JP2003144167A - Desulfurization method using recombinant microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently desulfurize a sulfur-containing heterocyclic compound resistant to desulfurization by using a microorganism. SOLUTION: The desulfurization method of a sulfur-containing heterocyclic compound comprises the use of a recombinant microorganism containing a desulfurization enzyme gene at the downstream of a promoter composed of (a) a DNA expressed by the sequence No.1 (refer to the specification) and having promoter activity or (b) a DNA having promoter activity and hybridizable under a stringent condition with a DNA having a base sequence complimentary to the DNA expressed by the sequence No.1.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for desulfurizing a sulfur-containing heterocyclic compound using a microorganism. More specifically, by using a recombinant microorganism with improved desulfurization capacity, desulfurization of alkylated dibenzothiophenes and alkylated benzothiophenes contained in fossil fuel oil such as petroleum, and removal of sulfur that is a source of environmental pollution Regarding how to enable.

[0002]

2. Description of the Related Art Fossil fuel oils such as petroleum contain many kinds of sulfur compounds. When sulfur is released into the atmosphere, it has a serious effect on the environment. ing. The concentration of sulfur contained in various crude oils in the world is different, but it is about 1.13% on average, and typical sulfur compounds contained are mercaptans, sulfides,
Thiophenes. In each fraction obtained by distilling and fractionating crude oil, the heavier fraction has a higher proportion of thiophenes such as benzothiophene and dibenzothiophene.

As methods for desulfurizing sulfur-containing compounds, methods such as alkali cleaning, solvent cleaning and adsorption are known, but at present, hydrodesulfurization is the mainstream method. The hydrodesulfurization method is a method in which a sulfur compound in a petroleum fraction is reacted with hydrogen in the presence of a catalyst to remove sulfur as hydrogen sulfide. As the catalyst, a metal catalyst such as cobalt, molybdenum, nickel, or tungsten having alumina as a carrier is used. However, the hydrodesulfurization method can reduce the sulfur concentration of the entire fossil fuel, but cannot sufficiently desulfurize the sulfur-containing heterocyclic compound that is difficult to desulfurize. For example, looking at the sulfur compounds remaining in the gas oil after hydrodesulfurization, 94% of the 400 ppm gas oil was alkylated dibenzothiophenes and alkylated benzothiophenes, and 67% of the 45 ppm gas oil was alkylated dibenzothiophenes. It is reported to consist of thiophene and alkylated benzotiphene (Kenji Maruhashi, Chemical Engineering, 46 (9), 24-29 (2001)).

To completely remove such alkylated dibenzothiophenes and alkylated benzothiophenes by a hydrodesulfurization method, higher reaction temperature and pressure are required, and thus enormous equipment investment and operation cost are required. It is expected that On the other hand, as a method for desulfurizing such a sulfur-containing heterocyclic compound that is difficult to desulfurize, a desulfurization method using a microorganism has been studied. For example, Pseudomonas (P
seudomonas) CB1 (Isbister, JD and Kobylinski, E.
A. (Microbial desulfurization of coal, in Coal Scien
ce and Technology, Ser.9, p.627 (1985)), Rhodococcus rhodochrous IGTS8 (ATCC
53968) (Kilbane, JJ Resources, Conservationand Re
cycling, 3, 69-70 (1990)), Rhodokoccus erytythropolis KA2-5-1 strain (FERM
P-16277) (Kobayashi, M. Horiuchi, K., Yoshikawa, O.,
Hirasawa, K., Ishii, Y., Fujino, K., Sugiyama, H.and M
aruhashi, K., Biosci. Biotechnol. Biochem., 65 (2), 28
9-304, 2001), Corynebacteriums
p. SY-1 (Ohmori, T., Monna, L., Saiki, Y. and Kodam
a, T.Appl.Environ.Microbiol., 58,911-915, 1992),
Brevibacterium sp. DO (van Aff
erden, M., Schacht, S., Klein, J.andTruper, HG, Arc
h. Microbiol., 153, 324-328, 1990) and Arthrobacter K3b (Dahlberg, MD (1992) Third In
ternational Symposium on the Biological Processing
of Coal, May4-7, ClearwaterBeach, FL, pp.1-10.Electr
The desulfurization method using ic Power Research Institute, PaloAlto, CA.) has been reported so far. The microbial desulfurization method has an advantage that benzothiophenes and the like can be effectively desulfurized without requiring high temperature and pressure unlike the hydrodesulfurization method.

The microbial desulfurization method requires the production (culturing) of microbial cells of desulfurization microorganisms. However, desulfurization enzyme genes related to desulfurization are suppressed by expression of inorganic sulfur compounds and sulfur-containing amino acids in the culture medium (Mamie ZL, Szuires CH,
Monticello DJ, and Chils J.D., J. Bacteriol., 17
8, 6409-6418, 1996). Therefore, inexpensive inorganic sulfur compounds cannot be used as an essential sulfur source in bacterial cell production, and the sulfur concentration in the culture broth must be accurately maintained below a certain level. This is because the promoter controlling the expression of the desulfurase enzyme gene is inducible.
However, constitutively expressible promoters (for example, rRNA promoters) which are widely used at present have low activity and cannot be expected to improve protein production.

By the way, the gas oil after hydrodesulfurization contains a considerable amount of alkylated sulfur-containing heterocyclic compounds having 6 or more carbon atoms. However, a heterocyclic compound having an alkyl side chain with a large number of carbon atoms generally has low permeability to the cell membrane of microorganisms. Therefore, it has been reported that even if the desulfurizing enzyme in the bacterial cell has these desulfurizing ability, it cannot be desulfurized in the bacterial cell due to impediment of membrane permeation (Kobayashi, M., Horiuchi,
K., Yoshikawa, O., Hirasawa, K., Ishii, Y., Fujino, K.,
Sugiyama, H. and Maruhashi, K., Biosci. Biotechno
l. Biochem., 65 (2), 298-304, 2001). According to this report, alkylated dibenzothiophenes having 1 to 5 carbon atoms in the alkyl side chain are Rhodococcus erythropolis KA2-5-
Although it permeates through the bacterial cell membrane of one strain and is desulfurized, 4,6-dipropyldibenzothiophene, whose alkyl side chain has 6 carbon atoms, can be desulfurized in the cell-free extract but not in the bacterial cells. .

[0007] In the past, in Gram-negative bacteria, by mutating the fatty acid composition to make the cell membrane hydrophobic,
Report that the membrane permeability of hydrophobic substances is increased (Kobayash
i, H., Takami, H., Hirayama, H., Kobata, K., Usami,
R. and Horikoshi, KJ Bacteriology, 181 (15), 4493-
4498 (1999)). However, in Gram-positive bacteria, there is no report that the fatty acid composition of the cell membrane was altered by mutation or recombinant technology.

[0008]

DISCLOSURE OF THE INVENTION The present invention is to provide a method for effectively desulfurizing a sulfur-containing heterocyclic compound which is difficult to desulfurize under mild conditions using a microorganism.

[0009]

Means for Solving the Problems As a result of intensive studies, the present inventors succeeded in producing a recombinant microorganism having a high desulfurization ability by using a novel promoter which is constantly expressed and has a high activity. . Then, a method for efficiently desulfurizing a sulfur-containing heterocyclic compound that is difficult to desulfurize was established by using the recombinant microorganism, and the present invention was completed. That is, the present invention relates to the following (1) to (9).

(1) A method for desulfurizing a sulfur-containing heterocyclic compound using a recombinant microorganism containing a desulfurase enzyme gene downstream of a promoter composed of the DNA of (a) or (b) below. (a) D having the promoter activity shown in SEQ ID NO: 1
NA. (b) A DNA which hybridizes with a DNA having a base sequence complementary to the DNA represented by SEQ ID NO: 1 under stringent conditions and has a promoter activity.

(2) The method according to (1) above, wherein the desulfurase gene is a desulfurase gene that selectively cleaves the CS bond of a sulfur-containing heterocyclic compound. (3) the desulfurization enzyme gene is a desulfurization gene derived from Rhodococcus sp. IGTS8 strain, a desulfurization gene derived from Rhodococcus sp. KA2-5-1 strain, a degradation gene derived from Sphingomonas sp. 1
The method according to (2) above, which is at least one gene selected from the group consisting of a desulfurization gene derived from a strain and an A11-2 strain.

(4) The desulfurization enzyme gene is of the genus Rhodococcus
The desulfurization enzyme gene dszABCD derived from the KA2-5-1 strain,
(3) The method described. (5) The method according to any one of (1) to (4) above, wherein the recombinant microorganism uses a Nocardioform-type bacterium as a host.

(6) The recombinant microorganism is a genus Rhodococcus,
The method according to (1) to (4) above, which uses a microorganism belonging to any one selected from the group consisting of Mycobacterium and Gordonia as a host. (7) The sulfur-containing heterocyclic compound is at least one compound selected from the group consisting of benzothiophene, dibenzothiophene and derivatives thereof, (1) to (6) above
The method according to any one of 1.

(8) Any of (1) to (6) above, wherein the sulfur-containing heterocyclic compound is at least one compound selected from the group consisting of alkylated benzothiophenes and alkylated dibenzothiophenes. The method according to 1. (9) Rhodococcus erythropolis MC02, a recombinant microorganism containing the promoter shown in SEQ ID NO: 1 and a desulfurization enzyme gene
03 shares (FERM P-18595).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION A desulfurization method according to the present invention comprises a desulfurization enzyme gene downstream of a novel promoter derived from Rhodococcus erythropolis strain KA2-5-1 (FERMP-16277: Japanese Patent Laid-Open No. 11-9293). A method for desulfurizing a sulfur-containing heterocyclic compound using a modified microorganism.

1. Promoter The promoter used in the present invention is specifically as follows.
It consists of the DNA of (a) or (b). (a) D having the promoter activity shown in SEQ ID NO: 1
NA. (b) A DNA which hybridizes with a DNA having a base sequence complementary to the DNA represented by SEQ ID NO: 1 under stringent conditions and has a promoter activity.

The promoter is expressed constantly and with high activity in microorganisms classified into Nocardioform-type bacteria such as Rhodococcus and bacteria belonging to Mycobacterium and Gordonia. Therefore, the gene controlled by the promoter shows a high expression level regardless of the external environment. The promoter can be obtained, for example, as follows.

(1) Screening of promoter The presence of a promoter can be confirmed by using an appropriate reporter gene. That is, since the reporter gene is expressed only when the target promoter is inserted in the upstream region of the 5'side, or conversely, when the reporter gene is inserted in the downstream region of the 3'side of the promoter, the presence of the promoter can be identified. To be done.

Screening of the promoter can be carried out by a known method using the reporter gene. For example, a method of preparing a genomic library of a microorganism whose promoter is to be isolated, randomly inserting this into the 5 ′ upstream region of the reporter gene, and determining by the expression of the reporter gene can be mentioned. . The reporter gene used is not particularly limited as long as it can be expressed in the host.

(2) Insertion of Reporter Gene Using Transposome In the present invention, the insertion of the reporter gene is preferably performed using a transposome described in detail below. A transposome is a DNA protein complex of transposon and transposase, and can insert the DNA on the transposon into the chromosome of many microorganisms (Hoffman, LM, Jendrisak, JJ, Meis, R.
J., Coryshin, IY and Rezhikof, SW Genetica, 10
8, 19-24 (2000)). The method using a transposome is already known in the art as a powerful tool for inserting DNA into a chromosome, for example, pMOD1 (Air Brown Co.).
Methods using the like are known.

The transposon is also called a transposable genetic element and has a unique ability (metastatic ability) to move to a new position in the chromosome of the host organism. Therefore, unlike the chromosomal homologous recombination method, a DNA region having homology between the factor and the target site is not required. In addition, due to the structure of the transposon, only the nucleotide sequences at both ends of DNA are necessary for the basic function as a transposon, that is, transposition, and various functional factors such as antibiotics are present in the region between the nucleotide sequences at both ends. An arbitrary DNA sequence such as a marker gene such as a resistance gene and a restriction enzyme cleavage site can be incorporated.

Therefore, for example, if a reporter gene having a promoter region deleted is inserted into a transposon and randomly inserted into the chromosome of the target microorganism, the reporter gene is expressed only when inserted under the control of the promoter. . That is, the presence of the promoter sequence can be confirmed by the expression of the reporter gene.
At this time, if the culture conditions such as the culture medium and temperature of the microorganism are controlled, it is also possible to select only the promoter that functions under the desired conditions.

In the above method, it is preferable to insert an appropriate marker gene together with a reporter gene in order to facilitate selection. As the marker gene,
Examples thereof include drug resistance genes such as kanamycin resistance gene, ampicillin resistance gene and neomycin resistance gene. Inserting the transposon into the chromosome
Known transformation, transduction, conjugal transfer
g) or electroporation. Transposon insertion recombinants can be selected, for example, by the above-mentioned marker gene on the transposon.

As a preferred embodiment of the above-mentioned steps,
For example, the following examples can be given. First, pQBI63 (Takara Shuzo Co., Ltd.) with the promoter region deleted was rsGFP (red-
Using the shift Green Fluorescent Protein gene as a reporter gene, a transposon in which a kanamycin resistance factor is incorporated as a marker downstream is prepared. Next, transposon is transformed into transposome, and Rhodococcus
Introduced into Erythropolis KA2-5-1 strain. The transposon insertion recombinant of interest contained approximately 10 kanamycin sulfate.
Selection can be carried out by culturing on LB agar medium containing 0 mg / l or more, preferably 200 mg / l.

(3) Selection of Recombinant and Cloning The intended insertion recombinant, that is, the selection of the microorganism in which the transposon is inserted under the control of the promoter, can be confirmed by the functional expression of the reporter gene.
For example, when the rsGFP gene is used as a reporter, it is only necessary to visually select strains that exhibit fluorescent green color under ultraviolet light.

Next, chromosomal DNA is extracted from the selected strain, cleaved with an appropriate restriction enzyme, and then incorporated into an appropriate cloning vector for cloning. The vector to be used is not particularly limited, but Escherichia coli cloning vectors, particularly pUC18, pUC19, pBlueScriptII and the like are preferable. The DNA fragment containing the target promoter can be easily obtained by selecting a microorganism showing the phenotypes of physiological markers of both the vector and the transposon.

(4) Determination of the nucleotide sequence of the promoter The nucleotide sequence adjacent to the transposon can be determined, for example, by the chain termination method (Sanger FS et al Proc. Natl. Aca
d.Sci., USA, 75: 5463-5467 (1977)). Based on the determined nucleotide sequence, for example, GE
The predicted promoter site (base sequence) can be analyzed by the score calculation method using NETYX-MAC / ATSQ v3.0 and GENETYX-MAC v8.0.

The promoter thus obtained has the nucleotide sequence specified by SEQ ID NO: 1 and has a length of about 350 bp.
And had a decomposition property against restriction enzymes as shown in Table 1. However, the promoter used in the present invention is not limited to such a sequence. DNA having a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 1
DN that hybridizes with stringent conditions
A is also included in the promoter as long as it has promoter activity.

The term "stringent conditions" means
For example, conditions for removing DNA that does not hybridize with the DNA of SEQ ID NO: 1 by using 0.5 × SSC buffer at 65 ° C., more preferably, hybridization with DNA of SEQ ID NO: 1 by using 0.1 × SSC buffer at 55 ° C. It refers to the conditions for removing non-soybean DNA. Further, the promoter has 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more homology to the nucleotide sequence represented by SEQ ID NO: 1 at the DNA level. As long as the gene also has promoter activity,
Shall be included. Hereinafter, this promoter is referred to as the KAP1 promoter.

[0030]

[Table 1]

2. Recombinant Microorganism (1) Desulfurase enzyme gene The recombinant microorganism of the present invention is a transformant containing a KAP1 promoter and a desulfurase enzyme gene in a functional manner. Here, the term "functional aspect" means an aspect in which the KAP1 promoter can function as a promoter for expressing the target protein. Therefore, at least the desulfurase gene is contained downstream of the KAP1 promoter sequence.

The desulfurase enzyme gene includes not only a gene encoding a desulfurase enzyme protein but also a gene relating to desulfurase. The desulfurization enzyme gene is preferably an enzyme gene that selectively cleaves the CS bond of the sulfur-containing heterocyclic compound. In particular, an enzyme gene capable of producing a corresponding hydroxybiphenyl derivative from alkylated dibenzothiophenes is preferable. Examples of the gene include desulfurization gene (CSPiddinton, BRKovacevich and L. Rambosek, App) derived from Rhodococcus IGTS8 strain.
L. Environ. Microbiol. 61, 468-475 (1995)), a desulfurization gene (K. Hirasawa, YI) derived from Rhodococcus KA2-5-1 strain.
shii, M. Kobayashi, K. Koizumi and K. Maruhashi, Bio
sci. Biotechnol. Biochem., 65 (2), 239-246, 2001),
Degradation gene (WO 98/45446) derived from Sphingomonas genus AD109 strain, Paenibacillus
Desulfurization genes from genus A11-1 and A11-2 (FERM BP-60
25, FERM BP-26) and the like. Among them, the desulfurization enzyme gene dszABCD (SEQ ID NO: 6) derived from Rhodococcus KA2-5-1 strain is most preferable.

(2) Host microorganisms The host microorganisms are not particularly limited, but include microorganisms classified as Nocardioform type bacteria such as Rhodococcus, bacteria belonging to the genus Mycobacterium, genus Gordonia, or substantially these. It is necessary that the KAP1 promoter is a microorganism that can be expected to function, such as a bacterium having equivalent bacteriological properties.

The nocardioform-type bacteria are
33 in the classification system of bacteria adopted by Bergey's Manual
In one of the sections of the
ia), Rhodococcus and the like.
In this classification system, unlike conventional hierarchical classification, bacteria are classified according to their characteristics such as cell morphology, reaction to enzymes, Gram stainability, energy metabolism and the like. Therefore, it is expected that a common promoter can function within the same section.

(3) Recombinant vector (expression vector) The KAP1 promoter and the desulfurase gene can be inserted using an expression vector containing the promoter and the desulfurase gene. The vector used is not particularly limited as long as it can be replicated in the host, and for example, a plasmid, phage, virus, YAC or the like can be used, but transposon or transposome is particularly preferable.

The expression vector contains a KAP1 promoter and a gene encoding a desulfurase enzyme protein (desulfurase enzyme gene), or a suitable terminator sequence in a functional manner. In the expression vector, in addition to the above-mentioned essential sequences, within the range of not impairing its purpose and effect,
Other genes and sequences can be included. Examples of the genes and sequences include genes involved in replication of plasmid vectors, marker genes used for selection of recombinants, nucleotide sequences for substitution in homologous recombination vectors, and repetitive DNA sequences of transposon vectors. .

As a preferred embodiment of the present invention, an expression vector using a transposon can be prepared as follows. First, in a transposon encapsulated in a plasmid that can be replicated in E. coli such as pMOD, a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and downstream of it are desulfurase enzyme gene (dszABCD gene) and antibiotic resistance factor, etc. Incorporate a marker gene for recombinant selection. Next, the transposon portion is cleaved with an appropriate restriction enzyme and isolated by agarose gel electrophoresis or the like. By reacting the isolated transposon DNA fragment with transposase, a transposome that can be used as an expression vector for expressing a desulfurase gene in a host is prepared.

(4) Transformation of Microorganism Transformation of a microorganism can be achieved, for example, by introducing the expression vector into a host microorganism by a known method.
The introduction method is not particularly limited, and any available method such as lipofection method or electroporation method may be used.

[0039] 3. Production of Cell of Recombinant Microorganism (Culture) The recombinant microorganism is cultured to produce a cell to be subjected to a desulfurization method. The culture may be carried out according to the usual culture method for microorganisms. The form of culture may be solid culture or liquid culture, but liquid culture is preferable. As the nutrient source of the medium, those commonly used are widely used. Any carbon compound may be used as the carbon source, and for example, glucose, sucrose, lactose, succinic acid, citric acid, acetic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, organic nutrient substances such as peptone, polypeptone, meat extract, soybean flour, and casein hydrolyzate are used. As the sulfur source, any usable inorganic sulfur compound may be used, and for example, a sulfate or the like is used. In addition, phosphate, carbonate, magnesium, calcium,
Potassium, sodium, iron, manganese, zinc, molybdenum, tungsten, copper, vitamins and the like are used as necessary. The culturing is carried out at a temperature and pH at which the microorganism can grow, and it is preferable to carry out the culturing under optimal culturing conditions for the microorganism used. Generally, the pH of the medium is adjusted to a suitable pH, such as pH 6-8.
And aerobically at a suitable temperature, for example about 30 ° C., under shaking or aeration conditions.

[0040] 4. Desulfurization method of the sulfur-containing heterocyclic compound, the desulfurization method according to the present invention, after culturing the recombinant microorganism in a medium, the resting cells obtained by harvesting operation such as centrifugation from the resulting culture It can be carried out by contacting with a sulfur-containing heterocyclic compound. Specifically, desulfurization using resting cells is performed as follows, for example.

First, resting cells are prepared. A fresh medium is inoculated with an appropriate amount of inoculum, for example 1-2% by volume. As the medium, the above-mentioned medium can be used. As inoculum,
Bacteria in any state from the early logarithmic growth phase to the stationary phase may be used, and preferably those in the late logarithmic growth phase are used. The amount of inoculum can be increased or decreased as needed. Then, the cells are cultivated at pH 6-9 at about 30 ° C for 1 to 2 days with reciprocating or rotary shaking. The medium used is not particularly limited, but NKII medium shown in Table 3 described later is preferable.

Then, the cells are separated and collected and washed to obtain resting cells. The cell collection may be carried out in any state from the early logarithmic growth phase to the stationary phase of the cultured cells, but it is preferably carried out in the midlog to late logarithmic growth phase. In addition, the bacteria can be collected by any method such as filtration, sedimentation separation, etc. in addition to centrifugation. For washing the bacterial cells, any buffer solution such as physiological saline, phosphate buffer, Tris buffer or the like can be used, and if necessary, the bacterial cells may be washed with water.

Desulfurization by resting cells is carried out by adding a light oil or a sulfur-containing heterocyclic compound as a substrate to an oil phase to a cell suspension prepared by suspending resting cells in an appropriate buffer and reacting. By. The buffer solution used is not particularly limited, and any buffer solution can be used. The pH of the buffer solution is not particularly limited, but pH 6 to 7 is preferable. Also, instead of the buffer,
Water or a medium may be used. The concentration of the bacterial cell suspension is OD
₆₆₀ Is preferably between 1 and 100 and can be increased or decreased as needed. The amount of oil in the bacterial suspension is between 0.01 and 100 in the oil-water ratio,
It can be added if necessary, but an oil / water ratio of 1 to 10 is preferable. The reaction temperature is not particularly limited, but is 30 to 42 ° C.
Is preferable, and 35-40 degreeC is more preferable. The reaction time may be increased or decreased as necessary, but 1 to 2 hours is preferable.

5. Sulfur-Containing Heterocyclic Compound According to the desulfurization method of the present invention, the sulfur-containing heterocyclic compound contained in petroleum fossil fuel oil can be desulfurized. Especially KA
The insertion of the P1 promoter causes the recombinant microorganism to express high levels of the desulfurase gene under its control regardless of the external environment. Therefore, by selecting the desulfurizing enzyme to be inserted, it is possible to efficiently desulfurize the hardly desulfurizing benzothiophene and dibenzothiophenes. In addition, as described below, select a host microorganism or a microorganism after transformation that has a cell membrane that is highly permeable to highly alkylated (including alkyl side chains with 6 or more carbon atoms) sulfur-containing heterocyclic compounds. By doing so, the hardly desulfurized alkylated benzothiophenes and alkylated dibenzothiophenes can be suitably desulfurized. Such alkylated benzothiophenes include 2-methyldibenzothiophene, 3-methylbenzothiophene, 5-methyldibenzothiophene,
Methylbenzothiophene, 5-methylbenzothiophene,
2-ethylthiophene and the like are exemplified. As alkylated dibenzothiophene, 4-methyldibenzothiophene,
4-ethyldibenzothiophene, 4-propyldibenzothiophene, 4-butyldibenzothiophene, 4-pentyldibenzothiophene, 4-hexyldibenzothiophene, 4,6-
Examples thereof include dimethylbenzothiophene, 4,6-diethyldibenzothiophene, 4,6-dipropyldibenzothiophene, 3,4,6-trimethyldibenzothiophene and 3-propyl, 4,8-dimethyldibenzothiophene.

6. Rhodococcus erythropolis MC0203
Strain The present invention is a preferred example of the recombinant microorganism used in the desulfurization method of the present invention, as Rhodococcus erythropolis MC0203.
Offer stock. The MC0203 strain is a recombinant microorganism in which the KAP1 promoter and dszABCD gene are inserted into the oil-resistant Rhodococcus erythropolis MC1109 strain isolated from soil. This MC0203 strain is a patent biological deposit center of National Institute of Advanced Industrial Science and Technology (1-1, Higashi, Tsukuba City, Ibaraki Prefecture).
1 Central 6), dated November 12, 2001, FERM P-18595.
Has been deposited as.

The MC0203 strain can efficiently desulfurize the hardly desulfurizable alkylated sulfur-containing heterocyclic compound by the introduced promoter and desulfurization enzyme gene. However, the MC0203 strain is not limited to this, and can also desulfurize a highly alkylated sulfur-containing heterocyclic compound having high desulfurization resistance. This is because the MC0203 strain has a membrane composition with a low methylated higher fatty acid content (Table 5), and therefore the intracellular permeability of hydrophobic substances is high. This change in the membrane composition of the MC0203 strain is presumably due to the fact that the insertion position of the transposon was in the vicinity of the fatty acid methylase-related region, and therefore some deficiency occurred in the fatty acid methylase. Thus, among the recombinant microorganisms used in the present invention, if a transformant having a membrane composition with high permeability of hydrophobic substances is selected,
It becomes possible to more effectively desulfurize the highly alkylated sulfur-containing heterocyclic compound which is difficult to desulfurize. As described above, the MC0203 strain highly expresses desulfurization enzyme regardless of the external environment, and can efficiently desulfurize a sparingly desulfurizable alkylated sulfur-containing heterocyclic compound. Therefore, as a desulfurizing bacterium on an industrial level,
It can be applied to a desulfurization method that complements hydrodesulfurization.

[0047]

EXAMPLES The present invention will now be specifically described with reference to examples, but the scope of the present invention is not limited to the ranges of these examples. Example 1 Preparation of Promoter Searching Transposon A transposon for searching for a promoter sequence from Rhodococcus erythropolis KA2-5-1 strain was prepared as follows.

(1) Preparation of transposon DNA fragment pMOD1 (air.
A transposon DNA fragment was prepared from Brown).
First, using pMOD1 as a template, PCR reaction was performed with the following primers. The primer is designed so that the restriction enzyme cleavage site PvuII in the transposon contained in pMOD1 is replaced with StuI and the restriction enzyme recognition site for HpaI is introduced on the 3'side.

Primer: Forward: 5-CCTAGGCCTGTCTCTTATACACATCTCAACCATCATCGAT
G-3 (SEQ ID NO: 2) Reverse: 5-CTTAAGGCCTGTCTCTTATACACATCTCGTTAACCCTGAA
GC-3 (SEQ ID NO: 3) Next, a part of the reaction solution was subjected to 1.5% agarose gel electrophoresis, and it was confirmed that a gene-amplified fragment (target transposon DNA fragment) of about 130 bp was obtained.

(2) Preparation of pMODSt The vector pBluescript II SK (+) (Stratagene) derived from Escherichia coli was reacted with the restriction enzymes SacI and KpnI at 37 ° C. for 1 hour to cut the plasmid DNA. Add an equal volume of phenol-chloroform to this solution and mix well.
After centrifugation at 17,000 g for 10 minutes at 0 ° C, the upper layer was collected. To the collected upper layer, an equal volume of 3M sodium acetate aqueous solution and 2.5 volumes of ethanol were added, and the deposited precipitate was recovered by centrifugation, and TE buffer solution [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA was added. : pH 8.0]. 0.1 volume of Blunting Buffer, 0.1 volume of K in the collected solution
OD DNA Polymerase (manufactured by Toyobo Co., Ltd.) was added and reacted at 72 ° C. for 5 minutes to smooth the cut surface of the DNA restriction enzyme.

Equal volumes of the pMOD1 DNA reaction solution prepared in (1) (including transposon DNA fragment) and the solution containing pBluescriptII KS (+) DNA fragment were mixed, and Ligation high (manufactured by Toyobo Co., Ltd.) was added in an amount of 2 times. After stirring well, it was incubated at 16 ° C for 60 minutes. The above reaction solution was added to competent cells of Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.), allowed to stand at 0 ° C for 1 hour, and then heat-treated at 42 ° C for 1 minute to prepare an SOC medium [tryptone 2
%, Yeast extract 0.5%, 0.025M sodium chloride, 0.0025
M potassium chloride, 0.01M magnesium sulfate, 0.01M magnesium chloride, 0.02M glucose: pH 7.4]
Shake at ℃ for 1 hour. A plasmid was extracted from the obtained Escherichia coli and its nucleotide sequence was analyzed with 310 type genetic analyzer.
(Perkins Elmer) was used to analyze and select the plasmid into which the transposon DNA fragment prepared in (1) had been inserted. This plasmid was named pMODSt.

(3) Construction of pTnKSt The pK18mobsacB vector for utilizing the kanamycin resistance gene as an antibiotic resistance marker of transposon
(ATCC 87097 Justin ACPowell & John AcArcher "Mo
lecular characterization of a Rhodococcus ohp oper
on "Antonie van Leeuwenhoek 74: 175-188, 1998), a DNA fragment containing the marker gene sequence was prepared. That is, PCR reaction was performed using pK18mobsacB as a template and the following primers.

Primer: Forward: 5-CTAGCTTCACGCTGCCGCAAGCACTCAGGGCGC-3 (SEQ ID NO: 4) Reverse: 5-CGAACCCCAGAGTCCCGCTCAGAAGAACTCGTC-3 (SEQ ID NO: 5) A part of the reaction solution was subjected to 1.5% agarose gel electrophoresis,
It was confirmed that a gene-amplified fragment of about 1.2 Kb was obtained.

PMODst was reacted with the restriction enzyme HpaI at 37 ° C. for 1 hour to cut the plasmid DNA. Add an equal volume of phenol-chloroform to this solution and mix well.
Centrifugation was performed at 00 g for 10 minutes, and the upper layer was collected. To the collected upper layer, 0.1 volume of 3M sodium acetate aqueous solution and 2.5 volume of ethanol were added, and the deposited precipitate was collected by centrifugation, and TE buffer [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA: pH8 .0]. above
Equal volumes of a solution containing the pK18mobSacB DNA reaction product and a solution containing the pMODst DNA fragment were mixed, 2 volumes of Ligation high (manufactured by Toyobo Co., Ltd.) was added, and the mixture was stirred well and then incubated at 16 ° C for 60 minutes.

The above reaction solution was added to a competent cell of Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) and left still at 0 ° C. for 1 hour.
After heat treatment at ℃ for 1 minute, SOC medium [tryptone 2%, yeast extract 0.5%, 0.025M sodium chloride, 0.0025M potassium chloride, 0.01M magnesium sulfate, 0.01M magnesium chloride, 0.02M glucose: pH7.4] In addition, it was shaken at 37 ° C for 1 hour. Next contains 200ppm of kanamycin sulfate
It was smeared on LB agar medium [tryptone 1%, yeast extract 0.5%, sodium chloride 0.5%, bacto agar 1.5%]. Plasmids were extracted from E. coli that showed kanamycin resistance, and the nucleotide sequences of these plasmids were analyzed with 310 type genetic analyzer.
(Perkins Elmer) was used to analyze and select the plasmid with the desired modification. This plasmid is called pT
It was named nKSt.

(4) pQ so that the insertion of the insertion promoter sequence of the reporter gene (rsGFP) can be visually confirmed.
RsGFP (red-shift Green Fluore) from BI63 (Takara Shuzo)
scent Protein) gene was inserted as a reporter gene. First, pQBI63 was digested with restriction enzymes XbaI and BamHI at 37 ° C for 1
After reacting for a period of time and cleaving, the product was subjected to 0.7% agarose gel electrophoresis, and a DNA fragment of about 800 bp was subjected to GFX PCR and Gel band Purification.
A cation kit (Amersham Pharmacia) was used to collect and prepare rsGFP. Repeat this rsGFP DNA fragment solution
Equal volumes of the pTnKStDNA solution cleaved with XbaI and BamHI were mixed, 2 volumes of Ligation high (manufactured by Toyobo Co., Ltd.) were added, and the mixture was stirred well and then incubated at 16 ° C for 60 minutes.

The above reaction solution was added to a competent cell of Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) and left still at 0 ° C. for 1 hour.
After heat treatment at ℃ for 1 minute, SOC medium [tryptone 2%, yeast extract 0.5%, 0.025M sodium chloride, 0.0025M potassium chloride, 0.01M magnesium sulfate, 0.01M magnesium chloride, 0.02M glucose: pH7.4] In addition, it was shaken at 37 ° C for 1 hour. Next contains 200ppm of kanamycin sulfate
The above reaction solution was spread on LB agar medium [tryptone 1%, yeast extract 0.5%, sodium chloride 0.5%, bacto agar 1.5%]. Plasmids were extracted from the obtained Escherichia coli, and the nucleotide sequences of these plasmids were analyzed using a 310 type genetic analyzer (Perkins Elmer) to select plasmids into which rsGFP had been inserted. This plasmid was named pTnKgfp.

The pTnKgfp thus prepared was digested with the restriction enzyme StuI for 1 hour at 37 ° C. and digested, and then subjected to 0.7% agarose gel electrophoresis to obtain a DNA fragment of about 2.2 Kb by GFX PCR.
It was recovered using an andGel band Purification kit (manufactured by Amersham Pharmacia). An equal volume of the transposome solution was added to the recovered DNA solution, 2 volumes of 100% glycerol was further added and mixed well, and then incubated at room temperature for 20 minutes. The prepared promoter searching transposon was named TnKgfp.

Example 2 Isolation of Promoter from Rhodococcus erythropolis KA2-5-1 Strain (1) Introduction of Reporter Gene by Transposon TnKgfp 100 ml of sterilized LB medium in a 500 ml Erlenmeyer flask was placed in 100 ml of Rhodococcus. Inoculated with erythropolis KA2-5-1 strain, 30
It was cultured at ℃ for 24 hours. The resulting culture is at 4 ° C and 10,000 rpm
The precipitate was washed twice with sterilized water, and then suspended in a 10% glycerol aqueous solution so that the turbidity at a measurement wavelength of 660 nm was 40.

The TnK prepared in Example 1 was added to 80 μl of the above suspension.
1 μl of gfp solution was added, and treated with Gene Pulser II (Bio-Rad) under the conditions of 25 μF, 400Ω, and 1.5 kV / cm. 0.42 ml of SOC medium was added to the treated solution, and the mixture was cultured at 30 ° C. for 3 hours. The obtained culture solution was added to 100m of kanamycin sulfate.
The mixture was applied to a basal medium (A-KmASA medium) containing g / l, ammonium sulfate 0.1 mM and agar powder 15 g / l, and cultured at 30 ° C. for 48 hours. As the basal medium, the composition described in JP-A No. 11-181446, "Method for producing desulfurization active microorganism" was used. Bacteria that formed colonies in the A-KmASA medium were observed under UV irradiation of 302 nm, and colonies that exhibited a fluorescent green color specific to rsGFP were selected.

Selected colonies were treated with ammonium sulfate 0.1 m.
2 ml of basal medium containing 100 mg / l of M and kanamycin was inoculated with 1 ears of white bacterium, and cultured at 30 ° C. for 72 hours. Culture at 4 ℃, 1
After centrifugation at 7,000 g for 5 minutes, the precipitated bacterial cells were collected. Chromosomal DNA was separated and purified from the collected cells using ISOPLANT (manufactured by Nippon Gene). The entire amount of the purified chromosomal DNA was reacted with the restriction enzyme EcoRI at 37 ° C for 16 hours to cut the chromosomal DNA. The cleaved chromosomal DNA was used for GFX PCR and Gel ban.
d Purification kit (manufactured by Amersham Pharmacia) was used for purification.

(2) Cloning of promoter and determination of nucleotide sequence On the other hand, vector pBluescript II SK (+) derived from E. coli
(Stratagene) was reacted with the restriction enzyme EcoRI at 37 ° C. for 1 hour to cut the plasmid DNA. Add an equal volume of phenol-chloroform to this solution and mix well.
After centrifugation at 17,000 g for 10 minutes at 0 ° C, the upper layer was collected. To the collected upper layer, 0.1 volume of 3M sodium acetate aqueous solution and 2.5 volume of ethanol were added, and the deposited precipitate was collected by centrifugation, and TE buffer [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA: pH8. Dissolved in 0]. Ligation high (Toyobo Co., Ltd.) was prepared by mixing equal volumes of the above chromosomal DNA reaction product and a solution containing pBluescript II KS (+) DNA fragment.
After adding 2 volumes and stirring well, it incubated at 16 degreeC for 60 minutes.

The above reaction solution was added to competent cells of Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) and allowed to stand at 0 ° C. for 1 hour.
After heat treatment at ℃ for 1 minute, SOC medium [tryptone 2%, yeast extract 0.5%, 0.025M sodium chloride, 0.0025M potassium chloride, 0.01M magnesium sulfate, 0.01M magnesium chloride, 0.02M glucose: pH7.4] In addition, it was shaken at 37 ° C for 1 hour. 0.1 ml of the obtained culture solution was added to kanamycin 2
It was applied to LB medium containing 5 mg / l and agar 15 g / 1, and cultured at 37 ° C for one day. Plasmids were extracted from the obtained Escherichia coli and the nucleotide sequences of these plasmids were analyzed with 310 type genetic analyzer.
(Perkins Elmer). The obtained nucleotide sequence data are GENETYX-MAC / ATSQ v3.0 and GENETY
It was analyzed using X-MAC v8.0. About 35 obtained as a result of analysis
KAP1 with a DNA sequence highly homologous to the 0 bp E. coli promoter
The plasmid containing KAP1 was named pKAP1.

EcoRI and XbaI were added to the pKAP1 DNA solution, and the mixture was incubated at 37 ° C. for 1 hour. Add an equal volume of phenol-chloroform to this solution and mix well.
After centrifugation at 17,000 g for 10 minutes at 0 ° C, the upper layer was collected. To the collected upper layer, 0.1 volume of 3M sodium acetate aqueous solution and 2.5 volume of ethanol were added, and the deposited precipitate was collected by centrifugation, and TE buffer [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA: pH8. 0] to give a KAP1 DNA fragment solution.

Example 3 Preparation of Recombinant Transposon Containing Alkylated Dibenzothiophene Desulfurization Gene dszAB encoding an alkylated dibenzothiophene desulfase from Rhodococcus erythropolis KA2-5-1 strain
A recombinant transposon containing the CD gene group was prepared as follows. The decomposition reaction of alkylated benzothiophene into 2-hydroxybenzothiophene (2-HBP) by the dszABCD gene product is shown in FIG.

(1) Preparation of pSKABC Recombinant plasmid pRKPPBB (Kazuaki Hirasaw containing the dszABCD gene of Rhodococcus erythropolis KA2-5-1 strain
a et.al., Biosci. Biotechnol. Biochem., 65 (2) 239-
246 (2001)) and added DraI and XbaI solutions,
Incubated at ℃ for 1 hour. The reaction solution obtained is 0.8
Separate the DNA using% agarose gel electrophoresis,
The kb DNA fragment was subjected to GFX PCR and Gel band Purification ki.
DNAs containing dszABCD were recovered using t (Amersham Pharmacia), dissolved in TE buffer [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA: pH8.0].
It was made into fragments. 0.1 volume of Blunting Buffer in the collected solution,
72 volumes of KOD DNA Polymerase (manufactured by Toyobo Co., Ltd.)
The reaction was performed at 5 ° C for 5 minutes to smooth the cut surface of the DNA restriction enzyme.

On the other hand, the SmaI and EcoRV solutions were added to a solution containing pBluescript II SK (+) (Stratagene), and 37
Incubated at ℃ for 1 hour. Add an equal volume of phenol-chloroform to this solution and mix well.
After centrifugation at 00g for 10 minutes, the upper layer was collected. To the collected upper layer, 0.1 volume of 3M sodium acetate aqueous solution and 2.5 volume of ethanol were added, and the deposited precipitate was recovered by centrifugation, and TE buffer [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA: pH8.0], pBluescript
II SK (+) / SmaI / EcoRV fragment. Equal volumes of a solution containing the above DNA fragment containing dszABCD and pBluescript II SK (+) / SmaI / EcoRV fragment were mixed, 2 volumes of Ligation high (manufactured by Toyobo Co., Ltd.) were added, and after stirring well, 16 ° C, 1 Incubated for hours.

The above reaction solution was added to a competent cell of Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) and left still at 0 ° C. for 1 hour.
After heat treatment at ℃ for 1 minute, use SOC medium [tryptone 2%, yeast extract 0.5%, 0.025M sodium chloride, 0.0025M potassium chloride, 0.01M magnesium sulfate, 0.01M magnesium chloride, 0.02M glucose: pH7.4]. In addition, it was shaken at 37 ° C for 1 hour. Plasmids were extracted from the obtained Escherichia coli and the nucleotide sequences of these plasmids were analyzed as 310 type genetic ana
A lyzer (manufactured by Perkin Elmer) was used to select the plasmid into which the desired DNA was inserted. This plasmid is called pSKA
It was named BC.

(2) Preparation of DNA fragment containing dszABCD pBluescript II SK (+) (Stratagene) was dszABCD.
Recombinant plasmid containing the gene pSKABC solution was added and Xba
I and Hind III solutions were added and incubated at 37 ° C. for 1 hour to incorporate the dszABCD gene group into the Xba I and Hind III sites of pBluescript II SK (+). The reaction mixture was electrophoresed on 0.8% agarose gel, and a DNA fragment of about 5.0 kb was subjected to GFX.
It was recovered using PCR and Gel band Purification kit (manufactured by Amersham Pharmacia), and TE buffer [0.025M tris (hydroxymethyl) aminomethane, 0.025M EDTA: pH
8.0] to obtain a DNA fragment containing dszABCD.

(3) Preparation of TnKst / EcoRI / HindIII fragment On the other hand, E was added to a DNA solution containing the transposon vector pTnKst.
The coRI and HindIII solutions were added, and the mixture was incubated at 37 ° C for 1 hour. An equal volume of phenol-chloroform was added to this solution and mixed well, followed by centrifugation at 4 ° C. and 17000 g for 10 minutes to collect the upper layer. To the collected upper layer, 0.1 volume of 3M sodium acetate aqueous solution and 2.5 volume of ethanol were added, and the precipitated precipitate was collected by centrifugation and the TE buffer solution [0.025M
Tris (hydroxymethyl) aminomethane, 0.025M ED
It was dissolved in TA: pH 8.0] and used as a TnKst / EcoRI / Hind III fragment.

(4) Preparation of recombinant transposon TnKAPDS DNA fragment solution containing dszABCD obtained as described above,
Equal volumes of the pRHK1 / EcoRI / HindIII DNA fragment solution and the KAP1 DNA fragment solution obtained in Example 2 were mixed together to form Liga.
Add 3 volumes oftion High (Toyobo Co., Ltd.), mix well, and then add 1
Incubated at 6 ° C for 30 minutes.

The above reaction solution was added to a competent cell of Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) and left still at 0 ° C. for 1 hour.
After heat treatment at ℃ for 1 minute, SOC medium [trypton 2%, yeast extract 0.5%, 0.025M sodium chloride, 0.0025M potassium chloride, 0.01M magnesium sulfate, 0.01M magnesium chloride, 0.02M glucose: pH7.4] In addition, 37 ℃, 1
The culture was shaken for a period of time. 0.1 ml of the obtained culture solution was applied to LB medium containing 25 mg / l of kanamycin and 15 g / 1 of agar, and 37
Cultivated at ℃ for one day. The obtained culture colonies were inoculated into 1.5 ml of LB medium containing 25 mg / l of kanamycin, and GFX micro Pla
The plasmid DNA prepared using the smid kit (Amersham Pharmacia) was dissolved in 50 μl TE buffer to give a pTnKAPDS gene solution. The pTnKAPDS gene solution thus prepared was reacted with a restriction enzyme StuI at 37 ° C for 1 hour, and then subjected to 0.7% agarose gel electrophoresis, and a DNA fragment of about 6 Kb was subjected to GFX PCR.
It was collected using an and Gel band Purification kit (manufactured by Amersham Pharmacia). An equal volume of transposome solution was added to the recovered DNA solution, 2 volumes of 100% glycerol was further added and mixed well, and then incubated at room temperature for 20 minutes. The prepared recombinant transposon was named TnKAPDS.

Example 4 Rhodococcus by TnKAPDS
Transformation of erythropolis MC1109 strain (1) Acquisition and identification of Rhodococcus erythropolis MC1109 strain Put 1 g of soil in a 500 ml Erlenmeyer flask, add 50 ml of fresh light oil with no observed contamination and add LB medium of the same volume at 30 ° C Ten
Incubated for days. 1 m of the obtained light oil-containing culture solution
l as seeds, in a 500 ml Erlenmeyer flask, 50 ml of fresh light oil
An equal volume of LB medium was added, and 1 ml of the inoculum was inoculated into this and incubated at 30 ° C for 10 days. The same subculture was repeated 3 times. The culture solution after subculture was applied to an LB agar plate to isolate a single colony, which was designated as MC1109 strain.
The MC1109 strain was identified for the items shown in Table 2 and identified as Rhodococcus erythropolis. As is clear from Table 2, the MC1109 strain has no oxidase activity,
It has no desulfurization ability. This Rhodococcus erythropolis MC1109 strain was designated as FERM P-18594 on November 12, 2001 at the Patent Biological Depository Center (1-1-1, Higashi 1-1-chome, Tsukuba City, Ibaraki Prefecture), National Institute of Advanced Industrial Science and Technology. Has been deposited.

[0074]

[Table 2]

(2) Transformation with TnKAPDS 100 ml of sterilized LB medium contained in a 500 ml Erlenmeyer flask was inoculated with Rhodococcus erythropolis MC1109 strain at 30 ° C.
The cells were cultured for 24 hours. The resulting culture solution at 4 ° C, 10,000 rpm,
After centrifugation for 10 minutes, the precipitate was washed twice with sterilized water and suspended in a 10% glycerol aqueous solution so that the turbidity at a measurement wavelength of 660 nm was 40.

To 80 μl of the suspension, 1 μl of a TnKAPDS transposon solution was added and treated with Gene Pulser II (Bio-Rad) under the conditions of 25 μF, 400Ω and 1.5 kV / cm. 0.42 ml of SOC medium was added to the treated solution, and the mixture was cultured at 30 ° C. for 3 hours. The obtained culture solution was centrifuged at 4 ° C. and 10,000 g, the solution portion was discarded, and the suspension was resuspended in an equal volume of NKII medium (Table 3). The resulting bacterial suspension was placed in a 100 ml Erlenmeyer flask and the N
An equal volume of tetradecane containing 300 mg / l of 4,6-diethyldibenzothiophene was added to 10 ml of KII medium, and cultured at 30 ° C. for 72 hours. The culture solution was diluted with 25 mg / l dibenzothiophene and 15 agar.
Apply to dibenzothiophene agar NKII medium containing g / l,
It was obtained as a single colony and named TnKAPDS transformant Rhodococcus erythropolis MC0203 strain.

This Rhodococcus erythropolis MC0203
The strain has been deposited at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology as FERM P-18595 as of November 12, 2001.

[0078]

[Table 3]

[Example 5] Desulfurization activity test for alkylated dibenzothiophenes by MC0203 strain (1) Test method 2 ml of NKII medium shown in Table 3 was inoculated with 1 strain of white bacterium, Rhodococcus erythropolis MC0203 strain. 1 ml of the culture cultivated at 30 ° C. for 24 hours was transferred to 100 ml of a medium having the same composition as above, and further cultured at 30 ° C. for 48 hours. The obtained culture solution was added to 4
After centrifuging at 10,000 rpm for 5 minutes, the resulting precipitate is
The cells were washed twice with 10 mM phosphate buffer solution of 7.2 and suspended in 0.2 M phosphate buffer solution (pH 7.2) containing 100 ppm MgCl ₂ and 10 g / L glucose to prepare reaction cells. Measurement wavelength of this reaction cell
The turbidity at 660 nm was 30.

The desulfurization activity reaction was carried out by adding 1 ml containing 5 ml of reaction cells.
In a 00 ml Erlenmeyer flask, add 1 each of 4-hexyldibenzothiophene or 4,6-dipropyldibenzothiophene.
Equal volume (5 ml) of n-tetradecane dissolved at a concentration of 4 mM
In addition, reciprocal shaking was performed at 37 ° C and 150 spm. Using Rhodococcus erythropolis KA2-5-1 strain as a control,
The same desulfurization activity test was conducted.

(2) Measurement of desulfurization activity The desulfurization activity was measured by measuring the amount of alkylated dibenzothiophene in tetradecane by high performance liquid chromatography under the following conditions. The results are shown in Fig. 1. HPLC analysis conditions: Column: ODS-80Ts (Tosoh), 4.6mmI.D.x25cm Eluent: A --- 5% acetonitrile aqueous solution (containing 1% phosphoric acid) B --- acetonitrile (containing 1% phosphoric acid) A 25% B; 75%, Flow rate: 1.0 ml / min Injection volume: 10 μL Column temperature: 40 ° C Detector: Multi-wavelength detector PD8020 (Tosoh) (quantitative wavelength: 246 nm) (3) Results As is clear from Fig. 1. In addition, the concentration of alkyldibenzothiophene decreased with time in MC0203 strain, and the carbon number of MC0203 strain decreased.
It was shown that dibenzothiophene with 6 alkyl side chains can be desulfurized. On the other hand, no desulfurization activity was observed in the control KA2-5-1 strain. This is KA2-5-1
In the MC0203 strain, PK expression was suppressed in the MC0203 strain, whereas in the strain, the high concentration of inorganic sulfate in the culture solution suppressed the expression of the desulfurase enzyme gene.
It is considered that the desulfurase gene was constitutively and highly expressed by the A1 promoter regardless of the sulfate concentration.

[Example 6] Desulfurization activity test of F light oil by MC0203 strain (1) Test method 2 ml of NKII medium shown in Table 3 was inoculated with 1 white ear of Rhodococcus erythropolis MC0203 strain, and 30 1 ml of the culture cultivated at 24 ° C. for 24 hours was transferred to 100 ml of a medium having the same composition as above, and further cultured at 30 ° C. for 48 hours. The obtained culture solution was added to 4
Centrifuge at 10,000 rpm for 5 minutes at 10 ° C and precipitate to 10 mM at pH 7.2.
After washing twice with a phosphate buffer, the cells were suspended in a 0.2 M phosphate buffer (pH 7.2) containing 100 ppm MgCl ₂ and 10 g / L glucose to give a microbial cell for reaction. The turbidity of this reaction cell at a measurement wavelength of 660 nm was 60. The desulfurization activity reaction was performed by adding 1/4 volume of F gas oil to a 100 ml Erlenmeyer flask containing 8 ml of reaction cells, and
Reciprocal shaking was performed at 0 ° C and 150 spm.

(2) Measurement of desulfurization activity Desulfurization activity was measured by measuring the sulfur compounds in the F-gas oil before and after the desulfurization reaction with GC-AED (ANTEK 7000V type, ANTEK,
Houston, Texas), Okumura, K. and Suzuki,
M., J. Chomato. Sci., 35, 417-422, 1997. The results are shown in Figure 2.

(3) Results As is clear from FIG. 2, each peak showing sulfur compounds in the chromatographic affair is reduced in F gas oil after 20 hours of the desulfurization reaction. From this, the Rhodococcus of the present invention
It was shown that Erythropolis strain MC0203 can desulfurize most of alkylated dibenzothiophene and alkylated benzothiophene contained in gas oil after hydrodesulfurization.

Example 7 Comparison of Fatty Acid Composition of MC1109 Strain and MC0203 Strain 1. GC analysis of fatty acid composition MC1109 strain and MC0203 strain bacterial cells were collected from each plate 200 mg of dried bacterial cells, using 15% (w / v) sodium hydroxide solution 20 ml in 25% (v / v) methanol. Saponified. Next, commercially available 5% (w / v) anhydrous hydrochloric acid-methanol was added to 2 ml to methylate the fatty acid, and then 1.5 ml of hexane (for chromatograph) was used to extract the produced methyl ester four times. . All extracts were combined, washed with the same amount of water, dehydrated with anhydrous sodium sulfate, and then added with 50-100 μl of acetonitrile (for chromatograph), followed by gas chromatography (GC body (5890 SERISE type, AED ( Model 5921A, Hewlett-P
Fatty acids were analyzed by ackard, Wilmington, DE).
The results are shown in Tables 4 and 5.

[0086]

[Table 4]

[0087]

[Table 5]

2. Results As shown in Tables 4 and 5, compared with the host strain MC1109, the fatty acid composition of MC0203 strain was higher in saturated fatty acid palmitic acid C _{16: 0. In} particular, MC1109 strain contained 3% of all fatty acid components. The second highest content. On the other hand, tuberculostearic acid (Tuberculostearic aci
d) C _{18: 0 10} methyl was hardly detected in MC0203 strain. Furthermore, MC0203 strain not only contains C18 fatty acids but also C16
Also in C17, the amount of fatty acid methylated at the 10th position is M
It was about 1/10 of C1109 strain.

In particular, in the MC0203 strain, C _{18: 0 10 methyl} (one in which the 10-position of stearic acid was methylated) was decreased,
_{18: 1 ω9c} (oleic acid) is increased. This means that MC0203 strain is
It is suggested that this is a transposon mutant strain in which the fatty acid methyltransferase gene cannot be expressed. The melting points of oleic acid and stearic acid are 14 ℃ and 68 ℃, respectively.
Therefore, it is considered that the fatty acid having a low melting point increased and the fatty acid having a high melting point decreased, whereby the cell membrane permeability of the alkylated sulfur-containing heterocyclic compound was improved in MC0203 strain.
Thus, the improvement of desulfurization effect due to the introduction of promoter and desulfurization enzyme gene and the change of membrane composition act synergistically, and MC02
It is thought that this has brought excellent desulfurization capacity to 03 strain.

Example 8 Production of Recombinant Microorganisms Using Mycobacterium and Gordonia Bacteria (1) Transformation with TnKAPDS Mycobacterium sp was added to 100 ml of sterilized LB medium in a 500 ml Erlenmeyer flask. . (Mycobacterium sp.) 10403 strain
(NCIMB10403), as well as another 500 ml Erlenmeyer flask with Gordonia rubropertinc
tus) 13382 strain (NCIMB13382) was inoculated and cultured at 30 ° C. for 24 hours. The obtained culture solution was centrifuged at 4 ° C, 10,000 rpm for 10 minutes, the precipitate was washed twice with sterilized water, and then suspended in a 10% glycerol aqueous solution so that the turbidity at a measurement wavelength of 660 nm was 40. .

To 80 μl of the above suspension, 1 μl of TnKAPDS transposon solution was added and treated with Gene Pulser II (Bio-Rad) under the conditions of 25 μF, 400Ω and 1.5 kV / cm. 0.42 ml of SOC medium was added to the treated solution, and the mixture was cultured at 30 ° C. for 3 hours. The resulting culture was centrifuged at 4 ° C. and 10,000 g to discard the solution portion, and resuspended in an equal volume of NKII medium (Table 3). The resulting bacterial suspension was placed in a 100 ml Erlenmeyer flask and the N
An equal volume of tetradecane containing 300 mg / l of 4,6-diethyldibenzothiophene was added to 10 ml of KII medium, and cultured at 30 ° C. for 72 hours. The culture solution is dibenzothiophene 25 mg / l and agar 15
Apply to dibenzothiophene agar NKII medium containing g / l,
The TnKAPDS transformants of Mycobacterium sp. Were named 403GR strains, and the Gordonia ruboropatincus transformants were designated as 382GR strains.

(2) Degradation of DBT by transformed cells in the presence of sulfate ion Magnesium sulfate heptahydrate 500 mg / l and kanamycin 100 m
g / l, 2 ml of A medium containing 100 mg / l of dibenzothiophene, 1
White fungus ear amount of Rhodococcus erythropolis MC0203
The strains, the 403GR strain and the 382GR strain were inoculated and cultured at 30 ° C. and 110 rpm for 4 days. To the obtained culture broth, 0.1 ml of a 6N hydrochloric acid aqueous solution and an equal volume of ethyl acetate were added, and after extraction with stirring, the ethyl acetate layer was analyzed by gas chromatography. As a result, the decomposition rate of dibenzothiophene of MC203 strain was 100% and 2HBP.
Was 40 mg / l. Moreover, the dibenzothiophene decomposition rate of the 403GR strain is 100%, the production amount of 2HBP is 8 mg / l, and the dibenzothiophene decomposition rate of the 382GR strain is 100%, 2HBP.
Was 16 mg / l.

[0093]

According to the present invention, fossil fuel oil such as petroleum containing a sulfur-containing heterocyclic compound such as alkylated dibenzothiophene or alkylated benzothiophene can be effectively desulfurized under mild conditions. You can

[0094]

[Sequence list]                               SEQUENCE LISTING <110> JAPAN COOPERARION CENTER, PETROLEUM <120> A Process for Desulfurization using Transgenic Microorganism <130> P01-0768 <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 355 <212> DNA <213> Rhodococcus erythropolis <400> 1 gaattcgagc tcggtacggc cgcgacgtgg ctgttgctga ccgtcctggc cgtactcctg 60 gccgagcaac cacgcttccg gcgcgccttg caacgggaga tggtggtcta ccgcgaagaa 120 accaaccact tgcaggcgac cattcagcac aattttttct gtcttgacag atttgattgt 180 cggtgggatg gttttctcat gttggaagtt gcagtcatcg atcgtcccgc agctgccgag 240 gccgcgctcg atccgatccg ccaacggatt ctggctgaac tggcagtccc ggcatcagca 300 tcctcgctgg caccccgagt gggcttgacg cgccagaaga tcaattcgat ctaga 355 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 2 cctaggcctg tctcttatac acatctcaac catcatcgat g 41 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 cttaaggcct gtctcttata cacatctcgt taaccctgaa gc 42 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 ctagcttcac gctgccgcaa gcactcaggg cgc 33 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 cgaaccccag agtcccgctc agaagaactc gtc 33 <210> 6 <211> 4448 <212> DNA <213> Rhodococcus erythropolis <400> 6 aaaggacgca tacgcgatga ctcaacaacg acaaatgcat ctggccggtt tcttctcggc 60 cggcaatgtg actcatgcac atggggcgtg gcggcacacg gacgcgtcga atgactttct 120 gtcggggaag tactaccaac acatcgcccg tactctggag cgcggcaagt tcgatctgtt 180 gtttctgcct gacgggttgg ccgtcgagga cagctacggg gacaacctgg acaccggtgt 240 cggcctgggc gggcagggtg cagtcgcctt ggagccggcc agtgtggtcg caaccatggc 300 cgcggtgacc gagcacctgg gtcttggggc aaccatttcg gcgacctact atcccccgta 360 tcacgttgct cgggtgttcg cgacgctcga tcagttgtca gggggtcggg tgtcctggaa 420 cgtcgtcacc tcgctcaacg acgctgaagc gcgcaacttc ggcattaatc agcatctgga 480 acacgacgcc cgctatgacc gcgccgatga gttcttggaa gcggtcaaga aactctggaa 540 cagctgggac gaggacgccc tcgtgctgga caaggcggcc ggcgtgttcg ccgatcccgc 600 gaaggtgcac tacgtcgatc accacgggga gtggctgaat gtgcgcggac ctctgcaggt 660 accgcgttca cctcagggtg agccggtgat cctgcaggcc ggcctgtcgc cccggggtcg 720 gcgcttcgcc gggaagtggg ccgaggccgt cttcagtctt gcacccaacc tcgaggtgat 780 gcaggccacc taccagggca tcaaagccga ggtcgacgct gcggggcgcg atcccgatca 840 gacgaaaatc ttcaccgccg tgatgccggt actcggcgaa agccaggcgg tggcacagga 900 acgactggaa tatctcaaca gtctggtcca tccggaagtg ggactgtcga cgctatccag 960 tcacaccggc atcaacctgg cggcgtaccc tctcgacact ccgatcaagg acatcctgcg 1020 ggatctgcag gatcggaatg tcccgacgca actgcacatg ttcgccgccg caacgcacag 1080 cgaagagctc acgctggcgg aaatgggtcg gcgctatgga accaacgtgg ggttcgttcc 1140 tcagtgggcc ggtaccgggg agcagatcgc tgacgagctg atccgccact tcgagggcgg 1200 cgccgcggat ggtttcatca tctctccggc cttcctgccg ggctcctacg acgagttcgt 1260 cgaccaggtg gttccggttc tgcaggatcg cggctacttc cgcaccgagt accagggcaa 1320 cactctgcgc gaccacttgg gtctgcgcgt accacaactg caaggacaac cttcatgaca 1380 agccgcgtcg accccgcaaa ccccggttca gaactcgatt ccgccatccg cgacacactg 1440 acctacagca actgcccggt acccaacgct ctgctcacgg catcggaatc gggcttcctc 1500 gacgccgccg gcatcgaact cgacgtcctc agcggccagc agggcacggt tcatttcacc 1560 tacgaccagc ctgcctacac ccgttttggg ggtgagatcc cgccactgct cagcgagggg 1620 ttgcgggcac ctgggcgcac gcgtctactc ggcatcaccc cgctcttggg gcgccagggc 1680 ttctttgtcc gcgacgacag cccgatcaca gcggccgccg accttgccgg acgtcgaatc 1740 ggcgtctcgg cctcggcaat tcgcatcctg cgcggccagc tgggcgacta cctcgagttg 1800 gatccctggc ggcaaacgct ggtagcgctg ggctcgtggg aggcgcgcgc cttgttgcac 1860 acccttgagc acggtgaact gggtgtggac gacgtcgagc tggtgccgat cagcagtcct 1920 ggtgtcgatg ttcccgctga gcagctcgaa gaatcggcga ccgtcaaggg tgcggacctc 1980 tttcccgatg tcgcccgcgg tcaggccgcg gtgttggcca gcggagacgt tgacgccctg 2040 tacagttggc tgccctgggc cggggagttg caagccaccg gggcccgccc agtggtggat 2100 ctcggcctcg atgagcgcaa tgcctacgcc agtgtgtgga cggtcagcag cgggctggtt 2160 cgccagcgac ctggccttgt tcaacgactg gtcgacgcgg ccgtcgacgc cgggctgtgg 2220 gcacgcgatc attccgacgc ggtgaccagc ctgcacgccg cgaacctggg cgtatcgacc 2280 ggagcagtag gccagggctt cggcgccgac ttccagcagc gtctggttcc acgcctggat 2340 cacgacgccc tcgccctcct ggagcgcaca cagcaattcc tgctcaccaa caacttgctg 2400 caggaacccg tcgccctcga tcagtgggcg gctccggaat ttctgaacaa cagcctcaat 2460 cgccaccgat aggaacatcc gcatgacact gtcacctgaa aagcagcacg ttcgaccacg 2520 cgacgccgcc gacaacgatc ccgtcgcggt tgcccgtggg ctagccgaaa agtggcgagc 2580 caccgccgtc gagcgtgatc gcgccggggg ttcggcaaca gccgagcgcg aagacctgcg 2640 cgcgagcggc ctgctgtcgc tcctcgtccc gcgcgaatac ggcggctggg gcgcagactg 2700 gcccaccgcc atcgaggtcg tccgcgaaat cgcggcagcc gatggatctt tgggacacct 2760 gttcggatac cacctcacca acgccccgat gatcgaactg atcggctcgc aggaacaaga 2820 agaacacctg tacacccaga tcgcgcagaa caactggtgg accggaaatg cctccagcga 2880 gaacaacagc cacgtgctgg actggaaggt cagcgccacc ccgaccgaag acggcggcta 2940 cgtgctcaat ggcacgaagc acttctgcag cggcgccaag gggtcggacc tgctgttcgt 3000 gttcggcgtc gtccaggatg attctccgca gcagggtgcg atcattgctg ccgctatccc 3060 gacatcgcgg gctggcgtta cgcccaacga cgactgggcc gccatcggca tgcggcagac 3120 cgacagcggt tccacggact tccacaacgt caaggtcgag cctgacgaag tgctgggcgc 3180 gcccaacgcc ttcgttctcg ccttcataca atccgagcgc ggcagcctct tcgcgcccat 3240 agcgcaattg atcttcgcca acgtctatct ggggatcgcg cacggcgcac tcgatgccgc 3300 cagggagtac acccgtaccc aggcgaggcc ctggacaccg gccggtattc aacaggcaac 3360 cgaggatccc tacaccatcc gctcctacgg tgagttcacc atcgcattgc agggagctga 3420 cgccgccgcc cgtgaagcgg cccacctgct gcagacggtg tgggacaagg gcgacgcgct 3480 cacccccgag gaccgcggcg aactgatggt gaaggtctcg ggagtcaaag cgttggccac 3540 caacgccgcc ctcaacatca gcagcggcgt cttcgaggtg atcggcgcgc gcggaacaca 3600 tcccaggtac ggtttcgacc gcttctggcg caacgtgcgc acccactccc tgcacgaccc 3660 ggtgtcctac aagatcgccg acgtcggcaa gcacaccttg aacggtcaat acccgattcc 3720 cggcttcacc tcctgaggat ctgaggcgct gatcgaggcc gaggccaccg cgcggccgag 3780 tcgcgaatcg cccgccgata ctcagcttct ccatacggat ccgcggccgc cgaattcgat 3840 aattcaggaa catccatgtc tgacaagccg aatgccgttt ccagccacac cacccccgac 3900 gtccccgaag tagcggcgac gcccgagttg tccaccggca tctgcgccgg tgactaccgc 3960 gctgcgcttc gccgccaccc cgccggtgtc accgtcgtga ccctcgattc gggtaccggc 4020 ccggtgggtt tcaccgccac ctcgttctcg tccgtctccc tcgagccgcc gctcgtctcg 4080 ttcaacatcg cggagacgtc gtcgagcatc aatgcactca aggcagccga gtccttggtg 4140 atccaccttc tcggcgaaca tcagcagcat ctggcccagc gctttgcgcg tagtgccgat 4200 cagcgttttg cagacgagtc actgtgggca gtgctcgaca ccggggaacc ggtgctgcac 4260 ggcaccccca gctggatgcg cgtcaaggtc gaccagctga tccctgtcgg cgaccacacg 4320 ctggtcatcg gactcgtcac gcgggttcac gccgaagaag acgacgaatc cgctgccgcg 4380 ccgctgctct accacgaggg caagtactac cgcccgactc cgttaggtca ataggaatta 4440 tcaagctt 4448

[0095]

[Sequence list free text] SEQ ID NO: 2: primer SEQ ID NO: 3: primer SEQ ID NO: 4: primer SEQ ID NO: 5: primer

[Brief description of drawings]

FIG. 1 is a graph showing the results of a desulfurization reaction test on alkyl DBT by Rhodococcus erythropolis MC0203 strain.

FIG. 2 is a graph showing the results of a desulfurization activity test of F light oil by Rhodococcus erythropolis MC0203 strain.
(A: Before desulfurization b: After desulfurization)

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kenji Maruhashi             1-6-1 Nishikubo, Shimizu City, Shizuoka Prefecture             Yoneju A-201 F-term (reference) 4B024 AA17 BA08 CA02 DA05 HA20                 4B050 CC03 DD02 EE10 LL10                 4B065 AA01X AA01Y AA36X AA38X                       AA38Y AB01 AC14 AC20                       BA02 CA27 CA54 CA56

Claims (9)

[Claims] 1. A method for desulfurizing a sulfur-containing heterocyclic compound, which uses a recombinant microorganism containing a desulfurase enzyme gene downstream of a promoter composed of the following DNA (a) or (b). (a) D having the promoter activity shown in SEQ ID NO: 1
NA. (b) A DNA which hybridizes with a DNA having a base sequence complementary to the DNA represented by SEQ ID NO: 1 under stringent conditions and has a promoter activity. 2. The method according to claim 1, wherein the desulfurase enzyme gene is a desulfurase enzyme gene that selectively cleaves a CS bond of a sulfur-containing heterocyclic compound. 3. The desulfurization enzyme gene is Rhodococcus IG
Desulfurization gene from TS8 strain, desulfurization gene from Rhodococcus sp.KA2-5-1 strain, Sphingomonas sp. AD10
Degradation genes from 9 strains, Paenibacillus
The method according to claim 2, which is at least one gene selected from the group consisting of desulfurization genes derived from the genera A11-1 and A11-2. 4. The desulfurization enzyme gene is Rhodococcus sp. KA
The desulfurization enzyme gene dszABCD derived from the 2-5-1 strain, 4.
The method described. 5. The method according to claim 1, wherein the recombinant microorganism uses a Nocardioform-type bacterium as a host. 6. The method according to claim 1, wherein the recombinant microorganism uses a microorganism belonging to any one selected from the group consisting of Rhodococcus, Mycobacterium and Gordonia as a host. The method described. 7. The method according to claim 1, wherein the sulfur-containing heterocyclic compound is at least one compound selected from the group consisting of benzothiophene, dibenzothiophene and derivatives thereof. 8. The method according to claim 1, wherein the sulfur-containing heterocyclic compound is at least one compound selected from the group consisting of alkylated benzothiophenes and alkylated dibenzothiophenes. Method. 9. A recombinant microorganism containing the promoter of SEQ ID NO: 1 and a desulfurization enzyme gene, Rhodococcus erythropolis MC0203 strain (FERM P-18595).