Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-12-28
2002-10-08
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S248000, C435S253600
Reexamination Certificate
active
06461839
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a microorganism belonging to the genus Labyrinthula having the ability to produce long chain polyunsaturated fatty acids, a culture of the microorganism, and a method of producing long chain polyunsaturated fatty acids having a carbon number of 20 or more and having two or more unsaturated bonds (herein, polyunsaturated fatty acid having two or more unsaturated bonds is referred to as “PUFA”, and the above-mentioned polyunsaturated fatty acid having a chain length of 20 or more is referred to as “LCPUFA”) using the microorganism.
BACKGROUND OF THE INVENTION
LCPUFAs such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have attracted attention for their various physiological activities within, and recently their use in functional foods, and the pharmaceutical field. However, these LCPUFAS are not contained at all in general vegetable oils, only DHA and EPA are found in fish oils such as sardine and tuna. Since various components of LCPUFAs have characteristic physiological functions, regulation of their compositions and contents are important for application in various fields. However, as a technique for this purpose, there is only a method employing enzymes for concentration and purification of DHA from fish oil, and there is no other converting means for regulation of its composition and content. Under these circumstances, there is a need to develop a variety of supply sources of LCPUFA-containing oil having a characteristic LCPUFA composition. One of these important methods is a method of production using microorganisms. Until now, in the production of &ggr;-linolenic acid, arachidonic acid and the like which are not present in general vegetable oil, a method comprising culturing Mortierella and collecting oil containing these PUFAs from these molds, has been employed. Further, methods have been developed which use marine microorganisms such as Thraustochytrium or Schizochytrium for the production of DHA, which is a LCPUFA and the object of the present invention.
In the case where LCPUFA is produced by a microorganism in this manner, a method of culturing where precursor fatty acids or oils are used as carbon sources is promising. Since using an oil or fatty acid which is a PUFA precursor is energetically favorable, there is the possibility that higher production efficiency can be obtained than in the case where sugars such as glucose are used as initial raw materials. Further, by converting known vegetable oils such as soybean oil or palm oil into PUFA-containing oil, the added value of these oils can be greatly increased. It may also be possible to modify physical properties such as boiling point by altering the fatty acid composition.
It has long been known that some microorganisms can be cultured with oil as a carbon source. There are many examples of such cultures of yeast and bacteria and the like. However, the object of these cultures was primarily the microbes themselves, i.e. cultures directed to the production of proteins. It is also known that Mortierella molds have the ability to convert &agr;-linolenic acid, which is a component found in vegetable oil, into eicosapentaenoic acid.
However, until now there has not been found any microorganism having the ability to convert oil or fatty acid as a carbon source, into LCPUFA, having a carbon number of 20 or more and two or more unsaturated bonds, such as DHA. Even with the production method using microorganisms such as Thraustochytrium which have the ability to produce DHA, sugars such as glucose must be used as a carbon source. In the case where oil or fatty acid is used as a carbon source, while these are incorporated by the microorganisms, desaturation or elongation of chain length hardly occurs. As a result, while there is an increase of microbes yielded, there is the disadvantage that the amount of produced LCPUFA including DHA is extremely reduced.
Further, the Labyrinthula of the present invention, does not fundamentally grow other than on the solid surface of a agar medium, and need to be monoxenic culture with other bacteria or yeast as feed for the sub-cubture. Since no effective method of culturing able to be used in the production of LCPUFA such as DHA has yet been found, screening for a strain with high LCPUFA productivity or examination of culture conditions for LCPUFA production has not been conducted until now.
Conventionally, there has been no microorganisms which can grow with oil as a carbon source, as well as can convert the oil incorporated by the microorganism to LCPUFA including DHA, nor a method for culturing such a microorganism. Thus, the object of this invention is to provide a microorganism having the ability to convert vegetable oil and the like which does not contain LCPUFA into oil containing LCPUFA such as DHA, a method of culturing this microorganism, and a method of producing LCPUFA-containing oil.
BRIEF SUMMARY OF THE INVENTION
The present inventors examined the ecological characteristics etc. of Labyrinthula among marine fungi as one microorganism which comprises LCPUFA such as DHA, and at the same time conducted research on an isolation/culture method exploiting these ecological characteristics. As a result, with respect to Labyrinthula, which is conventionally difficult to grow efficiently and stably, the inventors have found a efficient and selective isolation method of Labyrinthula from a variety of isolation sources such as seaweed and the fallen leaves of mangroves, wherein the method involves placing sample leaves on agar medium, on which Moraxella marine bacterium had been spread and grown. The inventors have already obtained Japanese Patent No. 2976027 in respect of this method. Further, at the same time, the inventors examined the application of oil and the like as a carbon source as a method for efficiently producing LCPUFA including DHA, and have studied the culture with oil and the like of various marine fungi capable of producing DHA including Labyrinthula obtained from various isolation sources, as well as examined the screening and culture conditions of a microorganism having the ability to convert such carbon source to oil which contains LCPUFA such as DHA, thereby completing the present invention.
That is, one aspect of the present invention is a microorganism belonging to the genus Labyrinthula that is able to be cultured with oil or fatty acid as a carbon source, and has the ability to produce long chain polyunsaturated fatty acid. Labyrinthula sp S3-2 is provided as a concrete example of this microorganism.
Further, another aspect of the present invention is a culture of microorganisms belonging to the genus Labyrinthula that can be obtained by culturing the above microorganism in a medium containing oil or fatty acid as a carbon source.
Further, another aspect of the present invention is a method for producing long chain polyunsaturated fatty acid comprising the steps of culturing the above microorganism in a medium containing oil or fatty acid as a carbon source, and collecting long chain polyunsaturated fatty acid from the culture.
Further, another aspect of the present invention is a method for reforming conventional plant oils by converting the oil to long chain polyunsaturated fatty acid by culturing a microorganism of the genus Labyrinthula, with oil as a carbon source.
The above-mentioned long chain polyunsaturated fatty acid includes fatty acids having a carbon number of 20 or more and two or more unsaturated bonds, for example, DHA.
DETAILED DESCRIPTION OF THE INVENTION
1. The Microorganism to be Used in the Present Invention
The microorganism to be used in the present invention is an isolated strain of Labyrinthula collected by the method described hereafter, and may be any strain which will grow with oil or fatty acid as a carbon source and has the ability to convert these to LCPUFA including DHA.
The oils as carbon source includes soybean oil, palm oil, olive oil, safflower oil and the like and fatty acids includes saturated and unsaturated fatty acids such
Kurane Ryuichiro
Nakahara Toro
Yamaoka Masakazu
Yokochi Toshihiro
Agency of Industrial Science and Technology
Srivastava Kailash C.
Tate Christopher R.
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