(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`FP7
`
`||||||IlllllllllIllllllllllIlllllllllllll|||||||ll||lllllllllllllllllllllllllllllllllllllllllll
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`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`18 June 2009 (18.06.2009)
`
`(51) International Patent Classification:
`CIZP 7/64 (2006.01)
`
`
`
`(21) International Application Number:
`PC’l‘fU82008/086485
`
`(22) International Filing Date:
`11 December 2008 (11.12.2008)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`61/007,333
`
`11 December 2007 (11.12.2007)
`
`US
`
`(74)
`
`(81)
`
`(10) International Publication Number
`
`WO 2009/076559 A1
`
`Agents: RUBE, Daniel et al.; Morrison & Foerster LLP,
`12531 High Bluff Dirve, Suite 100, San Diego, CA 92130-
`2040 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
`EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
`IL, 1N, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT,
`RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY. TJ,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL,
`NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for all designated States except US): SYN-
`THETIC GENOMICS, INC. [US/US]; 11149 N. Torrey
`Pines Rd, Suite 100, La Jolla, CA 92037 (US).
`
`(84)
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): ROESSLER, Paul,
`Gordon [US/US]; 11149 N. Torrey Pines Rd, Suite 100,
`La Jolla, CA 92037 (US). CHEN, You [CN/US]; 11149
`N. Torrey Pines Rd, Suite 100, La Jolla, CA 92037 (US).
`LIU, Bo [CN/US]; 11149 N. Torrey Pines Rd, Suite 100,
`La Jolla, CA 92037 (US). DODGE, Corey, Neal [US/US];
`11149 N. Torrey Pines Rd, Suite 100, La Jolla, CA 92037
`(US).
`
`Published:
`with international search report
`
`
`
`(54) Title: SECRETION OF FATTY AICDS BY PHOTOSYNTHETIC MICROORGANISMS
`
`(57) Abstract: Recombinant photosynthetic microorganisms that convert inorganic carbon to secreted fatty acids are described.
`Methods to recover the secreted fatty acids from the culture medium without the need for cell harvesting are also described.
`
`
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`02009/076559A1llllllIlllllllllIlllllIllllIlllllllllllllllllllllllllllllllllllllllIllll|||lllll|ll|||||l|ll|||
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`

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`WO 2009/076559
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`PCT/U52008/086485
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`SECRETION OF FATTY ACIDS BY PHOTOSYNTHETIC MICROORGANISMS
`
`Cross—Reference to Related Applications
`
`[0001] This application claims benefit of provisional application 61/007,333 filed
`
`11 December 2007. The contents of this application are incorporated herein by reference.
`
`Technical Field
`
`[0002] This invention relates to photosynthetic microorganisms that convert inorganic
`
`carbon to fatty acids and secrete them into the culture medium, methods of production of
`
`fatty acids using such organisms, and uses thereof. The fatty acids may be used directly or
`
`may be further modified to alternate forms such as esters, reduced forms such as alcohols, or
`
`hydrocarbons, for applications in different industries, including fuels and chemicals.
`
`Background Art
`
`[0003] Photosynthetic microorganisms, including eukaryotic algae and cyanobacteria,
`
`contain various lipids, including polar lipids and neutral lipids. Polar lipids (e.g.,
`
`phospholipids, glycolipids, sulfolipids) are typically present in structural membranes
`
`whereas neutral lipids (e.g., triacylglycerols, wax esters) accumulate in cytoplasmic oil
`
`bodies or oil globules. A substantial research effort has been devoted to the development of
`
`methods to produce lipid-based fuels and chemicals from photosynthetic microorganisms.
`
`Typically, eukaryotic microalgae are grown under nutrient—replete conditions until a certain
`
`cell density is achieved, after which the cells are subjected to growth under nutrient-
`
`deficient conditions, which often leads to the accumulation of neutral lipids. The cells are
`
`then harvested by various means (e.g., settling, which can be facilitated by the addition of
`
`flocculants, followed by centrifugation), dried, and then the lipids are extracted from the
`
`cells by the use of various non-polar solvents. Harvesting of the cells and extraction of the
`
`lipids are cost—intensive steps. It would be desirable to obtain lipids from photosynthetic
`
`microorganisms without the requirement for cell harvesting and extraction.
`
`[0004] PCT publication numbers W02007/ 136762 and W02008/l 19082 describe the
`
`production of biofuel components using microorganisms. These documents disclose the
`
`production by these organisms of fatty acid derivatives which are, apparently, short and long
`
`chain alcohols, hydrocarbons, fatty alcohols and esters including waxes, fatty acid esters or
`
`

`

`WO 2009/076559
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`PCT/U82008/086485
`
`fatty esters. To the extent that fatty acid production is described, it is proposed as an
`
`intermediate to these derivatives, and the fatty acids are therefore not secreted. Further,
`
`there is no disclosure of converting inorganic carbon directly to secreted fatty acids using a
`
`photosynthetic organism grown in a culture medium containing inorganic carbon as the
`
`primary carbon source. The present invention takes advantage of the efficiency of
`
`photosynthetic organisms in secreting fatty acids into the medium in order to recover these
`
`valuable compounds.
`
`[0005] The invention includes the expression of heterologous acyl—ACP thioesterase
`
`(TE) genes in photosynthetic microbes. Many of these genes, along with their use to alter
`
`lipid metabolism in oilseeds, have been described previously. Genes encoding the proteins
`
`that catalyze various steps in the synthesis and further metabolism of fatty acids have also
`
`been extensively described.
`
`[0006] The two functional classes of plant acyl-ACP thioesterases (unsaturated fatty
`
`acid—recognizing Fat A versus saturated fatty acid-recognizing FatB) can be clustered based
`
`on amino acid sequence alignments as well as function. FatAs show marked preference for
`
`18:1—ACP with minor activity towards 18:0— and 16:0—ACPs, and FatBs hydrolyze primarily
`
`saturated acyl—ACPs with chain lengths that vary between 8—16 carbons. Several studies
`
`have focused on engineering plant thioesterases with perfected or altered substrate
`
`specificities as a strategy for tailoring specialty seed oils.
`
`[0007] As shown in Figure 1, fatty acid synthetase catalyzes a repeating cycle wherein
`
`malonyl-acyl carrier protein (ACP) is condensed with a substrate, initially acetyl-CoA, to
`
`form acetoacetyl-ACP, liberating C02. The acetoacetyl-ACP is then reduced, dehydrated,
`
`and reduced further to butyiyl-ACP which can then itself be condensed with malonyl-ACP,
`
`and the cycle repeated, adding a 2-carbon unit at each turn. The production of free fatty
`
`acids would therefore be enhanced by a thioesterase that would liberate the fatty acid itself
`
`from ACP, breaking the cycle. That is, the acyl—ACP is prevented from reentering the cycle.
`
`Production of the fatty acid would also be encouraged by enhancing the levels of fatty acid
`
`synthetase and inhibiting any enzymes which result in degradation or further metabolism of
`
`the fatty acid.
`
`[0008] Figure 2 presents a more detailed description of the sequential formation of acy1~
`
`ACPs of longer and longer chains. As shown, the thioesterase enzymes listed in Figure 2
`
`liberate the fatty acid from the ACP thioester.
`
`[0009] Taking advantage of this principle, Dehesh, K., et al., The Plant Journal (1996)
`
`9:167-172, describe “Production of high levels of octanoic (8:0) and decanoic (10:0) fatty
`
`2
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`

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`WO 2009/076559
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`PCT/U52008/086485
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`acids in transgenic canola by overexpression of ChFatBZ, a thioesterase cDNA from CLlphea
`
`hookeriana.” Dehesh, K., et al., Plant Physiology (1996) 1102203-210, and report “Two
`
`novel thioesterases are key determinants of the bimodal distribution of acyl chain length of
`
`Cup/tea palustris seed oil.”
`
`[0010] Voelker, T., et al., Science (1992) 257:72—74, describe “Fatty acid biosynthesis
`
`redirected to medium chains in transgenic oilseed plants.” Voelker, T., and Davies, M.,
`
`Journal of Bacteriology (1994) 176:7320-7327, describe “Alteration of the specificity and
`
`regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium—chain
`
`acyl—acyl carrier protein thioesterase.”
`
`Disclosure of the Invention
`
`[0011] The present invention is directed to the production of recombinant
`
`photosynthetic microorganisms that are able to secrete fatty acids derived from inorganic
`
`carbon into the culture medium. Methods to remove the secreted fatty acids from the
`
`culture medium without the need for cell harvesting are also provided. It is anticipated that
`
`these improvements will lead to lower costs for producing lipid—based fuels and chemicals
`
`from photosynthetic microorganisms.
`
`In addition, this invention enables the production of
`
`fatty acids of defined chain length, thus allowing their use in the formulation of a variety of
`
`different products, including fuels and chemicals.
`
`[0012] Carbon dioxide (which, along with carbonic acid, bicarbonate and/or carbonate
`
`define the term “inorganic carbon”) is converted in the photosynthetic process to organic
`
`compounds. The inorganic carbon source includes any way of delivering inorganic carbon,
`
`optionally in admixture with any other combination of compounds which do not serve as the
`
`primary carbon feedstock, but only as a mixture or carrier (for example, emissions from
`
`biofuel (e.g., ethanol) plants, power plants, petroleum-based refineries, as well as
`
`atmospheric and subterranean sources).
`
`[0013] One embodiment of the invention relates to a culture of recombinant
`
`photosynthetic microorganisms, said organisms comprising at least one recombinant
`
`expression vector encoding at least one exogenous acyl-ACP thioesterase, wherein the at
`
`least one exogenous acyl—ACP thioesterase preferentially liberates fatty acid chains
`
`containing 6 to 20 carbons from these ACP thioesters. The fatty acids are formed from
`
`inorganic carbon as their carbon source and the culture contains substantially only inorganic
`
`carbon as a carbon source. The presence of the exogenous thioesterase will increase the
`
`secretion levels of desired fatty acids by at least 2-4 fold.
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`

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`WO 2009/076559
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`PCT/U52008/086485
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`[0014]
`
`Specifically, in one embodiment, the invention is directed to a cell culture of
`
`a recombinant photosynthetic microorganism where the microorganism has been modified
`
`to contain a nucleic acid molecule comprising at least one recombinant expression system
`
`that produces at least one exogenous acyl—ACP thioesterase, wherein said acyl-ACP
`
`thioesterase preferentially liberates a fatty acid chain that contains 6-20 carbons, and
`
`wherein the culture medium provides inorganic carbon as substantially the sole
`
`carbon source and wherein said microorganism secretes the fatty acid liberated by the acyl-
`
`ACP thioesterase into the medium.
`
`In alternative embodiments, the thioesterase
`
`preferentially liberates a fatty acid chain that contains 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
`
`17, 18, 19 or 20 carbons.
`
`[0015]
`
`In other aspects, the invention is directed to a method to produce fatty acids of
`
`desired chain lengths by incubating these cultures and recovering these secreted fatty acids
`
`from the cultures. In one embodiment, the recovery employs solid particulate adsorbents to
`
`harvest the secreted fatty acids. The fatty acids thus recovered can be further modified
`
`synthetically or used directly as components of biofuels or chemicals.
`
`Brief Description of the Drawings
`
`[0016] Figure l is a diagram of the pathway of fatty acid Synthesis as is known in the
`
`art.
`
`[0017] Figure 2 is a more detailed diagram of the synthesis of fatty acids of multiple
`
`chain lengths as is known in the art.
`
`[0018] Figure 3 is an enzymatic overview of fatty acid biosynthesis identifying
`
`enzymatic classes for the production of various chain length fatty acids.
`
`[0019] Figure 4is a schematic diagram of a recovery system for fatty acids from the
`
`medium.
`
`[0020] Figure 5 shows an experimental system based on the principles in Figure 4.
`
`[0021] Figure 6 shows representative acyl—ACP thioesterase from a variety of
`
`organisms.
`
`Modes of Carrying Out the Invention
`
`[0022] The present invention provides photosynthetic microorganisms that secrete fatty
`
`acids into the culture medium, along with methods to adsorb the fatty acids from the culture
`
`medium and collect them for processing into fuels and chemicals. The invention thereby
`
`

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`WO 2009/076559
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`PCT/US2008/086485
`
`eliminates or greatly reduces the need to harvest and extract the cells, resulting in
`
`substantially reduced production costs.
`
`[0023] Figure 2 is an overview of one aspect of the invention. As shown in Figure 2,
`
`carbon dioxide is converted to acetyl-CoA using the multiple steps in the photosynthetic
`
`process. The acetyl-CoA is then convelted to malonyl—COA by the action of acetyl—CoA
`
`carboxylase. The malonyl—CoA is then converted to malonyl-ACP by the action of malonyl-
`
`CoAzACP transacylase which, upon progressive action of fatty acid synthetase, results in
`
`successive additions of two carbon units. In one embodiment of the invention, the process is
`
`essentially halted at carbon Chain lengths of 6 or 8 or 10 or 12 or 14 or 16 or 18 carbons by
`
`supplying the appropriate thioesterase (shown in Figure 2 as FatB). To the extent that
`
`further conversions to longer chain fatty acids occur in this embodiment, the cell biomass
`
`can be harvested as well. The secreted fatty acids can be converted to various other forms
`
`including, for example, methyl esters, alkanes, alkenes, alpha-olefins and fatty alcohols.
`
`Thioesterases {Acyl—ACP TEs)
`
`[0024]
`
`In order to effect secretion of the free fatty acids, the organism is provided at
`
`least one expression system for at least one thioesterase that operates preferentially to
`
`liberate fatty acids of the desired length. Many genes encoding such thioesterases are
`
`available in the alt. Some of these are subjects of U.S. patents as follows:
`
`[0025] Examples include U.S. Patent 5298421, entitled “Plant medium-chain—preferrin g
`
`acyl-ACP thioesterases and related methods,” which describes the isolation of an acyl-ACP
`
`thioesterase and the gene that encodes it from the immature seeds of Umbellularia
`
`californica. Other sources for such thioesterases and their encoding genes include U.S.
`
`Patent 5,304,481, entitled “Plant thioesterase having preferential hydrolase activity toward
`
`C12 acyl—ACP substrate,” U.S. Patent 5,344,771, entitled “Plant thioesterases,” U.S. Patent
`
`5,455 ,167, entitled “Medium-chain thioesterases in plants,” U.S. Patent 5,512,482, entitled
`
`“Plant thioesterases,” U.S. Patent 5,530,186, entitled “Nucleotide sequences of soybean
`
`acyl-ACP thioesterase genes,” U.S. Patent 5,639,790, entitled “Plant medium-chain
`
`thioesterases,” U.S. Patent 5,667,997, entitled “C8 and C10 medium—chain thioesterases in
`
`plants,” U.S. Patent 5,723,761, entitled “Plant acyl—ACP thioesterase sequences,” U.S.
`
`Patent 5,807,893, entitled “Plant thioesterases and use for modification of fatty acid
`
`composition in plant seed oils,” U.S. Patent 5,850,022, entitled “Production of myristate in
`
`plant cells,” U.S. Patent 5,910,631, entitled “Middle chain-specific thioesterase genes from
`
`Cuphea lanceolara,” U.S. Patent 5,945,585, entitled “Specific for palmitoyl, stearoyl and
`
`

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`WO 2009/076559
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`PCT/U82008/086485
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`oleoyl—alp thioesters nucleic acid fragments encoding acyl—ACP thioesterase enzymes and
`
`the use of these fragments in altering plant oil composition,” US. Patent 5,955,329, entitled
`
`“Engineering plant thioesterases for altered substrate specificity,” U.S. Patent 5 ,955 ,650,
`
`entitled “Nucleotide sequences of canola and soybean palmitoyl—ACP thioesterase genes and
`
`their use in the regulation of fatty acid content of the oils of soybean and canola plants,” and
`
`U.S. Patent 6,331,664, entitled “Acyl—ACP thioesterase nucleic acids from maize and
`
`methods of altering palmitic acid levels in transgenic plants therewit
`
`.”
`
`[0026] Others are described in the open literature as follows:
`
`[0027] Dormann, P. et al., Planra (1993) 189:425—432, describe “Characterization of
`
`two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for
`
`medium—chain and oleoyl—acyl carrier protein.” Dormann, P., et al., Biochimica Biophysica
`
`Acra (1994) 1212:134-136, describe “Cloning and expression in Escherichia call of a CDNA
`
`coding for the oleoyl-acyl carrier protein thioesterase from coriander (Coriandrum sativum
`
`L.).” Filichkin, S., et (11., European Journal of Lipid Science and Technology (2006)
`
`108:979—990, describe “New FATB thioesterases from a high-laurate Cuphea species:
`
`Functional and complementation analyses.” Jones, A., et al, Plant Cell (1995) 72359—371,
`
`describe “Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of
`
`plant acyl—ACP thioesterases.” Knutzon, D. S., et al., Plant Physiology (1992) 100:1751-
`
`l758, describe “Isolation and characterization of two safflower oleoyl-acyl carrier protein
`
`thioesterase cDNA clones.” Slabaugh, M., et al, The Plant Journal (1998) 13:611-620,
`
`describe “Condensing enzymes from Cuphea wrighrii associated with medium chain fatty
`
`acid biosynthesis.”
`
`[0028] Additional genes, not previously isolated, that encode these acyl-ACP TEs can
`
`be isolated from plants that naturally contain large amounts of medium—chain fatty acids in
`
`their seed oil, including certain plants in the Lauraceae, Lythraceae, Rutaceae, Ulmaceae,
`
`and Vochysiaceae families. Typically, the fatty acids produced by the seeds of these plants
`
`are esterified to glycerol and retained inside the cells. The seeds containing the products can
`
`then be harvested and processed to isolate the fatty acids. Other sources of these enzymes,
`
`such as bacteria may also be used.
`
`[0029] The known acyl—ACP TEs from plants can be divided into two main classes,
`
`based on their amino acid sequences and their specificity for acyl—ACPs of differing chain
`
`lengths and degrees of unsaturation. The “FatA” type of plant acyl—ACP TE has preferential
`
`activity on oleoyl-ACP, thereby releasing oleic acid, an lS—carbon fatty acid with a single
`
`double bond nine carbons distal to the carboxyl group. The “FatB” type of plant acyl—ACP
`
`6
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`WO 2009/076559
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`PCT/US2008/086485
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`TE has preferential activity on saturated acyl-ACPs, and can have broad or narrow chain
`
`length specificities. For example, FatB enzymes from different species of Cuphea have
`
`been shown to release fatty acids ranging from eight carbons in length to sixteen carbons in
`
`length from the corresponding acyl-ACPS. Listed below in Table l are several plant acyl-
`
`ACP TEs along with their substrate preferences. (Fatty acids are designated by standard
`
`shorthand notation, wherein the number preceding the colon represents the acyl chain length
`
`and the number after the colon represents the number of double bonds in the acyl Chain.)
`
`Table 1
`
`Plant Acyl-ACP Thioesterase
`
`Garcinia mangosmna FatA
`18 1 and 18:0
`
`Coriandrum sativum FatA
`
`, Cuphea hookeriana FatBl
`
`1210to 16:0
`:‘Cuphea wrightii FatBl
`I 8:0 and 10:0
`H
`:CLlphea palustris FatBl
`3":140snd.1.§..9,-fi.i§i
`FLIP/WPCIWW.1??th 7
`iiib‘téiéto ..............
`9C’i’rafiééééléiiial'léi FtBl
`
`5‘aaazzgza‘aa“gnaw;agar“““"‘*-"15;5""""""""""
`
`j Ulmus americana FatBl
`8:0 and 10:0
`
`V
`L
`
`if
`
`i
`
`I
`
`i
`
`i
`
`[0030] The enzymes listed in Table 1 are exemplary and many additional genes
`
`encoding acyl-ACP TEs can be isolated and used in this invention, including but not limited
`
`to genes such as those that encode the following acy1«ACP TEs (referred to by GenPept
`
`Accession Numbers):
`
`CAA52069.1, CAA52070.1, CAA54060.1, CAA85387.1, CAA85388.1, CAB60830.1,
`CAC19933.1, CAC19934.1, CAC39106.1, CAC80370.1, CAC80371.1, CAD32683.1,
`CAL50570.1, CAN60643.1, CAN81819.1, CAOl7726.l, CAO42218.1, CAO65585.1,
`CAO68322.1, AAA33019.1, AAA33020.1, AAB51523.1, AAB51524.1, AAB51525.1,
`AAB71729.1, AAB71730.1, AAB71731.1, AAB88824.1, AAC49001.1, AAC49002.1,
`AAC49179.1, AAC49180.1, AAC49269.1, AAC49783.1, AAC49784.1, AAC72881.1,
`AAC72882.1, AAC72883.1, AAD01982.1, AAD28187.1, AAD33870.1, AAD42220.2,
`AAG35064.1, AAG43857.1, AAG43858.1, AAG43859.1, AAG43860.1, AAG43861.1,
`AAL15645.1, AAL77443.1, AAL77445.1, AAL79361.1, AAMO9524.1, AAN17328.1,
`
`AAQ08202.1, AAQO8223.1, AAX51636.1, AAX51637.1, ABB71579.1, ABB71581.1,
`ABC47311.1, ABD83939.1, ABE01139.1, ABH11710.1, AB118986.1, AB120759.1,
`AB120760.1, ABL85052.1, ABU96744.1, EAY74210.1, EAY86874.1, EAY86877.1,
`EAY86884.1, EAY99617.1, EA201545.1, EAZO9668.1, EA212044.1, EAZZ3982.1,
`EAZ37535.1, EAZ45287.1, NP_001047567.1, NP_001056776.1, NP_001057985.1,
`NP_001063601.1, NP__001068400.1, NP_172327.1, NP_189147.1, NP_193041.1,
`
`7
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`WO 2009/076559
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`PCT/US2008/086485
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`XP_001415703.1, Q39473, Q39513,Q41635,Q42712,Q9SQ13, NP_189147.1,
`AAC49002, CAA52070.1, CAA52069.1, 1930411, CAC39106, CA017726,
`AAC72883, AAA33020, AAL79361, AAQ08223.1, AAB51523, AAL77443,
`AAA33019, AAG35064, and AAL77445.
`
`Additional sources of acyl—ACP TEs that are useful in the present invention include:
`
`Arabidopsis thaliana (At); Bradyrhizobium japonicum (Bj); Brassica napus (Bn);
`
`Cinnamonum camphorum (CC); Capsicum chinense (Cch); Cuphea hookeriana (Ch);
`
`Cup/tea lanceolata (Cl); Cuphea palustris (Cp); Coriandmm sativum (Cs); Carthamus
`
`linctorius (Ct); Cuphea wrightii (Cw); Elaeis guineensis (Eg); Gossypium hirsutum (Gh);
`
`Garcinia mangostana (Gm); Helianthus (anIMS (Ha); Iris germanica (Ig); Iris tectorum (It);
`
`Myrisricafragrans (Mt); Triticum aestivum (Ta); Ulmus Americana (Ua); and Umbellularia
`
`calzfornica (Uc). Exemplary TEs are shown in Figure 6 with corresponding NCBI
`
`accession numbers.
`
`[0031]
`
`In one embodiment, the present invention contemplates the specific production
`
`of an individual length of medium—chain fatty acid, for example, predominently producing
`
`C8 fatty acids in one culture of recombinant photosynthetic microorganisms. In another
`
`embodiment, the present invention contemplates the production of a combination of two or
`
`more different length fatty acids, for example, both C8 and C10 fatty acids in one culture of
`
`recombinant photosynthetic microorganisms.
`
`[0032]
`
`Illustrated below are manipulations of these art—known genes to construct
`
`suitable expression systems that result in production of effective amounts of the
`
`thioesterases in selected recombinant photosynthetic organisms. In such constructions, it
`
`may be desirable to remove the portion of the gene that encodes the plastid transit peptide
`
`region, as this region is inappropriate in prokaryotes. Alternatively, if expression is to take
`
`place in eukaryotic cells, the appropriate plastid transit peptide encoding region to the host
`
`organism may be substituted. Preferred codons may also be employed, depending on the
`
`host.
`
`Other Modifications
`
`[0033]
`
`In addition to providing an expression System for one or more appropriate acyl—
`
`ACP TE genes, further alterations in the photosynthetic host may be made. For example,
`
`the host may be modified to include an expression system for a heterologous gene that
`
`encodes a B—ketoacyl synthase (KAS) that preferentially produces acyl—ACPs having
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`medium chain lengths. Such KAS enzymes have been described from several plants,
`
`including various species of Cuphea. See Dehesh, K., et 51]., The Plant Journal (1998)
`
`15:383—390, describe “KAS IV: a 3—ketoacyl—ACP synthase from Cuphea sp. is a medium
`
`chain specific condensing enzyme”; Slabaugh, M., er (11., The Plant Journal (1998) 13:611-
`
`620), and would serve to increase the availability of acyl-ACP molecules of the proper
`
`length for recognition and cleavage by the heterologous medium-chain acyl-ACP TE.
`
`Another example is that the photosynthetic host cell containing a heterologous acyl-ACP TE
`
`gene may be further modified to include an expression system for a heterologous gene that
`
`encodes a multifunctional acetyl-CoA carboxylase or a set of heterologous genes that
`
`encode the various subunits of a multi—subunit type of acetyl—CoA carboxylase. Other
`
`heterologous genes that encode additional enzymes or components of the fatty acid
`
`biosynthesis pathway could also be introduced and expressed in acyl-ACP TE—containing
`
`host cells.
`
`[0034] The photosynthetic microorganism may also be modified such that one or more
`
`genes that encode beta-oxidation pathway enzymes have been inactivated or downregulated,
`
`or the enzymes themselves may be inhibited. This would prevent the degradation of fatty
`
`acids released from acyl-ACPs, thus enhancing the yield of secreted fatty acids.
`
`In cases
`
`where the desired products are medium—chain fatty acids, the inactivation or downregulation
`
`of genes that encode acyl—CoA synthetase and/or acyl-CoA oxidase enzymes that
`
`preferentially use these chain lengths as substrates would be beneficial. Mutations in the
`
`genes encoding medium-chain-specific acyl-CoA synthetase and/or medium-chain-specific
`acyl—COA oxidase enzymes such that the activity of the enzymes is diminished would also
`
`be effective in increasing the yield of secreted fatty acids. An additional modification
`
`inactivates or down—regulates the acyl-ACP synthetase gene or inactivates the gene or
`
`protein. Mutations in the genes can be introduced either by recombinant or non-recombinant
`
`methods. These enzymes and their genes are well known, and may be targeted specifically
`
`by disruption, deletion, generation of antisense sequences, generation of ribozymes or other
`
`recombinant approaches known to the practitioner. Inactivation of the genes can also be
`
`accomplished by random mutation techniques such as UV, and the resulting cells screened
`
`for successful mutants. The proteins themselves can be inhibited by intracellular generation
`
`of appropriate antibodies or intracellular generation of peptide inhibitors.
`
`[0035] The photosynthetic microorganism may also be modified such that one or more
`
`genes that encode storage carbohydrate or polyhydroxyalkanoate (FHA) biosynthesis
`
`pathway enzymes have been inactivated or down—regulated, or the enzymes themselves may
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`be inhibited. Examples include enzymes involved in glycogen, starch, or chrysolaminaiin
`
`synthesis, including glucan synthases and branching enzymes. Other examples include
`
`enzymes involved in PHA biosynthesis such as acetoacetyl-CoA synthase and PHA
`
`synthase.
`
`Expression Systems
`
`[0036] Expression of heterologous genes in cyanobacteria and eukaryotic algae is
`
`enabled by the introduction of appropriate expression vectors. For transformation of
`
`cyanobacteria, a variety of promoters that function in cyanobacteria can be utilized,
`
`including, but not limited to the lac, tac, and trc promoters and derivatives that are inducible
`
`by the addition of isopropyl B-D—l-thiogalactopyranoside (IPTG), promoters that are
`
`naturally associated with transposon- or bacterial chromosome—borne antibiotic resistance
`
`genes (neomycin phosphotransferase, chloramphenicol acetyltransferase, spectinomycin
`
`adenyltransferase, etc.), promoters associated with various heterologous bacterial and native
`
`cyanobacterial genes, promoters from viruses and phages, and synthetic promoters.
`
`Promoters isolated from cyanobacteria that have been used successfully include the
`
`following:
`
`secA (secretion; controlled by the redox state of the cell)
`rbc (Rubisco operon)
`psaAB (PS Ireaction center proteins; light regulated)
`psbA (D1 protein of PSII; light—inducible)
`
`[0037] Likewise, a wide variety of transcriptional terminators can be used for expression
`
`vector construction. Examples of possible terminators include, but are not limited to, psbA,
`
`psaAB, rbc, secA, and T7 coat protein.
`
`[0038] Expression vectors are introduced into the cyanobacterial strains by standard
`
`methods, including, but not limited to, natural DNA uptake, conjugation, electroporation,
`
`particle bombardment, and abrasion with glass beads, SiC fibers, or other particles. The
`
`vectors can be: 1) targeted for integration into the cyanobacterial chromosome by including
`
`flanking sequences that enable homologous recombination into the chromosome, 2) targeted
`
`for integration into endogenous cyanobacterial plasmids by including flanking sequences
`
`that enable homologous recombination into the endogenous plasmids, or 3) designed such
`
`that the expression vectors replicate within the chosen host.
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`[0039] For transformation of green algae, a variety of gene promoters and terminators
`
`that function in green algae can be utilized, including, but not limited to promoters and
`
`terminators from Chlamydomonas and other algae, promoters and terminators from viruses,
`
`and synthetic promoters and terminators.
`
`[0040] Expression vectors are introduced into the green alga] strains by standard
`
`methods, including, but not limited to, electroporation, particle bombardment, and abrasion
`
`with glass beads, SiC fibers, or other particles. The vectors can be 1) targeted for site-
`
`specific integration into the green algal chloroplast chromosome by including flanking
`
`sequences that enable homologous recombination into the chromosome, or 2) targeted for
`
`integration into the cellular (nucleus-localized) chromosome.
`
`[0041] For transformation of diatoms, a variety of gene promoters that function in
`
`diatoms can be utilized in these expression vectors, including, but not limited to:
`
`l) promoters from Thalassiosira and other heterokont algae, promoters from viruses, and
`
`synthetic promoters. Promoters from Thalassiosira psemlonana that would be suitable for
`
`use in expression vectors include an alpha-tubulin promoter (SEQ ID NO:1), a beta-tubulin
`
`promoter (SEQ ID N02), and an actin promoter (SEQ ID N0:3). Promoters from
`
`Phaeodactylum tricomutum that would be suitable for use in expression vectors include an
`
`alpha~tubulin promoter (SEQ ID NO:4), a beta—tubulin promoter (SEQ ID NO:5), and an
`
`actin promoter (SEQ ID N016). These sequences are deduced from the genomic sequences
`
`of the relevant organisms available in public databases and are merely exemplary of the
`
`wide variety of promoters that can be used. The terminators associated with these and other
`
`genes, or particular heterologous genes can be used to stop transcription and provide the
`
`appropriate signal for polyadenylation and can be derived in a similar manner or are known
`
`in the art.
`
`Expression vectors are introduced into the diatom strains by standard methods, including,
`
`but not limited to, electroporation, particle bombardment, and abrasion with glass beads,
`
`SiC fibers, or other particles. The vectors can be 1) targeted for site-specific integration into
`
`the diatom chloroplast chromosome by including flanking sequences that enable
`
`homologous recombination into the chromosome, or 2) targeted for integration into the
`
`cellular (nucleus—localized) chromosome.
`
`Host Organisms
`
`[0042] The host cells used to prepare the cultures of the invention include any
`
`photosynthetic organism which is able to convert inorganic carbon into a substrate that is in
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`turn converted to fatty acid derivatives. These organisms include prokaryotes as well as
`
`eukaryotic organisms such as algae and diatoms.
`
`[0043] Host organisms include eukaryotic algae and cyanobacteria (blue-green algae).
`
`Representative algae include green algae (chlorophytes), red algae, diatoms, prasinophytes,
`
`glaucophytes, chlorarachniophytes, euglenophytes, chromophytes, and dinoflagellates. A
`
`number of cyanobacterial species are known and have been manipulated using molecular
`
`biological techniques, including the unicellular cyanobacteria Synechocystis sp. PCC6 803
`
`and Synechococcus elongates PCC7942, whose genomes have been completely sequenced.
`
`[0044] The following genera of cyanobacteria may be used: one group includes
`
`Chamaesiphon
`Chroococcus
`
`Cyanobacterium
`Cyanobium
`Cyanothece
`
`Dactylococcopsis
`Gloeobacter
`
`Gloeocapsa
`Gloeothece
`
`Microcystis
`
`[0045] Another group includes
`
`Cyanocyszis
`Dermocarpella
`Stanieria
`
`Xenococcus
`
`Ch