Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1999-07-23
2001-03-13
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C536S023100
Reexamination Certificate
active
06200788
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the amino acid sequence of a synthetase capable of synthesizing fatty acids in plants and the structure of DNA related to the same, that is to say, a fatty acid synthesizing enzyme protein having a specific amino acid sequence, and a gene coding for it. Cells can be transformed with such a gene and chimera genes in which an appropriate regulatory sequence (regulatory gene) has been inserted to control the amounts of saturated and unsaturated fatty acids in the cell.
BACKGROUND ART
Fatty acid synthases are known to be divided into two types; the enzymes in animals and yeasts are fatty acid synthetase complexes (FAS) in which a variety of enzymes are wholly linked as a complex having a single function (type I), while those in higher plant cells and procaryotes are of such type that each of the enzymes become independently disconnected outside the organisms (type II). An acyl carrier protein (ACP) which is a soluble protein is required for the synthesis of a fatty acid with the enzyme type II, and the fatty acids are synthesized as an acyl-ACP. The final product of the synthesis system is palmitoyl-ACP. The palmitoyl-ACP is further converted into stearoyl-ACP by chain elongation before desaturation with a soluble fatty acid desaturase (stearoyl-ACP desaturase) to lead to oleoyl-ACP. Palmitic acid and oleic acid are incorporated into polar lipid, and then the latter is further desaturated (J. Ohlrogge and J. Browse (1995) Lipid Biosynthesis. The Plant Cell, 7, p. 957-970).
The chain elongation enzyme which catalyzes the chain elongation from palmitoyl-ACP to stearoyl-ACP produces stearoyl-ACP from palmitoyl-ACP, malonyl-ACP and NADPH (J. Ohlrogge and J. Browse (1995) Lipid Biosynthesis. The Plant Cell, 7, p. 957-970). These reactions describe the total scheme of a series of enzyme reactions for the production of a stearoyl-thioester by the reduction, dehydration and further reduction of the condensation product of a palmitoyl-thioester and the C2 unit.
Lipid biosynthesis in plants has been studied very extensively (Browse et al., Annu. Rev. Plant Physiol. Mol. Biol. (1991) 42: 467-506). It has been elucidated from these researches that the production of stearic acid starting from palmitic acid in plant cells is a reaction catalyzed by the enzyme &bgr;-ketoacyl-ACP synthetase II (KASII). However, there has been described in the aforementioned publication the isolation of neither the enzyme KASII or its gene, and thus their sequences remain unknown.
Three isozymes of KAS have been found in chloroplast in plants. Among the two isozymes other than KASII, the isozyme KASIII catalyzes the initiation of the synthesis of an acyl chain, while the isozyme KASI catalyzes the elongation reaction of an acyl chain to the palmitoyl-ACP with 16 carbon atoms. A variety of mutants of enzymes involved in the lipid synthesis of plants have been isolated from Arabidopsis, among which the enzymes responsible for the desaturation reaction have been studied extensively. There have also been described for the KASII, the mutant of which has been designated as fab1. In this mutant, the KASII enzyme activity was lowered to 65%, and thus the palmitic acid content increased by 7% in leaves and 3% in roots (Wu et al., Plant Physiol. (1994) 106: 143-150). As regards the complete purification of the enzyme KASII, genes have been cloned from a castor bean (
Ricinus communis;
Japanese Patent Laid-Open Publication No. 500234/1994) and soybean (
Glycine max;
Japanese Patent Laid-Open Publication No. 501446/1995) on the basis of the amino acid sequences of the limitedly degraded peptide of the purified enzyme. In the above described publications, as regards the changes of fatty acids by transformation with these genes, the C16 fatty acid content on the expression of the gene of the castor bean in
E. coli
was decreased by ca. 20% thus corresponding to a little over 30% of the total fatty acid content, while on introducing the soybean gene into canola the palmitic acid content in the seeds was decreased by 0.8% and on introducing the gene into tobacco the palmitic acid content in the leaves was decreased by ca. 2%. In this connection, no sequence exhibiting distinct homology has been curiously found between the genes of the castor bean and soybean.
By the way, it has been known that in membrane lipids constituting biomembrane, the phase transition temperature varies primarily depending on the unsaturation degrees of the fatty acid linked to the lipid, and as a result the chilling resistance of the organism also varies. It is thought that the unsaturation degree of the membrane lipid is effectively increased with an enzyme such as fatty acid acyltransferase (PCT/JP 92/00024 (PCT/WO 92/13082)), fatty acid desaturase (PCT/JP 94/02288 (PCT/WO 95/18222)).
DISCLOSURE OF THE INVENTION
In consideration of the above described situations, the object of the present invention is to provide a gene of a protein having an enzyme activity which makes it possible to regulate or control the content of saturated fatty acids and unsaturated fatty acids in plant cells or microorganism cells and an enzyme protein as the expression product.
It is believed that if there is a protein having such an enzyme activity that the decrease of the enzyme activity responsible for the synthesis of fatty acids leads to the increase of the palmitic acid content in lipids of cells while the increase of enzyme activity leads to the increase of content of the fatty acids with 18 or more carbon atoms, the unsaturated fatty acid contents in the lipids is possibly increased for example as a result of the increase of content of the fatty acids with 18 or more carbon atoms due to the increase of the enzyme activity.
The present inventors have conducted earnest researches in order to solve the above described problems, and as a result, successfully isolated a gene which codes for an enzyme &bgr;-ketoacyl-ACP synthetase II (KASII) from cyanobacterium (
Anacystis nidulans
), and found that the introduction of the gene into
E. coli
confers the KASII producing ability whereby fatty acids having extended in chain length increase. The present invention has been accomplished on the basis of the finding.
That is, the present invention relates to the protein which has an amino acid sequence represented by SEQ ID NO. 2 or substantially the same amino acid sequence as the one represented by SEQ ID NO. 2 and exhibit the KASII enzyme activity.
The present invention also relates to the KASII enzyme gene coding for the protein which has an amino acid sequence represented by SEQ ID NO. 2 or substantially the same amino acid sequence as the one represented by SEQ ID NO.2 and exhibit the KASII enzyme activity.
Furthermore, the present invention relates to the recombinant vector containing the gene and the cells in which the gene has been introduced.
REFERENCES:
patent: 6-500234 (1994-01-01), None
patent: 6-504439 (1994-05-01), None
patent: 7-501446 (1995-02-01), None
patent: 92/03564 (1992-03-01), None
patent: 92/13082 (1992-08-01), None
patent: 93/10240 (1993-05-01), None
patent: 95/18222 (1995-07-01), None
Worsham, L., et al., “Early Catalytic Steps ofEuglena gracilisChloroplast Type II Fatty Acid Synthase,” Biochem. Biophys. Acta., vol. 1170, No. 1, pp. 62-71 (1993).
Garwin, J.L., et al., “Structural, Enzymatic, and Genetic Studies of &bgr;-Ketoacyl-Acyl Carrier Protein Synthases I and II ofEscherichia coli,” Journal of Biological Chemistry, vol. 255, No. 24, pp. 11949-11956 (1980).
Magnuson, K., et al., “The Putative fabJ Gene ofEscherichia coliFatty Acid Synthesis in the fabF Gene,” Journal of Bacteriology, vol. 177, No. 12, pp. 3593-3595 (1995).
Siggaard-Andersen, M., et al., “The fabJ-encoded &bgr;-ketoacyl-[acyl carrier protein] Synthase IV fromEscherichia coliis Sensitive to Cerulenin and Specific for Short-Chain Substrates,” Proc. Natl. Acad. Sci. USA, vol. 91, No. 23, pp. 11027-11031 (1994).
Shen, Z., et al., “Isolation ofVibrio harveyiAcyl Carrier Protein and the fabG, acpP, and fabF Genes Involved in Fat
Ferri Stefano R.
Toguri Toshihiro
Achutamurthy Ponnathapu
Foley & Lardner
Fronda Christian L.
Kirin Beer Kabushiki Kaisha
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