Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-01-05
2004-02-17
Murthy, Ponnathapu Achuta (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S252300, C435S252800, C435S253500, C435S320100, C436S023000
Reexamination Certificate
active
06692945
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for the production of polyhydroxyoctanoate in substantial amounts using recombinant
Streptomyces lividans
TK64. More particularly it relates to a method involving construction of a multifunctional
Escherichia coli
—Streptomyces conjugative shuttle vector, development of a recombinant vector designated as pCAB218, which is used to transform
Streptomyces lividans
TK64, such that it is capable of producing polyhydroxyoctanoate (PHO) in substantial amounts when grown in a conventional mineral medium.
BACKGROUND OF THE INVENTION
The citations in this specification are incorporated herein by reference to form a part of this application.
Synthetic polymers have become an integral part of our day to day life. These compounds like polyvinylchloride, polyhomopropylene, polyethylene and others are produced from fossil resources. They have many desirable properties including durability and resistance to degradation. They are used to a very high extent in the packaging industry, and once their useful life is over, are partially recycled, end in landfills, or are burnt in order to eliminate the solid waste. As far as total mass of plastic waste is concerned, these nondegradable plastics accumulate in the environment at a rate of over 25 million tonnes per year [Lee S. Y.,
Biotechnol. Bioeng.,
49 (1995), 1-14]. Recently, the problems concerning the global environment and solid waste management has created much interest in the development of biodegradable plastics, which must still retain the desired properties of conventional synthetic plastics. Some of the biodegradable plastic materials under development include polyhydroxyalkanoates (PHAs), polylactides, aliphatic polyesters, polysaccharides and the copolymers and/or blends of these [Byrome, D. (ed.), (1991),
Biomaterials
: novel materials from biological sources. Stockton, N.Y. 125-213].
Prior Art Methods
During the past 10 years polyhydroxyalkanoates (PHAs) as a class of biopolymers have globally experienced a great increase in research and development efforts. These are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as cytoplasmic granular inclusions in the cells in response to nutrient limitation. Polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and polyhydroxybutyrate-co-valerate (PHB-V) are by far the most widely and thoroughly characterized of the PHAs [Steinbüchel, A. and Schlegel, H. G.,
Mol. Microbiol.,
5 (1991) 30-37]. The nature of the polymer is determined by the carbon source supplied in the growth medium. Thus,
Ralstonia eutropha
grown in a medium containing glucose produces PHB. The use of propionic acid/or valeric acid as the sole carbon source in the growth medium for
Chromobacterium violaceum
yields PHV [Doi, Y. Tamaki, A., Kunioka, M. and Soga, K.,
Appl. Microbiol. Biotechnol.,
28, (1988), 330-334; Steinbüchel, A., Debzi, E. M., Marchessault, R. H. and Timm, A.
Appl. Microbiol. Biotechnol.,
39, (1993), 443-449]. Addition of propionic acid/or valeric acid to the glucose containing growth medium leads to the production of random copolymer PHB-V by
Ralstonia eutropha
as reported by Steinbüchel, A., In Byrom, D.
ed. Biomaterials: novel materials from biological sources
. Stocton, N.Y., (1991) pp 124-213. This is possible because of the broad substrate specificity of the bacterial enzymes involved in PHA synthesis [Doi, Y. Kunioka, M., Nakamura, Y. and Soga, K.,
Macromolecules,
21, (1988),2722-2727]. The polymer synthesis is by the sequential action of three enzymes. The first enzyme of the pathway, &bgr;-ketothiolase, coded by gene phaA, catalyzes the reversible condensation of two acyl-CoA moieties to form &bgr;-ketoacyl-CoA. Acetoacetyl-CoA reductase, coded by phaB gene, subsequently reduces &bgr;-ketoacyl-CoA to D(-)-&bgr;-hydroxyacyl-CoA, which in turn is polymerized by the action of the enzyme PHA synthase, coded by phaC gene, to form PHA. In
Ralstonia eutropha
, the structural genes for PHA synthesis are organized in a single operon designated as phaCAB
Re
, coding for PHA synthase, &bgr;-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase respectively.
Pseudomonas oleovorans
a Gram negative bacterium grown on aliphatic carbon sources such as alkanes, alkanols and alkanoic acids produces PHAs of various medium chain length &bgr;-hydroxyalkanoic acids [Lageween, R. G., Huisman, G. W., Preustig, H,. Ketelaar, P., Eggnik, G. and Wuholt, B.
Appl. Environ. Microbiol.,
54, (1988), 2924-2932]. The drawback is that this organism needs to be cultivated on octanoic acid, a very expensive aliphatic acid, to produce a homopolymer of &bgr;-hydroxyoctanoate [Timm, A., Wiese, S. and Steinbüchel, A.
Appl. Microbiol. Biotechnol.,
40 (1994) 669-675]. There are, however, no reports suggesting the use of an alternative and cheap carbon source for the production of polyhydroxyoctanoate (PHO) by any microorganism.
Non-pathogenic soil bacteria Streptomyces species are well known for their ability to synthesize antibiotics [Berdy, J.,
Process Biochem
., October/November(1980) 28-35]. These are also reported to synthesize and accumulate polyhydroxyalkanoates (PHAs) in very small quantities [Kannan, L. V. and Rehacek, Z.,
Ind. J. Biochem.,
7 (1970) 126-129].
Applications of recombinant DNA technology in Streptomyces are on the rise [Yang, R., Hu, Z., Deng, Z. and Li, J.,
Shengwu Gongcheng Xuebao,
14 (1998) 6-12; Ikeda, K., Suzuki, K., Yoshioka, H., Miyamoto, K., Masujima, T. and Sugiyama, M.,
FEMS Microbiol. Lett.,
168 (1998) 196-199]. Since the fermentation technology is well worked out with Streptomyces species, it is desirable to exploit
Streptomyces lividans
TK64 for the production of polyhydroxyoctanoate (PHO) using an alternate and cheap carbon source. This will, however, require genetic modification of the organism. The basic DNA constructions, gene modifications and genetic manipulations will have to be first made in
Escherichia coli
, and later introduced into the Streptomyces species. This necessitates the design, construction and preparation of multifunctional shuttle or conjugative plasmid vectors which allow assembly, construction and cloning of genes along with their regulatory sequences in
Escherichia coli
and later introduction into the Streptomyces species by polyethylene glycol (PEG) mediated DNA uptake [Hopwood, D. A., Bibb, M. I., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. and Scrempf, H.,
Genetic Manipulation Of Streptomyces; A Laboratory Manual.
John Innes Foundation, Norwich, England, 1985] or through conjugation with
Escherichia coli
[Mazodier, P., Petter, R. and Thompson, C.,
J.Bacteriol.,
171 (1989) 3583-3585].
The drawback of the most often used
Escherichia coli
—Streptomyces sp. shuttle vectors is the lack of their conjugative capability [Wehmeier, U. F.,
Gene,
165 (1995) 149-150; Morino, T. and Takahashi, H.,
Actinomycetologica,
12 (1998) 37-39]. The PEG mediated transformation of Streptomyces species also suffers from the drawback of low frequency of transformation when plasmid DNA of
Escherichia coli
origin is used [Rao, R. N., Richardson, M. A. and Kuhstoss, S. A.
Methods Enzymol,
153 (1987) 166-198]. The available
Escherichia coli
—Streptomyces sp. conjugative vectors suffer from the drawback of providing only one or two unique restriction endonuclease cloning sites thus severely limiting cloning options [Mazodier, P., Petter, R. and Thompson, C., J.
Bacteriol.,
171 (1989) 3583-3585; Voeykova, T., Emelyanova, L., Tabakov, V. and Mkrtumyan, N., FEMS
MicroBiol. Lett.,
162 (1998) 47-52].
Thus, there is a need in the prior art to develop a method whereby polyhydroxy octanoate can be readily and efficiently produced using
Escherichia coli
. To overcome the aforementioned drawbacks in t
Mahishi Lata Hanamantrao
Ramachander Turaga Venkata Naga
Rawal Shuban Kishen
Tripathi Gyanendra
Council of Scientific & Industrial Research
Fronda Christian L.
Murthy Ponnathapu Achuta
Smith , Gambrell & Russell, LLP
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