Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-02-20
2001-10-23
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C536S023100, C536S023200, C536S024100
Reexamination Certificate
active
06306639
ABSTRACT:
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 &mgr;mol 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
Zymom
Coleman John Robert
De Deng Ming
Woods Robert Paul
Achutamurthy Ponnathapu
Enol Energy Inc.
Michael & Best & Friedrich LLP
Tung Peter P.
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