JP4258577B2 - Process for producing poly-3-hydroxyalkanoic acid - Google Patents

Description

【００２０】
【発明の効果】
以上詳しく説明したとおり、この出願によって、Ｐ（３ＨＡ）の遺伝子工学的手法による製造方法と、この製造方法に用いる遺伝子組換え生物が提供される。これらの発明によって、高純度のポリ−３−ヒドロキシアルカン酸を簡便かつ大量に、しかも安価に製造することが可能となる。
【図面の簡単な説明】
【図１】この発明に用いることのできるｐＪＲＤＴrc１の構成を示した模式図である。
【図２】実施例で構築した脂肪酸合成酵素遺伝子発現ベクターの挿入遺伝子の関係を示した模式図である。[0001]
BACKGROUND OF THE INVENTION
This application relates to a method for producing poly-3-hydroxyalkanoic acid and a genetically modified organism that produces this poly-3-hydroxyalkanoic acid in large quantities. More specifically, this application relates to a method for producing high-purity poly-3-hydroxyalkanoic acid useful as an alternative plastic and the like in a simple and large amount at low cost, and a novel organism used in this method.
[0002]
[Prior art]
Poly-3-hydroxyalkanoic acid (hereinafter sometimes referred to as “P (3HA)”) is a general term for compounds in which two or more of 3-hydroxyalkanoic acids are polymerized by an ester bond or the like. Examples include 3-hydroxybutanoic acid and poly-3-hydroxyhexanoic acid.
[0003]
In recent years, P (3HA) has been promising for use in alternative plastics and the like. However, since production by chemical synthesis is costly, a genetic engineering production method using a bioreactor or the like is being studied.
In the production of P (3HA) by this genetic engineering method, the host organism is transformed by introducing a P (3HA) polymerase enzyme gene derived from another organism into a host organism such as E. coli that does not accumulate P (3HA). An attempt has been made to cultivate this transformant in a medium containing a carbon source such as glucose. This is because it is considered that P (3HA) non-accumulating organisms such as E. coli do not accumulate P (3HA) because they do not produce the polymerization enzyme.
[0004]
However, since the ability to accumulate P (3HA) in the transformant cannot be increased only by introducing a polymerization enzyme gene into a P (3HA) non-accumulating organism, the non-accumulation of P (3HA) in E. coli and the like is It is speculated that this is due not only to the fact that the organism does not produce P (3HA) polymerizing enzyme, but also because the ability to produce 3-hydroxyacyl CoA, which is a substrate for poly-3-hydroxyalkanoic acid polymerizing enzyme, is extremely low. ing. Thus, it has been reported that a fatty acid synthase gene for promoting the production of 3-hydroxyacyl CoA together with a P (3HA) polymerase enzyme gene is introduced into a host organism (Williams et al .: Protein Expr. Purif. 7: 203-211, 1996). In this conventional method, a mutant gene of fatty acid synthase derived from rat and a poly-3-hydroxyalkanoic acid polymerase enzyme gene derived from Alcaligenes eutrophus are introduced into insect (Spodoptera frugiperda) cells and cultured in insect cell culture medium for 3 days. As a result, 600 mg of dried cells and 1 mg of poly-3-hydroxyalkanoic acid were successfully obtained per liter of medium.
[0005]
[Problems to be solved by the invention]
However, in the conventional method such as Williams et al., The obtained P (3HA) is very small amount of 0.17% per dry cell weight and is not suitable for mass production on an industrial scale. In addition, since the host organism is an insect cell, there is a problem that a special technique is required for the culture.
[0006]
The invention of this application has been made in view of the circumstances as described above, and solves the problems of the conventional method, and can produce high-purity P (3HA) simply, in large quantities, and at low cost. It is an object of the present invention to provide a new P (3HA) production method.
Another object of the invention of this application is to provide a genetically modified organism used in this production method.
[0007]
[Means for Solving the Problems]
As an invention for solving the above-mentioned problems, this application firstly includes a poly-3-hydroxyalkanoic acid synthase gene and a microorganism-derived fatty acid synthase gene fabH , fab D, fab G, acpP , fab F. Characterized in that one or more of the above are introduced into E. coli and transformed, the transformed E. coli is grown in the presence of a carbon source, and poly-3-hydroxyalkanoic acid is isolated and purified from the grown E. coli. Secondly, a poly-3-hydroxyalkanoic acid synthase gene and a microorganism-derived fatty acid synthase gene fabH , fab D, fab G, acpP and fab F A poly-3-hydride which is transformed by introducing it into the E. coli, grown in the presence of a carbon source, and isolated and purified poly-3-hydroxyalkanoic acid from the grown E. coli The manufacturing method of Kishiarukan acid, the third, the microorganism to provide the first or second manufacturing method is Escherichia coli.
[0008]
Also this application, the fourth, E. coli poly-3 and hydroxyalkanoic acid synthase gene, derived from a microorganism of the fatty acid synthase gene fabH, fab D, fab G, acpP, one or more of the fab F Fifth, the transformed Escherichia coli produced by introduction into a poly-3-hydroxyalkanoic acid synthase gene and a fatty acid synthase gene fabH , fab D, fab G, acpP and fab F derived from microorganisms Sixth, the transformed E. coli produced by introduction into E. coli is provided as the fourth or fifth transformed E. coli wherein the microorganism is E. coli.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
As the P (3HA) polymerase enzyme gene used in the method of the present invention, genes derived from various P (3HA) accumulating organisms can be used. Such genes include, for example, the P (3HA) polymerase enzyme gene phaCAC (Fukui et al., J. Bacteriol. 179: 4821-4830, 1997) derived from Aeromonas caviae and the P (3HA) polymerase enzyme gene derived from Alcaligenes eutrophus. phbCAe (Peoples et al., J. Biol. Chem. 262: 15298-15303, 1989), Pseudomonas aeruginosa-derived polymerase enzyme gene phaC1Pa (Timm et al., Eur. J. Biochem. 209: 15-30, 1992) Etc.
[0011]
Alternatively, using these known genes as probes, a P (3HA) polymerase enzyme gene isolated from genomic DNA of an arbitrary species can also be used.
These P (3HA) polymerase enzyme genes can be incorporated into an expression vector for microorganisms, and a transformed microorganism can be produced by introducing the recombinant vector into a microorganism such as Escherichia coli according to a standard method. Expression vectors include known microorganism expression vectors having origins, promoters, ribosome binding sites, gene cloning sites, terminators, etc. that can be replicated in microorganisms (for example, pUC systems, pBluescript II, pET expression are used as expression vectors for E. coli). System, pGEX expression system) and the like. In addition, the plasmid vector pJRDDTrc1 (FIG. 1) newly constructed by the inventors of this application can also be used. As shown in FIG. 1, this pJRDTrc1 is a novel vector constructed by combining commercially available pTrc99A and pJRD215, and the P (3HA) polymerase gene phaCAC and the like are surely linked to the multicloning site. Can do. The fatty acid synthase gene used in the method of the present invention is a known gene derived from a eukaryote or a microorganism. For example, a fatty acid synthase gene fab (Battner et al., Science 277: 1453-1474, 1997) derived from Escherichia coli, a fatty acid synthase gene fab derived from Bacillus subtilis (Kunst et al., Nature 390: 249-256, 1997), etc., and these can be used alone or in combination.
[0012]
Such a fatty acid synthase gene (s) can be incorporated into a known microorganism expression vector and introduced into a host cell in the same manner as the P (3HA) polymerase enzyme gene.
The P (3HA) polymerase enzyme expression vector and the fatty acid synthase expression vector constructed as described above are introduced into the same host microorganism by known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method. Then, a microorganism transformed with both genes can be isolated using drug resistance by the resistance gene in the expression vector.
[0013]
P (3HA) can be accumulated in the microorganism body by growing the transformed microorganism produced in this manner in the presence of a carbon source, for example, by performing gene expression induction treatment as necessary. As the carbon source, saccharides such as glucose and fructose, or fatty acids can be used without limitation.
The accumulated P (3HA) can be isolated and purified by a known method. For example, in the case of Escherichia coli, cells are collected from the culture solution by centrifugation or the like, lyophilized, the dried cells are acid-methanol decomposed, and P (3HA) is isolated from the decomposed product by chloroform extraction. be able to. And it can refine | purify by various chromatography etc.
[0014]
Examples Hereinafter, the present invention will be described in more detail and specifically, but the present invention is not limited to the following examples.
[0015]
【Example】
Example 1
A P (3HA) polymerase enzyme gene (phaCAC gene derived from Aeromonas caviae FA440 strain) was incorporated into pJRDDTrc1 to construct a P (3HA) polymerase enzyme expression vector (FIG. 1). On the other hand, the fatty acid synthase genes fabH, fabD, fabG, acpP, and fabF genes derived from Escherichia coli HB101 were successively cloned into pUC118. As shown in FIG. 2, a fragment containing fabH, fabD, was cloned from this clone. A fragment containing the entire gene group and a fragment containing the entire gene group were cut out and incorporated into pBluescriptII KS (+) to construct fatty acid synthase gene expression vectors (pXbH1.0, pEEv1.6 and pXbSac5.0, respectively).
[0016]
Either a P (3HA) polymerase enzyme expression vector and a fatty acid synthase gene were combined and introduced into Escherichia coli HB101 strain at the same time to obtain transformed Escherichia coli.
After culturing these transformed bacteria in LB (Luria-Bertani) medium at 30 ° C. for 12 hours, IPTG (Isopropyl-β-D (−)-thiogalactopyranoside) as a gene expression inducer was added to a final concentration of 1 mM. And cultured for 4 hours. Glucose (final concentration 10 g / liter) was then added and further cultured for 24 hours.
[0017]
After completion of the culture, the culture solution is centrifuged to collect the cells, freeze-dried, the dried cells are digested with acid-ethanol, extracted with chloroform, and then the yield of P (3HA) and its composition ratio are determined by gas chromatography. Detected.
As a comparative control, a recombinant E. coli into which only the phaCAC gene was introduced was also cultured in the same manner and examined for the presence of P (3HA).
[0018]
The results are as shown in Table 1. From the transformed bacterium introduced with P (3HA) polymerizing enzyme gene and fatty acid synthase gene group, 3HB homopolymer with a monomer unit of R-3-hydroxybutanoic acid: 100% was obtained. No poly-3-hydroxyalkanoic acid was detected from the transformed bacterium introduced with only the phaCAC gene. In addition, the yield of 3HB was the highest at 10.3% per dry cell weight with all of the fabH, fabD, fabG, acpP and fabF gene groups introduced, followed by fabH (6.3%) and fabD (4.6%). there were.
[0019]
[Table 1]

[0020]
【The invention's effect】
As described in detail above, this application provides a method for producing P (3HA) by genetic engineering techniques and a genetically modified organism used in this production method. These inventions make it possible to produce high-purity poly-3-hydroxyalkanoic acid simply, in large quantities, and at low cost.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the structure of pJRDDTrc1 that can be used in the present invention.
FIG. 2 is a schematic diagram showing the relationship of inserted genes of the fatty acid synthase gene expression vector constructed in Example.

Claims (6)

Poly-3 and hydroxyalkanoic acid synthase gene, by introducing microorganism-derived fatty acid synthase gene fabH, fab D, fab G, acpP, one or more of the fab F to transform Escherichia coli, the transformant A method for producing poly-3-hydroxyalkanoic acid, which comprises growing transformed E. coli in the presence of a carbon source, and isolating and purifying poly-3-hydroxyalkanoic acid from the grown E. coli . The poly-3-hydroxyalkanoic acid synthase gene and the microorganism-derived fatty acid synthase genes fabH , fab D, fab G, acpP and fab F are introduced into E. coli for transformation, and the transformed E. coli is present in the presence of a carbon source. And producing poly-3-hydroxyalkanoic acid from the grown Escherichia coli by isolation and purification . The method according to claim 1 or 2, wherein the microorganism is Escherichia coli. Poly-3 and hydroxyalkanoic acid synthase gene, derived from a microorganism of the fatty acid synthase gene fabH, fab D, transformation is produced by fab G, ACPP, one or more of the fab F is introduced into E. coli E. coli . Poly-3-hydroxyalkanoic acid synthase gene and microorganism-derived fatty acid synthase gene fabH , fab D, fab G, acpP and fab A transformed E. coli produced by introducing F into E. coli. 6. The transformed Escherichia coli according to claim 4 or 5, wherein the microorganism is Escherichia coli.

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