of various fatty acids as a percentage of total C16 and C18 fatty acids for A. adeninivorans strain NS554 and Y. lipolytica strain NS276 comprising various elongase genes. A. adeninivorans strain NS554 comprises a Δ12 desaturase knockout mutation and Y. lipolytica strain NS276 comprises an ELO1 knockout mutation.

FIG. 16 depicts the percentage of C16 and C18 fatty acids that are either C16 or C18 fatty acids for an Y. lipolytica strain comprising an ELO1 knockout and the addition of various elongase genes. Each elongase gene was added to Y. lipolytica strain NS276, which comprises an ELO1 desaturase knockout and is shown as a control.

FIG. 17 depicts the percentage of C16 and C18 fatty acids that are either C16 or C18 fatty acids for A. adeninivorans strain NS557 further comprising an ELO1 gene from Y. lipolytica. The parent strain A. adeninivorans NS557 comprises a Δ12 desaturase knockout mutation and expresses Y. lipolytica DGA1. Strain NS557 was analyzed as a control, and the horizontal line marks the C18 percentage of this strain.

FIG. 18 depicts the percentage of C16 and C18 fatty acids for fatty acids comprising various chain lengths and saturation levels for A. adeninivorans strain NS776, described in Example 14.

FIG. 19 is a flowchart that shows the order in which various genetic modifications were introduced into Y. lipolytica strain NS18, resulting in strains NS987, NS988, NS991, NS992, NS993, and NS994, which are described in Example 15.

FIG. 20 depicts the percentage of C16 and C18 fatty acids that comprise various chain lengths and levels of saturation for various Y. lipolytica strains, which are described in Example 16.

FIG. 21 depicts the percentage of C16 and C18 fatty acids that comprise various chain lengths and levels of saturation for various Y. lipolytica strains, which are described in Example 16. Additionally, the total lipid content of each strain is shown as % dry cell weight (“total lipids”).

DETAILED DESCRIPTION

Definitions

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.
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 an enzyme in a cell may reduce the amount of the enzyme in a cell or reduce the efficiency of the enzyme. A knockout reduces the amount of a protein in the cell. Alternatively, a mutation to a gene may reduce the efficiency of its protein product with little effect on the amount of the protein in the cell. Mutations that reduce the efficiency of an enzyme 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 protein 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 the activity of a protein may instead affect the transcription or translation of the gene. For example, mutation of an enhancer or promoter can reduce the activity of a protein by reducing its expression. Mutating or deleting the non-coding portions of a gene, such as its introns, may also reduce transcription or translation. Additionally, mutations to the upstream regulators of a gene may affect the activity of its protein product; for example, the over-expression of one or more repressors may decrease the activity of a protein, and a knockout or mutation of one or more activators may similarly decrease the activity of a protein.
A genetic modification that increases the activity of a protein in a cell may increase the amount of the protein in the cell or increase the efficiency of the protein (e.g., the efficiency of an enzyme). For example, the genetic modification may simply insert an additional copy of the protein into the cell such that the additional copy is transcribed and translated into additional functional protein. The added gene can be native to the host organism or from a different organism. Alternatively, mutating or deleting the non-coding portions of a gene, such as its introns, may also increase translation. A native gene can be altered by adding a new promoter that causes more transcription. Similarly, enhancers may be added to the gene to increase transcription, or silencers may be mutated or deleted from the gene to increase transcription. Mutations to a native gene's coding region might also increase the activity of the protein, 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 the activity of a protein by increasing the expression of the protein, and a knockout or mutation of one or more repressors may similarly increase the activity of the protein.
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 protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein, e.g., the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159, which include fewer amino acids than the full length protein, and exhibit at least one activity of the protein. Similarly, biologically-active portions of a protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein, e.g., the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159, which include fewer amino acids than the full length protein, and exhibit at least one activity of the protein. A biologically-active portion of a protein 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, 696, 697, 698, 699, 700 or more amino acids. Typically, biologically-active portions comprise a domain or motif having a catalytic activity, such as catalytic activity for producing stearic acid, oleic acid, or linoleic acid. A biologically-active portion of a protein includes portions of the protein 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 an enzyme 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% or higher activity relative to the full-length enzyme. A biologically-active portion of a protein may include portions of a protein that lack a domain that targets the protein to a cellular compartment. A biologically active portion of a DGA1 protein can be a polypeptide which is, for example, 262 amino acids in length.
The term “DGAT1” refers to a gene that encodes a type 1 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA2 protein.
The term “DGAT2” refers to a gene that encodes a type 2 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA1 protein.
“Diacylglyceride,” “diacylglycerol,” and “diglyceride,” are esters comprised of glycerol and two fatty acids.
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 type 3 diacylglycerol acyltransferases (DGA3) and all homologs that catalyze the above-mentioned reaction.
The terms “diacylglycerol acyltransferase, type 1” and “type 1 diacylglycerol acyltransferases” refer to DGA2 and DGA2 orthologs.
The terms “diacylglycerol acyltransferase, type 2” and “type 2 diacylglycerol acyltransferases” refer to DGA1 and DGA1 orthologs.
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.
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.
“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.
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.
The term “enzyme” as used herein refers to a protein that can catalyze a chemical reaction.
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.
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.
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.
The term “genetic modification” refers to the result of a transformation. Every transformation causes a genetic modification by definition.
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).
“Inducible promoter” is a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus.
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.
“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.
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.
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.
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.
The acronym “ORF” stands for open reading frame.
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 parent the cell's genome.
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.
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 “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.
The term “protein” refers to molecules that comprise an amino acid sequence, wherein the amino acids are linked by peptide bonds.
“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.
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.
The term “substantially identical” refers to a nucleotide or amino acid sequence that encodes a biologically-active portion of a protein, which has 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 sequence identity with a reference sequence.
For enzymes, a substantially identical sequence typically retains the enzymatic activity of the reference sequence. For example, a sequence is substantially identical to a reference sequence if it encodes an enzyme that has between 10% and 1,000% of the enzymatic activity of the reference enzyme.
“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.
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.
The terms “triacylglyceride,” “triacylglycerol,” “triglyceride,” and “TAG” are esters comprised of glycerol and three fatty acids.
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, TGL4, and TGL3/4.
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
A. Overview
In certain embodiments of the invention, a microorganism is genetically modified to change its lipid composition, e.g., to increase its oleic acid content (FIG. 1 ).
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, Aspegillus, 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.
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.
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.
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. In certain embodiments 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.
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)).
B. Homologous Recombination
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.
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.
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.
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.
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.
C. Vectors and Vector Components
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.
1. Control Sequences
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.
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.
The promoter used to express a gene can be the promoter naturally linked to that gene or a different promoter.
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, e.g., substantially, transcription of an operably linked gene that is transcribed at a low level.
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)).
2. Genes and Codon Optimization
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.
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.
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.
C. Transformation
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.
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)).
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 Cells, Nucleic Acids, and Methods
A. Transformed Cell
In some embodiments, the transformed 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. In some embodiments, the cell is a yeast. Those with skill in the art will recognize that many forms of filamentous fungi produce yeast-like growth, and the definition of yeast herein encompasses such cells.
The cell may be selected from the group consisting of Arxula, Aspegillus, 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 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.
In certain embodiments, the cell is Saccharomyces cerevisiae, Yarrowia lipolytica, or Arxula adeninivorans.
In some embodiments, the cell is a yeast, fungus, or yeast-like algae. The cell may be selected from thraustochytrids (Aurantiochytrium) and achlorophylic unicellular algae (Prototheca).
In certain embodiments, the transformed cell comprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or more lipid as measured by % dry cell weight. In some embodiments, the transformed cell comprises C18 fatty acids at a concentration of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or higher as a percentage of total C16 and C18 fatty acids in the cell. In some embodiments, the transformed cell comprises oleic acid at a concentration of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or higher as a percentage of total C16 and C18 fatty acids in the cell.
B. Nucleic Acids and Methods for Increasing the Activity of a Protein
The genes of the invention may comprise conservative substitutions, deletions, and/or insertions while still encoding a protein that has activity. For example, 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.
Proteins may comprise conservative substitutions, deletions, and/or insertions while still maintaining activity. Conservative substitution tables are well known in the art (Creighton, Proteins (2d. ed., 1992)).
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, OH), Quick Change Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis, and other site-directed mutagenesis protocols.
To determine the percent identity of two amino acid sequences or 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.
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.
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.
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.
An alignment of selected sequences in order to determine “% identity” between two or more sequences is performed using for example, the CLUSTAL-W program.
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).
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′→3′direction.
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.
Amino acid and nucleotide sequences may be derived from oleaginous organisms having 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.

TABLE 1

List of oleaginous fungi with reported
lipid contents of about 50% and above.
Fungi with reported high lipid content

Aspergillus terreus

Aurantiochytrium limacinum

Claviceps purpurea

Cryptococcus albidus

Cryptococcus curvatus

Cryptococcus ramirezgomezianus

Cryptococcus terreus

Cryptococcus wieringae

Cunninghamella echinulata

Cunninghamella japonica

Leucosporidiella creatinivora

Lipomyces lipofer

Lipomyces starkeyi

Lipomyces tetrasporus

Mortierella isabellina

Prototheca zopfii

Rhizopus arrhizus

Rhodosporidium babjevae

Rhodosporidium paludigenum

Rhodosporidium toruloides

Rhodotorula glutinis

Rhodotorula mucilaginosa

Tremella enchepala

Trichosporon cutaneum

Trichosporon fermentans

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 protein. A native protein may be overexpressed by modifying the upstream transcription regulators of the gene that encodes the protein, for example, by increasing the expression of a transcription activator or decreasing the expression of a transcription repressor. Alternatively, the promoter of a native gene may be substituted with a constitutively active or inducible promoter by recombination with an exogenous nucleic acid.
In some embodiments, a genetic modification that increases the activity of a protein comprises transformation with a nucleic acid that comprises a gene that encodes the protein.
The gene may be native to the cell or from a different species. In certain embodiments, the gene is inheritable to the progeny of a transformed cell. In some embodiments, the gene is inheritable because it resides on a plasmid. In certain embodiments, the gene is inheritable because it is integrated into the genome of the transformed cell.
1. Increasing the Activity of a Δ9 Desaturase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification increases the activity of a Δ9 desaturase protein in the cell. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a Δ9 desaturase protein in the cell.
The nucleic acid may encode a Δ9 desaturase gene. In some embodiments, the gene is Δ9. In some embodiments, the gene is from Arxula adeninivorans, Gloeophyllum trabeum, Microbotryum violaceum, Puccinia graminis, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula graminis, or Yarrowia lipolytica. The gene may be from Arxula adeninivorans or Puccinia graminis.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:4; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:112; or SEQ ID NO:114.
The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:4; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:112; or SEQ ID NO:114. In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:8 or SEQ ID NO:14. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:8 or SEQ ID NO:14.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:3; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:111; or SEQ ID NO:113, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:3; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:111; or SEQ ID NO:113. In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:7 or SEQ ID NO:13, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:13.
The nucleic acid that comprises a gene encoding a Δ9 desaturase protein may comprise a nucleotide sequence set forth in SEQ ID NO:4; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:112; or SEQ ID NO:114. In other embodiments, the gene is substantially identical to SEQ ID NO:4; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:112; or SEQ ID NO:114 and the nucleotide sequence encodes a protein that retains the Δ9 desaturase activity of a protein encoded by SEQ ID NO:3; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:111; or SEQ ID NO:113, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The Δ9 desaturase protein may have an amino acid sequence set forth in SEQ ID NO:3; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:111; or SEQ ID NO:113. In other embodiments, the Δ9 desaturase protein is substantially identical to SEQ ID NO:3; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:111; or SEQ ID NO:113, and retains the functional activity of the protein of SEQ ID NO:3; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:111; or SEQ ID NO:113, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of an elongase, diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous Δ9 desaturase protein and (2) a knockout mutation in the native Δ9 desaturase gene.
2. Increasing the Activity of an Elongase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification increases the activity of an elongase protein in the cell. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of an elongase protein in the cell.
The nucleic acid may encode an elongase gene. In some embodiments, the gene is ELO1 or ELO2. In some embodiments, the gene is from Arxula adeninivorans, Rattus norvegicus, Saccharomyces cerevisiae, or Yarrowia lipolytica.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:6; SEQ ID NO:108; SEQ ID NO:156; SEQ ID NO:158; or SEQ ID NO:160. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:6; SEQ ID NO:108; SEQ ID NO:156; SEQ ID NO:158; or SEQ ID NO:160.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:5; SEQ ID NO:107; SEQ ID NO:155; SEQ ID NO:157; or SEQ ID NO:159, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:5; SEQ ID NO:107; SEQ ID NO:155; SEQ ID NO:157; or SEQ ID NO:159.
The nucleic acid that comprises a gene encoding an elongase protein may comprise a nucleotide sequence set forth in SEQ ID NO:6; SEQ ID NO:108; SEQ ID NO:156; SEQ ID NO:158; or SEQ ID NO:160. In other embodiments, the gene is substantially identical to SEQ ID NO:6; SEQ ID NO:108; SEQ ID NO:156; SEQ ID NO:158; or SEQ ID NO:160 and the nucleotide sequence encodes a protein that retains the elongase activity of a protein encoded by SEQ ID NO:5; SEQ ID NO:107; SEQ ID NO:155; SEQ ID NO:157; or SEQ ID NO:159, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The elongase protein may have an amino acid sequence set forth in SEQ ID NO:5; SEQ ID NO:107; SEQ ID NO:155; SEQ ID NO:157; or SEQ ID NO:159. In other embodiments, the elongase protein is substantially identical to SEQ ID NO:5; SEQ ID NO:107; SEQ ID NO:155; SEQ ID NO:157; or SEQ ID NO:159, and retains the functional activity of the protein of SEQ ID NO:5; SEQ ID NO:107; SEQ ID NO:155; SEQ ID NO:157; or SEQ ID NO:159, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that increases the activity of an elongase protein and (2) a genetic modification that decreases the activity native Δ12 desaturase gene. Similarly, the transformed cell may comprise (1) a genetic modification that increases the activity of an elongase protein and (2) a genetic modification that increases the activity of a diacylglycerol acyltransferase protein.
3. Increasing the Activity of an Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification increases the activity of an acyltransferase protein in the cell.
a. Increasing the Activity of a Type 1 Diacylglycerol Acyltransferase
In some embodiments, the acyltransferase protein is a type 1 diacylglycerol acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a type 1 diacylglycerol acyltransferase protein in the cell.
The nucleic acid may encode a type 1 diacylglycerol acyltransferase gene. In some embodiments, the gene is DGAT1. In some embodiments, the gene is from Arxula adeninivorans, Yarrowia lipolytica, Rhodosporidium toruloides, Lipomyces starkeyi, Aspergillus terreus, Claviceps purpurea, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricornutum, Pichia guilliermondii, Rhodotorula graminis, Rhodosporidium toruloides, Trichoderma virens, and Chaetomium globosum. For example, the gene may be from Claviceps purpurea.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:94; SEQ ID NO:98; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:144; SEQ ID NO:146; SEQ ID NO:148; or SEQ ID NO:150. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:94; SEQ ID NO:98; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:144; SEQ ID NO:146; SEQ ID NO:148; or SEQ ID NO:150. In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:38. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:38.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:93; SEQ ID NO:97; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; or SEQ ID NO:149, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:93; SEQ ID NO:97; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; or SEQ ID NO:149. In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:37, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:37.
The nucleic acid that comprises a gene encoding a type 1 diacylglycerol acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:94; SEQ ID NO:98; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:144; SEQ ID NO:146; SEQ ID NO:148; or SEQ ID NO:150. In other embodiments, the gene is substantially identical to SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:94; SEQ ID NO:98; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:144; SEQ ID NO:146; SEQ ID NO:148; or SEQ ID NO:150 and the nucleotide sequence encodes a protein that retains the diacylglycerol acyltransferase activity of a protein encoded by SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:93; SEQ ID NO:97; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; or SEQ ID NO:149, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The type 1 diacylglycerol acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:93; SEQ ID NO:97; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; or SEQ ID NO:149. In other embodiments, the type 1 diacylglycerol acyltransferase protein is substantially identical to SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:93; SEQ ID NO:97; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; or SEQ ID NO:149, and retains the functional activity of the protein of SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:93; SEQ ID NO:97; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; or SEQ ID NO:149, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise a genetic modification that increases the activity of a DGA1 protein and a genetic modification that increases the activity of a DGA2 protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous DGA2 protein and (2) a knockout mutation in the native DGAT1 gene. Similarly, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous DGA2 protein and (2) a genetic modification that decreases the activity of a native Δ12 desaturase protein.
b. Increasing the Activity of a Type 2 Diacylglycerol Acyltransferase
In some embodiments, the acyltransferase protein is a type 2 diacylglycerol acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a type 2 diacylglycerol acyltransferase protein in the cell.
The nucleic acid may encode a type 2 diacylglycerol acyltransferase gene. In some embodiments, the gene is DGAT2. In some embodiments, the gene is from Aspergillus terreus, Aurantiochytrium limacinum, Arxula adeninivorans, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Phaeodactylum tricornutum, Pichia guilliermondii, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, or Yarrowia lipolytica. The gene may be from Yarrowia lipolytica.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:52; SEQ ID NO:96; SEQ ID NO:100; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; or SEQ ID NO:142. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:52; SEQ ID NO:96; SEQ ID NO:100; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; or SEQ ID NO:142. In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:20. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:20.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:51; SEQ ID NO:95; SEQ ID NO:99; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; or SEQ ID NO:141, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:51; SEQ ID NO:95; SEQ ID NO:99; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; or SEQ ID NO:141. In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:19, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:19.
The nucleic acid that comprises a gene encoding a type 2 diacylglycerol acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:52; SEQ ID NO:96; SEQ ID NO:100; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; or SEQ ID NO:142. In other embodiments, the gene is substantially identical to SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:52; SEQ ID NO:96; SEQ ID NO:100; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; or SEQ ID NO:142, and the nucleotide sequence encodes a protein that retains the type 2 diacylglycerol acyltransferase activity of a protein encoded by SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:51; SEQ ID NO:95; SEQ ID NO:99; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; or SEQ ID NO:141, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The type 2 diacylglycerol acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:51; SEQ ID NO:95; SEQ ID NO:99; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; or SEQ ID NO:141. In other embodiments, the type 2 diacylglycerol acyltransferase protein is substantially identical to SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:51; SEQ ID NO:95; SEQ ID NO:99; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; or SEQ ID NO:141, and retains the functional activity of the protein of SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:51; SEQ ID NO:95; SEQ ID NO:99; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; or SEQ ID NO:141, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise a genetic modification that increases the activity of a DGA1 protein and a genetic modification that increases the activity of a DGA2 protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous DGA1 protein and (2) a knockout mutation in the native DGAT2 gene. Similarly, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous DGA1 protein and (2) a genetic modification that decreases the activity of a native Δ12 desaturase protein.
c. Increasing the Activity of a Type 3 Diacylglycerol Acyltransferase
In some embodiments, the acyltransferase protein is a type 3 diacylglycerol acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a type 3 diacylglycerol acyltransferase protein in the cell.
The nucleic acid may encode a type 3 diacylglycerol acyltransferase gene. In some embodiments, the gene is DGAT3. In some embodiments, the gene is from Ricinus communis or Arachis hypogaea.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:152 or SEQ ID NO:154. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:152 or SEQ ID NO:154.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:151 or SEQ ID NO:153, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:151 or SEQ ID NO:153.
The nucleic acid that comprises a gene encoding a type 3 diacylglycerol acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:152 or SEQ ID NO:154. In other embodiments, the gene is substantially identical to SEQ ID NO:4; SEQ ID NO:8; SEQ ID SEQ ID NO:152 or SEQ ID NO:154, and the nucleotide sequence encodes a protein that retains the type 3 diacylglycerol acyltransferase activity of a protein encoded by SEQ ID NO:151 or SEQ ID NO:153, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The type 3 diacylglycerol acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:151 or SEQ ID NO:153. In other embodiments, the type 3 diacylglycerol acyltransferase protein is substantially identical to SEQ ID NO:151 or SEQ ID NO:153, and retains the functional activity of the protein of SEQ ID NO:151 or SEQ ID NO:153, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
d. Increasing the Activity of a Glycerol-3-Phosphate Acyltransferase
In some embodiments, the acyltransferase protein is a glycerol-3-phosphate acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a glycerol-3-phosphate acyltransferase protein in the cell.
The nucleic acid may encode a glycerol-3-phosphate acyltransferase gene. In some embodiments, the gene is SCT1. In some embodiments, the gene is from Arxula adeninivorans, Phaeodactylum tricornutum, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula graminis, Saccharomyces cerevisiae, or Yarrowia lipolytica. The gene may be from Arxula adeninivorans or Saccharomyces cerevisiae.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:18; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:116; or SEQ ID NO:118. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:18; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:116; or SEQ ID NO:118. In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:42 or SEQ ID NO:44. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:42 or SEQ ID NO:44.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:17; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:115; or SEQ ID NO:117, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:17; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:115; or SEQ ID NO:117. In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:41 or SEQ ID NO:43, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:41 or SEQ ID NO:43.
The nucleic acid that comprises a gene encoding a glycerol-3-phosphate acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:18; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:116; or SEQ ID NO:118. In other embodiments, the gene is substantially identical to SEQ ID NO:18; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:116; or SEQ ID NO:118, and the nucleotide sequence encodes a protein that retains the glycerol-3-phosphate acyltransferase activity of a protein encoded by SEQ ID NO:17; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:115; or SEQ ID NO:117, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The glycerol-3-phosphate acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:17; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:115; or SEQ ID NO:117. In other embodiments, the glycerol-3-phosphate acyltransferase protein is substantially identical to SEQ ID NO:17; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:115; or SEQ ID NO:117, and retains the functional activity of the protein of SEQ ID NO:17; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:115; or SEQ ID NO:117, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or phospholipid:diacylglycerol acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous glycerol-3-phosphate acyltransferase protein and (2) a knockout mutation in a native SCT1 gene. Similarly, the transformed cell may comprise (1) a genetic modification that increases the expression of a glycerol-3-phosphate acyltransferase protein and (2) a genetic modification that decreases the activity of a native Δ12 desaturase protein.
e. Increasing the Activity of a Phospholipid:Diacylglycerol Acyltransferase
In some embodiments, the acyltransferase protein is a phospholipid:diacylglycerol acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a phospholipid:diacylglycerol acyltransferase protein in the cell.
The nucleic acid may encode a phospholipid:diacylglycerol acyltransferase gene. In some embodiments, the gene is LRO1. In some embodiments, the gene is from Arxula adeninivorans or Yarrowia lipolytica
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:106 or SEQ ID NO:110. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:106 or SEQ ID NO:110.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:105 or SEQ ID NO:109, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:105 or SEQ ID NO:109.
The nucleic acid that comprises a gene encoding a phospholipid:diacylglycerol acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:106 or SEQ ID NO:110. In other embodiments, the gene is substantially identical to SEQ ID NO:106 or SEQ ID NO:110, and the nucleotide sequence encodes a protein that retains the phospholipid:diacylglycerol acyltransferase activity of a protein encoded by SEQ ID NO:105 or SEQ ID NO:109, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The phospholipid:diacylglycerol acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:105 or SEQ ID NO:109. In other embodiments, the phospholipid:diacylglycerol acyltransferase protein is substantially identical to SEQ ID NO:105 or SEQ ID NO:109, and retains the functional activity of the protein of SEQ ID NO:105 or SEQ ID NO:109, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
f. Increasing the Activity of a Glycerol-3-Phosphate/Dihydroxyacetone Phosphate Sn-1 Acyltransferase
In some embodiments, the acyltransferase protein is a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein in the cell.
The nucleic acid may encode a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase gene. In some embodiments, the gene is GPT2. In some embodiments, the gene is from Saccharomyces cerevisiae, Naumovozyma dairenensis, Torulaspora delbrueckii, or Naumovozyma castellii.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; or SEQ ID NO:126. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; or SEQ ID NO:126.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; or SEQ ID NO:125, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; or SEQ ID NO:125.
The nucleic acid that comprises a gene encoding a glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; or SEQ ID NO:126. In other embodiments, the gene is substantially identical to SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; or SEQ ID NO:126, and the nucleotide sequence encodes a protein that retains the glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase activity of a protein encoded by SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; or SEQ ID NO:125, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; or SEQ ID NO:125. In other embodiments, the glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein is substantially identical to SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; or SEQ ID NO:125, and retains the functional activity of the protein of SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; or SEQ ID NO:125, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein and (2) a knockout mutation in a native GPT2 gene.
g. Increasing the Activity of a Sn-2 Acylglycerol Fatty Acyltransferase
In some embodiments, the acyltransferase protein is a sn-2 acylglycerol fatty acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a sn-2 acylglycerol fatty acyltransferase protein in the cell.
The nucleic acid may encode a sn-2 acylglycerol fatty acyltransferase gene. In some embodiments, the gene is SLC1 or SLC4. In some embodiments, the gene is from Arxula adeninivorans, Saccharomyces cerevisiae, Phaeodactylum tricornutum, Rhodosporidium toruloides, Rhodotorula minuta, Rhodotorula graminis, or Yarrowia lipolytica.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:60; SEQ ID NO:62; 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; or SEQ ID NO:78. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:60; SEQ ID NO:62; 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; or SEQ ID NO:78.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:59; SEQ ID NO:61; 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; or SEQ ID NO:77, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:59; SEQ ID NO:61; 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; or SEQ ID NO:77.
The nucleic acid that comprises a gene encoding a sn-2 acylglycerol fatty acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:60; SEQ ID NO:62; 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; or SEQ ID NO:78. In other embodiments, the gene is substantially identical to SEQ ID NO:60; SEQ ID NO:62; 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; or SEQ ID NO:78, and the nucleotide sequence encodes a protein that retains the sn-2 acylglycerol fatty acyltransferase activity of a protein encoded by SEQ ID NO:59; SEQ ID NO:61; 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; or SEQ ID NO:77, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The sn-2 acylglycerol fatty acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:59; SEQ ID NO:61; 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; or SEQ ID NO:77. In other embodiments, the sn-2 acylglycerol fatty acyltransferase protein is substantially identical to SEQ ID NO:59; SEQ ID NO:61; 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; or SEQ ID NO:77, and retains the functional activity of the protein of SEQ ID NO:59; SEQ ID NO:61; 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; or SEQ ID NO:77, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
h. Increasing the Activity of a Lysophosphatidic Acid Acyltransferase
In some embodiments, the acyltransferase protein is a lysophosphatidic acid acyltransferase protein. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a lysophosphatidic acid acyltransferase protein in the cell.
The nucleic acid may encode a lysophosphatidic acid acyltransferase gene. In some embodiments, the gene is LOA1. In some embodiments, the gene is from Arxula adeninivorans, Saccharomyces cerevisiae, or Yarrowia lipolytica.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:80; SEQ ID NO:82; or SEQ ID NO:84. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:80; SEQ ID NO:82; or SEQ ID NO:84.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83.
The nucleic acid that comprises a gene encoding a lysophosphatidic acid acyltransferase protein may comprise a nucleotide sequence set forth in SEQ ID NO:80; SEQ ID NO:82; or SEQ ID NO:84. In other embodiments, the gene is substantially identical to SEQ ID NO:80; SEQ ID NO:82; or SEQ ID NO:84, and the nucleotide sequence encodes a protein that retains the lysophosphatidic acid acyltransferase activity of a protein encoded by SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The lysophosphatidic acid acyltransferase protein may have an amino acid sequence set forth in SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83. In other embodiments, the lysophosphatidic acid acyltransferase protein is substantially identical to SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83, and retains the functional activity of the protein of SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
4. Increasing the Activity of a Phosphatidate Phosphatase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification increases the activity of a phosphatidate phosphatase protein in the cell. The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that increases the activity of a phosphatidate phosphatase protein in the cell.
The nucleic acid may encode a phosphatidate phosphatase gene. In some embodiments, the gene is PAH1. In some embodiments, the gene is from Arxula adeninivorans, Saccharomyces cerevisiae, or Yarrowia lipolytica.
In some embodiments, the nucleic acid comprises a nucleotide sequence that has at least 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 sequence homology with the sequence set forth in SEQ ID NO:54; SEQ ID NO:56; or SEQ ID NO:58. The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:54; SEQ ID NO:56; or SEQ ID NO:58.
In some embodiments, the nucleic acid encodes an amino acid sequence having at least 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 sequence homology with the sequence set forth in SEQ ID NO:53; SEQ ID NO:55; or SEQ ID NO:57, or a biologically active portion thereof. The nucleic acid may encode the amino acid sequence set forth in SEQ ID NO:53; SEQ ID NO:55; or SEQ ID NO:57.
The nucleic acid that comprises a gene encoding a phosphatidate phosphatase protein may comprise a nucleotide sequence set forth in SEQ ID NO:54; SEQ ID NO:56; or SEQ ID NO:58. In other embodiments, the gene is substantially identical to SEQ ID NO:54; SEQ ID NO:56; or SEQ ID NO:58, and the nucleotide sequence encodes a protein that retains the phosphatidate phosphatase activity of a protein encoded by SEQ ID NO:53; SEQ ID NO:55; or SEQ ID NO:57, yet differs in nucleotide sequence, e.g., due to natural allelic variation or mutagenesis.
The phosphatidate phosphatase protein may have an amino acid sequence set forth in SEQ ID NO:53; SEQ ID NO:55; or SEQ ID NO:57. In other embodiments, the phosphatidate phosphatase protein is substantially identical to SEQ ID NO:53; SEQ ID NO:55; or SEQ ID NO:57, and retains the functional activity of the protein of SEQ ID NO:53; SEQ ID NO:55; or SEQ ID NO:57, yet differs in amino acid sequence, e.g., due to natural allelic variation or mutagenesis.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
C. Nucleic Acids and Methods for Decreasing the Activity of a Native Protein
In some embodiments, the transformed oleaginous cell comprises a genetic modification that decreases the activity of a native protein. Such genetic modifications may affect a protein that regulates the transcription of the native protein, 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 the native protein and alter other gene expression profiles that shift the cellular equilibrium toward increased oleic acid accumulation. Alternatively, the genetic modification may be the introduction of an interfering nucleic acid, such as a small interfering RNA, or a nucleic acid that encodes an interfering nucleic acid. In other embodiments, the genetic modification consists of the homologous recombination of a nucleic acid and the regulatory region of a gene that encodes the native protein. The regulatory region of the gene may include an operator, promoter, sequences upstream from the promoter, enhancers, and/or sequences downstream of the gene.
In some embodiments the transformed oleaginous cell comprises a genetic modification consisting of a homologous recombination event. In certain embodiments, the transformed cell comprises a genetic modification consisting of a homologous recombination event between a native gene and a nucleic acid. Thus, the genetic modification deletes the native gene, prevents its transcription, or prevents the transcription of a gene that can be translated into a fully-active protein. A homologous recombination event may mutate or delete a portion of a native gene. For example, the homologous recombination event may mutate one or more residues in the active site of a native enzyme, thereby reducing the efficiency of the enzyme or rendering it inactive. Alternatively, the homologous recombination event may affect post-translational modification, folding, stability, or localization within the cell. In some embodiments, the homologous recombination event replaces the promoter with a promoter that drives less transcription. In other embodiments, the homologous recombination event mutates the promoter to impair its ability to drive transcription. In certain embodiments, the genetic modification is a knockout mutation.
A knockout mutation may delete one or more genes. Additionally, the knockout mutation may substitute a native gene with an exogenous gene that encodes a different protein. The exogenous 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 native gene such that the native gene's promoter drives transcription of the exogenous 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)).
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 native gene, and in the second event, a nucleic acid encoding the remaining portion of the gene recombines with the native 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.
In certain embodiments, the gene encodes a marker protein, such as 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.
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.
Different approaches may be used to knockout a gene in a yeast cell (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 genes in other species, such as Arxula adeninivorans.
In some embodiments, a genetic modification decreases the expression of a native 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.
In some embodiments, a genetic modification decreases the efficiency of a native protein 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.
In some embodiments, a genetic modification decreases the activity of a native protein 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.
1. Decreasing the Activity of a Native Δ12 Desaturase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native Δ12 desaturase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native Δ12 desaturase protein in the cell. The nucleic acid may be capable of recombining with a native Δ12 desaturase gene and/or a nucleotide sequence in the regulatory region of a native Δ12 desaturase gene. In some embodiments, the native Δ12 desaturase protein is encoded by the Δ12 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native Δ12 desaturase protein has the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the cell is Yarrowia lipolytica, and the native Δ12 desaturase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:2.
In certain embodiments, the cell is Arxula adeninivorans and the native Δ12 desaturase protein has the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the cell is Arxula adeninivorans, and the native Δ12 desaturase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:50.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native Δ12 desaturase protein and (2) a genetic modification that increases the activity of a Δ9 desaturase protein. Similarly, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native Δ12 desaturase protein and (2) a genetic modification that increases the activity of a diacylglycerol acyltransferase protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
2. Decreasing the Activity of a Native Δ9 Desaturase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native Δ9 desaturase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native Δ9 desaturase protein in the cell. The nucleic acid may be capable of recombining with a native Δ9 desaturase gene and/or a nucleotide sequence in the regulatory region of a native Δ9 desaturase gene. In some embodiments, the native Δ9 desaturase protein is encoded by the Δ9, OLE1, or FAD1 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native Δ9 desaturase protein has the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the cell is Yarrowia lipolytica, and the native Δ9 desaturase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:4.
In certain embodiments, the cell is Arxula adeninivorans and the native Δ9 desaturase protein has the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the cell is Arxula adeninivorans, and the native Δ9 desaturase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:8.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase (e.g., an exogenous Δ9 desaturase), elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native Δ9 desaturase protein and (2) a genetic modification that consists of transformation with a nucleic acid encoding a Δ9 desaturase protein, e.g., a Δ9 desaturase protein from a different species. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
3. Decreasing the Activity of a Native Diacylglycerol Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native diacylglycerol acyltransferase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native diacylglycerol acyltransferase protein in the cell. The nucleic acid may be capable of recombining with a native diacylglycerol acyltransferase gene and/or a nucleotide sequence in the regulatory region of a native diacylglycerol acyltransferase gene. In some embodiments, the native diacylglycerol acyltransferase protein is encoded by the DGAT1 or DGAT2 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native diacylglycerol acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:19 or SEQ ID NO:93. In some embodiments, the cell is Yarrowia lipolytica, and the native diacylglycerol acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:20 or SEQ ID NO:94.
In certain embodiments, the cell is Arxula adeninivorans and the native diacylglycerol acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:51 or SEQ ID NO:103. In some embodiments, the cell is Arxula adeninivorans, and the native diacylglycerol acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:52 or SEQ ID NO:104.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase (e.g., an exogenous diacylglycerol acyltransferase), sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native diacylglycerol acyltransferase protein and (2) a genetic modification that consists of transformation with a nucleic acid encoding a diacylglycerol acyltransferase protein, e.g., a diacylglycerol acyltransferase protein from a different species. Similarly, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native diacylglycerol acyltransferase protein and (2) a genetic modification that increases the activity of a Δ9 desaturase protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase (e.g., a different diacylglycerol acyltransferase), native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native DGA1 protein and (2) a genetic modification that decreases the activity of a native DGA2 protein. Similarly, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native diacylglycerol acyltransferase protein and (2) a genetic modification that decreases the activity of a native Δ12 desaturase protein.
4. Decreasing the Activity of a Native Triacylglycerol Lipase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native triacylglycerol lipase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native triacylglycerol lipase protein in the cell. The nucleic acid may be capable of recombining with a native triacylglycerol lipase gene and/or a nucleotide sequence in the regulatory region of a native triacylglycerol lipase gene. In some embodiments, the native triacylglycerol lipase is encoded by the TGL3, TGL3/4, or TGL4 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native triacylglycerol lipase protein has the amino acid sequence set forth in SEQ ID NO:91. In some embodiments, the cell is Yarrowia lipolytica, and the native triacylglycerol lipase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:92.
In certain embodiments, the cell is Arxula adeninivorans and the native triacylglycerol lipase protein has the amino acid sequence set forth in SEQ ID NO:85; SEQ ID NO:87; or SEQ ID NO:89. In some embodiments, the cell is Arxula adeninivorans, and the native triacylglycerol lipase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:86; SEQ ID NO:88; or SEQ ID NO:90.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native triacylglycerol lipase protein and (2) a genetic modification that increases the activity of a diacylglycerol acyltransferase protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
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 oleic acid. 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. Triacylglycerol lipase knockouts are described in WO 2015/168531 and U.S. Ser. No. 61/987,098 (both of which are incorporated by reference).
In some embodiments, the triacylglycerol lipase is TGL3. In other embodiments, the triacylglycerol lipase is TGL3/4 or TGL4.
The TGL3 gene in Y. lipolytica encodes the triacylglycerol lipase protein TGL3 (SEQ ID NO:91). SEQ ID NO:92 contains the TGL3 nucleotide sequence, 100 upstream nucleotides, and 100 downstream. Thus, the SEQ ID NO:92 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.
Knockout cassettes SEQ ID NOs: 167 and 168 are capable of recombining with the native TGL3 gene in Y. lipolytica. Thus, in some embodiments, the nucleic acids encoded by SEQ ID NOs: 167 and 168 may be used to generate a triacylglycerol lipase knockout mutation in Y. lipolytica. SEQ ID NOs: 167 and 168 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:167 nor SEQ ID NO:168 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.
Knockout cassette SEQ ID NO:167 may be prepared by amplifying a hygromycin resistance gene hph (SEQ ID NO:162) with primer NP1798 (SEQ ID NO:165) and primer NP656 (SEQ ID NO:164). Knockout cassette SEQ ID NO:50 may be prepared by amplifying a hygromycin resistance gene hph (SEQ ID NO:162) with primer NP655 (SEQ ID NO:163) and primer NP1799 (SEQ ID NO:166).
Different approaches may be used to design nucleic acids that reduce the activity of TGL3 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 reduce triacylglycerol lipase activity in other species. For example, these methods may be used to reduce the activity of the TGL3 gene of Arxula adeninivorans (SEQ ID NO:86), the TGL3/4 gene of Arxula adeninivorans (SEQ ID NO:88), or the TGL4 gene of Arxula adeninivorans (SEQ ID NO:90). Similarly, these methods are generally applicable to reduce the activity of a protein in yeast and other organism.
5. Decreasing the Activity of a Native Sn-2 Acylglycerol Fatty Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native sn-2 acylglycerol fatty acyltransferase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native sn-2 acylglycerol fatty acyltransferase protein in the cell. The nucleic acid may be capable of recombining with a native sn-2 acylglycerol fatty acyltransferase gene and/or a nucleotide sequence in the regulatory region of a native sn-2 acylglycerol fatty acyltransferase gene. In some embodiments, the native sn-2 acylglycerol fatty acyltransferase protein is encoded by the SLC1 or SLC4 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native sn-2 acylglycerol fatty acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:59 or SEQ ID NO:65. In some embodiments, the cell is Yarrowia lipolytica, and the native sn-2 acylglycerol fatty acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:60 or SEQ ID NO:66.
In certain embodiments, the cell is Arxula adeninivorans and the native sn-2 acylglycerol fatty acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:61 or SEQ ID NO:63. In some embodiments, the cell is Arxula adeninivorans, and the native sn-2 acylglycerol fatty acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:62 or SEQ ID NO:64.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase (e.g., an exogenous sn-2 acylglycerol fatty acyltransferase), lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
6. Decreasing the Activity of a Native Lysophosphatidic Acid Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native lysophosphatidic acid acyltransferase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native lysophosphatidic acid acyltransferase protein in the cell. The nucleic acid may be capable of recombining with a native lysophosphatidic acid acyltransferase gene and/or a nucleotide sequence in the regulatory region of a native lysophosphatidic acid acyltransferase gene. In some embodiments, the native lysophosphatidic acid acyltransferase protein is encoded by the LOA1 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native lysophosphatidic acid acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:83. In some embodiments, the cell is Yarrowia lipolytica, and the native lysophosphatidic acid acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:84.
In certain embodiments, the cell is Arxula adeninivorans and the native lysophosphatidic acid acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:81. In some embodiments, the cell is Arxula adeninivorans, and the native lysophosphatidic acid acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:82.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase (e.g., an exogenous lysophosphatidic acid acyltransferase), phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
7. Decreasing the Activity of a Native Phosphatidate Phosphatase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native phosphatidate phosphatase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native phosphatidate phosphatase protein in the cell. The nucleic acid may be capable of recombining with a native phosphatidate phosphatase gene and/or a nucleotide sequence in the regulatory region of a native phosphatidate phosphatase gene. In some embodiments, the native phosphatidate phosphatase protein is encoded by the PAH1 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native phosphatidate phosphatase protein has the amino acid sequence set forth in SEQ ID NO:57. In some embodiments, the cell is Yarrowia lipolytica, and the native phosphatidate phosphatase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:58.
In certain embodiments, the cell is Arxula adeninivorans and the native phosphatidate phosphatase protein has the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, the cell is Arxula adeninivorans, and the native phosphatidate phosphatase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:56.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase (e.g., an exogenous phosphatidate phosphatase), phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native glycerol-3-phosphate acyltransferase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase.
8. Decreasing the Activity of a Native Glycerol-3-Phosphate Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native glycerol-3-phosphate acyltransferase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native glycerol-3-phosphate acyltransferase protein in the cell. The nucleic acid may be capable of recombining with a native glycerol-3-phosphate acyltransferase gene and/or a nucleotide sequence in the regulatory region of a native glycerol-3-phosphate acyltransferase gene. In some embodiments, the native glycerol-3-phosphate acyltransferase is encoded by the SCT1 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native glycerol-3-phosphate acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:17. In some embodiments, the cell is Yarrowia lipolytica, and the native glycerol-3-phosphate acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:18.
In certain embodiments, the cell is Arxula adeninivorans and the native glycerol-3-phosphate acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the cell is Arxula adeninivorans, and the native glycerol-3-phosphate acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:44.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase (e.g., an exogenous glycerol-3-phosphate acyltransferase). For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native glycerol-3-phosphate acyltransferase protein and (2) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous glycerol-3-phosphate acyltransferase protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native phospholipid:diacylglycerol acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native glycerol-3-phosphate acyltransferase protein and (2) a genetic modification that decreases the activity of a native Δ12 desaturase protein.
9. Decreasing the Activity of a Native Phospholipid:Diacylglycerol Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native phospholipid:diacylglycerol acyltransferase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native phospholipid:diacylglycerol acyltransferase protein in the cell. The nucleic acid may be capable of recombining with a native phospholipid:diacylglycerol acyltransferase gene and/or a nucleotide sequence in the regulatory region of a native phospholipid:diacylglycerol acyltransferase gene. In some embodiments, the native phospholipid:diacylglycerol acyltransferase protein is encoded by the LRO1 gene.
In certain embodiments, the cell is Yarrowia lipolytica and the native phospholipid:diacylglycerol acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:109. In some embodiments, the cell is Yarrowia lipolytica, and the native phospholipid:diacylglycerol acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:110.
In certain embodiments, the cell is Arxula adeninivorans and the native phospholipid:diacylglycerol acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:105. In some embodiments, the cell is Arxula adeninivorans, and the native phospholipid:diacylglycerol acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:106.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase (e.g., an exogenous phospholipid:diacylglycerol acyltransferase), glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or glycerol-3-phosphate acyltransferase. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase, and/or native glycerol-3-phosphate acyltransferase.
10. Decreasing the Activity of a Native Glycerol-3-Phosphate/Dihydroxyacetone Phosphate Sn-1 Acyltransferase
In some aspects, the invention relates to a transformed cell comprising a genetic modification, wherein the genetic modification decreases the activity of a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein. In some embodiments, the genetic modification is a knockout mutation.
The genetic modification may be transformation with a nucleic acid. In certain embodiments, the invention relates to a method of modifying the lipid content of a cell, comprising transforming the cell with a nucleic acid that decreases the activity of a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein in the cell. The nucleic acid may be capable of recombining with a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase gene and/or a nucleotide sequence in the regulatory region of a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase gene. In some embodiments, the native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein is encoded by the GPT2 gene.
In certain embodiments, the cell is Saccharomyces cerevisiae and the native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein has the amino acid sequence set forth in SEQ ID NO:119. In some embodiments, the cell is Saccharomyces cerevisiae, and the native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein is encoded by the nucleotide sequence set forth in SEQ ID NO:120.
In some embodiments, the transformed cell further comprises a genetic modification that increases the activity of a Δ9 desaturase, elongase, diacylglycerol acyltransferase, sn-2 acylglycerol fatty acyltransferase, lysophosphatidic acid acyltransferase, phosphatidate phosphatase, phospholipid:diacylglycerol acyltransferase, glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase (e.g., an exogenous glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase), and/or glycerol-3-phosphate acyltransferase. For example, the transformed cell may comprise (1) a genetic modification that decreases the activity of a native glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein and (2) a genetic modification that consists of transformation with a nucleic acid that encodes an exogenous glycerol-3-phosphate/dihydroxyacetone phosphate sn-1 acyltransferase protein. In some embodiments, the transformed cell further comprises a genetic modification that decreases the activity of a native Δ9 desaturase, native Δ12 desaturase, native diacylglycerol acyltransferase, native triacylglycerol lipase, native sn-2 acylglycerol fatty acyltransferase, native lysophosphatidic acid acyltransferase, native phosphatidate phosphatase, native phosphatidate phosphatase, and/or native glycerol-3-phosphate acyltransferase.
D. Products
In certain embodiments, the transformed cells 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, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapenteaenoic acid, docosapentaenoic acid, eicosadienoic acid, and/or eicosatrienoic acid.
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, α-linolenic acid, arachidonic acid, eicosapenteaenoic acid, erucic acid, or docosahexaenoic acid.
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.
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.
Exemplification
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.

Example 1: Method to Increase the Activity of a DGA1 Protein (DGAT2 Gene)

Exemplary nucleic acid constructs for overexpressing DGA1 were described in U.S. Ser. No. 61/943,664 (hereby incorporated by reference). FIG. 2 shows expression construct pNC243 used for overexpression of the R. toruloides DGA1 gene NG66 (SEQ ID NO:22) 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 DGAT2 gene and for the Nat1 gene, used as a marker for selection with nourseothricin (NAT).
DGA1 expression constructs were randomly integrated into the genome of Y. 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 μg/mL NAT.
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 Natl cassette, or due to a negative effect of the site of DGA1 integration on DGA1 expression. All transformants had a significant increase in lipid content.
In certain experiments, the effect of native R. toruloides DGA1 overexpression on lipid production in Y. lipolytica was not as high as the effect of synthetic versions of R. toruloides DGAT2 genes that did not contain introns. This result may indicate that the gene splicing of the R. toruloides DGAT2 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.
The skilled artisan will recognize that similar methods may be used to increase the activity of other proteins in a range of organisms.

Example 2: Method to Decrease the Activity of a Native Triacylglycerol Lipase Protein

Exemplary nucleic acid constructs for knocking out the Y. lipolytica TGL3 gene while overexpressing the DGAT2 gene were described in WO 2015/168531 and U.S. Ser. No. 61/987,098 (both of which are incorporated by reference). The TGL3 gene was knocked out of 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: 92) 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: 162) using primer pairs NP1798-NP656 and NP655-NP1799 (SEQ ID NOs: 163-166). The resulting PCR fragments (SEQ ID NOs: 167 & 168) were co-transformed into NS18 and NS281 according to the protocol developed in WO 2014/182657 and U.S. Ser. No. 61/819,746 (both of which are 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.
The skilled artisan will recognize that similar methods may be used to decrease the activity of other proteins in a range of organisms.

Example 3: Decreasing Δ12 Desaturase Activity in Y. lipolytica

Δ12 desaturase is responsible for the production of linoleic acid through the desaturation of oleic acid, converting C18:1 fatty acid to C18:2. The J12 gene (SEQ ID NO:2) was deleted in Y. Lipolytica strain NS18 to produce strain NS419. Lipid accumulation was induced in NS419 and lipid composition was analyzed. Deletion of the J12 gene led to a complete elimination of linoleic acid production and a concomitant increase in oleic acid (FIG. 3 ).

Example 4: Increasing Δ9 Desaturase Activity in Y. lipolytica

Δ9 desaturase is responsible for the production of oleic acid through the desaturation of stearic acid, converting C18:0 fatty acid to C18:1. The Δ9 gene (SEQ ID NO:4) was overexpressed in Y. Lipolytica strain NS18 to produce strain NS441. Lipid accumulation was induced and lipid composition was analyzed. Overexpression of the z9 gene led to an increase in oleic (and palmitoleic) acids (FIG. 4 ).

Example 5: Increasing Elongase Activity in Y. lipolytica

Elongases extend the carbon chain of fatty acids beyond the length produced by fatty acid synthases. YALI0F06754 (SEQ ID NO:6) was identified as a Y. lipolytica gene with limited homology to S. cerevisiae elongases. Although this gene has not been annotated as an elongase, its function was assessed in Y. lipolytica, and YALI0F06754 was found to play a role in the elongation of C16 to C18 fatty acids. YALI0F06754 was thus termed ELO1.
The deletion of ELO1 in Y. lipolytica strain NS18 led to a decrease in C18 levels (FIG. 5A). The ELO1 knockout in Y. lipolytica strain NS18 was named NS276. In contrast, overexpression of ELO1 led to an increase in C18 levels (FIG. 5B), and specifically, the overexpression of ELO1 increased oleic acid levels (FIG. 5C). ELO1 overexpression was performed in strain NS452, which overexpresses Y. lipolytica DGA1 and Claviceps purpurea DGA2 and contains a J12 desaturase deletion, resulting in strain NS477.

Example 6: Switching Δ9 Desaturase Specificity in Y. lipolytica

The native Δ9 desaturase of Y. lipolytica uses both C16 and C18 saturated fatty acids as substrates. Exogenous Δ9 enzymes were screened for higher C18 specificity by introducing the genes (SEQ ID NOs: 8, 10, 12, 14, & 16) as the sole Δ9 activity in Y. lipolytica. This was achieved by first deleting Y. lipolytica Δ9 in NS18 to produce strain NS418. NS418 required supplementation with unsaturated fatty acids, such as oleic acid and/or Tween-80, for growth due to the absence of Δ9 activity. The exogenous z9 genes were then inserted into the native locus through targeted integration and selected for the ability to grow without supplementation. Expression of Δ9 enzymes from the source organisms shown here in the absence of the native enzyme resulted in a switch in substrate specificity to overwhelmingly C18:0 substrate, thus reducing C16:1 content to minimal levels. Δ9 enzymes from A. adeninivorans (SEQ ID NO:8) and Puccinia graminis (SEQ ID NO:14) resulted in the highest oleic acid levels (FIG. 6 ).

Example 7: Decreasing Acyltransferase Activity in Y. lipolytica

When a native acyltransferase activity exhibits substrate preference for fatty acids, deletion of the gene can affect fatty acid composition. The glycerol acyltransferase SCT1 (SEQ ID NO:18) was deleted in NS18 to produce strain NS563. Lipid accumulation was induced and lipid composition was analyzed. Deletion of SCT1 led to an increase in the oleic acid lipid fraction (FIG. 7 ).

Example 8: Increasing Acyltransferase Activity in Y. lipolytica

The overexpression of an acyltransferase can improve total lipid levels to achieve high lipid yields. It is important that the overexpressed acyltransferase have a desirable substrate specificity to maintain or increase the oleic acid content of the cell. Overexpression can be in the wild-type acyltransferase background or in a strain that comprises a deletion of a native acyltransferase. The type 2 diacylglycerol acyltransferases from various species (SEQ ID NOs: 20, 22, 24, 26, 28, & 30) were expressed in NS18. The DGAT2 gene from Y. lipolytica, which encodes the DGA1 protein, resulted in the highest oleic acid levels (FIG. 8 ). Similarly, type 1 diacylglycerol acyltransferases from different species (SEQ ID NOs: 32, 34, 36, 38, & 40) were expressed in NS281 (made by overexpressing R. toruloides DGA1 in NS18). The DGA2 gene from C. purpurea resulted in the highest oleic acid levels (FIG. 9 ). Additionally, glycerol-3-phosphate acyltransferases from different species (SEQ ID NOs: 18, 42, 44, 46, & 48) were expressed in a strain carrying deletions of native SCT1 and Δ12 genes (NS564). The SCT1 genes from S. cerevisiae and A. adeninivorans resulted in the highest oleic acid levels (FIG. 10 ).

Example 9: Decreasing Δ12 Desaturase Activity in A. Adeninivorans

The Δ12 gene (SEQ ID NO:50) was deleted from A. adeninivorans strain NS252 (ATCC 76597) to produce strain NS478. Lipid accumulation was induced and lipid composition was analyzed. Deletion of Δ12 led to a complete elimination of linoleic acid production and a concomitant increase in oleic acid (FIG. 11 ).

Example 10: Increasing Acyltransferase Activity in A. Adeninivorans

As in Y. lipolytica, overexpression of an acyltransferase in A. adeninivorans can improve total lipid levels to achieve high lipid yields. It is important that the overexpressed acyltransferase have a desirable substrate specificity to maintain or increase the oleic acid content of the cell. Overexpression can be in the wild-type acyltransferase background or a strain deleted for a native acyltransferase. Type 2 diacylglycerol acyltransferases from different species (SEQ ID NOs: 20, 22, & 52) were expressed in A. adeninivorans strain NS252. The DGA1 gene from Y. lipolytica (SEQ ID NO:20) resulted in the highest oleic acid levels (FIG. 12 ).

Example 11: Increasing Δ9 Desaturase Aactivity in Y. Lipolytica

The Y. lipolytica Δ9 desaturase gene was overexpressed in strain NS477, which is described in Example 5, resulting in strain NS551. Strain NS551 expresses Y. lipolytica DGA1, C. purpurea DGA2, Y. lipolytica ELO1, Y. lipolytica Δ9, and comprises a Δ12 knockout. This strain contains approximately 87% oleic acid as a percentage of total C16 and C18 fatty acids. FIG. 13 provides an overview of the bioengineering of strain NS551.

Example 12: Increasing elongase activity in A. adeninivorans

Elongase genes from A. adeninivorans (AaELO1, SEQ ID NO:108; AaELO2, SEQ ID NO:160), Y. lipolytica (Yl ELO1, SEQ ID NO:6), S. cerevisiae (ScELO1, SEQ ID NO:158), and R. norvegicus (rELO2, SEQ ID NO:156) were expressed in A. adeninivorans strain NS554 via random genomic integration of a linear expression cassette. NS554 carries deletion of the Δ12 desaturase gene (SEQ ID NO:50) in wild-type A. adeninivorans.
The expression of AaELO1, YlELO1, and rELO2 all increased the C18 fatty acid content of the cells, suggesting increased elongation of C16 fatty acids (FIG. 14 ). AaELO1, YlELO1, and rELO2 expression in NS554 also increased oleic acid content (FIG. 15 ).

Example 13: Switching elongase specificity in Y. lipolytica

Elongase genes from A. adeninivorans (AaELO1, SEQ ID NO:108; AaELO2, SEQ ID NO:160), Y. lipolytica (Yl ELO1, SEQ ID NO:6), S. cerevisiae (ScELO1, SEQ ID NO:158), and R. norvegicus (rELO2, SEQ ID NO:156) were expressed in Y. lipolytica strain NS276 via random genomic integration of a linear expression cassette. NS276 carries deletion of the ELO1 gene (SEQ ID NO:6) in wild-type Y. lipolytica. Expression of rELO2 increased the C18 fatty acid content of the cells, suggesting increased elongation of C16 fatty acids (FIG. 16 ). Additionally, rELO2 expression in NS276 also increased oleic acid content (FIG. 15 ).

Example 14: Increasing elongase activity in A. adeninivorans

The elongase 1 gene from Y. lipolytica (Yl ELO1, SEQ ID NO:6) was expressed in A. adeninivorans strain NS557 via random genomic integration of a linear expression cassette. NS557 carries a deletion of the Δ12 desaturase gene (SEQ ID NO:50) in wild-type A. adeninivorans and the Y. lipolytica gene for DGA1 (SEQ ID NO:20). Most transformants produced increased C18 fatty acids, suggesting that C16 fatty acids were elongated by the elongase (FIG. 17 ). Ninety-five total isolates were screened, and FIG. 17 depicts representative results. The top performing isolate was named NS776, and its lipid composition was further analyzed. Approximately 87% of the C16 and C18 fatty acids in strain NS776 were oleic acid (FIG. 18 ).

Example 15: Combinations of Genetic Modifications in Y. lipolytica

Various combinations of genetic modifications were introduced into Y. lipolytica. The strategy for introducing some of modifications is shown in FIG. 19 . Wild type Y. lipolytica strain NS18 was used as the parent strain (obtained from ARS Culture Collection, NRRL #YB 392). Strain NS804 was prepared from strain NS18 by first deleting the SCT1 gene (SEQ ID NO:18) and then adding the SCT1 gene from A. adeninivorans (SEQ ID NO:44). Strain NS809 was prepared from strain NS804 by first deleting the Δ9 desaturase gene (SEQ ID NO:4) and then adding the Δ9 desaturase gene from Puccinia graminis (SEQ ID NO:14). Strain NS810 was prepared from strain NS804 by first deleting the Δ9 desaturase gene (SEQ ID NO:4) and then adding the Δ9 desaturase gene from A. adeninivorans (SEQ ID NO:8).
Strain NS813 was prepared from strain NS18 by deleting the Δ12 desaturase gene (SEQ ID NO:2), overexpressing the DGA1 gene from Y. lipolytica (SEQ ID NO:20), adding the DGA2 gene from C. purpurea (SEQ ID NO:38), and adding the ELO2 gene from R. norvegicus (SEQ ID NO:156).
Strain NS814 was prepared from strain NS18 by deleting the Δ12 desaturase gene (SEQ ID NO:2), deleting the Δ9 desaturase gene (SEQ ID NO:4), adding the Δ9 desaturase gene from A. adeninivorans (SEQ ID NO:8), deleting the SCT1 gene (SEQ ID NO:18), adding the SCT1 gene from A. adeninivorans (SEQ ID NO:44), and adding the ELO2 gene from R. norvegicus (SEQ ID NO:156).
The fatty acid profiles for strains NS18, NS804, NS809, NS810, NS813, and NS814 are shown in FIG. 20 . Each modified strain produced more oleic acid (C18:1) than the wild type strain.
Strain NS968 was prepared from strain NS809 by deleting the Δ12 gene (SEQ ID NO:2). Strain NS975 was prepared from strain NS968 by adding the ELO2 gene from R. norvegicus (SEQ ID NO:156), and adding the DGA1 gene from R. toruloides (SEQ ID NO:22). Strains NS992, NS993, and NS994 are three isolates prepared from strain NS975 by adding the Δ9 desaturase gene from Puccinia graminis (SEQ ID NO:14), adding an additional copy of the DGA1 gene from R. toruloides (SEQ ID NO:22), and adding the DGA2 gene from C. purpurea (SEQ ID NO:38).
Strain NS812 was prepared from strain NS810 by deleting the Δ12 gene (SEQ ID NO:2). Strain NS969 was prepared from strain NS812 by adding the Δ9 desaturase gene from Puccinia graminis (SEQ ID NO:14), adding the DGA1 gene from R. toruloides (SEQ ID NO:22), adding the ELO2 gene from R. norvegicus (SEQ ID NO:156), and adding the DGA2 gene from C. purpurea (SEQ ID NO:38).
Strain NS662 was prepared from strain NS18 by deleting the Δ12 gene (SEQ ID NO:2), deleting the SCT1 gene (SEQ ID NO:18), and adding the SCT1 gene from A. adeninivorans (SEQ ID NO:44).
The fatty acid profiles for strains NS18, NS804, NS809,NS968, NS975, NS992, NS993, NS994, NS810, NS812, NS969, NS987, NS988, NS551 (described in Example 11), and NS622 are shown in FIG. 21 . Each modified strain produced more oleic acid (C18:1) than the wild type strain. Additionally, each strain modified with the DGA2 gene from C. purpurea (SEQ ID NO:38) comprised more lipids than the wild type NS18 strain.

INCORPORATION BY REFERENCE

Each of the patents, published patent applications, and non-patent references cited herein is hereby incorporated by reference in its entirety.

EQUIVALENTS

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.

Claims

1-20. (canceled)

21. A method for producing a product, wherein the product is a lipid, the method comprising cultivating a modified yeast cell thereby producing the lipid, wherein the modified yeast cell comprises:

a first genetic modification, wherein the first genetic modification comprises a knockout mutation of a native Δ12 desaturase protein;

a second genetic modification, wherein the second genetic modification increases the expression of an elongase protein, a diacylglycerol acyltransferase protein, or a glycerol-3-phosphate acyltransferase protein; and

a third genetic modification, wherein the third genetic modification comprises a nucleic acid encoding an exogenous fungal Δ9 desaturase protein or an exogenous Δ9 desaturase protein that comprises increased specificity for C18 a fatty acid relative to a native Δ9 desaturase protein of the modified yeast cell.

22. The method of claim 21, wherein the second genetic modification comprises a nucleic acid encoding an elongase protein, a diacylglycerol acyltransferase protein, or a glycerol-3-phosphate acyltransferase protein.

23. The method of claim 21, wherein the exogenous fungal Δ9 desaturase protein is a Puccinia graminis, Arxula adeninivorans or Microbotryum violaceum Δ9 desaturase protein.

24. The method of claim 21, wherein the yeast cell is 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.

25. The method of claim 21, wherein the yeast cell is selected from the group consisting of Arxula adeninivorans, Saccharomyces cerevisiae, and Yarrowia lipolytica.

26. The method of claim 21, wherein the yeast cell comprises at least 50% lipid as measured by % dry cell weight.

27. The method of claim 21, wherein the yeast cell comprises oleic acid at a concentration of at least 70% as a percentage of total C16 and C18 fatty acids in the yeast cell.

28. The method of claim 21, wherein the second genetic modification increases the expression of a glycerol-3-phosphate acyltransferase protein.

29. The method of claim 28, wherein the glycerol-3-phosphate acyltransferase protein is a native protein.

30. The method of claim 22, wherein the second genetic modification comprises a nucleic acid encoding an exogenous glycerol-3-phosphate acyltransferase protein.

31. The method of claim 21, wherein the second genetic modification increases the expression of an elongase protein.

32. The method of claim 21, wherein the second genetic modification increases the expression of a diacylglycerol acyltransferase protein.

33. The method of claim 21, wherein the yeast cell comprises a knockout mutation of a native Δ9 desaturase protein.

34. The method of claim 22, wherein the glycerol-3-phosphate acyltransferase protein comprises an Arxula adeninivorans glycerol-3-phosphate acyltransferase, a Saccharomyces cerevisiae glycerol-3-phosphate acyltransferase, or a glycerol-3-phosphate acyltransferase comprising amino acid sequence having 90% sequence identity to the sequence encoded by SEQ ID NO: 44 or 42.

35. The method of claim 22, wherein the elongase protein is an exogenous elongase protein.

36. The method of claim 22, wherein the elongase protein comprises a Rattus norvegicus elongase, a Yarrowia lipolytica elongase, an Arxula Adeninivorans elongase, or an elongase comprising an amino acid sequence having 90% sequence identity to the sequence encoded by SEQ ID NO: 156, 6, or 108.

37. The method of claim 22, wherein the diacylglycerol acyltransferase protein is an exogenous diacylglycerol acyltransferase protein.

38. The method of claim 22, wherein the diacylglycerol acyltransferase protein comprises a Rhodosporidium toruloides diacylglycerol acyltransferase, a Yarrowia lipolytica diacylglycerol acyltransferase, an Arxula adeninivorans diacylglycerol acyltransferase, a Claviceps purpurea diacylglycerol acyltransferase or a diacylglycerol acyltransferase comprising an amino acid sequence having 90% sequence identity to the sequence set forth in SEQ ID NO: 22, 20, 52, or 38.

39. The method of claim 21, wherein the exogenous Δ9 desaturase protein is a fungal Δ9 desaturase protein.

40. The method of claim 21, wherein the Δ9 desaturase protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by SEQ ID NO: 14, 8 or 12.

41. The method of claim 21, wherein the second genetic modification comprises a nucleic acid encoding (a) a diacylglycerol acyltransferase type 1 protein, (b) a diacylglycerol acyltransferase type 2 protein, or (c) a diacylglycerol acyltransferase type 1 protein and a diacylglycerol acyltransferase type 2 protein.

42. The method of claim 21, wherein the second genetic modification comprises one or more nucleic acids encoding an elongase protein, a diacylglycerol acyltransferase protein, and a glycerol-3-phosphate acyltransferase protein.

43. The method of claim 42, wherein (a) the elongase protein comprises a Rattus norvegicus elongase, a Yarrowia lipolytica elongase, or an elongase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 156, 6 or 108; (b) the diacylglycerol acyltransferase protein comprises a Rhodosporidium toruloides diacylglycerol acyltransferase, a Yarrowia lipolytica diacylglycerol acyltransferase, an Arxula adeninivorans diacylglycerol acyltransferase, a Claviceps purpurea diacylglycerol acyltransferase, or a diacylglycerol acyltransferase comprising an amino acid sequence having 90% sequence identity to the amino acid sequence set forth in SEQ ID Nos. 22, 20, 52, or 38; and (c) the glycerol-3-phosphate acyltransferase protein comprises an Arxula adeninivorans glycerol-3-phosphate acyltransferase or a glycerol-3-phosphate acyltransferase comprising an amino acid sequence having 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 44 or 42.