JP5578598B2 - Novel aromatic ring hydroxylase and its gene - Google Patents

Description

  The present invention introduces and expresses an aromatic ring hydroxylase that is one of the enzymes that play the most important role in the degradation of various aromatic compounds (aromatic rings), a gene encoding the enzyme, and this gene group. The present invention relates to a method for producing a hydroxylated aromatic compound using the microorganisms.

  Many aromatic compounds are toxic to cells and become pollutants in the environment. Many microorganisms that decompose and assimilate these have been separated in nature, and many have been separated so far. Under aerobic conditions, the decomposition of aromatic compounds is initiated by the initial oxygenation reaction, and is also decomposed to water and carbon dioxide via catechol, a common intermediate metabolite, but aromatic ring hydroxylation, which is the initial enzyme, It has been clarified that enzymes play the most important role in the degradation of aromatic compounds (Non-Patent Documents 1 and 2).

  Substance conversion processes such as the production and decomposition of low-molecular compounds that utilize microorganisms have higher reaction specificity (substrate characteristics, steric characteristics, and position specificity) and mild conditions (room temperature) than conventional chemical conversion processes. It is characterized by being capable of reacting at normal pressure) and being difficult to produce harmful by-products. In particular, the oxidation reaction is required to be converted to an enzyme process that can replace a harsh chemical process, and the search for an oxidase is actively performed.

  Aromatic compounds are widely used as raw materials for pharmaceuticals and chemicals. Introducing a hydroxyl group into an aromatic ring is a starting point for introducing various functional groups into an aromatic compound, and is an especially important process in the substance conversion process of an aromatic compound. To date, research on aromatic ring hydroxylase has been widely carried out, but aromatic ring hydroxylase is usually divided into three main subunits, hydroxylase (α-subunit, β-subunit and γ-subunit). It is common to exhibit a function as a very complex multicomponent enzyme (ferredoxin) and ferredoxin reductase (ferredoxin reductase) in addition to the unit (non-patent document 3). And Patent Documents 1 and 2).

  However, such a complex subunit structure and molecular complex are generally not suitable for industrial use, and there is a strong demand for a protein that can exhibit an equivalent function with a simple protein composed of a single polypeptide chain. Therefore, a novel hydroxylase composed of a single component different from the multicomponent complex enzymes known so far is very useful in the production of hydroxylated aromatic compounds.

Harayama et al., (1997) Polycyclic aromatic hydrocarbon bioremediation design. Curr. Opin. Biotechnol. 8, 268-273. Furukawa et al., (2004) Biphenyl dioxygenases: Functional versatilities and directed evolution. J. Bacteriol. 186, 5189-5196. Gibson et al., (2000) Aromatic hydrocarbon dioxygenases in environmental biotechnology. Curr. Opin. Biotechnol. 11, 236-243. WO2004 / 050875 JP2000-69968

  An object of the present invention is to solve the above-mentioned problems of the prior art, and a novel aromatic ring hydroxylase consisting of a simple protein of a single polypeptide chain and its gene have been found, and an aromatic ring is obtained by biological techniques. The present invention intends to provide a new means for industrially performing hydroxylation.

  If the present inventors obtain a novel single-component hydroxylase, introduce it into a microorganism using an appropriate vector, and obtain a transformant carrying this gene in the cell, DNA fragment encoding a novel hydroxylase consisting of a single component from a DNA library using activated sludge as an environmental sample. Succeeded in getting. Then, by transforming a microorganism using a recombinant vector containing this DNA fragment and culturing the obtained transformant, it was found that a hydroxylated aromatic compound was produced, and the present invention was It came to complete.

That is, the present invention is as follows.
(1) comprising the amino acid sequence represented by SEQ ID NO: 1, or comprising the amino acid sequence having one or several amino acid additions, deletions or substitutions in the amino acid sequence represented by SEQ ID NO: 1, and an aromatic ring A protein having hydroxylase activity.

(2) A gene encoding the protein according to (1) above.

(3) including the base sequence represented by SEQ ID NO: 2 or including a base sequence having one or several nucleotide additions, deletions or substitutions in the base sequence represented by SEQ ID NO: 2 and an aromatic ring A gene encoding a protein having hydroxylase activity.

(4) A gene that hybridizes under stringent conditions with a DNA having the base sequence represented by SEQ ID NO: 2 or a base sequence complementary to the base sequence and encodes a protein having an aromatic ring hydroxylase.

(5) comprising the amino acid sequence represented by SEQ ID NO: 3, or comprising the amino acid sequence having one or several amino acid additions, deletions or substitutions in the amino acid sequence represented by SEQ ID NO: 3, and an aromatic ring A protein having hydroxylase activity.

(6) A gene encoding the protein according to (5) above.

(7) including the base sequence represented by SEQ ID NO: 4 or including the base sequence having one or several nucleotide additions, deletions or substitutions in the base sequence represented by SEQ ID NO: 4 and an aromatic ring A gene encoding a protein having hydroxylase activity.

(8) A gene that hybridizes under stringent conditions with a DNA having the base sequence represented by SEQ ID NO: 4 or a base sequence complementary to the base sequence and encodes a protein having an aromatic ring hydroxylase.

(9) A recombinant vector comprising the gene according to any one of (2) to (4) or (6) to (8) above.

(10) A microorganism containing the recombinant vector according to (9).

(11) The microorganism according to (10) above, wherein the microorganism is Escherichia coli.

(12) Water characterized by culturing the microorganism according to (10) or (11) above in a medium containing an aromatic compound, and collecting the hydroxylated aromatic compound from the culture or the cells. A method for producing an oxidized aromatic compound.

  In general, the aromatic ring hydroxylase generally functions as a very complex multi-component complex enzyme. When these genes are introduced into a recombinant microorganism for expression, all of the component genes must be expressed. If for any reason the expression of any one of the genes decreases, hydroxylation is accompanied accordingly. In the presence of a component gene whose ability is reduced and is not expressed at all, hydroxylation ability is completely lost. In addition, since each component enzyme has optimum reaction conditions (temperature, pH, etc.), it is difficult to set reaction conditions in which all component enzymes are optimum, and efficient hydroxylation. Setting conditions for the reaction is very difficult. Since the aromatic ring hydroxylase protein provided by the present invention is a simple protein consisting of a single polypeptide chain, the conditions for gene expression and optimization of hydroxylation are greatly facilitated. Furthermore, since the total length of the aromatic ring hydroxylase gene is shortened, high efficiency can be expected in genetic engineering experiments such as plasmid construction and microbial introduction. For the above reasons, microorganisms that retain the gene encoding the protein have dramatically facilitated various work and optimization conditions, and simplified and efficient hydroxylation of aromatic compounds. Become.

A specific example of the aromatic ring hydroxylase protein of the present invention is a DNA-derived protein obtained by screening an environmental DNA library constructed from activated sludge, and a single polypeptide chain that functions as an aromatic ring hydroxylase Of 397 or 512 amino acids. The amino acid sequences are shown in SEQ ID NOs: 1 and 3 in the sequence listing.
In addition, the aromatic ring hydrogenase protein of the present invention is not limited to those shown in SEQ ID NOS: 1 and 3, but in the amino acid sequences shown in SEQ ID NOS: 1 and 3, addition or deletion of one or several amino acids Or a protein having an amino acid sequence having a substitution and having a slight mutation such as having an aromatic ring hydroxylase activity. Such mutations include not only natural mutations but also artificial mutations such as mutations or mutations using, for example, genetic engineering techniques.

  The gene for the aromatic ring hydroxylase protein of the present invention is not particularly limited as long as it encodes the protein having the amino acid sequence shown by SEQ ID NOs: 1 and 3, or the mutant protein thereof, but specific examples thereof. As shown in SEQ ID NOS: 2 and 4 in the Sequence Listing, the base sequence has a base sequence, or one or several nucleotides are added, deleted or substituted in the base sequence shown in SEQ ID NOS: 2 and 4 above. And a protein that encodes a protein having an aromatic ring hydroxylase activity, and further hybridizes under stringent conditions with a gene having the base sequence shown in SEQ ID NOs: 2 and 4 or a complementary sequence thereof. It may be a soybean that encodes a protein having an aromatic ring hydroxylase activity.

  Such a gene can be obtained by, for example, screening a gene derived from a bacterium by using hybridization between DNA proteins. In the present invention, “stringent conditions” refers to conditions under which only specific hybridization occurs and non-specific hybridization does not occur.

The gene of the present invention can be obtained, for example, by the following procedure.
The aromatic ring hydroxylase gene of the present invention collects DNA from environmental samples such as soil, rivers and ocean water or sediments, activated sludge, etc. that are considered to contain aromatic rings, and physically, enzymatically or chemically. The sheared DNA is ligated to a vector such as a plasmid, phage, fosmid, etc., and a host microorganism is transformed with the vector to prepare a microbial library containing the recombinant vector. Here, focusing on the fact that a catechol 2,3-dioxygenase gene that catalyzes a continuous metabolic reaction is known in the vicinity of the aromatic ring hydroxylase, the target gene is retained. A clone can be selected. That is, when catechol is sprayed on a microbial library and a yellow-colored clone is selected, it is found that the catechol 2,3-dioxygenase gene is contained in the clone. Using this as an index, the library can be screened to obtain adjacent aromatic ring hydroxylases.

In addition, chemical synthesis can also be performed based on the base sequences shown in SEQ ID NOs: 2 and 4.
Furthermore, as described above, an aromatic ring hydroxylase gene can be obtained by screening whether or not the thus obtained gene and other genes hybridize under stringent conditions. Alternatively, an aromatic ring hydroxylase gene comprising a mutant protein can also be obtained by using a known mutagenesis method for the obtained gene.

By inserting the aromatic ring hydroxylase gene of the present invention into an appropriate expression vector and introducing it into a microorganism for expression, a microorganism that retains the ability to hydroxylate an aromatic ring can be produced. Examples of the vector that can be used include a pUC vector, and examples of the host microorganism include E. coli JM109.
The above aromatic ring hydroxylase gene is introduced into microorganisms such as Escherichia coli, and then mixed and cultured with various aromatic compounds using the expressed recombinant microorganism, thereby introducing hydroxyl groups specifically into these aromatic compounds. can do.
According to the method of the present invention, for example, catechol is obtained as a conversion product when phenol is used as a substrate. However, the aromatic compound produced by the method of the present invention is not limited to catechol, and the substrate is toluene. In addition to aromatic compounds such as styrene, cresol, and chlorobenzene, polycyclic aromatic compounds such as naphthalene, phenanthrene, and anthracene, and heteroaromatic compounds such as 2-phenylindole and 1-phenylpyrazole can be given.
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Example 1]
Isolation and Analysis of Aromatic Hydroxylase Gene (1) Preparation of Environmental DNA Library Environmental DNA extracted from activated sludge was digested using End-Repair Enzyme attached to CopyControl Fosmid Library Production Kit (EPICENTRE). Smoothing was performed. Thereafter, DNA having an average chain length of about 33-48 kb was fractionated from the gel by pulse field electrophoresis. DNA was purified from the fragment excised using β-agarase (NEB).

  Subsequently, a ligation reaction was performed using T4 DNA ligase with the prepared insert DNA fragment and the pCC1FOS fosmid vector (EPICENTRE). After vector ligation, in vitro packaging was performed using MaxPlant Lambda Packaging Extracts (EPICENTRE). EPI300 (EPICENTRE) was used as the host E. coli. Escherichia coli transformed with λ phage was selected by chloramphenicol resistance in an LB agar medium containing 12.5 μg / mL chloramphenicol.

  E. coli colonies were picked and cultured in 1 mL of 2 × YT medium containing 12.5 μg / mL chloramphenicol at 37 ° C. for 18 hours at 1200 rpm. Next, 200 μL of the E. coli culture solution from which the colonies were collected was transferred to a new 800 μL 2 × YT medium, mixed, cultured at 37 ° C. for 30 minutes while shaking at 250 rpm, and then 1 μL of Copy Control Induction Solution ( EPICENTRE) was added to increase the fosmid vector copy number of the host E. coli EPI300. Furthermore, after culturing Escherichia coli for 2 hours at 37 ° C. while shaking vigorously at 1200 rpm, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.5). Thereafter, 150 μL of BugBuster Plus Benzoate Nuclease (Novagen) was added to lyse E. coli cells. Subsequently, the supernatant separated by centrifugation was obtained as a cell extract.

  Next, 5 μL of catechol (Tokyo Kasei Co., Ltd.) was added to 100 μL of the cell extract at a final concentration of 0.5 mM, and the reaction was carried out at 25 ° C. while mixing at 250 rpm. After 1 and 16 hours, fosmid clones were selected that showed yellow color due to the formation of 2-hydroxymuconate semialdehyde by degradation of catechol.

(2) Determination of base sequence Fosmid DNA was extracted from a clone showing catechol-degrading activity using FosmidMAX DNA Purification Kit (EPICENTRE). Subsequently, the DNA was randomly fragmented using a DNA fragmentation apparatus (Gene machines), fractionated by agarose gel electrophoresis, and then agarose fragments containing about 2 kb to 3 kb of DNA were excised. After DNA was purified from the excised agarose fragment, the DNA fragment was smoothed using T4 DNA Polymerase (Takara Bio Inc.). The blunted DNA fragment was subjected to a vector ligation reaction with 50 ng of pUC118 / HincII / BAP that had been dephosphorylated, followed by ethanol precipitation and dissolution in 10 μL of sterile water.

  A part of the prepared library was introduced into E. coli by electroporation (Gene pulser, BIO RAD) using 1 μL of ligation solution and 25 μL of host E. coli DH10B (Invitrogen). A transformant was obtained by culturing overnight at 37 ° C. in an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, and 40 μg / mL X-gal.

  288 E. coli white colonies were randomly picked using Q-Bot (GENETIX). Each LB medium containing 120 μL of 100 μg / mL ampicillin dispensed in a 96-well plate was inoculated, and cultured at 37 ° C. overnight. A template for sequencing was prepared using TempliPhiDNA Sequencing Template Amplification Kit (GE Healthcare) in the culture medium. The reaction of TempliPhi followed the attached instruction manual.

  Next, the nucleotide sequence was analyzed using DNA prepared by TempliPhi as a template. The sequence reaction was performed with BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystem). PCR and purification were performed according to the attached instruction manual, and analysis was performed using ABI3730XL (Applied Biosystem). Artemis (Sanger Center) was used for the analysis of the base sequence.

  As a result of the homology analysis of the base sequence, as shown in FIG. 1, in addition to the catechol 2,3-dioxygenase gene, the aromatic ring hydroxylase gene is contained in the inserted fragment of the fosmid clone showing catechol-degrading activity. Were present. The amino acid sequences of the aromatic ring hydroxylase genes are shown in SEQ ID NOs: 1 and 3, and the DNA base sequences are shown in SEQ ID NOs: 2 and 4, respectively.

[Example 2]
Expression of aromatic ring hydroxylase and confirmation of its activity An experiment was conducted to confirm that the DNA of SEQ ID NO: 2 obtained in Example 1 above encodes a hydroxylase. First, a DNA fragment containing an aromatic ring hydroxylase gene and a catechol 2,3-dioxygenase gene was amplified by PCR. PHE-F (SEQ ID NO: 5) was synthesized as a primer at the 5 ′ end, and PHE-R (SEQ ID NO: 6) was synthesized as a primer at the 5 ′ end. PCR was carried out for a total of 25 cycles, with one cycle consisting of 98 ° C for 10 seconds, 60 ° C for 5 seconds, and 72 ° C for 2 minutes. The PCR product was purified and further treated with the restriction enzyme XbaI. This insert DNA was mixed with a pUC19 vector treated with HincII and XbaI so that the total amount was 10 μL, and a ligation reaction was performed. The DNA was collected, inserted into E. coli strain JM109 by the heat shock method, cultured on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, 40 μg / mL X-gal at 37 ° C. overnight. A conversion was obtained.

  The obtained Escherichia coli transformant was streaked on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, and further 100 μM phenol, and cultured at 37 ° C. overnight. As a result, a yellow colony was obtained. Analysis was performed with a spectrophotometer, and the obtained yellow compound was confirmed to be 2-hydroxymuconate semialdehyde having a maximum absorption wavelength at 375 nm. Accordingly, the gene of SEQ ID NO: 2 encodes an aromatic ring hydroxylase consisting of a single component, which converts phenol into catechol, and catechol 2 is present by catechol 2,3-dioxygenase present downstream. -Hydroxymuconate It was confirmed that it was converted to semialdehyde.

As a comparison, a DNA fragment containing only the catechol 2,3-dioxygenase gene using EDO-F (SEQ ID NO: 7) as a 5'-end primer and PHE-R (SEQ ID NO: 6) as a 3'-end primer Was amplified by PCR. The Escherichia coli transformant obtained in the same manner as described above was streaked on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, and 100 μM phenol, and cultured at 37 ° C. overnight. The presenting colony was not obtained. On the other hand, as a result of spraying catechol, colonies showing a yellow color were obtained.
From the above, it was revealed that the gene of SEQ ID NO: 2 encodes a novel aromatic ring hydroxylase consisting of a single component. Furthermore, it has been clarified that a hydroxylated compound is produced by introducing a gene encoding this aromatic ring hydroxylase.

Example 3
Expression of Aromatic Hydroxylase and Confirmation of Its Activity An experiment was conducted to confirm that the DNA of SEQ ID NO: 4 obtained in Example 1 encodes a hydroxylase. First, a DNA fragment containing an aromatic ring hydroxylase gene and a catechol 2,3-dioxygenase gene was amplified by PCR. PHE2-F (SEQ ID NO: 8) was synthesized as a primer at the 5 ′ end, and PHE2-R (SEQ ID NO: 9) was synthesized as a primer at the 5 ′ end. PCR was carried out for a total of 25 cycles, with one cycle consisting of 98 ° C for 10 seconds, 60 ° C for 5 seconds, and 72 ° C for 2 minutes. The PCR product was purified and further treated with the restriction enzyme KpnI. The insert DNA and the pUC19 vector treated with KpnI were mixed so that the total amount was 10 μL, and ligation reaction was performed. The DNA was collected, inserted into E. coli strain JM109 by the heat shock method, cultured on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, 40 μg / mL X-gal at 37 ° C. overnight. A conversion was obtained.

  The obtained Escherichia coli transformant was streaked on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, and further 100 μM phenol, and cultured at 37 ° C. overnight. As a result, a yellow colony was obtained. Analysis was performed with a spectrophotometer, and the obtained yellow compound was confirmed to be 2-hydroxymuconate semialdehyde having a maximum absorption wavelength at 375 nm. Accordingly, the gene of SEQ ID NO: 2 encodes an aromatic ring hydroxylase consisting of a single component, which converts phenol into catechol, and catechol 2 is present by catechol 2,3-dioxygenase present downstream. -Hydroxymuconate It was confirmed that it was converted to semialdehyde.

For comparison, a DNA fragment containing only the catechol 2,3-dioxygenase gene using EDO-F (SEQ ID NO: 7) as a 5′-end primer and PHE2-R (SEQ ID NO: 9) as a 3′-end primer Was amplified by PCR. The Escherichia coli transformant obtained in the same manner as described above was streaked on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, and 100 μM phenol, and cultured at 37 ° C. overnight. The presenting colony was not obtained. On the other hand, as a result of spraying catechol, colonies showing a yellow color were obtained.
From the above, it was revealed that the gene of SEQ ID NO: 4 encodes a novel aromatic ring hydroxylase consisting of a single component. Furthermore, it has been clarified that a hydroxylated compound is produced by introducing a gene encoding this aromatic ring hydroxylase.

It is a figure which shows the positional relationship of the aromatic-ring hydroxylase gene and catechol 2,3-dioxygenase gene which were obtained from environmental DNA, and the conversion mode of phenol.

Claims (7)

The amino acid sequence shown in SEQ ID NO: 3 is included, or the amino acid sequence shown in SEQ ID NO: 3 contains an amino acid sequence having one or several amino acid additions, deletions or substitutions, and phenol is converted to catechol A protein having an aromatic ring hydroxylase activity. A gene encoding the protein according to claim 1 . Contains the base sequence shown in SEQ ID NO: 4 or contains the base sequence shown in SEQ ID NO: 4 with one or several nucleotide additions, deletions or substitutions, and converts phenol into catechol A gene encoding a protein having an aromatic ring hydroxylase activity. A recombinant vector comprising the gene according to claim 2 or 3 . A microorganism containing the recombinant vector according to claim 4 . The microorganism according to claim 5 , wherein the microorganism is Escherichia coli. A method for producing catechol , comprising culturing the microorganism according to claim 5 or 6 in a medium containing phenol and collecting catechol from the culture or the fungus body.