Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-02-08
2004-08-03
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S069100, C435S320100, C435S471000, C435S252340, C435S253300, C435S041000, C435S173800, C536S023100, C536S023200, C536S023700
Reexamination Certificate
active
06770464
ABSTRACT:
BACKGROUND OF THE INVENTION
The present application is a national stage application of PCT/DE95/01279, filed on Sep. 15, 1995.
The present invention relates to a process for the production of poly(hydroxyl acids) by means of recombinant bacteria which contain and express at least one fragment of the gene of poly(hydroxy fatty acid) synthase from
Thiocapsa pfennigii
and which are selected from the group comprising:
Pseudomonas putida
GPp 104 (pHP1014::E156).
Alcaligenes eutrophus
PHB 4 (pHP1014::E156),
Pseudomonas putida
GPp104 (pHP1014::B28+) [DSM #9417] and Alcaligenes eutrophus PHB 4 (pHP1014:B28+) [DSM #9418], whereby the bacteria are cultivated in a mineral medium under aerobic conditions, whereby one offers the bacteria at least one substrate carbon source which is selected from the group consisting of: levulinic acid, salts of levulinic acid, esters of levulinic acid, lactones of levulinic acid, substituted levulinic acid or, as the case may be, its derivatives: 5-hydroxyhexanoic acid, its salts, esters and lactones: 4-hydroxyheptanoic acid, its salts, esters and lactones: 4-hydroxyoctanoic acid, its salts, esters and lactones; their halogenated derivatives us well as their mixtures; one incubates the bacteria for a certain time with the carbon source; and one isolates the poly(hydroxyl fatty acid) polymers that have been synthesized by the bacteria:
a recombinant bacterial strain characterized by the feature that the bacterial strain is selected from the group which comprises
Pseudomonas putida
GPp104 (pHP1014::B28+) [DSM # 9417] and
Alcaligenes eutrophus
PHB 4 (pHP1014:B28+) [DSM #9418]:
a poly(hydroxyl fatty acid) produced by any one of the previously described processes:
and a DNA fragment which codes for a pha E component and a pha C component of the poly(hydroxyl fatty acid) synthase from Thiocapsa pfennigii characterized by the feature that it has at least the nucleotide sequence of sequence sections
180
through
1280
(phaE) and
1322
through
2392
(phaC) of the DNA sequence SEQ ID NO:1.
In this age of increasing environmental awareness, there are increasing attempts in industry and science to produce biodegradable polymers. In this regard, these new types of environmentally compatible polymers should essentially have the same properties as those polymers which, for decades, have been prepared via organic chemical synthesis.
In particular in this connection, the ability to process the new types of biodegradable polymers ought to be provided in a similar manner to the processing of conventional plastics using the same methods such as, for example, extrusion, injection molding, injection compression, foaming, etc.
A big disadvantage of organically synthesized plastics is, however, that many of these plastics have enormous biological half-lives or, as the case may be, they cannot be disposed of in garbage dumps or in garbage incineration plants in a non-harmful manner but, rather, aggressive gases are frequently produced such as, for example, in the case of poly(vinyl chloride) which liberates hydrogen chloride gas during incineration.
A first step in the direction of success with environmentally compatible materials was achieved by means of synthetic substances, e.g. the paraffin-like polymers polyethylene and polypropylene since these essentially release only CO
2
and water on incineration.
In addition, many attempts have also been made by means of so-called replaceable raw materials such as, e.g. plants that contain a lot of polysaccharide such as potatoes, corn, wheat, beans, peas or similar materials, to obtain the naturally occurring polysaccharides in these plants and to prepare polymers from them which are usable in plastics technology and which are biodegradable.
However, in the case of such polymer materials comprising replaceable raw materials, one is essentially relying on the natural quality of the polymers that occur in these higher plants and only the relatively complex processes of classical cultivation and modern gene technology offer themselves for modification at the genetic level.
An essential further step in the direction of naturally occurring polymers, which are very similar to synthetic thermoplastics, was brought about by the discovery of poly(3-hydroxybutyric acid) by Lemoigne in 1926 [Lemoigne, M. (1926) Products of the dehydration and polymerization of &bgr;-oxybutyric acid,
Bull. Soc. Chim. Biol
. (Paris) 8: 770-782]. The discovery by Lemoigne can be considered to have paved the way for the further development of modern poly(hydroxy fatty acids) which are also designated polyhydroxyalkanoates and represent chemically linear esters of hydroxy fatty acids and hence, ultimately, polyesters.
In the eighties and, especially, in the last five years, further hydroxy fatty acids have been described as components of the poly(hydroxy fatty acids) (PHF) that occur in nature. In this connection, the hydroxyl group of these PHF is usually located in the 3′ position. The aliphatic side chains are either saturated or singly or doubly unsaturated. They are thus non-branched or branched and they can be substituted by functional groups such as, for example, halogen atoms, preferably bromine, iodine and chlorine, or cyano groups, ester groups, carboxyl groups or even cyclic aliphatic groups and even aromatic. In some hydroxy fatty acids, the hydroxyl group is also located in the 4′ or 5′ position.
Poly(hydroxy fatty acids) have been detected previously in gram positive and gram negative groups of bacteria, aerobic and anaerobic groups of bacteria, heterotrophic and autotrophic groups of bacteria, eubacteria and archaebacteria and in anoxygenic and oxygenic photosynthetic groups of bacteria and therefore in virtually all important groups of bacteria. Thus the capability of synthesizing such polyesters apparently does not represent any specially demanding or rare biochemical metabolism. Biosynthesis of the PHF usually sets in when a usable source of carbon is present in excess with the simultaneous deficiency of another nutrient component. In this way, a nitrogen deficiency, a phosphorus deficiency, a sulfur deficiency, an iron deficiency, a potassium deficiency, a magnesium deficiency or an oxygen deficiency can trigger PHF synthesis in bacteria [Anderson, A. J. and Dawes, E. A. (1990) Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates,
Microbial. Rev.
54: 450-472; Steinbüchel, A. (1991) Polyhydroxyalkanoic acids: In: D. Byrom (editor) Biomaterials, Macmillan Press, New York, pages 123-213]. In most bacteria, PHF are deposited in the form of inclusions or grana in cytoplasm, whereby the dry mass of the cell can amount to up to a proportion of 95% by weight.
In eukaryotes, only poly(3-hydroxybutyric acid) has previously been demonstrated as the single PHF. This polyester arises in yeasts such as, for example,
Saccharomyces cerevisiae
, various plants, e.g. cauliflower, various organs from animals, e.g. the liver and also in humans, e.g. in blood plasma [Reusch, R N. 1992, Biological complexes of polyhydroxybutyrate,
FEMS Microbiol. Rev.
103: 119-130]. However, in contradistinction to prokaryotes, the proportion of poly(3-hydroxybutyric acid) in eukaryotes is maximally 0.1% by weight. Inclusions in the form of grana, in the manner in which they occur in prokaryotes, are not known in eukaryotes. As a rule, the eukaryotic PHF are not usually present in free form, either but the polyester is present either linked to other proteins or in the form of a complex which spans the cytoplasm membrane together with calcium ions and polyphosphate molecules.
Thus, only the production of PHF in bacteria is of interest for industrial biotechnological purposes.
The biosynthesis of PHF in bacteria can be subdivided into three phases.
In phase I, the carbon source, which is offered to the bacteria in the medium, is first taken up in the bacterial cells. Either special uptake transportation
Liebergesell Mathias
Pries Andreas
Steinbüchel Alexander
Valentin Henry
Leffers Gerry
Metabolix Inc.
Pabst Patent Group LLP
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