DNA encoding methymycin and pikromycin

Details DNA encoding methymycin and pikromycin DNA encoding methymycin and pikromycin

1998-06-26

2001-07-24

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Bacteria or actinomycetales; media therefor

C435S252300, C435S252330, C435S320100, C435S183000, C536S023200, C536S023700, C536S023100

Reexamination Certificate

active

06265202

ABSTRACT:

BACKGROUND OF THE INVENTION
Polyhydroxyalkanoates (PHAs) are one class of biodegradable polymers. The first identified member of the PHAs thermoplastics was polyhydroxybutyrate (PHB), the polymeric ester of D(−)-3-hydroxybutyrate. The biosynthetic pathway of PHB in the gram negative bacterium
Alcaligenes eutrophus
is depicted in FIG.
1
. PHAs related to PHB differ in the structure of the pendant arm, R (FIG.
2
). For example, R═CH
3
in PHB, while R═CH
2
CH
3
in polyhydroxyvalerate, and R═(CH
2
)
4
CH
3
in polyhydroxyoctanoate.
The genes responsible for PHB synthesis in
A. eutrophus
have been cloned and sequenced. (Peoples et al.,
J. Biol. Chem.,
264, 15293 (1989); Peoples et al.,
J. Biol. Chem.,
264, 15298 (1989)). Three enzymes: &bgr;-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), and PHB synthase (phbC) are involved in the conversion of acetyl-CoA to PHB. The PHB synthase gene encodes a protein of M
r
=63,900 which is active when introduced into
E. coli
(Peoples et al.,
J. Biol. Chem.,
264, 15298 (1989)).
Although PHB represents the archetypical form of a biodegradable thermoplastic, its physical properties preclude significant use of the homopolymer form. Pure PHB is highly crystalline and, thus, very brittle. However, unique physical properties resulting form the structural characteristics of the R groups in a PHA copolymer may result in a polymer with more desirable characteristics. These characteristics include altered crystallinity, UV weathering resistance, glass to rubber transition temperature (T
g
), melting temperature of the crystalline phase, rigidity and durability (Holmes et al., EPO 00052 459; Anderson et al.,
Microbiol. Rev.,
54, 450 (1990)). Thus, these polyesters behave as thermoplastics, with melting temperatures of 50-180° C., which can be processed by conventional extension and molding equipment.
Traditional strategies for producing random PHA copolymers involve feeding short- and long-chain fatty acid monomers to bacterial cultures. However, this technology is limited by the monomer units which can be incorporated into a polymer by the endogenous PHA synthase and the expense of manufacturing PHAs by existing fermentation methods (Haywood et al.,
FEMS Microbiol. Lett.,
57, 1 (1989); Poi et al.,
Int. J. Biol. Macromol.,
12, 106 (1990); Steinbuchel et al., In:
Novel Biomaterials from Biological Sources.
D. Byron (ed.), MacMillan, N.Y. (1991); Valentin et al.,
Appl. Microbiol. Biotechnical,
36, 507 (1992)).
The production of diverse hydroxyacylCoA monomers for homo- and co-polymeric PHAs also occurs in some bacteria through the reduction and condensation pathway of fatty acids. This pathway employs a fatty acid synthase (FAS) which condenses malonate and acetate. The resulting &bgr;-keto group undergoes three processing steps, &bgr;-keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit. However, this pathway provides only a limited array of PHA monomers which vary in alkyl chain length but not in the degree of alkyl group branching, saturation, or functionalization along the acyl chain.
The biosynthesis of polyketides, such as erythromycin, is mechanistically related to formation of long-chain fatty acids. However, polyketides, in contrast to FASs, retain ketone, hydroxyl, or olefinic functions and contain methyl or ethyl side groups interspersed along an acyl chain comparable in length to that of common fatty acids. This asymmetry in structure implies that the polyketide synthase (PKS), the enzyme system responsible for formation of these molecules, although mechanistically related to a FAS, results in an end product that is structurally very different than that of a long-chain fatty acid.
Because PHAs are biodegradable polymers that have the versatility to replace petrochemical-based thermoplastics, it is desirable that new, more economical methods be provided for the production of defined PHAs. Thus, what is needed are methods to produce recombinant PHA monomer synthases for the generation of PHA polymers.
SUMMARY OF THE INVENTION
The present invention provides a method of preparing a polyhydroxyalkanoate synthase. The method comprises introducing an expression cassette into a non-plant eukaryotic cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate synthase, e.g., a polyhydroxybutyrate synthase, operably linked to a promoter functional in the non-plant eukaryotic cell. The DNA molecule may be obtained from a bacterium such as
Alcaligenes eutrophus.
The DNA molecule encoding the polyhydroxyalkanoate synthase is then expressed in the cell. Thus, another embodiment of the invention provides a purified recombinant polyhydroxybutyrate synthase isolated from a host cell which expresses the synthase.
Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a eukaryotic cell. The first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the eukaryotic cell, e.g., an insect cell. The inactivation preferably is via a mutation in the catalytic site of the dehydrase. The second expression cassette comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the eukaryotic cell. The expression cassettes may be on the same or separate molecules. The DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer.
Another embodiment of the invention is a baculovirus expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in an insect cell. Preferably, the nucleic acid molecule is obtained from a bacterium, e.g.,
Alcaligenes eutrophus.
The present invention also provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell. The nucleic acid molecule comprises a plurality of DNA segments. Thus, the nucleic acid molecule comprises at least a first and a second DNA segment. No more than one DNA segment is derived from the eryA gene cluster of
Saccharopolyspora erythraea.
The first DNA segment encodes a first module and the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. The source of at least one DNA segment is preferably bacterial DNA. It is preferred that the first DNA segment encodes the first module form the vep gene cluster and the second DNA segment encodes module 7 from the tyl P gene cluster. The nucleic acid molecule may optionally further comprise a third DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second nucleic acid molecule encoding a polyhydroxyalkanoate synthase may be introduced into the host cell.
Also provided is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module. No more than one DNA segment is derived from the eryA gene cluster of
Saccharopolyspora erythraea.
Also, it is preferred that no more than one module is derived from the gene cluster from
Streptomyces hygroscopicus
that encodes rapamycin or the gene cluster that encodes spiramycin. Together the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase. A preferred embodiment of the invention employs a first DNA segment derived from the vep gene cluster of Streptomyces. Another preferred embodiment of the invention employs a second DNA segment derived from the tyl gene cluster of Streptomyces. A further preferred embodiment of the isolated DNA molecule of the invention includes a DNA s

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