Abstract:
The invention relates to the genetic modification of Cyanobacteria for the production of ethanol, and more particularly, to the genetic modification of Cyanobacteria by incorporating the genetic information encoding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh).

Description:
This application is a continuation-in-part of Ser. No. 08/801,331, filed Feb. 19, 1997, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     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 
     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. 
     A number of alternatives for the production of ethanol from living organisms have been investigated using microorganisms. 
     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. 
     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. No. 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. 
     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. 
     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) 
     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. 
     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 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     In another aspect of the present invention there is provided a genetically modified Cyanobacteria capable of producing ethanol produced according to the following steps: 
     a. selecting an appropriate promoter; 
     b. ligating said promotor to pdc and adh encoding DNA sequence; 
     c. cloning said ligated promoter and said pdc and adh encoding DNA into an appropriate construct; 
     d. transforming the construct into the Cyanobacteria 
     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. 
     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. 
     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.    
     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. 
     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. 
     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. 
     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.    
     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. 
     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: 
     a. harvesting cells of the Cyanobacteria; 
     b. adding the construct to the harvested Cyanobacteria cells; 
     c. incubating the construct and the Cyanobacteria cells such that the construct is transformed into the Cyanobacteria cells; 
     d. plating the incubated constructs and Cyanobacteria cells on plates containing ampicillin and incubating under appropriate growth conditions; 
     e. selecting the transformed ampicillin resistant Cyanobacteria cells. 
     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 
     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. 
     FIG. 1 is an illustration of the map of the plasmid pLOI295 containing the DNA fragments encoding for pdc and adh. 
     FIG. 2 is an illustration of the map of the plasmid construct pCB4-Rpa. 
     FIG. 3 is an illustration of the map of the plasmid construct pCB4-LRpa. 
     FIG. 4 is an illustration of the map of the plasmid construct pCB4-LR(TF)pa. 
     FIG. 5 is an illustration of the map of the plasmid construct pCB4-CPpa. 
     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. 
     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. 
     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. 
     FIG. 9 is a description of the pdc gene identified as SEQ ID. No. 5. 
     FIG. 10 is a description of the adh gene identified as SEQ. ID. No. 6. 
     FIG. 11 is a description of the CI-PL promoter identified as SEQ. ID. No. 7. 
    
    
     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 
     Cyanobacteria are photosynthetic bacteria which require light, inorganic elements, water and a carbon source, generally CO 2 , to metabolise and grow. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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.    
     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. 
     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. 
     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. 
     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. 
     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. O. 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 
     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./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. 
     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 Vidulans” , 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 
     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 
     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. 
     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 
     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-CI857 (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 ligated 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 
     Each of the four constructs of Examples 1, 2, 3 and 4 were incorporated into the Synechococcus PCC 7942. 
     The constructs of Examples 1, 2, 3 and 4 were incorporated into the Synechococcus is 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. 
     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 
               
               
                   
               
               
                   
                 PDC 
                   
                   
                   
               
               
                   
                 Activity 
                   
                   
                   
               
               
                   
                 nmol · 
                 ADH Activity 
                 Ethanol 
                 Ethanol 
               
               
                   
                 min. −1  · 
                 nmol · 
                 Conc. 
                 Conc. 
               
               
                   
                 mg −1   
                 min. −1  · mg −1   
                 in medium 
                 in μmoL · mg −1   
               
               
                 Constructs 
                 SP 1   
                 SP 
                 (μM) 3   
                 Chlorophyll 
               
               
                   
               
             
             
               
                 pCB4 4   
                 ND 2   
                 ND 
                 ND 
                 ND 
               
               
                 pCB4- 
                 130 
                 168 
                 1370 
                 274 
               
               
                 Rpa 
               
               
                 pCB4- 
                 136 
                 168 
                 1540 
                 308 
               
               
                 LRpa 
               
               
                 pCB4- 
                 234 
                 168 
                 1710 
                 342 
               
               
                 LR(TF)pa 
               
               
                   
               
               
                   1 SP, soluble protein.  
               
               
                   2 ND, not detectable.  
               
               
                   3 Represents ethanol concentration in medium following 21 days growth in batch culture at a final cell density of OD 730 1.5. This OD represents approximately 5 × 10 8  cells.ml −1 . Values in table are an underestimation of ethanol concentration as some ethanol is lost from the unsealed culture vessels during aeration. Concentrations approximating 5 mM can be achieved following 28 days of growth.  
               
               
                   4 Synechococcus PCC 7942 cells transformed with the shuttle vector pCB4 alone.  
               
             
          
         
       
     
     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. 
     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 
               
               
                   
                 (μmol · 
               
               
                 Temperature 
                 mg −1  chlorophyll) 
                 (μmol · mg −1  chlorophyll per hr) 
               
               
                   
               
               
                 30 
                 16(0.9) 
                 0.33 
               
               
                 42 
                 82(8.9) 
                 1.70 
               
               
                   
               
             
          
         
       
     
     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. 
     
       
         
           
             7 
           
           
             
               29 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
              1
GCTGAATTCA TGTCGTCTCT CCCTAGAGA                                       29 
           
           
             
               29 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
              2
GCTGAATTCA TGTCGTCTCT CCCTAGAGA                                       29 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
              3
GGACTCGAGG ATCCCCAAAT GGCAA                                           25 
           
           
             
               29 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
              4
GCATGAATTC TTATACTGTC GGTACCTAT                                       29 
           
           
             
               1905 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
              5
TATCGCTCAT GATCGCGACA TGTTCTGATA TTTTCCTCTA AAAAAGATAA AAAGTCTTTT     60
CGCTTCGGCA GAAGAGGTTC ATCATGAACA AAAATTCGGC ATTTTTAAAA ATGCCTATAG    120
CTAAATCCGG AACGACACTT TAGAGGTTTC TGGGTCATCC TGATTCAGAC ATAGTGTTTT    180
GAATATATGG AGTAAGCAAT GAGTTATACT GTCGGTACCT ATTTAGCGGC GCTTGTCCAG    240
ATTGGTCTCA AGCATCACTT CGCAGTCGCG GGCGACTACA ACCTCGTCCT TCTTGACAAC    300
CTGCTTTTGA ACAAAAACAT GGAGCAGGTT TATTGCTGTA ACGAACTGAA CTGCGGTTTC    360
AGTGCAGAAG GTTATGCTCG TGCCAAAGCG GACGCAGCAG CCGTCGTTAC CTACAGCGTC    420
GGTGCGCTTT CCGCATTTGA TGCTATCGGT GGCGCCTATG CAGAAAACCT TCCGGTTATC    480
CTGATCTCCG GTGCTCCGAA CAACAATGAT CACGCTGCTG GTCACGTGTT GCATCACGCT    540
CTTGGCAAAA CCGACTATCA CTATCAGTTG GAAATGGCCA AGAACATCAC GGCCGCAGCT    600
GAAGCGATTT ACACCCCAGA AGAAGCTCCG GCTAAAATCG ATCACGTGAT TAAAACTGCT    660
CTTCGTGAGA AGAAGCCGGT TTATCTCGAA ATCGCTTGCA ACATTGCTTC CATGCCCTGC    720
GCCGCTCCTG GACCGGCAAG CGCATTGTTC AATGACGAAG CCAGCGACGA AGCTTCTTTG    780
AATGCAGCGG TTGAAGAAAC CCTGAAATTC ATCGCCAACC GCGACAAAGT TGCCGTCCTC    840
GTCGGCAGCA AGCTGCGCGC AGCTGGTGCT GAAGAAGCTG CTGTCAAATT TGCTGATGCT    900
CTCGGTGGCG CAGTTGCTAC CATGGCTGCT GCAAAAAGCT TCTTCCAGAA GAAAACCGCA    960
TTACATCGGT ACCTCATGGG TGAAGTCAGC TATCCGGGCG TTGAAAAGAC GATGAAAGAA   1020
GCCGATGCGG TTATCGCTCT GGCTCCTGTC TTCAACGACT ACTCCACCAC TGGTTGGACG   1080
GATATTCCTG ATCCTAAGAA ACTGGTTCTC GCTGAACCGC GTTCTGTCGT CGTTAACGGC   1140
GTTCGCTTCC CCAGCGTTCA TCTGAAAGAC TATCTGACCC GTTTGGCTCA GAAAGTTTCC   1200
AAGAAAACCG GTGCTTTGGA CTTCTTCAAA TCCCTCAATG CAGGTGAACT GAAGAAAGCC   1260
GCTCCGGCTG ATCCGAGTGC TCCGTTGGTC AACGCAGAAA TCGCCCGTCA GGTCGAAGCT   1320
CTTCTGACCC CGAACACGAC GGTTATTGCT GAAACCGGTG ACTCTTGGTT CAATGCTCAG   1380
CGCATGAAGC TCCCGAACGG TGCTCGCGTT GAATATGAAA TGCAGTGGGG TCACATCGGT   1440
TGGTCCGTTC CTGCCGCCTT CGGTTATGCC GTCGGTGCTC CGGAACGTCG CAACATCCTC   1500
ATGGTTGGTG ATGGTTCCTT CCAGCTGACG GCTCAGGAAG TCGCTCAGAT GGTTCGCCTG   1560
AAACTGCCGG TTATCATCTT CTTGATCAAT AACTATGGTT ACACCATCGA AGTTATGATC   1620
CATGATGGTC CGTACAACAA CATCAAGAAC TGGGATTATG CCGGTCTGAT GGAAGTGTTC   1680
AACGGTAACG GTGGTTATGA CAGCGGCGCT GGTAAAGGCC TGAAGGCTAA AACCGGTGGC   1740
GAACTGGCAG AAGCTATCAA GGTTGCTCTG GCAAACACCG ACGGCCCAAC CCTGATCGAA   1800
TGCTTCATCG GTCGTGAAGA CTGCACTGAA GAATTGGTCA AATGGGGTAA GCGCGTTGCT   1860
GCCCGCCAAC AGCCGTAAGC CTGTTAACAA GCTCCTCTAG TTTTT                   1905 
           
           
             
               1747 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
              6
AAAGGCAAAA TCGGTAACCA CATCTCAATT ATTAAACAAT ACTTCATAAT AAAAAGACAA     60
CTTTTTCATA ATTTGCATAA GTCTTGATGT AAAAAATACA TATTTAGAAA GAACAAGCAG    120
CCTTGCTCAT CACCGCTGTC GCGAGTAGAA AAATCTCGGC TTTCAGAAAA AGAGGCCGCT    180
TCGTTAAACA GACTATAAAT GTGCTGGAAT AAAGCGAACC CCTTGATCTG ATAAAACTGA    240
TAGACATATT GCTTTTGCGC TGCCCGATTG CTGAAAATGC GTAAAAGGTG ATTTTACTCG    300
TTTTCAGGAA AAACTTTGAG AAAACGTCTC GAAAACGGGA TTAAAACGCA AAAACAATAG    360
AAAGCGATTT CGCGAAAATG GTTGTTTTCG GGTTGTTGCT TTAAACTAGT ATGTAGGGTG    420
AGGTTATAGC TATGGCTTCT TCAACTTTTT ATATTCCTTT CGTCAACGAA ATGGGCGAAG    480
GTTCGCTTGA AAAAGCAATC AAGGATCTTA ACGGCAGCGG CTTTAAAAAT GCGCTGATCG    540
TTTCTGATGC TTTCATGAAC AAATCCGGTG TTGTGAAGCA GGTTGCTGAC CTGTTGAAAG    600
CACAGGGTAT TAATTCTGCT GTTTATGATG GCGTTATGCC GAACCCGACT GTTACCGCAG    660
TTCTGGAAGG CCTTAAGATC CTGAAGGATA ACAATTCAGA CTTCGTCATC TCCCTCGGTG    720
GTGGTTCTCC CCATGACTGC GCCAAAGCCA TCGCTCTGGT CGCAACCAAT GGTGGTGAAG    780
TCAAAGACTA CGAAGGTATC GACAAATCTA AGAAACCTGC CCTGCCTTTG ATGTCAATCA    840
ACACGACGGC TGGTACGGCT TCTGAAATGA CGCGTTTCTG CATCATCACT GATGAAGTCC    900
GTCACGTTAA GATGGCCATT GTTGACCGTC ACGTTACCCC GATGGTTTCC GTCAACGATC    960
CTCTGTTGAT GGTTGGTATG CCAAAAGGCC TGACCGCCGC CACCGGTATG GATGCTCTGA   1020
CCCACGCATT TGAAGCTTAT TCTTCAACGG CAGCTACTCC GATCACCGAT GCTTGCGCCT   1080
TGAAGGCTGC GTCCATGATC GCTAAGAATC TGAAGACCGC TTGCGACAAC GGTAAGGATA   1140
TGCCAGCTCG TGAAGCTATG GCTTATGCCC AATTCCTCGC TGGTATGGCC TTCAACAACG   1200
CTTCGCTTGG TTATGTCCAT GCTATGGCTC ACCAGTTGGG CGGCTACTAC AACCTGCCGC   1260
ATGGTGTCTG CAACGCTGTT CTGCTTCCGC ATGTTCTGGC TTATAACGCC TCTGTCGTTG   1320
CTGGTCGTCT GAAAGACGTT GGTGTTGCTA TGGGTCTCGA TATCGCCAAT CTCGGTGATA   1380
AAGAAGGCGC AGAAGCCACC ATTCAGGCTG TTCGCGATCT GGCTGCTTCC ATTGGTATTC   1440
CAGCAAATCT GACCGAGCTG GGTGCTAAGA AAGAAGATGT GCCGCTTCTT GCTGACCACG   1500
CTCTGAAAGA TGCTTGTGCT CTGACCAACC CGCGTCAGGG TGATCAGAAA GAAGTTGAAG   1560
AACTCTTCCT GAGCGCTTTC TAATTTCAAA ACAGGAAAAC GGTTTTCCGT CCTGTCTTGA   1620
TTTTCAAGCA AACAATGCCT CCGATTTCTA ATCGGAGGCA TTTGTTTTTG TTTATTGCAA   1680
AAACAAAAAA TATTGTTACA AATTTTTACA GGCTATTAAG CCTACCGTCA TAAATAATTT   1740
GCCATTT                                                             1747 
           
           
             
               7922 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
              7
GGCGGAGTAA AAAGAGGAGC CCGGCGTCAT CTTTTGTTAC CCGCCAAACA AAACCCAAAA     60
ACAACCCATA CCCAACCCAA TAAAACACCA AAACAAGACA AATAATCATT GATTGATGGT    120
TGAAATGGGG TAAACTTGAC AAACAAACCC ACTTAAAACC CAAAACATAC CCAAACACAC    180
ACCAAAAAAA CACCATAAGG AGTTTTATAA ATGTTGGTAT TCATTGATGA CGGTTCAACA    240
AACATCAAAC TACAGTGGCA GGAAAGCGAC GGAACAATTA AACAGCACAT TAGCCCGAAC    300
AGCTTCAAAC GCGAGTGGGC AGTCCCTTTT GGTGATAAAA AGGTCTTTAA CTACACACTG    360
AACGGCGAAC AGTATTCATT TGATCCAACC AGCCCGGATG CTGTAGTCAC AACCAATATC    420
GCATGGCAAT ACAGCGACGT TAATGTCGTT GCAGTGCATC ACGCCTTACT GACCAGTGGT    480
CTGCCGGTAA GCGAAGTGGA TATTGTTTGC ACACTTCCTC TGACAGAGTA TTACGACAGA    540
AATAACCAAC CCAATACGGA AAATATTGAG CGTAAGAAAG CAAACTTCCG GAAAAAAATT    600
ACATTAAATG GCGGGGATAC ATTCACAATA AAAGATGTAA AAGTCATGCC TGAATCTATA    660
CCGGCAGGTT ATGAAGTTCT ACAAGAACTG GATGAGTTAG ATTCTTTATT AATTATAGAT    720
CTCGGGGGCA CCACATTAGA TATTTCTCAG GTAATGGGGA AATTATCGGG GATCAGTAAA    780
ATATACGGAG ACTCATCTCT TGGTGTCTCT CTGGTTACAT CTGCAGTAAA AGATGCCCTT    840
TCTCTTGCGA GAACAAAAGG AAGTAGCTAT CTTGCTGACG ATATAATCAT TCACAGAAAA    900
GATAATAACT ATCTGAAGCA ACGAATTAAT GATGAGAACA AAATATCAAT AGTCACCGAA    960
GCAATGAATG AAGCACTTCG TAAACTTGAG CAACGTGTAT TAAATACGCT CAATGAATTT   1020
TCTGGTTATA CTCATGTTAT GGTTATAGGC GGTGGCGCAG AATTAATATG CGATGCAGTA   1080
AAAAAACACA CACAGATTCG TGATGAACGT TTTTTCAAAA CCAATAACTC TCAATATGAT   1140
TTAGTTAACG GTATGTATCT CATAGGTAAT TAATGATGGA CAAGCGCAGA ACCATTGCCT   1200
TCAAACTAAA TCCAGATGTA AATCAAACAG ATAAAATTGT TTGTGATACA CTGGACAGTA   1260
TCCCGCAAGG GGAACGAAGC CGCCTTAACC GGGCCGCACT GACGGCAGGT CTGGCCTTAT   1320
ACAGACAAGA TCCCCGGACC CCTTTCCTTT TATGTGAGCT GCTGACGAAA GAAACCACAT   1380
TTTCAGATAT CGTGAATATA TTGAGATCGC TATTTCCAAA AGAGATGGCC GATTTTAATT   1440
CTTCAATAGT CACTCAATCC TCTTCACAAC AAGAGCAAAA AAGTGATGAA GAGACCAAAA   1500
AAAATGCGAC GAAGCTAATA AAATTAATTC AATTATTATT GAGTTCCCTT TATCCACTAT   1560
CAGGCTGGAT AAAGGGAACT CAATCAAGTT ATTTTCTTAC CAGTCATTAC ATAATCGTTA   1620
TTATGAAATA ATCGTTTGCA CTGTCTCTGT TATTCAGGCA ATTTCAATAA AGGCACTTGC   1680
TCACGCTCTG TCATTTTCTG AAACTCTTCA TGCTGCATTT CGCAGGTGGC ACTTTTCGGG   1740
GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC   1800
TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA   1860
TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG   1920
CTCACCCAGA AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG   1980
GTTACATCGA ACTGGATCTC AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC   2040
GTTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGTTG   2100
ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT   2160
ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG   2220
CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC   2280
CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT   2340
GGGAACCGGA GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGCAG   2400
CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC   2460
AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC   2520
TTCCGGCTGG CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA   2580
TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG   2640
GGAGTCAGGC AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA   2700
TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAGCTT   2760
GAATTAATTC CCGGAAGAGA GTCAATTCAG GGTGGTGAAT ATGAAACCAG TAACGTTATA   2820
CGATGTCGCA GAGTATGCCG GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC   2880
CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA   2940
CATTCCCAAC CGCGTGGCAC AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC   3000
CACCTCCAGT CTGGCCCTGC ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC   3060
CGATCAACTG GGTGCCAGCG TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG   3120
TAAAGCGGCG GTGCACAATC TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC   3180
GCTGGATGAC CAGGATGCCA TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT   3240
TCTTGATGTC TCTGACCAGA CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC   3300
GCGACTGGGC GTGGAGCATC TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG   3360
CCCATTAAGT TCTGTCTCGG CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG   3420
CAATCAAATT CAGCCGATAG CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA   3480
ACAAACCATG CAAATGCTGA ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA   3540
TCAGATGGCG CTGGGCGCAA TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA   3600
TATCTCGGTA GTGGGATACG ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTCAAC   3660
CACCATCAAA CAGGATTTTC GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT   3720
CTCTCAGGGC CAGGCGGTGA AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA   3780
AACCACCCTG GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT   3840
GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG   3900
TCGAAAAACT TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA   3960
TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAATAAGAT   4020
GATCTTCTTG AGATCGTTTT GGTCTGCGCG TAATCTCTTG CTCTGAAAAC GAAAAAACCG   4080
CCTTGCAGGG CGGTTTTTCG TATGATACAG GAGTAAAACC GCCGAAGCCC GGCGTAAGCC   4140
GGTACTGATT GATAGATTTC ACCTTACCCA TCCCCAGCCC TGCCAGACCA TACCCGCTTT   4200
CAGCCATGAG AGAGCTTCTG TGCGCGGTCG GAGTGGTCCC GACGAGGGTT TACCCGAAGT   4260
CGGGGCGTGT CTCCGCGTTA GCGGGCCGTG AGGGCCGCTT ACGAGCGTGT ACTGAGAACT   4320
TCCAGCGAGA AGACTGACAG CGATGAAGAT GTAGTTACAA CATTCATAAT TAAAAGCGAC   4380
TCTGTTCCGG CCCTTTGGGC CGGGGCGGGG CCGCTTTTCA GTTATGAGGG AGGGGCTTTG   4440
TGGTTTCGGT TCTGCGCTGG ACCGGGGTTT TTCTGGAGGT TGTTTTTGTG TGTTGTAACT   4500
AAAGTGGCTC CGGTCGGGGC CCGCCGCTTG CGGTGGGAGG TGCATATCTG TCTGTCCACA   4560
GGACAGGCAG TGAATAGGTT TTCTTTTTAA ATGAATGTAA TTAAGTAGTT TAAAGGAGAT   4620
ATAAACAGGT GTTTAAAAGA TACATTGCAC CCTGTAAGAC TGGCGGCTGG CGCTTTATGA   4680
CATGAACGGT TGTAACCTTA TGGGGAAGTC CCTTGCAGTT AAATGTGGAT AAGCAAAATT   4740
CCCCGTCGCT GAGGCGTATT TTGTATTAAA AACAGGGGGA ATCGGATGCT CCAGAAGGTG   4800
GATGATGAGA TTGTTTTTTG CATGCGACGC TGTTTTTTTG TGCACCGGCG GGCTTCAGGC   4860
GTGCGGATGC CTCCGGCGCA GGCCGGATTA TTCTGAGGAG ATCACTTTCA GGGAGAAGCT   4920
GTGGCCAGCC GGCTGTAATT GCGGTTACGT GACAGAATCA TGCGCTCCTT CACACGACGC   4980
TCCACTTCGC GTTTTACCGC CTCACCATTA GCAGTGAAGC GTCCTTCCGA GATTTCACGC   5040
GTCAGCTGCC GTTTCACTAG GGTGACGATA TCCTGACGTT CTCTGTTCGC ATCACGACGC   5100
GCACGGGCAC GTTTTATTCC ACGGGACTGA AGCTCTGTCT GGTAACTGCG GAAACGCTCA   5160
CGCACAAAAC GCCAGGCTTT CGCTATCAGC TCATCCATAC CCAGGGTATC CAGCCCCTGC   5220
TTTTTGCGCT GTTTGTTTTC CCATTCAACA CGACTGCGGC GCGCAGCTGC CACTGCATCC   5280
TCAGACACAT CAAGGGCAGC AAACAGAGCC AGTGTGAACG TGATGTCGGT CGGAATGTAG   5340
CACCCGATAA GCGGGTCATA TTCCGTCTGG TAGGTAATCA GTCCCAGCTC TGACAGGAAC   5400
GTCAGGGCCC GGGTGGCACG GGTGATGGAG AGTTTTCCTG CACCGGACTC TGTCGCCAGT   5460
CCGCACTCAA TGGCCAGTGT GGTGATGGAA CACTGGACGC GGTTGGCCAG CGGGTCATAG   5520
TGGAAACACA GCCCCTGCAG CAGCGCATCA ATAGCCCGTC GACGCAGCAC CGGTGGCATG   5580
CGCCGACGCA GACCACGGGA ACGGGCATGC GCCACATGAA TGGCGAAATC AAAACGGGAG   5640
GTGAGGCCCA CCGCCTTTTC CATCGGTTTT TCGCGGAACT TCGGCGTTCC GGCACCTTCA   5700
CGGGGAGTGA ACACCGGATT CGGGTTCTTT ACCTGGCGGT AATACGTTTG GTGAAGATCA   5760
GTCACACCAT CCTGCACTTA CAATGCGCAG AAGGAGCGAG CACAGAAAGA AGTCTTGAAC   5820
TTTTCCGGGC ATATAACTAT ACTCCCCGCA TAGCTGAATT GTTGGCTATA CGGTTTAAGT   5880
GGGCCCCGGT AATCTTTTCG TACTCGCCAA AGTTGAAGAA GATTATCGGG GTTTTTGCTT   5940
TTCTGGCTCC TGTAAATCCA CATCAGAACC AGTTCCTTGC CACCTTACGG CGTGGCAGCC   6000
ACAAAATTCC TTAAACGATC AGTAATCTAG CTAGCTACGC CACAAAGTAA AGTCTTTTAC   6060
TTTAGTATAT CCAGTCTCTG CAGTTCATCT TTGATGATTT TCTCAACGAA CTGAGCCTGT   6120
GTTATCCCCT CTCTCTCGCA GTACTCAACC ATGAGATCGA TCTTTCAGAG GATTTTTGAC   6180
AAAAACTTTT ATCTCTTTGT GTGTAAGACG TTTTCTTGCA ACAGCGGCCA TTTGTTTCTC   6240
AGAGTCAGTC ATAGGCTTAC CTCTGCGCAC AAACCGCTTT TGACTCAATG AGGAAGTCAC   6300
TGCATTTTCT GTCTGCGACA TCTCGCCTCC TCAATACTCA AACAGGGATC GTTTCGCAGA   6360
GGATACTACA GTTTTTTGAA ATCAGCAACT TGAGAATTGT GACGAAGATC TTTAGCTGTC   6420
TTGGTTTGCC CAAAGCGCAT TGCATAATCT TTCAGGGTTA TGCGTTGTTC CATACAACCT   6480
CCTTAGTACA TGCAACCATT ATCACCGCCA GAGGTAAAAT AGTCAACACG CACGGTGTTA   6540
GATATTTATC CCTTGCGGTG ATAGATTTAA CGTATGAGCA CAAAAAAGAA ACCATTAACA   6600
CAAGAGCAGC TTGAGGACGC ACGTCGCCTT AAAGCAATTT ATGAAAAAAA GAAAAATGAA   6660
CTTGGCTTAT CCCAGGAATC TGTCGCAGAC AAGATGGGGA TGGGGCAGTC AGGCGTTGGT   6720
GCTTTATTTA ATGGCATCAA TGCATTAAAT GCTTATAACG CCGCATTGCT TACAAAAATT   6780
CTCAAAGTTA GCGTTGAAGA ATTTAGCCCT TCAATCGCCA GAGAAATCTA CGAGATGTAT   6840
GAAGCGGTTA GTATGCAGCC GTCACTTAGA AGTGAGTATG AGTACCCTGT TTTTTCTCAT   6900
GTTCAGGCAG GGATGTTCTC ACCTAAGCTT AGAACCTTTA CCAAAGGTGA TGCGGAGAGA   6960
TGGGTAAGCA CAACCAAAAA AGCCAGTGAT TCTGCATTCT GGCTTGAGGT TGAAGGTAAT   7020
TCCATGACCG CACCAACAGG CTCCAAGCCA AGCTTTCCTG ACGGAATGTT AATTCTCGTT   7080
GACCCTGAGC AGGCTGTTGA GCCAGGTGAT TTCTGCATAG CCAGACTTGG GGGTGATGAG   7140
TTTACCTTCA AGAAACTGAT CAGGGATAGC GGTCAGGTGT TTTTACAACC ACTAAACCCA   7200
CAGTACCCAA TGATCCCATG CAATGAGAGT TGTTCCGTTG TGGGGAAAGT TATCGCTAGT   7260
CAGTGGCCTG AAGAGACGTT TGGCTGATCG GCAAGGTGTT CTGGTCGGCG CATAGCTGAT   7320
AACAATTGAG CAAGAATCTT CATCGAATTA GGGGAATTTT CACTCCCCTC AGAACATAAC   7380
ATAGTAAATG GATTGAATTA TGAAGAATGG TTTTTATGCG ACTTACCGCA GCAAAAATAA   7440
AGGGAAAGAT AAGCCTAGTG CTACTTGAGG GTATACCGCA AGAATATACG CAAGCGTCAG   7500
GATAGCTGCC AAAGCCGCAA GGAATTTACC AACCTTCTTA AACATAAAGT GTCTCCTTAT   7560
AAACGCAGAA AGGCCCACCC GAAGGTGAGC CAGTGTGATT ACATTTTCTC TTGAGGGTTG   7620
TCCTCGGTGC CACGGAACAT TACGAACGAT GGGTGCCGCA AAGAGCCATC AGGTGTTTCC   7680
TCCATGTAGC TAATTTGACA CGCCCAGCCA TCGTAAGGGT TAATAGTAAT TCGAGCTCGG   7740
TACCCGGGGA TCCTCTAGAG CTCGAGGCCT CATATGGATC CACGTGAATT CGTAATCATG   7800
TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC   7860
GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTACTAGAG   7920
TC                                                                  7922