WO2010019813A2

WO2010019813A2 - Production of fatty actds by genetically modified photosynthetic organisms

Info

Publication number: WO2010019813A2
Application number: PCT/US2009/053772
Authority: WO
Inventors: Michael Mendez; Bryan O'neill; Michael Burkart; Craig Behnke; Soyan Lieberman; Vince Bielinski; Yan Poon
Original assignee: Sapphire Energy Inc
Current assignee: Sapphire Energy Inc
Priority date: 2008-08-13
Filing date: 2009-08-13
Publication date: 2010-02-18
Anticipated expiration: 2011-02-13
Also published as: WO2010019813A3

Abstract

The present invention relates to compositions that facilitate the production of enzymes by genetically modified photosynthetic microorganisms wherein the enzymes contribute to the synthesis of fatty acids, lipids or oils. In some embodiments the compositions are one or more vectors that encode one or more polypeptides that increase production of fatty acids, lipids or oils. The vectors may comprise promoters and other nucleic acid sequences configured for expression of the polypeptides in the chloroplast of non-vascular, photosynthetic organisms. In some embodiments the compositions are genetically altered photosynthetic microorgansims or genetically altered chloroplasts. The present invention provides methods of producing one or more proteins, including fatty acid synthesizing enzymes in a photosynthetic microorganism. In some embodiments the present invention is a method of preparing a fuel. The methods disclosed herein allow for the production of crude or refined biofuels, including ethanol.

Description

PRODUCTION OF FATTY ΛCΪDS BY GENETICALLY MODIFIED PHOTOSYNTHETIC ORGANISMS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/088.591, filed August 13. 2008. which is herein incorporated by reference in its entirety for all purposes.

INCORPORATION BY REFERENCE

[0002] All publications and patent applicat ions mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

BACKGROUND

[0003] Fuel is becoming increasingly more expensive. In addition, fuel production by traditional means is associated with the generation of pollutants and global wanning. There is an increasing need in the industry to find cheaper, safer, and more environmentally unharmful ways So generate fuels. Genetic engineering of noii-piiotosynlhetic bacteria and yeast provides one means to produce crude fuels, proteins & nucleic acids, however currently available methods are limited. Byproducts produced by some bacteria often contaminate fuel sources. Thus, even where bacteria can be used to produce the biological material, additional steps such as puri fication or refining may be required to obtain biologically active material and/or bio-fuel Furthermore, the use of non-photosynthetic bacteria requires the addition of costly sugar or other organic carbon sources to feed the recombinant organism.

[0004] Thus, a need exists for methods to conveniently and economically produce fuels. Disclosed herein are novel compositions and methods used for the production of fuel from biological material utilizing genetically altered, photosynthef ic microorganisms. Also disclosed herein are novel compositions and methods used for the production of fatty acids, lipids or oils.

SUMMARY

[0005] One aspect of the present disclosure provides a method for increasing production of fatty acids and in particular fatty acids that are 10 to 18 carbons in length or 32 to I S carbons in length. The method comprises transforming a non-vascular photosynthetic organism with a nucleotide sequence encoding a protein involved in fatty acid synthesis. Exemplary proteins useful in the practice of the disclosed methods include, but are not limited to of acyl-CoA ligases. fatty acyl-CoA desaturases, methylmalonyl-CoA mutases, ketoacyl synthases, methylketone synthases, acetyl-CoA carboxylases, ketoreductases, malonyltransferases, dehydratases, acyl CoA synthetases and enoylreductases. In some embodiments, the nucleotide sequence is an exogenous nucleotide sequence. In other embodiments, transfonnation results in an increase in the number of copies of the endogenous nucleotide sequence. [0006] One particular embodiment provides a method for increasing production of a ClO to ci 8 fatty acid, such as myristic acid, in a noil- vascular photosyntheSic organism by transforming a plasSid of the organism, such as a chloroplast, with a vector comprising a nucleotide sequence encoding an acyl carrier protein thioest erase and expressing the thioesterase such that production of the ClO to C18 fatty acids is increased. Particular nucleotide sequences that can be used include one or more of SEQ ID NO: 20, 22, 30. 32 and 34, encoding proteins SEQ ID NO: 21 , 23, 31, 33 and 35, respectively

[0007] In another particular embodiment, plastids, for example chloroplasts, of a non-vascular photosyiithetic organism are transformed with a vector comprising a nucleotide sequence encoding a 3-keto-acyl-ACP synthase 11 (KASIi). The KASH is then expressed such that fatty acid production of the non-vascular photo synthetic organism is increased. An exemplary 3-keto-acyl-ACP synthase Iϊ useful in the practice of the disclosed method is SEQ ID NO: 29. [0008] Still another particular embodiment, provides a method for increasing production of fatty acids in a nonvascular photosynthetic organism by transforming a plastid of the organism, such as a chloroplast, wish a vector comprising a nucleoside sequence encoding an Acyl-CoA synthetase and expressing the synthetase such Shat production of fatty acids by the organism is increased. Particular nucleoside sequences that can be used include SEQ ID NO: 22 that encodes She protein of SEQ ID NO: 23

[0009] In any of the embodiments, the production of the CiO to CIS fatty acid may be increased at least 10%, at least 25%, at least 50%, at least 75%. at least 100%, at leasS 200%, at least 500% or at least 600%, In other embodiments, the production of the ClO So C14 fatty acid may be increased between about 10% and about 1000%,. for example about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%. about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, or about 1000%.

[0010] In any of the above embodiments, the vector may further comprise a promoter operably linked to the coding sequence. The promoter may be a constitutive or inducible promoter, Any constitutive or inducible promoter functional in the plastid of a non -vascular photosynthetic organism may be used. Examples of such promoters are known in the art. and non-limit ing examples are provided herein. The above vectors may also comprise a 5'UTR. In particular embodiments, the 5'UTR is one obtained from a plastid. Examples of suitable 5'UTRs include, but are not limited to, the atpA 5'UTR, the psbC 5'UTR, the psbD 5'UTR, the psbA 5'UTR, the rbcL 5'UTR and the 16s rRNA 5'UTR. Additionally, the vector may also comprise a ribosomal binding site. The vector may also comprise a 3'UTR, and in particular a 3^'UTR from a coding region of a plastid. Examples of suitable 3'UTRs include the psbA and rbcL 3'UTRs from C. reinhardtti.

[001 !] Any non-vascular photosynthetic organism can be used in practicing the methods disclosed herein, for example, cyanophyta, prochlorophyta, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta, crypto phyta, cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta. phaeophyta, and phytoplankton. ϊn particular embodiments, the non-vascular photosynthetic organism used in practicing the methods disclosed herein are of the genus Clilanivdomonas, Dunaliella, Hematococcus, Synechocystis, Sviiecliococcus, or Athrospira. In more particular embodiments, the organism is C. reinhardtii, D. salina, and H. pluvalis. [0012] In one embodiment of the methods disclosed herein, the organism is homoplasmic for the transforming vector. ϊn other embodiments, the vectors used in practicing the present methods further comprise a selection marker. Suitable selection markers are well known in the art and include those disclosed herein. [00 S3] In the practice of some of the methods described herein, the nucleotide sequence of the vector is altered to reflect the codors bias of the piastid which is transformed with the vector.

[00 ! 4] The present disclosure also provides a vector comprising: (a) a nucleic acid encoding at least one polypeptide which produces or increases production of one or more fatty acid(s); (b) a promoter configured for expression of said nucleic acid in a chloroplast of a non-vascular, photosynthetic organism: and (c) at least one nucleic acid for integration of at least a portion of said vector into a chloroplast genome. In some embodiments, insertion of the vector into a chloroplast genome does not disrupt photosynthetic capability of said chloroplast. In other embodiments, the at least one polypeptide is selected from the group of acetyl-CoA carboxylase, ketoreductase, thioesterase, malony {transferase, dehydratase. acyi-CoA iigase, ketoacyisynthase, acyl CoA synthetase, enoylreductase and a desaturase. A vector of the present disclosure may further comprise a selectable marker. In some embodiments, the non-vascular, photosynthetic organism for which a promoter is configured for expression is selected from the group consisting of: cyauophyta, prochloropiiyta, rhodophyta, chlorophyta, heterokontophyta, Sribophyta. glaucopiiyta. chlorarachniophytes, euglenophyta, euglenoids, iiaptophyia, chrysophyta, cryptophyta, cryptomonads. dinophyta, diαoflagellata, pyπnnesiophyta, bacillariophyta, xanthophyta. eustigmatophyta. raphidophyta. phaeophyta, and phytoplankton. The vectors of the present disclosure may be capable of stable transformation in C. reinhardtii, D. salinct or H. pluvalis. ϊn other instances, the nucleic acid encoding the at least one polypeptide is biased for a non-vascular photosynthet ic microorganism. The vector may comprise a nucleic acid encoding one of the sequences in Table L [0015] Another aspect provides a vector comprising a nucleic acid encoding any of the sequences in Table 1 . Also provided is a host cell comprising a vector wherein said vector comprises (a) a nucleic acid encoding at least one polypeptide which produces or increases production of one or snore fatty acid(s); (b) a promoter configured for expression of said nucleic acid in a chloroplast of a non -vascular, photosynthetic organism; and (c) at least one nucleic acid for integration of at least a portion of the vector into a chloroplast genome. Insertion of the vector into a chloroplast genome may not disrupt photosynthetic capability of the chloroplast. The at least one polypeptide encoded by a vector may be selected from the group of acetyϊ-CoA carboxylase, ketoreductase, thioesterase, malonyltransferase, dehydratase, acyl-CoA Iigase, ketoacyisynthase, acyl CoA synthetase, enoylreductase and a desaturase. The vector ma}? further comprise a selectable marker. The vector may be capable of stable transformation in C. reinhardtii, D. sahna or H. plurahs. In some embodiments, the nucleic acid encoding said at least one polypeptide is biased for a nonvascular photosynthetic microorganism. Tn other embodiments, the vector comprises a nucleic acid encoding any of the sequences in Table 1. The host cell may be homoplasmic for the nucleic acid encoding at least one polypeptide. The host cell can be cyanophyta, prochiorophyta, rhodophyta, chlorophyta, hcterokontophyta, tribophyta, giaucopbyta, chlorarachniophytes. euglenophyta, euglenoids, haptophyta, chrysophyta, cryptophyta. cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta. phaeophyta, or phytoplankton. In some embodiments, the host cell is C. reinhardtii, D. salina or H. pluvalis or a bacterium of the genus Synechocystis. the genus Synechococcus, or the genus Athrospira. Integration of a vector of the present disclosure may cause the host cell to lose photosynthetic capability after insertion of the vector. In some embodiments, the host cell produces at least one naturally occurring fatty acid at levels grater than a wild-type strain of the same organism. t>] Presented herein is a host cell comprising at least two copies of a nucleic acid encoding a sequence of Table 1. In some embodiments, the host cell is a non-vascular photosynthetic organism, including C. reinhardtii, D. salina or //. phtvaiis. The host cell may be homoplasmic.

[00 ! 7] Also provided is a chloroplast comprising one or more exogenous nucleic acid(s) encoding at least one polypeptide which produces or increases production of one or more fatty acid(s), wherein the chloroplast is from a nonvascular photosynthetic organism. The chloroplast may be homoplasmic. The polypeptide may be acetyl-CoA carboxylase, ketoreductase, thioesterase, malonyltransferase, dehydratase. acyl-CoA ligase, ketoacyisynthase, acyl CoA synthetase, enoylreductase or a desaturase.

[0018] Another aspect provides a non-vascular, photosynthetic organism comprising a chloroplast comprising one or more exogenous nucleic acid(s) encoding as least one polypeptide which produces or increases production of one or more fatty acid(s), wherein the chloroplast is from a noii-vascular photosynthetic organism. [0019] One method of She present disclosure provides for preparing a fatty acid comprising the steps of: (a) transforming a non-vascular, photosynthetic organism to produce the fast}' acid, and: (b) collecting the fatty acid. The transforming step may comprise transforming the organism with a vector encoding a plurality of distinct fatty acid synthetic enzymes. In some embodiments, the transformation is a chloroplast transformation. The collecting step may comprise one or more of the following steps: (a) harvesting the transformed organism; (b) mechanically disrupting she organism; or (c) chemically disrupting the organism, In some embodiments, 1he organism is an alga. [0020] Another aspect provides a method of preparing a fuel composition comprising: (a) transforming a non- vascular photosynthetic organism to produce a fatty acid; (b) collecting the fatty acid, and (c) treating she fatty acid, thereby producing a fuel.

SUMMARY OF THE FIGURES

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

2] Figure 1 illustrates transformation of alga cells, selection, confirmation, and scaling of production of enzymes. Figure 2 is a graphic representation of a fatty acid synthesis pathway..

14] Figure 3 is a graphic representation of designs of vectors useful in embodiments of the present disclosure.

S] Figure 4 illustrates primer pairs for PCR screening of transformants and expected band profiles for wild-type, heteroplasmic. and homoplasmic strains.

5] Figure 5 shows the presence of the introduced sequence identified and the degree of homoplasmy. Gels labeled "Genomic PCR" show the results of PCR performed on genomic chloroplast DNA to determine the degree of homoplasmy as described herein. The presence of a single band indicates that the organism is homoplasmic. Gels labeled "Gene specific^''^" show the presence of the introduced nucleotide sequence by the SEQ TD NO encoded. Primers were designed to produce products of the same size. [0027] Figure 6 is a Western blot showing expression of the various enzymes identified by SEQ ID NO. As described herein, enzymes were purified by FLAG resin affinity chromatography and detected using an anti-FLAG antibody.

8] Figure 7 shows an approximate 6 fold increase in myristic acid production by C. reinhardth strain 137c transformed with SEQ ΪD NO: 20 as determined by Covance analysis.

Figure 8 shows 1he increase in percent Iota] relative lipid content (TRLC) of C. reinhardtii strain 137c transformed wish various thioesterases as designated by SLQ ΪD NO. Daia were analyzed by one-way ANOVA followed by Dunnetfs test for significant difference from unrransformεd controls. A * indicates a significant difference at p<0.05. Percent TRLC is the percentage of the cell that is composed of fatty acids as determined by summing the concentrations of the individual fatty acids and dividing by the weight of biomass. 137c is an untransformcd control. Strains FA7, FA35, FA53, AF59, FA60 and FA63 express SEQ ΪD NOs 21, 27, 29, 3 i , 33 and 35, respectively.

Figure 9 shows a Western blot showing expression of a thioεst erase (SEQ ΪD NO: 2 1) and a 3-keto acyl -ACP- synthasε ΪI (SEQ ID NO; 29) in the same organism. Enzymes were purified using FLAG resin chromatography and detected using an anti -FLAG antibody.

Figure 10 shows the successful integration of nucleotide sequences encoding an acyl CoA synthetase (SEQ ΪD NO: 22). a thioεsterase (SEQ ΪD NO: 20) and a methylketoue synthase (SEQ ID NO: 24) in Synεchocystis. WT indicates untransformed cells. Five different strain transformed with SEQ ID NO: 24 were screened.

32] Figure 11 is a Western blot showing expression of an acyl CoA synthetase (SEQ ID NO: 23 ), a thioesterase (SEQ ID NO: 21) and a methylkεtonε synthase (SEQ ID NO: 25) in Synechocystis. Crude lysates were prepared and proteins detected using an anti-FLAG antibody. WT corresponds to untransformed (wild type) cell. Con is a positive control expressing an unrelated 25 kDa FLAG tagged protein. Arrows indicate proteins

DETAILED DESCRIPTION

[0033] The present disclosure involves the use of genetic manipulation of photosynthetic organisms (e.g., nonvascular organisms such as algae and cyanobacteria) to produce non-naturally occurring fatty acids, lipids and/or oils or to increase production of naturally occurring fatty acids, lipids anώ'or oils. Although these products are naturally produced by most organisms, fatty acids in particular may be highly useful as feedstocks for biofuel production. As such, the compositions and methods disclosed herein may be useful for the production of biofuels, for example by collecting the fatty acids, lipids or oils, by refining these products into a combustible fuel (e.g.. diesel fuel, gasoline, jet fuel, octane, propane, etc.),

[0034] The present disclosure relates to compositions that facilitate the production of enzymes by genetically modified photosynthetic microorganisms wherein the enzymes contribute to the synthesis of fatty acids, lipids or oils. In some embodiments, the compositions are one or more vectors that encode one or more polypeptides that increase production of fatty acids. The vectors may comprise exogenous nucleic acid sequences. The vectors may comprise promoters configured for expression of the polypeptides in the chloroplast of non- vascular, photosynthetic organisms. Non- vascular refers So the absence of vascular tissues such as xylem and phloem. Non-limiting examples of uon-vasular photosyntetic microorganisms include algae (e.g. red algae, green algae), protists (such as euglena) and bacteria (such as cyanobacSeria). Some more specific non-limiting examples of nou- vascular photosynthetic organisms include cyanophyta, proehloropbyta, rhodophyta. chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta, cryptophyta, cryptomonads. dinophyta, dinoflagellata, pyπnnesiophyta, bacillariophyta, xanthophyta. eustigmatopliyta. raphidophyta. phaeophyta, and phytoplankton. The vectors of the disclosure may comprise nucleic acid sequences that facil itate integration of at least a portion of the vector into a chloroplast genome wherein insertion and integration into the chloroplast genome does not disrupt the photsynthetic capability of the chloroplast,

[0035] As disclosed herein, molecular biology and genetic engineering techniques are u1 ilized to create enzyme- expressing and/or enzymat ic pathway -expressing photosynthetic strains for large scale production of active enzymes. The enzymes produced by the vectors may be anabolic enzymes or catabohc enzymes. Anabolic enzymes produced by said vectors may contribute to the synthesis of fatty acids, lipids or oils and may be part a biosynfhetic pathway for fatty acid synthesis. Non limiting examples of such anabolic enzymes include acctyl-CoA carboxylase, kctorcduetase, thiocsterasc, malonyltransfcrase, dehydratase, acyl-Coa ligase, ketoacylsynthase, acyl CoA synthetase, enoylreductasc and a desatnrase. Catabolic enzymes produced by the vectors ma}? aid in the biodcgradation of bio mass to provide nutrition to the organism and to provide substrates for the synthesis of fatty acids, lipids or oils.

[0036] The present disclosure also relates to a plastid (e.g., a chloroplast) comprising one or more exogenous nucleic acid(s) encoding at least one polypeptide which produces or increases production of one or more fatty acids. A chloroplast may also refer to a photosynthetic cyanobacterial cndosymbiont (e.g. the chloroplast-like organelles found in Pauhnella chromatophora).

[0037] The present disclosure also relates to a method for preparing fatty acids, lipids or oils comprising transforming a non-vascular photosynthetic organism with one of the vectors disclosed herein and collecting said fatty acids, lipids or oils, in some aspects the present disclosure relates to compositions and methods to produce fuels, using enzymes produced by non- vascular photosynthetic organisms.

[0038] One approach to construction of a genetically manipulated photosynthetic microorganism is diagramed as a flow chart in FlG. 1. As can be seen, photosynthetic cells (e.g., CMainydomonas reinhardtii, Diinalieila salina, Hematococcus pluralis) are transformed with a nucleic acid which encodes a gene of interest, typically an enzyme that contributes to the production of a fatty acid, lipid or oil. Iu some embodiments, a transformation may introduce nucleic acids into any plastid of the host cell. Transformed cells are typically plated on selective media following introduction of exogenous nucleic acids. This method may also comprise several steps for screening. Initially, a screen of primary transformants is typically conducted to determine which clones have proper insertion of the exogenous nucleic acids. Clones which show the proper integration may be patched and re-screened to ensure genetic stability. Such methodology ensures that the transformants contain the genes of interest. In many instances, such screening is performed by polymerase chain reaction (PCR); however, any other appropriate technique known in the art may be utilized. Many different methods of PCR are known in the art (e.g., nested PCR. real time PCR). For any given screen, one of skill in the art will recognize that PCR components may be varied to achieve optimal screening results. For example, magnesium concentration may need to be adjusted upwards when PCR is performed on disrupted alga cells as many such organisms have magnesium chelators. Tn such instances, magnesium concentration may need to be adjusted upward, or downward (compared to the standard concentrat ton in commercially available PCR kits) by 0. i , 0.2, 0.3, 0,4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1,1, 1 ,2, 1.3, 1.4, 1.5, 1.6, 1 .7, 1 .8, 1.9, or 2.0 raM. Thus, after adjusting, final magnesium concentration in a PCR reaction may be, for example 0.7, 0.8, 0.9, 1.0. 1.1. 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 3.8, 1.9. 2.0. 2.1, 2.2, 2.3, 2.4. 2.5. 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3. 3.4. 3.5 mM or higher. Particular examples are utilized in the examples described herein; however, one of skill in the art will recognize that other PCR techniques may be substituted for the particular protocols described. Following screening for clones with proper integration of exogenous nucleic acids, typically clones are screened for the presence of the encoded protein. Protein expression screening typically is performed by Western blot analysis and/or enzyme activity assays.

[0039] Following confirmation of nucleic acid integration and/or protein expression, selected clones may be scaled up for production of fatty acids, lipid, oils and/or biofuels, first in smaller volumes of 1. 2, 3, 4, 5. 6. 7, 8, 9, 10, 11, 12, 13. 14, 15. 16. 17, 18, 19, 20, 21. 22. 23, 24, 25, 26. 27. 28, 29, 30, 31. 32. 33, 34, 35, 36, 37. 38. 39, 40, 41, 42. 43. 44, 45, 46, 47. 48. 49, 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. 91. 92, 93, 94, 95. 96. 97, 98, 99, 100 or more liters. Following initial scaling up, larger scale production may be performed in larger quantities. One example of a partially closed bioreactor system is shown in FlG. 1, step 6, However, growth of the transformed strains for production of fatty acids, lipid, oils or biofuel can also be accomplished in man-made structures such as ponds, aqueducts, reservoirs and/or landfills. Alternately, the strains can also be grown directly in naturally occurring bodies of water, e.g., in ocean, sea. lakes, or rivers,,

[0040] The present disclosure contemplates making enzymes shaf contribute to the production of fatty acids, lipids, or oils by transforming host cells (e.g., alga cells such as C. reiυhardtii, D. salina, H. pluvalis and cyanobacterial cells) and/or organisms comprising host cells wish nucleic acids encoding one or more different enzymes. In some embodiments 1he enzymes that contribute so the production of fatty acids, lipids or oils are anabolic enzymes. Some examples of anabolic enzymes shat contribute to the synthesis of fatty acids include, bsrt are not limited so, acetyl-CoA carboxylase, kεtoreductase, tbioesterasε, malonyl transferase, dehydratase, acyl-CoA Isgase, ketoacylsynthasε, acyl CoA synthetase, enoylreductase and a dεsaturase. In some embodiments the enzymes are catabolic or biodegrading enzymes. In some embodiments, a single enzyme is produced.

[004 !] Some host cells may be transformed with multiple genes encoding one or more enzymes. For example, a single transformed cell may contain exogenous nucleic acids encoding enzymes that make up an entire fatty acid synthesis pathway. One example of a pathway might include genes encoding an acetyl CoA carboxylase, a malonyltransfεrase, a ketoacylsyntbasc, and a tbioestcrase (FIG 4). Cells transformed with entire pathways and/or enzymes extracted from them, can synthesize complete fatty acids or intermediates of the fatty acid synthesis pathway. In some embodiments constructs may contain multiple copies of the same gene, and/or multiple genes encoding the same enzyme from different organisms, and'or multiple genes with mutations in one or more parts of the coding sequences.

[0042] Following transformation with enzyme-encoding constructs, the host cells and/or organisms are grown. The enzymes then lead to the production of fatty acids, lipids or oils that may be collected from the organisms/cells. Collection may be by any means known in the art. including, but not limited to concentrating cells, mechanical or chemical disruption of cells, and purification of enzymes from cell cultures and-'or cell lysates. Cells and/or organisms can be grown and then the product(s) collected by any means. One method of extracting the product is by harvesting the host cell or a group of host cells and then drying the host cefl(s). The product(s) from the dried host cell(s) are then harvested by crashing the cells to expose the enzyme.

[0043] Synthesis of fatty acids, lipids or oils can also be accomplished by engineering a cell to express an accessory molecule or modulation molecule. In certain embodiments, the accessory molecule is an enzyme that produces a substrate utilized by a fatty acid synthesizing enzyme. In some embodiments the accessory or modulation molecule contributes to the growth or nourishment of the biomass.

[0044] In some instances, expression or production of a fatty acid synthesizing enzyme is inducible. Such inducible expression may comprise use of one or more activatable promoters controlling expression of nucleic acids encoding at least one enzyme that contributes to the production of a fatty acid, lipid or oil. Inducible promoters can activate the production of fatty acid producing enzymes, for example, after the biomass has grown to sufficient density or reached certain maturity. One non-limiting example is the TeS-On expression system. In this example, the Tet-On expression system allows tetracycline- or doxycycline-regulated gene expression under She control of the rsTA (reverse tetracycline-coutrolled trausactivator) protein and the Tet operaSor DiSlA sequence (tetO), both of which are regulator}' elements derived from the Escherichia coli tetracycline resistance operou. When a vector containing the gene of interest preceded by a TRE (tetracycline response element, which consists of repeats of the tetO sequence) is introduced into a cell line stably expressing 1he rtTA, expression of the gene can be controlled by 1he level of doxycycline in the culture medium. The rtTA can only bind to the TRE and initiate transcription of the gene in the presence of doxycycline, Io some embodiments, expression or production of an enzyme that contributes to the production of a fatty acid, lipid or oil is regulated by a repressor. One non-limiting example of a repressor system is the Tet -Off transactivator (ITA ) wherein gene transcription is activated in the absence of doxycycline. Expression of enzymes can be made inducible by any suitable method or by any suitable inducible expression system,

[0045] A method of the disclosure can be performed by introducing a recombinant nucleic acid molecule into a chloroplast wherein the recombinant nucleic acid molecule includes a first polynucleotide, which encodes at least one polypeptide (i.e., 1 , 2, 3, 4, or more). In some embodiments, a polypeptide is operatively linked to a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and/or subsequent polypeptide. For example several enzymes in a biosynthesis pathway ma}? be linked, cither directly or indirectly, such that products produced by one enzyme in the pathway, once produced, are in close proximity to the next enzyme in the pathway. For transformation of chloroplasts, one major benefit of the present disclosure is the utilization of a recombinant nucleic acid construct which contains both a selectable marker and one or morc genes of interest. Typically, transformation of chloroplasts is performed by co- transformation of chloroplasts with two constructs: one containing a selectable marker and a second containing the gene(s) of interest. Typically, secondary screening for the gene(s) of interest is performed by Southern blot (see, e.g. PCT/US2007/072465).

[0046] The present disclosure may utilize at least one recombinant nucleic acid construct which contains both a selectable marker and one or more genes of interest. In some embodiments constructs are engineered that allow for a PCR-based screening method in which transformants can be screened using a combination of primers specific for the insert and wild-type sequences (FIG, 3), This methodology provides an advantageous rapid screening process . For example, selection of transformants receiving unlinked markers inherently yields a lower percentage of clones with the transgenes. Because of this, the likelihood of obtaining homoplasmic lines from a primary transformation is low. By linking the marker and the gene(s) of interest the likelihood of obtaining transgenic clones with the transgene, especially homoplasmic clones, is improved on the first pass. Specific PCR protocols for screening transformants are detailed in the Examples below. One of skill in the art will recognize that these protocols may be modified to provide quantitative analysis of transformants using any suitable method. For example, different ratios of primers for a particular reaction may be utilized to compare insert copy number to a control reaction. Such variation may be performed where the multiplex reactions (FlG. 3) are run concurrently or separately.

[0047] Determination of insert copy number may be important where an optimal level of expression of the exogenous geue(s) of interest is, in part, determined by gene copy number. For example, transformation of an alga host cell (e.g., C. reinhardiii, D. sauna. II. phivalis) which results in incorporation of the exogenous nucleic acid in less Shan half of the copies of the chloroplast genomes in a cell may yield little or no detectable expression of She geue(s) of inSerest. Alternately, incorporation of exogenous nucleic acid in all the copies of the chloroplast genomes in a cell may yield little or no detectable expression of the gene(s) of interest where Shere are few initial copies of the genome (e.g., quantitative PCR analysis will allow for exclusion of homoplasmic clones which have low insert copy number, and thus may not have sufficiently high production of the gene and/or polypeptide of interest). In other embodiments, there may be an optimum level of incorporation of exogenous nucleic acid. Iu some instances, exogenous DMA may encode a protein which, whether through transcriptional, transnational, or other control mechanisms, is optimally produced when it is present in a particular range of copy number. Thus, determining the copy number of such exogenous DNA, for example by quantitative PCR, may allow select ton and/or production of transformed organisms which produce protein(s) of interest as an efficient level.

[0048] Additionally, recombinant nucleic acid molecules may be opeτatively linked to a second and/or subsequent nucleotide sequence. For example, the nucleotide sequences encoding enzymes of a fatty acid synthesis pathway are operativεly linked such that expression of these sequences may be controlled with a single inducing stimulus or controlled by a single transcriptional activator. Such systems are similar to bacterial operons (e.g., the Escherichia coil Lac operon). However, the groupings of operatively linked nucleotide sequences disclosed herein are synthetic and designed to function in plant plastids and arc preferably incorporated into the chloroplast genome. [0049] As used herein, the term 'Operatively linked" means that two or more molecules are positioned with respect to each other such that they act as a single unit and affect a function attributable to one or both molecules or a combination thereof. For example, a polynucleotide encoding a polypeptide can be operatively linked to a transcriptional or translationai regulatory element, in which case the clement confers its regulatory effect on the polynucleotide similarly to the way in which the regulatory element would affect a polynucleotide sequence with which it normally is associated with in a cell. A first polynucleotide coding sequence also can be operatively linked to a second (or more) coding sequence such that a chimeric polypeptide can be expressed from the operatively linked coding sequences. 'The chimeric polypeptide produced from such a construct can be a fusion protein, in which the two (or more) encoded peptides are translated into a single polypeptide, i.e., are covalently bound through a peptide bond, either directly or with a short spacer region.

[0050] In chloroplasts, regulation of gene expression generally occurs after transcription, and often during translation initiation. This regulation is dependent upon the chloroplast translational apparatus, as well as nuclear-encoded regulatory factors (see Barkan and Goldschmidt-Clermont. Biochemie 82:559-572. 2000: Zerges. Biockemie 82:583- 601. 2000). The chloroplast translational apparatus generally resembles that in bacteria; chioropiasts contain 70S ribosomes; have niRN As that lack 5^! caps and generally do not contain 3^! poly-adenylated tails (Hams et al., Microbiol Rev. 58:700-754, 1994); and translation is inhibited in chloroplasts and in bacteria by selective agents such as chloramphenicol.

[0051] An isolated ribonucleotide sequence may be obtained by any suitable method, including, but not limited to being chemically synthesized, generated using an enzymatic method, (e.g., generated from a DNA or RNA template using a DNA dependent RNA polymerase or an RNA dependent RNA polymerase). A DNA template encoding the ribonucleotide of the disclosure can be chemically synthesized, can be isolated from a naturally occurring DiSlA molecule, or can be derived from a naturally occurring DiSlA sequence that is modified to have the required characteristics,

[0052] The term "polynucleotide" or "nucleotide sequence^" or "nucleic acid molecule^*' is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, She terms include RNA and DNA. which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the terms as used herein include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthet ic polynucleotides, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR), It should be recognized that the different terms are used only for convenience of discussion so as to distinguish, for example, different components of a composition, except that the term "synthetic polynucleotide" as used herein refers to a polynucleotide that has been modified to reflect chloroplast codon usage.

[0053] In general, the nucleotides comprising a polynucleotide are naturally occurring dεoxyribonucleot ides, such as adenine, cytosine, guanine or thymine linked to 2'-dεoxyribose, or ribonucleotides such as adenine, cytosinε, guanine or uracil linked to ribose. Depending on the use, however, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Nucleotide analogs are well known in the art and commercially available, as arc polynucleotides containing such nucleotide analogs (Lin et al., Nm-L Acids Res, 22:5220-5234, 1994; Iellinek et al., Biochemistry 34: 11363-1 ! 372, 1995; Pagratis et al., Nature Biotechnol. 15:68-73, 1997). Generally, a phosphodiester bond links the nucleotides of a polynucleotide, however other bonds, including a thiodiester bond, a phosphorothioate bond, a pcptide-likc bond and any other bond known in the art may be utilized to produce synthetic polynucleotides (Tarn et al., Nucl. Acids Res. 22:977-986, 1994; Ecker and Crookc, BioTechnobgy 13:351360, 1995).

[0054] A polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template, in comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally are chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al.. supra. 1995). Polynucleotides useful for practicing the methods disclosed herein may be isolated from any organism. Non-limiting examples of such enzymes and their sources are shown in Table 3. Such polynucleotides may be isolated and/or synthesized by any means known in the art. including, but not limited to cloning, sub-cloning, and PCR.

[0055] One or more codons of an encoding polynucleotide can be biased to reflect an organism's preferred codon usage. Most amino acids are encoded by two or more different (degenerate) codons, and it is well recognized that various organisms utilize certain codons in preference to others. Such preferential codon usage, which also is utilized in chloroplasts, is referred to herein as codon bias or chloroplast codon bias where favored by chloroplasts. The codon bias of Chlamydomonas reinhardiii has been reported. See U. S. Publication Application 2004/0014374, [0056] The Eemi "biased,^" when used in reference to a codon. means that the sequence of a codon in a polynucleotide has been changed such thai the codoii is one that is used preferentially in the target which the bias is for. e.g.. alga cells, chloroplasts. A polynucleotide that is biased for chloroplast codon usage can be synthesized de novo, or can be genetically modified using routine recombinant DMA techniques, for example, by a site directed mutagenesis method, to change one or more codons such that they are biased for chloroplast codon usage. Chloroplast codon bias can be variously skewed in different plants, including, for example, in alga chloroplasts as compared to tobacco. Generally, the chloroplast codon bias selected reflects chloroplast codon usage of She plant which is being transformed with She nucleic acids. For example, where C. reinhardtii is the host, the chloroplast codon usage is biased to reflect alga chloroplast codon usage (about 74.6% AT bias in the third codon posh ton).

[0057] One method of the disclosure can be performed using a polynucleotide that encodes a first polypeptide and at least a second polypeptide, As such, the polynucleotide can encode, for example, a first polypeptide and a second polypeptide; a first polypept tde, a second polypeptide, and a third polypeptide; etc. Furthermore, any or all of the encoded polypeptides can be the same or di fferent. The polypept ides expressed in chloroplasts of the rnicroalga C. reinhardtii may be assembled to form funct ional polypept ides and protein complexes, As such, a method of the disclosure provides a means to produce functional protein complexes, including, for example, dimers, trimers, and tetramcrs, wherein the subunits of the complexes can be the same or different (e.g., homodimers or heterodimers, respectively).

[0058] The term "'recombinant nucleic acid molecule" is used herein to refer to a polynucleotide that is manipulated by human intervention. A recombinant nucleic acid molecule can contain two or more nucleotide sequences that are linked in a manner such that the product is not found in a cell in nature, Tn particular, the two or more nucleotide sequences can be operativcly linked and, for example, can encode a fission polypeptide, or can comprise an encoding nucleotide sequence and a regulator}' element. A recombinant nucleic acid molecule also can be based on, but manipulated so as to be different, from a naturally occurring polynucleotide, (e.g. biased for chloroplast codon usage, insertion of a restriction enzyme site, insertion of a promoter, insertion of an origin of replication). A recombinant nucleic acid molecule may further contain a peptide tag (e.g.. His-ό tag), which can facilitate identification of expression of the polypeptide in a cell. Additional tags include, for example: a FLAG epitope, a c-mye epitope; biotin; and glutathione S-transferase. Such tags can be detected by any method known in the art (e.g., anti-tag antibodies. streptavidin). Such tags may also be used to isolate the operatively linked polypeptide(s), for example by affinity chromatography. Nucleic acids

The compositions herein comprise nucleic acids which encode one or more different enzymes that contribute to the synthesis of fatty acids, lipids or oils. These fatty acids, lipids or oils may be used as biofuels or as feedstocks for biofuel production. The compositions herein may also comprise nucleic acids which encode one or more different fatty acid synthesis modulating agents and vectors of such nucleic acids. The nucleic acids can be heterologous to a photosynthetic host cell to which they are inserted. The vector can include one or a plurality of copies of the nucleic acids which encode she enzymes and/or one or a plurality of copies of the nucleic acids which encode the fatty acid synthesis modulating agents. When using a plurality of copies, at least 2, 3, 4. 5, 6 7. 8, 9, or 10 copies of the nucleic acids (e.g., encoding a single enzyme) can be inserted into a single vector. This allows for an increased level of Sheir production in She host cell.

[0061] Recombinant nucleic acid molecules useful in the methods disclosed herein can be contained in a vector. Furthermore, where the method is performed using a second (or more) recombinant nucleic acid molecules, the second recombinant nucleic acid molecule also can be contained in a vector, which can, but need not, be the same vector as that containing She first recombinant nucleic acid molecule. The vector can be any vector useful for introducing a polynucleotide iτt1o a chloroplast and, preferably, includes a nucleotide sequence of chloroplast genomic DNA that is sufficient to undergo homologous recombination with chloroplasi genomic DNA, for example, a nucleot ide sequence comprising about 400 to 1500 or more substantially contiguous nucleotides of chloroplast genomic DNA, Chloroplasi vectors and methods for selecting regions of a chloroplast genome for use as a vector are well known (see, for example. Bock, J. MoI. Biol. 312:425-438, 2001; see, also, Staυb and Maliga, Plant Cell 4:39-45, 1992; Kavanagh el aS,, Genetics 152; U 1 1 - 1 122, 1999, each of which is incorporated herein by reference).

[0062] In some instances, such vectors comprise promoters. Any suitable promoter obtained from any source may be used (e.g., viral, plant, bacterial, fungal, protist, animal). The promoters contemplated herein can be specific to photosynthetic organisms, non-vascular photosynthetic organisms, and vascular photosynthetic organisms (e.g., algae, flowering plants). As used herein, the term '"non-vascular photosynthetic organism," refers to any macroscopic or microscopic organism, including, but not limited to, algae, cyanobacteria and photosynthetic bacteria, which does not have a vascular system such as that found in higher plants. In some instances, the nucleic acids above arc insetted into a vector that comprises a promoter of a photosynthetic organism, e.g., algae. The promoter can be a promoter for expression in a chloroplast and/or other plastid. In some instances, the nucleic acids arc chloroplast based. Examples of promoters contemplated for insertion of any of the nucleic acids herein into the chloroplast include those disclosed in US Application No. 2004/0014174. The promoter can be a constitutive promoter or an inducible promoter, A promoter typically includes necessary nucleic acid sequences near the start site of transcription, (e.g., a 'TATA element). [0063] Some vectors of the present disclosure may include a sequence comprising a ribosomal binding site (RBS). Such RBSs can be chemically synthesized, or can be isolated from a naturally occurring nucleic acid molecule (e.g., isolation from a chloroplast gene), In addition, to an RBS, embodiments with a 5'UTR can include transcriptional regulatory elements such as a promoter. As with RBSs utilized, a 5'UTR may be chemically synthesized, or can be isolated from a naturally occurring nucleic acid molecule. Non-limiting examples of 5'UTRs which may be used include, but art not limited to, an atpA 5'UTR; a psbC 5'UTR. a psbD 5XJTR, a psbA 5'UTR. a rbcL 5^"UTR and/or a

.1 ">_ 16S rRNA 5'L¹TR. A ribonucleotide sequence may further include an initiation codon, (e.g., an AUG codon), operatively linked to an RBS. Initiation codons may be endogenous (e.g.. naturally occurring in a cloned gene) or can be synthetic (e.g., inserted in a linker polypeptide or PCR primer).

[0064] Placement of an RBS results in robust translation in plant chloroplasts (see U.S. Application 2004/0014174, incorporated herein by reference). Chloroplast-expressed polypeptides do not proceed through cellular compartments typically traversed by polypeptides expressed from a nuclear gene. Therefore, chloroplast-expressed polypeptides are not typically subject to certain post-translational modifications such as glycosylation. As such, the polypeptides and protein complexes produced by some methods described herein can be expected So be produced without such post- translational modification.

[0065] A "constitutive'^'1 promoter is a promoter that is active under most environmental and developmental conditions. Non-limiting examples of constitutive promoters include the atpA promoter, the psbC promoter, the psbD promoter, the psbA promoter, the rbcL promoter and the 16s rRNA promoter. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. Examples of inducible promoters/regulatory elements include, for example, a uitrate-inducible promoter (Back et al. Plant MoI. Biol. 17:9 (1991)), or a ligiit-inducible promoter, (Feinbaum et al. MoI Gen. Genet. 226:449 ( 1991); Lam and Chiia, Science 248:471 i 1990)). or a heat responsive promoter (Mυller et al., Gene I U : 165-73 (1992)).

[0066] The entire chloroplast genome of C. reinhardtii is available to Ihe public on the world wide web, at the URL "biology.duke.edυ/chlaray_genotne/- chloro.html" (see "view complete genome as text file" link and "maps of the chloroplast genome" Sink), each of which is, incorporated herein by reference (J. Maul, J. W, Lilly, and D. B, Stern, unpublished results; revised Jan. 28, 2002; to be published as GenBank Ace. No. AF396929), Generally, the nucleotide sequence of the chloroplasi genomic DNA is selected such that it is not a portion of a gene, including a regulatory sequence or coding sequence, particularly a gene that, if disrupted due to the homologous recombination event, would produce a deleterious effect with respect to the chloropiast, for example, for replication of the chloroplast genome, or to a plant cell containing the chloroplast. In this respect, the website containing the C. reirikarJni chloroplast genome sequence also provides maps showing coding and non-coding regions of the chloroplast genome, thus facilitating selection of a sequence useful for constructing a vector of the disclosure. For example, the chloroplast vector, p322 is a clone extending from the Eco (Eco Rl) site at about position 143.1 kb to the Xho (Xho I) site at about position 148.5 kb (sec, world wide web, at the URL "biology.duke.edu/chlamy genome/chloro.htrm"', and clicking on "maps of the chloroplast genome" link, and "140-150 kb" link; also accessible directly on world wide web at URL "biology.dukc.edu/chlam- y/chioro/chlorol40.html").

[0067] A vector utilized in the practice of the disclosure also can contain one or more additional nucleotide sequences that confer desirable characteristics on the vector, including, for example, sequences such as cloning sites that facilitate manipulation of the vector, regulator}' elements that direct replication of the vector or transcription of nucleotide sequences contain therein, sequences that encode a selectable marker, and the like. As such, the vector can contain, for example, one or more cloning sites such as a multiple cloning site, which can, but need not. be positioned such that a heterologous polynucleotide can be inserted into the vector and operatively linked to a desired element. The vector also can contain a prokaryote origin of replication (on), for example, an E. coli ori or a cosmid on, thus allowing passage of the vector in a prokaryote host cell, as well as in a plant chloroplast. as desired. S8] A regulatory element, as the term is used herein, broadly refers to a nucleotide sequence that regulates the transcription or translation of a polynucleotide or the localization of a polypeptide to which it is operatively linked. Examples include, but are not limited to, an RBS, a promoter, enhancer, transcription terminator, an initiation (start) codon. a splicing signal for intron excision and maintenance of a correct reading frame, a STOP codon. an amber or ochre codon, an ΪRES. Additionally, a ceil compartmentalization signal (i.e., a sequence that targets a polypeptide to the cytosol. nucleus, chloroplast membrane or ceil membrane). Such signals are well known in the art and have been widely reported (see, e.g., U.S. Pat. No. 5,776.689).

[0069] A vector or other recombinant nucleic acid molecule may include a nucleoside sequence encoding a selectable marker. The term or "selectable marker" refers to a polynucleotide (or encoded polypeptide) that confers a detectable phenotype. A selectable marker generally encodes a detectable polypeptide, for example, a green fluorescent protein or an enzyme such as liiciferase, which, when contacted with an appropriate agent (a particular wavelength of light or luciferin, respectively) generates a signal that can be detected by eye or using appropriate instrumentation (Giacomin. Plant Sd. 116:59-72. 1996; Scikantha, J. Bacterial. 178:121. 1996: Gerdes, FEBS Lett. 389:44-47, 1996; see. also. Jefferson, EMBO J. 6:3901-3907. 1997. fl-glucuronidase). A selectable marker generally is a molecule that, when present or expressed in a cell, provides a selective advantage (or disadvantage) So the cell containing the marker, for example, the ability to grow in the presence of an agent that otherwise would kill the cell.

[0070] A selectable marker can provide a means to obtain prokaryotic cells or εukaryotic cells or both that express the marker and, therefore, can be useful as a component of a vector of the disclosure (see, for example, Bock, supra, 2001), Examples of selectable markers include, but are not limited to, those that confer antimetabolite resistance, for example, dihydrofoiate reductase, which confers resistance to methotrexate fReiss, Plant Physiol. {Life Sci, Adv.) 13: 143-149. 1994); neomycin phosphotransferase, which confers resistance to the aminoglycosides neomycin, kanaraycin and paromycin (Hεrrera-Estrella, EMBO J. 2:987-995, 1983), hygro, which confers resistance to hygroraycin (Marsh. Gene 32:481-485, 1984), trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988); mannose-6-phospiiate isomerase which allows cells to utilize mannose (WO 94/20627); ornithine decarboxylase, which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine (DFMO; McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.); and deaminase from Aspergillus rerreus, which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59:2336-2338, 1995). Additional selectable markers include those that confer herbicide resistance, for example, phosphinothricin acctyltransferasc gene, which confers resistance to phospliinothricin (White ct al., Nucl. Acids Res. i 8:1062, 1990; Spencer ct al., Theor. Appl. Genet. 79:625-631 , 1990), a mutant EPSPV-syntbase. which confers glyphosate resistance (Hinchee et al., BioTeclmology 91 :915-922, 1998), a mutant acetolactate synthase, which confers imidazolione or sulfonylurea resistance (Lee et al., EMBO J. 7:1241-1248, 1988), a mutant psbA, which confers resistance to atrazine (Smeda et al., Plant Physiol. 103:93 1-917, 1993j. or a mutant protoporphyrinogen oxidase (see U.S. Pat. No. 5,767.373), or other markers conferring resistance to an herbicide such as glufosinate. Selectable markers include polynucleotides that confer dihydro folate reductase (DHFR) or neomycin resistance for eukaryotic cells and tetracycline; ampicillin resistance for prokaryotes such as E. coli: and bleomycin, gentamycin, glyphosate, hygromycin, kanamycin, methotrexate, phleomycin. phosphinotricin, spectinomycin, streptomycin, sulfonamide and sulfonylurea resistance in plants (see, for example. Maliga ef a]., Methods in Plant Molecular Biology, Cold Spring Harbor Laboratory Press, 1995, page 39).

[0071] Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. In addition, reporter genes have been used in the chloroplast of C. reinhardtii, but, in most cases very low amounts of protein were produced. Reporter genes greatly enhance the ability to monitor gene expression in a number of biological organisms. In chloroplasts of higher plants, β-glucuronidase (uidA, Staub and Maliga. EMBO J. 12:601-606, 1993). neomycin phosphotransferase (nptll, Carrer et a!.. MoL Gen. Genet. 243 :49-56, 1993), adenosyl-3-adenyltransf- erase f aadA, Svab and Maliga. Proc. Nail. Acad. Sd., USA 90:913-917. 1993), and she Aequorea victoria GFP (Sidorov et al., Plant J. 19:209-216. 1999) have been used as reporter genes (Heifetz, Biochemie 82:655-666. 2000), Each of these genes has attributes thai make them useful reporters of chloroplast gene expression, such as ease of analysis, sensitivity, or the ability to examine expression in situ. Based upon these studies, other heterologous proteins have been expressed in the chloroplasts of higher plants such as Bacillus thuringiensis Cry toxins, conferring resistance to insect herbivores (Ko ta et al,, Proc. Natl. Acad. ScL, USA 96: 1840-1845. 1999), or human somatotropin (Staub et al.. Nat. Biotechnol. 18:333-338. 2000), a potential biopharmaceutical. Several reporter genes have been expressed in the chloropiast of She eukaryotic green alga. C. reinhardtii. including aadA (Goldschmidt- Cksmoni , Nad. Acids Res. 19:4083-4089 1991; Zerges and Rochaix, MoL Cell Biol. 14:5268-5277, 1994), uidA (Sakamoto et al,, Proc. Natl. Acrid. Sa,, USA 90:477-501 , 19933, Ishikura et al,, /. Biosci. Bioetig, 87:307-3 14 1999), Renill a luciferase (Minko et al., MoL Gen. Genet. 262:421 -425, 1999) and Ihe amino glycoside phosphotransferase from Acinetobacter baunianii, aptιA6 fBatεman and Purton, MoL Gen. Genet 263:404-410, 2000).

[0072] In some instances, Ihe vectors described herein will contain elements such as an E. coli or S. cerevisiae origin of replication. Such features, combined with appropriate selectable markers, allows for the vector to be "shuttled" between the target host cell and she bacterial and/or yeast cell. The ability to passage a shuttle vector in a secondary' host may allow for more convenient manipulation of the features of the vector. For example, a reaction mixture containing the vector and putative inserted polynucleotides of interest can be transformed into prokaryote host cells such as E. coli, amplified and collected using routine methods, and examined to identify vectors containing an insert or construct of interest, Tf desired, the vector can be further manipulated, for example, by performing site directed mutagenesis of the inserted polynucleotide, then again amplifying and selecting vectors having a mutated polynucleotide of interest. A shuttle vector then can be introduced into plant cell chloroplasts, wherein a polypeptide of interest can be expressed and, if desired, isolated according to a method of the disclosure.

[0073] A polynucleotide or recombinant nucleic acid molecule can be introduced into plant chloroplasts using any suitable method. A polynucleotide can be introduced into a cell by a variety of methods, which are well known in the art and selected, in part, based on the particular host cell. For example, the polynucleotide can be introduced into a plant cell using a direct gene transfer method such as electroporation or microprojectiie mediated (biolistic) transformation using a particle gun, or the "glass bead method,'¹ or by pollen-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme- degraded embryogenic callus (Potrykus, Ann. Rev. Plant. Physiol. Plant MoI. Biol. 42:205-225, 1991). [0074] The term "'exogenous^"' is used herein in a comparative sense to indicate that a nucleotide sequence (or polypeptide) being referred to is from a source other than a reference source, or is linked to a second nucleotide sequence (or polypeptide) with which it is not normally associated, or is modified such that it is in a form that is not normally associated with a reference material. For example, a polynucleotide encoding an enzyme of the disclosure is heterologous with respect to a nucleotide sequence of a plant chloroplast, as are the components of a recombinant nucleic acid molecule comprising, for example, a first nucleotide sequence operatively linked to a second nucleotide sequence, as is a mutated polynucleotide introduced into a chloroplast where the mutant polynucleotide is not normally found in the chloroplast. Additionally a nucleotide sequence which has been altered to contain a purification tag or moiety, for example a FLAG tag, is considered an exogenous nucleotide sequence.

[0075] Methods for nuclear and plastid transformation are routine and well known for introducing a polynucleotide into a plant cell chloroplast f see U.S. Pat. Nos. 5,451,513, 5,545.817, and 5.545.818; WO 95/16783; McBride et al, Proc. Nati. Acad, ScL, USA 91:7301-7305. 1994). Iu some embodiments, chloroplast transformation involves introducing regions of chloroplast I)NA flanking a desired nucleotide sequence, allowing for homologous recombination of the exogenous DiSlA into She target chloroplast genome. In some instances one to 1.5 kb flanking nucleotide sequences of chloroplast genomic DNA may be used. Using this method, point mutations in the chloroplast 16S rRNA and rpsl2 genes, which confer resistance to spectinomycin and streptomycin, can be utilized as selectable markers for transformation (Svab et al.. Proc. Natl. Acad Sd., USA 87:8526-8530, 1990), and can result in stable bomoplasmic Iransforrnants, at a frequency of approximately one per 100 bombardments of target leaves. [0076] Mi crop reject tie mediated transformation also can be used to introduce a polynucleotide into a plant cell chloroplast (Klein et al,, Nature 327:70-73. 1987), This method utilizes rnkτoproject iles such as gold or tungsten, which are coated with the desired polynucleotide by precipitation with calcium chloride, spermidine or polyethylene glycol. The rnicroprojectile particles are accelerated at high speed into a plant t issue using a device such as the BIOLISTIC PD- 1000 part icle gun (BioRad; Hercules Cali f.). Methods for the transformation using biolistic methods are well known in the art (see, e.g.: Christou, Trends in Plant Science 1 :423-431, 1996). Microprojεctilε mediated transformation has been used, for example, to generate a variety of transgenic plant species, including cotton, tobacco, corn, hybrid poplar and papaya. Important cereal crops such as wheat, oat, barley, sorghum and rice also have been transformed using rnicroprojectile mediated delivery (Duan ct al., Nature Biotech. 14:494-498, 1996; Shimamoto, Curr. Opin. Biotech. 5: 158-362, 1994). The transformation of most dicotyledonous plants is possible with the methods described above. Transformation of monocotyledonous plants also can be transformed using, for example, biolistic methods as described above, protoplast transformation, elcctropo ration of partially pcrmcabilizcd cells, introduction of DNA using glass fibers, the glass bead agitation method, and the like.

[0077] Transformation frequency may be increased by replacement of recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, including, but not limited to the bacterial aadA gene (Svab and Maliga, Proc. Natl. Acad. ScL, USA 90:913-917, 1993). Approximately 15 to 20 cell division cycles following transformation are generally required to reach a homoplastidie state. It is apparent to one of skill in the art that a chloroplast may contain multiple copies of its genome, and therefore, the term "homoplasmic" or "liomoplasmy" refers to the state where all copies of a particular locus of interest are substantially identical, Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. [0078] The methods of the present disclosure are exemplified using the microalga. One non-limiting example of microalga is C. reinhardiii. The use of microalgae to express a polypeptide or protein complex according to a methods disclosed herein provides the advantage that large populations of the microalgae can be grown, including commercially (Cyanoteeh Corp.; Kailua-Kona Hl), thus allowing for production and, if desired, isolation of large amounts of a desired product. However, the ability to express, for example, functional mammalian polypeptides, including protein complexes, in the chlorop lasts of any plant allows for production of crops of such plants and, therefore, the ability to conveniently produce large amounts of the polypeptides. Accordingly, the methods of the disclosure can be practiced using any plant having cliloroplasts. including, for example, macroalgae, for example, marine algae and seaweeds, as well as plants that grow in soil.

[0079] The term "plant" is used broadly herein to refer Eo a eukaryotic organism containing plastids, particularly chloroplasts, and includes any such organism at any stage of development, or to part of a plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed, and a plantlet. A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or a cultured cell, or can be part of higher organized unit, for example, a plant tissue, plant organ, or plant Thus, a plant cell can be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. As such, a seed, which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered plant cell for purposes of this disclosure. A plant tissue or plant organ can be a seed, protoplast, callus, or any other groups of plant cells that is organized into a structural or functional unit. Particularly useful parts of a plant include harvestable parts and parts useful for propagation of progeny plants. A harvestable part of a plant can be any useful part of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots, and the like. A part of a plant useful for propagation includes, for example, seeds, fruits, cutt ings, seedlings, tubers, root stocks, and the like.

[0080] A method of the disclosure can generate a plant containing chloroplasts that arc genetically modified to contain a stably integrated polynucleotide (Hagcr and Bock, Appl. Microbiol. Biolechnol. 54:302-310, 2000). Accordingly, the present disclosure further provides a transgenic ftransplastomic) plant, e.g. C. vemharάtii, which comprises one or more chloroplasts containing a polynucleotide encoding one or more heterologous polypeptides, including polypeptides that can specifically associate to form a functional protein complex.

[0081] Examples of enzymes that contribute to the production of fatty acids, lipids or oils are shown in Table I . Non- limiting examples of enzymes that contribute to the production of fatty acids, lipids or oils include: acctyl-CoA carboxylase (ACC), ketoreductasc (KR), thiocstcrase (TE), malonyltransfera.se (MT), dehydratase (DH), acyl-CoA ligase (ACL). ketoacyl synthase (KAS), enoylreductase (ER) acyl CoA synthetase, and a desaturase (DESAT), One of skill in the art will recognize that these enzymes are only a partial list of enzymes which could be used in the present invention.

The present disclosure also contemplates a host cell transformed with one or more of the nucleic acids herein. ϊn preferred embodiments, the host cell is photosynthetic. In some cases, the host cell is photo synthetic and nonvascular, ϊn other cases, the host cell is photosynthetic and vascular. The host cell can be eukaryotic or prokaryotic. 4] The host ceil is transfected with a vector as described herein (e.g.. a vector comprising one or more fatty acid synthesizing enzymes and/or one or more modulating agents). The vector may contain a plastid promoter or a nucleic promoter for transfecting a chioroplast or other plastid of the host cell. The vector may also encode a fusion protein or agent that selectively targets the vector product to the chioroplast or other plastid. Transfection of a host cell can occur using any method known in the art.

[0085] A host organism is an organism comprising a host cell. In certain embodiments, the host organism is photosynthetic. A photosynthetic organism is one that naturally photosynthesizes (has a plastid) or that is genetically engineered or otherwise modified to be photosynthetic. In some instances, a photosynthetic organism may be transformed with a construct of the disclosure which renders all or part of the photosynthetic apparatus inoperable, ϊn some instances it is nou- vascular and photosynthetic. The host cell can be prokaryotic. Examples of some prokaryotic organisms of the present disclosure include, but are not limited to cyanobacteria (e.g., Synechococcus, Synechocystis. Alhrospirά). The host organism can be unicellular or multicellular, ϊn most embodiments, She host organism is eukaryotic (e.g. green algae). Examples of organisms contemplated herein include, but are not limited to, rhodophyta. chlorophyta. heterokontophyta. tribophyta. glaucophyta, chlorarachniophytes. eugleuoids, haplophyta. cryptomonads. dinoflagellata, and phytoplankton.

[0086] A host organism may be grown under conditions which permit photosynthesis, however, this is not a requirement (e.g., a host organism may be grown in the absence of light ), In some instances, the host organism may be genetically modified in such a way that photosynthetic capability is diminished and/or destroyed. Tn growth conditions where a host organism is not capable of photosynthesis (e.g., because of the absence of light and/or genetic modification), typically, the organism will be provided with the necessary nutrients to support growth in the absence of photosynthesis. For example, a culture medium in (or on) which an organism is grown, may be supplemented with any required nutrient, including an organic carbon source, nitrogen source, phosphorous source, vitamins, metals, lipids, nucleic acids, micronutrients, or an organism-specific requirement. Organic carbon sources include any source of carbon which the host organism is able to metabolize including, but not limited to, acetate, simple carbohydrates (e.g., glucose, sucrose, lactose), complex carbohydrates (e.g., starch, glycogen), proteins, and lipids. One of skill in the art will recognize that not all organisms will be able to sufficiently metabolize a particular nutrient and that nutrient mixtures may need to be modified from one organism to another in order to provide the appropriate nutrient mix. [0087] A host organism can be grown on land, e.g., ponds, aqueducts, landfills, or in closed or partially closed bioreactor systems. The host organisms herein can also be grown directly in water, e.g., in ocean, sea, on lakes, rivers, reservoirs, etc. Tn embodiments where algae are mass-cultured, the algae can be grown in high density photobioreactors Methods of mass-culturing algae are known. For example, algae can be grown in high density photobioreactors (see, e.g., Lee et al, Biotech. Bioengineering 44: 1161-1167, 1994) and other bioreactors (such as those for sewage and waste water treatments) (e.g., Sawayama et al, Appl. Micro. Biotech., 41 :729-731. 1994). Additionally, algae may be mass- cultured to remove heavy metals (e.g., Wilkinson, Biotech. Letters, 13 :861-864, 3989), hydrogen (e.g., U.S. Patent Application Publication No, 20030162273). and pharmaceutical compounds

[0088] In some cases, host organism(s) are grown near facilities generating flue gas or CO₇; (e.g.. electrical generating plants, concrete plants, oil refineries, other industrial facilities, cities, highways, etc.). As such, the methods herein contemplate business methods for selling carbon credits to ethanol plants or other facilities generating CO₂ while producing fatty acids, lipids, oils, or other fuels or fuel feedstocks by the modified organisms described herein. Fuel Production

990] The fatty acids or fuels produced using the methods herein may be subsequently refined into various fuel products (e.g. the fatty acids, lipids and/or oils may be used as feedstocks for fuel production). In one instance, the methods herein comprise expressing a gene encoding a fatty acid synthesis enzyme in a photosynthetic organism (e.g.. non-vascular). The methods further comprise utilizing the fatty acid synthesis enzyme to produce fatty acids, lipids or oils. Iu some embodiments, the method may further involve refining the fatty acids, lipids or oils to produce a biofuel. The final product (e.g.. octane) may then be mixed wish one or more other fuels.

[0091] The disclosure relates to a method of producing a biofuel comprising a least one vector encoding at least one fatty acid synthesis enzyme in a photosynthetic organism. The method may comprise use of a least one vector encoding multiple enzymes of an anabolic enzymatic pathway in at least one photosynthetic organism. Iu some embodiments the methods comprise extracting the product (e.g. fatty acid, lipid or oil) from the transformed organism. The product produced by one or more of the methods of the present disclosure may be a biofuel or crude biofuel. Iu some embodiments, the method comprises further refining the product.

[0092] In some embodiments, the biofuel is an oil, fatty acid, lipid or mixture of such, gasoline or other combustible fuels. A biofueS of the present disclosure may resemble crude oil (e.g., a mix of hydrocarbons). In some instances, the fatty acids, lipids, and/or oils described herein can be used directly as fuels. Tn other instances, these products serve as feedstocks which can be treated or refined, converting them into liquid hydrocarbon fuel and chemicals using any appropriate technology (e.g., catalytic cracking, use of alkaline catalyst). Many such methodologies are known in the art including, but not limited to, hydrolysis, decarboxylation, dehydration, isomerization, cyclization, aromatization, catalytic cracking, use of alkaline catalysts, acid-catalyzed pretreatmεnt followed by alkaline catalyzed transcsterificarion, thermal cracking (e.g., pyrolysis), use of equilibrium catalysts and fluid catalytic cracking. See, e.g., Dupain et al,, Applied Catalysis B: Environmental 72(1-2): 44-61 (2007); Oo i et al,, Biomass and Bioenergy 27(5):477- 84 (2004); Bhatia, S., Reaction Kinetics and Catalysis Letters, 84(2); 295-302 (2005); Canakci, ct al., Transactions of the ASAE, 46(4); 945-54 (2003); PCT Pub. No. WO/2007/068097; U.S. Pat. No. 7,288,685.

[0093] Various processes have been used to treat or crack fatty acids, lipids and oils. Among these, direct pyrolysis or catalytic cracking carried out at high temperatures (350-700⁰C) and near atmospheric pressure is one approach. Biofuel production via transesterification processes can also be used. Biofuel can also be further processed using conventional upgrading and post refining processes that typically include one or more of the following unit operations: catalytic cracking, isomerization, aromatization. and hydrogenation. High pressure processes involving hydrolysis, transesterification and/or pyrolysis can also be utilized. Another approach for converting fatty acids, lipids anώ'or oils into biofuels involves the use of catalytic bydrothermolysis. See, e.g., U.S. App. No. 11/857937. This process involves the use of hot. compressed water in conjunction with cracking, hydrolysis, decarboxylation, dehydration, isomerization. cyclization, recombination and/ or aromatization for conversion of fatty acids, lipids and/or oils to biofuels. [0094] In some instances, prior to refining, contaminants such as alkali metals may be removed, for example, by contacting the fatty acids, lipids and/or oils with an ion-exchange resin (e.g., Amberlyst™-15). using a mild acid wash (e.g., using sulfuric acid, hydrochloric acid, acetic acid, etc.). or using alumina guard beds with or without demetallation catalysts. The conditions at which such a reactor is operated are well known m the art.

[0095] Reference is made herein to various materials and methodologies known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et a!.. "Molecular Cloning: A Laboratory Manual". 2d ed., Cold Spring Harbor Laboratory Press. Plainview, N. Y., 1989; Kaufman et a!., eds., "Handbook of Molecular and Cellular Methods in Biology and Medicine", CRC Press, Boca Raton, 1995; and McPherson. ed., "Directed Mutagenesis: A Practical Approach". ΪRL Press, Oxford. 1991. Standard reference literature teaching general methodologies and principles of yeast genetics useful for selected aspects of the invention include, bus are not limited to" Sherman et al. "Laboratory Course Manual Methods in Yeast Genetics". Cold Spring Harbor Laboratory, Cold Spring Harbor, N, Y., 1986 and Guthne et al., ^''Guide to Yeast Genetics and Molecular Biology". Academic, New York, 1991.

[0096] Where a range of values is provided, it is understood that each intervening value, to She tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a sSated range and any other stated or intervening value in that sSated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed, subject to any specifically excluded limit in the stated range. Where the slated tange includes one or both of the limits, ranges excluding cither or both of those included limits are also included.

EXAMPLES 7] The following examples merely illustrate the invention disclosed herein, but do not limit it.

Example 1. Production of thioesterase in C. remhardiii

In this example nucleic acids encoding thioesterase (TFi from C reinhcirJni, U. cahjornica and C caniphora are introduced into C. rewhaniui. Specifically, the nucleic acids encode truncated versions of thioesterases (SEQ ID NOs: 21. 27, 31 , 33 and 35), for example, snyristoyl-ACP-speesfie thioestcrases. The Transforming DNA is shown graphically in FIG, 3A. In this instance the gene encoding a thioesterase (SEQ ID NO: 20, 26, 30. 32 and 34) is the segment labeled "'Transgene,'^" which is regulated by the 5^" IJTR and promotes' sequence for the pshD gene from C reuihardth, and the 3^" IJTR for thopsbA gene from C reinhcirJni. The segment labeled "Resistance Marker^'' ss the kanamycin resistance encoding gene from bacteria, which ss regulated by the 5' IJTR and promoter sequence for the arpA gene from C. reinhaniui and the 3^' UTR sequence for the rhcL gene from from C. reinharJtii. The transgene cassette is targeted to the 3HB locus of C reinhcirJni via the segments labeled "Homology A^'' and "Homology B,^'' which are identical to sequences of DNA flanking the 3HB locus on the 5' and 3¹ sides, respectively. All DNA manipulations carried out in the construction of this transforming DNA are essentially as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen et al.. MeIh. Enzymol 297. 192-208. 1998.

[0099] For these experiments, all transformations are carried out on C. reinhardtii strain 137c (mt+). Cells are grown to late log phase (approximately 7 days) in the presence of 0.5 niM 5-ftuorodeoxyuridine in TAP medium (Gorman and Levme. Proc. Null. Acad. Sd.. USA 54: 1665-3 b69, 3965, which is incorporated herein by reference) at 23°C under constant illumination of 450 Lux on a rotary shaker set at 100 rpm. Fifty ml of cells are harvested by centrifugation at 4,000xg at 23⁰C for 5 min. The supernatant is decanted and cells resuspended in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment (Cohen et a!., supra. 1998). All transformations are carried out under kanamycin selection (100 μg/ml) in which resistance is conferred by the gene encoded by the segment in FlG. 3A labeled "Resistance Marker.^"' (Chlamydomonas Stock Center, Duke University).

[0010Oj PCR is used to identify transformed strains. For PCR analysis. lϋ⁶ algae cells (from agar plate or liquid culture) are suspended in 10 mM EDTA and heated to 95°C for 10 minutes, then cooled to near 23"C. A PCR cocktail consisting of reaction buffer. MgC12, dNTPs, PCR primer ρair(s) (T^' able 2 & a gene specific reverse primer: also shown graphically in FIG. 4), DNA polymerase, and water is prepared. Algae Iy sate in EDTA is added to provide a template for the reaction. The magnesium concentration is varied So compensate for amount and concentration of algae lysate and EDTA added. Additionally, genomic DNA from algal cells is used (Promega Genomic DNA Purification Kit) as the template for PCR reactions, if algal iysates yield unclear results. Annealing temperature gradients are employed to determine optimal annealing temperature for specific primer pairs.

[00101J To identify strains that contain the thioesterase gene, a primer pair is used in which one primer anneals to a site within the 5"UTR and She other primer anneals within the Shioesterase coding segment (See Table 2). Desired clones are those that yield a PCR product of expected size. To determine the degree to which the endogenous gene locus is displaced (Tietεroplasmic vs. hornoplasrnic), a PCR reaction consisting of two sets of primer pairs is employed fin the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by 1he expression vector, so should produce a product of expected size in all cases. This reaction confirms that Ihe absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited the PCR reaction. The number of cycles used is >30 to increase sεnsit ivity. The most desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak-intensity endogenous locus products relative to the control reaction, FIG S shows the successful production of several homoplamic transgenetic clones using various constructs.

[00102] To ensure that the presence of the thioestcrase-encoding gene leads to expression of a thioesterase protein, a Western blot is performed. Approximate!}' 5x 10^s algae cells are collected from TAP liquid media by centrifuging cells at 3.000 rpm at 20°C for 5 minutes. Cells are resuspended in cold lysis buffer (TBS buffer pH 7.4 (25 mM Tris-HCl, 137 mM NaCl, 2,7 mM KCl ) supplemented with EDTA-free protease inhibitors (Roche)). Cells are lysed by mechanical disruption of membranes by sonication. Samples were centrifugεd to remove debris and insoluble components. Affinity purification of the FLAG-tagged thioesterase enzymes is performed by incubation at 4°C with anti-FLAG resin. The resin is washed several times, and bound proteins εlυtεd using 0, 1 M Glycine pH 3.0. Eluted protein is mixed 3: 1 with loading buffer (XT Sample Buffer; Bio-Rad) containing β-mεrcaptoetbanol. Samples are then heated to 95'^"C for 5 min, and cooled to near 23°C. Proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is incubated with blocking solution (StartingBlock; ThermoSciεntific) at 23⁰C for 30 min, followed by incubation with horseradish peroxidasε-linked anti-FLAG antibody (diluted 1 :4000 in TBST + 2.5% horse serum) at 4⁰C for 12 hours, and washed three times with TBST, Proteins are visualized by incubating with horseradish peroxidase substrate (SupεrSignal West Dura; TherraoSciεntific) and chεrailuminεscent detection. Fig 6 shows successful expression of several thioesterase genes in C. reiiihardtii . Fig. 8 shows the increase in total relative lipid content (% TRLC) in various strains transformed with nucleotide sequences encoding thioesterases. [00103] Cultivation of C. reϊnhardtύ transformants for expression of thioesterase is carried out in liquid HSM medium at 5% CO₂ at 28⁰C under constant illumination of 5,000 Lux on a rotary shaker set at 300 rpm, unless stated otherwise. Cultures are maintained at a density of 1x10' cells per ml for at least 48 hr prior to harvest.

[00 ! 04] To identify the lipid composition of strains expressing thioesterase, a 100 L culture is grown in HSM media. Algae are harvested Eo yield approximately 100 g of wet paste, which is then frozen at -80^cC until solid. The frozen pellet is placed into a 300 micron vacuum to lyophilize the algae for at least 24 hours. Cells are then sent to a third party vendor (Covance) for standard analysis of fatty acid content using the published protocol (Association of Oil Chemists Society (AOCS) method Ceib-89: Fatty Acid Composition by GLC). Additionally, the lipid content and composition is measured using Flame Ionization Detection (FlD). Per sample, 20 mg of lyophilized biomass is incubated with 500 μl of 0.5N Potassium Hydroxide solution in methanol, and 200 μl of tetrahydrofuran containing 0.05% butylated hydroxy toluene. 0.4 μg of internal fatty acid standards (C13:0 Trilridecanoin. C2i:0 Methyl Heneicosanoatej are added for quantitative analysis of fatty acids. Samples are incubated at 80⁰C for 60 minutes. After cooling, 500 μl of a 10% solution of boron trifhioridc in methanol is added to samples and incubated an additional 5 minutes at 80⁰C. Samples arc incubated with 500 μl of heptane and 300 μl of sodium chloride saturated in water. Samples are then mixed and centrifuged for 20 minutes at 3900 tpra, resulting in an aqueous phase containing isolated fatty acid methyl esters (FAME). These samples were 1hen run on 1he GC-FID according to standard operat ing procedure to quantify and identify FAMEs. Fig. 7 shows the results of transformation of C reinhardth with SEQ ID NO: 20 which resulted in an approximately 6 fold increase in Ihe production of rπyrisl ic acid.

[00105] Results from the GC-FID are reported as concentration of each individual fatty acid, calculated according to the following equation;

C) fmg/gl == (Ms * Ai * Fm * Fa )/(As * M*Fs)

Ci = Concentration of individual FA.

Ms = Mass of internal standard added to the sample. Ms is equal to 2 mg.

Ai= Area of individual FAME in the sample.

Fm = Correction factor of FAMEi to corresponding FAi, the approximation of this factor is equal to 1 for now (Fm=

0.86-0.96).

Fa = Conversion factor to mg/^'g. Fa is equal to 1000.

M — Mass of dry biomass or oil sample,

As — Area of the internal standard.

Fs — Relative response factor for each FAME compared with Cl 3:0 for which Fs=LO

The percent relative lipid content (%TRLC) is calculated using the following equation:

% Relative total lipid content = £ Ci * Fb * Fn Fb — Conversion factor to %. This factor is equal to 0.1.

Fn — Correction factor of FAMEi to the corresponding TAGi, the approximation of this factor is equal to 1 (Fn- 0.988-

0.9961

£xanτple_.2._.Production _.of_. ΑcjχhCoA lig^seJnXlχemlιgrώji

In this example a nucleic acid encoding acyl-CoA iigase (ACL) from Arctbiposis ihaiiana is introduced into C. reinhardiii. Transforming DMA is shown graphically in FlG, 3A. In this instance the gene encoding acyl-CoA Iigase is the segment labeled "Transgene," which is regulated by the 5^* UTR and promoter sequence for the psbD gene from C. reinhardtii, psbC gene from C. reinhardiii. atpA gene from C. reinhardiii. oτpsbA gene from C. reinhardtii and the 3 ^* UTR for the psbA gene from C reinhardiii. The segment labeled ''Resistance Marker^" is She kauamycin resistance encoding gene from bacteria, which is regulated by the 5' UTR and promoter sequence for the aipA gene from C. reinhardiii and the 3' UTR sequence for the rbcL gene from from C. reinhardtii. The transgene cassette is targeted to the 3HB locus of C. reinhardiii via the segments labeled "Homology A" and "Homology B," which are identical to sequences of DNA flanking the 3HB locus on the 5' and 3^" sides, respectively. All DNA manipulations carried ouf in the construct ion of 1his transforming DNA are essentially as described by Sarnbrook εt al,, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen el aS,, Meth. Enzymol, 297, 192-208, 1998. [00107] For these experiments, all transformat ions are carried out on C. reinhardiii strain 137c (rat+). Cells are grown to Sase log phase (approximately 7 days) in the presence of 0,5 mM 5-fϊuorodeoxyuridine in TAP medium (Gorman and Lεvinε, Proc. NaIi. Acad. Sd., USA 54: 1665- 1669, 1965, which is incorporated herein by reference) at 23⁰C under constant illumination of 450 Lux on a rotary shaker set at 100 rpm. Fifty ml of cells are harvested by centrifugation at 4,000xg at 23"C for 5 min. The supernatant is decanted and cells resuspcnded in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment (Cohen et al., supra, i 998), AU transformations arc carried out under kanamycin selection (100 ug/ml) in which resistance is conferred by the gene encoded by the segment in FIG. 3Λ labeled "Selection Marker." (Cblamydomonas Stock Center, Duke University).

[00108] PCR is used to identify transformed strains. For PCR analysis, I O⁶ algae ceils (from agar plate or liquid culture) are suspended in 10 mM F.DTA and heated to 95°C for i 0 minutes, then cooled to near 23°C. A PCR cocktail consisting of reaction buffer, MgC12, dNTPs, PCR primer pair(s) (Table 2 & a gene specific reverse primes'), DNA polymerase, and water is prepared. Algae lysate in EDTA is added to provide a template for the reaction. The magnesium concentration is varied to compensate for amount and concentration of algae lysate and EDTA added. Annealing temperature gradients are employed to determine optimal annealing temperature for specific primer pairs. [00109] To identify strains that contain the acyl-CoA Iigase gene, a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the acyl-CoA Iigase coding segment. Desired clones are those that yield a PCR product of expected size. To determine the degree to which the endogenous gene locus is displaced (heteroplasmic vs. homoplasmicj, a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in ail cases. 'This reaction confirms that the absence of a PCR product from the endogenous iocus did not result from cellular and/or other contaminants that inhibited the PCR reaction. Concentrations of the primer pairs are varied so that both reactions work in the same tube; however, the pair for the endogenous locus is 5X the concentration of the constant pair. The number of cycles used was >30 to increase sensitivity. The most desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak-intensity endogenous locus products relative to the control reaction, [00 ! 1Oj To ensure that the presence of the acyl-CoA ligase-encoding gene leads to expression of a acyl-CoA ligase protein, a Western blot is performed. Approximately lxiθ^s algae cells are collected from TAP agar medium and suspended in 0.5 ml of lysis buffer (Bυgbuster: Novagen). Ly sate is mixed 3: 1 with loading buffer (XT Sample Buffer; Bio-Rad). Samples are then heated to 95°C for 5 mill, cooled to near 23 "C, and insoluble proteins are removed by centrifugation. Soluble proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is blocked with TBST +^■ 5% dried, nonfat milk at 23^UC for 30 min, incubated with horseradish-linked anti- FLAG antibody (diluted 1 :2,500 in TBST ⁺ 5% dried, nonfat milk) at 4⁰C for 12 hours, and washed three times with ¹TBST. Proteins are visualized with chemiliiminescent detection. Positive results from multiple clones show that expression of She acyl-CoA ligase gene in C. reinhardtii cells results in production of the protein. [00111] Cultivation of C. reinhardtii transformants for expression of acyl-CoA ligase is carried out in liquid HSM medium at 23⁰C under constant illumination of 5.000 Lux on a rotary shaker set at 100 rpra, unless stated otherwise. Cultures are maintained at a density of 1 xlO' cells per ml for at least 48 hr prior to harvest.

Example 3. Production of fatry acyl-CoA desatυrase in C. reinhardtii

[00112] In this example a nucleic acid encoding fatty acyl-CoA desaturase (DESAT) from Arabiposis ihaliana is introduced into C. reinhardtii. Transforming DNA is shown graphically in FIG. 3A. Tn this instance the gene encoding fatty acyl-CoA desaturase is the segment labeled "Transgene," which is regulated by the 5' UTR and promoter sequence for the/λsfeD gene from C. reinhardtii, psbC gene from C. reinhardtii, atpA gene from C. reinhardtii, or psbA gene from C. reinhardtii and the 3' UTR for the psbA gene from C. reinhardtii. The segment labeled "'Resistance Marker" is the kanamycin resistance encoding gene from bacteria, which is regulated by the 5' UTR and promoter sequence for the arpA gene from C. reinhardtii and the 3^' UTR sequence for the rbcL gene from from C. reinhardtii. The transgene cassette is targeted to the 3HB iocus of C. reinhardtii via the segments labeled "Homology A" and "Homology B," which are identical to sequences of DNA flanking the 3HB locus on the 5' and 3' sides, respectively. All DNA manipulations carried out in the construction of this transforming DNA are essentially as described by Sambrook et al,. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen et al., Meth. Enzymol. 297. 192-208. 1998.

[001 ! 3] For these experiments, all transformations are carried out on C. reinhardtii strain 137c (mt+). Cells are grown to late log phase (approximately 7 days) in the presence of 0.5 niM 5-fluorodeoxyuridine in TAP medium (Gorman and Levine, Proc. Natl. Acad. Sd.. USA 54:1665-3669, 3965, which is incorporated herein by reference) at 23 °C under constant illumination of 450 Lux on a rotary shaker set at 100 rpm. Fifty ml of cells are harvested by centrifugation at 4,000xg at 23"C for 5 min. The supernatant is decanted and cells resuspended in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment (Cohen et al., supra, 1998). All transformations are carried out under kanamycin selection (100 μg/ml) in which resistance is conferred by the gene encoded by the segment in FlG. 3A labeled "Resistance Marker.^"' (Chlamydomonas Stock Center, Duke University).

[00114] PCR is used to identify transformed strains. For PCR analysis. iϋ⁶ algae cells (from agar plate or liquid culture) are suspended in 10 mM EDTA and heated to 95°C for 10 minutes, then cooled to near 23"C. A PCR cocktail consisting of reaction buffer. MgC12, dNTPs, PCR primer ρair(s) (T^' able 2 & a gene specific reverse primer), DNA polymerase, and water is prepared. Algae lysate in EDTA is added to provide a template for the reaction. The magnesium concentration is varied to compensate for amount and concentration of algae lysate and EDTA added. Annealing temperature gradients are employed Eo determine optimal annealing temperature for specific primer pairs. [OOllSj To identify strains that contain the fatty acyl-CoA desaturase gene, a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the fatty acyi-CoA desaturase coding segment. Desired clones are those that yield a PCR product of expected size. To determine the degree to which the endogenous gene locus is displaced iheteroplasmic vs. homoplasmic), a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region thai is not targeted by the expression vector, so should produce a product of expected size in all cases. This reaction confirms that She absence of a PCR product from Ihe endogenous locus did not result from cellular and/or other contaminants that inhibited 1he PCR reaction. Concentrat ions of the primer pairs are varied so that both reactions work in the same tube; however, the pair for the endogenous locus is 5X the concentration of the constant pair. The number of cycles used was >30 to increase sεnsit ivity. The most desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak- intensity endogenous locus products relative to the control reaction,

[00116] To ensure that the presence of the fatty acyl-CoA desaturasε-εncoding gene leads to expression of a fatty acyl- CoA desaturase protein, a Western blot is performed. Approximately 1 x 30^b algae cells are collected from TAP agar medium and suspended in 0.5 ml of lysis buffer (Bugbuster; Novagen). Lysate is mixed 3: i with loading buffer (XT Sample Buffer; Bio-Rad). Samples are then heated to 95 "C for 5 min, cooled to near 23°C, and insoluble proteins are removed by centrifugation. Soluble proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is blocked with TBST ÷ 5% dried, nonfat milk at 23⁰C for 30 min, incubated with horseradish-linked anti-FLAG antibody (diluted 1 :2,500 in TBST + 5% dried, nonfat milk) at 4°C for i 2 hours, and washed three times with TBST. Proteins are visualized with chcmilumincscent detection. Positive results from multiple clones show that expression of the fatty acyl-CoA desaturase gene in C. veinharάtlϊ cells results in production of the protein. [001 ! 7] Cultivation of C. reinhardiiϊ transformants for expression of fatty acyl-Co A desaturase is carried out in liquid HSM medium at 23⁰C under constant illumination of 5,000 Lux on a rotary shaker set at 300 rpm, unless stated otherwise. Cultures are maintained at a density of 3 xlO' cells per ml for at least 48 hr prior to harvest.

Example 4. Production of methylmalonyl-CoA mutase in C. reinhardiii

[00118] In this example a nucleic acid encoding methylmalonyl-CoA mutase (MMCM) from Streptomyces cinnamonensis is introduced into C. reinhardiii. Transforming DTs'A is shown graphically in FIG. 3A. ϊn this instance the gene encoding metbylmalonyl-CoA mutase is the segment labeled "Transgene,^"' which is regulated by the 5^" UTR and promoter sequence for the pshD gene from C reinhardiii, psbC gene from C. reinhardiii, atpA gene from C. reinharάύi, or psbΛ gene from C reinhardtii and the 3' UTR for the psbA gene from C. reinhardtii. The segment labeled "Resistance Marker" is the kanamyein resistance encoding gene from bacteria, which is regulated by the 5' UTR and promoter sequence for the atpA gene from C. reinhardtii and the 3' UTR sequence for the rbcL gene from from C. reinhardtii. The transgene cassette is targeted to the 3HB locus of C. reinhardtii via the segments labeled "Homology A" and "Homology B,'^" which are identical to sequences of DNA flanking the 3HB locus on the 5' and T sides, respectively. AU DNA manipulations carried out in the construction of this transforming DNA are essentially as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen et al.. MeIh. Enzymol. 297, 192-208, 1998.

[00119] For these experiments, all transformations are carried out on C. reinhardtii strain 137c (mt+). Cells are grown to IaEe log phase (approximately 7 days) in She presence of 0,5 mM 5-fluorodeoxyuridine in TAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669. 1965. which is incorporated herein by reference) at 23⁰C under constant illumination of 450 Lux on a rotary shaker set at 100 rpm. Fifty ml of cells are harvested by centrifugation at 4,0G0xg at 23 "C for 5 min. The supernatant is decanted and cells resuspended in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment i Cohen et al.. supra. 1998). All transformations are carried out under kanamyein selection i 100 μg/ml) in which resistance is conferred by She gene encoded by the segment in FlG, 3 A labeled "Resistance Marker," (Chlaraydomonas Stock Center, Duke University).

[00120] PCR is used to identify transformed strains. For PCR analysis, iθ^fc algae cells (from agar plate or liquid culture) are suspended in 10 mM EDTA and heated to 95°C for i0 minutes, then cooled Io near 23°C. A PCR cocktail consisting of reaction buffer, MgC12, dNTPs, PCR primer ρair(s) (Table 2 & a gene specific reverse primer), DNA polymerase, and water is prepared. Algae lysale in EDTA is added Io provide a template for the reaction. The magnesium concentration is varied so compensate for amount and concentration of algae lysale and EDTA added. Annealing temperature gradients are employed so determine optimal annealing lemperature for specific primer pairs, [00121] To identify strains that contain the methylmalonyl-CoA mutase gene, a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the methylmalonyl-CoA mntase coding segment. Desired clones arc those that yield a PCR product of expected size. To determine the degree to which the endogenous gene locus is displaced (heteroplas ic vs. homoplasmic), a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in ail cases. This reaction confirms that the absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited the PCR reaction. Concentrations of the primer pairs are varied so that both reactions work in the same tube: however, the pair for the endogenous locus is 5X the concentration of the constant pair. The number of cycles used was >30 to increase sensitivity. The most desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak-intensity endogenous locus products relative to the control reaction.

[00122] To ensure that the presence of the methylmalonyl-CoA mutase -encoding gene leads to expression of a methylmalonyl-CoA mutase protein, a Western blot is performed. Approximately lxiO⁸ algae cells are collected from TAP agar medium and suspended in 0.5 ml of lysis buffer (Bugbuster: Novagen). Lysate is mixed 3: 1 with loading buffer (XT Sample Buffer; Bio-Rad). Samples are then heated to 95°C for 5 min. cooled to near 23°C, and insoluble proteins are removed by centrifugation. Soluble proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is blocked with TBST + 5% dried, nonfat milk at 23⁰C for 30 min, incubated with horseradish-linked anti-FLAG antibody (diluted 1 :2,500 in TBST + 5% dried, nonfat milk) at 4⁰C for 12 hours, and washed three times with TBST. Proteins are visualized with cliemiluminescent detection. Positive results from multiple clones show that expression of the metbylmalonyl-CoA mutase gene in C. reinhardtii cells results in production of the protein.

[00123 j Cultivation of C. reinhardtii transformants for expression of methylmalonyi-CoA mutase is carried out in liquid HSM medium at 23⁰C under constant illumination of 5.000 Lux on a rotary shaker set at 100 rpm, unless stated otherwise. Cultures are maintained at a density of 1x10 ' cells per ml for at least 48 hr prior to harvest. [00Ϊ24] To determine if the metliylnialonyl-CoA mutase produced by transformed algae cells is functional, metliylmaloiiyl-CoA mutase activity is tested using a suitable assay. iiMifflSlεAJioductiqn of kritoacyXs\Ηth^e_in_C_rg/«^fl£dW/

[00125] In this example a nucleic acid f SEQ ID NO: 28) encoding 3-ketoacyl ACP synthase Ll (KAS IL) from R. comminis (SEQ ID NO: 29) is introduced into C. reinhardtii, Transforming DNA is shown graphically in FIG. 3A. Iu this instance the gene encoding kεioacyl synthase is the segment labeled "Transgene," which is regulated by the 5' UTR and promoter sequence for thepsbD gene from C. reinhardtii, and the 3^' UTR for thspsbΛ gene from C. reinhardtii. The segment labeled "Resistance Marker" is the kanarnycin resistance encoding gene from bacteria, which is regulated by the 5^' UTR and promoter sequence for the atpA gene from C. reinhardtii and the 3^' UTR sequence for the rbcL gene from from C. reinhardtii. The transgene cassette is targeted to the 3HB locus of C. reiυhardtii via the segments labeled ''Homology A" and "Homology B,'^' which are identical Io sequences of DNA flanking the 3HB locus on the 5' and 3' sides, respectively. AU DNA manipulations carried out in the construction of this transforming DNA are esseni tally as described by Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press i 989) and Cohen et al., Meth. Enzymol. 297, 192-208, 1998.

[00126| For these experiments, all transformations are carried out on C. reinhardtii strain 137c (mt+). Ceils are grown to late log phase (approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridinc in TAP medium (Gorman and Lcvinc, Proc. Natl. Acad. ScL, USA 54: i 665- 1669, 1965, which is incorporated herein by reference) at 23°C under constant illumination of 450 Lux on a rotary shaker set at 100 φm. Fifty ml of cells are harvested by centrifugation at 4,000xg at 23^UC for 5 min. The supernatant is decanted and cells rcsuspendcd in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment (Cohen et al,, supra, i 998), All transformations are carried out under kanamycin selection (100 μg/ml) in which resistance is conferred by the gene encoded by the segment in FIG. 3 A labeled "Resistance Marker. " (Chlamydomonas Stock Center. Duke University).

[00127] PCR is used to identify transformed strains. For PCR analysis, 10⁶ algae cells (from agar plate or liquid culture) are suspended in 10 mM EDTA and heated to 95°C for 10 minutes, then cooled to near 23 "C. A PCR cocktail consisting of reaction buffer, MgC12. dNTPs. PCR primer pair(s) (Table 2 & a gene specific reverse primer; also shown graphically in FlG. 4). DNA polymerase, and water is prepared. Algae lysate in EDTA is added to provide a template for the reaction. The magnesium concentration is varied to compensate for amount and concentration of algae lysate and EDTA added. Additionally, genomic DNA from algal cells is used (Promega Genomic DNA Purification Kit) as the template for PCR reactions, if algai lysates yield unclear results. Annealing temperature gradients are employed to determine optimal annealing temperature for specific primer pairs.

[00 ! 28] To identify strains that contain the 3-ketoacyl ACP synthase gene, a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the ketoacyl synthase coding segment. Desired clones are those that yield a PCR product of expected size, 'TO determine the degree to which the endogenous gene locus is displaced (heteroplasmic vs. homoplasmic), a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in all cases. This reaction confirms that the absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited She PCR reaction. The number of cycles used was >J0 to increase sensitivity. The most desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak-intensity endogenous locus products relative to the control reaction. Results are shown in Fig. 5,

[00Ϊ29J To ensure that the presence of the ketoacyl synthase -encoding gene leads to expression of a 3-ketoacyl ACP synthase protein, a Western blot is performed. Approximately 5xiO^s algae cells are collected from TAP liquid media by centrifuging cells at 3,000 rprn at 20⁰C for 5 minutes. Cells are rεsuspended in cold lysis buffer CTBS buffer pH 7,4 (25 niM Tris-HCl, 137 mM NaCl. 2.7 niM KCl) supplemented with EDTA-free protease inhibitors (Roche)). Cells are lysed by mechanical disruption of membranes by souication. Samples were cenSrifuged to remove debris and insoluble components. A ffinity purification of the FLAG-tagged ketoacyl enzymes is performed by incubation at 4°C with anti- FLAG resin. The resin is washed several times, and bound proteins eiuted using 0,LM Glycine pH 3,0. Eluted protein was mixed 3; 1 with loading buffer (XT Sample Buffer; Bio-Rad) containing β-raercaptoethanol. Samples are then heated to 95°C for 5 min. and cooled to near 23°C. Proteins are separated by SDS-PAGE. followed by transfer to PVDF membrane. The membrane is incubated with blocking solution (StartingBlock; ThermoSeientifϊc) at 23⁰C for 30 min, followed by incubation with horseradish-linked anti-FLAG antibody (diluted 1 :4000 in TBST ⁺ 2.5% horse serum) at 4°C for 12 hours, and washed three times with TBST. Proteins are visualized by incubating with horseradish peroxidase substrate (SuperSignal West Dura; ThermoScientifϊc) and chemiluminescent detection. Fig. 6 shows that expression of the ketoacyl synthase gene in C. reinhardiii cells results in production of the protein,

[00130] Cultivation of C reinhardiii transformants for expression of ketoacyl synthase is carried out in liquid HSM medium at 5% CO_? at 28⁰C under constant illumination of 5,000 Lux on a rotary shaker set at 100 rpm, unless stated otherwise. Cultures are maintained at a density of 1x10' cells per ml for at least 48 hr prior to harvest.

[00131] In this example a nucleic acid encoding tnεthylkεtonε synthase (MKS) from Lycopβrsicon hirsututn/. glabratwn is introduced into C. reinhardiii . Transforming DNA is shown graphically in FIG. 3A. In this instance the gene encoding mεthylkεtonε synthase is the segment labeled "Transgεnε," which can is regulated by the 5' UTR and promoter sequence for the psbD gene from C. reinhardiii, psbC gene from C. reinhardth, atpA gene from C. reinhardiii, or psbA gene from C reinhardiii and the 3^" UTR for the psbA gene from C. reinhardiii. The segment labeled "Resistance Marker^"' is the kanamycin resistance encoding gene from bacteria, which is regulated by the 5' UTR and promoter sequence for the atpA gene from C reinhardiii and the 3^" UTR sequence for the rbcL gene from from C. reinhardtii. The transgene cassette is targeted to the 3HB locus of C. reinhardtii via the segments labeled "Homology A" and "Homology B,'^" which are identical to sequences of DNA flanking the 3HB locus on the 5' and T sides, respectively. All DNA manipulations carried out in the construction of this transforming DNA are essentially as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen et al.. MeIh. Enzymol. 297, 192-208, 1998.

[00132] For these experiments, all transformations are carried out on C. reinhardtii strain 137c (mt+). Cells are grown to late log phase (approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine in TAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669. 1965. which is incorporated herein by reference) at 23⁰C under constant illumination of 450 Lux on a rotary shaker set at 100 rpm. Fifty ml of cells are harvested by cenSrifugaSion at 4,000xg at 23 "C for 5 min. The supernatant is decanted and cells resuspended in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment i Cohen et al.. supra. 1998). All transformations are carried out under kanamycin selection i 100 μg/ml) in which resistance is conferred by She gene encoded by the segment in FlG, 3 A labeled '"Resistance Marker.^*' (Chlamydomouas Stock Center, Duke University).

[Q0133] PCR is used to identify transformed strains. For PCR analysis. 10^fc algae cells (from agar plate or liquid culture) are suspended in 10 mM ED¹TA and healed to 95°C for 10 minutes, then cooled to near 23°C. A PCR cocktail consisting of reaction buffer, MgC12, dNTPs, PCR primer ρair(s) (Table 2 & a gene specific reverse primer), DNA polymerase, and water is prepared. Algae lysale in EDTA is added Io provide a template for the reaction. The magnesium concentration is varied to compensate for amount and concentration of algae lysale and EDTA added. Annealing temperature gradients are employed to determine optimal annealing temperature for specific primer pairs, [00134] To identi fy strains that contain the methylketone synthase gene, a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the methylketone synthase coding segment. Desired clones are those that yield a PCR product of expected size. To determine the degree to which the endogenous gene locus is displaced (heteroplas ic vs. homoplasmic), a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in ail cases. This reaction confirms that the absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited the PCR reaction. Concentrations of the primer pairs are varied so that both reactions work in the same tube; however, the pair for the endogenous locus is 5X the concentration of the constant pair. The number of cycles used was >30 to increase sensitivity. The most desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak- intensity endogenous locus products relative to the control reaction.

[00135] To ensure that the presence of the methylketone synthase -encoding gene leads to expression of a methylketone synthase protein, a Western blot is performed. Approximately 1x10^" algae cells are collected from TAP agar medium and suspended in 0.5 ml of lysis buffer (Bugbuster; Novagen). Lysate is mixed 3:1 with loading buffer (XT Sample Buffer; Bio-Rad). Samples are then heated to 95°C for 5 min, cooled to near 23 °C, and insoluble proteins are removed by centrifugation. Soluble proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is blocked with TBST + 5% dried, nonfat milk at 23⁰C for 30 min. incubated with horseradish-linked anti- FLAG antibody (diluted 1 :2,500 in TBST^' + 5% dried, nonfat milk) at 4°C for 12 hours, and washed three times with TBST. Proteins are visualized with ehemiluminescent detection. Positive results from multiple clones show that expression of the methylketone synthase gene in C. reinhardtii cells results in production of the protein. [00136] Cultivation of C. reinhardtii transformants for expression of methylketone synthase is carried out in liquid HSM medium at 23⁰C under constant illumination of 5,000 Lux on a rotary shaker set at 100 rpm, unless stated otherwise. Cultures are maintained at a density of 1x10 ' cells per ml for at least 48 hr prior to harvest.

Bxamrjle li_.C_.Oiisjrisction o^^^_^

S_.licQ_.dirig_.ge_.nes

[00137J In this example a strain containing multiple enzyme-encoding genes using a single vector is described. Transforming DNA comprising more Shan one is shown in FIG. 3B. In this instance she gene (SEQ ID NO: 20) encoding a thioesterase (SIiQ ID NO: 21) is the segment labeled "Transgene 1," which is regulated by the 5^" UTR and promoter sequence for ihcpsbD gene from C reinhardtii, and the 3' UTR for the psbA gene from C. reinhardtii. The segment labeled "Transgene 2^*' is the gene f SEQ ID NO: 28) encoding a 3-ketoacyl ACP synthase (SEQ ID NO: 29), which is regulated by the 5' UTR and promoter sequence for ihepsbD gene from C. reinhardtii, and the 3^" UTR for the psbA gene from C. reinhardtii. The segment labeled "Resistance Marker" is the kanamyciu resistance encoding gene from bacteria, which is regulated by the 5^' UTR and promoter sequence for the aipA gene from C. reinhardϋi and the 3^" UTR sequence for the rbcL gene from from C. reinhardtii. The transgene cassette is targeted to the 3HB locus of C. reinhardtii via the segments labeled "Homology A" and "Homology B," which are identical to sequences of DNA flanking the 3HB locus on the 5^" and 3' sides, respectively. AlS DNA manipulations carried out in the construction of this transforming DNA are essentially as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen et al, MeAh. Enzymoi. 297, 192-208, 1998.

[00138] For these experiments, all transformat ions are carried out on C. reinhardtii strain 137c (rnt+). Cells are grown to late log phase (approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridinc in TAP medium (Gorman and Lcvinc, Proc. Natl. Acad. Sd., USA 54: 1665-1669, 1965, which is incorporated herein by reference) at 23⁰C under constant illumination of 450 Lux on a rotary shaker set at 100 rpm. Fifty ml of cells are harvested by centrifugation at 4,000xg at 23^UC for 5 min. The supernatant is decanted and cells resuspcnded in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment (Cohen et al,, supra, i 998), All transformations are carried out under kanamycin selection (100 fig/ml) in which resistance is conferred by the gene encoded by the segment in FIG. 3B labeled "Resistance Marker." (Cblamydomonas Stock Center, Duke University).

[00139] PCR is used to identify transformed strains. For PCR analysis, I O⁶ algae cells (from agar plate or liquid culture) are suspended in 10 mM EDTA and heated to 95°C for 10 minutes, then cooled to near 23 "C. A PCR cocktail consisting of reaction buffer, MgC12. dNTPs. PCR primer pair(s) (Table 2 & a gene specific reverse primer), DNA polymerase, and water is prepared. Algae Iy sate in EDTA is added to provide a template for the reaction. The magnesium concentration is varied to compensate for amount and concentration of algae lysate and EDTA added. Additionally, genomic DNA from algal cells is used (Pro mega Genomic DNA Purification Kitj as the template for PCR reactions, if algal lysates yield unclear results. Annealing temperature gradients are employed to determine optimal annealing temperature for specific primer pairs. To identify strains that contain the gene encoded by "Transgene 1.^"' a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the thioesterase coding segment. Desired clones are those that yield a PCR product of expected size. To identify strains that contain the gene encoded by "Transgene 2," a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the ketoacyl synthase coding segment. Desired clones are those that yield a PCR product of expected size. To determine the degree to which the endogenous gene locus is displaced (heteroplasmic vs. homoplasmic), a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in all cases. This reaction confirms that the absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited the PCR reaction. The number of cycles used is >J0 to increase sensitivity. The most desired clones are those that yield a product for the constant region but not for She endogenous gene locus. Desired clones are also those that give weak-intensity endogenous locus products relative to the control reaction.

[00141 j To ensure that the presence of the trausgenes leads to expression of She desired proteins, a Western blot is performed. Approximately 5xiO^s algae cells are collected from TAP liquid media by centrifuging cells at 3,000 rpm at 20⁰C for 5 minutes. Cells, are resuspended in cold lysis bu ffer (TBS buffer pH 7.4 (25 tnM Trts-HCl, 137 rnM NaCl, 2.7 mM KCl) supplemented with EDTA-free protease inhibitors (Roche)). Cells are lysed by mechanical disruption of membranes by sonication. Samples are centrifuged to remove debris and insoluble components. Affinity purification of the FLAG-tagged transgene enzymes is performed by incubation at 4°C with anti-FLAG resin. The resin is washed several times, and bound proteins eluted using Q. IM Glycine pH 3.0. EluSed protein is mixed 3: 1 with loading buffer (XT Sample Buffer; Bio-Rad) containing β-rnercaptoethanol. Samples are then healed to 95 °C for 5 min, and cooled to near 23 °C. Proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is incubated with blocking solution (StartingBlock; Thermo Scientific) at 23⁰C for 30 min. followed by incubation with horseradish-linked auti-FLAG antibody (diluted 1 :4000 in TBST + 2.5% horse serum) at 4⁰C for 12 hours, and washed three times with TBST. Proteins are visualized by incubating with horseradish peroxidase substrate I SuperSignal West Dura: TiiermoScientific) and chemiluminescent detection. Positive results from multiple clones show that expression of the Srangenes in C reinhardiii cells results in production of the desired proteins. Fig. 9

[00142] Cultivation of C reinhardiii transformants is carried out in liquid HSM medium at 5% CO_? at 28"C under constant illumination of 5,000 Lux on a rotary shaker set at 100 rpm, unless stated otherwise. Cultures are maintained at a density of 1x10' cells per ml for at least 48 hr prior to harvest.

encoding genes

[00143] In this example a strain containing multiple enzyme-encoding genes using multiple vectors is described. Transforming DNA is shown in FIG. 3C. In this instance the gene encoding Ihioesterase is the segment labeled "Transgene 1 ,'^" which is regulated by 1he 5^" UTR and promoter sequence for ihe/w&D gene from C. reinhardiii, psbC gene from C. reinhardtii, ciipA gene from C. reinhardtii, or psb A gene from C. reinhardiii and the 3' UTR for lhepsb.4 gene from C. reinhardtii. The segments labeled "Transgene 2" is Ihe gene encoding one of the following enzymes: acyl-CoA Isgase, fatty acyl-CoA desaturase, rnethylmalonyl-CoA mutase, ketoacyl synthase, or rnetbylketone synthase, which is regulated by the 5' UTR and promoter sequence for XhepsbD gene from C remhardtii, psbC gene from C. reinhardπi, atpA gene from C. reinhardπi, or psbA gene from C remhardtii and the 3' UTR for the psbA gene from C. reinliardni. The segment labeled "Resistance Marker" is the kanamycin resistance encoding gene from bacteria, which is regulated by the 5' UTR and promoter sequence for the aψA gene from C remhardtii and the 3¹ UTR sequence for the rbcL gene from from C. remhardtii. The transgene cassette is targeted to the 3HB locus of C remhardtii via the segments labeled "Homology A'^" and "Homology B,'^" which are identical to sequences of DNA flanking the 3HB locus on the 5^" and 3' sides, respectively.

[00144] The segment labeled 'Transgene 3^*' is the gene encoding acyl-CoA ligase, fatty acyl-CoA desaturase, melhylmakmyl-CoA mutase, ketoacyl synthase, or methylketone synthase, which is regulated by the 5' UTR and promoter sequence for ihcpsbD gene from C re inhardϋi, psbC gene from C^" remhardtii, atpA gene from C reinhardiii, oipsbΛ gene from C. reinhardth and the 3' UTR for lhcpsbA gene from C. remhardtii. The segment labeled '"Trausgene 4" is the gene encoding acyl-CoA ligase, fatty acyl-CoA desaturase, methylmalonyl-CoA mutase. ketoacyl synthase, or methylketone synthase, which is regulated by the 5^* UTR and promoter sequence for lkcpsbD gene from C remhardtii, psbC gene from C remhardtii, aipA gene from C remhardtii, or pshA gene from C^" reinhardiii and the 3' UTR for lhcpsbA gene from C. reinhardth. The segment labeled "Resistance Marker" is the streptomycin resistance encoding gene from bacteria, which is regulated by Ihe 5^" UTR and promoter sequence for the atpA gene ftora C rcuihardiπ and the 3 ^' UTR sequence for the rbcL gene ftom from C reinhardth The transgene cassette is targeted Io the 322 loci of C reinhardni via the segments labeled "Homology C" and ''Homology D," which arc identical to sequences of DNA flanking the 322 loci on the 5' and 3^" sides, respectively. As will be recognized, all transgenes may be different or some may be the same, depending on the number of copies of an individual transgene ate desired. All DNA manipulations carried out in the construction of this transforming DNA are essentially as described by Sambrook ct ai, Molecular Cloning, A Labotatoty Manual (Cold Spring Harbor Laboratory Press 1989) and Cohen et aj., Meth. Enzymol 297. 192-208. 1998.

[00145| For these experiments, all transformations are carried out on C. remhardtii strain 137c (rnt+). Cells are grown to late log phase (approximately 7 days) in the presence of 0.5 mM 5-fiuorodeoλyuπdinc in TAP medium (Gorman and Lcvinc. Proc. Nutl Acad. SL i... USA 54: 1665-1669, ] 065, which is incorporated herein by reference) at 23°C under constant illumination of 450 Lux on a rotary shaker set at 100 φm. Fifty ml of cells are harvested by centrifugation at 4,000xg at 23^UC for 5 min. The supernatant is decanted and cells resuspcnded in 4 ml TAP medium for subsequent chloroplast transformation by particle bombardment (Cohen et al., supra, i 998), All Transformations arc carried out under kanamycin or streptomycin selection (100 μg/ml or 50 μg/ml, respectively) in which resistance is conferred by the genes encoded by the segment in FlG. 3C labeled "Selection Marker.^"' (Chlamydomonas Stock Center, Duke University).

[00146] PCR is used to identify transformed strains. For PCR analysis, 10⁶ algae cells (from agar plate or liquid culture) are suspended in 10 mM EDTA and heated to 95°C for 10 minutes, then cooled to near 23 "C. A PCR cocktail consisting of reaction buffer, MgC12. dJNTΪPs. PCR primer pair(s) (Table 2 & a gene specific reverse primer;), DNA polymerase, and water is prepared. Algae Iy sate in EDTA is added to provide a template for the reaction. The magnesium concentration is varied to compensate for amount and concentration of algae lysate and EDTA added. Annealing temperature gradients are employed to determine optimal annealing temperature for specific primer pairs. To identify strains that contain the gene encoded by each transgene. a primer pair is used in which one primer anneals to a site within the 5'UTR and the other primer anneals within the thioesterase, acyl-CoA ligase, fatty acyl-€oA desaturase. methylmalonyl-CoA mutase, ketoacyl synthase, and/or methylketone synthase coding segment. Desired clones are those that yield a PCR product of expected size,

[00148 J To determine the degree to which the endogenous 3HB gene locus is displaced (hεteroplasmic vs. homoplasmicj. a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in all cases. This reaction confirms thai the absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited the PCR reaction. To determine the degree to which She endogenous 322 gene loci are displaced I heteroplastic vs. homoplasmic), a PCR reaction consisting of two sets of primer pairs are employed (in the same reaction). The first pair of primers amplifies the endogenous locus targeted by the expression vector. The second pair of primers amplifies a constant, or control region that is not targeted by the expression vector, so should produce a product of expected size in all cases. This reaction confirms that She absence of a PCR product from the endogenous locus did not result from cellular and/or other contaminants that inhibited the PCR reaction. Concentrat ions of the primer pairs are varied so that both reactions work in the same tube; however, the pair for the endogenous, locus is 5X the concentration of the constant pair. The number of cycles used was >30 Io increase sensil ivity. The roost desired clones are those that yield a product for the constant region but not for the endogenous gene locus. Desired clones are also those that give weak- intensity endogenous locus products relative to the control reaction,

[00149] To ensure that the presence of the transgεnεs leads to expression of the desired proteins, a Western blot is performed. Approximately 1 x 10^b algae cells are collected from TAP agar medium and suspended in 0,5 ml of lysis buffer (Biigbustcr; Novagen). Lysate is mixed 3: 1 with loading buffer (XT Sample Buffer; Bio-Rad). Samples are then heated to 95°C for 5 min, cooled to near 23° C, and insoluble proteins arc removed by ccntrifugation. Soluble proteins are separated by SDS-PAGE, followed by transfer to PVDF membrane. The membrane is blocked with TBST ÷ 5% dried, nonfat milk at 23⁰C for 30 min, incubated with horseradish-linked anti-FLAG antibody (diluted 1 :2,500 in TBST + 5% dried, nonfat milk) at 4⁰C for 12 hours, and washed three times with TBST, Proteins are visualized with chemihiminescent detection. Positive results from multiple clones show that expression of the trangenes in C. reinhardtii ceils results in production of the desired proteins.

[00150] Cultivation of C. reinhardtii transfbrmants is carried out in liquid HSM medium at 23°C under constant illumination of 5,000 Lux on a rotary shaker set at 100 rpm, unless stated otherwise. Cultures are maintained at a density of lxlθ^? cells per ml for at least 48 hr prior to harvest.

Example 9. Expression of fatty acid biosynthetic genes in Sγnechocγstis

1^*00151] In this example nucleic acids (SEQ ID ^"NO: 22) encoding acyl-CoA synthetase from Arabidopsis thaliana (SEQ TD NO: 23), a thioesterase gene from C reinhardtii (SEQ {D NOs: 20 and 21), or a methylketone synthase 1 (MKS 1 SEQ {D NOs: 24 and 25) from L hirsiitum are introduced into Synechocyslis (PCC 6803), Transforming DNA is shown graphically in FIG. 3 A with the gene encoding any of the three above enzymes as the segment labeled "Transgene.^"' A vector (pCB) was constructed for the use in the deliver}', insertion, and expression of the exogenous proteins in PCC 6803. The regions of genomic sequence selected for homologous recombination are two regions flanking the dispensable gene ycβ2, a component of the photosystem II. Homology region "'A" of FlG. 3A is a 907 bp long portion of the hypothetical gene slr2141. Homology region "B" is a 945 bp long portion spanning the hypothetical gene ss 13829. The segment labeled "Resistance Marker" is the 1110 bp kanamycin resistance fragment KmR from the vector pAMIA A 107 bp DNA element 3' downstream of the S-layer gene from L. hrevis (accession number Z145250) was amplified by PCR and is used as a transcriptional terminator of the synthetic gene. The 5'UTR region is a truncated version of the hi A 5^"UTR.

[00152] The vector is transformed into PCC 6803 via natural transformation. Synechocysiis f PCC 6803) cells are grown in liquid G medium under constant illumination (90 rnicroeinsSeins) and in 5% CO₂ with shaking. Once the culture reaches an OD_^0O of 0.8. the cells are harvested by centrifugation. the pellet washed once with fresh BG-11 medium, then resu spend ed at a concentration of IxIO^' cells/mL in BG-11. 400ul of cell suspension is transferred to an Eppendorf tube and mixed 400 ug of DNA. Water control is used to ensure clearing of cells on selective plates. The cell/DNA suspension is incubated overnight in the dark with no shaking. The next day, the cells are spread on solid G medium plates with 5ug/mL kanamycin. After drying, the plates are exposed to low levels of light ( 30 microcinsteins) and 5% CO;; for 48 hours, after which She plates are exposed to levels of light similar to flask growth conditions. Over the course of 14 days, the background cells clear and small clones appear on 1he select ion plate. Clones are picked and patched onto a new plate of solid G medium containing 5 ug/raL of kanaraycin. The patch plates are incubated under light and 5% CO 2 for 5 days, after which enough biomass had accumulated to screen,

[00153] PCR is used to identify transformed strains. For PCR analysis, patches are lightly touched and the biomass transferred directly to the PCR reaction mixture consisting of buffer, dNTPs, PCR primer pairs, DNA polymerase, and water as described in previous examples. To identify strains that possessed a transgene. primer pairs comprised of internal DNA sequences from the transgene were utilized and are listed in Table 2.

[00154] To ensure that the presence of transgene results in expression of protein, a Western blot is performed. Biomass is diluted into 1 ml. of liquid G medium containing 5 ug/mL of kanamycin and grown with light and 5% CO2 at 28"C. After 48 hours, 750 ul of the culture is transferred to an Eppendorf tube and the ceils harvested by centrifugation for 5 minutes at 14,000 rpm. Controls include the preparation of lysates from wild-type, non-transformed Synechocysiis cells. The ceil pellets are resuspended in 100 ul of 1 x PBS and flash frozen in an cthanol/dry ice bath. After freezing, the tubes are incubated for I minute in a 95⁰C heating block to thaw. The cells arc then subjected to sonication (6 sec at 30%, 2 iterations). After lysis, 12.5 ul of 4x SDS sample buffer containing 2% beta-mercapoethanol is added to the lysate, and the samples incubated for 5 minutes at 95⁰C. After heating, the samples arc allowed to cool to room temperature, then vortexed briskly for 30 seconds. The samples are subjected to a short centrifugation and 20ul of lysate loaded onto a 10% aerylamide gel. The samples are separated by electrophoresis at 100 V for 90 minutes, and protein then transferred to PVDF. The blot is blocked by incubating at room temp for 30 minutes at room temperature with Starting Block (Thermo Scientific), After blocking, the blot is probed with an anti-FLAG antibody coupled to alkaline phosphatase (M2-AP, Sigma Aldrich) at a dilution of 1: 1000 for 90 minutes at room temperature. After probing, the blot is washed 4 times with excess TBS-T (1% Tween-2ϋj at room temp. The blot is exposed using the Lumiphos substrate (Pierce Biotechnology) by incubating the blot for 5 minutes with substrate and imaged using a chemiluminesence filter. Alternatively, the blot is visualized using the 3 -step TMP (3,3',5,5'-tetramethyJbenzidine) colormetrie substrate (Thermo Scientific) by incubating the blot for 5 mintues with the substrate. Results of the Western blot are shown in FIG. 11.

Table 3. Amino Acid Se uences of Enz mes Useful for Fatt Acid S nthesis

Table 2. PCR primers.

Primers for Chlamydomonas

Primers for Synechocyslis

Various_* modifications, processes, as well as numerous structures that may be applicable herein will be apparent. Various aspects, features or embodiments may have been explained or described in relation to understandings, beliefs, theories, underlying assumptions, and/or working or prophetic examples, although it will be understood that any particular understanding, belief, theory, underlying assumption, and/ or working or prophetic example is not limiting. Although the various aspects and features may have been described with respect to various embodiments and specific examples herein, it will be understood that any of same is not limiting with respect to the full scope of the appended claims or other claims that may be associated with this application.

Claims

What is claimed is:

1. A method of increasing production of a ClO to Cl 8 fatty acid in a non-vascular photosynthetic organism comprising transforming a plastid of said non-vascular photosynthetic organism with a vector comprising a nucleotide sequence encoding an acyl carrier protein thioesterase and expressing said thioesterase, wherein production of a ClO to Cl 8 fatty acid in said organism is increased.

2. The method of claim 1, wherein said thioesterase is selected from the group consisting of SEQ ID NO: 21, 27, 31, 33 and 35.

3. The method of claim 1 or 2. wherein said production of a fatty acid is increased by at least 100%.

4. The method of claim 3, wherein said production of a fatty acid is increased by at least 200%.

5. The method of claim 3, wherein said production of a fatty acid is increased by at least 500%.

6. The method of claim 3, wherein said production of a fatty acid is increased by between 100% and 1000%,

7. The method of claim 1 , wherein said thioesterase is a myristoyl acyl carrier protein thioesterase,

8. The method of any one of the preceding claims, wherein the fatty acid is C 14 fatty acid,

9. The method of claim 8, wherein said C14 fatty acid is myristic acid.

10. The method of any one of claims 1 to 9, wherein said vector further comprises a promoter.

11. The method of claim 10, wherein said promoter is a constitutive promoter.

12. The method of claim 10, wherein said promoter is an inducible promoter.

13. The method of claim 3 1, wherein said constitutive promoter is selected from the group consisting of an atpA promoter, a psbC promoter, a psbD promoter, a psbA promoter, a rbcL promoter and a 16s rRNA promoter.

14. The method of claim 32, wherein said inducible promoter is selected from the group consisting of a nitrate inducible promoter, a light inducible promoter and a heat inducible promoter.

15. The method of any one of claims 10 to 14, wherein said vector further comprises a 5'UTR.

16. The method of claim 15. wherein said 5^"UTR is selected from at least one of the group consisting of an atpA 5^"UTR. a psbC 5^"UTR. a psbD 5'UTR, a psbA 5'UTR, a rbcL 5'UTR and a 36s rRNA 5'UTR.

17. The method of any one of claims 10-16, wherein said vector further comprises a ribosomal binding site,

18. The method of any one of claim 10-17, wherein said vector further comprises a 3 ' UTR.

19. The method of claim 18. wherein said 3" UTR is a psbA 3"UTR from C. reinhardlu or a rbcL 3^" UTR from C reinhardiii.

20. The method of any one of she preceding claims , wherein said non- vascular photo symhetic organism is a member of a genus from the group consisting of Chlctmydomonas, Dimalielia, Hematococcus, Synechocystis. Synechococcus, and Aihrospira.

21. The method of claim 20. wherein said piiotosynthetic organism is selected from she group consisting of C. reinliardtii, D, saiina, and H. pluvahs.

22. The method of claim 1 , wherein said plastid is a chloroplast.

23. The method of claim 22, wherein said nucleoside sequence is biased to reflect codon usage in a chloroplast.

24. The method of claim 23, wherein said bias reflects codon usage in a chloroplasi from an organism of a genus from the group consisting of ^' Chlainydomonas, Dwicdieila, Heniatococcus, Synechocystis, Synechococcus, and Athrospira.

25. The method of any one of the preceding claims, wherein said nucleotide sequence is an exogenous nucleotide sequence.

26. A method of increasing production of fatty acid in a non-vascular photosynthetic organism comprising transforming a pjastid of said non-vascular photosynthetic organism with a vector comprising a nucleotide sequence encoding a 3-keto-acyl-ACP synthase Iϊ and expressing said 3-keto-acyl-ACP synthase lϊ, wherein production of said fatty acid in said organism is increased.

27. The method of claim 26, wherein said fatty acid is a ClO to CIS fatty acid.

28. The method of claim 27, wherein said fatty acid is a C18 fatty acid.

29. The method of any one of claims 26 to 28, wherein the 3-keto-acyl-ACP synthase II is SEQ ID NO: 29,

30. The method of any one of claims 26 to 29, wherein said vector further comprises a promoter.

31. The method of claim 30. wherein said promoter is a constitutive promoter.

32. The method of claim 30. wherein said promoter is an inducible promoter.

33. The method of claim 31. wherein said constitutive promoter is selected from the group consisting of an atpA promoter, a psbC promoter, a psbD promoter, a psbA promoter, a rbcL promoter and a 16s rRNA promoter.

34. The method of claim 32. wherein said inducible promoter is selected from the group consisting of a nitrate inducible promoter, a light inducible promoter and a heat inducible promoter.

35. The method of any one of claims 26 to 34. wherein said vector further comprises a 5"UTR.

36. The method of claim 35, wherein said 5'UTR is selected from at least one of the group consisting of an atpA 5"UTR, a psbC 5"UTR, a psbD 5'UTR. a psbA 5'UTR, a rbcL 5'UTR and a 16s rRNA 5'UTR.

37. The method of any one of claims 26- 36, wherein said vector further comprises a ribosomal binding site.

38. The method of any one of claim 26- 37. wherein said vector further comprises a 3^' UTR.

39. The method of claim 38, wherein said 3' UTR is a psbA 3'1JTR from C. reinharcilii or a rbcL 3' IJTR from C. reinhardtii.

40. The method of any one of claims 26 to 39, wherein said non-vascular photosynthetic organism is a member of a genus from the group consisting of Chlamydomonas, Dunnliella, Hemarococcus, Synechocysiis, Synechococcus, and Athrospira.

41. The method of claim 40, wherein said photosynthetic organism is selected from the group consisting of C reinhardtii, D. set Una. and //. pluvalis.

42. The method of claim 26, wherein said plastid is a chloroplast.

43. The method of claim 42, wherein said nucleotide sequence is biased to reflect codon usage in a chloroplast.

44. The method of claim 43. wherein said bias reflects codon usage m a chloroplast from an organism of a genus from the group consisting of Chlamydomonas, Dunaliella, llematococcus, Sγiiechocystis. Synechococcus, and Afhi'ospira.

45. The method of any one of claims 26-44, wherein said nucleotide sequence is an exogenous nucleotide sequence.

46. A method for increasing production of fatty acids in a non-vascular photo&ynthetic organism comprising transforming a plasSid of said non- vascular photo&ynthetic organism with a first nucleic acid sequence encoding a myristoyl acyl carrier protein Slnoesterase and a second nucleic acid sequence encoding a 3-keto-acyl-ACP synthase II, expressing said first and said second nucleic acid sequences, wherein expression of said first and second nucleic acid sequences increases fatty acid production in said non-vascular photosyntheSic organism.

47. The method of claim 46. wherein said Shioesterase is selected from the group consisting of SEQ ID NO: 21. 27. 31, 33. and 35

48. The method of claim 46, wherein said 3-kcto-acyi-ACP synthase II is SΗQ ID NO: 29.

49. The method of claim 46, wherein said first and said second nucleic acid sequences arc in a single vector.

50. The method of claim 46, wherein said first and said second nucleic acid sequences arc sn different vectors.

51. The method of claim 49, wherein said vector comprises a single promoter operahly linked to sasd nucleic acid sequences.

52. The method of claim 49, wherein said first nucleic acid sequence is operably linked a first promoter and sasd second nuclesc acid sequence is operably linked to a second promotes' and wherein said first and said second promoter ma}? be the same or different.

53. The method of claim 50, wherein in each of said different vectors further comprises a promoter.

54. The method of claim 53, wherein said promoter is the same for each of said different vectors.

55. The method of claim 53, wherein said promoter is different for each of said different vectors.

56. The method of claim 51 or 54, wherein sasd promoter is a constitutive promoter.

57. The method of claim 51 or 54, wherein sasd promoter is an inducible promoter.

58. The method of claim 52 or 55. wherein said promoters are constitutive promoters.

59. The method of claim 52 or 55, wherein said promoters are inducible promoters.

60. The method of claim 52 or 55, wherein one of said promoters is constitutive and the other is inducible.

61. The method of any one of claims 58, 59 or 60. wherein said constitutive promoter is selected from the group consisting of an atpA promoter, a psbC promoter, a psbD promoter, a psbA promoter, a rbcL promoter and a 16s rRNA promoter.

62. The method of any one of claims 57, 59 or 60, wherein said inducible promoter is selected from the group consisting of nitrate inducible promoter, a light inducible promoter and a heat inducible promoter.

63. The method of claims 49 and 50, wherein said vector or vectors further comprise a 5' UTR.

64. The method of claim 63, wherein said 5'UTR is selected from at least one of the group consisting of an atpA 5"UTR, a psbC 5"UTR, a psbD 5'UTR. a psbA 5'UTR, a rbcL 5'UTR and a 16s rRNA 5'UTR.

65. The method of claims 49 or 50. wherein said vector further comprises a ritaosomal binding site,

66. The method of 49 or 50, wherein said vector further comprises a 3^' UTR.

67. The method of claim 66, wherein said 3' UTR is a psbA 3'UTR from C. reinharcilii or a rbcL 3' UTR from C. reinhardtii.

68. The method of any one of claims 46 to 67, wherein said non-vascular photosynthetic organism is a member of a genus from the group consisting of Chlamydomonas, Dunnliella, Hemarococcus, Synechocysiis, Synechococcus, and Athrospira.

69. The method of claim 68, wherein said photosynthetic organism is selected from the group consisting of C. reinhardtii, D. set Una. and //. pluvalis.

70. The method of claim 46, wherein said plastid is a chloroplast.

73. The method of claim 70, wherein said first nucleotide sequence and said second nucleotide sequence are biased to reflect codon usage in a chloroplast.

-4")-

72. The method of claim 71. wherein said bias reflects codon usage in a chloroplast from an organism of a genus from the group consisting of ^' Chlamydomonas, Dunaliella, Hematococcus, Sγiiechocystis. Synechococcus, and Athrospira.

73. The method of any one of claims 46-72, wherein said nucleotide sequence is an exogenous nucleotide sequence.

74. A method of increasing production of fatty acid in a non- vascular photosynthetic organism comprising transforming a plasSid of said non-vascular photosynthetic organism with a vector comprising a nucleotide sequence encoding an Acyl-CoA synthetase and expressing said Acyl-CoA synthetase, wherein production of said fatty acid in said organism is increased.

75. The method of claim 74. wherein said fatty acid is a ClO to C18 fatty acid.

76. The method of claim 75. wherein said fatty acid is a ClO to C 16 fatty acid.

77. The method of any one of claims 74 to 76, wherein the Acyl-CoA synthetase is SEQ ID NO: 23,

78. The method of any one of claims 74 to 77, wherein said vector further comprises a promoter.

79. The method of claim 78, wherein said promoter is a constitutive promoter,

80. The method of claim 78, wherein said promoter is an inducible promoter.

81. The method of claim 79, wherein said constitutive promoter is selected from the group consisting of an atpA promoter, a psbC promoter, a psbD promoter, a psbA promoter, a rbcL promoter and a 16s rRNA promoter.

82. The method of claim 80, wherein said inducible promoter is selected from the group consisting of a nitrate inducible promotes', a light inducible promoter and a heat inducible promoter.

83. The method of any one of claims 74 to 82, wherein said vector further comprises a 5'UTR.

84. The method of claim 83, wherein said 5'UTR is selected from at least one of the group consisting of an atpA 5'UTR, a psbC 5'UTR, a psbD 5'UTR, a psbA 5^"UTR. a rbcL 5^"UTR and a 16s rRNA 5'UTR.

85. The method of any one of claims 74-84, wherein said vector further comprises a ribosomal binding site.

86. The method of any one of claims 74-86, wherein said vector further comprises a 3' UTR,

87. The method of claim 86. wherein said 3' L¹TR is a psbA 3'UTR from C. rcinhurdtn or a rbcL 3^" UTR from C. reinliardni.

88. The method of any one of claims 74 to 87. wherein said non-vascular photosynthetic organism is a member of a genus from the group consisting of Chlamydomonas, Dimaϊiclla, llematococcus, Sγiiechoeystis, Synechococcus, and Afhi'ospira.

89. The method of claim 88. wherein said piiotos.ynlfa.etic organism is selected from the group consisting of C reinhardiii, D sσlina, and 11. phivalis.

90. The method of claim 74. wherein said piastid is a chioropiast,

91. The method of claim 90. wherein said nucleotide sequence is biased to reflect codon usage in a chioropiast.

92. The method of claim 9 L wherein said bias reflects codon usage in a cbloroplasl from an organism of a genus from lhe group consisting of Chictiny domtmas, Dunaliella, Ilemcttococnis, Syiiechocvstis. Synechococcus, and Aihrospira,

93. The method of any one of claims 73-92, wherein said nucleotide sequence is an exogenous nucleotide sequence.

94. A method of increasing production of fatty acids in a non-vascular photosynthetic organism comprising transforming a piastid of said non-vascular photosynthetic organism with a vector comprising a nucleotide scqtience encoding at least one protein selected from the group consisting of an acyl-CoA ligase, a fatty acyl-CoA dcsaturase, a methylmalonyl-CoA mutase, a ketoacyl synthase, a methylketone synthase, an acetyl-CoA carboxylase, a ketoreductase, a rnaionyltransferase, a dehydratase, and an enoylrcduetase, wherein production of said fatty acids in said organism is increased.

95. The method of claim 94, wherein said vector further comprises a promoter.

96. The method of claim 95, wherem said promoter is a constitutive promoter.

97. The method of claim 95, wherem said promoter is an inducible promoter,

98. The method of claim 96, wherem said constitutive promoter ss selected from the group consisting of an atpA promoter, a psbC promoter, a psbD promoter, a psbA promoter, a rbcL promoter and a 16s rRNA promoter.

99. The method of claim 97. wherein said inducible promoter is selected from the group consisting of nitrate inducible promoter, a light inducible promoter and a heat inducible promoter.

100. The method of any one of claims 94 to 99, wherein said vector further comprises a 5'UTR.

101. The method of claim 100, wherein said 5'UTR is selected from at least one of the group consisting of an atpA 5^"UTR. a psbC 5^"UTR. a psbD 5'UTR, a psbA 5'UTR, a rbcL 5'UTR and a 36s rRNA 5'UTR.

102. The method of any one of claims 94-101. wherein said vector further comprises a ribosomal binding site.

103. The method of any one of claim 94-102, wherein said vector further comprises a 3' UTR.

104. The method of claim 103, wherein said 3' UTR is a psbA 3'UTR from C. reinharώii or a rbcL 3' UTR from C reinhardiii.

105. The method of any one of claims 94 to 104, wherein said non -vascular photosynihetic organism is a member of a genus from the group consisting of Chlamydomojias, DimalieUa, Heniatococciis, Syneciiocvstis, Synechococcus, and AUirospira,

106. The method of claim 105, wherein said photosynthεlic organism is selected from the group consisting of C. reinliardtn^' , D, saiina, and H. pluvahs.

107. The method of claim 94, wherein said plastid is a chloroplast.

108. The method of claim 307, wherein said nucleotide sequence is biased to reflect codon usage in a chloroplast.

109. The method of claim 308, wherein said bias reflects codon usage in a chloroplast from an organism of a genus from the group consisting of ^' Chlainydomonas, Dwicdieila, Heniarococcus, Synechocystis, Synechococcus, and Athrospira.

130. The method of any one of claims 94-109, wherein said nucleotide sequence is an exogenous nucleotide sequence.

13 1. A method of inducing or increasing production of methylketones in a non-vascular photosynthetic organism comprising transforming said non-vascular photosynthetic organism with a vector comprising a nucleotide sequence encoding a methylketone synthase, wherein production of said methylketones in said organism is induced or increased.

132. The method of claim 3 11, wherein said methylketone synthase is a methylketone synthase 1. 1 13, The method of claim 112, wherein said methylketone synthase 1 is encoded by SEQ ID NO: 25.

1 14, The method of claim 111, wherein said nucleotide sequence is introduced mto a plastsd of said non-vascular photosynthetie organism.

1 15, The method of claim 114, wherein said plastid is a chloroplast.

116, The method of claim 111, wherein said vector further comprises a promoter,

117, The method of claim 116, wherein said promoter is a constitutive promoter.

118, The method of claim l ib, wherein said promoter is an inducible promoter.

119, The method of any one of claims 111 to 118, wherein said vector further comprises a 5'UTR.

120, The method of any one of claims 1 1 l - l 19, wherein said vcctot further comprises a ribosomal binding site.

12 I , The method of any one of claim 1 1 1 - 120, wherein said vcctot further comprises a 3' UTR,

122. The method of any one of claims H O to 121 , wherein said non-vascular photosynthclic organism ss a member of a genus from the group consisting of Chlamvdoinonas, Duυahella, Hematococcus, Synechocγ<(h; Sviiechococcns, and Λihraspira.

123. The method of claim 322. wherein said non-vascular photosynthetie plant is a member of the genus Synechotyslis

124. The method of any one of the preceding claims, wherein said non-vascular photosynthetie organism is homoplastic for said exogenous nucleotide sequence or sequences.