JP2002142779A - Recombinant microorganism having action of decreasing malodor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To construct a stable recombinant microorganism by transducing, into a microorganism, a gene capable of degrading hydrogen sulfide that is a typical compound that may cause malodor of feces, and to continuously decrease the malodor of feces by orally administrating the microorganism to an animal to make the microorganism inhabit in its intestine. SOLUTION: The objective recombinant microorganism having an activity of removing hydrogen sulfide was created, wherein sulfide-quinone oxidoreductase gene sqr is operably integrated into a gene region, which does not give any effect to its proliferation by a promoter of Escherichia coli, of the chromosome of E. coli.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the production of a genetically modified microorganism that reduces the odor of feces of livestock, poultry, or pets. More specifically, the present invention relates to the production of genetically modified microorganisms that reduce the odor of feces and improve the environment by removing hydrogen sulfide, which is a major cause of odors in feces.

[0002]

2. Description of the Related Art In recent years, in the livestock industry, the breeding environment has become overcrowded in order to improve the productivity of livestock and poultry such as cattle, pigs and chickens. Under such circumstances, odor caused by feces excreted by livestock and poultry is a serious problem in terms of living environment. In recent years, since the pet boom, in general households, pets have been actively bred indoors, and the odor of feces excreted by pets has become a problem.

[0003] The main compounds responsible for the fecal odor are:
Ammonia, hydrogen sulfide, short-chain fatty acids, and the like produced by spoilage fermentation of proteins by harmful bacteria living in the intestine (Shahak, Y. et al .: FEBS Le).
tt. , 299: 127-130, 1992). Therefore, if these compounds remaining in the intestinal contents of animals can be artificially decomposed, it will be an effective means for reducing the above-mentioned odor in animal husbandry. Thus, sulfide-quinone oxidoreductase (sulfide-quinone o)
xidoreductase (SQR) gene sqr
And the ubiquinone-8 (UQ-8) gene were transformed into E. coli E. coli .
Attempts have been made to increase the SQR active sulfide by introducing it into the E. coli cytoplasm and expressing it (Shibata, He.
t al. : J. Biosci. Bioengin. ,
88: 244-249, 1999). However, a problem has arisen in that enzymatic conditions that allow a series of electron transfer reactions in cells must be satisfied.

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to a stable recombinant recombinant gene obtained by introducing a gene capable of decomposing ammonia, hydrogen sulfide, and the like, which are typical compounds that cause odor in feces. By constructing microorganisms (hereinafter also referred to as "recombinants") and orally giving the microorganisms to animals and allowing them to settle in the intestine, the odor of feces is permanently reduced, and the intestinal flora The task is to improve

[0005]

As the first step, an attempt was made to remove hydrogen sulfide, one of the malodorous substances remaining in the animal intestinal tract. Rho , one of the red non-sulfur bacteria
doctor capsulatus (Schut
z, M .; et al. : J. Biol. Chem. , 2
72: 9890-9894, 1997) was used to construct the recombinant of the present invention. R. cap
The sqr gene isolated from Sulatus was 1,27
It has an open reading frame of 5 bp and 42
It encodes an SQR consisting of 5 amino acids and having a molecular weight of 55 kDa (Schuetz, M. et al .: J. Bio).
l. Chem. , 272: 9890-9894, 199.
717; Schuetz, M .; et al. : J. Ba
cterol. , 6516-6523, 1999 2
3). This sqr gene was transformed into R. Capsulatus was isolated, and many or several of them were introduced into the cytoplasm of Escherichia coli or into the chromosome using a plasmid vector.

[0006] As a result, the SQR activity was increased by the recombinant containing the sqr gene with the lac promoter or the tac promoter, as well as by the sqr gene with the similar promoter.
It was significantly increased in recombinants containing 2-3 qr genes in the chromosome. In these recombinants, ta
SQR activity expression by the c promoter was stronger than that of the lac promoter. The degree of sulfide removal is
It increased linearly with increasing SQR enzyme activity. This linearity was lost when sulfide removal exceeded 700 nmol / mg cell protein / min. During the same reaction period,
Oxygen consumption also increased with increasing SQR enzyme activity.

[0007] When these results are combined, t
A few copies of the sqr gene integrated with the ac promoter were most preferred for sulfide removal and recombinant growth. As a result of culturing the recombinant up to 160 generations, a recombinant WL2 in which two copies of the sqr gene were incorporated together with the lac promoter, a recombinant WL3 in which three copies were incorporated, and two sqr genes together with the tac promoter were introduced into the chromosome. The recombinant WT2 had the same SQR activity as that of the primary culture. A large number of plasmids incorporating the sqr gene (each about 300
And WT20), the SQR activity decreased below the detection limit.

[0008] That is, the present invention provides (1) one or more isolated sulfide-quinone oxidoreductase genes sqr that can be operable with a promoter of a microorganism at a gene region that does not affect the growth in the chromosome of the microorganism. (2) a recombinant microorganism having a LacZ gene in a gene region that does not affect growth, (3) a recombinant microorganism having a promoter that is a tac promoter, A microorganism, (4) a recombinant microorganism of (1) or (3) having at least two copies of the sqr gene operable by a tac promoter, and (5) a recombinant of (1) wherein the sqr gene is derived from a red non-sulfur bacterium. Microorganisms, (6) Red non-sulfur bacteria are Rhodobacter
the recombinant microorganism of (5), which is capsulatus ;
(7) The recombinant microorganism of (1), wherein the microorganism is Escherichia coli W3110 strain, and (8) an effect of reducing the odor of excreted feces of livestock containing any of the recombinant microorganisms of (1) to (7) as an active ingredient. Feed, (9) the feed of (8), wherein the livestock is a pig, and (10) the recombinant of any one of (1) to (8) for producing a feed having an effect of reducing bad smell of excreted feces of livestock Use of microorganisms, (11) Domestic animals are pigs (1
0).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The chromosomal DNA containing the sqr gene used in the present invention can be isolated from a microorganism exhibiting SQR activity by a conventional method (Shibata, He.
t al. : J. Biosci. Bioengin. ,
88: 244-249, 1999). Examples of the microorganisms include, for example, P. denitrific
and (Schneider, A. and Fried
rich, C.E. G. FIG. FEBS Lett. , 350: 61
-65, 1994), C.I. vinosum (Reina
rtz, M .; et al. : Arch. Microbi
ol. , 170: 59-68, 1998), C.I. lim
icola (Shahak, Y. et al .: FEB
S Lett. , 299: 127-130, 1992).
And the like.

Chromosomal DNA containing the isolated sqr gene
Is used as a template to perform a PCR primer reaction to amplify the sqr gene. For design of sense and antisense primers for PCR, restriction enzyme sites are added in advance for convenient subcloning.

The DNA sequence of the sqr gene of the present invention is described, for example, in the literature (Schuetz, M. et al .:
J. Biol. Chem. , 272: 9890-989.
4, 1997), and a DNA sequence functionally equivalent to this DNA sequence is also included in the present invention. "Functionally equivalent DNA sequence"
The term refers to a DNA sequence that expresses an enzyme that has the biological function of SQR, for example, sulfide removal activity. For example, mutations that do not affect the biological function of SQR can be introduced into the DNA sequence. Generally, the nucleic acid sequence encoding a polypeptide can vary widely without affecting the function of the expression product. It is thought that an amino acid sequence having substantial homology (an amino acid sequence having a homology of more than 50% of the whole) can already exert the same function.

In recent years, software for analyzing various sequences has been released on the Internet, and by using them, specific sequences have sufficient homology to be functionally equivalent. It has become possible to predict whether or not. Therefore, those skilled in the art will understand that sqr
Certain portions of the gene can be modified to obtain an expression product that is functionally equivalent to SQR.

[0013] The microorganism having sulfide removal activity of the present invention contains a gene having SQR activity and a promoter that activates the gene in a chromosome that does not affect the growth of the microorganism. Plasmids that cannot replicate outside the chromosome of the recipient strain are known to rejoin the chromosome if they have a homologous sequence to the host chromosome (Hamilton, CM et al .: J. Am. ba
cteriol, 171: 4617-4622,198
9).

[0014] In view of the above, an expression vector which can be operated based on a rapid change in temperature and which can select between opposing gene conversion and plasmid removal is used. For example, E. coli β-galactosidase gene (La
As a vector for incorporating the sqr gene into the cZ gene, pBRINT-Ts (Borgne, S.L.
L. et al. : Gene, 223: 213-21
9, 1998) will be described as an example. The introduction into Escherichia coli is performed, for example, by the method of Hanahan (D. Hanahan, J. Mol. Biol., 16).
6: 557, 1983) or electroporation (WJ Dower, et al .: Nucl.
eic Acids Res. , 16: 6127, 19
88).

This vector contains the LacZ gene, kanamycin gene (Km), chloramphenicol (Cm) or gentamicin (Gm) as a selectable marker (antibiotic resistance) gene, temperature-sensitive replicon Ts, and a multiple cloning site ( MCS). The characteristics of these vectors are: 1) the integration site into the chromosome is in the LacZ gene, and blue-white selection on a solid medium containing X-Gal and IPTG is possible; 2) MCS is an antibiotic resistance gene cassette ( Km, Cm, or Gm)
The insertion of the cloned fragment can be easily monitored;
3) Carbenicillin (C
4) pSC instead of the ori of pBR322, which replicates at 30 ° C. but cannot replicate at 37 ° C.
Having a temperature sensitive (Ts) replicon from 101,
And the like. Lac in pBRINT-Ts derivative
The Z gene fragment and the Ts replicon are
By allowing direct selection of foreign gene integration into the Z gene, and subsequently increasing the culture temperature to 44 ° C.,
Extrachromosomal plasmids can be removed.

That is, transformants are selected based on the resistance to Cb present in the vector portion and the resistance to an antibiotic (Km, Cm or Gm) inserted into the LacZ gene. Incubate at 44 ° C. on a plate containing X-Gal, IPTG, and the appropriate selection agent (antibiotic). Large colonies of antibiotic resistance appear, which are due to the LacZ present on the chromosome.
This indicates that the plasmid was integrated into the gene.
In one recombination event, a clone is produced that is resistant not only to the resistance marker inserted in the LacZ gene but also to the resistance marker encoded in the vector portion, whereas in an allelic recombination event, It is necessary to examine the susceptibility to Cb on white colonies, as they only show colony production showing the resistance marker inserted in the LacZ gene.

The colonies selected above are also sensitive to Cb at the permissive temperature of 30 ° C., which indicates that the plasmid has been lost. The absence of these vectors in the cells can be confirmed by isolating the plasmid from these colonies, developing on an agarose gel, and staining with ethidium bromide (EtdBr). Furthermore, whether the sqr gene was properly integrated into the LacZ gene present on the chromosome,
DNA can be extracted from the transformant, fragmented with restriction enzymes, separated by electrophoresis, and confirmed by Southern hybridization using an sqr gene probe.

In the homologous recombination used in the present invention, sqr
The gene is E. coli. Since it was integrated into the lacZ gene of the E. coli chromosome, the disruption of other genes present on the chromosome can be avoided. Further, the integrated gene is stably retained. As the host E. coli used for the transformation, in addition to the above-mentioned strain W3110, the homologous recombination gene re
recA1 strains DH1, DH5, DH10B with cA,
JM109 or XL-Blue may be used.

By the way, in human and animal intestines, 100
It constitutes trillion and 100 species of intestinal flora (intestinal flora). However, E. coli is not a major intestinal bacterium. Thus, intestinal resident bacteria are more effective as targets for incorporating the sqr gene. Intestinal resident bacteria having a lactose operon are suitable as the microorganisms into which the sqr gene of the present invention is to be integrated. There is a restriction system problem, but it is possible in principle. Therefore, the recombinant of the present invention
Including these intestinal resident bacteria.

In this invention, the integration of multiple copies of the sqr gene into the chromosome, the correlation between SQR activity and sulfide removal, and the stability of the chromosome-integrated sqr gene have been elucidated. However, the Km-gene is DN
Left in the A sequence. To remove genes resistant to antibiotics, for example, a recombination system (Morales, FM et al.) Into which FRT-recombinase (flippase) is introduced.
al. : J. Bacteriol. , 181: 714
3-7148, 1999) can be effectively used.

In the present invention, hydrogen sulfide which is one of the main malodorous substances remaining in the intestinal contents was targeted.
Another major malodorous substance is the oxidase gene for ammonia (amoA, amoB (Mctavish, He.
t al. : J. Bacteriol. , 175: 24
36-2444, 1993), amoC (Klotz,
M. G. FIG. , Et al. : FEMS Microbio
l. Lett. , 150: 65-73, 1997), h
ao (Sayavedra-soto, LA et al.
al. : J. Bacteriol. , 176: 504-
510, 1994)) can be produced by a person skilled in the art based on the method of the present invention. Technical modifications therefor are included in the present invention. Therefore, the present invention also includes a recombinant containing the oxidase gene for ammonia in the chromosome.

The bacteria in the culture solution are separated and washed according to a conventional method (eg, centrifugation). Separated cells concentrate comprises E. coli ¹⁰ 7 ^~10 9 / g, the feed, body weight
0.1 to 10 mL / kg per day, preferably 1 to 4 m
Add L. Alternatively, after adding a protective agent (for example, skim milk, lactose, starch, etc.), the pH may be adjusted, and the resultant may be used as a cell preparation prepared by drying the water to 4% or less with a vacuum freeze dryer or the like. The preparation can be in the form of powder, granules, pellets, or tablets.

The recombinant bacterium of the present invention may be added to livestock feed as it is, or may be used in combination with other nutrients such as oligosaccharides, zeolites, catechins and polyphenols. Good.

[0024]

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] Construction of chromosome integration vector For construction of all plasmids, E. coli E. coli was used . coli strain X
L1Blue-MRF 'was used. Escherichia coli is cultured in LB medium, and if necessary, carbenicillin (Cb) 50 μl is used.
g / ml and 30 μg / ml of kanamycin (Km).

1) Preparation of sqr gene The sqr gene can be prepared by the method described in the literature (Shibata,
H. et al. : J. Biosci. Bioengi
n. , 88: 244-249, 1999). R. A chromosomal DNA containing the sqr gene was extracted from capsulatus DSM155 by a conventional method, and using this as a template, the sqr gene was amplified by PCR using a sense primer (SEQ ID NO: 1) and an antisense primer (SEQ ID NO: 2). PCR cycle Initial 94 ° C, 3 minutes 1 cycle Thermal denaturation 98 ° C, 20 seconds Annealing 58 ° C, 1 minute DNA synthesis 68 ° C, 2 minutes After completion of 30 cycles from thermal denaturation to DNA synthesis 72 ° C, 10 minutes 1 cycle PCR product is Nde Cleavage with I (present at the start codon) and BamHI (incorporated in the antisense primer) gave the sqr gene fragment (1.3 kbp).

2) Preparation of plasmid having sqr gene under control of lac promoter Plasmid pKF18 (Ha
Shimoto, T .; , Et al. : Gene, 16
7: 333-334, 1995) with Nde I and Ba.
Cleavage with mHI, the sqr gene fragment (1.3 kb)
Ligation with p) resulted in plasmid pFSQ containing the sqr gene expressed by the lac promoter. This pFSQ was digested with ApaL I and blunt-ended with T4 polymerase, followed by Sac I or Xb
aI digestion and sqr downstream of the lac promoter.
A DNA fragment (1.7 kbp) containing the gene was obtained. Plasmid pSTV28 (Amersham Pharma
Cia Biotech) with Sac I and Sma
C. and ligated with the above DNA fragment (1.7 kbp) to obtain plasmid pV-LSQ. This pV
-LSQ contains the sqr gene under the control of the lac promoter from pFSQ. Also, pBluescript
t Sk (+) is digested with Sma I and Xba I, ligated with the above DNA fragment (1.7 kbp),
The plasmid pSc-LSQ was obtained.
Sq under the control of the lac promoter from pFSQ
r gene.

3) Preparation of plasmid having sqr gene under control of tac promoter pKK223-3 (Amer
sham Pharmacia Biotech)
Was digested with EcoR I and Pst I, and the above sqr
After blunt-ending with T4 polymerase together with the gene fragment (1.3 kbp), ligation was performed to obtain plasmid pK-TSQ. This pK-TSQ contains the sqr gene expressed by the tac promoter. This p
K-TSQ was digested with Pvu I and BamHI, and pSTV28 was digested with Sma I, blunted with T4 polymerase, and ligated to obtain plasmid pV-TSQ. This pV-TSQ is pK-TS
Sqr controlled by the tac promoter from Q
Including genes.

4) Construction of Chromosome Integration Vector In order to construct a chromosome integration vector having sqr, pSc-L
SQ is cut with Eco RV and BamHI to obtain a sqr gene fragment (1.7 kbp) controlled by a lac promoter.
Got. Also, pV-TSQ is converted to Pvu I and Bam
Cleavage with HI resulted in a sqr gene fragment (2.5 kbp) controlled by the tac promoter. These DNA fragments 1 to 3 are converted into a p-type having a temperature-sensitive replication origin.
INT-Ts / Km (Borgne, SL et a
l. : Gene, 233: 213-219, 1998).
Multi cloning site (MCS)
pINT-LSQ, an expression vector for homologous recombination having a sqr gene controlled by a lac promoter, introduced by the pletandem insertion method
1, pINT-LSQ2 and pINT-LSQ3, t
Expression vectors pINT-TSQ1 and pINT-TSQ2 for homologous recombination having the sqr gene controlled by the ac promoter were obtained (FIG. 1).

[Example 2] Preparation of chromosome integration of sqr gene Expression vectors pINT-LSQ1, pINT-LSQ
2, pINT-LSQ3, pINT-TSQ1, and p
INT-TSQ2 was replaced with W which is considered to be close to a wild strain of E. coli.
3110. W3110 is 100 mM KNO ₃ , 1% glycerol, 0.2% in Davis minimal medium
The cells were cultured in a medium supplemented with casamino acid under nitrogen gas. W
3110 was cultured at 30 ° C. for 40 hours to obtain a strain having the above vector. Pick one colony and add 2 mL
The cells were cultured in an LB medium at 30 ° C. for 12 hours. This culture solution was diluted 1,000 times (about 2 × 10 ⁶ cells / m
L), cultured on a Km plate containing X-gal and IPTG at 44 ° C. for 20 hours. The resulting white colony is
Planted on a plate. After culturing this at 44 ° C. for 4 hours, in order to select a strain that has undergone homologous recombination by double crossover, it is subcultured on a Cb plate and sensitized to Cb (Cb
The strain of ^S ) was selected. Further, this strain was subcultured on a Km plate, cultured at 44 ° C. for 24 hours, and a strain containing the sqr gene on the chromosome was selected. This strain was subcultured on a Cb plate, and it was confirmed that the plasmid had been deleted since the bacteria did not grow. Furthermore, DNA was extracted from these finally obtained strains, and subjected to Southern hybridization analysis using a PCR DIG probe synthesis kit and a DIG nucleic acid detection kit (Boehringer Mannheim KK). Has been incorporated. The probe was constructed at nucleotides 477 to 1149 of the sqr gene (J. Biol. Che.
m. , 272: 9890-9894, 1997.
3. Of DIG (Digox)
igenin labelling kit: Boeh
ringer Mannheim Biochemical
a company). That is, the lac promoter,
A recombinant having 1-3 copies of the sqr gene downstream of this promoter was obtained. These recombinants were named WL1, WL2, and WL3, respectively. Also,
A recombinant having the tac promoter and one and two copies of sqr downstream of this promoter was obtained. These recombinants were named WT1 and WT2, respectively. Incubation of these recombinants at 44 ° C. on plates containing X-gal and IPTG resulted in 10
Large white colonies appeared at a frequency of ^-3, of which 0.5 to 5.0% had homologous recombination. The recombination frequency decreased as the inserted sequence extended.

Example 3 Propagation of Recombinants Recombinants WL1, W having the sqr gene controlled by the lac promoter in the Escherichia coli W3110 chromosome
The growth of L2 and WL3 was similar to that of a control strain with only the Km marker in the chromosome.
The density of the growing recombinant bacterium reached a maximum after about 6 hours with the given medium and culture conditions (FIG. 2). On the other hand, the growth of the recombinants WT1 and WT2 having the sqr gene controlled by the tac promoter in the Escherichia coli W3110 chromosome showed the same tendency as in the case of the lac promoter, and the control having the Km marker alone in the chromosome (FIG. 4). In addition, the growth of the recombinants WT20-30 having 20-30 plasmids pK-TSQ carrying the sqr gene in the cytoplasm is due to W
Growth was delayed compared to T1, WT2, or control strains (FIG. 4).

Example 4 Sulfide Removal by Recombinant To measure the sulfide removal activity of SQR, NaS was used as a substrate.
₂ was used. Reactions were performed at 25 ° C. unless otherwise specified, and sulfide was measured by the methylene blue method (Truepe).
r, H .; G. FIG. and Schlegel, H .; G. FIG. : A
ntonie van Leeuwen hook, 3
0: 225-238, 1964). The composition of the reaction solution was 20 mM Tris-HCl (pH 7.5),
0.5% NaCl, 5 mM MgCl ₂ , 1 mM Ca
When Cl ₂ and 200 μM Na ₂ S (substrate) were used and the cells were used, the cells were suspended at 1.5 mg protein / 100 mL. 10 mL of these reaction solutions are added to 30
After taking the sample in a mL-volume test tube, sealing it tightly with a butyl rubber stopper, placing the test tube aside, shaking it at 100 rpm for 10 minutes,
_The reaction was started by the addition of 2S. The remaining sulfide is
2% (CH ₃ C) containing diamine and FeCl ₃ solution
OO) ₂ Zn and measured at OD ₆₇₀ . The sulfide removal activity was expressed as nmol sulfide oxidized / cell protein 1 mg / min).

The protein concentration was measured using a DC protein assay kit (Biolad).
The bacterial protein concentration was determined by the method of Herbert et al. (Her
bert, D .; et al: Methods in M
microbiology Vol. 5B, p209-3
44, Academic Press, New York
k, 1971). Sqr controlled by lac promoter in chromosome
Recombinant WL2 having 1 to 3 genes or WL3
When culturing was carried out, the sulfide removal activity tended to increase until the stationary phase with the lapse of the culturing time (FIG. 3). WL
The sulfide removal activity of 3 was higher than those of WL2 and WL1. The activity of WL10 having about 10 pV-LSQs extrachromosomally showed almost constant activity regardless of the culture time. The activity of WL300, which has about 300 pSc-LSQs extrachromosomally, is due to the addition of IPTG,
There was a slight decrease in both cases where no additive was added. WL1, WL2,
The culture times at which the sulfide removal of WL3, WL10, WL300 (with IPTG added), and WL300 (without IPTG added) were maximized were 7, 6, 7, 8, 4, and 4, respectively.
And the sulfide removal activity at that time was 36, 112, 141, 337, 316, and 3 respectively.
It was 24 nmol / mg cell protein / mg. WL
The activity of 3 was almost half that of the recombinant with multiple copies of the sqr gene in the cytoplasm.

On the other hand, when WT1 or WT2 having the sqr gene controlled by the tac promoter in the chromosome was cultured, the sulfide removing activity of WT1 tended to increase gradually with the elapse of the culture time. (FIG. 5). The activity of WT2 was higher than that of WT1, but, unlike WT1, it remained almost constant over time. WT1 with about 10 pV-TSQ
WT20 with 0 and 20-30 pK-TSQs
Showed high sulfide removal activity at an early stage of the culture period, but then decreased and showed almost constant activity in the stationary phase. WT1, WT2, WT10, WT2
0 (with no IPTG) and WT20 (without IPTG)
The culture time at which the sulfide removal activity of
8, 7, 2, 2, and 3 hours, at which time the sulfide removal activity was 240, 310, 42, respectively.
3, 394 and 372 nmol / mg cell protein /
mg. In conclusion, WT2 grows without any delay in the growth of control cells (FIG. 4), has almost half the sulfide removal activity of WT20 in mid-log phase, and sq
This was extremely satisfactory in that cells having multiple copies of the r gene had activities close to the sulfide removal activity in the stationary phase.

[Example 5] Relationship between SQR activity and oxygen consumption 1) Preparation of crude extract of recombinant cells Cells obtained by centrifuging the culture solution (4,000 xg, 10 minutes, 4 ° C) Was washed once with 25 mM phosphate buffer (pH 7.4) containing 5 mM EDTA, and then centrifuged again under the same conditions to collect cells. After suspending the collected cells in the above buffer, the cells were sonicated (12 seconds × 2 times, 30 seconds apart) to pulverize the cells. For the measurement of SQR activity, 25 μL /% of 10% (v / v)
mL was added and incubated for 1 hour on ice to solubilize the SQR protein. After the incubation, the supernatant (crude extract) obtained by centrifugation (10,000 × g, 10 minutes, 4 ° C.) was used for SQR activity measurement.

2) Measurement of SQR activity The SQR activity is determined by decyl-UQ catalyzed by SQR.
Was measured at a wavelength of 275 nm. The reaction temperature was 25 ° C., and the reaction solution composition (3 mL) was 25 mM phosphate buffer (pH 6.5), 0.01% Thesit, 50%
It was prepared to be μM decyl-UQ, 100 μM Na ₂ S, and the above crude extract (10 μg protein / mL). The reaction was started by the addition of Na ₂ S.
The amount of decrease in absorption at a wavelength of 275 nm was determined by comparing the decyl-UQ with a molar extinction coefficient of 15 (Morton, RA: Bioch).
emistry of Quinones, p23-2
4, 1965).

3) Measurement of oxygen consumption due to sulfide removal The oxygen consumption due to sulfide removal was measured using an oxygen electrode (Iijima Denshi Kogyo, GU-BM). Reaction solution and Na ₂
The same S concentration as that used in the measurement of the sulfide removal activity was used. However, the cell concentration was 0.15 mg protein / mL. 2.2 mL of the reaction solution was used. After adding the cells, the mixture was kept at 25 ° C. for 10 minutes, and then the reaction was started by adding Na ₂ S.

4) SQR Activity and Oxygen Consumption The relationship between the SQR activity of the recombinant and the oxygen consumption due to the removal of sulfide was examined in each of the middle logarithmic growth phase, the late logarithmic growth phase, and the stationary phase. . When the copy number of the sqr gene is 10 or less, the lac promoter or tac
Recombinants with the sqr gene under the control of the promoter increased oxygen consumption with increasing SQR activity when the copy number of the sqr gene was 10 or less.
That is, the effect of introducing the sqr gene was clearly observed. SQR activity generally showed a decreasing trend as the culture stage progressed. Oxygen consumption also decreased at the end of the culture stage. Oxygen consumption increased almost linearly with increasing SQR activity until mid-log phase. SQR activity is about 700 nmol /
In the case of mg cell protein / min or less, the oxygen consumption is S
Increased in proportion to QR activity. However, when SQR activity exceeded 900 nmol / mg bacterial cell protein / min, oxygen consumption was no longer affected by SQR activity (FIG. 6). In this reaction, the molecular ratio of oxidized sulfide to consumed oxygen was approximately 2: 1.

Example 6 Stability of Chromosome-Integrated Strains The survival of recombinant cells in which the sqr gene was introduced into the cytoplasm or chromosome was examined on a non-selective medium up to 160 generations. The bacterial solution obtained by culturing the strain having the sqr gene on the chromosome and the strain having the plasmid having the sqr gene in a selective medium for 14 hours (18 hours for WL300 and WT20) was defined as the “0” generation (t = 0). This is 10 ³ CFU / m
After being prepared in a non-selective medium so as to be L, the cells were cultured until the stationary phase. At this point, about 20 generations have been cultured. Further, such culture was repeated seven times, and finally, about 160
Cultured to generations. The stability of the integrated strain was examined by performing sqr activity at t = 0 and t = 160, oxygen consumption accompanying sulfide removal activity, and Southern hybridization analysis.

WL30 with plasmid pSc-LSQ
WT20 cells having 0 and pK-TSQ lost the SQR activity, and the sulfide removal activity also fell below the detection limit.
WL1 with one copy of the sqr gene under control of the lac promoter in the chromosome, WL2 with two copies, WL3 with three copies, and WT1 and two copies of the sqr gene with one copy under the control of the tac promoter in the chromosome. WT2 cells have the same SQ as the 0 generation
R activity and sulfide removal activity were retained (Table 1). The sqr sequence in the chromosome of the above-mentioned 160 generation recombinant cells is:
Confirmed by Southern blotting.

[0041]

[Table 1]

[0042]

The recombinant having the activity of removing hydrogen sulfide of the present invention is added to livestock feed to remove hydrogen sulfide, which remains in the intestine and is a main cause of malodor, and as a result, excreted feces It can be expected that odor is reduced. Further, when used in combination with other deodorant substances (for example, oligosaccharides, zeolites, catechins, etc.), a further deodorant effect can be expected.

[0043]

[Sequence list]

[Brief description of the drawings]

FIG. 1. 1-3 copies of the sqr gene are found in E. coli W31.
FIG. 3 is a diagram showing the construction of a W3110 derivative integrated into chromosomes of 10 strains. The sqr gene fragment is EcoR V
(PINT-LSQ1), BamHI (pINT-L
SQ2), XbaI (pINT-LSQ3), EcoR
I (pINT-TSQ1) or SmaI (pI
After NT-TSQ2) digestion, the ends were blunted with T4 polymerase and introduced one by one into the multicloning site (MCS) of pINT-Ts / Kmr.

FIG. 2 shows the growth pattern of a W3110 derivative with several copies of the sqr gene under the control of the lac promoter. Sqr with 1 mM (final concentration) IPTG
Gene expression was induced. Copy number is manfactor
ure's report (QIAGENProduct
Guide, 1999). □: 1 copy, ●: 2 copies, △: 3 copies, ■: about 10 copies,
◆: 300-500 copies, ○: 300-500 copies
^* . ^* : The medium does not contain IPTG.

FIG. 3 shows sulfide removal of W3110 derivative whole cell extract with several copies of the sqr gene under the control of the lac promoter. □: 1 copy, ●: 2 copies, △: 3 copies, ■: about 10 copies, ◆: 300-5
00 copies, ：: 300 to 500 copies ^* . ^* : The medium does not contain IPTG.

FIG. 4 shows the proliferation pattern of a W3110 derivative having several copies of the sqr gene under the control of the tac promoter. Sqr with 1 mM (final concentration) IPTG
Gene expression was induced. Copy number is manfactor
ure's report (QIAGENProduct
Guide, 1999). Δ: 1 copy, ○: 2 copies, □: 10 copies, ●: 20 to 30 copies, Δ: 20 to 30 copies ^* . ^* : The medium does not contain IPTG.

FIG. 5 shows sulfide removal of W3110 derivative whole extract having several copies of sqr gene under the control of tac promoter. △: 1 copy, ○: 2 copies,
□: 10 copies, ●: 20-30 copies, Δ: 20-3
0 copies ^* . ^* : The medium does not contain IPTG.

FIG. 6 is a graph showing the relationship between the SQR enzyme activity and the amount of oxygen consumed in the crude extract of whole cells. Low SQR activity is
L1, WL2, WL3, WT1 and WT2 were obtained. High SQR activity is particularly significant in late log phase WT1
0 and WT20 strains. □: middle logarithmic growth phase, ○: late logarithmic growth phase, Δ: stationary phase.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12N 9/02 C12R 1:01) (C12N 15/09 ZNA (C12N 1/20 E C12R 1:01) ) C12R 1:19) (C12N 1/20 (C12N 1/21 C12R 1:19) C12R 1:19) (C12N 1/21 C12N 15/00 ZNAA C12R 1:19) C12R 1:01) F term (reference) ) 2B005 EA01 2B150 AA01 AA03 AA05 AA06 AB12 AC01 AC37 DC13 DD11 DH21 4B024 AA10 AA17 BA08 CA03 DA06 EA04 FA02 FA03 FA04 GA11 4B050 CC04 DD02 LL10 4B065 AA01Y AA26X AB01 AC14 BA02 CA43 CA54

Claims (11)

[Claims] 1. A hydrogen sulfide-removing activity in which one or more isolated sulfide-quinone oxidoreductase genes sqr are operably integrated with a promoter of a microorganism in a gene region that does not affect growth in the chromosome of the microorganism. A recombinant microorganism having: 2. A gene region that does not affect growth is LacZ.
The recombinant microorganism according to claim 1, which is a gene. 3. The recombinant microorganism according to claim 1, wherein the promoter is a tac promoter. 4. The recombinant microorganism according to claim 1, which has at least two copies of the sqr gene operable by a tac promoter. 5. The recombinant microorganism according to claim 1, wherein the sqr gene is derived from a red non-sulfur bacterium. 6. The red non-sulfur bacterium is Rhodobacte.
The recombinant microorganism according to claim 5, which is r capsulatus . 7. The recombinant microorganism according to claim 1, wherein the microorganism is Escherichia coli strain W3110. 8. A feed containing the recombinant microorganism according to any one of claims 1 to 7 as an active ingredient and having an action of reducing foul odor of excreted feces of livestock. 9. The feed according to claim 8, wherein the livestock is a pig. 10. Use of the recombinant microorganism according to any one of claims 1 to 8 for producing a feed having an action of reducing malodor of feces excreted by livestock. 11. Use according to claim 10, wherein the livestock is a pig.