JP2005073563A - Method for obtaining polyhydroxyalkanoate synthase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently obtaining an active polyhydroxyalkanoate synthase by expressing a large amount of the polyhydroxyalkanoate synthase by host cells without forming an inclusion body. <P>SOLUTION: Cells to coexpress the polyhydroxyalkanoate synthase and a molecular chaperone are cultured and the polyhydroxyalkanoate synthase and the molecular chaperone are coexpressed in the cells. Consequently, the ratio of formation of an inclusion body in host cells is reduced and the ratio of an enzyme molecule functioning as an enzyme to the molecular weight of an expressed enzyme is increased. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell capable of simultaneously expressing a polyhydroxyalkanoate synthase and a molecular chaperone, a method for obtaining a polyhydroxyalkanoate synthase using the same, and a polyhydroxyalkanoate synthase obtained by the method. More specifically, cells transformed with both a vector capable of expressing a gene encoding a polyhydroxyalkanoate synthase and a vector capable of expressing a gene encoding a molecular chaperone, and polyhydroxyalkanoate synthesis using the same The present invention relates to a method for obtaining an enzyme, and a polyhydroxyalkanoate synthase obtained by the method.

  In recent years, it has been found that polyhydroxyalkanoates (hereinafter sometimes abbreviated as PHA) containing various 3-hydroxyalkanoates other than 3-hydroxybutyrate as monomer units are produced by microorganisms. It has begun to attract attention as a polymer that can overcome the brittleness and impart functionality.

  This biosynthesis of PHA is based on (R) -3-hydroxyacyl CoA generated from various alkanoic acids as raw materials via various metabolic pathways in the body (for example, β-oxidation system and fatty acid synthesis pathway). It is carried out by an enzymatic polymerization reaction. The enzyme that catalyzes this polymerization reaction is called PHA synthase (also called PHA polymerase or PHA synthase).

  More recently, attempts have been made to synthesize PHA in a cell-free system (in vitro) by removing the PHA synthase from the cells. PHA synthesis in a cell-free system (in vitro) using this PHA synthase is different from extracting PHA produced by microorganisms and molding it to form a structure. It can be applied to construction and is highly expected as an innovative micro molding technology that has never existed before. In order to put PHA synthesis into practical use in this cell-free system (in vitro), it is indispensable to obtain a large amount of PHA synthase. It has been studied to produce a large amount of the desired PHA synthase by transforming the host cell and expressing the gene at a high level in the host cell.

  However, when PHA synthase is expressed in large quantities in the host cell, the PHA synthase forms aggregates called inclusion bodies in the host cell, and as a result, an active enzyme cannot be obtained. Has occurred. The formation of such inclusion bodies is not limited to PHA synthase, but is a phenomenon often observed when various foreign proteins are highly expressed by host cells. This is because a large amount of foreign protein is produced in the cell, so that the host cell's inherent protein folding (folding) function is not applied to all foreign proteins. This is thought to be due to the accumulation of the protein in the cell. From the above, the formation of inclusion bodies is a major issue for mass production of useful proteins by recombinants.

  Conventionally, methods for avoiding the formation of inclusion bodies include methods such as reducing the concentration of inducers for foreign protein expression, shortening the induction time for expression, and lowering the culture temperature. However, none of them led to a decrease in the production efficiency of the target protein, and the effect could not be expected with certainty.

  In addition, in order to regenerate the inactivated denatured protein into an active protein with a normal structure that forms inclusion bodies, the abnormal structure of the denatured protein is rewound into the correct structure (refolding). Is being considered. For example, there is a method in which a denatured protein is once denatured with a strong denaturing agent such as guanidine hydrochloride and then the concentration of the denaturing agent is decreased by a method such as dilution or dialysis to thereby unwind the protein. However, with such a method, it is not always possible to rewind all the proteins to the original correct structure, and the rewinding efficiency is usually low, so that it is difficult to efficiently obtain an active protein.

  In recent years, there has been increased interest in protein folding or refolding with molecular chaperones. Molecular chaperones are mainly a group of heat shock proteins, and have been found because their expression is induced when cells are exposed to environmental stress such as high temperature, and they have the effect of preventing the denaturation of other proteins. These exist widely in both prokaryotes and eukaryotes, and in particular, a group called GroE, DnaK, DnaJ, and GrpE and a protein called a trigger factor are known as molecular chaperones produced from E. coli.

  By utilizing the folding or refolding function of these molecular chaperones, foreign proteins that are normally expressed as inclusion body proteins in host cells can be co-expressed with foreign proteins and molecular chaperones in the same cell. There has been reported a method of expressing the protein without insolubilizing it. Examples of solubilized transglutaminase by co-expression of DnaJ (JP-A-8-308654: Patent Document 1) and various foreign proteins such as interferon and the like by co-expression of DnaK, DnaJ and GrpE Has been proposed (Japanese Patent Laid-Open No. 11-9274: Patent Document 2).

  By the way, studies have been reported that the effect of folding or refolding action by the chaperone as described above varies depending on the type of protein (Hockney, R. C. (1994) Trends Biotechnol. 12, 456-463: Non-Patent Document 1). This is because the structure varies depending on the type of protein, and the method of folding varies accordingly. Therefore, in order to correctly fold a foreign protein with a chaperone, a chaperone depends on each foreign protein. It is indispensable to individually examine the type and co-expression conditions.

Here, regarding the previous PHA synthase, folding or refolding by such chaperone has not been studied, and whether co-expression with chaperone is effective in preventing inclusion body formation of PHA synthase, and So far, it has not been known what co-expression with chaperones is effective in preventing inclusion body formation.
JP-A-8-308654 Japanese Patent Laid-Open No. 11-9274 Hockney, RC (1994) Trends Biotechnol. 12, 456-463

  An object of the present invention is to provide a method for efficiently obtaining an active polyhydroxyalkanoate synthase by expressing a large amount of polyhydroxyalkanoate synthase by a host cell without forming inclusion bodies.

  The present invention relates to a polyhydroxyalkanoate synthase characterized by culturing a cell capable of co-expressing a polyhydroxyalkanoate synthase and a molecular chaperone, and co-expressing the polyhydroxyalkanoate synthase and the molecular chaperone in the cell. The acquisition method is provided.

  Further, the present invention is characterized in that it is co-transformed by both a vector capable of expressing a gene encoding a polyhydroxyalkanoate synthase and a vector capable of expressing a gene encoding a molecular chaperone. Provide cells.

  The present invention also provides a polyhydroxyalkanoate obtained by culturing a cell capable of co-expressing a polyhydroxyalkanoate synthase and a molecular chaperone, and co-expressing the polyhydroxyalkanoate synthase and the molecular chaperone in the cell. Synthetic enzymes are provided.

  According to the present invention, in the production of polyhydroxyalkanoate synthase by microorganisms, it becomes possible to highly express polyhydroxyalkanoate synthase while suppressing the formation of aggregates, and active polyhydroxyalkanoate synthase can be efficiently produced. It became possible to acquire it.

Hereinafter, the present invention will be described in more detail.
<Preparation of PHA synthase gene expression vector>
An expression vector for PHA synthase gene can be constructed by inserting DNA encoding PHA synthase into an appropriate expression vector. The PHA synthase gene can be obtained by cloning from a microorganism producing PHA such as mcl-PHA or unusual-PHA. This PHA synthase gene cloning method can be performed according to a standard method.

  In addition to Pseudomonas aeruginosa as described above, PHA-producing microorganisms include Pseudomonas cichorii YN2 (Pseudomonas cichorii YN2) isolated by the present inventors and described in JP-A-2002-080571. Pseudomonas putida P91 strain (Pseudomonas putida P91) described in JP 2002-080571 A, Pseudomonas cichorii H45 strain (Pseudomonas cichorii H45) described in JP 2002-080571 A, JP 2002-080571 A. Pseudomonas jessenii P161 (Pseudomonas jessenii P161) described in JP No. 2001-78753, Burkholderia sp. OK3, JP 2001-78753, JP 2001 Burkholderia sp. OK4 strain described in Japanese Patent No. 69968 can be used. In addition to these microorganisms, microorganisms belonging to the genus Aeromonas sp., Comamonas sp., Etc. and producing mcl-PHA and unusual-PHA can also be used.

  The YN2 strain is deposit number FERM BP-7375, the P91 strain is deposit number FERM BP-7373, the H45 strain is deposit number FERM BP-7374, the P161 strain is deposit number FERM BP-7376, and the OK3 strain is FERM. As P-17370, OK4 strain as FERM P-17371, deposit of microorganisms in patent procedure (deposit based on the Budapest Treaty on International Approval for BP number), National Institute of Advanced Industrial Science and Technology patent biological deposit Deposited at the Center (Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology (formerly the Ministry of International Trade and Industry, Institute of Industrial Technology) Biotechnology Institute of Technology Patent Microorganism Depositary Center)

  The expression vector for constructing the PHA synthase gene expression vector may be appropriately selected from those in which a host-vector system has been established according to the host cell, such as a plasmid or a phage. Those that do not exhibit plasmid incompatibility, and phages that do not exhibit exclusivity with chaperone vectors can be used, and are not particularly limited. Furthermore, it is more preferable if it has a genetic trait (marker) such as antibiotic resistance or an appropriate restriction enzyme cleavage point that enables selection of host cells containing the vector.

  In addition, as a promoter for expressing PHA synthase, an appropriate promoter may be selected in consideration of the ratio of expression levels of PHA synthase and chaperone, but it is highly expressed to increase the production amount of PHA synthase. It is preferable that the promoter is capable of. A promoter capable of inducing the expression of PHA synthase with an appropriate chemical substance can also be used. This makes it possible to control the expression timing of PHA synthase according to the expression time of chaperone, and to increase the efficiency of folding or refolding of PHA synthase. As such a promoter, for example, a promoter such as lac, tac, trc and the like that can be induced by isopropyl-β-D-thiogalactopyranoside (IPTG) can be used.

  In addition, as described later, in order to easily purify PHA synthase using affinity, it has an affinity adsorption function such as histidine residue and glutathione-S-transferase (GST) at the N-terminus and C-terminus of PHA synthase. A method in which a “tag” is bound, PHA synthase is expressed in the form of a fusion protein with the tag, and the fusion protein is bound to an insoluble material having affinity for the tag is preferably used. In order to fuse with a synthase, a base sequence encoding such a tag can be placed upstream or downstream of the PHA synthase inserted into the vector.

  A method for inserting a DNA encoding a PHA synthase into the above expression vector may be performed by a conventional method. For example, a PCR product of a PHA synthase gene and an expression vector are treated with a restriction enzyme, and then both are ligated. It can be constructed by joining.

<Preparation of molecular chaperone expression vector>
A molecular chaperone expression vector can be constructed by inserting a DNA encoding a molecular chaperone into an appropriate expression vector. Any molecular chaperone can be used as long as it can prevent inclusion body formation of the expressed PHA synthase. For example, the molecular chaperone team of DnaK-DnaJ-GrpE, DnaK-DnaJ-GrpE and GroES-GroEL Of these two molecular chaperone teams, GroES-GroEL and two molecular chaperone teams of trigger factors, it is preferable to use any set of molecular chaperone groups.

  As the molecular chaperone gene to be used, an appropriate one can be selected from conventionally cloned genes. Moreover, what has been cloned by itself from an appropriate species can also be used. For example, some molecular chaperones have consensus sequences with very high homology among microorganisms, such as GroEL, and the target molecular chaperone DNA is cloned by PCR using DNA encoding these consensus sequences as a primer. be able to.

  An expression vector for constructing a molecular chaperone expression vector may be appropriately selected from plasmids, phages, and the like in which a host-vector system has been established according to the host cell. If it is a plasmid, a PHA synthase vector And those that do not exhibit plasmid incompatibility, and phages that do not exhibit exclusivity with a PHA synthase vector can be used, and are not particularly limited. Furthermore, it is more preferable if it has a genetic trait (marker) such as antibiotic resistance or an appropriate restriction enzyme cleavage point that enables selection of host cells containing the vector.

  As a promoter for expressing a molecular chaperone, an appropriate promoter may be selected in consideration of the expression level ratio of PHA synthase and chaperone. However, in order to fold or refold a large amount of PHA synthase to be expressed. It is preferable that the promoter is capable of high expression. A promoter capable of inducing molecular chaperone expression by an appropriate chemical substance can also be used. This makes it possible to control the expression time of the molecular chaperone according to the expression time of the PHA synthase, and to increase the efficiency of folding or refolding of the PHA synthase. For example, it is preferable to use araB having L-Arabinose as an inducing chemical substance, Pzt1 having Tetracyclin as an inducing chemical substance, or the like.

  As a method for inserting a DNA encoding a molecular chaperone into the above expression vector, a conventional method may be used. For example, a PCR product of a molecular chaperone gene and an expression vector are treated with a restriction enzyme, and then both are combined by a ligation reaction. Can be built.

<Host cell>
A host cell for producing PHA synthase may be any host cell that can be transformed with both a PHA synthase expression vector and a molecular chaperone expression vector and can co-express PHA synthase and molecular chaperone. In particular, Escherichia coli can be suitably used as a host cell because it can be easily cultured at high density, the research on host-vector systems is most advanced, and many high expression vectors have been developed.

<Transformation>
A technique well known to those skilled in the art can be used as a method for transforming a host cell with both a PHA synthase expression vector and a molecular chaperone expression vector to obtain a cotransformed cell. For example, an electroporation method or a calcium chloride method can be used.

  The order of introducing the PHA synthase expression vector and the molecular chaperone expression vector into the host cell should be any order considering that the transformation efficiency varies depending on the type of host cell and vector used. In either case, for example, a PHA synthase expression vector is introduced first, a molecular chaperone expression vector is introduced first, or both are introduced simultaneously. After transformation with a PHA synthase expression vector, transformation efficiency decreases when transformed with a molecular chaperone expression vector. In such a case, an antibiotic introduced as a resistance marker was added to the molecular chaperone expression vector. After culturing in a liquid medium, it is preferable to select a colony formed on a solid medium to which antibiotics have been applied.

<Co-expression of PHA synthase and molecular chaperone>
A method for co-expressing a PHA synthase and a molecular chaperone may be performed by culturing a host cell co-transformed with both a PHA synthase expression vector and a molecular chaperone expression vector.
Any host cell culture method may be used as long as it satisfies the expression level and expression ratio of PHA synthase and molecular chaperone within the range of conditions suitable for the growth of the host cell. For example, when Escherichia coli is used as a host cell, it can be carried out by culturing aerobically at a culture temperature of 25 to 37 ° C. using LB medium or the like.

  In addition, when an antibiotic resistance gene is added to the expression vector, the corresponding antibiotic may be added to the medium in order to selectively culture only the host cells carrying the expression vector. Examples of such antibiotics include kanamycin, ampicillin, tetracycline, chloramphenicol, streptomycin and the like. However, when an inducible promoter is used in the expression vector of PHA synthase or molecular chaperone, it is desirable to avoid antibiotics that can be used as these inducers.

  In addition, when using a vector that requires induction for expression of at least one of PHA synthase and molecular chaperone, by adding an inducer corresponding to the expression to the target expression period, Can be controlled. Here, it is preferable that the expression time of the molecular chaperone coincides with or precedes the expression time of the PHA synthase. The molecular chaperone is expressed at least earlier than the PHA synthase, so that the expressed PHA synthase can be folded into the correct structure by the chaperone before forming the inclusion body.

  Further, the expression level of the molecular chaperone may be an amount that can suppress the formation of inclusion bodies of PHA synthase, and if possible, the molecular chaperone functional unit is preferably larger than the number of molecules of the PHA synthase expressed. .

<Extraction of PHA synthase>
After expressing a desired amount of PHA synthase by the above culture, the PHA synthase may be extracted from the host cell by a commonly used method. For example, the cultured host cells are collected by centrifugation, washed with a buffer solution, etc., and then suspended again in a buffer solution, etc., and then commonly used methods such as French press, freeze-thawing, ultrasonic disruption, lysozyme and various interfaces. After disrupting the cells with an activator or the like, the crude supernatant containing PHA synthase can be obtained by collecting the centrifuged supernatant.

  Further, the target PHA synthase may be purified from the crude enzyme by various methods. For example, the purified enzyme can be obtained by combining one or a plurality of means such as affinity chromatography, ion exchange chromatography, and gel filtration. In particular, a “tag” having an affinity adsorption function such as a histidine residue or glutathione-S-transferase (GST) is bound to the N-terminus or C-terminus of PHA synthase, and PHA synthase in the form of a fusion protein with the tag. It can be more easily purified by expressing it and binding the fusion protein to an insoluble material having affinity for this tag. In order to separate only the PHA synthase from this fusion protein, it is possible to use a method such as cleaving with a protease such as thrombin or blood coagulation factor Xa, lowering the pH, or adding a high concentration of imidazole as a binding competitor. it can. Alternatively, when the tag contains intein as in the case of using pTYB1 (manufactured by New Englan Biolab) as an expression vector, it can be cleaved as a reducing condition with dithiothreitol or the like. In addition to the above, chitin-binding domain (CBD), maltose-binding protein (MBP), thioredoxin (TRX) and the like can also be used to form a fusion protein that can be purified by affinity chromatography.

  The purified enzyme can be used by appropriately adding stabilizers and activators such as metal salts, glycerin, dithiothreitol, EDTA, bovine serum albumin (BSA) as necessary.

<Evaluation of PHA synthase expression level>
The expression levels of PHA synthase and molecular chaperone can be evaluated using protein measurement methods known to those skilled in the art. For example, PHA synthase and molecular chaperone in a crude enzyme solution obtained by crushing host cells can be separated using gel electrophoresis such as SDS-PAGE and detected by Coomassie blue staining or silver staining. . Furthermore, the expression level can be compared from the staining level of each protein band separated on the gel. Using this method, the folding effect of PHA synthase by the co-expressed molecular chaperone can be evaluated. For example, the lysate of host cells is separated into soluble and insoluble components by centrifugation, etc., and SDS-PAGE is performed for each, and PHA synthesis in soluble and insoluble components is determined from the amount of PHA synthase band staining. A comparison of enzyme amounts, that is, the solubilization degree of PHA synthase can be evaluated. In addition, the expression level of PHA synthase can be quantified using a conventionally used protein quantification method for a purified PHA synthase. For example, a BCA method, a Bradford method, a Lowry method, a biuret method, a turbidimetric method, an ultraviolet absorption spectrum method, or the like can be used.

<Method for measuring activity of PHA synthase>
By measuring the activity of the obtained PHA synthase, the degree of folding of the expressed PHA synthase can be evaluated. For the activity measurement of PHA synthase, a conventionally reported method can be used. For example, CoA released in the process of polymerizing 3-hydroxyacyl-CoA to PHA by the catalytic action of PHA synthase, The measurement can be carried out by the following method based on the principle of measurement by coloring with 5,5′-dithiobis- (2-nitrobenzoic acid). Reagent 1: Bovine serum albumin (Sigma) dissolved in 0.1 M Tris-HCl buffer (pH 8.0) at 3.0 mg / ml, Reagent 2: 3-hydroxyacyl-CoA in 0.1 M Tris-HCl buffer ( Dissolve 3.0 mM in pH 8.0), Reagent 3: 10 mg / ml of trichloroacetic acid in 0.1 M Tris-HCl buffer (pH 8.0), Reagent 4: 5,5′-dithiobis- (2-nitro Dissolve 2.0 mM of benzoic acid in 0.1 M Tris-HCl buffer (pH 8.0). First reaction (PHA synthesis reaction): Add 100 μl of reagent 1 to 100 μl of sample (enzyme) solution, mix, and preincubate at 30 ° C. for 1 minute. Here, 100 μl of reagent 2 is added and mixed, and incubated at 30 ° C. for 1 to 30 minutes. Then, reagent 3 is added to stop the reaction. Second reaction (color development reaction of free CoA): Centrifugation (15,000 × g, 10 minutes) of the first reaction liquid that has stopped the reaction, 500 μl of reagent 4 was added to 500 μl of this supernatant, and 10 minutes at 30 ° C. After incubation, measure the absorbance at 412 nm. Calculation of enzyme activity: The amount of enzyme that releases 1 μmol of CoA per minute is defined as 1 unit (U). The 3-hydroxyacyl-CoA used for this activity measurement may be prepared by a method described later, as long as a PHA monomer expected to be synthesized by an enzyme is selected as appropriate.

<PHA synthesis by PHA synthase>
The PHA that can be synthesized by the PHA synthase obtained by the present invention can be any PHA that can be synthesized by a microorganism that is the source of the PHA synthase. As described above, a PHA synthase is an enzyme that catalyzes the final stage in a PHA synthesis reaction system in an organism, and therefore any PHA that is known to be synthesized in an organism can be used as the enzyme. It is synthesized under the catalytic action of. Therefore, PHA can be synthesized in vitro by allowing 3-hydroxyacyl CoA corresponding to the desired PHA to act on the PHA synthase obtained in the present invention.

  Specific examples of such PHA include PHA containing at least monomer units represented by the following chemical formulas [1] to [10].

(However, the monomer unit is at least one selected from the group consisting of monomer units in which the combination of R1 and a is any of the following:

  A monomer unit in which R1 is a hydrogen atom (H) and a is an integer from 3 to 10, R1 is a halogen atom and a is any integer from 1 to 10, and R1 is a chromophore A is a monomer unit in which any one of integers from 1 to 10, R1 is a carboxyl group, and a monomer unit in which a is any one of integers from 1 to 10, R1 is

A monomer unit in which a is any integer from 1 to 7. )

(However, b in the formula represents either 0-7 integer, R2 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5 and -C ₃ F ₍ Represents any one selected from the group consisting of ₇ )

(However, c in the formula represents any of integers from 1 to 8, R3 is hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5 and -C ₃ F ₍ Represents any one selected from the group consisting of ₇ )

(Wherein d represents any of 0 to 7 integers, R4 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5 and -C ₃ F ₍ Represents any one selected from the group consisting of ₇ )

(Wherein in e represents any of integers from 1 to 8, R5 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5, -C 3 F ₇ represents one selected from the group consisting of —CH ₃ , —C ₂ H ₅ and —C ₃ H _7. )

(However, f in the formula represents any integer from 0 to 7.)

(In the formula, g represents an integer of 1 to 8.)

(Wherein in h represents any of 1 to 7 integer, R6 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -COOR '} , - SO 2 R ", - CH 3, _{-C 2 H 5, -C 3 H} 7, -CH (CH 3) 2 and -C (CH ₃₎ represents any one selected from the group consisting of _3,
Here, R ′ is any one of a hydrogen atom (H), Na, K, —CH ₃ , —C ₂ H ₅ , and R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ and — Either OC ₂ H ₅ )

(Wherein i represents an integer of 1 to 7, and R7 is selected from the group consisting of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —COOR ′, and —SO ₂ R ″). Any one of the
Here, R ′ is any one of a hydrogen atom (H), Na, K, —CH ₃ , —C ₂ H ₅ , and R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ and — Either OC ₂ H ₅ )

(However, j in the formula represents any integer from 1 to 9.)
Specific examples of the halogen atom include fluorine, chlorine, bromine and the like. The chromophore is not particularly limited as long as the 3-hydroxyacyl CoA form can be catalyzed by PHA synthase. However, considering steric hindrance during polymer synthesis, 3- In the hydroxyacyl-CoA molecule, it is desirable that a methylene chain having 1 to 5 carbon atoms is present between the carboxyl group to which CoA is bonded and the chromophore. Further, a colored structure can be obtained if the light absorption wavelength of the chromophore is in the visible region, and can be used as various electronic materials even if the light absorption wavelength is outside the visible region. Examples of such chromophores include nitroso, nitro, azo, diarylmethane, triarylmethane, xanthene, acridine, quinoline, methine, thiazole, indamine, indophenol, lactone, aminoketone, hydroxyketone, stilbene, azine, ocasazine, Examples include thiazine, anthraquinone, phthalocyanine, and indigoid.

  In addition, PHA composed of a random copolymer or a block copolymer containing a plurality of the above monomer units can be synthesized, and control of physical properties of PHA using the characteristics of each monomer unit or the functional group contained, and addition of a plurality of functions, It is possible to develop a new function using interaction between functional groups. If necessary, further chemical modification or the like can be performed after the synthesis of PHA or during the synthesis.

  Specific examples of 3-hydroxyacyl-CoA used as a substrate for PHA as described above include 3-hydroxyacyl-CoA represented by the following chemical formulas [11] to [20].

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, wherein the combination of R1 and a is at least one selected from the following group, and the chemical formula [ It corresponds to R1 and a in the monomer unit represented by 1].
A monomer unit in which R1 is a hydrogen atom (H) and a is an integer from 3 to 10, R1 is a monomer unit in which a is a halogen atom and a is an integer from 1 to 10, and R1 is a chromophore A is a monomer unit in which any one of integers from 1 to 10, R1 is a carboxyl group, and a monomer unit in which a is any one of integers from 1 to 10, R1 is

A monomer unit in which a is any integer from 1 to 7. )

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, b represents one of integers from 0 to 7 corresponding to b in the monomer unit represented by the chemical formula [2], and R2 represents It consists of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —CF ₃ , —C ₂ F ₅ and —C ₃ F ₇ corresponding to R 2 in the monomer unit represented by the chemical formula [2]. Represents any one selected from the group.)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, c represents an integer of 1 to 8 corresponding to c in the monomer unit represented by the chemical formula [3], and R3 represents It consists of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —CF ₃ , —C ₂ F ₅ and —C ₃ F ₇ corresponding to R 3 in the monomer unit represented by the chemical formula [3]. (Represents any one selected from the group.)

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, d represents an integer of 0 to 7 corresponding to d in the monomer unit represented by the chemical formula [4], and R4 represents It consists of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —CF ₃ , —C ₂ F ₅ and —C ₃ F ₇ corresponding to R 4 in the monomer unit represented by the chemical formula [4]. Represents any one selected from the group.)

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, e represents an integer of 1 to 8 corresponding to e in the monomer unit represented by the chemical formula [5], and R5 represents Hydrogen atom (H), halogen atom, —CN, —NO ₂ , —CF ₃ , —C ₂ F ₅ , —C ₃ F ₇ , − corresponding to R 5 in the monomer unit represented by the chemical formula [5] It represents one selected from the group consisting of CH ₃ , —C ₂ H ₅ and —C ₃ H _7. )

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, and f represents an integer of 0 to 7 corresponding to f in the monomer unit represented by the chemical formula [6].)

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, and g represents an integer of 1 to 8 corresponding to g in the monomer unit represented by the chemical formula [7].)

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, h represents an integer of 1 to 7 corresponding to h in the monomer unit represented by the chemical formula [8], and R6 represents Hydrogen atom (H), halogen atom, —CN, —NO ₂ , —COOR ′, —SO ₂ R ″, —CH ₃ , —C ₂ corresponding to R 6 in the monomer unit represented by the chemical formula [8] And represents any one selected from the group consisting of H ₅ , —C ₃ H ₇ , —CH (CH ₃ ) ₂ and —C (CH ₃ ) ₃ ;
Here, R ′ is any one of a hydrogen atom (H), Na, K, —CH ₃ , —C ₂ H ₅ , and R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ and — Either OC ₂ H ₅ )

(Wherein -SCoA represents coenzyme A bonded to alkanoic acid, i represents an integer of 1 to 7 corresponding to i in the monomer unit represented by the chemical formula [9], and R7 represents Any selected from the group consisting of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —COOR ′, and —SO ₂ R ″ corresponding to R7 in the monomer unit represented by the chemical formula [9] Or one
Here, R ′ is any one of a hydrogen atom (H), Na, K, —CH ₃ , —C ₂ H ₅ , and R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ and — Either OC ₂ H ₅ )

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, and j represents an integer of 1 to 9 corresponding to j in the monomer unit represented by the chemical formula [10].)
As specific examples of the chromophore and the halogen atom in the above formulas [11] to [15] and [18] to [19], those mentioned above can be similarly mentioned.

These 3-hydroxyacyl CoAs can be synthesized by, for example, an in vitro synthesis method using an enzyme, an in vivo synthesis method using a living organism such as a microorganism or a plant, a chemical synthesis method, or the like. Can be used. Particularly, the enzyme synthesis method is a method generally used for the synthesis of the substrate, and the following reaction using a commercially available acyl CoA synthetase (acyl CoA ligase, EC 6.2.1.3),
3-hydroxyalkanoic acid + CoA → (acyl CoA synthetase) → 3-hydroxyacyl CoA
(Eur. J. Biochem., 250, 432-439 (1997), Appl. Microbiol. Biotechnol., 54, 37-43 (2000), etc.) are known. In the synthesis step using an enzyme or an organism, a batch-type synthesis method may be used, or continuous production may be performed using an immobilized enzyme or an immobilized cell.

  PHA synthesized by PHA synthase can be subjected to composition analysis of monomer units by a gas chromatography-mass spectrometer (GC / MS) or a nuclear magnetic resonance spectrometer (NMR).

  Further, as a method for simply confirming the synthesis of PHA by PHA synthase, a method newly developed by the present inventors and combined with Nile Blue A staining and fluorescence microscope observation can be used. As a result of intensive studies on a method for easily determining PHA synthesis by a PHA synthase, the present inventors have developed a drug that specifically binds to PHA and emits fluorescence. Environ. Microbiol. , 44, 238-241 (1982), Nile Blue A, which is reported to be used for simple discrimination of PHA production in microbial cells (in vivo), The present invention has been completed by finding that it can also be used for determination of PHA synthesis in a cell-free system. That is, in this method, after filtering a Nile Blue A solution of a predetermined concentration, it is mixed with a reaction solution containing PHA, and observed while irradiating excitation light of a certain wavelength with a fluorescence microscope. By fluorescing and observing this, PHA synthesis by PHA synthase can be easily determined.

  The molecular weight of PHA synthesized by PHA synthase can be measured by gel permeation chromatography (GPC) or the like, and has a number average molecular weight of about 1,000 to 10,000,000.

  In addition, PHA synthesized by the PHA synthase obtained by the present invention is an isotactic polymer generally composed only of the R isomer.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the best mode of the present invention, but the technical scope of the present invention is not limited to these examples. In the following, “%” is based on mass unless otherwise specified.
<Example 1>
YN2 strain in 100 ml LB medium (1% polypeptone (Nippon Pharmaceutical Co., Ltd.), 0.5% yeast extract (Difco), 0.5% sodium chloride, pH 7.4) at 30 ° C overnight After culturing, chromosomal DNA was separated and recovered by the method of Marmer et al. The obtained chromosomal DNA was completely digested with the restriction enzyme HindIII. PUC18 was used as a vector and digested with the restriction enzyme HindIII. After terminal dephosphorylation (Molecular Cloning, 1,572, (1989); published by Cold Spring Harbor Laboratory), DNA Ligation Kit Ver. Using II (Takara Shuzo Co., Ltd.), the cleavage site (cloning site) of the vector and the HindIII complete degradation fragment of the chromosomal DNA were ligated. Using a plasmid vector incorporating this chromosomal DNA fragment, Escherichia coli HB101 strain was transformed to prepare a DNA library of YN2 strain. Next, in order to select a DNA fragment containing the PHA synthase gene of the YN2 strain, a probe for colony hybridization was prepared. Oligonucleotides consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized (Amersham Pharmacia Biotech Co., Ltd.), and PCR was performed using this oligonucleotide as a primer and chromosomal DNA as a template. A DNA fragment that had been PCR amplified was used as a probe. The labeling of the probe was performed using a commercially available labeling enzyme system AlkPhosDirect (manufactured by Amersham Pharmacia Biotech Co., Ltd.). Using the resulting labeled probe, an E. coli strain having a recombinant plasmid containing the PHA synthase gene was selected from the chromosomal DNA library of the YN2 strain by colony hybridization. By recovering the plasmid from the selected strain by the alkaline method, a DNA fragment containing the PHA synthase gene could be obtained. The gene DNA fragment obtained here was recombined into a vector pBBR122 (Mo Bi Tec) containing a broad host range replication region that does not belong to any of the incompatibility groups IncP, IncQ, or IncW. When this recombinant plasmid was transformed into Pseudomonas chicoryai YN2ml strain (strain lacking PHA synthesis ability) by electroporation, the PHA synthesis ability of the YN2ml strain was restored and showed complementarity. Therefore, it was confirmed that the selected gene DNA fragment contains a PHA synthase gene region that can be translated into PHA synthase in Pseudomonas chicory eye YN2ml strain.

  The base sequence of this DNA fragment was determined by the Sanger method. As a result, it was confirmed that the determined nucleotide sequence contained the nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4, which respectively encode peptide chains. Among these, the PHA synthase gene of SEQ ID NO: 3 was subjected to PCR using chromosomal DNA as a template, and the full length of the PHA synthase gene was prepared again. That is, an upstream primer (SEQ ID NO: 5) and a downstream primer (SEQ ID NO: 6) for the PHA synthase gene having the base sequence represented by SEQ ID NO: 3 were synthesized (Amersham Pharmacia Biotech Co., Ltd.).

  Using these primers, PCR was performed using the previously prepared PHA synthase gene as a template to amplify the full length of the PHA synthase gene (LA-PCR kit; manufactured by Takara Shuzo Co., Ltd.). Next, the obtained PCR amplified fragment and the expression vector pTrc99A were cleaved with the restriction enzyme HindIII and dephosphorylated (Molecular Cloning, 1, 572, 1989; Cold Spring Harbor Laboratory published). A DNA fragment containing the full length of the PHA synthase gene excluding unnecessary nucleotide sequences at both ends at the cleavage site of pTrc99A was prepared using a DNA ligation kit Ver. It connected using II (made by Takara Shuzo Co., Ltd.).

  Escherichia coli HB101 (Takara Shuzo) was transformed with the obtained recombinant plasmid by the calcium chloride method. The obtained recombinant was cultured, the recombinant plasmid was amplified, and the recombinant plasmid was recovered. The recombinant plasmid holding the gene DNA of SEQ ID NO: 3 was designated as pYN2-C1. E. coli (Escherichia coli HB101fB fadB deficient strain) was transformed with pYN2-C1 by the calcium chloride method to obtain a recombinant E. coli strain, pYN2-C1 recombinant strain carrying this recombinant plasmid.

  Next, for pYN2-C1, an oligonucleotide (SEQ ID NO: 7) serving as an upstream primer and an oligonucleotide (SEQ ID NO: 8) serving as a downstream primer were respectively designed and synthesized (Amersham Pharmacia Biotech ( stock)). Using this oligonucleotide as a primer, PCR was performed using pYN2-C1 as a template to amplify the full length of the PhaC1 gene having a BamHI restriction site upstream and an XhoI restriction site downstream (LA-PCR kit; manufactured by Takara Shuzo Co., Ltd.) .

  Plasmid pGEX-6P-1 (manufactured by Amersham Pharmacia Biotech Co., Ltd.) in which the PCR amplification product of the purified PhaC1 gene was digested with BamHI and XhoI, and the base sequence encoding glutathione-S-transferase was placed upstream of the restriction enzyme site The PhaC1 expression vector fused with the GST tag was constructed. E. coli (JM109) was transformed with this vector. Confirmation of strains transformed with the GST-fused PhaC1 expression vector is performed using DNA fragments obtained by treating plasmid DNA prepared in large quantities using Miniprep (Wizard Minipreps DNA Purification Systems, PROMEGA) with BamHI and XhoI. It was.

The strain transformed with the GST-fused PhaC1 expression vector was pre-cultured overnight in 5 ml of LB medium containing 100 μg / ml of ampicillin, and then 2 ml thereof was added to 200 ml of LB medium containing 100 μg / ml of ampicillin, Shake culture was performed at 37 ° C. and 170 rpm until OD ₆₀₀ = 0.5 to 0.6. The culture was immediately cooled to 4 ° C and centrifuged (8,000 xg, 10 minutes, 4 ° C) to remove the supernatant. After washing and centrifuging twice with sterilized water cooled to 4 ° C, centrifuging (8,000 × g, 10 minutes, 4 ° C) and removing the supernatant, sterilized water of the same weight as the pellet is added to the pellet. Added and resuspended.

  PG-KJE8 (manufactured by Takara Bio Inc.), a plasmid vector constructed to express a molecular chaperone group consisting of two molecular chaperone teams of DnaK-DnaJ-GrpE and GroES-GroEL in 100 μl of this bacterial suspension Add and mix 10 ng amount, inject into a cooled electroporation cuvette (Gene Pulser Cuvette; manufactured by BIO RAD), and attach this cuvette to the electroporation device (Gene Pulser II; manufactured by BIO RAD) A voltage was applied under conditions of 2.5 kv, 25 μF, and 200Ω. This treated bacterial solution was added to 1 ml of SOC medium and cultured with shaking at 37 ° C. for 1 hour. 100 μl of this culture solution was spread on an LB agar medium containing 100 μg / ml ampicillin and 20 μg / ml chloramphenicol, and cultured overnight at 37 ° C. to confirm the appearance of colonies. As described above, a strain in which a GST-fused PhaC1 expression plasmid and DnaK-DnaJ-GrpE and groES-groEL expression plasmids were co-transformed was obtained.

The resulting strain was precultured overnight in 20 ml of LB medium containing 100 μg / ml of ampicillin and 20 μg / ml of chloramphenicol, and then 10 ml of the resulting strain was 100 μg / ml of ampicillin, 20 μg / ml of chloramphenicol, arabinose Add to 1 L of LB medium containing 4 mg / ml and tetracycline 10 ng / ml, shake culture at 30 ° C, 120 rpm, add IPTG when OD ₆₀₀ = 0.5 (final concentration 20 μg / ml), The culture was further continued for 3 hours.

The cultured bacterial solution is collected (8,000 × g, 10 minutes, 4 ° C.), and 40 ml of 4 ° C. PBS buffer solution (8 g NaCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₍₄ , 0.2 g KCl, 1 L purified water). The cells were crushed with a French press and centrifuged (15,000 × g, 10 minutes, 4 ° C.) to separate soluble and insoluble components. When both soluble and insoluble components were subjected to Coomassie staining after SDS-PAGE, a band having a molecular weight of 87 kD corresponding to PhaC1 fused with GST was confirmed for both the soluble and insoluble components. The amount ratio of the band of GST-fused PhaC1 in this soluble component and insoluble component is approximately 5: 2, and the solubility of GST-fused PhaC1 is improved by co-expression with a molecular chaperone compared to Comparative Example 1 described later. I understood it. In addition, in the soluble components, bands having molecular weights corresponding to DnaK, DnaJ, GrpE, groES, and groEL were confirmed.

  Next, the soluble component of this cell disruption solution, that is, GST-fused PhaC1 present in the crude enzyme, was purified with glutathione Sepharose 4B (manufactured by Amersham Pharmacia Biotech Co., Ltd.). After washing 500 μl of 75% glutathione sepharose slurry 3 times with 1 ml PBS (8,000 × g, 1 minute, 4 ° C.), it was added to the crude enzyme solution and gently shaken at room temperature. As a result, GST-fused PhaC1 was adsorbed to glutathione sepharose. After adsorption, the mixture was centrifuged (500 × g, 1 minute, 4 ° C.) to collect glutathione sepharose and washed 3 times with 1 ml of PBS at 4 ° C. Further, the cells were washed with Clearvage buffer (6.1 g Tris-HCl, 8.7 g NaCl, 0.37 g EDTA, 0.15 g Dithiothreitol, 0.1 g Nonidet, 1 L purified water, PH7) at 5 ° C. and resuspended in 200 μl of Clearvage buffer at 5 ° C. To this was added 20 μl of PreScission protease (Amersham Pharmacia Biotech Co., Ltd., 5U) and treated at 5 ° C. for 15 hours to cut out only PhaC1 from glutathione sepharose. Further, the treatment solution was passed through glutathione sepharose washed with PBS to remove PreScission protease. The flow-through fraction was further dialyzed to obtain about 300 μl of a PhaC1 Tris-HCl solution (Tris-HCl; 6.05 g Tris, 1 L purified water, pH 8.0). When this purified enzyme solution was subjected to SDS-PAGE, a 61 kDa single band corresponding to the molecular weight of PhaC1 was confirmed.

  The concentration of the obtained purified enzyme was quantified with a micro BCA protein quantification reagent kit (Pierce Chemical Co., Ltd.), and further converted into a yield per culture solution. In addition, the activity of the purified enzyme was determined by the above method using (R) -3-hydroxyoctanoyl CoA (prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)) as a monomer. Measured and converted to enzyme activity per mg of enzyme. The results are shown in Table 1. From the results, it was found that the method of the present invention yielded a PHA synthase having an activity with good yield.

  Next, synthesis of PHA was examined using this purified enzyme. 10 μl of the above purified enzyme solution was mixed with 10 μl of a 27.3 mg / ml (R) -3-hydroxyoctanoyl CoA Tris-HCl solution and 10 μl of a 3 mg / ml bovine serum albumin (manufactured by Sigma) Tris-HCl solution, 30 It was shaken gently at 24 ° C. for 24 hours.

  Collect 10 μl of the above reaction solution on a slide glass, add 10 μl of 1% Nile Blue A aqueous solution, mix on the slide glass, place a cover glass, and place a fluorescence microscope (330-380 nm excitation filter, 420 nm long-pass absorption) (Filter, manufactured by Nikon Corporation) was observed. As a result, particles emitting fluorescence were confirmed, and it was found that PHA was synthesized by PhaC1 according to this example.

  As a control, 10 μl of Tris-HCl solution was mixed with 27 μg / ml (R) -3-hydroxyoctanoyl CoA Tris-HCl solution 10 μl, 3 mg / ml bovine serum albumin (manufactured by Sigma) Tris-HCl solution 10 μl, After gently shaking at 30 ° C. for 24 hours, the sample was similarly stained with Nile Blue A and observed with a fluorescence microscope. As a result, particles emitting fluorescence were not confirmed.

  Further, a part of the PHA particles was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), vacuum-dried, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA. . The extract was filtered through a membrane filter with a pore size of 0.45 μm, and the molecular weight was determined by gel permeation chromatography (GPC; Tosoh HLC-8020, column; Polymer Laboratory PLgel MIXED-C (5 μm), solvent; chloroform, column temperature; 40 ° C. The result measured by polystyrene conversion is shown in FIG. As a result, Mn = 36,000 and Mw = 70,000.

<Example 2>
PKJE7 (Takara), a plasmid vector constructed to express the molecular chaperone group consisting of the molecular chaperone team of DnaK-DnaJ-GrpE in the JM109 strain transformed with the GST-fused PhaC1 expression vector obtained in Example 1. Bio product) was transformed by electroporation in the same manner as in Example 1.

The resulting strain was precultured overnight in 5 ml of LB medium containing 100 μg / ml of ampicillin and 20 μg / ml of chloramphenicol, and then 2 ml of the resulting strain was 100 μg / ml of ampicillin, 20 μg / ml of chloramphenicol, Add to 1 L of LB medium containing 4 mg / ml arabinose, shake culture at 30 ° C and 120 rpm, add IPTG when OD ₆₀₀ = 0.5 (final concentration 20 μg / ml), and further culture for 3 hours Continued.

  Next, as in Example 1, the cultured cells were crushed and centrifuged to separate soluble and insoluble components. When both soluble and insoluble components were subjected to Coomassie staining after SDS-PAGE, a band having a molecular weight of 87 kD corresponding to GST-fused PhaC1 was confirmed for both the soluble and insoluble components. The amount ratio of GST-fused PhaC1 band in this soluble component and insoluble component is approximately 3: 1. Compared to Comparative Example 1 described later, the co-expression with the molecular chaperone improves the solubility of GST-fused PhaC1. I found out. In addition, molecular weight bands corresponding to DnaK, DnaJ, and GrpE were mainly observed in the soluble components.

  Next, the soluble component of this cell disruption solution, that is, the GST-fused PhaC1 present in the crude enzyme was purified in the same manner as in Example 1. When this purified enzyme was subjected to SDS-PAGE, a 61 kDa single band corresponding to the molecular weight of PhaC1 was confirmed.

  The concentration of the obtained purified enzyme was quantified in the same manner as in Example 1 and further converted into a yield per culture solution. Further, the activity of the purified enzyme was measured in the same manner as in Example 1 and converted to the enzyme activity per amount of enzyme (mg). The results are shown in Table 1. From the results, it was found that the method of the present invention yielded a PHA synthase having an activity with good yield.

  Next, using this purified enzyme, PHA synthesis was examined in the same manner as in Example 1. As a result, fluorescent particles were confirmed, and it was found that PHA was synthesized by PhaC1 of this example.

  Furthermore, as a result of measuring the molecular weight by gel permeation chromatography in the same manner as in Example 1 using a part of the PHA particles, Mn = 38,000 and Mw = 75,000.

<Example 3>
A plasmid vector configured to express a molecular chaperone group consisting of two molecular chaperone teams of GroES-GroEL and trigger factor in the JM109 strain transformed with the GST-fused PhaC1 expression vector obtained in Example 1 pG-Tf2 (manufactured by Takara Bio Inc.) was transformed by electroporation in the same manner as in Example 1.

The resulting strain was precultured overnight in 5 ml of LB medium containing 100 μg / ml of ampicillin and 20 μg / ml of chloramphenicol, and then 2 ml of the resulting strain was 100 μg / ml of ampicillin, 20 μg / ml of chloramphenicol, Add to 1 L of LB medium containing 10 ng / ml of tetracycline, shake culture at 30 ° C and 120 rpm, add IPTG when OD ₆₀₀ = 0.5 (final concentration 20 µg / ml), and further culture for 3 hours Continued.

  Next, as in Example 1, the cultured cells were crushed and centrifuged to separate soluble and insoluble components. When the soluble component and the insoluble component were subjected to Coomassie staining after SDS-PAGE, a band having a molecular weight of 87 kD corresponding to GST-fused PhaC1 was confirmed for both the soluble component and the insoluble component. The amount ratio of the GST-fused PhaC1 band in this soluble component and insoluble component is approximately 4: 1. Compared to Comparative Example 1 described later, the solubility of GST-fused PhaC1 is improved by co-expression with the molecular chaperone. I found out. In addition, molecular weight bands corresponding to groES, groEL, and tig were mainly observed in the soluble components.

  Next, the soluble component of this cell disruption solution, that is, the GST-fused PhaC1 present in the crude enzyme was purified in the same manner as in Example 1. When this purified enzyme was subjected to SDS-PAGE, a 61 kDa single band corresponding to the molecular weight of PhaC1 was confirmed.

  The concentration of the obtained purified enzyme was quantified in the same manner as in Example 1 and further converted into a yield per culture solution. Further, the activity of the purified enzyme was measured in the same manner as in Example 1 and converted to the enzyme activity per amount of enzyme (mg). The results are shown in Table 1. From the results, it was found that the method of the present invention yielded a PHA synthase having an activity with good yield.

  Next, using this purified enzyme, PHA synthesis was examined in the same manner as in Example 1. As a result, fluorescent particles were confirmed, and it was found that PHA was synthesized by PhaC1 of this example.

  Furthermore, as a result of measuring the molecular weight by gel permeation chromatography in the same manner as in Example 1 using a part of the PHA particles, Mn = 32,000 and Mw = 69,000.

<Comparative Example 1>
The JM109 strain transformed with the GST-fused PhaC1 expression vector obtained in Example 1 was precultured overnight in 5 ml of LB medium containing 100 μg / ml of ampicillin, and then 2 ml thereof was treated with 100 μg / ampicillin. Add to 200 ml of LB medium containing 100 ml, and incubate with shaking at 30 ° C and 120 rpm. When OD ₆₀₀ = 0.5, add IPTG (final concentration 20 µg / ml), and continue culturing for another 3 hours. It was.

  Next, as in Example 1, the cultured cells were crushed and centrifuged to separate soluble and insoluble components. When both soluble and insoluble components were subjected to Coomassie staining after SDS-PAGE, a band having a molecular weight of 87 kD corresponding to PhaC1 fused with GST was confirmed for both the soluble and insoluble components. The amount ratio of the GST-fused PhaC band between the soluble component and the insoluble component was approximately 1: 8, indicating that most of the expressed GST-fused PhaC1 formed insoluble inclusion bodies.

  Next, the soluble component of this cell disruption solution, that is, the GST-fused PhaC present in the crude enzyme was purified in the same manner as in Example 1. When this purified enzyme was subjected to SDS-PAGE, a slight 61 kDa band corresponding to the molecular weight of PhaC1 was confirmed.

  The concentration of the obtained purified enzyme was quantified in the same manner as in Example 1 and further converted into a yield per culture solution. Further, the activity of the purified enzyme was measured in the same manner as in Example 1 and converted to the enzyme activity per amount of enzyme (mg). The results are shown in Table 1. From the results, it was found that when the molecular chaperone was not coexpressed with PHA synthase, the yield of the obtained PhaC1 was low and the enzyme activity per enzyme amount was also low.

  Next, using this purified enzyme, PHA synthesis was examined in the same manner as in Example 1. As a result, it was found that only 10% or less of weakly fluorescent particles were confirmed as compared with Example 1, and the amount of PHA synthesized was reduced.

<Comparative example 2>
The JM109 strain transformed with the GST-fused PhaC1 expression vector obtained in Example 1 is a plasmid vector, pGro7 (Takara Bio (Takara Bio)), which is configured to express a molecular chaperone group consisting of a molecular chaperone team of GroES-GroEL. Co., Ltd.) was transformed by electroporation in the same manner as in Example 1.

The resulting strain was precultured overnight in 5 ml of LB medium containing 100 μg / ml of ampicillin and 20 μg / ml of chloramphenicol, and then 2 ml of the resulting strain was 100 μg / ml of ampicillin, 20 μg / ml of chloramphenicol, Add to 1 L of LB medium containing 4 mg / ml arabinose, shake culture at 30 ° C, 120 rpm, add IPTG when OD ₆₀₀ = 0.5 (final concentration 20 μg / ml), and further culture for 3 hours Continued.

  Next, as in Example 1, the cultured cells were crushed and centrifuged to separate soluble and insoluble components. When the soluble component and the insoluble component were subjected to Coomassie staining after SDS-PAGE, a band having a molecular weight of 87 kD corresponding to GST-fused PhaC1 was confirmed for both the soluble component and the insoluble component. The amount ratio of the GST-fused PhaC1 band between the soluble component and the insoluble component was approximately 2: 3. It was found that more than half of the expressed GST-fused PhaC1 formed insoluble inclusion bodies. In addition, molecular weight bands corresponding to groES and groEL were mainly observed in the soluble components.

  Next, the soluble component of this cell disruption solution, that is, the GST-fused PhaC1 present in the crude enzyme was purified in the same manner as in Example 1. When this purified enzyme was subjected to SDS-PAGE, a 61 kDa single band corresponding to the molecular weight of PhaC1 was confirmed.

  The concentration of the obtained purified enzyme was quantified in the same manner as in Example 1 and further converted into a yield per culture solution. Further, the activity of the purified enzyme was measured in the same manner as in Example 1 and converted to the enzyme activity per amount of enzyme (mg). The results are shown in Table 1. The results showed that the yield of PhaC1 obtained was low and the enzyme activity per enzyme amount was low depending on the type of molecular chaperone co-expressed with PHA synthase.

  Next, using this purified enzyme, PHA synthesis was examined in the same manner as in Example 1. As a result, it was found that only 10% or less of weakly fluorescent particles were confirmed as compared with Example 1, and the amount of PHA synthesized was reduced.

<Comparative Example 3>
The JM109 strain transformed with the GST-fused PhaC1 expression vector obtained in Example 1 was subjected to pTf16 (manufactured by Takara Bio Inc.), a plasmid vector configured to express a molecular chaperone of the trigger factor alone. Transformation was performed by electroporation as in Example 1.
The resulting strain was precultured overnight in 5 ml of LB medium containing 100 μg / ml of ampicillin and 20 μg / ml of chloramphenicol, and then 2 ml of the resulting strain was 100 μg / ml of ampicillin, 20 μg / ml of chloramphenicol, Add to 1 L of LB medium containing 4 mg / ml arabinose, shake culture at 30 ° C, 120 rpm, add IPTG when OD ₆₀₀ = 0.5 (final concentration 20 μg / ml), and further culture for 3 hours Continued.

  Next, as in Example 1, the cultured cells were crushed and centrifuged to separate soluble and insoluble components. When the soluble component and the insoluble component were subjected to Coomassie staining after SDS-PAGE, a band having a molecular weight of 87 kD corresponding to GST-fused PhaC1 was confirmed for both the soluble component and the insoluble component. The amount ratio of the GST-fused PhaC1 band between the soluble component and the insoluble component was approximately 1: 3, indicating that most of the expressed GST-fused PhaC1 forms insoluble inclusion bodies. In addition, a molecular weight band corresponding to tig was mainly observed in the soluble component.

  Next, the soluble component of this cell disruption solution, that is, the GST-fused PhaC1 present in the crude enzyme was purified in the same manner as in Example 1. When this purified enzyme was subjected to SDS-PAGE, a 61 kDa single band corresponding to the molecular weight of PhaC1 was confirmed.

  The concentration of the obtained purified enzyme was quantified in the same manner as in Example 1 and further converted into a yield per culture solution. Further, the activity of the purified enzyme was measured in the same manner as in Example 1 and converted to the enzyme activity per amount of enzyme (mg). The results are shown in Table 1. The results showed that the yield of PhaC1 obtained was low and the enzyme activity per enzyme amount was low depending on the type of molecular chaperone co-expressed with PHA synthase.

  Next, using this purified enzyme, PHA synthesis was examined in the same manner as in Example 1. As a result, it was found that only 10% or less of weakly fluorescent particles were confirmed as compared with Example 1, and the amount of PHA synthesized was reduced.

<Example 4>
Using the PhaC1 purified enzyme obtained in Example 1, synthesis of PHA was examined using (R) -3-hydroxy-5-phenylvaleryl CoA as a monomer. As a monomer, 28.3 mg / ml (R) -3-hydroxy-5-phenylvaleryl CoA (3-hydroxy-5-phenylvaleric acid obtained by hydrolyzing 3-hydroxyphenylvalerate obtained by Reformatsky reaction) And prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)) PHA was synthesized in the same manner as in Example 1 except that 10 μl of Tris-HCl solution was used. investigated. As a result, particles emitting fluorescence were confirmed, and it was found that PHA was synthesized by PhaC1 of this example even when (R) -3-hydroxy-5-phenylvaleryl CoA was used as a monomer.

  Furthermore, the result of having measured the molecular weight by gel permeation chromatography in the same manner as in Example 1 using a part of the PHA particles is shown in FIG. As a result, Mn = 37,000 and Mw = 69,000. Moreover, UV absorption was confirmed in this polymer (there is a slight shift in the development time due to the flow path length between the RI and UV detector), and it was speculated that the monomer having a phenyl group was introduced into the PHA. .

<Example 5>
Using the PhaC1 purified enzyme obtained in Example 1, synthesis of PHA was examined using (R, S) -3-hydroxy-5-phenoxyvaleryl CoA as a monomer. As a monomer, 28.8 mg / ml (R, S) -3-hydroxy-5-phenoxyvaleryl CoA (3-hydroxy-5-phenoxy ester obtained by Reformatsky reaction of 3-phenoxypropanal and ethyl bromoacetate was used. After hydrolyzing the herbic acid ester to obtain 3-hydroxy-5-phenoxyvaleric acid, prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)) Using 10 μl of Tris-HCl solution The synthesis of PHA was examined in the same manner as in Example 1. As a result, fluorescent particles were confirmed, and it was found that PHA was synthesized by PhaC1 of this example even when (R, S) -3-hydroxy-5-phenoxyvaleryl CoA was used as a monomer. It was.

  Furthermore, the result of having measured the molecular weight by gel permeation chromatography in the same manner as in Example 1 using a part of the PHA particles is shown in FIG. As a result, Mn = 65,000 and Mw = 172,000. Moreover, UV absorption was confirmed in this polymer (there is a slight shift in the development time due to the flow path length between the RI and UV detector), and it was speculated that the monomer having a phenyl group was introduced into the PHA. .

FIG. 3 is a diagram showing the results of GPC analysis of PHA in Example 1. It is a figure which shows the GPC analysis result of PHA in Example 4. FIG. 6 is a diagram showing the GPC analysis result of PHA in Example 5.

Claims (9)

  A method for obtaining a polyhydroxyalkanoate synthase, comprising culturing cells capable of co-expressing a polyhydroxyalkanoate synthase and a molecular chaperone, and co-expressing the polyhydroxyalkanoate synthase and the molecular chaperone in the cell.   The method according to claim 1, wherein in the co-expression of the polyhydroxyalkanoate synthase and the molecular chaperone in the cell, the expression start time of the molecular chaperone coincides with or precedes the expression start time of the polyhydroxyalkanoate synthase.   The method according to claim 1 or 2, wherein the cell is a cell transformed with both a vector capable of expressing a gene encoding a polyhydroxyalkanoate synthase and a vector capable of expressing a gene encoding a molecular chaperone.   The method according to any one of claims 1 to 3, wherein the molecular chaperone is a molecular chaperone group consisting of a molecular chaperone team of DnaK-DnaJ-GrpE.   The method according to any one of claims 1 to 3, wherein the molecular chaperone is a molecular chaperone group consisting of two molecular chaperone teams, DnaK-DnaJ-GrpE and GroES-GroEL. The method according to any one of claims 1 to 3, wherein the molecular chaperone is a molecular chaperone group composed of two molecular chaperone teams of GroES-GroEL and a trigger factor. The method according to any one of claims 1 to 5, wherein the cell is Escherichia coli. A cell characterized by being cotransformed with both a vector capable of expressing a gene encoding a polyhydroxyalkanoate synthase and a vector capable of expressing a gene encoding a molecular chaperone. A polyhydroxyalkanoate synthase obtained by culturing a cell capable of co-expressing a polyhydroxyalkanoate synthase and a molecular chaperone and co-expressing the polyhydroxyalkanoate synthase and the molecular chaperone in the cell.

Images