AU779434B2 - Genetically modified cyanobacteria for the production of ethanol - Google Patents

Description

Our Ref:7659630 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Enol Energy Inc.

Suite 202 80 Tiventon Court Markham Ontario L3R 0G4 Canada Address for Service: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Invention Title: Genetically modified cyanobacteria for the production of ethanol The following statement is a full description of this invention, including the best method of performing it known to me:- P:\WPDOCS\HjwSpcs\7659630div.doc-15/11/01 -1- GENETICALLY MODIFIED CYANOBACTERIA FOR THE PRODUCTION OF ETHANOL.

This application is a divisional of Australian patent application No. 62014/98 Field of 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 Syiiechococcus 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 Saccharomvees and bacteria Zymomonas, which is a facultative anaerobic.

20 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. Patent 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 i 25 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. Patent 4,350,765 to Chibata et al. describes a method of producing S ethanol in a high concentration by using an immobilized Saccharomvees or Zymomonas and a nutrient culture broth containing a fermentative sugar. U.S. Patent 4,413,058 to Arcuri et al. describes a new strain of Zvmomonas 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 15 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.

*oool.

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 2Q 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 Cvanobacteria 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 acetaldehvde and acetaldehyde to ethanol, respectively. In particular, Synechococcus is a preferred Cvanobacteria of the present invention.

In a preferred embodiment, transformed Synechococcus produce ethanol in recoverable quantities of at least 1.7 4mol 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.

5 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 S: 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 *i 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.

or a gene sequence that encodes the pdc enzyme and is capable of expression in Cyanobacteria.

4 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 ofpCB4-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 'i 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. M 15393 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 Zvmomonas mobilis wherein said promoter is activated only once a particular 5 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 pLO 1295 plasmid, or other suitable source ofpdc and adh enzymes, according to the following steps: 9 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 Cvanobacteria 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 Zvmomonas mobiles pLOI295 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.

Figure 1 is an illustration of the map of the plasmid pLOI295 containing the DNA fragments encoding forpdc and adh.

Figure 2 is an illustration of the map of the plasmid construct pCB4-Rpa.

Figure 3 is an illustration of the map of the plasmid construct pCB4-LRpa.

Figure 4 is an illustration of the map of the plasmid construct pCB4-LR(TF)pa.

Figure 4 is an illustration of the map of the plasmid construct pCB4-LR(TF)pa.

20 Figure 5 is an illustration of the map of the plasmid construct pCB4-CPpa.

S*Figure 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.

Figure 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.

25-11-04: 9:56AM:DAVIES COLLISON CAVE :1 2200#1/2 :612 92621080 10/ 26 7 Figure 8 is an illustration of the induction time of Synechococcus FCC 7942 versus ethanol production in Synechococcus FCC 7942 in cells transformed with pCB4-Rpa.

Figure 9 is a description of the pdc gene idniidas SEQ ID. No. Figure 10 Is a description of the adh gene identified as SEQ. ID. No. 6.

Figure I1 IIs 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 plasmaid constructs in the figures as follows: AMAPR (ainpicillin resistant); PDC (pynuvate dcalrboxylase); ADH- (alcohol dehydrogenase); ATG (start codon); L (IacZ promoter); R (rbc..LS promoter); R' (EcoRi); B (HamHl); S (Sall); X (Xbal);. )UP (XbaV/^uIl fusion); XhIB (XhoI/BamHIl fusion); T (transcription terminator) and CI-PL (temperature inducible gene and bacterial phage left-ward promoter).

Detailed Description Cyanobacteria arc photosynthetic bacteria which require light, inorganic elements, water and a carbon source, generally C0 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 cyanobacreria described and exemplified herein are unicellular.

The initial product of photosynthetic fixation of CO 2 is 3-phosphoglycerate- 3-.

phosphoglyceratc is used in thte Calvin Cycle to regenerate ribulose-l.5-biphosphatc, which is the acceptor of There are two major branching points where the intermediates of the Calvin Cycle are connected to other metabolic pathways. At one point, fr-uctose- 6-phosphate is converted into glucose-6-phosphate and glucosc- 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, COMS ID No: SBMI-01012913 Received by IP Australia: Time 11:19 Date 2004-11-25 8 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 1phosphate into a molecule of fructosel,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 1.5 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.

SThe 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.

15 By incorporating the genetic material encoding the pdc and adh enzymes into the S: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 Cvanobacteria. E. coli is more like Z. mobilis which depends on feed stock for growth and ethanol production. There are other sources ofpdc 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 (PL) 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 PL promoter. Therefore, the expression ofpdc-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.

25-11-04: 9:56AM:DAVIES COLLISON CAVE 612 9 2 62 10 0 I I 2 6

II

In the examples below, Synechococcus PCC 7942, which is a unicellular cyanobacteria 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, Florida, 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 Figure 1. In particular, the DNA segment excised from the pLOI295 plasmid includes the pdc sequence starting at -46bp (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. MI5394.

Example 1: pCB4-Rpa The pCB4-Rpa construct is driven by a promoter obtained from the rbcLS operon of the cyanobacteriun 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 S* to the rbcL gene sequence obtained from the cyanobacterium Anacystis nidulan 6301, a strain S genetically similar to Synechococcus PCC 7942. (Shinozaki C. et al. (1983) "Molecular cloning and sequence analysis of the Cyanobacteria gene for the large subunit of bisphosphate carboxylase-oxygenase." Proc Natl Acad Sci USA 80:4050-4054). The PCR reaction mixture (1001l) contained 0.5/AM of each primer, 0.4 mM dNTP, 10 ng genomic DNA from Svnechococcus sp. PCC 7942 and 2 units of Vent, DNA plolymerase (New England Biolabs) in I x reaction buffer: 10mM KCI, 10mM (NH.XSO,, 20mM Tris-HCI (pH 8.8 at 25*C), 2mM MgCI, and 0.1% Triton X-100. PCR reactions were carried out in PTC-IOOTM COMS ID No: SBMI-01012913 Received by IP Australia: Time 11:19 Date 2004-11-25 12 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 0 C/1.5 min, 72 0 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.

A 3.2 kbp EcoRI-SalI DNA fragment containing thepdc-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 Figure 1. A 3.6 kbp BamHI DNA fragment containing the rbcLS promoter region and the pdcadh 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 Figure 2.

Example 2 pCB4-LRpa 1 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, California, 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 ofpCB4 resulting in the vector construct pCB4-LRpa. The vector construct pCB4-LRpa is illustrated in Figure 3.

13 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 Xhol 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 0 C/1.5 min, 72°C/3.5 min; 30 cycles of 93 0 C/1 min, 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-Xhol 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 1 frame was replaced by TCT to code the same amino acid (serine). This new plasmid was Sdigested with XhoI, the cut sites blunt ended with Klenow fragment from DNA poll, 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 XbaL The resulting plasmid is designated pCB4-LR(TF)pa and is illustrated in Figure 4.

Example 4 pCB4-CPpa The vector pCB4-Rpa was digested with Xbal, 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 PL promoter sequence, collectively termed the cI-PL gene sequence (EMBL Accession No. L05669; Sanger et al.

14 Nucleotide sequence of the bacteriophage lambda DNA. 1982, J. Mole. Biol. 162: 729-773) and identified as SEQ. ID. No. 7. The PL promoter had been isolated from the plasmid pHUB2- CI857 (Gruber et al. (1991)) "Escherichia coli-Anacystis nidulans plasmid shuttle vectors containing the PL 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 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 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 X 108 cells per ml. To one ml of this cell solution is added the appropriate plasmid construct DNA to a final concentration of 2ig. ml. 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 itg.ml' ampicillin. Plates are incubated under the standard growth conditions (30 0

C.

light intensity of 100 -pmol photons. m 2 .s Ampicillin resistant colonies were visible in 7-10 days.

•The genetically modified Synechococcus PCC 7942 were grown, bubbling with air at and a light intensity of 100 CiE.M- 2 in liquid BG-l1 medium containing 5 ug.ml"' 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 ofpdc, 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 Figure 8.

Table 2 illustrates the ethanol production for Example 4. Figures 6 and 7 illustrate the pdc activity and adh expression, respectively, for Example 4. The activity ofpdc 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 forpdc and adh activity and ethanol production for the 0 constructs of Examples 1-3 are illustrated in Table 1.

Table 1

C

4 3* Constructs PDC Activity ADH Activity Ethanol Conc. Ethanol Cone.

nmol.min.-'.mg nmol.min.'.mg'' in medium in .moL.mg' SP' SP 3 Chlorophyll pCB4 4

ND

2 ND ND ND pCB4-Rpa 130 168 1370 274 pCB4-LRpa 136 168 1540 308 pCB4- 234 168 1710 342 LR(TF)pa_ 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 OD3 0 1.5. This OD represents approximately 5x10 8 cells.ml-'. 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.

16 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 Figure 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."'.mg-' SP, which is about 20-fold higher than in the strain harboring the shuttle vector pCB4-Rpa which had apdc activity of approximately 130 nmol.min.-'.mg-' SP as can be seen in Figure 6 and Table 1, respectively.

0 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 /umol 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 ,g 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.

0 for 4 different samples.

I

2 .2

C.

9**

C.

Temperature Ethanol Cone. Rate of Ethanol Synthesis (umol.mg-chlorophyll) (/mol.mg-'chlorophyll per hr) 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.

P.%WPDOCSwjwSpmk765963div.dC- IS/[ -17- The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

0* 00 0 *00 0**00 Page(s) 3S--36 are claims pages they appear after the sequence listing SEQUENCE

LISTING

GENERAL

INFORMATION:

APPLICANT:

NAME: Enol Energy Inc.

STREET: 202-80.Tiverton Court CITY: Markham STATE: Ontario COUNTRY:

CANADA

POSTAL CODE (ZIP): L3R 0G4 NAME: Robert Paul Woods STREET: 11 Albert Street CITY: Markham STATE: Ontario COUNTRY:

CANADA

POSTAL CODE (ZIP): L3P 2T3 NAME: John Robert Coleman STREET: 2 Glencrest Coulevard CITY: Toronto STATE: Ontario COUNTRY:

CANADA

POSTAL CODE (ZIP): M4B I L3 NAME: Ming De Deng STREET: 132 Hollyberry Trail CITY: North York STATE: Ontario COUNTRY: CANADA POSTAL CODE (ZIP): M2H 2P1 (ii) TITLE OF INVENTION: Genetically Modified Cyanobacteria for the Production of Ethanol, the Constructs and Method Thereof .*i (iii) NUMBER OF SEQUENCES: 7 (iv) COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE

CHARACTERISTICS:

LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCTGAATTCA TGTCGTCTCT CCCTAGAGA 29 INFORMATION FOR SEQ ID NO: 2: SEQUENCE

CHARACTERISTICS:

LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCTGAATTCA TGTCGTCTCT CCCTAGAGA 29 S: INFORMATION FOR SEQ ID NO: 3: SEQUENCE

CHARACTERISTICS:

LENGTHz 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GGACTCGAGG ATCCCCAAAT GGCAA INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCATGAATTC TTATACTGTC GGTACCTAT 29 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1905 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATCGCTCAT GATCGCGACA TGTTCTGATA TTTTCCTCTA AAAAAGATAA AAAGTCTTTT CGCTTCGGCA GAAGAGGTTC ATCATGAACA AAAATTCGGC ATTTTAAAA 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 21 AGTGCAGAAG GTTATGCTCG TGCCAAAGCG GACGCAGCAG CCGTCGTTAC CTACAGCGTC 420 GGTGCGCTTT CCGCATTTGA TGCTATCGGT GGCGCCTATG CAGAAAACCT TCCGGTTFATC 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 GGA-AGTGTTC 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 TTFFFI 1905 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 1747 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (i)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAAGGCAAAA TCGGTAACCA CATCTCAATT ATTAAACAAT ACTTCATAAT AAAAAGACAA CTTTTTCATA ATTTGCATAA GTCTTGATGT AAAAAATACA TATTTAGAAA GAACAAGCAG 120 CCTTGCTCAT CACCGCTGTC GCGAGTAGAA AAATCTCGGC TTTCAGAAAA AGAGGCCGCT 180 TCGTTAAACA GACTATAAAT GTGCTGGAAT AAAGCGAACC CCTTGATCTG ATAA-AACTGA 240 TAGACATATFI GCTFITGCGC TGCCCGATTG CTGAAAATGC GTAAAAGGTG ATTTTACTCG 300 TTTTCAGGAA AAACTTTGAG AAAACGTCTC GAAAACGGGA TTAAAACGCA AAAACAATAG 360 AAAGCGATTr CGCGAAAATG GTTGTTTCG GGTTGTTGCT TTAAACTAGT ATGTAGGGTG 420 AGGTTATAGC TATGGCTTCT TCAACTTTT ATATTCCTTT CGTCAACGAA ATGGGCGAAG 480 GTTCGCTTGA AAAAGCAATC AAGGATCTTA ACGGCAGCGG CTTTAAAAAT GCGCTGATCG 540 TTTCTGATGC TTTCATGAAC AAATCCGGTG TTGTGAAGCA GGTTGCTGAG CTGTTGAAAG 600 CACAGGGTAT TAATTCTGCT GTTTATGATG GCGTTATGCC GAACCCGACT GTTACCGCAG 660 TTCTGGAAGG CCTITAAGATC 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 CA.ACGCTGTT CTGCTTCCGC ATGTTCTGGC TTATAACGCC TCTGTCGTTG 1320 CTGGTCGTCT GAAAGACGTT GGTGTTGCTA TGGGTCTCGA TATCGCCAAT CTCGGTGATA 1380 AAGAAGGCGC AGAAGCCACC ATTCAGGCTG TTCGCGATCT GGCTGCTFTCC ATTGGTATTC 1440 CAGCAAATCT GACCGAGCTG GGTGCTAAGA AAGAAGATGT GCCGCTTCTT GCTGACCACG 1500 CTCTGAAAGA TGCTTGTGCT CTGACCAACC CGCGTCAGGG TGATCAGAAA GAAGTTGAAG 1560 AACTCTTCCT GAGCGCTTTC TAATTTCAAA ACAGGAAAAC GGTTTTCCGT CCTGTCTTGA 1620 TTTTCA.AGCA AACAATGCCT CCGATTTCTA ATCGGAGGCA 'TTTGTTTfG TTTATTGCAA 1680 AAACAAAAAA TATTGTTACA AATTFFFACA GGCTATTAAG CCTACCGTCA TAAATAATTT 1740 GCCATTT -1747 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 7922 base pairs TYPE: nucleic acid STRANTDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGCGGAGTAA AAAGAGGAGC CCGGCGTCAT CTTTTGTTAC

CCGCCA-AACA

AAACCCAAAA ACAACCCATA CCCAACCCAA TAAAACACCA AAACAAGACA

AATAATCATT

GATTGATGGT 120 TGAAATGGGG TAAACTTGAC AAACAAACCC ACTTAAAACC

CAA-AACATAC

CCAAACACAC 180 ACC.AAAAAA 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 TATTfGTTTGC ACACTTCCTC

TGACAGAGTA

TTACGACAGA 540 AATA-ACCAAC CCAATACGGA AAATATTrGAG CGTAAGAAAG

CAAACTTCCG

GAAAAAAATT 600 ACATTAAATG GCGGGGATAC ATTCACA-ATA 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 TTTTT7CAAAA CCAATAACTC TCAATATGAT 1140 TTAGTTAACG GTATGTATCT CATAGGTAAT TAATGATGGA CAAGCGCAGA ACCATTGCCT 1200 TCAAACTAAA TCCAGATGTA AATCAAACAG ATAAAATTGT TTGTGATACA CTGGACAGTA 1260 TCCCGCAAGG GGAACGAAGC CGCCTTAACC GGGCCGCACT GACGGCAGGT CTGGCCTTrAT 1320 ACAGACAAGA TCCCCGGACC CCTTTGCFFIT TATGTGAGCT GCTGACGAAA GAAACCACAT 1380 TTTCAGATAT CGTGAATATA TTGAGATCGC TATTTCCAAA AGAGATGGCC GATTTTAA2Ff 1440 CTTCAATAGT CACTCAATCC TCTTCACAAC AAGAOCAAAA AAGTGATGAA GAGACCAAAA 1500 AAAATGCGAC GAAGCTAATA AAATTAATTC AATTATTATT GAGTTCCCTT TATCCACTAT 1560 CAGGCTGGAT AAAGGGAACT CAATCAAGTT ATTICTTAC CAGTCATTAC ATAATCGTTA 1620 TTATGAAATA ATCGTTGCA 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 A-ACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG 1980 GTTACATCGA ACTGGATCTC AACAGCGGTA AGATCCTTrGA GAGTTTTCGC CCCGAAGAAC 2040 GTTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGTTG 2 100 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 GGCATACCAA 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 TA.ACGTTATA 2820 CGATGTCGCA GAGTATGCCG GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGA-ACCAGGC 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 Gc3GTCACCAG 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 TCATTTTT.AA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA 3960 TGACCAAAAT CCCTTAACGT GAGTTTCGT TCCACTGAGC GTCAGACCCC GTAATAAGAT 4020 GATCTTCTTG AGATCGTTTT GGTCTGCGCG TAATCTCTTG CTCTGAAAAC GAAAAAACCG 4080 CCTTGCAGGG CGGTITTTTCG TATGATACAG GAGTAAAACC GCCGAAGCCC GGCGTAAGCC 4140 GGTACTGATT GATAGATTTC ACCTTACCCA TCCCCAGCCC TGCCAGACCA TACCCGCTTT 4200 CAGCCATGAG AGAGCTTCTG TGCGCGGTCG GAGTGGTCCC GACGAGGGTT TACCCGAAGT 4260 CGGGGCGTGT CTCCGCGTTA GCGGGCCGTG AGGGCCGCTT ACGAGGGTGT ACTGAGAACT 4320 TCCA.GCGAGA AGACTGACAG CGATGAAGAT GTAGTTACAA CA'ITCATAAT TAAAAGCGAC 4380 TCTGTTCCGG CCCTTTGGGC CGGGGCGGGG CCGCTTTTCA GTTATGAGGG AGGGGCTTTG 4440 TGGTTTCGGT TCTGCGCTGG ACCGGGGTTT TTCTGGAGGT TGTTTTGTG TGTTGTAACT 4500 AAAGTGGCTC CGGTCGGGGC CCGCCGCTTrG CGGTGGGAGG TGCATATCTG TCTGTCCACA 4560 GGACAGGCAG TGAATAGGTT TTCTT rTAA ATGAATGTAA TTAAGTAGTT TAAAGGAGAT 4620 ATAAACAGGT GTTTAA-AAGA TACATTGCAC CCTGTAAGAC TGGCGGCTGG CGCTTTATGA 4680 CATGAACGGT TGTAACCTTA TGGGGAAGTC CCTTGCAGTT AAATGTGGAT C AAGCAAAATT 4740 CCCCGTCGCT GAGGCGTATT TTGTATTAAA AACAGGGGGA ATCGGATGCT CCAGAAGGTG 4800 GATGATGAGA TTGTTTTTTG CATGCGACGC TGTTTTTTTG TGCACCGGCG GGCTTCAGGC 4860 GTGCGGATGC CTCCGGCGCA GGCCGGATTA TTCTGAGGAG ATCACTTTCA GGGAGAAGCT 4920 GTGGCCAGCC GGCTGTA-ATT GCGGTTACGT GACAGAATCA TGCGCTCCTT CACACGACGC 4980 TCCACTTCGC GTTTACCGC CTCACCATTA GCAGTGAAGC GTCCTTCCGA GATTTCACCC 5040 GTCAGCTGCC GTTTCACTAG GGTGACGATA TCCTGACGTT CTCTGTTCGC ATCACGACGC 5100 GCACGGGCAC GTTTTATTCC ACGGGACTGA AGCTCTGTCT GGTAACTGCG GAAACGCTCA 5 160 CGCACAAAAC GCCAGGCTTT CGCTATCAGC TCATCCATAC CCAGGGTATC CAGCCCCTGC 5220 T~FTTGCGCT GTTTGTTTTC CCATTCAACA CGACTGCGGC GCGCAGCTGC CACTGCATCC 5280 TCAGACACAT CAAGGGCAGC AAACAGAGCC AGTGTGAACG TGATGTCGGT CGGAATGTAG 53 CACCCGATAA GCGGGTCATA TTCCGTCTGG TAGGTAATCA GTCCCAGCTC TGACAGGAAC 5400 GTCAGGGCCC GGGTGGCACG GGTGATGGAG AGTTI'TCCTG CACCGGACTC TGTCGCCAGT 5460 CCGCACTCAA TGGCCAGTGT GGTGATGGAA CACTGGACGC GGTTGGCCAG CGGGTCATAG 5520 TGGAAACACA GCCCCTGCAG CAGCGCATCA ATAGCCCGTC GACGCAGCAC V 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 AATCTTTCG TACTCGCCAA AGTTGAAGAA GATTATCGGG GTTTTTGCTT 5940 TTCTGGCTCC TGTAAATCCA CATCAGAACC AGTTCCTTGC CACCTTACGG CGTGGCAGCC 6000 ACAAAATTCC TTAAACGATC AGTAATCTAG CTAGCTACGC CACAAAGTAA AGTCTTTTAC 6060 TTTAGTATAT CCAGTCTCTG CAGTFFCATCT TTGATGATTT TCTCAACGAA CTGAGCCTGT 6120 GTTATCCCCT CTCTCTCGCA GTACTCAACC ATGAGATCGA TCTTTCAGAG GATTTTTGAC 6180 AAAAACTTTTr ATCTCTTGT 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 TTrGGTTTGCC CAAAGCGCAT TGCATAATCT TTCAGGGTTA TGCGTTGTTC CATACAACCT 6480 CCTTAGTACA TGCAACCATT ATCACCGCCA GAGGTAAAAT AGTCAACACG CACGGTGTTA 6540 GATATTTATC CCTTGCGGTG ATAGATTTA-A CGTATGAGCA CAA.AAAAGAA ACCATTAACA 6600 CAAGAGCAGC TTGAGGACGC ACGTCGCCTT AAAGCAATTT ATGAAAAAAA GAAAAATGAA 6660 CTTGGCTTAT CCCAGGAATC TGTCGCAGAC AAGATGGGGA TGCGGCAGTC 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 CCAGACTrGG GGGTGATGAG 7140 TTTACCTTCA AGAAACTGAT CAGGGATAGC GGTCAGGTGT TTTTACAACC ACTAAACCCA 7200 CAGTACCCAA TGATCCCATG CAATGAGAGT TGTTCCGTTG TGGGGA.AAGT TATCGCTAGT 7260 CAGTGGCCTG AAGAGACGTT TGGCTGATCG GCAAGGTGTT CTGGTCGGCG CATAGCTGAT 7320 AACAATTGAG CAAGAATCTT CATCGAATTA GGGGAATTFT CACTCCCCTC AGAACATAAC 7380 ATAGTAAATG GATTGAATTA TGAAGAATGG TTTTTATGCG ACTTACCGCA GCAAAAATAA 7440 AGGGAAAGAT AAGCCTAGTG CTACTI'GAGG GTATACCGCA AGAATATACG CAAGCGTCAG 7500 GATAGCTGCC AAAGCCGCAA GGAATTTACC AACCTTCTTA AACATAAAGT GTCTCCTTAT 7560 AAACGCAGAA AGGCCCACCC GAAGGTGAGC CAGTGTGATT ACATTTTCTC a...: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 AAATrGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC 7860 GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTACTAGAG 7920 TC 7922

Claims (2)

1. 25-11-04; 9:56AM:DAVIES COLLISON CAVE :1 2200#1/2 :612 92621080 12/ 26
2. 34- The claims defining the invention are as follows: 1. Genetically modified unicellular Cyanobacteria containing a construct comprising DNA fragments encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) enzymhes obtained from the Zymomonas mobilis plasrnid pLOI295 wherein said unicellular Cyanobacteria are capable of producing ethanol in recoverable quantities of at least 1.7 umol ethanol per mg of chlorophyll per hour. 2. Genetically modified Cyanobacteia according to claim 1 wherein said Cyanobactenia is Synechococcus. 3. Genetically modified Cyanobacteria according to claim 2 wherein said Cyanobacteria is Synechococcus PCC 7942. 4. Genetically modified Cyanobacteria according to claim 1, whcrcin said construct is selected from the group consisting of pCB4-Rpa, pCB4-LRpa, pCB4-LR(Tf)pa and pCB4-CPpa. 5. Genetically modified Cyanobacteria according to claim 4, wherein said Cyanobacteria is Synechococcus. Genetically modified Cyanobactefia according to clati 1, wherein said construct is pCB4-Rpa. 7. Genetically modified Cyanobacteria according to claim 1, wherein said construct is selected from the group consisting of pCB4-LRpa and pCB4-LR(TF)pa. Genetically modified Cyanobacteria according to claim 1, wherein said construct is pCB4-CPpa. 9. Genetically modified Cyanobacteria according to claim I wherein said DNA fragment encoding the pdc enzyme comprises SEQ. ID NO. COMS ID No: SBMI-01012913 Received by IP Australia: Time 11:19 Date 2004-11-25 25-11-04: 9:56AM:DAVIES COLLISON CAVE :612 92621080 #13/ 26 RAMOMPMaT 7670rn30 dxlyl( WO 35 Genetically modified Cyanobacteria according to claim 1 wherein said DNA fragment encoding the adh enzyme comprises SEQ. ID NO. 6. 11. Genetically modified Cyanobacteria according to claim 1 wherein said construct is temperature inducible wherein said ethanol is produced in relatively quantifiable amounts after a particular temperature is reached. 12. Genetically modified Cyanobacteria according to claim I1I wherein said construct comprises the temperature inducible gene C1857. 13. Genetically modified Cyanobacteria according to claimi 12 wherein said construct contains the promoter CI-PL, SEQ. ED. NO. 7. 14. A process for producing genetically modified unicellular Cyanobacteria according to claim 1 comprising the following steps: a. selecting an appropriate promoter; ligating said promoter to said pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) enzyme encoding DNA fragments; cloning said ligated fragments comprising the promoter, pdc and adh sequences into an appropriate construct; transforming said construct into said unicellular Cyonobacteria. A process according to claim 14, wherein said construct flurther contains DNA fragments encoding ampicillmi resistance and step d. comprises the steps of: a. harvesting cells of said unicellular Cyanobacteria; b. adding said construct to said harvested Cyanobacreria cells; incubating said construct with said Cyanobacreria cells such that said d. construct is trasformed into said Cyanobactenia cells; d. plating said incubated constructs and Cyanobacteria cells on plates containing ainpicillin and incubating under appropriate growth conditions;, e. selecting said transformed ampicillin resistant Cyanobacteria cells. COMS IDNo: 5BMI-01012913 Received by IPAustralia: Time 11:19 Date 2004-11-25 25-11-04: 9:56AM:DAVIES COLLISON CAVE :612 92621080 14/ 26 rMWFUOC? M Igwpo= L7t )X dc-2y I S -36- 16. A process according to claim 15 wherein said Cyanobacteria is Synechococcus and said construct is selected from the group consisting of pCB4-Rpa, pCB4-LRpa, pCB4- LR(TF)pa and pCB4-CPpa. 17. A construct selected from the group consisting of pCB4-Rpa, pCB4-LRpa, pCB4- LR(TF)pa and pCB4-CPpa. 18. A process for producing ethanol which comprises the steps of: culturing in a culture medium unicellular Cyanobacteria, said unicellular Cyanobacteria containing a construct comprising DNA fragments encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) enzymes obtained from the Zymomonas mobilis pLOI295 plasmid; and accumulating ethanol in the culture medium in an amount of approximately 1.7 umol of ethanol per mg of chlorophyll per hour. 19. A process for producing ethanol according to claim 18 wherein the Cyanobacteria is Synechococcus and said construct is selected from the group consisting of pCB4-Rpa, S: pCB4-LRpa, pCB4-LR(TF)pa and pCB4-CPpa. a.. 20. A process for producing ethanol according to claim 18 wherein said construct further comprises encoding for a temperature inducible gene wherein said ethanol is produced only when a particular temperature is reached. 21. Genetically modified unicellular Cyanobacteria, use thereof in the production of ethanol or ethanol produced therefrom substantially as herein described with reference to the Examples. DATED this 25th day of November 2004 ENOL ENERGY INC. By its Patent Attorneys DAVIES COLLISON CAVE COMS ID No: SBMI-01012913 Received by IP Australia: Time 11:19 Date 2004-11-25