Abstract:
The invention provides a genetically modified Cyanobacteria having a construct comprising DNA fragments encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) enzymes obtained from the  Zymomonas mobilis  plasmid pLOI295. The Cyanobacteria are capable of producing ethanol in recoverable quantities of at least 1.7 μmol ethanol per mg of chlorophyll per hour.

Description:
FIELD OF INVENTION  
         [0001]    This invention relates to the genetic modification of Cyanobacteria for the production of ethanol. In particular, this invention relates to the genetic modification of Synechococcus by incorporating the genetic information encoding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh).  
         BACKGROUND  
         [0002]    Ethanol is an energy source which is particularly attractive because it can be utilized with little waste. In addition, ethanol derived from living organisms is an attractive alternative to petroleum based fuels because it is a renewable resource.  
           [0003]    A number of alternatives for the production of ethanol from living organisms have been investigated using microorganisms.  
           [0004]    The production of ethanol by microorganisms has, in large part, been investigated using the yeast Saccharomyces and bacteria Zymomonas, which is a facultative anaerobic. Both of these microorganisms contain the genetic information to produce enzymes pdc and adh, which enzymes are used to produce ethanol from pyruvate, a product of the glycolytic pathway.  
           [0005]    U.S. Pat. No. 4,242,455 to Muller et al. describes a continuous process in which an aqueous slurry of carbohydrate polymer particles, such as starch granules and/or cellulose chips, fibres, etc., are acidified with a strong inorganic acid to form a fermentable sugar. The fermentable sugar is then fermented to ethanol with at least two strains of Saccaromyces. U.S. Pat. 4,350,765 to Chibata et al. describes a method of producing ethanol in a high concentration by using an immobilized Saccharomyces or Zymomonas and a nutrient culture broth containing a fermentative sugar. U.S. Pat. No. 4,413,058 to Arcuri et al. describes a new strain of  Zymomonas mobilis  which is used to produce ethanol by placing the microorganism in a continuous reactor column and passing a stream of aqueous sugar through said column.  
           [0006]    PCT Application WO/88/09379 to Hartley et al. describes the use of facultative anaerobic thermophilic bacteria strains which produce ethanol by fermenting a wide range of sugars, including cellobiose and pentoses. These bacteria strains contain a mutation in lactate dehydrogenase. As a result, these strains which would normally produce lactate under anaerobic conditions, produce ethanol instead.  
           [0007]    In addition,  Escherichia coli  has been genetically altered to produce ethanol by inserting the genetic material encoding for the adh and pdc enzymes using the pLOI295 plasmid. The genetic material encoding the pdc enzyme was isolated from  Zymomonas mobilis . This altered  Escherichia coli  produces ethanol; however, it still requires a variety of organic substrates for bacterial metabolism and growth. (Ingram, et al. (1987), “Genetic Engineering of Ethanol Production in  Escherichia coli ” (Appl. Environ Microbiol. 53: 2420-2425)  
           [0008]    All of the above prior art describe microorganisms which utilize a carbohydrate/sugar substrate to produce ethanol. As such, these processes are costly because a feed substrate of carbohydrates/sugars is required in order for the microorganisms to be able to produce ethanol. Hence, the cost of these systems is a deterrent to the refinement and scale up of such systems for the production of ethanol.  
           [0009]    It is highly desirable to find a microorganism which can effectively produce ethanol wherein said microorganism requires minimal feed substrate.  
         SUMMARY OF THE PRESENT INVENTION  
         [0010]    In an aspect of the present invention, there is provided genetically modified photosynthetic Cyanobacteria which are capable of producing ethanol. The Cyanobacteria are genetically modified by the insertion of DNA fragments encoding the enzymes pdc and adh. Consequently, the enzymes pdc and adh are produced in vivo by the genetically modified Cyanobacteria; which enzymes convert pyruvate to acetaldehyde and acetaldehyde to ethanol, respectively. In particular, Synechococcus is a preferred Cyanobacteria of the present invention. In a preferred embodiment, transformed Synechococcus produce ethanol in recoverable quantities of at least 1.7 μmol of ethanol per mg of chlorophyll per hour.  
           [0011]    In a further aspect of the present invention, there is provided genetically modified Cyanobacteria which contain constructs comprising a temperature inducible gene so that the ethanol is produced only once a particular temperature is reached. In a particular embodiment, the construct comprises the CI857 temperature inducible gene. The CI857 temperature inducible gene maybe used in the form of the CI-PL promoter, EMBL Accessive No. L05669, SEQ. ID. No.7.  
           [0012]    In a further aspect of the present invention, there is provided genetically modified Cyanobacteria which contain constructs comprising DNA fragments encoding the pdc and adh enzymes obtained from the  Zymomonas mobilis  plasmid pLOI295.  
           [0013]    In a further aspect of the present invention, the Cyanobacteria is Synechococcus PCC 7942 or other transformable strains capable of producing ethanol when a construct comprising DNA fragments encoding pdc and adh enzymes from the pLOI295 plasmid is transformed into the Synechococcus.  
           [0014]    In a further aspect of the present invention, there is provided genetically modified Cyanobacteria containing constructs comprising DNA fragments from the  Zymomonas mobilis  plasmid pLOI295 encoding the pdc and adh enzymes wherein the DNA fragment encoding the pdc enzyme is listed in the European Molecular Biology Laboratories (“EMBL”) as Accession No. M15393 and as described in Conway et al. (1987) J. Bacterial 169: 949-954 SEQ. ID. No. 5, or a gene sequence that encodes the pdc enzyme and is capable of expression in Cyanobacteria.  
           [0015]    In a further aspect of the present invention, there is provided genetically modified Cyanobacteria containing constructs comprising DNA fragments from the  Zymomonas mobilis  plasmid pLOI295 encoding the pdc and adh enzymes wherein the DNA fragment encoding the adh enzyme is adh II listed in the EMBL as Accession No. M15394 and as described in Conway et al. (1987) J. Bacterial 169: 2591-2597, SEQ. ID. No. 6 or a gene sequence that encodes the adh enzyme and that is capable of expression in Cyanobacteria.  
           [0016]    In another aspect of the present invention there is provided a genetically modified Cyanobacteria capable of producing ethanol produced according to the following steps:  
           [0017]    a. selecting an appropriate promoter;  
           [0018]    b. ligating said promotor to pdc and adh encoding DNA sequence;  
           [0019]    c. cloning said ligated promoter and said pdc and adh encoding DNA into an appropriate construct;  
           [0020]    d. transforming the construct into the Cyanobacteria  
           [0021]    In a preferred embodiment the modified Cyanobacteria is a modified Synechococcus PCC 7942. Constructs produced according to these steps include constructs selected from the group consisting of pCB4-Rpa, pCB4-LRpa and pCB4-LR(TF)pa.  
           [0022]    In a further aspect of the present invention, there is provided a construct comprising a promoter from Synechococcus operatively linked to genes encoding pdc and adh enzymes from the  Zymomonas mobilis  pLOI295 plasmid.  
           [0023]    In a further aspect of the present invention there is provided a construct wherein the promoter comprises an rbcLS operon of Synechococcus. In another aspect the promoter further comprises a lacZ operon of  Escherichia coli.    
           [0024]    In a further aspect of the present invention there is provided a construct wherein the DNA fragments encoding the pdc and adh enzymes are listed in EMBL as Accession No. M15393 and M15394, SEQ. ID. Nos. 5 and 6, respectively, or analogous sequences thereof that include encoding for the pdc enzyme and the adh enzyme, respectively.  
           [0025]    In a further aspect of the present invention, there is provided constructs encoding the pdc and adh enzymes wherein the constructs include a temperature inducible gene CI857.  
           [0026]    In a further aspect of the invention, there is provided a promoter capable of being used in a construct encoding pdc and adh enzymes obtained from  Zymomonas mobilis , wherein the promoter comprises a rbcLS operon of Synechococcus.  
           [0027]    In a further aspect of the present invention, there is provided a promoter capable of being used in a construct encoding the pdc and adh enzymes obtained from  Zymomonas mobilis , wherein the promoter comprises a rbcLS operon of Synechococcus and a lacZ operon of  Escherichia coli.    
           [0028]    In a further aspect of the present invention there is provided a CI-PL promoter which is temperature inducible and is capable of being used in a construct encoding pdc and adh enzymes obtained from  Zymomonas mobilis  wherein said promoter is activated only once a particular temperature is reached.  
           [0029]    In a further aspect of the present invention there is provided a process for making genetically modified Cyanobacteria by incorporating a construct encoding the pdc and adh enzymes from the  Zymomonas mobilis  pL01295 plasmid, or other suitable source of pdc and adh enzymes, according to the following steps:  
           [0030]    a. harvesting cells of the Cyanobacteria;  
           [0031]    b. adding the construct to the harvested Cyanobacteria cells;  
           [0032]    c. incubating the construct and the Cyanobacteria cells such that the construct is transformed into the Cyanobacteria cells;  
           [0033]    d. plating the incubated constructs and Cyanobacteria cells on plates containing ampicillin and incubating under appropriate growth conditions;  
           [0034]    e. selecting the transformed ampicillin resistant Cyanobacteria cells.  
           [0035]    In a further aspect of the present invention, there is provided a process for producing ethanol using genetically modified Cyanobacteria which comprises the steps of: culturing in a culture medium Cyanobacteria, wherein the Cyanobacteria contains a construct comprising DNA fragments encoding pdc and adh enzymes obtained from the Zymomonas mobiles pL0I295 and accumulating ethanol in the culture medium. In a preferred embodiment, the process for producing ethanol includes a construct which comprises a temperature inducible gene and the process comprises the further step of increasing the temperature of the culture medium to induce expression of the pdc and adh genes. 
       
    
    
     DETAILED DESCRIPTION OF THE DRAWINGS  
       [0036]    The invention will now be better understood with reference to the following figures and examples, and corresponding description, which are illustrative of preferred embodiments of the invention. The invention should not be limited by the drawings.  
         [0037]    [0037]FIG. 1 is an illustration of the map of the plasmid pLOI295 containing the DNA fragments encoding for pdc and adh.  
         [0038]    [0038]FIG. 2 is an illustration of the map of the plasmid construct pCB4-Rpa.  
         [0039]    [0039]FIG. 3 is an illustration of the map of the plasmid construct pCB4-LRpa.  
         [0040]    [0040]FIG. 4 is an illustration of the map of the plasmid construct pCB4-LR(TF)pa.  
         [0041]    [0041]FIG. 5 is an illustration of the map of the plasmid construct pCB4-CPpa.  
         [0042]    [0042]FIG. 6 is an illustration of a graph of the incubation time of Synechococcus PCC 7942 cells transformed with the vector pCB4-CPpa. at 42 degrees Celsius versus the activity of pyruvate decarboxylase.  
         [0043]    [0043]FIG. 7 is an illustration of the induction of adh expression at 42 degrees Celsius for Synechococcus PCC 7942 as compared to  E. coli  and wild type Synechococcus.  
         [0044]    [0044]FIG. 8 is an illustration of the induction time of Synechococcus PCC 7942 versus ethanol production in Synechococcus PCC 7942 in cells transformed with pCB4-Rpa.  
         [0045]    [0045]FIG. 9 is a description of the pdc gene identified as SEQ ID. No.5.  
         [0046]    [0046]FIG. 10 is a description of the adh gene identified as SEQ. ID. No. 6.  
         [0047]    [0047]FIG. 11 is a description of the CI-PL promoter identified as SEQ. ID. No. 7. 
     
    
       [0048]    All like letter designations refer to the same sites on the different maps of the plasmid constructs in the figures as follows: AMP R  (ampicillin resistant); PDC (pyruvate decarboxylase); ADH (alcohol dehydrogenase); ATG (start codon); L (lacZ promoter); R (rbcLS promoter); R′ (EcoRI); B (BamHI); S (SalI); X (XbaI); X/P (XbaI/PvuII fusion); Xh/B (XhoI/BamHI fusion); T (transcription terminator) and CI-PL (temperature inducible gene and bacterial phage left-ward promoter).  
       DETAILED DESCRIPTION  
       [0049]    Cyanobacteria are photosynthetic bacteria which require light, inorganic elements, water and a carbon source, generally CO 2 , to metabolise and grow.  
         [0050]    Cyanobacteria are photosynthetic procaryotes which carry out oxygenic photosynthesis. The main product of the metabolic pathway of Cyanobacteria during aerobic conditions is oxygen and carbohydrate reserves.  
         [0051]    The initial product of photosynthetic fixation of CO 2  is 3-phosphoglycerate. 3-phosphoglycerate is used in the Calvin Cycle to regenerate ribulose-1,5-biphosphate, which is the acceptor of CO 2 . There are two major branching points where the intermediates of the Calvin Cycle are connected to other metabolic pathways. At one point, fructose-6-phosphate is converted into glucose-6-phosphate and glucose-phosphate, which are the substrates for the pentose phosphate pathway, the synthesis of cellulose (a major component of the cell wall) and the synthesis of glycogen (the major form of carbohydrate reserve). At the other branching point, 3-phosphoglycerate is converted into 2-phosphoglycerate, phosphoenolpyruvate and pyruvate in a sequence of reactions catalysed by phosphoglycerate mutase, enolase and pyruvate kinase, respectively. Pyruvate is directed to the partial TCA cycle for the synthesis of amino acids, nucleotides, etc. in aerobic conditions. Pyruvate is also the substrate for ethanol synthesis.  
         [0052]    To convert the carbohydrate reserves into ethanol, the carbohydrate reserves must be diverted to the glycolytic pathway. The presumed pathway for carbohydrate reserve metabolism in Cyanobacteria is through both the glycolytic pathway and the phosphogluconate pathway. For the purposes of ethanol formation, the glycolytic pathway is of primary importance. Although not well characterized in Cyanobacteria, glycogen is presumed to be metabolized into glucose 1—phosphate by a combination of glycogen phosphorylase and a 1,6-glycosidase. Phosphoglucomutase, phosphoglucoisomerase and phosphofructokinase convert glucose 1-phosphate into a molecule of fructose1,6-bisphosphate. This compound is cleaved by the action of aldolase and triose phosphate isomerase into two molecules of glyceraldehyde 3-phosphate. This compound is converted into pyruvate through sequential series of reactions catalysed by glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase, respectively.  
         [0053]    In some algae and Cyanobacteria strains, a small amount of ethanol is synthesized as a fermentation product under dark and anaerobic conditions (Van der Oost et al., 1989; Heyer and Krumbein, 1991). However, the dark-anaerobic fermentation process is generally operating at a very low level, only sufficient for the survival of the organisms under such stress conditions. The synthesis of ethanol under dark and anaerobic conditions is dependent on the degradation of glycogen reserve, as described above. Moreover, it has been found that ethanol synthesis under anaerobic conditions is totally inhibited by light. Thus, in photosynthetic microorganisms ethanol synthesis is not coupled with photosynthesis and can actually be inhibited by photosynthesis.  
         [0054]    Therefore, it has been observed that Cyanobacteria do not utilize CO 2  to produce ethanol. Furthermore, there are no known photosynthetic microorganisms, including genetically engineered photosynthetic microorganisms, which produce ethanol in relatively substantial amounts. A further complication is that some photosynthetic organisms have been shown to be inhibited by ethanol such that the addition of ethanol to the culture medium inhibits the expression of genes involved in photosynthesis.  
         [0055]    In the present invention, it has been found that Cyanobacteria can be successfully genetically engineered to utilize a direct flux of carbon from CO 2  to 3-phosphoglycerate, and to pyruvate, to produce a quantifiable amount of ethanol as opposed to utilizing a glycogen reserve as is done under anaerobic and dark conditions.  
         [0056]    It has been found that Cyanobacteria can be genetically modified by introducing genes encoding for the enzymes pdc and adh to produce ethanol. In particular, a pathway for ethanol synthesis has been created in Synechococcus PCC 7942, and this pathway is directly coupled with photosynthesis.  
         [0057]    By incorporating the genetic material encoding the pdc and adh enzymes into the Synechococcus genetic material, a Synechococcus capable of producing ethanol is created. It was surprisingly found that pdc and adh enzymes from an obligate anaerobe,  Z. mobilis , could be successfully inserted, expressed and be fully functional in Synechoccocus. Although pdc and adh enzymes from  Z. mobilis  had been transformed into  E. coli . As described in Ingram, et al. (1987), “Genetic Engineering of Ethanol Production in  Escherichia coli ” (Appl. Environ Microbiol. 53: 2420-2425),  E. coli  is a facultative anaerobic, it has an inducible adh gene and it is grown in a carbohydrate medium and said carbohydrates are used to produce ethanol. On the other hand, Cyanobacteria are photosynthetic organisms and are recalcitrant to taking up organic substances for any purpose, including growth or ethanol production. Hence,  E. coli  is a very different system than Cyanobacteria.  E. coli  is more like  Z. mobilis  which depends on feed stock for growth and ethanol production. There are other sources of pdc and adh enzymes, including  Saccharomyces cerevisciae.    
         [0058]    It has also been found that ethanol synthesis may compete with cell growth for the use of carbon. Therefore, it would be beneficial to have an inducible system for ethanol synthesis so that cell growth and ethanol synthesis could be carried out in two phases. During the first phase, Cyanobacteria cells are cultured under non-induced conditions, so that the cell culture can reach a high density and accumulate a large amount of carbohydrates. Ethanol synthesis is then induced in the second phase.  
         [0059]    In particular it was discovered that a temperature inducible system could be successfully developed to induce the production of ethanol in Cyanobacteria. A pdc-adh operon with the bacterial phage left-ward promoter (P L ) and a temperature sensitive repressor gene CI857 were employed to produce a temperature inducible system for producing ethanol in Cyanobacteria.  
         [0060]    It is believed that at a non-permissible temperature (low temperature, 30 degrees Celsius), the repressor binds to the operator sequence, and thus prevents RNA polymerase from initiating transcription at the P L  promoter. Therefore, the expression of pdc-adh genes is repressed. When the cell culture is transferred to a permissible temperature (37-42 degrees Celsius), the repressor can not bind to the operator. Therefore, RNA polymerase can initiate the transcription of the pdc-adh gene.  
         [0061]    The Examples below exemplify the four different constructs: pCB4-Rpa, pCB4-LRpa, pCB4-LR(TF)pa and pCB4-CPpa: the synthesis of these constructs; the incorporation of these constructs into Synechococcus PCC 7942 and the production of ethanol from said genetically modified Synechococcus. Other transformable strains of Synechococcus which are capable of producing ethanol when a construct containing DNA encoding the adh and pdc enzyme is transformed into the Synechococcus may also be used.  
         [0062]    In the examples below, Synechococcus PCC 7942, which is available from the Pasteur Culture Collection, Rue de Dr. Roux, Paris, France, was used. The genes encoding the pdc and adh enzymes of  Zymomonas mobilis  were excised from the pLOI295 plasmid, which is available from Dr. L. 0. Ingram, Dept. of Microbiology and Cell Science, University of Florida, Gainsville, Fla., U.S.A. 32611. (See also: Ingram et al., (1987) “Genetic Engineering of Ethanol Production in  Escherichia coli ” Appl. Environ Microbial 53: 2420-2425). A map of the pLOI295 plasmid is illustrated in FIG. 1. In particular, the DNA segment excised from the pLOI295 plasmid includes the pdc sequence starting at −46 bp (relative to the transcription start site) to a position +27 bp after the translation stop codon and is listed in EMBL as Accession No. M15393 and the DNA adh sequence starting from −31 bp up from the ATG initiation codon to +164 bp after the translation stop codon, which is listed in EMBL as Accession No. M15394.  
       EXAMPLE 1  
     pCB4-Rpa  
       [0063]    The pCB4-Rpa construct is driven by a promoter obtained from the rbcLS operon of the cyanobacterium Synechococcus PCC 7942. The promoter sequence from the rbcLS operon was amplified from Synechococcus PCC 7942 by the polymerase chain reaction (PCR) using the forward primer identified as SEQ ID No. 1 (containing a BamHI site) and the reverse primer identified as SEQ ID No. 2 (containing an EcoRI site). These primers were designed according to the rbcL gene sequence obtained from the cyanobacterium  Anacystis nidulan  6301, a strain genetically similar to Synechococcus PCC 7942. (Shinozaki K. et al. (1983) “Molecular cloning and sequence analysis of the Cyanobacteria gene for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase.” Proc Natl Acad Sci USA 80:4050-4054). The PCR reaction mixture (100 μl) contained 0.5 μM of each primer, 0.4 mM dNTP, 10 ng genomic DNA from Synechococcus sp. PCC 7942 and 2 units of Vent R  DNA plolymerase (New England Biolabs) in 1× reaction buffer: 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 8.8 at 25° C.), 2 mM MgCl 2  and 0.1% Triton X-100. PCR reactions were carried out in PTC-100TM Programmable Thermal Controller (MJ Research, Inc.) by using the temperature cycles as follows: 93° C./3 min; 30 cycles of 93° C./1 min, 62° C./1.5 min, 72° C./0.5 min; 72° C./5. The PCR product of expected size was cloned into the BamHI-EcoRI sites of the plasmid pBlueScript SK (Stratagene Inc.) to generate a plasmid designated pRBCp.  
         [0064]    A 3.2 kbp EcoRI-SalI DNA fragment containing the pdc-adh sequence from  Zymomonas mobilis  was isolated from the pLOI295 plasmid and ligated into the corresponding sites of pRBCp to generate the plasmid pRpa. The pLOI295 plasmid map is illustrated in the map in FIG. 1. A 3.6 kbp BamHI DNA fragment containing the rbcLS promoter region and the pdc-adh sequences were then excised from pRpa and ligated into the BamHI site of the shuttle vector pCB4 (Gendel et al., (1983) “Shuttle Cloning Vectors for the Cyanobacterium  Anacystis Nidulans ”, J. Bacteriol, 156: 148-154) resulting in the vector construct pCB4-Rpa. The shuttle vector pCB4 contains genes encoding ampicillan resistance. The vector construct pCB4-Rpa is illustrated in FIG. 2.  
       EXAMPLE 2  
     pCB4-LRpa  
       [0065]    A 3.6 kbp BamHI DNA fragment from pRpa was ligated into a modified version of pCB4. The modified version of pCB4 is constructed by ligating a 220 bp PvuII-BamHI DNA fragment from the plasmid pBS (Stratagene Inc., 11011 North Torrey Pines Road, La Jolla, Calif., U.S.A. 92037), which fragment contains the lacZ promoter region from  Escherichia coli , into the modified XbaI-BamHI sites of the pCB4 multi-cloning site. (Soltes-Rak E et al. (1993) “Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942.” Appl Environ Microbio. 59: 2404-2410). The 3.6 kbp DNA fragment is then ligated into the modified version of pCB4 resulting in the vector construct pCB4-LRpa. The vector construct pCB4-LRpa is illustrated in FIG. 3.  
       EXAMPLE 3  
     pCB4-LR(TF)pa  
       [0066]    The pdc-adh coding region is driven by a combination of the rbcLS and lacZ promoter regions, as in pCB4-LRpa, but in this construct the  Zymomonas mobilis  pdc ribosome binding site and start codon have been removed and replaced with the corresponding DNA region of the rbcL sequence from Synechococcus PCC 7942 to generate a translation fusion product.  
         [0067]    The pdc-adh DNA segment in pLOI295 plasmid is amplified and modified by PCR using the forward primer identified as SEQ ID No. 3 (containing an EcoRI site) and reverse primer identified as SEQ ID No. 4 (containing BamHI and XhoI sites). The PCR reaction mixture was as described above for Example 1. The temperature cycles were as follows: 93° C./5 min; 4 cycles of 93° C./1 min, 56° C./1.5 min, 72° C./3.5 min; 30 cycles of 93° C./1 min, 65° C./1.5° C., 72° C./3.5 min; 72° C./5 min. The 3.1 kbp PCR product was then ligated into pRBCp at the EcoRI-XhoI sites (double-cut) to generate plasmid pR(TF)pa (TF as in Translation Fusion). The cloning for translation fusion generated an extra codon AAT (asparagine) immediately after the initiation codon and the original second codon, AGT in pdc open reading frame was replaced by TCT to code the same amino acid (serine). This new plasmid was digested with XhoI, the cut sites blunt ended with Klenow fragment from DNA polI, and then digested with XbaI. This DNA fragment containing rbc-(TF)pdc-adh was then ligated into pCB4-lac which had been prepared by digestion with BamHI, blunt ended with Klenow, and redigested with XbaI. The resulting plasmid is designated pCB4-LR(TF)pa and is illustrated in FIG. 4.  
       EXAMPLE 4  
     pCB4-CPpa  
       [0068]    The vector pCB4-Rpa was digested with XbaI, end-filled with Klenow fragment of DNA polymerase I and re-digested with EcoRI to delete the rbcLS promoter. The vector was then ligated to a PstI-EcoRI fragment containing the CI857 repressor gene and P L  promoter sequence, collectively termed the cI-PL gene sequence (EMBL Accession No. L05669; Sanger et al.  Nucleotide sequence of the bacteriophage lambda DNA.  1982, J. Mole. Biol. 162: 729-773) and identified as SEQ. ID. No. 7. The P L  promoter had been isolated from the plasmid pHUB2-C1857 (Gruber et al. (1991)) “ Escherichia coli - Anacystis nidulans  plasmid shuttle vectors containing the P L  promoter from bacteriophage lambda.” Curr. Microbio. 22:15-19). The vector was litigated by digestion with PstI, end-filling with Klenow and a second digestion with EcoRI. The recombinant plasmid is designated as pCB4-CPpa.  
       EXAMPLE 5  
     Genetically Modified Synechococcus PCC 7942  
       [0069]    Each of the four constructs of Examples 1, 2, 3 and 4 were incorporated into the Synechococcus PCC 7942.  
         [0070]    The constructs of Examples 1, 2, 3 and 4 were incorporated into the Synechococcus PCC 7942 using a standard protocol as set out in Golden SS et al. (1987) “Genetic engineering of the Cyanobacteria chromosome” Methods Enzymol 153: 215-231 and in S. S. Golden and L. A. Sherman, J. Bacteriology 158:36 (1984), incorporated herein by reference. Briefly, cells of Synechococcus PCC 7942 are harvested by centrifugation and re-suspended in BG-11 medium at a concentration of 2-5×10 8  cells per ml. To one ml of this cell solution is added the appropriate plasmid construct DNA to a final concentration of 2 μg. ml −1 . Cells are incubated in the dark for 8 hours followed by a 16 h light incubation prior to plating on BG-11 plates containing 1 μg.ml −1  ampicillin. Plates are incubated under the standard growth conditions (30° C. light intensity of 100 μmol photons. m −2 .s −1 ). Ampicillin resistant colonies were visible in 7-10 days.  
         [0071]    The genetically modified Synechococcus PCC 7942 were grown, bubbling with air at 30 and a light intensity of 100 μE.M −2 .s −1  in liquid BG-11 medium containing 5 μg.ml −1  ampicillin (Soltes-Rak E et al. (1993) “Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942.” Appl Environ Microbio. 59: 2404-2410) The activity of pdc, adh and the production of ethanol were measured as set out in Table 1 below for Examples 1, 2 and 3. The ethanol production for Example 3 is also illustrated in FIG. 8. Table 2 illustrates the ethanol production for Example 4. FIGS.  6  and 7 illustrate the pdc activity and adh expression, respectively, for Example 4. The activity of pdc was measured by determining the rate of pyruvic acid dependent reduction of NAD +  with yeast with adh as the coupling enzyme as previously described in Conway et al., J. Bacteriology 169:2591-2597 (1987). Adh was measured for Examples 1, 2 and 3 by determining the rate of ethanol dependent NADH oxidation as described in Neale et al., Eur. J. Biochem. 154: 119-124 (1986). Ethanol was assayed using a standard Ethanol Assay kit obtained from Boehringer Mannheim Canada, Laval, Quebec. The results of the tests for pdc and adh activity and ethanol production for the constructs of Examples 1-3 are illustrated in Table 1.  
                               TABLE 1                                   Eth-   Ethanol                   anol   Conc.                   Conc.   in           PDC Activity   ADH Activity   in   μmoL.mg −1             nmol.min. − .mg −1     nmol.min. − .mg −1     medium   Chlor-       Constructs   SP 1     SP   (μM) 3     ophyll                   pCB4 4     ND 2     ND   ND   ND       pCB4-Rpa   130   168   1370   274       pCB4-   136   168   1540   308       LRpa       pCB4-   234   168   1710   342       LR(TF)pa                                                  
 
         [0072]    Synechococcus PCC 7942 cells were transformed with the vector pCB4-CPpa. The transformed cells were first grown at 30 degrees Celsius as set out above and then transferred to 42 degrees Celsius for 48 hours. Cells were harvested at intervals to assay pdc activity. As shown in FIG. 6, pdc activity was induced at 42 degrees, reaching a 20-fold increase at 48 hours after the temperature shift. Surprisingly, the pdc activity induced at 42 degrees Celsius with the pCB4-CPpa vector after 48 hours was approximately 2000 nmol.min. −1 .mg −1  SP, which is about 20-fold higher than in the strain harboring the shuttle vector pCB4-Rpa which had a pdc activity of approximately 130 nmol.min. −1 mg −1  SP as can be seen in FIG. 6 and Table 1, respectively.  
         [0073]    The impact of temperature shift on ethanol synthesis was studied in liquid batch culture. The rate of ethanol synthesis at 42 degrees Celsius was 1.7 μmol ethanol per mg of chlorophyll per hour. As such, it was 5-times higher at 42 degrees than at 30 degrees Celsius, as can be seen in Table 2.  
                             TABLE 2                           Effect of temperature shift on Ethanol Synthesis       Synechococcus PCC 7942 cells transformed with the shuttle       vector pCB4-CPpa were first grown at 30 deg. Celsius in the       light, harvested at log phase and resuspended into a fresh       medium at a cell density of 4.3 μg chlorophyll per ml.       The resuspended cells were grown for 48 h in the light       at 30 deg. Celsius and 42 deg. Celsius, respectively. The       value in the brackets indicates the S.D. for 4 different samples.                Ethanol Conc.   Rate of Ethanol Synthesis       Temperature   (μmol.mg −1 chlorophyll)   (μmol.mg −1 chlorophyll per hr)               30   16(0.9)   0.33       42   82(8.9)   1.70                  
 
         [0074]    The above examples are intended to exemplify the invention. It is understood by the skilled workman in the art that various modifications and alterations may be made without departing from the scope of the invention and as set out in the claims attached hereto.   
     
       
       
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7 
 
           
           
             
               29 base pairs  
               nucleic acid  
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              1 

GCTGAATTCA TGTCGTCTCT CCCTAGAGA                                       29 

 
           
           
             
               29 base pairs  
               nucleic acid  
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GCTGAATTCA TGTCGTCTCT CCCTAGAGA                                       29 

 
           
           
             
               25 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              3 

GGACTCGAGG ATCCCCAAAT GGCAA                                           25 

 
           
           
             
               29 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              4 

GCATGAATTC TTATACTGTC GGTACCTAT                                       29 

 
           
           
             
               1905 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
              5 

TATCGCTCAT GATCGCGACA TGTTCTGATA TTTTCCTCTA AAAAAGATAA AAAGTCTTTT     60 

CGCTTCGGCA GAAGAGGTTC ATCATGAACA AAAATTCGGC ATTTTTAAAA ATGCCTATA     120 

CTAAATCCGG AACGACACTT TAGAGGTTTC TGGGTCATCC TGATTCAGAC ATAGTGTTT     180 

GAATATATGG AGTAAGCAAT GAGTTATACT GTCGGTACCT ATTTAGCGGC GCTTGTCCA     240 

ATTGGTCTCA AGCATCACTT CGCAGTCGCG GGCGACTACA ACCTCGTCCT TCTTGACAA     300 

CTGCTTTTGA ACAAAAACAT GGAGCAGGTT TATTGCTGTA ACGAACTGAA CTGCGGTTT     360 

AGTGCAGAAG GTTATGCTCG TGCCAAAGCG GACGCAGCAG CCGTCGTTAC CTACAGCGT     420 

GGTGCGCTTT CCGCATTTGA TGCTATCGGT GGCGCCTATG CAGAAAACCT TCCGGTTAT     480 

CTGATCTCCG GTGCTCCGAA CAACAATGAT CACGCTGCTG GTCACGTGTT GCATCACGC     540 

CTTGGCAAAA CCGACTATCA CTATCAGTTG GAAATGGCCA AGAACATCAC GGCCGCAGC     600 

GAAGCGATTT ACACCCCAGA AGAAGCTCCG GCTAAAATCG ATCACGTGAT TAAAACTGC     660 

CTTCGTGAGA AGAAGCCGGT TTATCTCGAA ATCGCTTGCA ACATTGCTTC CATGCCCTG     720 

GCCGCTCCTG GACCGGCAAG CGCATTGTTC AATGACGAAG CCAGCGACGA AGCTTCTTT     780 

AATGCAGCGG TTGAAGAAAC CCTGAAATTC ATCGCCAACC GCGACAAAGT TGCCGTCCT     840 

GTCGGCAGCA AGCTGCGCGC AGCTGGTGCT GAAGAAGCTG CTGTCAAATT TGCTGATGC     900 

CTCGGTGGCG CAGTTGCTAC CATGGCTGCT GCAAAAAGCT TCTTCCAGAA GAAAACCGC     960 

TTACATCGGT ACCTCATGGG TGAAGTCAGC TATCCGGGCG TTGAAAAGAC GATGAAAG     1020 

GCCGATGCGG TTATCGCTCT GGCTCCTGTC TTCAACGACT ACTCCACCAC TGGTTGGA     1080 

GATATTCCTG ATCCTAAGAA ACTGGTTCTC GCTGAACCGC GTTCTGTCGT CGTTAACG     1140 

GTTCGCTTCC CCAGCGTTCA TCTGAAAGAC TATCTGACCC GTTTGGCTCA GAAAGTTT     1200 

AAGAAAACCG GTGCTTTGGA CTTCTTCAAA TCCCTCAATG CAGGTGAACT GAAGAAAG     1260 

GCTCCGGCTG ATCCGAGTGC TCCGTTGGTC AACGCAGAAA TCGCCCGTCA GGTCGAAG     1320 

CTTCTGACCC CGAACACGAC GGTTATTGCT GAAACCGGTG ACTCTTGGTT CAATGCTC     1380 

CGCATGAAGC TCCCGAACGG TGCTCGCGTT GAATATGAAA TGCAGTGGGG TCACATCG     1440 

TGGTCCGTTC CTGCCGCCTT CGGTTATGCC GTCGGTGCTC CGGAACGTCG CAACATCC     1500 

ATGGTTGGTG ATGGTTCCTT CCAGCTGACG GCTCAGGAAG TCGCTCAGAT GGTTCGCC     1560 

AAACTGCCGG TTATCATCTT CTTGATCAAT AACTATGGTT ACACCATCGA AGTTATGA     1620 

CATGATGGTC CGTACAACAA CATCAAGAAC TGGGATTATG CCGGTCTGAT GGAAGTGT     1680 

AACGGTAACG GTGGTTATGA CAGCGGCGCT GGTAAAGGCC TGAAGGCTAA AACCGGTG     1740 

GAACTGGCAG AAGCTATCAA GGTTGCTCTG GCAAACACCG ACGGCCCAAC CCTGATCG     1800 

TGCTTCATCG GTCGTGAAGA CTGCACTGAA GAATTGGTCA AATGGGGTAA GCGCGTTG     1860 

GCCCGCCAAC AGCCGTAAGC CTGTTAACAA GCTCCTCTAG TTTTT                   1905 

 
           
           
             
               1747 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA (genomic)  
             
              6 

AAAGGCAAAA TCGGTAACCA CATCTCAATT ATTAAACAAT ACTTCATAAT AAAAAGACAA     60 

CTTTTTCATA ATTTGCATAA GTCTTGATGT AAAAAATACA TATTTAGAAA GAACAAGCA     120 

CCTTGCTCAT CACCGCTGTC GCGAGTAGAA AAATCTCGGC TTTCAGAAAA AGAGGCCGC     180 

TCGTTAAACA GACTATAAAT GTGCTGGAAT AAAGCGAACC CCTTGATCTG ATAAAACTG     240 

TAGACATATT GCTTTTGCGC TGCCCGATTG CTGAAAATGC GTAAAAGGTG ATTTTACTC     300 

TTTTCAGGAA AAACTTTGAG AAAACGTCTC GAAAACGGGA TTAAAACGCA AAAACAATA     360 

AAAGCGATTT CGCGAAAATG GTTGTTTTCG GGTTGTTGCT TTAAACTAGT ATGTAGGGT     420 

AGGTTATAGC TATGGCTTCT TCAACTTTTT ATATTCCTTT CGTCAACGAA ATGGGCGAA     480 

GTTCGCTTGA AAAAGCAATC AAGGATCTTA ACGGCAGCGG CTTTAAAAAT GCGCTGATC     540 

TTTCTGATGC TTTCATGAAC AAATCCGGTG TTGTGAAGCA GGTTGCTGAC CTGTTGAAA     600 

CACAGGGTAT TAATTCTGCT GTTTATGATG GCGTTATGCC GAACCCGACT GTTACCGCA     660 

TTCTGGAAGG CCTTAAGATC CTGAAGGATA ACAATTCAGA CTTCGTCATC TCCCTCGGT     720 

GTGGTTCTCC CCATGACTGC GCCAAAGCCA TCGCTCTGGT CGCAACCAAT GGTGGTGAA     780 

TCAAAGACTA CGAAGGTATC GACAAATCTA AGAAACCTGC CCTGCCTTTG ATGTCAATC     840 

ACACGACGGC TGGTACGGCT TCTGAAATGA CGCGTTTCTG CATCATCACT GATGAAGTC     900 

GTCACGTTAA GATGGCCATT GTTGACCGTC ACGTTACCCC GATGGTTTCC GTCAACGAT     960 

CTCTGTTGAT GGTTGGTATG CCAAAAGGCC TGACCGCCGC CACCGGTATG GATGCTCT     1020 

CCCACGCATT TGAAGCTTAT TCTTCAACGG CAGCTACTCC GATCACCGAT GCTTGCGC     1080 

TGAAGGCTGC GTCCATGATC GCTAAGAATC TGAAGACCGC TTGCGACAAC GGTAAGGA     1140 

TGCCAGCTCG TGAAGCTATG GCTTATGCCC AATTCCTCGC TGGTATGGCC TTCAACAA     1200 

CTTCGCTTGG TTATGTCCAT GCTATGGCTC ACCAGTTGGG CGGCTACTAC AACCTGCC     1260 

ATGGTGTCTG CAACGCTGTT CTGCTTCCGC ATGTTCTGGC TTATAACGCC TCTGTCGT     1320 

CTGGTCGTCT GAAAGACGTT GGTGTTGCTA TGGGTCTCGA TATCGCCAAT CTCGGTGA     1380 

AAGAAGGCGC AGAAGCCACC ATTCAGGCTG TTCGCGATCT GGCTGCTTCC ATTGGTAT     1440 

CAGCAAATCT GACCGAGCTG GGTGCTAAGA AAGAAGATGT GCCGCTTCTT GCTGACCA     1500 

CTCTGAAAGA TGCTTGTGCT CTGACCAACC CGCGTCAGGG TGATCAGAAA GAAGTTGA     1560 

AACTCTTCCT GAGCGCTTTC TAATTTCAAA ACAGGAAAAC GGTTTTCCGT CCTGTCTT     1620 

TTTTCAAGCA AACAATGCCT CCGATTTCTA ATCGGAGGCA TTTGTTTTTG TTTATTGC     1680 

AAACAAAAAA TATTGTTACA AATTTTTACA GGCTATTAAG CCTACCGTCA TAAATAAT     1740 

GCCATTT                                                             1747 

 
           
           
             
               7922 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA (genomic)  
             
              7 

GGCGGAGTAA AAAGAGGAGC CCGGCGTCAT CTTTTGTTAC CCGCCAAACA AAACCCAAAA     60 

ACAACCCATA CCCAACCCAA TAAAACACCA AAACAAGACA AATAATCATT GATTGATGG     120 

TGAAATGGGG TAAACTTGAC AAACAAACCC ACTTAAAACC CAAAACATAC CCAAACACA     180 

ACCAAAAAAA CACCATAAGG AGTTTTATAA ATGTTGGTAT TCATTGATGA CGGTTCAAC     240 

AACATCAAAC TACAGTGGCA GGAAAGCGAC GGAACAATTA AACAGCACAT TAGCCCGAA     300 

AGCTTCAAAC GCGAGTGGGC AGTCCCTTTT GGTGATAAAA AGGTCTTTAA CTACACACT     360 

AACGGCGAAC AGTATTCATT TGATCCAACC AGCCCGGATG CTGTAGTCAC AACCAATAT     420 

GCATGGCAAT ACAGCGACGT TAATGTCGTT GCAGTGCATC ACGCCTTACT GACCAGTGG     480 

CTGCCGGTAA GCGAAGTGGA TATTGTTTGC ACACTTCCTC TGACAGAGTA TTACGACAG     540 

AATAACCAAC CCAATACGGA AAATATTGAG CGTAAGAAAG CAAACTTCCG GAAAAAAAT     600 

ACATTAAATG GCGGGGATAC ATTCACAATA AAAGATGTAA AAGTCATGCC TGAATCTAT     660 

CCGGCAGGTT ATGAAGTTCT ACAAGAACTG GATGAGTTAG ATTCTTTATT AATTATAGA     720 

CTCGGGGGCA CCACATTAGA TATTTCTCAG GTAATGGGGA AATTATCGGG GATCAGTAA     780 

ATATACGGAG ACTCATCTCT TGGTGTCTCT CTGGTTACAT CTGCAGTAAA AGATGCCCT     840 

TCTCTTGCGA GAACAAAAGG AAGTAGCTAT CTTGCTGACG ATATAATCAT TCACAGAAA     900 

GATAATAACT ATCTGAAGCA ACGAATTAAT GATGAGAACA AAATATCAAT AGTCACCGA     960 

GCAATGAATG AAGCACTTCG TAAACTTGAG CAACGTGTAT TAAATACGCT CAATGAAT     1020 

TCTGGTTATA CTCATGTTAT GGTTATAGGC GGTGGCGCAG AATTAATATG CGATGCAG     1080 

AAAAAACACA CACAGATTCG TGATGAACGT TTTTTCAAAA CCAATAACTC TCAATATG     1140 

TTAGTTAACG GTATGTATCT CATAGGTAAT TAATGATGGA CAAGCGCAGA ACCATTGC     1200 

TCAAACTAAA TCCAGATGTA AATCAAACAG ATAAAATTGT TTGTGATACA CTGGACAG     1260 

TCCCGCAAGG GGAACGAAGC CGCCTTAACC GGGCCGCACT GACGGCAGGT CTGGCCTT     1320 

ACAGACAAGA TCCCCGGACC CCTTTCCTTT TATGTGAGCT GCTGACGAAA GAAACCAC     1380 

TTTCAGATAT CGTGAATATA TTGAGATCGC TATTTCCAAA AGAGATGGCC GATTTTAA     1440 

CTTCAATAGT CACTCAATCC TCTTCACAAC AAGAGCAAAA AAGTGATGAA GAGACCAA     1500 

AAAATGCGAC GAAGCTAATA AAATTAATTC AATTATTATT GAGTTCCCTT TATCCACT     1560 

CAGGCTGGAT AAAGGGAACT CAATCAAGTT ATTTTCTTAC CAGTCATTAC ATAATCGT     1620 

TTATGAAATA ATCGTTTGCA CTGTCTCTGT TATTCAGGCA ATTTCAATAA AGGCACTT     1680 

TCACGCTCTG TCATTTTCTG AAACTCTTCA TGCTGCATTT CGCAGGTGGC ACTTTTCG     1740 

GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCC     1800 

TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAG     1860 

TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTT     1920 

CTCACCCAGA AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGT     1980 

GTTACATCGA ACTGGATCTC AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGA     2040 

GTTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGT     2100 

ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGA     2160 

ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAG     2220 

CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGG     2280 

CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCG     2340 

GGGAACCGGA GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGC     2400 

CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCG     2460 

AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGC     2520 

TTCCGGCTGG CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGG     2580 

TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGAC     2640 

GGAGTCAGGC AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACT     2700 

TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAGC     2760 

GAATTAATTC CCGGAAGAGA GTCAATTCAG GGTGGTGAAT ATGAAACCAG TAACGTTA     2820 

CGATGTCGCA GAGTATGCCG GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAG     2880 

CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAAT     2940 

CATTCCCAAC CGCGTGGCAC AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTT     3000 

CACCTCCAGT CTGGCCCTGC ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGC     3060 

CGATCAACTG GGTGCCAGCG TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCC     3120 

TAAAGCGGCG GTGCACAATC TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTAT     3180 

GCTGGATGAC CAGGATGCCA TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTA     3240 

TCTTGATGTC TCTGACCAGA CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGT     3300 

GCGACTGGGC GTGGAGCATC TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCG     3360 

CCCATTAAGT TCTGTCTCGG CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACT     3420 

CAATCAAATT CAGCCGATAG CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTT     3480 

ACAAACCATG CAAATGCTGA ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAAC     3540 

TCAGATGGCG CTGGGCGCAA TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCG     3600 

TATCTCGGTA GTGGGATACG ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTCA     3660 

CACCATCAAA CAGGATTTTC GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAA     3720 

CTCTCAGGGC CAGGCGGTGA AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGA     3780 

AACCACCCTG GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTA     3840 

GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAA     3900 

TCGAAAAACT TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCT     3960 

TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAATAAG     4020 

GATCTTCTTG AGATCGTTTT GGTCTGCGCG TAATCTCTTG CTCTGAAAAC GAAAAAAC     4080 

CCTTGCAGGG CGGTTTTTCG TATGATACAG GAGTAAAACC GCCGAAGCCC GGCGTAAG     4140 

GGTACTGATT GATAGATTTC ACCTTACCCA TCCCCAGCCC TGCCAGACCA TACCCGCT     4200 

CAGCCATGAG AGAGCTTCTG TGCGCGGTCG GAGTGGTCCC GACGAGGGTT TACCCGAA     4260 

CGGGGCGTGT CTCCGCGTTA GCGGGCCGTG AGGGCCGCTT ACGAGCGTGT ACTGAGAA     4320 

TCCAGCGAGA AGACTGACAG CGATGAAGAT GTAGTTACAA CATTCATAAT TAAAAGCG     4380 

TCTGTTCCGG CCCTTTGGGC CGGGGCGGGG CCGCTTTTCA GTTATGAGGG AGGGGCTT     4440 

TGGTTTCGGT TCTGCGCTGG ACCGGGGTTT TTCTGGAGGT TGTTTTTGTG TGTTGTAA     4500 

AAAGTGGCTC CGGTCGGGGC CCGCCGCTTG CGGTGGGAGG TGCATATCTG TCTGTCCA     4560 

GGACAGGCAG TGAATAGGTT TTCTTTTTAA ATGAATGTAA TTAAGTAGTT TAAAGGAG     4620 

ATAAACAGGT GTTTAAAAGA TACATTGCAC CCTGTAAGAC TGGCGGCTGG CGCTTTAT     4680 

CATGAACGGT TGTAACCTTA TGGGGAAGTC CCTTGCAGTT AAATGTGGAT AAGCAAAA     4740 

CCCCGTCGCT GAGGCGTATT TTGTATTAAA AACAGGGGGA ATCGGATGCT CCAGAAGG     4800 

GATGATGAGA TTGTTTTTTG CATGCGACGC TGTTTTTTTG TGCACCGGCG GGCTTCAG     4860 

GTGCGGATGC CTCCGGCGCA GGCCGGATTA TTCTGAGGAG ATCACTTTCA GGGAGAAG     4920 

GTGGCCAGCC GGCTGTAATT GCGGTTACGT GACAGAATCA TGCGCTCCTT CACACGAC     4980 

TCCACTTCGC GTTTTACCGC CTCACCATTA GCAGTGAAGC GTCCTTCCGA GATTTCAC     5040 

GTCAGCTGCC GTTTCACTAG GGTGACGATA TCCTGACGTT CTCTGTTCGC ATCACGAC     5100 

GCACGGGCAC GTTTTATTCC ACGGGACTGA AGCTCTGTCT GGTAACTGCG GAAACGCT     5160 

CGCACAAAAC GCCAGGCTTT CGCTATCAGC TCATCCATAC CCAGGGTATC CAGCCCCT     5220 

TTTTTGCGCT GTTTGTTTTC CCATTCAACA CGACTGCGGC GCGCAGCTGC CACTGCAT     5280 

TCAGACACAT CAAGGGCAGC AAACAGAGCC AGTGTGAACG TGATGTCGGT CGGAATGT     5340 

CACCCGATAA GCGGGTCATA TTCCGTCTGG TAGGTAATCA GTCCCAGCTC TGACAGGA     5400 

GTCAGGGCCC GGGTGGCACG GGTGATGGAG AGTTTTCCTG CACCGGACTC TGTCGCCA     5460 

CCGCACTCAA TGGCCAGTGT GGTGATGGAA CACTGGACGC GGTTGGCCAG CGGGTCAT     5520 

TGGAAACACA GCCCCTGCAG CAGCGCATCA ATAGCCCGTC GACGCAGCAC CGGTGGCA     5580 

CGCCGACGCA GACCACGGGA ACGGGCATGC GCCACATGAA TGGCGAAATC AAAACGGG     5640 

GTGAGGCCCA CCGCCTTTTC CATCGGTTTT TCGCGGAACT TCGGCGTTCC GGCACCTT     5700 

CGGGGAGTGA ACACCGGATT CGGGTTCTTT ACCTGGCGGT AATACGTTTG GTGAAGAT     5760 

GTCACACCAT CCTGCACTTA CAATGCGCAG AAGGAGCGAG CACAGAAAGA AGTCTTGA     5820 

TTTTCCGGGC ATATAACTAT ACTCCCCGCA TAGCTGAATT GTTGGCTATA CGGTTTAA     5880 

GGGCCCCGGT AATCTTTTCG TACTCGCCAA AGTTGAAGAA GATTATCGGG GTTTTTGC     5940 

TTCTGGCTCC TGTAAATCCA CATCAGAACC AGTTCCTTGC CACCTTACGG CGTGGCAG     6000 

ACAAAATTCC TTAAACGATC AGTAATCTAG CTAGCTACGC CACAAAGTAA AGTCTTTT     6060 

TTTAGTATAT CCAGTCTCTG CAGTTCATCT TTGATGATTT TCTCAACGAA CTGAGCCT     6120 

GTTATCCCCT CTCTCTCGCA GTACTCAACC ATGAGATCGA TCTTTCAGAG GATTTTTG     6180 

AAAAACTTTT ATCTCTTTGT GTGTAAGACG TTTTCTTGCA ACAGCGGCCA TTTGTTTC     6240 

AGAGTCAGTC ATAGGCTTAC CTCTGCGCAC AAACCGCTTT TGACTCAATG AGGAAGTC     6300 

TGCATTTTCT GTCTGCGACA TCTCGCCTCC TCAATACTCA AACAGGGATC GTTTCGCA     6360 

GGATACTACA GTTTTTTGAA ATCAGCAACT TGAGAATTGT GACGAAGATC TTTAGCTG     6420 

TTGGTTTGCC CAAAGCGCAT TGCATAATCT TTCAGGGTTA TGCGTTGTTC CATACAAC     6480 

CCTTAGTACA TGCAACCATT ATCACCGCCA GAGGTAAAAT AGTCAACACG CACGGTGT     6540 

GATATTTATC CCTTGCGGTG ATAGATTTAA CGTATGAGCA CAAAAAAGAA ACCATTAA     6600 

CAAGAGCAGC TTGAGGACGC ACGTCGCCTT AAAGCAATTT ATGAAAAAAA GAAAAATG     6660 

CTTGGCTTAT CCCAGGAATC TGTCGCAGAC AAGATGGGGA TGGGGCAGTC AGGCGTTG     6720 

GCTTTATTTA ATGGCATCAA TGCATTAAAT GCTTATAACG CCGCATTGCT TACAAAAA     6780 

CTCAAAGTTA GCGTTGAAGA ATTTAGCCCT TCAATCGCCA GAGAAATCTA CGAGATGT     6840 

GAAGCGGTTA GTATGCAGCC GTCACTTAGA AGTGAGTATG AGTACCCTGT TTTTTCTC     6900 

GTTCAGGCAG GGATGTTCTC ACCTAAGCTT AGAACCTTTA CCAAAGGTGA TGCGGAGA     6960 

TGGGTAAGCA CAACCAAAAA AGCCAGTGAT TCTGCATTCT GGCTTGAGGT TGAAGGTA     7020 

TCCATGACCG CACCAACAGG CTCCAAGCCA AGCTTTCCTG ACGGAATGTT AATTCTCG     7080 

GACCCTGAGC AGGCTGTTGA GCCAGGTGAT TTCTGCATAG CCAGACTTGG GGGTGATG     7140 

TTTACCTTCA AGAAACTGAT CAGGGATAGC GGTCAGGTGT TTTTACAACC ACTAAACC     7200 

CAGTACCCAA TGATCCCATG CAATGAGAGT TGTTCCGTTG TGGGGAAAGT TATCGCTA     7260 

CAGTGGCCTG AAGAGACGTT TGGCTGATCG GCAAGGTGTT CTGGTCGGCG CATAGCTG     7320 

AACAATTGAG CAAGAATCTT CATCGAATTA GGGGAATTTT CACTCCCCTC AGAACATA     7380 

ATAGTAAATG GATTGAATTA TGAAGAATGG TTTTTATGCG ACTTACCGCA GCAAAAAT     7440 

AGGGAAAGAT AAGCCTAGTG CTACTTGAGG GTATACCGCA AGAATATACG CAAGCGTC     7500 

GATAGCTGCC AAAGCCGCAA GGAATTTACC AACCTTCTTA AACATAAAGT GTCTCCTT     7560 

AAACGCAGAA AGGCCCACCC GAAGGTGAGC CAGTGTGATT ACATTTTCTC TTGAGGGT     7620 

TCCTCGGTGC CACGGAACAT TACGAACGAT GGGTGCCGCA AAGAGCCATC AGGTGTTT     7680 

TCCATGTAGC TAATTTGACA CGCCCAGCCA TCGTAAGGGT TAATAGTAAT TCGAGCTC     7740 

TACCCGGGGA TCCTCTAGAG CTCGAGGCCT CATATGGATC CACGTGAATT CGTAATCA     7800 

TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAG     7860 

GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTACTAG     7920 

TC                                                                  7922