Escherichia coli having accession No. PTA 1579 and its use...

Details Escherichia coli having accession No. PTA 1579 and its use... Escherichia coli having accession No. PTA 1579 and its use...

2001-01-29

2004-06-29

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S071100, C435S243000, C435S252800, C435S252330, C435S320100, C435S132000, C536S023200

Reexamination Certificate

active

06756222

ABSTRACT:

FIELD OF INVENTION
The present invention relates to the field of recombinant deoxyribonucleic acid (DNA) technology. Specifically, the invention relates to identification of the genes responsible for poly-beta-hydroxybutyrate biosynthesis pathway from
Streptomyces aureofaciens
NRRL 2209, creation of a plasmid vector carrying the said gene and expression of this gene in
Escherichia coli
designated as NCIM 5128 and bearing ATCC Accession No. PTA 1579, which is used for synthesis of polyhydroxybutyrate in recoverable amounts of at least 60% of dry bacterial cell mass.
BACKGROUND OF THE INVENTION
Lemoigne in 1926 discovered the presence of PHB (poly-beta-hydroxybutyrate) in Bacillus. This has been reported to be present in a multitude of other bacterial genera, including Azotobacter, Alcaligenes, Psuedonomas, Rhizobium, Chromatium, Acinetobacter, Rhodospirillum and some species of cyanobacteria. It is also reported to be present in certain actinomycetes in very minute quantities. PHB is synthesized and stored by these microorganisms essentially as an energy source under stress conditions. PHB, a homopolymer of D-(−)-3-hydroxybutyrate, has properties comparable to synthetic polymers like polypropylene. PHB is commercially produced by fermentation technology using
Alcaligenes eutrophus
(
Ralstonia eutropha
) and is marketed under the brand name Biopol. In the present global environment awareness, as against synthetic polymers which are persistent by nature, PHB is bestowed with the property of biodegradability. Besides being used in packaging industry, PHB has also been used as a source of chiral centers for the organic synthesis of certain antibiotics, in drug delivery and bone replacement applications. The biosynthesis of PHB has been studied extensively in
Alcaligenes eutrophus, Rhodospirullum rubrum
, Pseudomonas species and
Azotobacter beijerinckii
. &bgr;-ketothiolase, the first enzyme in the pathway and coded for by the phaA gene, first catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. Acetoacetyl-CoA is then reduced to D-(−)-
3-
hydroxybutyryl-CoA by NADPH dependent acetoacetyl-CoA reductase which is coded for by phaB gene. D-(−)-3-hydroxybutyryl-CoA monomer is then polymerized to PHB by PHB synthase coded for by the phaC gene. PHB in the bacterial cell accumulates as cytoplasmic inclusions when growth of the bacteria in culture is limited by a nutrient other than a carbon source. It may be oxygen deprivation, nitrogen deprivation, phosphate limitation, sulfate limitation and magnesium limitation. Once the limiting conditions are relaxed, PHB is metabolized down to preinduction levels. It has been shown that both &bgr;-ketothiolase and acetoacetyl CoA reductase activities increase in response to PHB-stimulating limitation conditions.
Some of the U.S. Patents covering production and extraction of PHB from microorganisms include the following: U.S. Pat. No. 4,786,598 to Lafferty et al. discloses a two-stage fermentation process where PHB is produced using
Alcaligenes latus
, U.S. Pat. No. 4,705,604 to Vanlautem et al. discloses using 1,2 dichloroethane to simultaneously remove water from the bacterial suspension by azeotropic distillation and extract PHB from the cells, U.S. Pat. No. 4,477,654 to Holmes et al discloses limiting the nitrogen nutrient source to microbiologically accumulate 3-hydroxybutyrate polymers, U.S. Pat. No. 4,433,053 discloses a fermenting process for PHB accumulation using
A. eutrophus
where a nutrient required for growth is limited, U.S. Pat. No. 4,336,334 to Powell et al. shows a microbiological process for producing PHB using
Methylobacterium organophilum
, U.S. Pat. No. 4,358,583 to Walker et al. discloses extracting PHB by first flocculating the cells by heat or pH treatment then extracting with a suitable solvent, U.S. Pat. No. 4,138,291 to Lafferty discloses bacterial strains assimilating various carbon sources and converting them to PHB, U.S. Pat. No. 5,518,907 to Dennis discloses Cloning and expression in
Escherichia coli
of the
Alcaligenes eutrophus
H16 poly-beta-hydroxybutyrate biosynthetic pathway, U.S. Pat. No. 5,798,235 to Peoples, et. al., gene encoding bacterial acetoacetyl Co-A reductase and U.S. Pat. No. 5,650,555 to Somerville et. al. discloses transgenic plants producing polyhydroxyalkanoates. U.S. Pat. No. 5,512,456 to Dennis discloses method for the improved production and recovery of poly-beta-hydroxybutyrate from transformed
Escherichia coli
, U.S. Pat. No. 5,250,430 discloses Polyhydroxyalkanoate polymerase from
Zoogloea ramigera.
Among the prokaryotes, the actinomycetes constitute an important part of the microbial community responsible for the degradation and recycling of natural substrates. Accumulation of PHB has been reported from nine different strains of Streptomyces by Kannan and Rehacek (Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126-129, 1970). A possible role in thew biosynthesis of polyketide-derived phenolic metabolites such as actinorhodin or antimycin has been suggested by Kannan and Rehacek (Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126-129, 1970) and Packter and Flatman (Characterization of acetoacetyl-CoA reductase (3-oxoreductase) from
Streptomyces coelicolor
, its possible role in polyhydroxybutyrate biosynthesis. Biochem. Soc. Trans., 11:598-599, 1983).
Streptomyces aureofaciens
NRRL 2209 is a very poor accumulator of PHB and has been reported by Kannan and Rehacek (Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126-129, 1970) to accumulate PHB upto 1.10% of the dry cell mass. These bacteria are not as amenable as
Escherichia coil
to genetic manipulations and certainly are not as well characterized.
Escherichia coli
as a host cell has been exploited for producing molecules like the human growth hormone, insulin and interferon.
Thus, although polyhydroxybutyrate and other polyalkanoate biosynthesis genes have been isolated and characterized in various organisms such as
Ralstonia eutropha
(formerly known as
Alcaligenes entrophus
), there are several disadvantages in the use of these genes for production of polyalkanaoates on a large scale. For instance, when these genes are cloned into bacteria such as
E. coli
, the eventual production of alkanoate is not substantial. Further, there is no instance in the prior art wherein DNA fragments are isolated from actinomycetes and expressed in a heterogeneous host for production of polyhydroxybutyrate.
In order to make PHB production regulatable; a need exists for cloning of the PHB biosynthetic pathway from
Streptomyces aureofaciens
NRRL2209, its introduction and expression in
Escherichia coli.
OBJECTS OF THE INVENTION
The main object of the invention is to provide a method for cloning of genes coding for poly-beta-hydroxybutyrate biosynthesis pathway from
Streptomyces aureofaciens
NRRL2209 and expression thereof in
Escherichia coli.
Another object of the present invention is to identify the genomic sequences responsible for poly-beta-hydroxybutyrate biosynthesis pathway, from
Streptomyces aureofaciens
NRRL2209.
Yet another object of the present invention is to clone the genes responsible for poly-beta-hydroxybutyrate biosynthesis pathway, in a multicopy plasmid vector, thus creating a new vector which carries the nucleotide sequence responsible for the PHB synthesis pathway.
Yet another object of the present invention is to transform
Escherichia coli
with a multicopy plasmid vector for expression of the PHB biosynthesis pathway genes in the bacterial host.
Another object of the present invention is to produce PHB using transformed
Escherichia coli
in recoverable quantities.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method for isolation of genes coding for poly-beta-hydroxybutyrate (PHB) biosynthesis pathway from
Streptomyces aureofaciens
NRRL2209, expressing the said genes in
Escherichia coli
and producing PHB using the tra

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