Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2001-11-19
2003-02-04
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S252300, C435S320100, C435S135000, C435S141000, C435S142000, C536S023200, C536S023700, C530S300000, C530S350000
Reexamination Certificate
active
06514743
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polyester synthase, a gene coding for the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a process for producing polyester synthase by use of the transformant.
BACKGROUND OF THE INVENTION
Polyesters (e.g., poly-3-hydroxyalkanoic acid) biosynthesized by microorganisms are biodegradable plastics with thermoplasticity ranging widely from rigid matter to viscoelastic rubber.
Poly-3-hydroxybutanoic acid (P(3HB)) is a typical polyester consisting of C4 monomer units, but it is a rigid and brittle polymeric material, so its application is limited. Accordingly, various polyesters such as P(3HB-co-3HV) having (P(3HB)) copolymerized with a C5 monomer unit (3HV) by adding propionic acid etc. to the medium have been prepared and examined to alter the physical properties of the polyester. On the other hand, polyesters consisting of at least C6 monomer units are soft polymeric materials having plasticity.
Polyester-synthesizing microorganisms are roughly divided into 2 groups, that is, those synthesizing polyesters with C3-5 monomer units and those synthesizing polyesters with C6-14 monomer units. The former microorganisms possess a polyester synthase using C3-5 monomer units as the substrate, while the latter microorganisms possess a polyester synthase using C6-14 monomer units as the substrate. Therefore, polyesters with different properties are synthesized by the respective microorganisms.
However, the respective polyesters from such known microorganisms are different in substrate specificity, so with one kind of enzyme given, polyesters (copolymers) having various monomer unit compositions adapted to the object of use are difficult to synthesize.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a polyester synthase with monomer units having a wide range of carbon atoms as the substrate, a gene coding for the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a process for producing the polyester synthase by use of the transformant.
As a result of their eager research, the present inventors succeeded in cloning a polyester synthase gene from a microorganism belonging to the genus Pseudomonas isolated from soil, to arrive at the completion of the present invention.
That is, the present invention is a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a sequence where in said amino acid sequence, one or more amino acids are deleted, replaced or added, said polypeptide having polyester synthase activity.
Further, the present invention is a polyester synthase gene comprising DNA coding for said polypeptide. The DNA coding for the protein with polyester synthase activity includes, e.g., that of SEQ ID NO:2.
Further, the present invention is a polyester synthase gene comprising the nucleotide sequence of SEQ ID NO:3.
Further, the present invention is a recombinant vector comprising the polyester synthase gene.
Further, the present invention is a transformant transformed with said recombinant vector.
Further, the present invention is a process for producing polyester synthase wherein said transformant is cultured in a medium and polyester synthase is recovered from the resulting culture.
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention is described in detail.
(1) Cloning of the Polyester Synthase Gene
The polyester synthase gene of the present invention is separated from a microorganism belonging to the genus Pseudomonas.
First, genomic DNA is isolated from a strain having the polyester synthase gene. Such a strain includes, e.g., Pseudomonas sp. Any known methods can be used for preparation of genomic DNA. For example, Pseudomonas sp. is cultured in a bouillon medium and then its genomic DNA is prepared by the hexadecyl trimethyl ammonium bromide method (Current Protocols in Molecular Biology, vol. 1, page 2.4.3., John Wiley & Sons Inc., 1994).
The DNA obtained in this manner is partially digested with a suitable restriction enzyme (e.g., Sau3AI, BamHI, BglII etc.). It is then ligated into a vector dephosphorylated by treatment with alkaline phosphatase after cleavage with a restriction enzyme (e.g., BamHI, BglII etc.) to prepare a library.
Phage or plasmid capable of autonomously replicating in host microorganisms is used as the vector. The phage vector includes, e.g., EMBL3, M13, gt11 etc., and the plasmid vector includes, e.g., pBR322, pUC18, and pBluescript II (Stratagene). Vectors capable of autonomously replicating in 2 or more host cells such as
E. coli
and
Bacillus brevis,
as well as various shuttle vectors, can also be used. Such vectors are also cleaved with said restriction enzymes so that their fragment can be obtained.
Conventional DNA ligase is used to ligate the resulting DNA fragment into the vector fragment. The DNA fragment and the vector fragment are annealed and then ligated to produce a recombinant vector.
To introduce the recombinant vector into a host microorganism, any known methods can be used. For example, if the host microorganism is
E. coli,
the calcium chloride method (Lederberg, E. M. et al., J. Bacteriol. 119, 1072 (1974)) and the electroporation method (Current Protocols in Molecular Biology, vol. 1, page 1.8.4 (1994)) can be used. If phage DNA is used, the in vitro packaging method (Current Protocols in Molecular Biology, vol. 1, page 5.7.1 (1994)) etc. can be adopted. In the present invention, an in vitro packaging kit (Gigapack II, produced by Stratagene etc.) may be used.
To obtain a DNA fragment containing the polyester synthase gene derived from Pseudomonas sp., a probe is then prepared. The amino acid sequences of some polyester synthases have already been known (Peoples, O. P. and Sinskey, A. J., J. Biol. Chem., 264, 15293 (1989); Huisman, G. W. et al., J. Biol. Chem., 266, 2191 (1991); Pieper, U. et al., FEMS Microbiol. Lett., 96, 73 (1992); Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992), etc.). Well-conserved regions are selected from these amino acid sequences, and nucleotide sequences coding for them are estimated to design oligonucleotides. Examples of such oligonucleotides include, but are not limited to, the sequence 5′-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3′ (SEQ ID NO:4) reported by Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992).
Then, this synthetic oligonucleotide is labeled with a suitable reagent and used for colony hybridization of the above genomic DNA library (Current Protocols in Molecular Biology, vol. 1, page 6.0.3 (1994)).
The
E. coli
is screened by colony hybridization, and a plasmid is recovered from it using the alkaline method (Current Protocols in Molecular Biology, vol. 1, page 1.6.1 (1994)), whereby a DNA fragment containing the polyester synthase gene is obtained. The nucleotide sequence of this DNA fragment can be determined in, e.g., an automatic nucleotide sequence analyzer such as 373A DNA sequencer (Applied Biosystems) using a known method such as the Sanger method (Molecular Cloning, vol. 2, page 13.3 (1989)).
After the nucleotide sequence was determined by the means described above, the gene of the present invention can be obtained by chemical synthesis or the PCR technique using genomic DNA as a template, or by hybridization using a DNA fragment having said nucleotide sequence as a probe.
(2) Preparation of Transformant
The transformant of the present invention is obtained by introducing the recombinant vector of the present invention into a host compatible with the expression vector used in constructing said recombinant vector.
The host is not particularly limited insofar as it can express the target gene. Examples are bacteria such as microorganisms belonging to the genus Alcaligenes, microorganisms belonging to the genus Bacillus, bacteria such as
E. coli,
yeasts such as the genera Saccharomyces, Candida etc., and animal cells such as COS cells, CHO cells etc.
If microorganisms belonging to the genus Alcaligenes or bacteria such as
E. coli
are us
Doi Yoshiharu
Fukui Toshiaki
Matsusaki Hiromi
Fish & Richardson P.C.
Japan Science and Technology Corporation
Saidha Tekchand
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