JPH07265065A - Transformant of copolymer by synthetic gene and production of copolymer - Google Patents

Abstract

PURPOSE:To produce a copolymer comprising a 3-hydroxybutyrate and a 3- hydroxyhexanoate from inexpensive fats and oils or a fatty acid as a raw material in high yield. CONSTITUTION:This transformant is obtained by transfecting or transducing a vector plasmid containing a synthetic gene for a copolymer comprising a 3-hydroxybutyrate and a 3-hydroxyhexanoate into a host microbial cell, especially the microbial cell of the genus Aeromonas. Furthermore, this transformant is prepared by transfecting or transducing the vector plasmid in which a beta- ketothiolase gene or an acetoacetyl-CoA reductase gene or both are integrated into the host microbial cell. This method for producing the copolymer is to use the resultant transformant.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a copolymer polyester and a microorganism which fermentatively synthesizes the same, and to produce a plastic-like polymer which is decomposed by the action of a microorganism in a natural environment (soil, river, sea). It is about the method.
More specifically, it relates to a transformant having an improved ability to fermentatively synthesize a copolymer polyester, and further to a method for producing a copolymer polyester using the transformant.

[0002]

2. Description of the Related Art Up to now, it has been known that a large number of microorganisms accumulate polyester in cells. As a typical example,
It is poly-3-hydroxybutyrate (hereinafter referred to as P (3HB)), which is a homopolymer of 3-hydroxybutyrate (hereinafter referred to as 3HB), and many producing bacteria thereof are known (for example, Davis (Dawes)). ) Et al.
In Microbiology and Biophysics (Adv. Microbiol. Biophys.), 14: 135,
See 1973). P (3HB) is a plastic that biodegrades in the natural environment (hereinafter referred to as biodegradable plastic), but when viewed as a polymer material, it is a homopolymer and therefore has high crystallinity and brittle properties. However, it was not practically sufficient. As a method for overcoming such drawbacks, screening of a strain producing a copolymer was carried out. According to Japanese Patent Laid-Open No. 63-226291, Pseudomonas oleovorans ATCC29.
By giving an alkane as a carbon source to 347, a copolymer (hereinafter referred to as P (3HA)) having 3-hydroxyalkanoate (hereinafter referred to as 3HA) having a carbon number of 6 to 12 as a monomer unit can be fermented and synthesized. It was However, since P (3HA) is a copolymer having a wide distribution of carbon atoms of 6 to 12 and has a large number of side chain methylene groups, it has a physical property of being a gel-like polymer at room temperature. . Therefore, the melting point of the plastic material is too low.

On the other hand, it has been proposed to incorporate structurally different monomer units other than 3HB as the monomer units constituting the polyester. JP-A-57-150393, JP-A-58-69225, JP-A-63-269989, JP-A-64-
According to JP48821 and JP-A-1-156320, by using Alcaligenes eutrophus and supplying a carboxylic acid having an odd number of carbon atoms as a carbon source, for example, propionic acid or valeric acid, 3HB and β-hydroxyvarie are obtained. A copolymer (hereinafter referred to as P (3HB-CO-3HV)) with a rate (hereinafter referred to as 3HV) is obtained. Similarly, by providing 4-hydroxybutyric acid or γ-butyrolactone as a carbon source, a copolymer of 3HB and 4-hydroxybutyrate (4HB) (hereinafter referred to as P (3HB-
CO-4HB)) is obtained. And this P (3HB-CO-3HV) and P
(3HB-CO-4HB) improves the brittleness of P (3HB) by increasing the component composition of 3HV and 4HB, and has the property that it has a wide range of workability as a plastic. Is attracting attention as a flexible plastic. However, in order to fermentatively synthesize such a copolymer in a microorganism, a carbon source (usually glucose, sugars such as sucrose or acetic acid, methanol, ethanol, glycerin, etc.) for making a unit component of 3HB, Three
Intermediates for making the unit components of copolymers such as HV and 4HB (propionic acid, valeric acid when making 3HV, 1,4-butanediol, γ when making 4HB
-Butyrolactone) and must be added to the medium,
Moreover, since such a raw material is expensive, it has a drawback that the culture cost is high.

On the other hand, as a method for overcoming the disadvantages of P (3HB-CO-3HV) and P (3HB-CO-4HB), it has been proposed to use a strain of the genus Aeromonas.
According to Japanese Unexamined Patent Publication No. 5-93049, strains FA440 and OL-338 belonging to the genus Aeromonas are used, and 3-hydroxybutyrate and 3-hydroxybutyrate are used as a carbon source with a very inexpensive natural oil and fat.
-Hydroxyhexanoate copolymer (hereinafter P (3H
B-CO-3HHx) has been obtained. Since this copolymer has high plasticity and flexibility, P (3HB-
CO-3HV) and P (3HB-CO-4HB) are not only superior in polymer physical properties, but also do not require the addition of expensive raw materials (carbon source) to the medium, so production at low cost is possible. Was expected However, the main characteristic of Aeromonas strains is that polyester synthesis proceeds from 3-hydroxyacyl CoA, which is an intermediate metabolite of the β-oxidation pathway of fatty acids, and acetyl CoA, which is the main product of β-oxidation, is effective. Is not used for P.
It has been revealed that intracellular polyester accumulation is suppressed due to, for example, the low activity of HB-CO-3HHx) synthase. Therefore, there remains a problem that productivity is low and carbon source yield is low even if inexpensive natural oils and fats can be used as a carbon source. Therefore, an object of the present invention is to provide P (3HB-CO-3HHx) of Aeromonas sp.
P (3HB-CO-3H to enhance the synthase activity of
It is intended to provide a transformant transformed with the Hx) synthase gene or the like. Another object of the present invention is to use P (3HB-CO-3H) which uses such a transformant.
It is to provide a method for producing a copolymer such as Hx).

[0005]

Means for Solving the Problems Although the strains of the genus Aeromonas are capable of accumulating excellent polyesters, the present inventor says that they cannot be industrially used because of the low accumulation amount and carbon yield in the bacterial cells. Focusing on the shortcomings, we conducted diligent research in order to increase the accumulated amount in these cells and the carbon yield. As a result, we discovered that a transformant strain in which a group of genes involved in the polyester synthesis of Aeromonas was transferred into an appropriate host synthesized a significant amount of polyester and had a very good carbon yield. Then, the present invention was completed. That is, the gist of the present invention is (1) transformation obtained by transferring into a host cell a vector plasmid containing a synthetic gene of a copolymer composed of 3-hydroxybutyrate and 3-hydroxyhexanoate. Body, (2)
Furthermore, the transformant according to (1), which is obtained by transferring a vector plasmid in which the β-ketothiolase gene and / or acetoacetyl CoA reductase gene is incorporated into the host cells, and (3) the host strain is a strain of the genus Aeromonas. (1) or (2) characterized in that
The transformant described in 1., (4) The vector plasmid is p
LA2917, pLA2905, pLA2910, pL
A2901, pRK248, pRK290, pLAFR
1, pVK100, pVK101, pVK102 is selected from the group consisting of (1)
~ The transformant according to any one of (3), and (5)
By culturing the transformant described in any one of (1) to (4), a copolymer composed of 3-hydroxybutyrate and 3-hydroxyhexanoate is accumulated. A method for producing a polymer. The present invention will be described in detail below.

1. Method for Obtaining Transformant Transformed by Copolymer Synthetic Gene The following method is used to obtain the transformant of the present invention transformed by the P (3HB-CO-3HHx) synthetic gene. Is convenient. First, P (3HB-C
A gene library was prepared from a strain capable of synthesizing O-3HHx), and then the gene library was put into a synthetic ability-deficient mutant strain lacking the synthetic gene of P (3HB-CO-3HHx).
By selecting a strain in which the synthetic ability of (3HB-CO-3HHx) is recovered, the transformant of the present invention transformed with the synthetic gene of P (3HB-CO-3HHx) can be obtained.

In order to prepare a gene library from a strain capable of synthesizing P (3HB-CO-3HHx), chromosomal DNA is extracted and purified from a strain capable of synthesizing P (3HB-CO-3HHx) and cleaved with a restriction enzyme. After that, a DNA fragment of an appropriate length is separated and ligated with a vector plasmid.

The strain capable of synthesizing P (3HB-CO-3HHx) may be any strain of the genus Aeromonas, for example Aeromonas caviae FA440.
Examples thereof include Aeromonas hydrophila OL-338 strain.

As a method for extracting and purifying chromosomal DNA from a strain capable of synthesizing P (3HB-CO-3HHx),
For example, the method of Marmur et al. (Journal of Molecular Biology).
Biology), vol. 3, p. 208, 1961). The restriction enzyme used for cutting the chromosome may be any restriction enzyme corresponding to the vector plasmid, and can be easily obtained from Takara Shuzo, Pharmacia, Bio-Rad and the like. In addition, DNA of an appropriate length is extracted from the cleaved DNA fragment.
When using an ordinary vector, it means about 5000 to 20000 base pairs, and when using a cosmid or phage vector, it means about 15000 to 30000 base pairs.
As a method for extracting an appropriate length from a DNA fragment, a method using a sucrose density gradient or a method using an agarose gel (Moleculr Cloning
), 150 pages, Cold Spring Harbor Laboratory published (1
982))), for example, RK2
Vector plasmids having a region involved in replication of pRK229, pRK229, pRK248 (see Journal of Bacteriology, 141, 219, 1980), pRK290 (Proceeding National Academy Science of USA, Volume 77, 7
347, 1980), pLA2901, pLA29.
05, pLA2910, pLA2917 (Journal
Of Bacteriology, 161, 955, 198.
5 years), pLAFR1 (Gene, 18 volumes, 2
P. 89, 1982), pVK100, pVK10.
1, pVK102 (Plasmid), 8 volumes, 45
(Page, 1982), or a region related to the replication of RK2 that has been cut out with a restriction enzyme and processed.

The vector plasmid is cleaved with a restriction enzyme corresponding to the nucleotide sequence of the restriction enzyme used for fragmenting the chromosomal DNA, and then ligated with the chromosomal DNA fragment having an appropriate length. This ligation may be performed using T4 DNA ligase, for example, using a ligation kit (Takara Shuzo Co., Ltd.). This allows
It is possible to obtain a mixture (hereinafter referred to as a gene library) in which various gene fragments are ligated to a vector plasmid. Alternatively, instead of using a chromosomal DNA fragment of appropriate length in a gene library, P (3H
B-CO-3HHx) -synthesizing mRNA from a strain capable of synthesizing it, purifying the mRNA, and then synthesizing a cDNA fragment therefrom (molecular cloning (Molec
ular Cloning), Cold Spring Harbor Laboratory published (1
982))). Alternatively, the gene library can be purified in a large amount after once transforming or transducing Escherichia coli (Molecular Cloning).
g), Cold Spring Harbor Laboratory
(Cold Spring Harbor Laboratory) Publishing (1982)
reference).

Next, P (3HB-CO-3HHx)
In order to obtain a mutant strain deficient in the synthetic gene (hereinafter referred to as a synthetic ability deficient mutant strain), P (3HB-CO-3H
After mutating a strain capable of synthesizing Hx), the strain can grow in a medium containing an oil-and-fat-related substance having 6 or more carbon atoms as the sole carbon source, and P ( 3HB-CO-3HHx) in a medium capable of synthesizing P
A strain that cannot synthesize (3HB-CO-3HHx) may be selected.

Here, the mutagenesis treatment may be carried out by any method, for example, mutation by ultraviolet rays, X-rays, γ rays, N-
Any treatment such as treatment with a mutagen such as methyl-N′-nitro-N-nitrosoguadinine or ethylmethanesulfonic acid may be used.

The medium containing only an oil-and-fat-related substance having 6 or more carbon atoms as a carbon source is a medium containing only an oil-and-fat-related substance having 6 or more carbon atoms as a carbon source.
(Davis) minimal medium or M9 minimal medium supplemented with the minimum necessary vitamins and minerals for growth as the basic medium,
It is a medium in which glucose is replaced with a fat-and-oil-related substance having 6 or more carbon atoms. A medium capable of synthesizing P (3HB-CO-4HHx) by using an oil-and-fat-related substance having a carbon number of 6 or more as a sole carbon source does not contain any other than the oil-and-fat-related substance having a carbon number of 6 or more as a carbon source, and In contrast, a medium with a sufficiently limited nitrogen source, for example, Davis minimal medium or M9 minimal medium supplemented with the minimum necessary vitamins and minerals for growth is used as the basic medium, and glucose has 6 carbon atoms.
It is a medium in which nitrogen sources such as ammonium sulfate are reduced to ⅕ or less of the usual medium by replacing the above-mentioned fats and oils-related substances.

Natural fats and oils such as corn oil, soybean oil, safflower oil, sunflower oil, olive oil, coconut oil, palm oil, rapeseed oil, fish oil, whale oil, pig oil, etc. Fatty acids such as beef oil, eg, hexanoic acid, decanoic acid, octanoic acid, lauric acid, oleic acid, palmitic acid, linolenic acid, linoleic acid, myristic acid, etc., or esters of these fatty acids, or alcohols, eg, octanol , Lauryl alcohol, oleyl alcohol, palmityl alcohol, etc., and esters of these alcohols.

Regarding the strains subjected to the mutation treatment, P (3H
As a method for examining the presence or absence of (B-CO-3HHx) synthesis, for example, a method of staining with Sudan Black B (Archives of Biotechnology)
technology), 71, 283, 1970), a method of examining accumulation by a phase contrast microscope, or the like.
In addition, the mutant strain was subjected to a sucrose or Percoll concentration gradient method (Archives of Biotechnology).
Biotechnology), 143, 178, 1985).
Alternatively, the mutant strain lacking in synthetic ability may be concentrated before screening. And the copolymer P (3HB-CO-3HH
To obtain a transformant containing the synthetic gene of x),
After transforming or transducing the gene library into the synthetic capacity-deficient mutant, P (3HB-CO-3HHx) can be synthesized in a medium capable of synthesizing P (3HB-CO-3HHx) using only an oil-and-fat-related substance having 6 or more carbon atoms as the sole carbon source. -3HHx) -synthesizing ability-repaired bacterial strain (hereinafter referred to as synthetic-ability repairing strain) may be selected.

The method for transforming the gene library into the synthetic defective mutant strain may be a commonly used method, for example, calcium chloride method (Journal of Molecular Biology,
53, p. 159, 1970), Rubidium chloride method (Methods in Enz
ymology), 68, 253, 1979), low pH method (see Gene Operation Manual (Kodansha Scientific Publishing)) and the like. In addition, transduction when using a cosmid vector or a phage vector may be a commonly used method, for example, commercially available in
A vitro packaging kit (for example, manufactured by Takara Shuzo Co., Ltd.) can be used. In addition, the vector plasmids are trfA, trfB, which are present in the plasmid RK2,
When the rlx region is included, the joining method (Journal of Bacteriology, 161, 955, 1985, 1985)
(See year). P (3HB-CO-3HH) with oil and fat-related substances having 6 or more carbon atoms as the sole carbon source
The medium capable of synthesizing x) may be the same as the medium used for obtaining the mutant strain lacking the synthetic ability described above, and P (3HB-C
The method for investigating the presence or absence of the synthesis of O-3HHx) may be the same as the method used for obtaining the synthetic-deficiency mutant strain described above.

Further, β-ketothiolase is acetyl C
Acetoacetyl CoA was synthesized from oA, and acetoacetyl CoA reductase was converted from acetoacetyl CoA to β.
-Hydroxybutyl CoA is synthesized. This β-hydroxybutyl CoA is 3HB for P (3HB) synthesis.
It is a monomer unit of. Therefore, in cells having a low β-ketothiolase and / or acetoacetyl CoA reductase activity, acetyl CoA, which is synthesized in a large amount during the process in which the cells metabolize the carbon source, is converted into P (3HB-CO-3).
HHx) cannot be fully used as a monomer unit for 3HB in synthesis. On the other hand, P (3HB-CO-3HHx)
In addition to the synthetic gene, the transformant into which the β-ketothiolase and / or acetoacetyl CoA reductase gene had been introduced had an increased β-ketothiolase and / or acetoacetyl CoA reductase activity, and thus the acetyl CoA was reduced to P (3HB -CO-3HHx) can be sufficiently used as a monomer unit of 3HB for synthesis. Therefore, when a substance other than a carbon source having 6 or more carbon atoms is used as the only carbon source and the medium is cultured in a nitrogen source-limited medium, P (3
HB) can be accumulated in high concentration. These genes are located close to the P (3HB-CO-3HHx) synthetic gene. Therefore, among the synthetic ability repair strains described above, a transformant having a synthetic gene of P (3HB-CO-3HHx) and no β-ketothiolase and acetoacetyl CoA reductase genes and P (3 3HB-CO-3HHx), and a transformant having both a β-ketothiolase gene and an acetoacetyl CoA reductase gene, or one of them.

Finally, in order to select the transformant of the present invention, the synthetic ability-repairing strain was cultivated in a medium in which a substance other than the oil and fat-related substance having 6 or more carbon atoms was the only carbon source and the nitrogen source was restricted. After that, a strain that accumulates the polymer (3HB) at a high concentration may be selected. Examples of carbon sources other than the oil and fat-related substances having 6 or more carbon atoms include sugars such as glucose, sucrose, maltose, fructose, galactose,
Starch, starch hydrolysates, lower alcohols such as methanol, ethanol, propanol,
Butanol, etc., glycerin, organic acids such as acetic acid, acetoacetic acid, citric acid, succinic acid, tartaric acid, lactic acid,
Gluconic acid and the like can be mentioned.

The method of examining the amount of accumulation may be any of the methods already described, for example, a method of staining with Sudan Black B or a method of examining the accumulation with a microscope, or a method of recovering the polymer and measuring the weight thereof, for example, After completion of the culturing, the bacterial cells are washed with distilled water and methanol, and sterilized and dried, and the dried bacterial cells are subjected to an extraction treatment with chloroform or the like, and after centrifugation, the bacterial cell components are removed by filtration or the like. Alternatively, methanol may be added to the extract to precipitate and recover the copolymer. Furthermore, the β-ketothiolase and acetoacetyl CoA reductase activities of transformants may be directly measured (for example, Senia (Sen
ior) et al., Biochemical Journal
al Journal), 125, 55, 1971).

In the transformant using a strain of the genus Aeromonas as a host, most of the genes on the vector plasmid placed in the host have a large copy number inserted into the chromosome. Therefore, these transformants can be used directly for the production of P (3HB-CO-3HHx).

It is also possible to produce P (3HB-CO-HHx) in strains other than the genus Aeromonas. In this case, for example, after extracting and purifying the plasmid from the transformant, after putting it in E. coli, the plasmid was purified,
The plasmid may be put in the target host as it is or after being re-ligated to a vector that can be expressed in the target host.

The method for purifying a plasmid from the genus Aeromonas and the method for purifying a plasmid from Escherichia coli may be a conventional method, for example, the alkaline method (Methods in Enzymology, 100 volumes, 24).
3 pages, published in 1983) can be used. In addition, the method of inserting the plasmid into Escherichia coli or a target host is the same as the method for transforming the gene library into the synthetic ability-deficient mutant strain, for example, calcium chloride method, rubidium chloride method, low pH method, etc. Transduction method when using cosmid vector or phage vector, for example, commercially available in
Examples include a joining method such as a method using a vitro package kit.

The target host may be any microorganism as long as it is a microorganism capable of metabolizing oil-and-fat-related substances having 6 or more carbon atoms,
As the bacterium, a Gram-negative bacterium, for example, genus Esherihia, Pseudomonas
as) genus, Alcaligenes genus, Agrobacterium genus, Flavobacterium (Fla
Bobacterium) genus, Vibrio (Vibrio) genus, Enterobacter (Enterobacter) genus, Rhizobium (Rhizobium) genus, Gluconobacter (Gluconobacter) genus, such as gram-positive bacteria, for example, Bacillus (Bacillus) genus, Clostridium (Clostridium) genus, Lactobacillus
s) genus, Corynebacterium genus, Artherobacter genus, Streptococcus genus, etc., and actinomycetes such as Streptomyces genus, Actinomyces genus
Inomyces genus, Nocardia genus and the like. Alternatively, yeast or mold may be used.

2. Method for Culturing Copolymer and Extraction and Purification Method Using Transformant In order to fermentatively synthesize P (3HB-CO-3HHx), the transformant is provided with a carbon source containing an oil and fat-related substance having 6 or more carbon atoms. , A nutrient medium other than carbon sources, such as nitrogen sources, inorganic salts, or other organic nutrient sources, which is restricted (p
H is neutral to slightly alkaline), for example, a nitrogen source of 0.
By culturing aerobically in a medium limited to 5 to 8% at a culture temperature of 25 to 38 ° C for 1 to 4 days, P (3
HB-CO-3HHx) may be accumulated in the cells as granules, and then the copolymer may be recovered. Alternatively, in order to fermentatively synthesize P (3HB-CO-3HHx), the transformant is provided with a carbon source containing an oil-and-fat-related substance having 6 or more carbon atoms or a carbon source containing no oil-and-fat-related substance having 6 or more carbon atoms. , A medium that does not limit the nitrogen source, which is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources, the pH is neutral to slightly alkaline, and the culture temperature is in the range of 25 to 38 ° C.
After aerobically culturing for 1 to 4 days, a carbon source containing an oil-and-fat-related substance having 6 or more carbon atoms is given, and a nitrogen source that is a nutrient source other than the carbon source, an inorganic salt, or any other organic nutrient source, For example, in a medium in which the nitrogen source is limited, the pH is neutral to slightly alkaline, the culture temperature is in the range of 25 to 38 ° C, and aerobically 1 to 4
By culturing for one day, P (3HB-CO-3HHx)
The granules may be accumulated in the cells and then the copolymer may be recovered. In addition, the culture may be a semi-batch culture or continuous culture.

When limiting the nitrogen source as a nutrient source other than the carbon source, it is preferable to give the carbon source at least 5 times the weight of the nitrogen source. Further, the carbon source containing the oil or fat-related substance having 6 or more carbon atoms is composed of the oil or fat-related substance having 6 or more carbon atoms and the carbon source other than the oil or fat-related substance.
It may be appropriately changed according to the composition of B-CO-3HHx). The higher the amount ratio of oil-related substances having 6 or more carbon atoms, the more 3
A copolymer rich in HHx can be obtained.

As the oil-and-fat-related substances having 6 or more carbon atoms, as described above, natural oils and fats such as corn oil, soybean oil, safflower oil, sunflower oil, olive oil, coconut oil, palm oil, rapeseed oil, Fish oil, whale oil, pig oil, cow oil, etc., fatty acids, such as caproic acid, decanoic acid, octanoic acid, lauric acid, oleic acid, palmitic acid, linolenic acid, linoleic acid, myristic acid, etc., or esters of these fatty acids, Alternatively, alcohols such as octanol, lauryl alcohol, oleyl alcohol, palmityl alcohol and the like, or esters of these alcohols and the like can be mentioned.

Carbon sources other than the oil-and-fat-related substances having 6 or more carbon atoms include sugars such as glucose, sucrose,
Maltose, fructose, galactose, starch,
Lower alcohols such as starch hydrolysates, for example,
Methanol, ethanol, propanol, butanol and the like, glycerin and organic acids, for example, acetic acid, acetoacetic acid, propionic acid, citric acid, succinic acid, tartaric acid, lactic acid, gluconic acid and the like can be mentioned. As the nitrogen source, ammonium salts, for example, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium succinate, ammonium citrate, etc., nitrates, for example, sodium nitrate, potassium nitrate, etc., ammonia water, ammonia gas, urea, amino acids, peptides, Examples include nucleic acid-related substances. As the inorganic salt, a phosphate, for example,
Potassium phosphate, sodium phosphate, calcium phosphate, etc., potassium salt, such as potassium chloride, calcium salt, such as calcium chloride, magnesium salt, such as magnesium chloride, magnesium sulfate, sodium salt, such as sodium chloride, carbonic acid Examples thereof include manganese salts such as sodium, for example, manganese sulfate, manganese chloride, etc., and heavy metal salts, for example, chlorides, sulfates, nitric oxides, etc. of iron, zinc, copper, cobalt and the like.

Other organic nutrient sources include amino acids,
For example, glycine, alanine, serine, threonine, proline, aspartic acid, ornithine, proline, tyrosine and the like, peptides, for example, peptone, casamino acid, glycylglycine, alanylalanine, alanylglycine, vitamins, for example, Natural product-derived substances such as vitamin B1, vitamin C, vitamin B12 and biotin, for example, yeast extract, meat extract, malt, malt extract, corn steep liquor, soybean powder, rice bran, soybean protein decomposed product, casein decomposed product, etc. Nucleic acid-related substances, for example,
Examples include uracil, cytosine, thymine, adenine, purine, nucleotides composed of these and ribose, deoxyribose, and the like.

The fermentation-synthesized copolymer can be recovered from the cells by a conventional method. For example, after culturing, the bacterial cells are washed with distilled water and methanol, and sterilized and dried, and the dried bacterial cells are subjected to an extraction treatment with chloroform or the like, centrifuged, and then filtered to remove bacterial cell components. Then, methanol can be added to the obtained extract to precipitate and recover the copolymer.

[0030]

The present invention will be described below with reference to examples, but the present invention is not limited to these examples. Example 1 First, a mutant strain defective in polyester synthesis was obtained. As a strain of the genus Aeromonas, Aeromonas caviae (A
eromonas caviae) FA440 strain was used. Aeromonas caviae FA440 was subjected to N-methyl-N-nitrosoguanidine treatment (see Biotechnology Experimental Manual, page 66, Japan Society for Biotechnology, edited by Baifukan Publishing Co., Ltd.) for mutation. Then, MM (0.5% glucose, 0.01% magnesium sulfate, 0.3% dipotassium phosphate, 0.7% monopotassium phosphate, 0.1% ammonium sulfate) was used so that there were about 100 colonies per plate. , 0.01%
The yeast extract, pH 7.0) agar medium was coated with the cell suspension and incubated at 30 ° C. for 5 days. MM agar medium and MP for 10,000 colonies obtained
A (0.15% palmitic acid, 0.01% magnesium sulfate, 0.7% dipotassium phosphate, 0.3% monopotassium phosphate, 0.1% ammonium sulfate, 0.01% yeast extract, 1 ml / 100 ml Triton X-100) agar and MNP (0.15% palmitic acid, 0.01
% Magnesium sulfate, 0.7% dipotassium phosphate, 0.
3% monopotassium phosphate, 0.01% ammonium sulfate,
Each colony was replicated in 0.01% yeast extract, 1 ml / 100 ml Triton X-100, pH 7.0) agar medium. Then, the plate of the MNP agar medium was incubated at 30 ° C. for 5 days, and then the polyester was dyed by the Sudan Black B method. That is,
95% containing 200 mg / liter Sudan Black B
Pour 10 ml of the ethanol solution of each onto each plate, and
After standing for 0 minutes, the solution was removed and the plate was washed once with a 95% ethanol solution. As a result, 5 colonies that grew in MM medium and MPA medium and were not stained blue in MNP medium were obtained. They were observed with a phase contrast microscope (Olympus BH2, 1000 times), and it was confirmed that polyester did not accumulate. One of these polyester synthetic gene-deficient strains (synthetic ability-deficient mutant strain) is called AC004.

Example 2 Next, an Aeromonas gene library was prepared.
Aeromonas caviae FA440 strain was mixed with 200 ml of LB medium (1% yeast extract, 0.5% bactopeptone, 0.5% sodium chloride, 0.1% glucose, p.
After culturing in H7.2) at 30 ° C for 24 hours, 10000
The cells were collected by centrifugation at rpm for 10 minutes. Then, 50 ml of 0.01 M tris (hydroxymethyl) aminomethane-0.001 M disodium ethylenediaminetetraacetate solution (hereinafter referred to as TE solution)
Suspended in, and after adding 50 mg of lysozyme chloride, 4
It was left at 30 ° C. for 30 minutes. Further, 50 ml of TE solution containing 1% sodium lauryl sulphate is added,
It was left for 20 minutes at ℃. After cooling the liquid to room temperature, 10
0 ml phenol-chloroform solution (volume ratio 1:
After 1) was added and gently stirred, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was gently taken out with a Pasteur pipette. Then, instead of the phenol-chloroform solution (volume ratio 1: 1), the same operation was performed using 100 ml of chloroform solution, and the supernatant was gently taken out with a Pasteur pipette and placed in a beaker. Aerosol Cavier FA440 was obtained by winding the precipitated chromosomal DNA with a glass rod while gradually adding ethanol cooled to -20 ° C to the supernatant.
Was obtained.

The obtained chromosomal DNA was digested with the restriction enzyme Sau.
After partial digestion with 3AI, dialysis tube method (Molecular cloning, page 164, Cold Spring Harbor Laboratory (Cold Sp)
ring Harbar Laboratory) (see publication), 5-15
A kilobase pair DNA fragment was separated and purified. On the other hand, the vector plasmid is pLA2 which can be expressed in Aeromonas sp.
917 was used. This plasmid is used as a restriction enzyme BglII.
Cleavage with, dephosphorylation treatment (molecular cloning
(Molecular cloning), p. 133, Cold Spring Harbar Labora
tory)), and then ligated with the purified chromosomal DNA fragment (referred to as gene library solution).

Transformation was carried out using the calcium chloride method. That is, 1 platinum loop of the Escherichia coli DH1 strain was inoculated into 1 ml of LB medium, and cultured at 37 ° C. with shaking for 24 hours. And
Add 0.05 ml of culture medium to 5 ml of LB medium,
Incubation was continued for another 2 hours at ℃. After that, add 1000 to the culture solution.
After collecting the cells by centrifugation at 0 rpm for 10 minutes,
The suspension was suspended in 2 ml of a 0.1 ml / l calcium chloride solution and left at 4 ° C. for 2 hours. After that, 10,000 rpm again
After centrifuging at 10 minutes to collect the cells, 0.1mo
l / l calcium chloride-suspended in 0.01 ml / l tris (hydroxymethyl) aminomethane solution 0.2 ml, and added 0.01 ml gene library solution,
It was left for another 45 minutes. Then, after standing at 42 ° C. for 2 minutes, 1.8 ml of LB medium was added, and the mixture was allowed to stand at 37 ° C. for another 2 hours to collect 25 mg / m 2 of the bacterial cells.
It was spread on LB agar medium containing 1 liter of tetracycline and incubated at 37 ° C. for 24 hours. as a result,
About 10 ⁵ colonies resistant to tetracycline were obtained. From these obtained tetracycline-resistant colonies, the alkaline method (Methods in Enzymology, 100, 2
P.43, published in 1983), after roughly purifying the plasmid, followed by ultracentrifugation (molecular cloning).
(Molecular cloning), p. 150, Cold Spring Harbar Laboratories
(atory) publication) was used to purify the plasmid. In this way, a purified gene library was obtained.

Example 3 Aerosol obtained in Example 1 as a strain of Aeromonas
Anti-monas caviae AC004 strain
When I examined the resistance to biogenic substances,
Sensitive to kanamycin and to ampicillin
Showed sex. So, using tetracycline as a marker
Used. Aeromonas caviae AC004 fruit
The gene library prepared in Example 2 was used as the large library of Example 2.
As with the case of Enterobacter DH1, the trait was determined by the calcium chloride method.
After conversion, spread on LB medium containing tetracycline.
It was covered and incubated at 30 ° C. overnight. as a result,
10 ^FiveColonies resistant to about 4 tetracyclines
Got

Each of the obtained tetracycline-resistant colonies was replicated with a toothpick onto MM agar medium containing tetracycline and 10,000 MNP agar medium containing tetracycline. Plates of MM agar containing tetracycline were incubated at 30 ° C for 3 days and then stored in a refrigerator at 4 ° C. On the other hand, the plate of MNP agar medium containing tetracycline was incubated at 30 ° C. for 5 days, and
It was dyed by the above-mentioned Sudan Black method. As a result, 3 colonies stained blue with Sudan Black were obtained. Further, it was observed using a phase contrast microscope as in Example 1, and it was confirmed that polyester granules had accumulated. Thus, three colonies in which the fragment containing the P (3HB-CO-3HHx) synthetic gene was cloned were obtained. One colony of them was designated as AC118
Call it a stock.

Aeromonas caviae FA440, A
C004 and AC118 were added to 10 ml of LB medium (AC11
8 was cultivated with shaking in an Erlenmeyer flask containing LB medium containing tetracycline) at 30 ° C. for 24 hours. afterwards,
Each bacterium was collected and suspended in 1 ml of physiological saline, and 100 ml of M9G (0.6% disodium phosphate, 0.3% monopotassium phosphate, 0.05% sodium chloride, 0. (01% ammonium chloride, 0.024% magnesium chloride, 0.001% calcium chloride, 1% glucose, 0.01% yeast extract)
Culture was carried out at 0 ° C. for 48 hours with shaking. 1 ml in the obtained culture solution
The cells were centrifuged to collect the cells, and the accumulation state of the cells was examined by a phase contrast microscope. Bacteria obtained from the remaining 99 ml were collected, washed twice with distilled water, washed once with ethanol, collected, dried and then weighed. Then, the dried cells were suspended in 15 ml of chloroform and left at 50 ° C. for 2 hours. And Whatman No.
Filter with the filter paper No. 1 and further use a membrane filter (manufactured by Advantech, PTFE type T050A04
The cell residue was removed by filtration through 7A). Furthermore, 40 ml of methanol was added to this chloroform solution to precipitate the polyester. The precipitate was centrifuged to remove the supernatant, and then dried to obtain polyester.

After measuring the total weight of the polyester, 5 to 7 mg of the obtained polyester was dissolved in 1 ml of chloroform, 0.85 ml of methanol and 0.15 ml of concentrated sulfuric acid were added, and the bottle was tightly sealed and left at 100 ° C. for 140 minutes. By doing so, a methyl ester form of a polymer decomposition product was obtained.
This was sufficiently cooled with ice water at 0 ° C., 0.5 ml of distilled water saturated with ammonium sulfate was added, and the mixture was vigorously stirred. After standing to separate into two layers, the lower organic layer was carefully extracted and its composition was analyzed. Capillary gas chromatography was used for the analysis. The capillary gas chromatograph was performed using HP5890II (manufactured by Hewlett Packard). The column used is J & W
Fused Silica Capillary Column DB-
5 (column inner diameter 0.25 mm, liquid film thickness 0.25 μm, column length 30 m). In addition, the temperature is the initial temperature 60 ℃
At 30 ° C. for 30 minutes, then heated at a rate of 8 ° C./minute, and a final temperature of 240 ° C. for 3 minutes. The results obtained are shown in Table 1.
(Upper). In Table 1, only AC118 was able to accumulate P (3HB) with glucose as the sole carbon source. In addition, the method of Senior et al. (Biochemical Journal, 125, 55
Page, 1971), the β-ketothiolase activity and the acetoacetyl CoA reductase activity were measured. As a result, the AC118 strain had 2-fold and 1.5-fold higher activities than the FA440 strain, respectively. Therefore, A
The DNA fragment cloned into the C118 strain contains 3-
There are a synthetic gene of a copolymer composed of hydroxybutyrate and 3-hydroxyhexanoate, a β-ketothiolase gene, and an acetoacetyl-CoA reductase gene, and the corresponding enzyme depending on the expression of these genes. It can be concluded that the activity was increased.

[0038]

[Table 1]

Example 4 Aeromonas caviae FA440, AC004, A
C118 was shake-cultured for 24 hours at 30 ° C. in an Erlenmeyer flask containing 10 ml of LB medium (AC118 is LB medium containing tetracycline). After that, each of the bacteria was collected and suspended in 1 ml of physiological saline,
It was placed in 0 ml of MNP medium and cultured with shaking at 30 ° C. for 48 hours. 1 ml of the obtained culture solution was centrifuged to collect bacterial cells, and the accumulation status of the bacterial cells was examined by a phase contrast microscope. Bacteria obtained from the remaining 99 ml were collected, and the polyester was purified and analyzed in the same manner as in Example 3. The obtained results are shown in Table 1 (lower side).

The FA440 strain, which is the parent strain, has a very low polyester content and polyester yield, whereas
In the C118 strain, the polyester content reached 60%, and the polyester yield was significantly improved. And, the polyester content obtained in this experiment is
Not only is a method using utrophus used to give a carboxylic acid having an odd number of carbon atoms as a carbon source to obtain P (3HB-CO-3HV), but a very inexpensive oil-like substance is used as carbon. It was proved to be an extremely excellent method from the viewpoint of production cost as well.

The Aeromonas used in this Example
The caviae FA440 strain has been deposited with the Institute of Microbial Science and Technology of the Institute of Industrial Science (Microtechnical Lab) on June 11, 1991, and the deposit number is Micromachine Lab Article No. BP-3432. In addition, the Aeromonas caviae AC118 strain obtained in this example has been deposited at the Institute of Biotechnology, Institute of Industrial Science (Life Research Institute) on February 2, 1994, and the deposit number is the Life Science Article. It is No. BP-4544. In addition, plasmid pLA2917 was each deposited as American Type Culture Collection (ATCC) deposit number 37355.

[0042]

EFFECT OF THE INVENTION By using the transformant of the present invention and the production method of the present invention, a copolymer composed of 3-hydroxybutyrate and 3-hydroxyhexanoate can be used as a raw material from inexpensive oils and fats and fatty acids. It can be produced in high yield.

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Claims (5)

[Claims] 1. A transformant obtained by transferring into a host cell a vector plasmid containing a synthetic gene for a copolymer composed of 3-hydroxybutyrate and 3-hydroxyhexanoate. 2. The transformant according to claim 1, which is obtained by transferring a vector plasmid into which a β-ketothiolase gene and / or an acetoacetyl CoA reductase gene has been incorporated into host cells. 3. The transformant according to claim 1, wherein the host bacterium is a strain of the genus Aeromonas. 4. The vector plasmid is pLA2917,
pLA2905, pLA2910, pLA2901, p
RK248, pRK290, pLAFR1, pVK10
The transformant according to any one of claims 1 to 3, which is selected from the group consisting of 0, pVK101, and pVK102. 5. Culturing the transformant according to any one of claims 1 to 4 to accumulate a copolymer composed of 3-hydroxybutyrate and 3-hydroxyhexanoate. A method for producing a copolymer characterized by the following.