Abstract:
The invention provides an isolated photosynthetic microorganism with an altered lipid profile compared to its wild type or naturally occurring counterpart. The bacterium has been genetically modified such that the lipids produced are amenable to producing biodiesel fuel. Accordingly, such an isolated cyanobacterium contains a fatty acid, the length of which is less than C 16.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention relates to production of biofuels. 
       BACKGROUND OF THE INVENTION 
       [0002]    Biofuels are fuels that come from biological origin like plants, animals, or bacteria. Some bacteria capture energy in the form of sunlight and use it to carry out a variety of biochemical processes. Photosynthesis provides a mechanism for capturing solar energy to directly drive a myriad of chemical processes. After light energy is captured, it is converted to chemical energy and precursors in the form of carbohydrates through a series of enzymatic steps. Conversion of carbohydrate into useful molecules including fuels such as ethanol, butanol, or biodiesel also proceeds through specific enzymatic steps. It would be a major advance to construct microbes that efficiently harness photosynthesis to drive the biosynthesis of useful chemicals such as biofuels. 
       SUMMARY OF THE INVENTION 
       [0003]    The invention provides an isolated photosynthetic microbe with an altered lipid profile compared to its wild type or naturally occurring counterpart. For example, the photosynthetic microbe is a such as a cyanobacterium or blue-green algae, e.g.,  Synechococcus  sp. The bacterium has been genetically modified such that the lipids produced are amenable to producing biodiesel fuel. Accordingly, such an isolated cyanobacterium contains a fatty acid, the length of which is less than C16. At least 10% of the lipids in the microbe are less than 16 carbons (16C) in length, preferably, at least 25%, 50%, 75%, 90%, 95%, 99%, and up to 100% of the lipids are less than 16 carbons in length. For example, the chain length is 15, 14, 13, 12, 11, 10, 9, 8, or less. In a preferred embodiment, the length is C12, e.g., the fatty acid fatty acid comprises lauric acid (C 11 H 23 COOH). The microbes are used to produce a combustible biofuel. 
         [0004]    To achieve an altered lipid profile, the bacterium contains an enzyme derived from a species other that that of the host species. For example, the cyanobacterium contains a heterologous short or medium-chain acyl-acyl carrier protein thioesterase enzyme such as California bay tree thioesterase (CBT), I.E. FatB1 from  Cuphea palustris  (as well as all medium-chain specific enzymes from the genus  Cuphea ) or luxD from  Vibrio harveyi . An exemplary amino acid sequence is provided in SEQ ID NO:9 or a fragment thereof, and an exemplary nucleic acid sequence encoding the enzyme is SEQ ID NO:6 or a fragment thereof. Bacteria genetically altered in this manner produce shorter lipids compared to wild type or naturally-occurring bacteria. These shorter fatty acids are better suited to the production of biodiesel fuel, because unlike longer fatty acids that freeze or become viscous at low temperatures, these fatty acids have a higher cloud or gel point. Thus, the thioesterase-modified bacteria are a better source of fatty acids from which to make biodiesel fuel. A method of producing a biodiesel fuel is carried out by culturing the thioesterase-modified bacterium and extracting a medium chain fatty acid from the bacterium and/or culture medium using conventional chemical techniques, e.g., Huber et al., Chem Rev. 2006 September; 106(9):4044-98, for producing a biodiesel fuel. For example, the fatty acids obtained from the bacteria are esterified to yield a medium chain hydrocarbon biodiesel fuel composition. 
         [0005]    In an alternate approach, a single bacterium is used to produce biodiesel fuel. In that case, the bacterium is engineered to express additional heterologous proteins that are required to further process the medium chain fatty acids in the bacterium itself. Biodiesel synthesis is carried out by expression of elements of the  Zymomonas mobilis  ethanol fermentation pathway and the non-specific acyltransferase of  Acinetobacter baylyi , which esterifies fatty acids with ethanol. Accordingly, the cyanobacterium further comprises a heterologous wax synthase such as one containing the amino acid sequence of SEQ ID NO:7 (Wax ester synthase/acyl-CoA:diacylglycerol acyltransferase; WS/DGAT) or a fragment thereof. For example, the enzyme is produced by a nucleic acid comprising SEQ ID NO:8 or a fragment thereof. Other suitable wax synthase enzymes include mammalian (human or mouse) wax synthases (e.g., described in Cheng et al., J. Biol. Chem., Vol. 279, Issue 36, 37798-37807, Sep. 3, 2004; GenBank™ AY605053 (human) or AY611031 and AY611032 (mouse)) as well as Jojoba ( Simmondsia chinensis ) Synthase (e.g., described in Lardizabal et al., Plant Physiol, March 2000, Vol. 122, pp. 645-656; GenBank™ AB015479). 
         [0006]    The cyanobacterium may also comprise a nucleic acid encoding a heterologous pyruvate decarboxylase (PDC). An exemplary amino acid sequence is from  Zymomonas mobilis , SEQ ID NO:1 or a fragment thereof, and an exemplary nucleic acid sequence encoding the enzyme includes SEQ ID NO:2 or a fragment thereof. PDCs, e.g., PDC 1, 2, 5 or 6 from  Saccharomyces cerevisiae  (PDC1, GenBank™ NP — 01314.1; PDC5, GenBank™, NP — 013235.1; PDC6, GenBank™ NP — 011601.1). 
         [0007]    The bacterium may further include a heterologous alcohol dehydrogenase (ADHE) such as those including the amino acid sequence of SEQ ID NO:3 (from  Zymomonas  mobilis) or a fragment thereof. An exemplary nucleic acid comprises SEQ ID NO:4 or a fragment thereof. Other suitable ADHE enzymes include ADH4 or ADH7 from  Saccharomyces cerevisiae  (ADH4, GenBank™ NP — 011258.1; ADH7, GenBank™ NP — 010030.1). 
         [0008]    For example, the isolated cyanobacterium contains a nucleic acid encoding a heterologous thioesterase, wax synthase, pyruvate decarboxylase, and alcohol dehydrogenase, and a method of producing a biodiesel fuel is carried out by culturing the bacterium and extracting from the bacterium a medium chain hydrocarbon biodiesel fuel composition. Thus, the photosynthetic microbes described herein are useful to produce biodiesel, or a component or intermediate thereof, e.g., laurate ethyl ester, in a solar-powered manner utilizing atmospheric CO 2  as the carbon source. 
         [0009]    The invention provides isolated polynucleic acids containing genes or any fragment thereof. Isolated polynucleic acids of the invention are bound, conjugated to, incorporated into, or associated with gene delivery systems for insertion into host cells. Exemplary gene delivery systems include, but are not limited to, polymers, nanoparticles (gold, magnetic), lipids, liposomes, microspheres, proteins, and compounds. Furthermore, isolated polynucleic acids of the invention comprise genes or any fragment thereof and expression vectors, plasmids, markers, reporter genes, enhancers, promoters, repressors, recombination sites (such as loxP or Frt), and any other sequence used to insert or delete genes from engineered yeast and host cells of the invention. 
         [0010]    The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control. 
         [0011]    Other features, objects, and advantages of the invention will be apparent from the description and drawings. All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1  is a diagram of a nucleic acid construct for an organism that has been engineered to express a heterologous thioesterase. 
           [0013]      FIG. 2  is a diagram of a nucleic acid construct for an organism that has been engineered to express a heterologous PDC, heterologous ADHE, and a heterologous WSDGAT. 
           [0014]      FIG. 3  is a photograph of the results of a Western blot assay showing thioesterase expression in  Synechococcus . Two thioesterase FatB genes were expressed. 
           [0015]      FIG. 4  is a line graph showing engineered fatty acid production in stratin 651S expressing  U. californica  thioesterase. Fatty acid analysis of purified  Synechococcus  lipids shows that expression of the  U. californica  thioesterase alters the lipid profile and produces the shorter C12 fatty acid. 
           [0016]      FIG. 5  is a bar graph showing ethanol synthesis in  Synechococcus  using the  Zymomonas  pdc/adh pathway. Demonstration that ethanol synthesis is accomplished by expression of two genes form  Zymomonas.    
       
    
    
     DETAILED DESCRIPTION 
       [0017]    Liquid transportation fuels (e.g. ethanol or petroleum-based fuels) ultimately derive their energy from the photosynthetic conversion of sunlight into chemical energy. Earlier plant-based strategies have been plagued by the low efficiency with which sunlight is converted into useful molecules within the plant and in further ex vivo processing steps. To overcome this problem, photosynthetic microbes that more efficiently convert light into chemical energy were engineered to direct the chemienergetic flux of photosynthesis into optimized lipids and lipid derivatives. These molecules are harvested and used as a combustible fuel with little to no processing. Thus, the microbes described herein convert sunlight directly into biofuels. The methods permit harnessing and converting of solar energy into chemical energy in a single organism, thereby producing combustible fuels from sunlight. 
         [0018]    An exemplary microbe is a photosynthetic cyanobacterium that produces a short alkyl chain by commandeering the flow of electrons/reducing equivalents from Photosystem I into an artificial metabolic pathway. For example, a cyanobacterium is engineered to produce lauric acid ethyl ester (C 11 H 23 COOC 2 H 5 ) by forced regulated expression of one or more of the following four proteins: the California bay thioesterase, a wax synthase, pyruvate decarboxylase, and alcohol dehydrogenase. Described below are exemplary nucleic acid and amino acid sequences for each enzyme. 
         [0000]                      Zymomonas mobilis         Pyruvate decarboxylase       Swissprot ID: P06672       &gt;sp|P06672|PDC_ZYMMO Pyruvate decarboxylase       OS =  Zymomonas mobilis  GN = pdc PE = 1 SV = 1       (SEQ ID NO: 1)       MSYTVGTYLAERLVQIGLKHHFAVAGDYNLVLLDNLLLNKNMEQVYCC               NELNCGFSAEGYARAKGAAAAVVTYSVGALSAFDAIGGAYAENLPVIL               ISGAPNNNDHAAGHVLHHALGKTDYHYQLEMAKNITAAAEAIYTPEEA               PAKIDHVIKTALREKKPVYLEIACNIASMPCAAPGPASALFNDEASDE               ASLNAAVEETLKFIANRDKVAVLVGSKLRAAGAEEAAVKFADALGGAV               ATMAAAKSFFPEENPHYIGTSWGEVSYPGVEKTMKEADAVIALAPVFN               DYSTTGWTDIPDPKKLVLAEPRSVVVNGIRFPSVHLKDYLTRLAQKVS               KKTGALDFFKSLNAGELKKAAPADPSAPLVNAEIARQVEALLTPNTTV               IAETGDSWFNAQRMKLPNGARVEYEMQWGHIGWSVPAAFGYAVGAPER               RNILMVGDGSFQLTAQEVAQMVRLKLPVIIFLINNYGYTIEVMIHDGP               YNNIKNWDYAGLMEVFNGNGGYDSGAGKGLKAKTGGELAEAIKVALAN               TDGPTLIECFIGREDCTEELVKWGKRVAAANSRKPVNKLL            
Codon Optimized Sequence for  S. elongatus :
 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 2) 
               
               
                 ATGTCATATACTGTCGGTACTTATCTAGCTGAACGCTTGGTGCAAAT 
               
               
                   
               
               
                 TGGTCTGAAACACCACTTTGCAGTGGCAGGGGATTACAACCTAGTGC 
               
               
                   
               
               
                 TCTTGGATAACCTTTTGTTGAACAAAAACATGGAACAAGTGTATTGT 
               
               
                   
               
               
                 TGCAATGAGCTAAACTGTGGCTTTTCGGCGGAAGGGTACGCACGCGC 
               
               
                   
               
               
                 GAAGGGTGCTGCGGCGGCAGTCGTCACCTACTCTGTCGGCGCACTCT 
               
               
                   
               
               
                 CTGCGTTTGATGCTATCGGTGGCGCTTATGCGGAGAATCTCCCTGTC 
               
               
                   
               
               
                 ATCCTCATCTCCGGTGCGCCAAACAATAACGACCACGCTGCTGGGCA 
               
               
                   
               
               
                 CGTCCTCCACCATGCTCTCGGTAAAACAGATTATCATACCAACTTGA 
               
               
                   
               
               
                 GATGGCGAAAAACATAACCGCTGCTGCGGAGGCGATCTATACTCCAG 
               
               
                   
               
               
                 AAGAAGCGCCCGCAAAAATCGACCATGTAATTAAGACGGCATTGCGC 
               
               
                   
               
               
                 GAGAAAAAGCCGGTTTATCTCGAAATTGCTTGCAATATTGCTTCAAT 
               
               
                   
               
               
                 GCCCTGTGCTGCGCCCGGTCCTGCGTCGGCATTGTTCAACGATGAAG 
               
               
                   
               
               
                 CAAGCGATGAAGCAAGCCTGAACGCGGCGGTTGAAGAGACGCTGAAA 
               
               
                   
               
               
                 TTCATTGCAAACAGAGACAAAGTTGCTGTGTTGGTTGGTTCCAAGCT 
               
               
                   
               
               
                 GCGCGCTGCTGGGGCGGAAGAAGCGGCGGTGAAGTTTGCTGATGCTT 
               
               
                   
               
               
                 TGGGCGGAGCAGTGGCAACAATGGCTGCTGCTAAATCCTTTTTCCCT 
               
               
                   
               
               
                 GAGGAGAACCCACATTATATCGGCACCAGCTGGGGAGAGGTAAGCTA 
               
               
                   
               
               
                 TCCCGGCGTGGAAAAGACGATGAAAGAAGCGGACGCGGTGATTGCTC 
               
               
                   
               
               
                 TAGCTCCCGTTTTTAATGATTATTCCACGACGGGATGGACTGACATT 
               
               
                   
               
               
                 CCTGACCCCAAGAAACTGGTTTTGGCGGAACCTCGCAGCGTGGTTGT 
               
               
                   
               
               
                 GAACGGCATTCGATTCCCAAGTGTGCACCTGAAGGATTACCTCACCC 
               
               
                   
               
               
                 GCTTAGCTCAAAAAGTCAGCAAGAAGACCGGTGCGCTAGATTTCTTT 
               
               
                   
               
               
                 AAATCGCTGAACGCGGGTGAACTCAAGAAAGCTGCTCCGGCTGACCC 
               
               
                   
               
               
                 TTCTGCGCCCCTCGTGAACGCAGAAATCGCTCGTCAAGTGGAGGCGC 
               
               
                   
               
               
                 TGCTGACGCCGAATACGACTGTTATTGCTGAGACGGGCGATAGCTGG 
               
               
                   
               
               
                 TTTAATGCTCAGCGAATGAAACTGCCCAACGGGGCTCGCGTGGAATA 
               
               
                   
               
               
                 TGAAATGCAATGGGGCCATATTGGATGGTCTGTGCCTGCGGCTTTTG 
               
               
                   
               
               
                 GTTACGCAGTTGGTGCACCCGAACGGCGTAACATCTTGATGGTGGGG 
               
               
                   
               
               
                 GATGGGAGCTTCCAGCTTACCGCACAAGAGGTGGCTCAAATGGTGCG 
               
               
                   
               
               
                 CCTGAAATTGCCCGTGATTATCTTCCTGATTAACAATTACGGGTACA 
               
               
                   
               
               
                 CGATTGAAGTCATGATTCATGATGGCCCCTATAACAACATCAAGAAT 
               
               
                   
               
               
                 TGGGACTATGCTGGTCTGATGGAAGTCTTTAATGGTAACGGTGGCTA 
               
               
                   
               
               
                 CGATAGTGGCGCTGGGAAGGGCCTCAAGGCAAAGACGGGTGGGGAAC 
               
               
                   
               
               
                 TAGCGGAAGCAATTAAAGTCGCGTTGGCAAACACTGACGGGCCGACG 
               
               
                   
               
               
                 CTCATCGAATGTTTCATCGGTCGCGAGGATTGTACCGAAGAGCTTGT 
               
               
                   
               
               
                 GAAATGGGGGAAGCGGGTTGCTGCTGCGAATAGTCGTAAGCCGGTGA 
               
               
                   
               
               
                 ACAAACTGTTG 
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 
                   Zymomonas mobilis 
                 
               
               
                 Alcohol dehydrogenase 
               
               
                 Swissprot ID: P06758 
               
               
                 &gt;sp|P06758|ADH2_ZYMMO Alcohol dehydrogenase 2 
               
               
                 OS =  Zymomonas mobilis  GN = adhB PE = 1 SV = 3 
               
               
                 (SEQ ID NO: 3) 
               
               
                 MASSTFYIPFVNEMGEGSLEKAIKDLNGSGFKNALIVSDAFMNKSGV 
               
               
                   
               
               
                 VKQVADLLKAQGINSAVYDGVMPNPTVTAVLEGLKILKDNNSDFVIS 
               
               
                   
               
               
                 LGGGSPHDCAKAIALVATNGGEVKDYEGIDKSKKPALPLMSINTTAG 
               
               
                   
               
               
                 TASEMTRFCIITDEVRHVKMAIVDRHVTPMVSVNDPLLMVGMPKGLT 
               
               
                   
               
               
                 AATGMDALTHAFEAYSSTAATPITDACALKAASMIAKNLKTACDNGK 
               
               
                   
               
               
                 DMPAREAMAYAQFLAGMAFNNASLGYVHAMAHQLGGYYNLPHGVCNA 
               
               
                   
               
               
                 VLLPHVLAYNASVVAGRLKDVGVAMGLDIANLGDKEGAEATIQAVRD 
               
               
                   
               
               
                 LAASIGIPANLTELGAKKEDVPLLADHALKDACALTNPRQGDQKEVE 
               
               
                   
               
               
                 ELFLSAF 
               
             
          
         
       
     
       Nucleic Acid Sequence: 
       [0019]      
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 4) 
               
               
                 ATGGCGTCCAGTACCTTCTACATCCCTTTTGTCAATGAAATGGGTGA 
               
               
                   
               
               
                 GGGCAGCTTGGAAAAAGCGATCAAGGACCTGAACGGGTCGGGCTTTA 
               
               
                   
               
               
                 AAAACGCATTGATCGTTAGCGATGCGTTTATGAACAAGAGCGGCGTC 
               
               
                   
               
               
                 GTCAAACAAGTGGCAGATCTGCTGAAGGCGCAGGGGATCAATTCGGC 
               
               
                   
               
               
                 GGTTTATGACGGTGTCATGCCCAACCCCACCGTTACTGCTGTCTTGG 
               
               
                   
               
               
                 AAGGCTTGAAAATTCTCAAGGATAACAACTCTGATTTTGTCATTTCC 
               
               
                   
               
               
                 CTGGGCGGGGGATCGCCCCATGACTGCGCTAAAGCAATCGCGTTGGT 
               
               
                   
               
               
                 CGCTACCAACGGCGGTGAGGTTAAAGATTATGAGGGCATCGACAAGT 
               
               
                   
               
               
                 CGAAGAAACCCGCTCTGCCTTTGATGTCGATTAATACTACTGCTGGT 
               
               
                   
               
               
                 ACGGCATCGGAAATGACACGCTTCTGTATTATCACCGATGAAGTCCG 
               
               
                   
               
               
                 CCACGTGAAGATGGCTATCGTCGATCGACACGTTACTCCTATGGTGT 
               
               
                   
               
               
                 CGGTGAATGATCCCCTCCTGATGGTCGGCATGCCCAAAGGGCTGACG 
               
               
                   
               
               
                 GCTGCGACGGGCATGGACGCGCTGACGCATGCTTTCGAGGCATACAG 
               
               
                   
               
               
                 CAGTACCGCAGCGACACCGATCACCGATGCATGTGCACTCAAGGCTG 
               
               
                   
               
               
                 CTAGCATGATCGCTAAAAACTTGAAGACCGCTTGTGATAACGGCAAA 
               
               
                   
               
               
                 GACATGCCGGCACGTGAAGCTATGGCGTACGCACAATTTCTGGCTGG 
               
               
                   
               
               
                 CATGGCATTCAACAATGCTAGCTTGGGCTATGTCCACGCAATGGCGC 
               
               
                   
               
               
                 ACCAACTGGGTGGCTATTATAACCTCCCTCACGGAGTGTGCAATGCG 
               
               
                   
               
               
                 GTCCTGCTGCCCCATGTTCTCGCTTACAACGCGAGTGTCGTGGCTGG 
               
               
                   
               
               
                 GCGTCTGAAAGATGTGGGTGTGGCAATGGGTCTGGATATTGCTAATC 
               
               
                   
               
               
                 TGGGCGACAAAGAAGGAGCAGAAGCTACTATCCAGGCAGTGCGGGAT 
               
               
                   
               
               
                 TTGGCTGCAAGTATTGGCATTCCCGCTAACCTGACCGAATTGGGCGC 
               
               
                   
               
               
                 GAAGAAAGAGGACGTTCCTCTGCTGGCGGATCACGCGCTCAAGGATG 
               
               
                   
               
               
                 CTTGCGCTCTGACAAATCCGCGCCAAGGGGATCAGAAAGAAGTCGAG 
               
               
                   
               
               
                 GAGCTCTTCCTGAGTGCTTTC 
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 
                   Ubellularia californica 
                 
               
               
                 FatB thioesterase 
               
               
                 Swissprot ID: Q41635 
               
               
                 MATTSLASAFCSMKAVMLARDGRGMKPRSSDLQLRAGNAPTSLKMING 
               
               
                   
               
               
                 TKFSYTESLKRLPDWSMLFAVITTIFSAAEKQW TNLEWKPKPKLPQLL   
               
               
                   
               
               
                 
                   DDHFGLHGLVFRRTFAIRSYEVGPDRSTSILAVMNHMQEATLNHAKSV 
                 
               
               
                   
               
               
                 
                   GILGDGFGTTLEMSKRDLMWVVRRTHVAVERYPTWGDTVEVECWIGAS 
                 
               
               
                   
               
               
                 
                   GNNGMRRDFLVRDCKTGEILTRCTSLSVLMNTRTRRLSTIPDEVRGEI 
                 
               
               
                   
               
               
                 
                   GPAFIDNVAVKDDEIKKLQKLNDSTADYIQGGLTPRWNDLDVNQHVNN 
                 
               
               
                   
               
               
                 
                   LKYVAWVFETVPDSIFESHHISSFTLEYRRECTRDSVLRSLTTVSGGS 
                 
               
               
                   
               
               
                 
                   SEAGLVCDHLLQLEGGSEVLRARTEWRPKLTDSFRGISVIPAEPRV 
                 
               
               
                   
               
               
                 (SEQ ID NO: 5; entire sequence), (bold AA are 
               
               
                 part of expressed gene; SEQ ID NO: 9) 
               
             
          
         
       
     
       Nucleic Acid Sequence: 
       [0020]      
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 6) 
               
               
                 ATGACAAATCTGGAATGGAAGCCTAAACCCAAGCTCCCTCAATTGTTG 
               
               
                   
               
               
                 GATGACCACTTCGGGCTCCATGGCCTGGTCTTTCGACGGACCTTCGCG 
               
               
                   
               
               
                 ATTCGGTCGTATGAAGTCGGCCCCGACCGCAGTACCAGCATTTTGGCA 
               
               
                   
               
               
                 GTGATGAACCACATGCAGGAAGCGACGCTGAACCACGCTAAATCCGTG 
               
               
                   
               
               
                 GGCATTCTCGGCGACGGTTTTGGCACGACTTTGGAGATGAGTAAACGC 
               
               
                   
               
               
                 GATCTCATGTGGGTGGTCCGCCGAACACACGTTGCAGTCGAACGTTAC 
               
               
                   
               
               
                 CCCACCTGGGGTGATACCGTCGAAGTGGAGTGCTGGATTGGCGCGAGT 
               
               
                   
               
               
                 GGCAATAATGGCATGCGCCGTGATTTTCTGGTTCGCGATTGCAAAACA 
               
               
                   
               
               
                 GGTGAAATCCTGACGCGGTGTACATCGCTGAGCGTGTTGATGAACACT 
               
               
                   
               
               
                 CGTACACGACGACTGTCGACTATTCCCGATGAGGTGCGCGGTGAGATC 
               
               
                   
               
               
                 GGGCCCGCGTTCATTGATAACGTCGCAGTCAAGGATGATGAGATCAAA 
               
               
                   
               
               
                 AAGCTCCAGAAACTGAATGATTCTACGGCAGATTACATCCAAGGCGGG 
               
               
                   
               
               
                 CTCACCCCGCGCTGGAACGATTTGGACGTGAACCAGCATGTTAATAAC 
               
               
                   
               
               
                 CTGAAATATGTTGCGTGGGTCTTTGAGACGGTCCCTGATTCGATTTTT 
               
               
                   
               
               
                 GAGAGCCACCACATCAGCTCGTTCACCTTGGAATATCGGCGGGAGTGT 
               
               
                   
               
               
                 ACGCGCGATTCGGTGCTGCGTAGCCTGACTACCGTCTCGGGCGGTTCC 
               
               
                   
               
               
                 TCCGAAGCAGGACTCGTGTGCGATCATCTCCTGCAATTGGAGGGAGGA 
               
               
                   
               
               
                 TCCGAGGTGCTGCGAGCTCGAACTGAATGGCGCCCAAAACTGACCGAT 
               
               
                   
               
               
                 AGCTTTCGGGGGATTAGCGTCATTCCCGCTGAACCGCGCGTC 
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                   Acinetobacter sp . (strain ADP1) 
               
               
                 Wax ester synthase/acyl-CoA: diacylglycerol 
               
               
                 acyltransferase 
               
               
                 Swissprot ID: Q8GGG1 
               
               
                 (SEQ ID NO: 7) 
               
               
                 MRPLHPIDFIFLSLEKRQQPMHVGGLFLFQIPDNAPDTFIQDLVNDI 
               
               
                   
               
               
                 RISKSIPVPPFNNKLNGLFWDEDEEFDLDHHFRHIALPHPGRIRELL 
               
               
                   
               
               
                 IYISQEHSTLLDRAKPLWTCNIIEGIEGNRFAMYFKIHHAMVDGVAG 
               
               
                   
               
               
                 MRLIEKSLSHDVTEKSIVPPWCVEGKRAKRLREPKTGKIKKIMSGIK 
               
               
                   
               
               
                 SQLQATPTVIQELSQTVFKDIGRNPDHVSSFQAPCSILNQRVSSSRR 
               
               
                   
               
               
                 FAAQSFDLDRFRNIAKSLNVTINDVVLAVCSGALRAYLMSHNSLPSK 
               
               
                   
               
               
                 PLIAMVPASIRNDDSDVSNRITMILANLATHKDDPLQRLEIIRRSVQ 
               
               
                   
               
               
                 NSKQRFKRMTSDQILNYSAVVYGPAGLNIISGMMPKRQAFNLVISNV 
               
               
                   
               
               
                 PGPREPLYWNGAKLDALYPASIVLDGQALNITMTSYLDKLEVGLIAC 
               
               
                   
               
               
                 RNALPRMQNLLTHLEEEIQLFEGVIAKQEDIKTAN 
               
             
          
         
       
     
       Nucleic Acid Sequence: 
       [0021]      
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 8) 
               
               
                 ATGCGACCTCTGCACCCGATTGATTTTATCTTTCTCAGTCTGGAAAA 
               
               
                   
               
               
                 ACGCCAACAGCCCATGCATGTCGGCGGCCTCTTTCTCTTCCAGATCC 
               
               
                   
               
               
                 CAGATAATGCTCCCGATACCTTCATCCAGGATTTGGTCAATGACATC 
               
               
                   
               
               
                 CGCATTAGCAAGAGCATTCCGGTGCCCCCATTTAACAATAAACTCAA 
               
               
                   
               
               
                 TGGCCTGTTTTGGGATGAAGATGAGGAATTTGATCTGGATCACCACT 
               
               
                   
               
               
                 TTCGACATATTGCACTGCCCCACCCGGGTCGCATCCGGGAGCTGTTG 
               
               
                   
               
               
                 ATTTATATCAGCCAGGAGCACTCCACTCTGCTCGATCGCGCGAAGCC 
               
               
                   
               
               
                 ACTGTGGACCTGCAACATTATTGAGGGCATTGAGGGCAATCGGTTCG 
               
               
                   
               
               
                 CTATGTACTTCAAAATCCATCACGCGATGGTTGATGGGGTCGCTGGT 
               
               
                   
               
               
                 ATGCGCCTGATTGAGAAGTCGTTGAGTCATGACGTTACCGAGAAATC 
               
               
                   
               
               
                 TATCGTCCCGCCCTGGTGCGTGGAGGGGAAGCGCGCAAAGCGGCTGC 
               
               
                   
               
               
                 GAGAGCCGAAAACAGGTAAAATTAAGAAAATCATGAGCGGTATCAAG 
               
               
                   
               
               
                 AGCCAGCTGCAAGCTACGCCAACTGTGATCCAAGAGCTGTCCCAGAC 
               
               
                   
               
               
                 GGTCTTTAAAGATATTGGCCGCAACCCGGACCACGTGTCCTCGTTCC 
               
               
                   
               
               
                 AGGCTCCTTGCTCGATCCTGAACCAACGGGTGTCTAGCTCCCGACGG 
               
               
                   
               
               
                 TTTGCGGCACAGTCCTTCGACTTGGATCGATTTCGCAACATCGCTAA 
               
               
                   
               
               
                 GAGCCTGAACGTGACAATTAACGATGTTGTGCTGGCGGTCTGCAGTG 
               
               
                   
               
               
                 GAGCACTCCGGGCGTACCTCATGAGCCACAATAGCCTCCCGTCTAAA 
               
               
                   
               
               
                 CCGTTGATCGCTATGGTGCCCGCTAGCATTCGGAACGATGACAGTGA 
               
               
                   
               
               
                 TGTTAGCAACCGTATTACAATGATTCTCGCTAATCTGGCAACACATA 
               
               
                   
               
               
                 AAGATGATCCTCTGCAGCGCTTGGAAATCATCCGCCGCTCCGTCCAA 
               
               
                   
               
               
                 AATTCGAAACAACGATTCAAACGCATGACCAGCGATCAGATTTTGAA 
               
               
                   
               
               
                 TTATTCGGCTGTTGTTTACGGCCCAGCTGGACTCAACATTATTTCTG 
               
               
                   
               
               
                 GCATGATGCCTAAACGGCAAGCATTTAACCTGGTGATTTCGAACGTT 
               
               
                   
               
               
                 CCGGGTCCGCGCGAACCGTTGTACTGGAACGGCGCGAAACTGGATGC 
               
               
                   
               
               
                 GCTGTACCCCGCGTCGATCGTTCTGGATGGACAGGCTCTGAACATCA 
               
               
                   
               
               
                 CCATGACCAGTTATTTGGATAAGCTCGAGGTGGGCTTGATTGCGTGC 
               
               
                   
               
               
                 CGTAATGCTCTGCCACGCATGCAGAATCTGCTGACGCACCTGGAGGA 
               
               
                   
               
               
                 AGAGATTCAGCTGTTCGAGGGCGTCATTGCGAAACAGGAAGATATTA 
               
               
                   
               
               
                 AAACGGCTAAT 
               
             
          
         
       
     
         [0022]    Nucleic acids encoding the enzymes are delivered to the host bacterium via one or more plasmids such as those shown in  FIGS. 1-2 .  FIG. 1  shows a plasmid map of a construct for expression of the thioesterase enzyme, and  FIG. 2  shows a plasmid map for expression of PDC, ADHE, and WSDGAT. Alternatively, all 4 genes are includes in one plasmid or in separate individual plasmids. The sequences are then incorporated into the genome of the bacterium and expressed to produce the compositions of interest. 
         [0023]    The gene sequences and recombinant proteins described herein are isolated. The term “isolated”, as in isolated nucleic acid molecule or isolated bacterial cell, as used herein, refers to a molecule or cell that is separated from other molecules and/or cells which are present in the natural source of the molecule or cell. For example, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′- and 3′-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule is substantially free of other cellular material, or culture medium, or of chemical precursors or other chemicals. 
       A Cyanobacterial Chassis for Synthetic Biology 
       [0024]      Synechococcus elongatus  7942 is a model organism for both circadian rhythm and photosynthesis.  Synechococcus  is naturally transformable and will integrate recombinant DNA into its chromosome via homologous recombination. Genes and metabolic pathways are inserted and deleted easily, and  Synechococcus  is an suitable platform for synthetic biology. Other suitable photosynthetic microbes (microalgae) include  Gloeobacter violaceus  PCC 7421,  Anabaena variabilis  ATCC 29413,  Nostoc punctiforme  PCC 73102,  Nostoc  sp. PCC 7120,  Prochlorococcus marinus, Synechococcus elongatus  PCC 6301,  Synechococcus elongatus  PCC  7942 ,  Synechococcus  sp. WH 8102,  Synechocystis  sp. PCC 6803,  Thermosynechococcus elongates, Synechococcus  sp. PCC7002, and  Synechococcus  sp. NKBG042902. 
         [0025]    Methods were developed for culturing  Synechococcus , including construction of lighted incubators and optimizing the genetic manipulations necessary for synthetic biology. Using this platform, a promoter screen was carried out to develop a library of promoters to be used for protein expression. To perform this screen, a  Synechococcus  codon-optimized version of the yellow fluorescent protein was used a reporter. Codons were optimized to the known natural occurrence of protein-encoding sequences in  Synechococcus , e.g., codon usage website, http://www.kazusa.or.jp/codon/). By fusing promoters to this reporter, the timing (with regards to circadian rhythm) and protein expression level associated with each promoter was identified. The following exemplary promoters were identified as useful: (strong, PpplC, PapcA; medium, PrbcL; weak, Pfbp). Large fragments of DNA (˜10 kb) were integrated into the chromosome facilitating the integration of synthetic operons coding for entire metabolic pathways into the bacterium. 
       Medium-Chain Fatty Acid Production in Cyanobacteria 
       [0026]    Biodiesel is the collective term for long alkyl chain fatty acid molecules that have been esterified with a short-chain alcohol. Most often, this term refers to plant triacylglycerols that have been esterified ex vivo with methanol. Alkyl chain length has the most influence on biodiesel&#39;s chemical properties and being plant-derived biodiesel is generally longer (C16-24+) than either gasoline or petrodiesel (C6-C18). Therefore, biodiesel is slightly more energy dense than conventional petroleum fuels, but has the distinct disadvantage of a lower cloud point, the temperature at which solids begin to precipitate out of solution. This lower cloud point fundamentally limits the widespread usefulness of biodiesel. An improved biodiesel fuel is based on shorter chain fatty acids such as C12. 
         [0027]    Fatty acid synthesis is a series of condensation reactions in which two-carbon acetyl units from acetyl-CoA are sequentially added to a growing acyl chain. Synthesis in bacteria occurs in a number of protein complexes that function as an assembly line, continually passing the growing chain down the line, until the desired length is reached and the chain is cleaved from its carrier protein by an enzyme called thioesterase. The final chain length is determined by thioesterase, which can have short, medium, or long chain specificity. 
         [0028]    Bacterial fatty acid length is generally C16-18 and optimized for proper membrane fluidity at growth around 20-40° C. In plants, however, fatty acids are used as secondary metabolites including essential oils and waxes and chain length can vary from C6 to C24+. Due to evolutionary homology, plant and prokaryotic fatty acid synthesis pathways are structurally and functionally similar, and heterologously expressed plant enzymes, including thioesterases are functional in prokaryotes. 
         [0029]    Expression of the California bay tree thioesterase (CBT) in  E. coli  results in laurate (C12) being the predominant fatty acid produced. In CBT-expressing strains, overall fatty acid content was increased nearly 5-fold, with the major component being C12. Laurate overproduction was significant enough to result in its secretion to the medium, and laurate crystals were visible on agar culture plates. 
         [0030]    Cyanobacterial fatty acid synthesis is nearly identical that of to  E. coli  and results in mostly C16-C18 fatty acids. Due to this evolutionary similarity, the CBT functions similarly in  Synechococcus  fatty acid synthesis. A codon-optimized version of the CBT gene is used for expression in  Synechococcus . Because the CBT enzyme changes lipid content, CBT constructs are made under the control of transcriptional promoters (PrbcL, PpplC, PapcA, Pfbp) of differing expression strength. These constructs are integrated into the genome using homologous recombination. Optionally, the CBT enzyme is C-terminally Streptagged for protein expression via western blots. 
         [0031]    To analyze the effect of CBT in  Synechococcus , cultures expressing CBT are grown at increasing levels (under strong, medium, or week promoters) as described above. Cultures are grown in 500 mL of BG11 medium at 30° C. under 4000 lux of illumination and diurnal light cycles using lighted incubators. Samples are collected, and growth rate and lipid content are determined in cells and medium. Growth rate is analyzed by optical absorbance at 750 nm. Lipids are extracted by standard methods, e.g., chloroformmethanol extraction, and profiled and quantitated tandem gas chromatography and mass spectrometry (GC/MS). 
       Engineering a Cyanobacterium to Produce a Gasoline-Type Biofuel 
       [0032]      Synechococcus  was engineered to synthesize the medium-chain fatty acid lauric acid (C 11 H 23 COOH) by heterologous expression of medium-chain length specific thioesterase from the California bay tree to induce premature termination of fatty acid synthesis. In vivo biodiesel synthesis is carried out by expression of the  Zymomonas mobilis  ethanol fermentation pathway and the non-specific acyltransferase of  Acinetobacter baylyi , which esterifies fatty acids with ethanol. 
         [0000]    In Vivo Biodiesel Synthesis from Solar Energy 
         [0033]    As described above, biodiesel contains of fatty acids esterified with short chain alcohols. Constructs were made to synthesize biodiesel in the organism in which fatty acids are produced from solar energy. Described herein is a complete in vivo biodiesel synthesis pathway directly from metabolites produced during photosynthesis. 
         [0034]    Lipid-accumulating bacteria store fatty acid chains as wax esters and triaglycerols. One enzyme responsible for this process is wax ester synthase/acyl-coenzymeA:diaclyglycerol acyltransferase (WS/DGAT). The WS/DGAT gene from  Acinetbobacter baylyi  has a broad substrate specificity and esterifies fatty acids with a wide range of alcohols including ethanol. WS/DGAT is functional in gram-negative bacteria and in an engineered ethanol producing strain of  E. coli , WS/DGAT expression resulted in the accumulation of a variety of ethyl esters. For increased biodiesel production levels, WS/DGAT is optionally co-expressed along with the CBT enzyme. 
         [0035]    While  Synechococcus  does not normally produce the ethanol required for esterification, expression of the ethanol fermentation pathway from  Z. mobilis  results in the production of millimolar quantities of ethanol. The complete biodiesel synthesis pathway is as follows. The pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes from  Z. mobilis  are synthesized and codon-optimized for  Synechococcus . PDC and ADH are co-expressed, and the four promoters above are tested for optimal expression level. Cultures are grown in BG11 as described above. Expression of PDC and ADH in log-phase (OD750 0.2-0.7) cultures are verified by western blot analysis. Ethanol synthesis is analyzed in a time-course fashion from log-phase to stationary phase and quantitified using standard methods, e.g., an Ethanol Enzymatic BioAnalysis kit (Roche). 
         [0036]    The WS/DGAT gene from  Acinetobacter  baylyi is synthesized and codon-optimized for  Synechococcus  and integrated into the PDC/ADH bi-cistron for co-expression of all three enzymes. The cells are cultured using standard methods, and lipid/biodiesel is extracted using chloroformmethanol and analyzed via GC/MS. Various ethyl esters (e.g. C16 and C18) are obtained and used as standards for identification and quantitation. CBT is be incorporated using the optimal construct identified above and co-expressed with all genes. 
         [0037]    GC/MS analysis is carried out on lipids/esters isolated to verify the shift in biodiesel length. The constructs and methods described herein produce laurate ethyl ester. High levels of laurate ethyl ester are obtained while allowing cell growth and survival. 
       Example 1 
     Expression of C12 and C8 Thioesterases and Production of Medium Chain-Length Fatty Acids in Cyanobacteria 
       [0038]    A gene encoding the C12 thioesterase from the California bay tree ( U. californica ) was synthesized and expressed in  Synechococcus elongatus  7942 from a promoter containing the −35 region of the  E. coli  Trp operon promoter and the −10 region, including the lac repressor binding site. The  E. coli  lac repressor was also expressed in the strain from the lacI Q  promoter. The C12 thioesterase was expressed in with a His 6  tag and with optimized codons for expression in  S. elongatus  7942. Expression of the protein was verified by Western blot using an anti-His 6  antibody as a probe ( FIG. 3 ). Stably transformed  S. elongatus  7942 with an integrated C12 thioesterase gene were induced with IPTG, and total proteins from an induced and an uninduced culture were run on SDS-PAGE, blotted to nitrocellulose, and probed with the anti-His 6  antibody. A band with the predicted molecular weight of about 30,000 Daltons was observed from the induced culture, but not from the uninduced culture. 
         [0039]    A gene encoding a C8 thioesterase from  C. palustris  was expressed in  Synechococcus elongatus  7942 using the same general strategy and analogous DNA constructions. Expression of the C8 thioesterase protein containing a His 6  tag was confirmed by Western blot as described above. A band of about 30,000 Daltons was observed from an IPTG-induced culture, but not from a corresponding uninduced culture. 
         [0040]    To demonstrate the production of the C12 fatty acid, i.e. lauric acid, cells were solubilized and free fatty acids were converted to the corresponding methyl esters by incubation of the cell extract in methanol according to standard procedures. This procedure is useful in preparing samples for analysis by gas chromatography-mass spectrometry. The samples were then dissolved in hexane and analyzed by gas chromatography ( FIG. 4 ). The resulting chromatographic trace revealed the presence of both lauric acid (C12 carboxylic acid) and myristic acid (C14 carboxylic acid), which were not present in wild-type  S. elongatus  extracts prepared in the same manner. Mass spectrometric analysis confirmed the identity of these peaks. 
         [0041]    To demonstrate the production of ethanol in  S. elongatus , genes from  Zymomonas mobilis  encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) were expressed in  Synechococcus elongatus  7942 under the control of the hybrid Trp-Lac promoter described above. The lac repressor gene of  E. coli  was also expressed under the control of the lacI Q  promoter. The resulting strain was induced with IPTG and ethanol levels in the medium were measured. The induced strain produced about 675 micromolar ethanol, while under identical conditions the uninduced strain produced about 225 micromolar ethanol and the non-engineered parental  Synechococcus elongatus  7942 produced only about 30 micromolar ethanol, as assayed by standard enzymatic procedures ( FIG. 5 ). 
         [0042]    These results indicate that medium-chain fatty acids and ethanol are produced in engineered cyanobacterial cells. According to the invention, the C12 or C8 thioesterase is co-expressed with pyruvate decarboxylase and alcohol dehydrogenase, so that both a medium chain fatty acid and ethanol are produced in the same cell. Furthermore, the C12 or C8 thioesterase is co-expressed with pyruvate decarboxylase and alcohol dehydrogenase and also with a ‘wax synthase’, with the result that the ethyl ester of a C12 or C8 fatty acid is produced. The resulting ethyl esters are purified and used as fuels. 
         [0043]    While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.